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Background Melanoma is a malignant tumor that is characterized by high proliferation and aggressive metastasis. To address the efficient treatment of melanoma, the molecular mechanisms of the proto-oncogene, Rous sarcoma oncogene (Src), which is highly activated and promotes cell proliferation, migration, adhesion, and metastasis in melanoma, should be understood. Plectin has recently been identified as an Src-binding protein that regulates Src activity in osteoclasts. Plectin, a cytoskeleton-regulatory protein, is a candidate biomarker of certain tumors because of its high expression and the target of anti-tumor reagents such as ruthenium pyridinecarbothioamide, although the molecular mechanisms by which plectin works in melanoma are still unclear. In this study, we examined its pathological role in melanoma tumor formation. Methods We established plectin knock-out B16 cells, the mouse melanoma cell line, with CRISPR/Cas9 system. The expression of plectin and activity of Src were examined by western blotting analysis. The tumors were formed at 2 weeks after subcutaneous injection of B16 cells in nude mice and analyzed by Hematoxylin-Eosin (H-E) staining. Cell proliferation was evaluated by cell counting kit-8, expression of cyclin D1 and Ki-67. Cell aggregation and adhesion were assessed by spheroid formation and cell adhesion assay.Results Depletion of plectin induced low-density and sparce tumor formation by melanoma cells in vivo. In vitro experiments revealed that plectin-deficient melanomas exhibit reduced cell proliferation and suppressed cell-to-cell adhesion. Because Src activity is reduced in plectin-deficient melanomas, we examined the relationship between plectin and Src signaling. Src overexpression restored Src activity and rescued cell proliferation and cell-to-cell adhesion in plectin-deficient melanomas. Conclusion: These results suggest that plectin is required for tumor formation by promoting cell proliferation and cell-to-cell adhesion through Src signaling activity in melanoma cells.

DNA methylation is an epigenetic modification critical for the regulation of chromatin structure and gene expression during development and disease. The ten-eleven translocation (TET) enzyme family catalyzes the hydroxymethylation and subsequent demethylation of DNA by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Little is known about TET protein function due to a lack of pharmacological tools to manipulate DNA hydroxymethylation levels. In this study, we examined the role of TET-mediated DNA hydroxymethylation during BMP-induced C2C12 osteoblast differentiation using a novel cytosine-based selective TET enzyme inhibitor, Bobcat339 (BC339). Treatment of C2C12 cells with BC339 increased global 5mC and decreased global 5hmC without adversely affecting cell viability, proliferation, or apoptosis. Furthermore, BC339 treatment inhibited osteoblast marker gene expression and decreased alkaline phosphatase activity during differentiation. Methylated DNA immunoprecipitation and bisulfite sequencing showed that inhibition of TET with BC339 led to increased 5mC at specific CpG-rich regions at the promoter of Sp7, a key osteoblast transcription factor. Consistent with promoter 5mC marks being associated with transcriptional repression, luciferase activity of an Sp7-promoter-reporter construct was repressed by in vitro DNA methylation or BC339. Chromatin immunoprecipitation analysis confirmed that TET2 does indeed occupy the promoter region of Sp7. Accordingly, forced overexpression of SP7 rescued the inhibition of osteogenic differentiation by BC339. In conclusion, our data suggest that TET-mediated DNA demethylation of genomic regions, including the Sp7 promoter, plays a role in the initiation of osteoblast differentiation. Furthermore, BC339 is a novel pharmacological tool for the modulation of DNA methylation dynamics for research and therapeutic applications.

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., In this study, we show that VNUT (SLC17A9), a vesicular nucleotide transporter, regulates osteoblast differentiation in response to mechanical loading. Mechanical loading promotes adenosine triphosphate (ATP) exocytosis via VNUT. Extracellular ATP subsequently inhibits osteoblast differentiation via activation of P2X7 and/or P2Y2 receptors. Thus, VNUT modulation of purinergic signaling contributes to the regulation of bone metabolism by mechanical loading.

Bone invasion is a critical factor in determining the prognosis of oral squamous cell carcinoma (OSCC) patients. Transforming growth factor β (TGF-β) is abundantly expressed in the bone matrix and is involved in the acquisition of aggressiveness by tumors. TGF-β is also important to cytoskeletal changes during tumor progression. In this study, we examined the relationship between TGF-β signaling and cytoskeletal changes during bone invasion by OSCC. Immunohistochemical staining of OSCC samples from five patients showed the expression of p130Cas (Crk-associated substrate) in the cytoplasm and phosphorylated Smad3 expression in the nucleus in OSCC cells. TGF-β1 induced the phosphorylation of Smad3 and p130Cas, as well as epithelial–mesenchymal transition (EMT) accompanied by the downregulation of the expression of E-cadherin, a marker of epithelial cells, and the upregulation of the expression of N-cadherin, or Snail, a marker of mesenchymal cells, in human HSC-2 cells and mouse squamous cell carcinome VII (SCCVII) cells. SB431542, a specific inhibitor of Smad2/3 signaling, abrogated the TGF-β1-induced phosphorylation of p130Cas and morphological changes. Silencing p130Cas using an short hairpin RNA (shRNA) or small interfering RNA in SCCVII cells suppressed TGF-β1-induced cell migration, invasion, EMT and matrix metalloproteinase-9 (MMP-9) production. Compared with control SCCVII cells, SCCVII cells with silenced p130Cas strongly suppressed zygomatic and mandibular destruction in vivo by reducing the number of osteoclasts, cell proliferation and MMP-9 production. Taken together, these results showed that the expression of TGF-β/p130Cas might be a new target for the treatment of OSCC bone invasion.

Background/aim Geranylgeraniol (GGOH), a C20 isoprenoid naturally occurs in several foods. We previously reported that GGOH treatment reduced the expression levels of Atrogin-1 which is involved in skeletal muscle degradation and stimulates the myogenic differentiation of C2C12 myoblasts. However, the effect of GGOH supplementation on skeletal muscle metabolism in vivo is unknown. Materials and methods Skeletal muscle atrophy was induced by denervation. The expression levels of Atrogin-1 were assessed by western blotting or real time PCR. Results Intraoral administration of GGOH reduced the decrease in the cross-sectional area of muscle fibers and also suppressed the expression levels of Atrogin-1 in denervation induced muscle atrophy. Also, GGOH treatment suppressed the expression of Atrogin-1 and the decrease in skeletal muscle fiber size by glucocorticoid in vitro. Conclusion Intraoral administration of GGOH rescues denervation-induced muscle atrophy via suppression of Atrogin-1.

Podosome formation in osteoclasts is an important initial step in osteoclastic bone resorption. Mice lacking c-Src (c-Src-/- ) exhibited osteopetrosis due to a lack of podosome formation in osteoclasts. We previously identified p130Cas (Crk-associated substrate [Cas]) as one of c-Src downstream molecule and osteoclast-specific p130Cas-deficient (p130CasΔOCL-/- ) mice also exhibited a similar phenotype to c-Src-/- mice, indicating that the c-Src/p130Cas plays an important role for bone resorption by osteoclasts. In this study, we performed a cDNA microarray and compared the gene profiles of osteoclasts from c-Src-/- or p130CasΔOCL-/- mice with wild-type (WT) osteoclasts to identify downstream molecules of c-Src/p130Cas involved in bone resorption. Among several genes that were commonly downregulated in both c-Src-/- and p130CasΔOCL-/- osteoclasts, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization. Reduced Kif1c expression was observed in both c-Src-/- and p130CasΔOCL-/- osteoclasts compared with WT osteoclasts. Kif1c exhibited a broad tissue distribution, including osteoclasts. Knockdown of Kif1c expression using shRNAs in WT osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in p130CasΔOCL-/- osteoclasts but not c-Src-/- osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas (191 words). SIGNIFICANCE OF THE STUDY: We previously showed that the c-Src/p130Cas (Cas) plays an important role for bone resorption by osteoclasts. In this study, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization, as a downstream molecule of c-Src/p130Cas axis, using cDNA microarray. Knockdown of Kif1c expression using shRNAs in wild-type osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in osteoclast-specific p130Cas-deficient (p130CasΔOCL-/- ) osteoclasts but not c-Src-/- osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas.

Melanoma is malignant cancer characterized by high proliferation and aggressive metastasis. To address efficient treatment for melanoma, we should understand the molecular mechanisms for a proto-oncogene Src, which is highly activated and promotes cell proliferation, migration, adhesion, and metastasis in melanoma. We recently identified plectin as the Src binding protein and regulates Src activity in osteoclasts. Plectin, a cytoskeleton regulatory protein, is focused as the candidates of biomarker of certain tumors because of higher expression and the candidate of anti-tumor reagents such as ruthenium pyridinecarbothioamide although the molecular mechanisms how plectin works in melanoma is unclear. In this study, we examined the pathological role in melanoma tumor formation. Depletion of plectin induced low density and sparce tumor formation by melanoma cells in vivo. In vitro experiment revealed that plectin deficient melanomas reduced cell proliferation and suppressed cell-to-cell adhesion. Because Src activity was reduced in plectin deficient melanomas, we examined the relationship between plectin and Src signaling. Src overexpression that restored Src activity rescued cell proliferation and cell-to-cell adhesion of plectin deficient melanomas. These results suggest that plectin is required for tumor formation by promoting cell proliferation and cell-to-cell adhesion via Src signaling activity in melanoma.

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.

Skeletal tissue homeostasis is maintained via the balance of osteoclastic bone resorption and osteoblastic bone formation. Autophagy and apoptosis are essential for the maintenance of homeostasis and normal development in cells and tissues. We found that Bax-interacting factor 1 (Bif-1/Endophillin B1/SH3GLB1), involving in autophagy and apoptosis, was upregulated during osteoclastogenesis. Furthermore, mature osteoclasts expressed Bif-1 in the cytosol, particularly the perinuclear regions and podosome, suggesting that Bif-1 regulates osteoclastic bone resorption. Bif-1-deficient (Bif-1 -/- ) mice showed increased trabecular bone volume and trabecular number. Histological analyses indicated that the osteoclast numbers increased in Bif-1 -/- mice. Consistent with the in vivo results, osteoclastogenesis induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) was accelerated in Bif-1 -/- mice without affecting RANKL-induced activation of RANK downstream signals, such as NF-κB and mitogen-activated protein kinases (MAPKs), CD115/RANK expression in osteoclast precursors, osteoclastic bone-resorbing activity and the survival rate. Unexpectedly, both the bone formation rate and osteoblast surface substantially increased in Bif-1 -/- mice. Treatment with β-glycerophosphate (β-GP) and ascorbic acid (A.A) enhanced osteoblastic differentiation and mineralization in Bif-1 -/- mice. Finally, bone marrow cells from Bif-1 -/- mice showed a significantly higher colony-forming efficacy by the treatment with or without β-GP and A.A than cells from wild-type (WT) mice, suggesting that cells from Bif-1 -/- mice had higher clonogenicity and self-renewal activity than those from WT mice. In summary, Bif-1 might regulate bone homeostasis by controlling the differentiation and function of both osteoclasts and osteoblasts (235 words).

Melanoma, one of the most aggressive neoplasms, is characterized by rapid cell proliferation. Transducin-like Enhancer of Split (TLE) is an important regulator of cell proliferation via Histone deacetylase (HDAC) recruitment. Given that HDAC activity is associated with melanoma progression, we examined the relationship between TLE3, a TLE family member, and melanoma. TLE3 expression was increased during the progression of human patient melanoma (p < 0.05). Overexpression of Tle3 in B16 murine melanoma cells led to an increase in cell proliferation (p < 0.01) as well as the number of cyclinD1-positive cells. in vivo injection of mice with B16 cells overexpressing Tle3 resulted in larger tumor formation than in mice injected with control cells (p < 0.05). In contrast, siRNA-mediated knockdown of Tle3 in B16 cells or TLE3 in HMV-II human melanoma cells decreased proliferation (p < 0.01). Treatment of B16 cells with trichostatin A (2.5 μM), a class I and II HDAC inhibitor, prevented the effect s of Tle3 on proliferation. In conclusion, these data indicate that Tle3 is required, at least in part, for proliferation in the B16 mouse melanoma model.

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., Highlights • PPP1r18 suppresses osteoclast differentiation. • PPP1r18 suppresses c-fos phosphorylation and nuclear localization. • PPP1r18 suppresses NFAT via c-fos.

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 β-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.

Background/Aim: Geranylgeraniol (GGOH), a C20 isoprenoid naturally occurs in several foods. We previously reported that GGOH treatment reduced the expression levels of Atrogin-1 which is involved in skeletal muscle degradation and stimulates the myogenic differentiation of C2C12 myoblasts. However, the effect of GGOH supplementation on skeletal muscle metabolism in vivo is unknown. Materials and Methods: Skeletal muscle atrophy was induced by denervation. The expression levels of Atrogin-1 were assessed by western blotting or real time PCR. Results: Intraoral administration of GGOH reduced the decrease in the cross-sectional area of muscle fibers and also suppressed the expression levels of Atrogin-1 in denervation induced muscle atrophy. Also, GGOH treatment suppressed the expression of Atrogin-1 and the decrease in skeletal muscle fiber size by glucocorticoid in vitro. Conclusion: Intraoral administration of GGOH rescues denervation-induced muscle atrophy via suppression of Atrogin-1.

Bone morphogenetic protein (BMP) potentiates bone formation through the Smad signaling pathway in vitro and in vivo. The transcription factor nuclear factor κB (NF-κB) suppresses BMP-induced osteoblast differentiation. Recently, we identified that the transactivation (TA) 2 domain of p65, a main subunit of NF-κB, interacts with the mad homology (MH) 1 domain of Smad4 to inhibit BMP signaling. Therefore, we further attempted to identify the interacting regions of these two molecules at the amino acid level. We identified a region that we term the Smad4-binding domain (SBD), an amino-terminal region of TA2 that associates with the MH1 domain of Smad4. Cell-permeable SBD peptide blocked the association of p65 with Smad4 and enhanced BMP2-induced osteoblast differentiation and mineralization without affecting the phosphorylation of Smad1/5 or the activation of NF-κB signaling. SBD peptide enhanced the binding of the BMP2-inudced phosphorylated Smad1/5 on the promoter region of inhibitor of DNA binding 1 (Id-1) compared with control peptide. Although SBD peptide did not affect BMP2-induced chondrogenesis during ectopic bone formation, the peptide enhanced BMP2-induced ectopic bone formation in subcortical bone. Thus, the SBD peptide is useful for enabling BMP2-induced bone regeneration without inhibiting NF-κB activity.

Osteoclasts are multinuclear cells which maintain bone homeostasis by resorbing bone. During bone resorption, osteoclasts attach to the bone matrix via a sealing zone formed by an actin ring. Rous sarcoma oncogene (Src) is essential for actin ring formation and bone resorption. Recently, we demonstrated that plectin, a cytolinker protein, is a Src-binding protein in osteoclasts. However, the function of plectin in osteoclasts remains unknown. In this study, we demonstrated that shRNA knockdown of plectin in RAW 264.7 cells resulted in tartrate resistant acid phosphatase positive multinuclear cells (TRAP (+) MNCs) with impaired actin ring formation and bone resorption activity. Moreover, we found that in plectin-silenced TRAP (+) MNCs, Src and protein tyrosine kinase 2 beta (Pyk2), two critical kinases in osteoclastic bone resorption, were inactivated and microtubule polarity was disturbed. These results suggest that plectin plays a critical role in osteoclast biology by acting as a scaffold to facilitate Src and Pyk2 activation during microtubule organization.

The balance of bone formation by osteoblasts and bone resorption by osteoclasts is important for bone homeostasis. Pathologic high activity of osteoclasts and repression of bone osteoblastic bone formation result in bone metabolic diseases such as periodontal disease. To understand the mechanism of osteoblast and osteoclast function leads to establishment of therapy for bone metabolic disease. A tyrosine kinase Src deficient mice shows osteopetrosis because of defect bone resorbing activity and acceleration bone formation. This indicates Src is a key molecule to regulate bone resorption and bone formation. We discuss about the role of Src in osteoclast and osteoblast and entire bone metabolism.

Background The global incidence of diabetes mellitus (DM) has risen precipitously, even in middle- and low-income countries. Peroxisome proliferator-activated receptor γ (PPARγ) plays an important role in the control of cellular glucose metabolism. Activation of PPARγ beneficially results in increased insulin sensitivity. However, the expression of PPARγ is reduced by obesity and several nutritional factors. Here we examined the effect of geranylgeraniol (GGOH), a bioactive compound found naturally in fruits, vegetables, and grains, on the expression and activation of PPARγ. Materials and methods C3H10T1/2 mouse embryonic fibroblasts and 3T3-L1 pre-adipocytes were used as in vitro models of adipocyte differentiation and function. Quantitative reverse-transcriptase polymerase chain reaction, western blotting, Oil Red O staining, and luciferase assay were performed to respectively assess mRNA expression, protein levels, lipid droplet formation and transcriptional activity. Results GGOH increased the expression of PPARγ in adipocyte lineage cells. GGOH also enhanced adipogenesis induced by rosiglitazone, a thiazolidinedione class PPARγ agonist. Conclusion GGOH induces PPARγ expression and enhances the biological effects of a PPARγ agonist in adipocyte lineage cells.

Background Geranylgeraniol (GGOH) is a C20 isoprenoid found in fruits, vegetables, and grains, including rice. As a food substance, GGOH is categorized as 'Generally Recognized as Safe'. GGOH is an intermediate product in the mevalonate pathway and acts as a precursor to geranylgeranyl pyrophosphate. Materials and methods C2C12 mouse myoblasts derived from muscle satellite cells were used. Quantitative reverse-transcriptase polymerase chain reaction, western blotting analysis, and immunocytochemical analysis were performed to respectively assess mRNA expression, protein levels, and the number of myofibers. Results GGOH reduced the expression levels of skeletal muscle atrophy-related ubiquitin ligases in myofibers derived from C2C12 cells. GGOH induced myogenic differentiation of C2C12 cells via geranylgeranylation. GGOH did not adversely affect the proliferation of C2C12 cells. Conclusion GGOH induces myoblast differentiation in C2C12 cells.

Endochondral ossification is important for skeletal development. Recent findings indicate that the p65 (RelA) subunit, a main subunit of the classical nuclear factor-κB (NF-κB) pathway, plays essential roles in chondrocyte differentiation. Although several groups have reported that the alternative NF-κB pathway also regulates bone homeostasis, the role of the alternative NF-κB pathway in chondrocyte development is still unclear. Here, we analyzed the in vivo function of the alternative pathway on endochondral ossification using p100-deficient (p100−/−) mice, which carry a homozygous deletion of the COOH-terminal ankyrin repeats of p100 but still express functional p52 protein. The alternative pathway was activated during the periarticular stage in wild-type mice. p100−/− mice exhibited dwarfism, and histological analysis of the growth plate revealed abnormal arrangement of chondrocyte columns and a narrowed hypertrophic zone. Consistent with these observations, the expression of hypertrophic chondrocyte markers, type X collagen (ColX) or matrix metalloproteinase 13, but not early chondrogenic markers, such as Col II or aggrecan, was suppressed in p100−/− mice. An in vivo BrdU tracing assay clearly demonstrated less proliferative activity in chondrocytes in p100−/− mice. These defects were partly rescued when the RelB gene was deleted in p100−/− mice. Taken together, the alternative NF-κB pathway may regulate chondrocyte proliferation and differentiation to maintain endochondral ossification.

Deletion of c-Src, a ubiquitously expressed tyrosine kinase, results in osteoclast dysfunction and osteopetrosis, in which bones harden into “stone.” In contrast, deletion of the genes encoding other members of the Src family kinase (SFK) fails to produce an osteopetrotic phenotype. This suggests that c-Src performs a unique function in the osteoclast that cannot be compensated for by other SFKs. We aimed to identify the molecular basis of this unique role in osteoclasts and bone resorption. We found that c-Src, Lyn, and Fyn were the most highly expressed SFKs in WT osteoclasts, whereas Hck, Lck, Blk, and Fgr displayed low levels of expression. Formation of the podosome belt, clusters of unique actin assemblies, was disrupted in src−/− osteoclasts; introduction of constitutively activated SFKs revealed that only c-Src and Fyn could restore this process. To identify the key structural domains responsible, we constructed chimeric Src–Hck and Src–Lyn constructs in which the unique, SH3, SH2, or catalytic domains had been swapped. We found that the Src unique, SH3, and kinase domains were each crucial to establish Src functionality. The SH2 domain could however be substituted with Lyn or Hck SH2 domains. Furthermore, we demonstrate that c-Src’s functionality is, in part, derived from an SH3–proximal proline-rich domain interaction with c-Cbl, leading to phosphorylation of c-Cbl Tyr700. These data help clarify Src’s unique functionality in the organization of the cytoskeleton in osteoclasts, required for efficient bone resorption and explain why c-Src cannot be replaced, in osteoclasts, by other SFKs.

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.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the oral cavity and the head and neck region. Gingival squamous cell carcinomas (SCCs) frequently invade the maxilla or the mandibular bone and are associated with poor prognosis. Recent findings suggest that osteoclasts, rather than OSCC cells, mediate invasion to the bone. Nuclear factor-κB (NF-κB) is constitutively activated in OSCCs and is involved in promoting the invasive characteristics of OSCC. NF-κB activation is also important for receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. NF-κB inhibitors suppress proliferation and stimulate apoptosis of OSCC cells in vitro and in vivo, as well as inhibit matrix metalloproteinase (MMP) production in OSCC. Furthermore, NF-κB inhibitors have been shown to suppress osteoclastogenesis by reducing RANKL expression in animal models. Thus, inhibition of NF-κB activity may constitute a promising therapeutic approach to treat bone-invasive OSCC. In this review, we discuss recent findings, which suggest that bone invasion in OSCC is mediated via NF-κB signaling and may be successfully prevented by NF-κB inhibition.

Background Bone and cartilage are essential skeletal tissues, which not only function as structural basis of locomotive organs, but also regulate calcium homeostasis, phosphate metabolism, hematopoiesis, and glucose turnover. Several hormones and cytokines in cooperation with their downstream transcription factors regulate bone and cartilage development; therefore, it is important to understand the precise mechanisms of this regulation. Highlight Genetic studies in human and mouse have provided a wealth of information regarding the transcription factors implicated in bone and cartilage development. Moreover, innovative molecular cloning techniques identified several new transcription factors that play indispensable roles in controlling the development of bone and cartilage. The mechanisms controlling the expression of these transcription factors have been meticulously elucidated, so that the transcriptional network system, which seemed so complex and mysterious not so long ago, has become considerably clearer in recent years. Conclusion Recent advances in our knowledge about transcriptional network systems contributed to understanding the molecular underpinnings of regulation and pathological disease mechanisms in bone and cartilage.

Satellite cells (SCs) are skeletal muscle stem cells that proliferate in response to injury and provide myogenic precursors for growth and repair. Zfp423 is a transcriptional cofactor expressed in multiple immature cell populations, such as neuronal precursors, mesenchymal stem cells, and preadipocytes, where it regulates lineage allocation, proliferation, and differentiation., Satellite cells (SCs) are skeletal muscle stem cells that proliferate in response to injury and provide myogenic precursors for growth and repair. Zfp423 is a transcriptional cofactor expressed in multiple immature cell populations, such as neuronal precursors, mesenchymal stem cells, and preadipocytes, where it regulates lineage allocation, proliferation, and differentiation. Here, we show that Zfp423 regulates myogenic progression during muscle regeneration. Zfp423 is undetectable in quiescent SCs but becomes expressed during SC activation. After expansion, Zfp423 is gradually downregulated as committed SCs terminally differentiate. Mice with satellite-cell-specific Zfp423 deletion exhibit severely impaired muscle regeneration following injury, with aberrant SC expansion, defective cell cycle exit, and failure to transition efficiently from the proliferative stage toward commitment. Consistent with a cell-autonomous role of Zfp423, shRNA-mediated knockdown of Zfp423 in myoblasts inhibits differentiation. Surprisingly, forced expression of Zfp423 in myoblasts induces differentiation into adipocytes and arrests myogenesis. Affinity purification of Zfp423 in myoblasts identified Satb2 as a nuclear partner of Zfp423 that cooperatively enhances Zfp423 transcriptional activity, which in turn affects myoblast differentiation. In conclusion, by controlling SC expansion and proliferation, Zfp423 is essential for muscle regeneration. Tight regulation of Zfp423 expression is essential for normal progression of muscle progenitors from proliferation to differentiation.

Osteoclasts resorb bone by attaching on the bone matrix and forming a sealing zone. In Src-deficient mice, osteoclasts cannot form the actin ring, a characteristic actin structure that seals the resorbed area, and resorb hardly any bone as a result. However, the molecular mechanism underlying the role of Src in the regulation and organization of the actin ring is still unclear. We identified an actin-regulatory protein, protein phosphatase 1 regulatory subunit 18 (PPP1r18), as an Src-binding protein in an Src-, Yes-, and Fyn-deficient fibroblast (SYF) cell line overexpressing a constitutively active form of Src. PPP1r18 was localized in the nucleus and actin ring. PPP1r18 overexpression in osteoclasts inhibited terminal differentiation, actin ring formation, and bone-resorbing activity. A mutation of the protein phosphatase 1 (PP1)-binding domain of PPP1r18 rescued these phenotypes. In contrast, PPP1r18 knockdown promoted terminal differentiation and actin ring formation. In summary, we showed that PPP1r18 likely plays a role in podosome organization and bone resorption.

It is difficult to cope with the product surface inspections with complicated textures by using the conventional procedures such as basic banalization, morphology processing, etc. Then we propose a set of new local features which could be sensitive to the differences among neighboring ROI's and experimented successfully how the proposed features were effective.

Osteoclasts are cells that resorb the bone matrix and maintain bone and calcium homeostasis. An actin ring is a characteristic actin structure that is essential for bone resorption by osteoclasts. Tyrosine kinase Src deficient osteoclasts do not form actin rings; thus, Src is a key molecule for actin ring formation in osteoclasts. However, how Src regulates actin ring formation is not fully understood. We identified the cytolinker protein plectin as a Src-binding protein by immunoprecipitation and liquid chromatography tandem mass spectrometry. Plectin is a huge protein (>500 kDa) and regulates the cytoskeleton by binding to actin and tubulin. We assessed the expression and role of plectin in osteoclasts. Plectin was expressed and co-localized with Src close to the actin ring in osteoclasts. Moreover, plectin was tyrosine-phosphorylated by Src. Differentiation and actin ring formation were inhibited by downregulation of plectin. These results suggest an important role for plectin in osteoclast differentiation and actin ring formation through Src binding.

Endochondral ossification is temporally and spatially regulated by several critical transcription factors, including Sox9, Runx2, and Runx3. Although the molecular mechanisms that control the late stages of endochondral ossification (e.g. calcification) are physiologically and pathologically important, these precise regulatory mechanisms remain unclear. Here, we demonstrate that Osterix is an essential transcription factor for endochondral ossification that functions downstream of Runx2. The global and conditional Osterix-deficient mice studied here exhibited a defect of cartilage-matrix ossification and matrix vesicle formation. Importantly, Osterix deficiencies caused the arrest of endochondral ossification at the hypertrophic stage. Microarray analysis revealed that matrix metallopeptidase 13 (MMP13) is an important target of Osterix. We also showed that there exists a physical interaction between Osterix and Runx2 and that these proteins function cooperatively to induce MMP13 during chondrocyte differentiation. Most interestingly, the introduction of MMP13 stimulated the calcification of matrices in Osterix-deficient mouse limb bud cells. Our results demonstrated that Osterix was essential to endochondral ossification and revealed that the physical and functional interaction between Osterix and Runx2 were necessary for the induction of MMP13 during endochondral ossification.

Bone morphogenetic protein(s) (BMP) are very powerful cytokines that induce bone and cartilage formation. BMP also stimulate osteoblast and chondrocyte differentiation. During bone and cartilage development, BMP regulates the expression and/or the function of several transcription factors through activation of Smad signalling. Genetic studies revealed that Runx2, Osterix and Sox9, all of which function downstream of BMP, play essential roles in bone and/or cartilage development. In addition, two other transcription factors, Msx2 and Dlx5, which interact with BMP signalling, are involved in bone and cartilage development. The importance of these transcription factors in bone and cartilage development has been supported by biochemical and cell biological studies. Interestingly, BMP is regulated by several negative feedback systems that appear necessary for fine-tuning of bone and cartilage development induced by BMP. Thus, BMP harmoniously regulates bone and cartilage development by forming network with several transcription factors.

A tyrosine kinase, c-Src, that plays an indispensable role in ruffled border formation and bone resorption is constitutively active in osteoclasts. However, to date, the molecular mechanism underlying increased c-Src activity in osteoclasts is unknown. To address this, we first examined the expression levels and subcellular localization of Csk, a negative regulatory kinase for c-Src. We found that the expression level of Csk in osteoclasts was comparable with that of other tissues. However, in osteoclasts, Csk was hardly localized in lipid rafts, where c-Src is highly expressed. Interestingly, expression of Cbp, which recruits Csk into lipid rafts through physical interaction with Csk, was very low in osteoclasts compared with other tissues. To understand the importance of Cbp in osteoclasts, we introduced Cbp into osteoclasts using an adenovirus gene delivery system. Introduction of Cbp stimulated recruitment of Csk into lipid rafts and suppressed c-Src activity in a dose-dependent manner. Furthermore, introduction of Cbp markedly inhibited formation of actin rings and bone-resorbing activity in osteoclasts. In addition, treatment with RANKL and overexpression of TRAF6 or NFAT2 inhibited Cbp expression in the osteoclastogenic cell line RAW264.7 along with osteoclastic differentiation. NFAT2 overexpression also inhibited Cbp expression in spleen macrophages. Collectively, our results indicate that reduction in Cbp expression is responsible for maintaining high c-Src activity in osteoclasts. These findings contribute to an understanding of the unique regulatory system for c-Src in osteoclasts.

Genetic and cell biological studies have indicated that Indian hedgehog (Ihh) plays an important role in bone development and osteoblast differentiation. However, the molecular mechanism by which Ihh regulates osteoblast differentiation is complex and remains to be fully elucidated. In this study, we investigated the role of Ihh signaling in osteoblast differentiation using mesenchymal cells and primary osteoblasts. We observed that Ihh stimulated alkaline phosphatase (ALP) activity, osteocalcin expression, and calcification. Overexpression of Gli2- but not Gli3-induced ALP, osteocalcin expression, and calcification of these cells. In contrast, dominant-negative Gli2 markedly inhibited Ihh-dependent osteoblast differentiation. Ihh treatment or Gli2 overexpression also up-regulated the expression of Runx2, an essential transcription factor for osteoblastogenesis, and enhanced the transcriptional activity and osteogenic action of Runx2. Coimmunoprecipitation analysis demonstrated a physical interaction between Gli2 and Runx2. Moreover, Ihh or Gli2 overexpression failed to increase ALP activity in Runx2-deficient mesenchymal cells. Collectively, these results suggest that Ihh regulates osteoblast differentiation of mesenchymal cells through up-regulation of the expression and function of Runx2 by Gli2.

Tyrosine kinase c-Src plays an essential role in ruffled border formation and bone resorption in osteoclasts; however, it is unclear how c-Src controls ruffled border formation during bone resorption. To address this question, we investigated the role of cortactin, a c-Src substrate, in osteoclasts. We found that cortactin showed colocalization with c-Src and actin rings in osteoclasts. Overexpression of cortactin stimulated actin ring formation in RAW 264.7 cells. In contrast, overexpression of Csk inhibited tyrosine phosphorylation of cortactin and binding of cortactin to c-Src. More importantly, overexertion of a mutant cortactin strongly suppressed actin ring formation and bone resorbing activity in osteoclasts. Collectively, our data indicate that cortactin controls osteoclastic bone resorption by regulating actin organization.