Royal Jelly Enhances the Ability of Myoblast C2C12 Cells to Differentiate into Multilineage Cells.

Royal jelly (RJ) is recognized as beneficial to mammalian health. Multilineage differentiation potential is an important property of mesenchymal stem cells (MSCs). C2C12 cells have an innate ability to differentiate into myogenic cells. Like MSCs, C2C12 cells can also differentiate into osteoblast- and adipocyte-lineage cells. We recently reported that RJ enhances the myogenic differentiation of C2C12 cells. However, the effect of RJ on osteoblast or adipocyte differentiation is still unknown. Here in this study, we have examined the effect of RJ on the osteoblast and adipocyte differentiation of C2C12 cells. Protease-treated RJ was used to reduce the adverse effects caused by RJ supplementation. To induce osteoblast or adipocyte differentiation, cells were treated with bone morphogenetic proteins (BMP) or peroxisome proliferator-activated receptor γ (PPARγ) agonist, respectively. RNA-seq was used to analyze the effect of RJ on gene expression. We found that RJ stimulates osteoblast and adipocyte differentiation. RJ regulated 279 genes. RJ treatment upregulated glutathione-related genes. Glutathione, the most abundant antioxidative factor in cells, has been shown to promote osteoblast differentiation in MSC and MSC-like cells. Therefore, RJ may promote osteogenesis, at least in part, through the antioxidant effects of glutathione. RJ enhances the differentiation ability of C2C12 cells into multiple lineages, including myoblasts, osteoblasts, and adipocytes.

Nobiletin, a NF-κB signaling antagonist, promotes BMP-induced bone formation.

The NF-κB family of transcription factors plays an important role in skeletal development and bone homeostasis. In osteoblast cells, NF-κB signaling has been shown to suppress survival, proliferation, and differentiation. Furthermore, pharmacological suppression of NF-κB enhances osteoblast differentiation and bone formation. Thus, NF-κB antagonists are promising candidates as anabolic agents for enhancing bone mass. In this study, we describe the mechanism by which nobiletin, an inhibitor of NF-κB activity, regulates osteoblast differentiation and mineralization. We found that in MC3T3-E1 osteoblast cells, nobiletin inhibited a TNF-α responsive NF-κB luciferase reporter and also decreased the induction of classical NF-κB target genes by TNF-α. Consistent with this, nobiletin prevented TNF-α -mediated suppression of osteogenesis and potently enhanced the differentiation and mineralization of MC3T3-E1 cells. Likewise, in an in vivo BMP2-induced ectopic bone formation assay, nobiletin markedly enhanced ossicle bone volume. Western blotting and SMAD-responsive luciferase assays also demonstrated that NF-κB suppression of BMP signaling could be inhibited by nobiletin. Thus, our data suggest that mechanistically, nobiletin prevents the endogenous repression of BMP signaling by TNF-α, thereby enhancing osteoblast activity. In conclusion, nobiletin is a novel NF-κB antagonist that may be a useful anabolic agent for bone formation.

Factors Regulating or Regulated by Myogenic Regulatory Factors in Skeletal Muscle Stem Cells.

MyoD, Myf5, myogenin, and MRF4 (also known as Myf6 or herculin) are myogenic regulatory factors (MRFs). MRFs are regarded as master transcription factors that are upregulated during myogenesis and influence stem cells to differentiate into myogenic lineage cells. In this review, we summarize MRFs, their regulatory factors, such as TLE3, NF-κB, and MRF target genes, including non-myogenic genes such as taste receptors. Understanding the function of MRFs and the physiology or pathology of satellite cells will contribute to the development of cell therapy and drug discovery for muscle-related diseases.

Tumor necrosis factor alpha regulates myogenesis to inhibit differentiation and promote proliferation in satellite cells.

TNF-α and NF-κB signaling is involved in the wasting of skeletal muscle in various conditions, in addition to cancer cachexia. TNF-α and NF-κB signaling promotes the expression level of muscle RING finger protein 1, a ubiquitin ligase, causing muscle degradation. Several studies have indicated that of TNF-α and NF-κB signaling suppresses muscle differentiation by reducing the levels of MyoD protein. On the other hand, TNF-α and NF-κB is required for myoblast proliferation. Thus, the role of TNF-α and NF-κB signaling in the process of myogenesis and regeneration of skeletal muscle is not completely elucidated. Here, we reported that TNF-α reduced the width of single fibers of skeletal muscle in an organ culture model. TNF-α and p65 repressed the transactivation of MyoD and suppressed myoblast differentiation. In addition, TNF-α increased the number of satellite cells, and NF-κB signaling was promoted at the proliferation stage during skeletal muscle regeneration in vivo. TNF-α and NF-κB signaling regulate myogenesis to inhibit differentiation and promote proliferation in satellite cells.

Natural Compounds Attenuate Denervation-Induced Skeletal Muscle Atrophy.

The weight of skeletal muscle accounts for approximately 40% of the whole weight in a healthy individual, and the normal metabolism and motor function of the muscle are indispensable for healthy life. In addition, the skeletal muscle of the maxillofacial region plays an important role not only in eating and swallowing, but also in communication, such as facial expressions and conversations. In recent years, skeletal muscle atrophy has received worldwide attention as a serious health problem. However, the mechanism of skeletal muscle atrophy that has been clarified at present is insufficient, and a therapeutic method against skeletal muscle atrophy has not been established. This review provides views on the importance of skeletal muscle in the maxillofacial region and explains the differences between skeletal muscles in the maxillofacial region and other regions. We summarize the findings to change in gene expression in muscle remodeling and emphasize the advantages and disadvantages of denervation-induced skeletal muscle atrophy model. Finally, we discuss the newly discovered beneficial effects of natural compounds on skeletal muscle atrophy.

Protein phosphatase 1 regulatory subunit 18 suppresses the transcriptional activity of NFATc1 via regulation of c-fos.

The transcription factor NFATc1 and its binding partner AP-1 (a complex containing c-fos and c-Jun) play a central role in osteoclast differentiation. NFATc1 and AP-1 promote the expression of target genes such as Acp5, Ctsk and also auto-regulate NFATc1 expression as well. We previously reported that protein phosphatase 1 regulatory subunit 18 (PPP1r18) is a negative regulator of osteoclast bone resorption by inhibiting cell attachment to bone matrix. We also reported that PPP1r18 potentially regulates NFATc1 expression during osteoclast differentiation. To further explore this, in this study we have examined the effect of PPP1r18 on NFATc1 expression and activity by overexpressing PPP1r18 during the early stage of osteoclast differentiation. We found that PPP1r18 suppressed NFATc1 expression through inhibition of the transcriptional activity of NFATc1. Since PPP1r18 does not regulate NFATc1 directly, we next explored the involvement of AP-1. Our data showed that c-fos phosphorylation and nuclear localization were reduced by PPP1r18 overexpression. Further experiments showed that overexpression of c-fos together with PPP1r18 rescued NFATc1 expression and transcriptional activity. Moreover, c-fos activity inhibition by PPP1r18 was canceled by mutation of the phosphatase binding site of PPP1r18. Taken together, PPP1r18-regulated phosphatase activity targets c-fos phosphorylation and suppresses subsequent NFATc1 expression and activity.

Vignacyanidin Polyphenols Isolated from Vigna Angularis Bean Promote Osteoblast Differentiation.

BACKGROUND/AIM: An effective bone regenerative method needs to be established for the dental field. To identify a novel osteogenic factor for bone regeneration, we examined the effect of vignacyanidin (VIG) on osteoblastogenesis. MATERIALS AND METHODS: W20-17 cells, MC3T3-E1 cells, and primary cultured murine calvarial osteoblasts were used. Osteoblast differentiation was stimulated by ß-glycerophosphate, ascorbic acid, or bone morphogenetic protein (BMP)-4. Adipogenesis was induced using dexamethasone, 3-isobutyl-1-methylxanthine, insulin, and rosiglitazone. Differentiation or proliferation markers were determined using western blotting and/or the quantitative reverse transcription polymerase chain reaction. Adipogenic cells were visualized by Oil Red O staining. RESULTS: VIG treatment increased the expression of osteoblastic markers and alkaline phosphatase activity of osteoblast-lineage cells in a concentration-dependent manner. However, adipogenesis and cell proliferation were not affected by VIG. CONCLUSION: VIG treatment promoted osteoblast differentiation in osteoblast-lineage cells.

Daily Oral Administration of Protease-Treated Royal Jelly Protects Against Denervation-Induced Skeletal Muscle Atrophy.

Honeybees produce royal jelly (RJ) from their cephalic glands. Royal jelly is a source of nutrition for the queen honey bee throughout its lifespan and is also involved in fertility and longevity. Royal jelly has long been considered beneficial to human health. We recently observed that RJ delayed impairment of motor function during aging, affecting muscle fiber size. However, how RJ affects skeletal muscle metabolism and the functional component of RJ is as of yet unidentified. We demonstrate that feeding mice with RJ daily prevents a decrease in myofiber size following denervation without affecting total muscle weight. RJ did not affect atrophy-related genes but stimulated the expression of myogenesis-related genes, including IGF-1 and IGF receptor. Trans-10-hydroxy-2-decenoic acid (10H2DA) and 10-hydroxydecanoic acid (10HDAA), two major fatty acids contained in RJ. After ingestion, 10H2DA and 10HDAA are metabolized into 2-decenedioic acid (2DA) and sebacic acid (SA) respectively. We found that 10H2DA, 10HDAA, 2DA, and SA all regulated myogenesis of C2C12 cells, murine myoblast cells. These novel findings may be useful for potential preventative and therapeutic applications for muscle atrophy disease included in Sarcopenia, an age-related decline in skeletal muscle mass and strength.

VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation.

Osteoblasts release adenosine triphosphate (ATP) out of the cell following mechanical stress. Although it is well established that extracellular ATP affects bone metabolism via P2 receptors [such as purinergic receptor P2X7 (P2X7R) and purinergic receptor P2Y2 (P2Y2R)], the mechanism of ATP release from osteoblasts remains unknown. Recently, a vesicular nucleotide transporter [VNUT, solute carrier family 17 member 9 (SLC17A9)] that preserves ATP in vesicles has been identified. The purpose of this study was to elucidate the role of VNUT in osteoblast bone metabolism. mRNA and protein expression of VNUT were confirmed in mouse bone and in osteoblasts by quantitative real-time PCR (qPCR) and immunohistochemistry. Next, when compressive force was applied to MC3T3-E1 cells by centrifugation, the expression of Slc17a9, P2x7r, and P2y2r was increased concomitant with an increase in extracellular ATP levels. Furthermore, compressive force decreased the osteoblast differentiation capacity of MC3T3-E1 cells. shRNA knockdown of Slc17a9 in MC3T3-E1 cells reduced levels of extracellular ATP and also led to increased osteoblast differentiation after the application of compressive force as assessed by qPCR analysis of osteoblast markers such as Runx2, Osterix, and alkaline phosphatase (ALP) as well as ALP activity. Consistent with these observations, knockdown of P2x7r or P2y2r by siRNA partially rescued the downregulation of osteoblast differentiation markers, caused by mechanical loading. In conclusion, our results demonstrate that VNUT is expressed in osteoblasts and that VNUT inhibits osteoblast differentiation in response to compressive force by mechanisms related to ATP release and P2X7R and/or P2Y2R activity.