Postnatal Pdzrn3 deficiency causes acute muscle atrophy without alterations in endplate morphology.

PDZ domain-containing RING finger family protein 3 (PDZRN3) is expressed in various tissues, including the skeletal muscle. Although PDZRN3 plays a crucial role in the terminal differentiation of myoblasts and synaptic growth/maturation in myogenesis, the role of this molecule in postnatal muscles is completely unknown despite its lifelong expression in myofibers. In this study, we aimed to elucidate the function of PDZRN3 in mature myofibers using myofiber-specific conditional knockout mice. After tamoxifen injection, PDZRN3 deficiency was confirmed in both fast and slow myofibers of Myf6-CreERT2; Pdzrn3flox/flox (Pdzrn3mcKO) mice. Transcriptome analysis of the skeletal muscles of Pdzrn3mcKO mice identified differentially expressed genes, including muscle atrophy-related genes such as Smox, Amd1/2, and Mt1/2, suggesting that PDZRN3 is involved in the homeostatic maintenance of postnatal muscles. PDZRN3 deficiency caused muscle atrophy, predominantly in fast-twitch (type II) myofibers, and reduced muscle strength. While myofiber-specific PDZRN3 deficiency did not influence endplate morphology or expression of neuromuscular synaptic formation-related genes in postnatal muscles, indicating that the relationship between PDZRN3 and neuromuscular junctions might be limited during muscle development. Considering that the expression of Pdzrn3 in skeletal muscles was significantly lower in aged mice than in mature adult mice, we speculated that the PDZRN3-mediated muscle maintenance system might be associated with the pathophysiology of age-related muscle decline, such as sarcopenia.

Identification of Potential Blood-Based Biomarkers for Frailty by Using an Integrative Approach.

INTRODUCTION: Although frailty is a geriatric syndrome that is associated with disability, hospitalization, and mortality, it can be reversible and preventable with the appropriate interventions. Additionally, as the current diagnostic criteria for frailty include only physical, psychological, cognitive, and social measurements, there is a need for promising blood-based molecular biomarkers to aid in the diagnosis of frailty. METHODS: To identify candidate blood-based biomarkers that can enhance current diagnosis of frailty, we conducted a comprehensive analysis of clinical data, messenger RNA-sequencing (RNA-seq), and aging-related factors using a total of 104 older adults aged 65-90 years (61 frail subjects and 43 robust subjects) in a cross-sectional case-control study. RESULTS: We identified two candidate biomarkers of frailty from the clinical data analysis, nine from the RNA-seq analysis, and six from the aging-related factors analysis. By using combinations of the candidate biomarkers and clinical information, we constructed risk prediction models. The best models used combinations that included skeletal muscle mass index measured by dual-energy X-ray absorptiometry (adjusted p = 0.026), GDF15 (adjusted p = 1.46E-03), adiponectin (adjusted p = 0.012), CXCL9 (adjusted p = 0.011), or apelin (adjusted p = 0.020) as the biomarker. These models achieved a high area under the curve of 0.95 in an independent validation cohort (95% confidence interval: 0.79-0.97). Our risk prediction models showed significantly higher areas under the curve than did models constructed using only basic clinical information (Welch's t test p < 0.001). CONCLUSION: All five biomarkers showed statistically significant correlations with components of the frailty diagnostic criteria. We discovered several potential biomarkers for the diagnosis of frailty. Further refinement may lead to their future clinical use.

Association between Circulating Levels of CXCL9 and CXCL10 and Physical Frailty in Older Adults.

INTRODUCTION: Dysregulation of pro-inflammatory chemokines is considered a potential mechanism for the development of age-related medical conditions such as frailty. However, evidence linking circulating chemokines with frailty remains lacking. MATERIALS AND METHODS: We performed a case-control study including 48 cases and 48 controls aged 65-90 years, using the National Center for Geriatrics and Gerontology outpatient registry data. Cases were outpatients with physical frailty and low habitual daily activity. Controls were robust outpatients who performed habitual daily activities. The Japanese version of the Cardiovascular Health Study criteria was used to diagnose physical frailty, and the modified Baecke questionnaire was used to evaluate habitual daily activities. Serum CXCL9 and CXCL10 levels were measured using enzyme-linked immunosorbent assay. RESULTS: The median age (interquartile range) in cases and controls was 78 (73-83) and 76 (72-80) years, with the proportions of men were 47.9% and 43.8%, respectively. In the logistic regression model with adjustment for age, sex, and other confounding factors, the multivariable odds ratios (95% confidence intervals) for the highest versus lowest tertile of CXCL9 and CXCL10 levels were 7.90 (1.61-49.80) and 1.61 (0.42-6.30), respectively. However, we did not observe a linear association between CXCL9 levels and physical frailty components. DISCUSSION/CONCLUSION: Our preliminary data exhibit that circulating CXCL9 levels were positively associated with the odds of physical frailty. However, these findings lack evidence of a dose-response relationship between CXCL9 levels and physical frailty components. Further research with a larger sample size is required to confirm these findings.

Treatment of Cutaneous Ulcers with Multilayered Mixed Sheets of Autologous Fibroblasts and Peripheral Blood Mononuclear Cells.

BACKGROUND/AIMS: We have developed a mixed-cell sheet consisting of autologous fibroblasts and peripheral blood mononuclear cells with a high potency for angiogenesis and wound healing against refractory cutaneous ulcers in mouse and rabbit models. To increase the effectiveness of the mixed sheet, we developed a multilayered mixed sheet. METHODS: We assessed the therapeutic effects of multilayered sheets on cutaneous ulcers in mice. Growth factors and chemokines were assessed by enzyme-linked immunosorbent assay. Angiogenesis and fibroblast migration were measured by using tube formation and migration assays. Wound healing rate of cutaneous ulcers was evaluated in mice with diabetes mellitus. RESULTS: The concentration of secreted vascular endothelial growth factor, hepatocyte growth factor, transforming growth factor, C-X-C motif chemokine ligand (CXCL)-1, and CXCL-2 in multilayered sheets was much higher than that in single-layered mixed-cell sheets (single-layered sheets) and multilayered sheets of fibroblasts alone (fibroblast sheets). The supernatant in multilayered sheets enhanced angiogenic potency and fibroblast migration compared with single-layered and fibroblast sheets in an in vitro experiment. The wound healing rate in the multilayered sheet-treated group was higher compared with the no-treatment group (control) at the early stage of healing. Moreover, both vessel lumen area and microvessel density in tissues treated with multilayered sheets were significantly increased compared with tissues in the control group. CONCLUSION: Multilayered sheets promoted wound healing and microvascular angiogenesis in the skin by supplying growth factors and cytokines. Accordingly, our data suggest that multilayered sheets may be a promising therapeutic material for refractory cutaneous ulcers.

Microgravity influences maintenance of the human muscle stem/progenitor cell pool.

Microgravity induces skeletal muscle atrophy; however, the underlying mechanism is not clarified. In particular, the influence of microgravity on human skeletal muscle stem/progenitor cells (SMPCs) is not well understood. In this study, we used induced pluripotent stem cell-derived human SMPCs to investigate the effect of microgravity on maintenance of the stem/progenitor cell pool. Human SMPCs were induced by free-floating spherical aggregation culture, and derivatized-SMPC spheres were maintained in a microgravity condition (10-3 G) for 2 weeks using a clinostat rotation system. Microgravity culture deformed the SMPC spheres, with no signs of apoptosis. The most obvious change from microgravity culture was a significant decrease in the expression level of Pax7 in the SMPC spheres, with reduced numbers of myotubes in adhesion culture. Pax7 expression also decreased in the presence of the proteasome inhibitor MG132, indicating that the proteasomal degradation of Pax7 protein is not critical for its reduced expression in microgravity culture. Moreover, microgravity culture decreased the expression level of tumor necrosis factor receptor-associated factor 6 (TRAF6) and phosphorylation of its downstream molecule extracellular-related kinase (ERK) in SMPC spheres. Therefore, microgravity negatively regulates Pax7 expression in human SMPCs possibly through inhibition of the TRAF6/ERK pathway to consequently dysregulate SMPC pool maintenance. Overall, these results suggest that skeletal muscle atrophy is caused by microgravity-induced exhaustion of the stem cell pool.

Age-related increase in Wnt inhibitor causes a senescence-like phenotype in human cardiac stem cells.

Aging of cardiac stem/progenitor cells (CSCs) impairs heart regeneration and leads to unsatisfactory outcomes of cell-based therapies. As the precise mechanisms underlying CSC aging remain unclear, the use of therapeutic strategies for elderly patients with heart failure is severely delayed. In this study, we used human cardiosphere-derived cells (CDCs), a subtype of CSC found in the postnatal heart, to identify secreted factor(s) associated with CSC aging. Human CDCs were isolated from heart failure patients of various ages (2-83 years old). Gene expression of key soluble factors was compared between CDCs derived from young and elderly patients. Among these factors, SFRP1, a gene encoding a Wnt antagonist, was significantly up-regulated in CDCs from elderly patients (≥65 years old). sFRP1 levels was increased significantly also in CDCs, whose senescent phenotype was induced by anti-cancer drug treatment. These results suggest the participation of sFRP1 in CSC aging. We show that the administration of recombinant sFRP1 induced cellular senescence in CDCs derived from young patients, as indicated by increased levels of markers such as p16, and a senescence-associated secretory phenotype. In addition, co-administration of recombinant sFRP1 could abrogate the accelerated CDC proliferation induced by Wnt3A. Taken together, our results suggest that canonical Wnt signaling and its antagonist, sFRP1, regulate proliferation of human CSCs. Furthermore, excess sFRP1 in elderly patients causes CSC aging.

Therapeutic strategies for cell-based neovascularization in critical limb ischemia.

Critical limb ischemia (CLI) causes severe ischemic rest pain, ulcer, and gangrene in the lower limbs. In spite of angioplasty and surgery, CLI patients without suitable artery inflow or enough vascular bed in the lesions are often forced to undergo amputation of a major limb. Cell-based therapeutic angiogenesis has the potential to treat ischemic lesions by promoting the formation of collateral vessel networks and the vascular bed. Peripheral blood mononuclear cells and bone marrow-derived mononuclear cells are the most frequently employed cell types in CLI clinical trials. However, the clinical outcomes of cell-based therapeutic angiogenesis using these cells have not provided the promised benefits for CLI patients, reinforcing the need for novel cell-based therapeutic angiogenesis strategies to cure untreatable CLI patients. Recent studies have demonstrated the possible enhancement of therapeutic efficacy in ischemic diseases by preconditioned graft cells. Moreover, judging from past clinical trials, the identification of adequate transplant timing and responders to cell-based therapy is important for improving therapeutic outcomes in CLI patients in clinical settings. Thus, to establish cell-based therapeutic angiogenesis as one of the most promising therapeutic strategies for CLI patients, its advantages and limitations should be taken into account.

The c-MYC-ABCB5 axis plays a pivotal role in 5-fluorouracil resistance in human colon cancer cells.

c-MYC overexpression is frequently observed in various cancers including colon cancer and regulates many biological activities such as aberrant cell proliferation, apoptosis, genomic instability, immortalization and drug resistance. However, the mechanism by which c-MYC confers drug resistance remains to be fully elucidated. In this study, we found that the c-MYC expression level in primary colorectal cancer tissues correlated with the recurrence rate following 5-fluorouracil (5-FU)-based adjuvant chemotherapy. Supporting this finding, overexpression of exogenous c-MYC increased the survival rate following 5-FU treatment in human colon cancer cells, and knockdown of endogenous c-MYC decreased it. Furthermore, c-MYC knockdown decreased the expression level of ABCB5, which is involved in 5-FU resistance. Using a chromatin immunoprecipitation assay, we found that c-MYC bound to the ABCB5 promoter region. c-MYC inhibitor (10058-F4) treatment inhibited c-MYC binding to the ABCB5 promoter, leading to a decrease in ABCB5 expression level. ABCB5 knockdown decreased the survival rate following 5-FU treatment as expected, and the ABCB5 expression level was increased in 5-FU-resistant human colon cancer cells. Finally, using a human colon cancer xenograft murine model, we found that the combined 5-FU and 10058-F4 treatment significantly decreased tumorigenicity in nude mice compared with 5-FU or 10058-F4 treatment alone. 10058-F4 treatment decreased the ABCB5 expression level in the presence or absence of 5-FU. In contrast, 5-FU treatment alone increased the ABCB5 expression level. Taken together, these results suggest that c-MYC confers resistance to 5-FU through regulating ABCB5 expression in human colon cancer cells.

Hypoxic preconditioning reinforces cellular functions of autologous peripheral blood-derived cells in rabbit hindlimb ischemia model.

Peripheral blood mononuclear cell (PBMNC) is one of powerful tools for therapeutic angiogenesis in hindlimb ischemia. However, traditional approaches with transplanted PBMNCs show poor therapeutic effects in severe ischemia patients. In this study, we used autograft models to determine whether hypoxic pretreatment effectively enhances the cellular functions of PBMNCs and improves hindlimb ischemia. Rabbit PBMNCs were cultured in the hypoxic condition. After pretreatment, cell adhesion, stress resistance, and expression of angiogenic factor were evaluated in vitro. To examine in vivo effects, we autografted preconditioned PBMNCs into a rabbit hindlimb ischemia model on postoperative day (POD) 7. Preconditioned PBMNCs displayed significantly enhanced functional capacities in resistance to oxidative stress, cell viability, and production of vascular endothelial growth factor. In addition, autologous transplantation of preconditioned PBMNCs significantly induced new vessels and improved limb blood flow. Importantly, preconditioned PBMNCs can accelerate vessel formation despite transplantation on POD 7, whereas untreated PBMNCs showed poor vascularization. Our study demonstrated that hypoxic preconditioning of PBMNCs is a feasible approach for increasing the retention of transplanted cells and enhancing therapeutic angiogenesis in ischemic tissue.

Roles of pigment epithelium-derived factor in exercise-induced suppression of senescence and its impact on lung pathology in mice.

Senescent cells contribute to tissue aging and underlie the pathology of chronic diseases. The benefits of eliminating senescent cells have been demonstrated in several disease models, and the efficacy of senolytic drugs is currently being tested in humans. Exercise training has been shown to reduce cellular senescence in several tissues; however, the mechanisms responsible remain unclear. We found that myocyte-derived factors significantly extended the replicative lifespan of fibroblasts, suggesting that myokines mediate the anti-senescence effects of exercise. A number of proteins within myocyte-derived factors were identified by mass spectrometry. Among these, pigment epithelium-derived factor (PEDF) exerted inhibitory effects on cellular senescence. Eight weeks of voluntary running increased Pedf levels in skeletal muscles and suppressed senescence markers in the lungs. The administration of PEDF reduced senescence markers in multiple tissues and attenuated the decline in respiratory function in the pulmonary emphysema mouse model. We also showed that blood levels of PEDF inversely correlated with the severity of COPD in patients. Collectively, these results strongly suggest that PEDF contributes to the beneficial effects of exercise, potentially suppressing cellular senescence and its associated pathologies.

Lack of vitamin D signalling in mesenchymal progenitors causes fatty infiltration in muscle.

BACKGROUND: Recent studies have indicated the importance of muscle quality in addition to muscle quantity in sarcopenia pathophysiology. Intramuscular adipose tissue (IMAT), which originates from mesenchymal progenitors (MPs) in adult skeletal muscle, is a key factor affecting muscle quality in older adults, suggesting that controlling IMAT formation is a promising therapeutic strategy for sarcopenia. However, the molecular mechanism underlying IMAT formation in older adults has not been clarified. We recently found that the vitamin D receptor (VDR) is highly expressed in MPs in comparison to myotubes (P = 0.028, N = 3), indicating a potential role of vitamin D signalling in MPs. In this study, we aimed to clarify the role of vitamin D signalling in MP kinetics, with a focus on adipogenesis. METHODS: MPs isolated from mouse skeletal muscles were subjected to adipogenic differentiation conditions with or without vitamin D (1α,25(OH)2D3, 100 nM) for 7 days, and adipogenicity was evaluated based on adipogenic marker expression. For in vivo analysis, tamoxifen-inducible MP-specific VDR-deficient (VdrMPcKO) mice were newly developed to investigate whether lack of vitamin D signalling in MPs is involved in IMAT formation. To induce muscle atrophy, VdrMPcKO male mice were subjected to tenotomy of the gastrocnemius muscle, and then muscle weight, myofibre cross-sectional area, adipogenic marker expression, and fatty infiltration into the muscle were evaluated at 3 weeks after operation (N = 3-4). In addition, a vitamin D-deficient diet was provided to wild-type male mice (3 and 20 months of age, N = 5) for 3 months to investigate whether vitamin D deficiency causes IMAT formation. RESULTS: Vitamin D treatment nearly completely inhibited adipogenesis of MPs through Runx1-mediated transcriptional modifications of early adipogenic factors such as PPARγ (P = 0.0031) and C/EBPα (P = 0.0027), whereas VDR-deficient MPs derived from VdrMPcKO mice differentiated into adipocytes even in the presence of vitamin D (P = 0.0044, Oil-Red O+ area). In consistency with in-vitro findings, VdrMPcKO mice and mice fed a vitamin D-deficient diet exhibited fat deposition in atrophied (P = 0.0311) and aged (P = 0.0216) skeletal muscle, respectively. CONCLUSIONS: Vitamin D signalling is important to prevent fate decision of MPs towards the adipogenic lineage. As vitamin D levels decline with age, our data indicate that decreased vitamin D levels may be one of the causes of IMAT formation in older adults, and vitamin D signalling may be a novel therapeutic target for sarcopenia.

Significant role of bone marrow-derived cells in compensatory regenerative lung growth.

BACKGROUND: Extensive studies have attempted to clarify the contribution of bone marrow-derived cells to the regeneration of various organs, but not the lungs. We evaluated the role of bone marrow-derived cells in compensatory regenerative lung growth. METHODS: We induced regenerative lung growth by left pneumonectomy in adult C57BL/6 mice. To evaluate the role of bone marrow-derived cells in lung regenerative growth, green fluorescent protein (GFP)-positive, bone marrow-transplanted chimeric mice underwent inhibition of stromal-cell-derived factor (SDF)-1α/CXCR4 signaling by 7-d continuous administration of a CXCR4 antagonist after pneumonectomy. RESULTS: Left pneumonectomy resulted in a significant increase in lung dry weight, as well as an increase in lung volume, without enlargement of the alveolar air space. We observed GFP-positive cells 2.1-fold more frequently in the lungs of pneumonectomized mice versus sham-operated mice by immunohistochemistry (P = 0.001), although only a proportion of these accumulated cells possessed a pneumocyte-like appearance. Pneumonectomy induced a 1.4-fold increase in the SDF-1α level in the remaining lung at 7 d compared with sham-operated mice (P < 0.05), although pneumonectomy was not accompanied by histopathological lung injury. Blockade of SDF-1α/CXCR4 signaling resulted in a significant reduction in the accumulation of GFP-positive cells in the remaining lung at 7 d and prevented regenerative lung growth, as shown by a 10% reduction in lung dry weight at 14 d compared with control pneumonectomized mice (P < 0.05). CONCLUSIONS: Bone marrow-derived cells have a significant role in compensatory regenerative lung growth in an adult mouse model. Further evaluation to clarify molecular interactions between bone marrow-derived cells and pneumocytes should prove fruitful.

Isolation and in vitro propagation of human skeletal muscle progenitor cells from fetal muscle.

Skeletal muscle progenitor cells (SMPCs) are considered one of the most valuable cells for cell-based therapy targeting skeletal muscle. However, an efficient protocol for isolating and maintaining human myogenic progenitors in vitro has not been fully established. In this study, we demonstrate that human myogenic progenitors can be expanded and proliferated from human fetal muscles. Human SMPCs were prepared from fetal hind limb muscles and induced to proliferate as free-floating spheres termed myospheres in the medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Both myogenic progenitors and myoblast populations from human fetal muscles were effectively propagated in myospheres and passaged by a mechanical chopping. After expanding these spheres in culture, we tested whether myogenic progenitor cells can differentiate into multinucleated myotubes. The myospheres were dissociated, plated down on coverslips and cultured in the medium for terminal differentiation. We could confirm that the plated cells formed well-developed, multinucleated myotubes. This culture method using myospheres is an effective protocol to isolate and maintain SMPCs from human fetal skeletal muscles in culture.

RNA-Sequencing Analysis Identification of Potential Biomarkers for Diagnosis of Sarcopenia.

Sarcopenia is a geriatric disease associated with increased mortality and disability. Early diagnosis and intervention are required to prevent it. This study investigated biomarkers for sarcopenia by using a combination of comprehensive clinical data and messenger RNA-sequencing (RNA-seq) analysis obtained from peripheral blood mononuclear cells. We enrolled a total of 114 older adults aged 66-94 years (52 sarcopenia diagnosed according to the Asian Working Group for Sarcopenia 2019 consensus and 62 normal older people). We used clinical data which were not included diagnosis criteria of sarcopenia, and stride length showed significance by logistic regression analysis (Bonferroni corrected p = .012, odds ratio = 0.14, 95% confidence interval [CI]: 0.05-0.40). RNA-seq analysis detected 6 differential expressed genes (FAR1, GNL2, HERC5, MRPL47, NUBP2, and S100A11). We also performed gene-set enrichment analysis and detected 2 functional modules (ie, hub genes, MYH9, and FLNA). By using any combination of the 9 candidates and basic information (age and sex), risk-prediction models were constructed. The best model by using a combination of stride length, HERC5, S100A11, and FLNA, achieved a high area under the curve (AUC) of 0.91 in a validation cohort (95% CI: 0.78-0.95). The quantitative PCR results of the 3 genes were consistent with the trend observed in the RNA-seq results. When BMI was added, the model achieved a high AUC of 0.95 (95% CI: 0.84-0.99). We have discovered potential biomarkers for the diagnosis of sarcopenia. Further refinement may lead to their future practical use in clinical use.

Rb1 gene inactivation expands satellite cell and postnatal myoblast pools.

Satellite cells are well known as a postnatal skeletal muscle stem cell reservoir that under injury conditions participate in repair. However, mechanisms controlling satellite cell quiescence and activation are the topic of ongoing inquiry by many laboratories. In this study, we investigated whether loss of the cell cycle regulatory factor, pRb, is associated with the re-entry of quiescent satellite cells into replication and subsequent stem cell expansion. By ablation of Rb1 using a Pax7CreER,Rb1 conditional mouse line, satellite cell number was increased 5-fold over 6 months. Furthermore, myoblasts originating from satellite cells lacking Rb1 were also increased 3-fold over 6 months, while terminal differentiation was greatly diminished. Similarly, Pax7CreER,Rb1 mice exhibited muscle fiber hypotrophy in vivo under steady state conditions as well as a delay of muscle regeneration following cardiotoxin-mediated injury. These results suggest that cell cycle re-entry of quiescent satellite cells is accelerated by lack of Rb1, resulting in the expansion of both satellite cells and their progeny in adolescent muscle. Conversely, that sustained Rb1 loss in the satellite cell lineage causes a deficit of muscle fiber formation. However, we also show that pharmacological inhibition of protein phosphatase 1 activity, which will result in pRb inactivation accelerates satellite cell activation and/or expansion in a transient manner. Together, our results raise the possibility that reversible pRb inactivation in satellite cells and inhibition of protein phosphorylation may provide a new therapeutic tool for muscle atrophy by short term expansion of the muscle stem cells and myoblast pool.

Influence of vitamin D on sarcopenia pathophysiology: A longitudinal study in humans and basic research in knockout mice.

BACKGROUND: Vitamin D is an essential nutrient in musculoskeletal function; however, its relationship to sarcopenia remains ambiguous, and the mechanisms and targets of vitamin D activity have not been elucidated. This study aimed to clarify the role of vitamin D in mature skeletal muscle and its relationship with sarcopenia. METHODS: This epidemiological study included 1653 community residents who participated in both the fifth and seventh waves of the National Institute for Longevity Sciences, Longitudinal Study of Aging and had complete background data. Participants were classified into two groups: vitamin D-deficient (serum 25-hydroxyvitamin D < 20 ng/mL) and non-deficient (serum 25-hydroxyvitamin D ≥ 20 ng/mL); they underwent propensity-score matching for background factors (age, sex, height, weight, comorbidities, smoker, alcohol intake, energy intake, vitamin D intake, steps, activity, season and sarcopenia). Changes in muscle strength and mass over the 4-year period were compared. For basic analysis, we generated Myf6CreERT2 Vitamin D Receptor (VDR)-floxed (VdrmcKO ) mice with mature muscle fibre-specific vitamin D receptor knockout, injected tamoxifen into 8-week-old mice and analysed various phenotypes at 16 weeks of age. RESULTS: Grip strength reduction was significantly greater in the deficient group (-1.55 ± 2.47 kg) than in the non-deficient group (-1.13 ± 2.47 kg; P = 0.019). Appendicular skeletal muscle mass reduction did not differ significantly between deficient (-0.05 ± 0.79 kg) and non-deficient (-0.01 ± 0.74 kg) groups (P = 0.423). The incidence of new cases of sarcopenia was significantly higher in the deficient group (15 vs. 5 cases; P = 0.039). Skeletal muscle phenotyping of VdrmcKO mice showed no significant differences in muscle weight, myofibre percentage or myofibre cross-sectional area; however, both forelimb and four-limb muscle strength were significantly lower in VdrmcKO mice (males: forelimb, P = 0.048; four-limb, P = 0.029; females: forelimb, P < 0.001; four-limb, P < 0.001). Expression profiling revealed a significant decrease in expression of sarcoendoplasmic reticulum Ca2+ -ATPase (SERCA) 1 (P = 0.019) and SERCA2a (P = 0.049) genes in the VdrmcKO mice. In contrast, expression of non-muscle SERCA2b and myoregulin genes showed no changes. CONCLUSIONS: Vitamin D deficiency affects muscle strength and may contribute to the onset of sarcopenia. Vitamin D-VDR signalling has minimal influence on the regulation of muscle mass in mature myofibres but has a significant influence on muscle strength.

Bortezomib stabilizes NOXA and triggers ROS-associated apoptosis in medulloblastoma.

We have previously demonstrated that bortezomib, a 26S proteasome inhibitor, effectively inhibits medulloblastoma growth in vivo in a genetically engineered Ptch1, p53 mouse model; however, bortezomib is also associated clinically with severe peripheral neuropathy, which would be disadvantageous for patients with central nervous system malignancy. The purpose of this study was to determine the mechanism of bortezomib efficacy in medulloblastoma in order to replicate more specifically the therapeutic advantage of targeting the ubiquitin-proteosome system. In our studies of upstream components of the ubiquitin-proteasome system, we identified the pro-apoptotic protein NOXA as a post-translationally modified target that is stabilized by bortezomib and induces caspase cleavage in the context of reactive oxidative stress induced cell death. These preclinical results may apply to the sizable fraction of Shh-driven human medulloblastoma and perhaps other medulloblastoma subtypes, independent of p53 status.

Suppression of MyoD induces spontaneous adipogenesis in skeletal muscle progenitor cell culture.

The degree of intramuscular adipose tissue accumulation is one of the factors affecting meat quality. Accumulation of adipocytes is also observed under the pathological condition of skeletal muscle such as muscular dystrophy and sarcopenia. The origin of adipocytes seen in skeletal muscle is mesenchymal progenitor cells that can give rise to both adipocytes and fibroblasts. In the present study, we demonstrated that siRNA-mediated suppression of MyoD expression in rat skeletal muscle progenitor cell culture, which comprises both myogenic satellite cells and mesenchymal progenitor cells, resulted in diminished myotube formation and an unexpected spontaneous appearance of white adipocytes. Suppressing myomaker expression also resulted in complete absence of myotube formation without reducing MyoD expression, but no adipogenesis was seen in this scenario, indicating that decline in MyoD expression rather than decreased myotube formation is necessary to induce adipogenesis. In addition, spontaneous adipogenesis induced by suppressing MyoD expression in culture was inhibited by the conditioned medium from control culture, indicating that anti-adipogenic factor(s) are secreted from MyoD-positive myogenic cells. These results indicate the presence of regulatory mechanism on adipogenesis by myogenic cells.

High concentrations of HGF inhibit skeletal muscle satellite cell proliferation in vitro by inducing expression of myostatin: a possible mechanism for reestablishing satellite cell quiescence in vivo.

Skeletal muscle regeneration and work-induced hypertrophy rely on molecular events responsible for activation and quiescence of resident myogenic stem cells, satellite cells. Recent studies demonstrated that hepatocyte growth factor (HGF) triggers activation and entry into the cell cycle in response to mechanical perturbation, and that subsequent expression of myostatin may signal a return to cell quiescence. However, mechanisms responsible for coordinating expression of myostatin after an appropriate time lag following activation and proliferation are not clear. Here we address the possible role of HGF in quiescence through its concentration-dependent negative-feedback mechanism following satellite cell activation and proliferation. When activated/proliferating satellite cell cultures were treated for 24 h beginning 48-h postplating with 10-500 ng/ml HGF, the percentage of bromodeoxyuridine-incorporating cells decreased down to a baseline level comparable to 24-h control cultures in a HGF dose-dependent manner. The high level HGF treatment did not impair the cell viability and differentiation levels, and cells could be reactivated by lowering HGF concentrations to 2.5 ng/ml, a concentration that has been shown to optimally stimulate activation of satellite cells in culture. Coaddition of antimyostatin neutralizing antibody could prevent deactivation and abolish upregulation of cyclin-dependent kinase (Cdk) inhibitor p21. Myostatin mRNA expression was upregulated with high concentrations of HGF, as demonstrated by RT-PCR, and enhanced myostatin protein expression and secretion were revealed by Western blots of the cell lysates and conditioned media. These results indicate that HGF could induce satellite cell quiescence by stimulating myostatin expression. The HGF concentration required (over 10-50 ng/ml), however, is much higher than that for activation, which is initiated by rapid release of HGF from its extracellular association. Considering that HGF is produced by satellite cells and spleen and liver cells in response to muscle damage, local concentrations of HGF bathing satellite cells may reach a threshold sufficient to induce myostatin expression. This time lag may delay action of the quiescence signaling program in proliferating satellite cells during initial phases of muscle regeneration followed by induction of quiescence in a subset of cells during later phases.

IGF-1 receptor inhibition by picropodophyllin in medulloblastoma.

The insulin-like growth factor-1 receptor (Igf1r) is a multifunctional membrane-associated tyrosine kinase associated with regulation of transformation, proliferation, differentiation and apoptosis. Increased IGF pathway activity has been reported in human and murine medulloblastoma. Tumors from our genetically-engineered medulloblastoma mouse model over-express Igf1r, and thus this mouse model is a good platform with which to study the role of Igf1r in tumor progression. We hypothesize that inhibition of IGF pathway in medulloblastoma can slow or inhibit tumor growth and metastasis. To test our hypothesis, we tested the role of IGF in tumor growth in vitro by treatment with the tyrosine kinase small molecule inhibitor, picropodophyllin (PPP), which strongly inhibits the IGF pathway. Our results demonstrate that PPP-mediated downregulation of the IGF pathway inhibits mouse tumor cell growth and induces apoptotic cell death in vitro in primary medulloblastoma cultures that are most reflective of tumor cell behavior in vivo.