Chicago sky blue 6B inhibits α-synuclein aggregation and propagation.

Abnormal deposition of α-synuclein aggregates in Lewy bodies and Lewy neurites is the hallmark lesion in Parkinson's disease (PD). These aggregates, thought to be the culprit of disease pathogenesis, spread throughout the brain as the disease progresses. Agents that inhibit α-synuclein aggregation and/or spread of aggregates would thus be candidate disease-modifying drugs. Here, we found that Chicago sky blue 6B (CSB) may be such a drug, showing that it inhibits α-synuclein aggregation and cell-to-cell propagation in both in vitro and in vivo models of synucleinopathy. CSB inhibited the fibrillation of α-synuclein in a concentration-dependent manner through direct binding to the N-terminus of α-synuclein. Furthermore, both seeded polymerization and cell-to-cell propagation of α-synuclein were inhibited by CSB treatment. Notably, CSB alleviated behavioral deficits and neuropathological features, such as phospho-α-synuclein and astrogliosis, in A53T α-synuclein transgenic mice. These results indicate that CSB directly binds α-synuclein and inhibits its aggregation, thereby blocking α-synuclein cell-to-cell propagation.

Bone Generation Following Repeated Administration of Recombinant Bone Morphogenetic Protein 2.

BACKGROUND: The delivery of recombinant human bone morphogenetic protein 2 (rhBMP2) by using various carriers has been used to successfully induce bone formation in many animal models. However, the effect of multiple administration of rhBMP2 on bone formation and BMP2 antibody production has not been determined. Our aim was to examine the bone formation activity of rhBMP2 and serum levels of anti-BMP2 antibodies following the repeated administration of rhBMP2 in mice. METHODS: Absorbable collagen sponges or polyphosphazene hydrogels containing rhBMP2 were subcutaneously implanted or injected into one side on the back of six-week-old C57BL/6 mice. Three or 4 weeks later, the same amount of rhBMP2 was administered again with the same carrier into the subcutaneous regions on the other side of the back or into calvarial defects. The effects of a single administration of rhBMP2 on the osteoinductive ability in the ectopic model were compared with those of repeated administrations. In vivo ectopic or orthotopic bone formation was evaluated using microradiography and histological analyses. Serum concentrations of anti-rhBMP2 antibodies were measured by ELISAs. RESULTS: Re-administration of the same amount of rhBMP2 into the subcutaneous area showed a comparable production of ectopic bone as after the first administration. The bone forming ability of repeated rhBMP2 administrations was equal to that of single rhBMP2 administration. The administration of rhBMP2 into calvarial defects, following the first subcutaneous administration of rhBMP2 on the back, completely recovered the defect area with newly regenerated bone within 3 weeks. Repeated administration of rhBMP2 at 4-week intervals did not significantly alter the serum levels of anti-BMP2 antibodies and did not induce any inflammatory response. The serum obtained from rhBMP2-exposed mice had no effect on the ability of rhBMP2 to induce osteogenic gene expressions in MC3T3-E1. CONCLUSION: We suggest that the osteoinductive ability of rhBMP2 is not compromised by repeated administrations. Thus, rhBMP2 can be repeatedly used for bone regeneration at various sites within a short duration.

Macrophage-Stimulating Protein Enhances Osteoblastic Differentiation via the Recepteur d'Origine Nantais Receptor and Extracellular Signal-Regulated Kinase Signaling Pathway.

BACKGROUND: Macrophage-stimulating protein (MSP; also known as macrophage stimulating 1 and hepatocyte growth factor-like protein) has been shown to play a crucial role in calcium homeostasis and skeletal mineralization in zebrafish. However, the precise role of MSP in osteoblasts has not been elucidated. In this study, we investigated the effect of MSP on osteoblast differentiation of pre-osteoblast cells. METHODS: Osteoblast differentiation upon MSP treatment was evaluated by analyzing the osteogenic gene expression, alkaline phosphatase (ALP) activity, and mineralized nodule formation. To assess changes in the MSP-RON signaling pathway, knockdown of Ron gene was performed using siRNA and pharmacological inhibitor treatment. RESULTS: Expression of the tyrosine kinase receptor RON, a receptor of MSP, was found to be significantly increased during osteoblast differentiation. MSP treatment significantly upregulated the expression of osteogenic marker genes and remarkably increased ALP activity and mineralized nodule formation. Conversely, knockdown of Ron significantly attenuated the expression of osteogenic marker genes and ALP activity that were induced upon MSP treatment. Mechanistically, MSP treatment significantly enhanced the phosphorylation of extracellular signal-regulated kinase (ERK); however, additional treatment with the selective ERK inhibitor PD98059 attenuated the effect of MSP on osteoblast differentiation. CONCLUSIONS: Altogether, these results indicate that the MSP-RON axis is involved in promoting osteoblast differentiation via activation of the ERK signaling pathway.

CUEDC2 controls osteoblast differentiation and bone formation via SOCS3-STAT3 pathway.

The CUE domain-containing 2 (CUEDC2) protein plays critical roles in many biological processes, such as the cell cycle, inflammation, and tumorigenesis. However, whether CUEDC2 is involved in osteoblast differentiation and plays a role in bone regeneration remains unknown. This study investigated the role of CUEDC2 in osteogenesis and its underlying molecular mechanisms. We found that CUEDC2 is expressed in bone tissues. The expression of CUEDC2 decreased during bone development and BMP2-induced osteoblast differentiation. The overexpression of CUEDC2 suppressed the osteogenic differentiation of precursor cells, while the knockdown of CUEDC2 showed the opposite effect. In vivo studies showed that the overexpression of CUEDC2 decreased bone parameters (bone volume, bone area, and bone mineral density) during ectopic bone formation, whereas its knockdown increased bone volume and the reconstruction percentage of critical-size calvarial defects. We found that CUEDC2 affects STAT3 activation by regulating SOCS3 protein stability. Treatment with a chemical inhibitor of STAT3 abolished the promoting effect of CUEDC2 silencing on osteoblast differentiation. Together, we suggest that CUEDC2 functions as a key regulator of osteoblast differentiation and bone formation by targeting the SOCS3-STAT3 pathway. CUEDC2 manipulation could serve as a therapeutic strategy for controlling bone disease and regeneration.

Transmembrane protein 64 modulates prostate tumor progression by regulating Wnt3a secretion.

Wnt3a is a glycosylated ligand that activates the ß-catenin-dependent signaling pathway. Wnt signaling is also important in the prostate tumor microenvironment, and Wnt proteins secreted by the tumor stroma promote resistance to therapy. Bioactive Wnt3a production requires a number of dedicated factors in the secretory cell, but their coordinated functions are not fully understood. We previously reported transmembrane protein 64 (Tmem64) as a novel regulator of the Wnt/ß-catenin signaling pathway, which is correlated with ß-catenin regulation. In the present study, the role of Tmem64 in prostate cancer cells was investigated by modulating Wnt3a secretion. Overexpression of Tmem64 inhibited Wnt3a secretion and Lef/Tcf-sensitive transcription. By contrast, a Tmem64 mutation deleting the protein's transmembrane region restored Wnt3a secretion. Notably, Tmem64 protein and mRNA in PC3 cells were significantly overexpressed compared with that observed in LNCaP and DU145 cells. In a mouse metastasis model intracardially injected with PC3 cells, Tmem64 expression was downregulated in the metastatic spine and mandible lesions compared with in the primary injection regions. However, Wnt3a was strongly expressed in the metastatic spine and mandible lesions. Collectively, these findings suggest that Tmem64 is involved in the metastatic progression of prostate cancer cells by regulating Wnt3a secretion.

ATF3 mediates the inhibitory action of TNF-α on osteoblast differentiation through the JNK signaling pathway.

Tumor necrosis factor (TNF)-α, which is a proinflammatory cytokine, inhibits osteoblast differentiation under diverse inflammatory conditions. Activating transcription factor 3 (ATF3), which is a member of the ATF/cAMP response element-binding protein family of transcription factors, has been implicated in the regulation of cell proliferation and differentiation. However, the precise interactions between ATF3 and the TNF-α signaling pathway in the regulation of osteoblast differentiation remain unclear. In this study, we examined the role of ATF3 in the TNF-α-mediated inhibition of osteoblast differentiation and investigated the signaling pathways involved. The treatment of cells with TNF-α downregulated osteogenic markers, but significantly upregulated the expression of Atf3. The inhibition of Atf3 by small interfering RNAs rescued osteogenesis, which was inhibited by TNF-α. Conversely, the enforced expression of Atf3 enhanced the TNF-α-mediated inhibition of osteoblast differentiation, as revealed by the measurement of osteogenic markers and alkaline phosphatase staining. Mechanistically, TNF-α-induced Atf3 expression was significantly suppressed by the inhibition of the c-Jun N-terminal kinase (JNK) pathway. Furthermore, the overexpression of Atf3 did not affect the rescue effect that inhibiting TNF-α expression using a JNK inhibitor had on alkaline phosphatase activity and mineralization. Taken together, these results indicate that ATF3 mediates the inhibitory action of TNF-α on osteoblast differentiation and that the TNF-α-activated JNK pathway is responsible for the induction of Atf3 expression.

The Fam50a positively regulates ameloblast differentiation via interacting with Runx2.

Differentiated ameloblasts secret enamel matrix proteins such as amelogenin, ameloblastin, and enamelin. Expression levels of these proteins are regulated by various factors. To find a new regulatory factor for ameloblast differentiation, we performed 2D-PAGE analysis using mouse ameloblast lineage cell line (mALCs) cultured with mineralizing medium. Of identified proteins, family with sequence similarity 50 member A (Fam50a) was significantly increased during differentiation of mALCs. Fam50a protein was also highly expressed in secretory ameloblasts of mouse tooth germs. In mALCs cultures, forced expression of Fam50a up-regulated the expression of enamel matrix protein genes such as amelogenin, ameloblastin, and enamelin. In addition, up-regulation of Fam50a also increased ALP activity and mineralized nodule formation in a dose-dependent manner. In contrast, knockdown of Fam50a decreased expression levels of enamel matrix protein genes, ALP activity, and mineralized nodule formation. By fluorescence microscopy, endogenous Fam50a protein was found to be localized to the nucleus of ameloblasts. In addition, Fam50a synergistically increased Ambn transactivation by Runx2. Moreover, Fam50a increased binding affinity of Runx2 to Ambn promoter by physically interacting with Runx2. Taken together, these results suggest Fam50a might be a new positive regulator of ameloblast differentiation.

PHF20 positively regulates osteoblast differentiation via increasing the expression and activation of Runx2 with enrichment of H3K4me3.

Plant homeodomain finger protein 20 (PHF20), a methyl lysine effector protein, is a component MOF-NSL lysine acetyltranferase complex. Global deletion of PHF20 has shown spinal bone defects and reduced skeletal formation. However, the molecular basis of PHF20 involved in skeletal development has not been elucidated yet. The objective of this study was to determine the role of PHF20 in osteoblast differentiation and mineralization. Expression of PHF20 was gradually increased during osteoblast differentiation. Overexpression of PHF20 enhanced ALP activity and mineralized nodule formation as well as the expression of osteogenic markers including Runx2. In contrast, inhibition of PHF20 expression reduced osteoblast differentiation and mineralization. Mechanistically, PHF20 increased the promoter activity of osteogenic genes including Og2, Alp, and Bsp through direct association with Runx2. Moreover, PHF20 increased the enrichment of H3K4me3 on the promoter of Runx2 followed by increased Runx2 promoter activity. Interestingly, Bix-01294, a histone methylation inhibitor, decreased mineralized nodule formation through decreasing the levels of H3K4me3 and Runx2. Overexpression of PHF20 restored the Bix-01294 effects. Taken together, these results indicate that methyl lysine-binding protein PHF20 might be a novel regulator of osteoblast differentiation.

Chemical inhibitors of c-Met receptor tyrosine kinase stimulate osteoblast differentiation and bone regeneration.

The c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), have been recently introduced to negatively regulate bone morphogenetic protein (BMP)-induced osteogenesis. However, the effect of chemical inhibitors of c-Met receptor on osteoblast differentiation process has not been examined, especially the applicability of c-Met chemical inhibitors on in vivo bone regeneration. In this study, we demonstrated that chemical inhibitors of c-Met receptor tyrosine kinase, SYN1143 and SGX523, could potentiate the differentiation of precursor cells to osteoblasts and stimulate regeneration in calvarial bone defects of mice. Treatment with SYN1143 or SGX523 inhibited HGF-induced c-Met phosphorylation in MC3T3-E1 and C3H10T1/2 cells. Cell proliferation of MC3T3-E1 or C3H10T1/2 was not significantly affected by the concentrations of these inhibitors. Co-treatment with chemical inhibitor of c-Met and osteogenic inducing media enhanced osteoblast-specific genes expression and calcium nodule formation accompanied by increased Runx2 expression via c-Met receptor-dependent but Erk-Smad signaling independent pathway. Notably, the administration of these c-Met inhibitors significantly repaired critical-sized calvarial bone defects. Collectively, our results suggest that chemical inhibitors of c-Met receptor tyrosine kinase might be used as novel therapeutics to induce bone regeneration.

ATF3 modulates calcium signaling in osteoclast differentiation and activity by associating with c-Fos and NFATc1 proteins.

Activating transcription factor 3 (ATF3), a member of the ATF/cAMP response element-binding protein family of transcription factors, has been implicated in the regulation of cell proliferation and differentiation. However, whether ATF3 is involved in osteoclast differentiation and activity has not been well-studied. In the present study, we examined the role of ATF3 in osteoclast differentiation and function. ATF3 expression was down-regulated during RANKL-induced osteoclast differentiation. Overexpression of ATF3 in bone marrow-derived monocyte/macrophage lineage cells (BMMs) promoted osteoclast differentiation and activity and strongly induced the expression of osteoclast genes encoding nuclear factor of activated T-cells c1 (NFATc1) and tartrate-resistant acid phosphatase (TRAP) compared to that in the control group. In contrast, small interfering RNA-mediated knockdown of ATF3 prevented the formation of multinucleated osteoclasts and markedly abrogated the expression of osteoclast marker genes. Mechanistically, ATF3 synergistically enhanced c-Fos- or NFAT-mediated transcriptional activity of the NFATc1 or TRAP promoter, respectively. Furthermore, ATF3 physically interacted with c-Fos and NFATc1 and enhanced the binding affinity of c-Fos and NFATc1 to the promoters. Interestingly, ATF3 is involved in calcium signaling during osteoclastogenesis. Taken together, these results suggest that ATF3 is a new co-factor of c-Fos and NFATc1 to activate osteoclast differentiation and activity.

RCANs regulate the convergent roles of NFATc1 in bone homeostasis.

Activation of calcineurin-dependent nuclear factor of activated T cells c1 (NFATc1) is convergent for normal bone homeostasis. NFATc1 regulates both osteoclastogenesis and osteoblastogenesis. Here we investigated the roles of regulator of calcineurin (RCAN) genes in bone homeostasis. RCANs function as potent physiological inhibitors of calcineurin. Overexpression of RCANs in osteoclast precursor cells attenuated osteoclast differentiation, while their overexpression in osteoblasts enhanced osteoblast differentiation and function. Intriguingly, opposing effects of RCANs in both cell types were shown by blocking activation of the calcineurin-NFATc1 pathway. Moreover, the disruption of RCAN1 or RCAN2 in mice resulted in reduced bone mass, which is associated with strongly increased osteoclast function and mildly reduced osteoblast function. Taken together, RCANs play critical roles in bone homeostasis by regulating both osteoclastogenesis and osteoblastogenesis, and they serve as inhibitors for calcineurin-NFATc1 signaling both in vivo and in vitro.

STAT5 is a key transcription factor for IL-3-mediated inhibition of RANKL-induced osteoclastogenesis.

Among the diverse cytokines involved in osteoclast differentiation, interleukin (IL)-3 inhibits RANKL-induced osteoclastogenesis. However, the mechanism underlying IL-3-mediated inhibition of osteoclast differentiation is not fully understood. Here we demonstrate that the activation of signal transducers and activators of transcription 5 (STAT5) by IL-3 inhibits RANKL-induced osteoclastogenesis through the induction of the expression of Id genes. We found that STAT5 overexpression inhibited RANKL-induced osteoclastogenesis. However, RANKL did not regulate the expression or activation of STAT5 during osteoclast differentiation. STAT5 deficiency prevented IL-3-mediated inhibition of osteoclastogenesis, suggesting a key role of STAT5 in IL-3-mediated inhibition of osteoclast differentiation. In addition, IL-3-induced STAT5 activation upregulated the expression of Id1 and Id2, which are negative regulators of osteoclastogenesis. Overexpression of ID1 or ID2 in STAT5-deficient cells reversed osteoclast development recovered from IL-3-mediated inhibition. Importantly, microcomputed tomography and histomorphometric analysis revealed that STAT5 conditional knockout mice showed reduced bone mass, with an increased number of osteoclasts. Furthermore, IL-3 inhibited RANKL-induced osteoclast differentiation less effectively in the STAT5 conditional knockout mice than in the wild-type mice after RANKL injection. Taken together, our findings indicate that STAT5 contributes to the remarkable IL-3-mediated inhibition of RANKL-induced osteoclastogenesis by activating Id genes and their associated pathways.

FGF2 Stimulates COUP-TFII Expression via the MEK1/2 Pathway to Inhibit Osteoblast Differentiation in C3H10T1/2 Cells.

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an orphan nuclear receptor that regulates many key biological processes, including organ development and cell fate determination. Although the biological functions of COUP-TFII have been studied extensively, little is known about what regulates its gene expression, especially the role of inducible extracellular factors in triggering it. Here we report that COUP-TFII expression is regulated specifically by fibroblast growth factor 2 (FGF2), which mediates activation of the MEK1/2 pathway in mesenchymal lineage C3H10T1/2 cells. Although FGF2 treatment increased cell proliferation, the induction of COUP-TFII expression was dispensable. Instead, FGF2-primed cells in which COUP-TFII expression was induced showed a low potential for osteoblast differentiation, as evidenced by decreases in alkaline phosphatase activity and osteogenic marker gene expression. Reducing COUP-TFII by U0126 or siRNA against COUP-TFII prevented the anti-osteogenic effect of FGF2, indicating that COUP-TFII plays a key role in the FGF2-mediated determination of osteoblast differentiation capability. This report is the first to suggest that FGF2 is an extracellular inducer of COUP-TFII expression and may suppress the osteogenic potential of mesenchymal cells by inducing COUP-TFII expression prior to the onset of osteogenic differentiation.

Downregulation of Runx2 by 1,25-Dihydroxyvitamin D3 Induces the Transdifferentiation of Osteoblasts to Adipocytes.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) indirectly stimulates bone formation, but little is known about its direct effect on bone formation. In this study, we observed that 1,25(OH)2D3 enhances adipocyte differentiation, but inhibits osteoblast differentiation during osteogenesis. The positive role of 1,25(OH)2D3 in adipocyte differentiation was confirmed when murine osteoblasts were cultured in adipogenic medium. Additionally, 1,25(OH)2D3 enhanced the expression of adipocyte marker genes, but inhibited the expression of osteoblast marker genes in osteoblasts. The inhibition of osteoblast differentiation and promotion of adipocyte differentiation mediated by 1,25(OH)2D3 were compensated by Runx2 overexpression. Our results suggest that 1,25(OH)2D3 induces the transdifferentiation of osteoblasts to adipocytes via Runx2 downregulation in osteoblasts.

Betulinic acid synergically enhances BMP2-induced bone formation via stimulating Smad 1/5/8 and p38 pathways.

BACKGROUND: Healing of bone defects is a dynamic and orchestrated process that relies on multiple growth factors and cell types. Bone morphogenetic protein 2 (BMP2) is a key growth factor for bone healing, which stimulates mesenchymal stem cells to differentiate into osteoblasts. Betulinic acid (BetA) is a natural pentacyclic triterpenoid from plants. This study aimed to examine combinatory effects of BetA and BMP2 on ectopic bone generation in mice. RESULTS: In MC3T3-E1 preosteoblast culture, 10-15 µM of BetA increased the alkaline phosphatase (ALP) activity and expression levels of osteogenic marker genes without the decreased cell viability. In addition, BetA synergistically enhanced BMP2-induced gene expressions and mineralization with the enhancement of phosphorylation of Smad1/5/8 and p38. In an in vivo ectopic bone formation model, combination of BetA (50 µg) and BMP2 (3 µg) resulted in increases in the amount of new bone generation, compared with treatment with BMP2 alone. Histological studies showed that bone generation with cortical and trabecular structures was resulted from the combination of BetA and BMP2. CONCLUSION: BetA can enhance in vivo osteogenic potentials of BMP2, possibly via stimulating Smad 1/5/8 and p38 pathways, and combination of both agents can be considered as a therapeutic strategy for bone diseases.

GATA4 negatively regulates bone sialoprotein expression in osteoblasts.

GATA4 has been reported to act as a negative regulator in osteoblast differentiation by inhibiting the Dlx5 transactivation of Runx2 via the attenuation of the binding ability of Dlx5 to the Runx2 promoter region. Here, we determine the role of GATA4 in the regulation of bone sialoprotein (Bsp) in osteoblasts. We observed that the overexpression of Runx2 or Sox9 induced the Bsp expression in osteoblastic cells. Silencing GATA4 further enhanced the Runx2- and Sox9-mediated Bsp promoter activity, whereas GATA4 overexpression down-regulated Bsp promoter activity mediated by Runx2 and Sox9. GATA4 also interacted with Runx2 and Sox9, by attenuating the binding ability of Runx2 and Sox9 to the Bsp promoter region. Our data suggest that GATA4 acts as a negative regulator of Bsp expression in osteoblasts. [BMB Reports 2016; 49(6): 343-348].

Repair of Cranial Bone Defects Using rhBMP2 and Submicron Particle of Biphasic Calcium Phosphate Ceramics with Through-Hole.

Recently a submicron particle of biphasic calcium phosphate ceramic (BCP) with through-hole (donut-shaped BCP (d-BCP)) was developed for improving the osteoconductivity. This study was performed to examine the usefulness of d-BCP for the delivery of osteoinductive rhBMP2 and the effectiveness on cranial bone regeneration. The d-BCP was soaked in rhBMP2 solution and then freeze-dried. Scanning electron microscope (SEM), energy dispersive spectroscopy (EDS), and Raman spectroscopy analyses confirmed that rhBMP2 was well delivered onto the d-BCP surface and the through-hole. The bioactivity of the rhBMP2/d-BCP composite was validated in MC3T3-E1 cells as an in vitro model and in critical-sized cranial defects in C57BL/6 mice. When freeze-dried d-BCPs with rhBMP2 were placed in transwell inserts and suspended above MC3T3-E1, alkaline phosphatase activity and osteoblast-specific gene expression were increased compared to non-rhBMP2-containing d-BCPs. For evaluating in vivo effectiveness, freeze-dried d-BCPs with or without rhBMP2 were implanted into critical-sized cranial defects. Microcomputed tomography and histologic analysis showed that rhBMP2-containing d-BCPs significantly enhanced cranial bone regeneration compared to non-rhBMP2-containing control. These results suggest that a combination of d-BCP and rhBMP2 can accelerate bone regeneration, and this could be used to develop therapeutic strategies in hard tissue healing.

The Angiopoietin-1 Variant COMP-Ang1 Enhances BMP2-Induced Bone Regeneration with Recruiting Pericytes in Critical Sized Calvarial Defects.

Craniofacial bone defects are observed in a variety of clinical situations, and their reconstructions require coordinated coupling between angiogenesis and osteogenesis. In this study, we explored the effects of cartilage oligomeric matrix protein-angiopoietin 1 (COMP-Ang1), a synthetic and soluble variant of angiopoietin 1, on bone morphogenetic protein 2 (BMP2)-induced cranial bone regeneration, and recruitment and osteogenic differentiation of perivascular pericytes. A critical-size calvarial defect was created in the C57BL/6 mouse and COMP-Ang1 and/or BMP2 proteins were delivered into the defects with absorbable collagen sponges. After 3 weeks, bone regeneration was evaluated using micro-computed tomography and histologic examination. Pericyte recruitment into the defects was examined using immunofluorescence staining with anti-NG2 and anti-CD31 antibodies. In vitro recruitment and osteoblastic differentiation of pericyte cells were assessed with Boyden chamber assay, staining of calcified nodules, RT-PCR and Western blot analyses. Combined administration of COMP-Ang1 and BMP2 synergistically enhanced bone repair along with the increased population of CD31 (an endothelial cell marker) and NG2 (a specific marker of pericyte) positive cells. In vitro cultures of pericytes consistently showed that pericyte infiltration into the membrane pore of Boyden chamber was more enhanced by the combination treatment. In addition, the combination further increased the osteoblast-specific gene expression, including bone sialoprotein (BSP), osteocalcin (OCN) and osterix (OSX), phosphorylation of Smad/1/5/8, and mineralized nodule formation. COMP-Ang1 can enhance BMP2-induced cranial bone regeneration with increased pericyte recruitment. Combined delivery of the proteins might be a therapeutic strategy to repair cranial bone damage.

Effects of light-emitting diode irradiation on RANKL-induced osteoclastogenesis.

BACKGROUND AND OBJECTIVE: Bone homeostasis is maintained by a balance between osteoblastic bone formation and osteoclastic bone resorption, where intracellular reactive oxygen species (ROS) are crucial mediators of osteoclastogenesis. Recently, low-level light therapy (LLLT), a form of laser medicine used in various clinical fields, was shown to alleviate oxidative stress by scavenging intracellular ROS. The present study aimed to investigate the impact of 635 nm irradiation from a light-emitting diode (LED) on osteoclastogenesis from receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL)-stimulated mouse bone marrow-derived macrophages (BMMs). STUDY DESIGN/MATERIALS AND METHODS: The effects of LED irradiation on osteoclastogenesis were assessed in tartrate-resistant acid phosphatase (TRAP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell viability, and resorption pit formation, respectively. Quantitative real-time polymerase chain reaction (qPCR) and Western blot analyses were also performed to assess mRNA expression of osteoclastogenesis-related genes and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2), p38, and c-Jun-N-terminal kinase (JNK). NF-κB activity was assayed by luciferase reporter assay and Intracellular ROS generation was investigated by the 2',7'-dichlorodihydrofluorescein diacetate (H2 DCF-DA) detection method. RESULTS: LED irradiation significantly inhibited RANKL-mediated osteoclast differentiation from BMMs and mRNA expression of TRAP, osteoclast-associated immunoglobulin-like receptor (OSCAR), and dendrocyte-expressed seven-transmembrane protein (DC-STAMP). Exposure to LED light likewise significantly decreased RANKL-facilitated NF-κB activity, p38 and ERK phosphorylation and intracellular ROS generation, and increased gene expression of nuclear factor E2-related factor 2 (Nrf2). CONCLUSIONS: Taken together, the results presented herein show that LED irradiation downregulates osteoclastogenesis by reducing ROS production. Therefore, LED irradiation/LLLT might be useful as an alternative, conservative approach to osteoporosis management.

Transmembrane protein 64 reciprocally regulates osteoblast and adipocyte differentiation by modulating Wnt/ß-catenin signaling.

Age-related osteoporosis is associated with a reciprocal decrease in bone formation and an increase in adiposity in the bone marrow niche. We previously reported Transmembrane protein 64 (Tmem64) to be an important regulator of osteoclast function; however, its precise role in osteoblasts has not yet been established. Here, we showed that ablation of the Tmem64 gene in mice resulted in markedly increased osteoblast and reduced adipocyte differentiation from bone marrow-derived stromal cells (BMSCs). Conversely, Tmem64 overexpression inhibited osteogenesis and accelerated adipogenesis. Furthermore, BMSCs isolated from Tmem64 knockout mice formed a greater number of colony-forming unit-osteoblasts and a lower number of colony-forming unit-adipocytes than the wild type controls. Mechanistically, the expression level of ß-catenin, the key Wnt signaling molecule, increased significantly, and its nuclear translocation was enhanced in Tmem64-deficient cells. Introduction of Tmem64 significantly suppressed ß-catenin-mediated transcriptional activity in an in vitro co-transfection experiment as well as during an in vivo experiment involving BAT-Gal reporter mice. These results demonstrate that Tmem64 plays an important role in the regulation of mesenchymal lineage allocation by modulating Wnt/ß-catenin signaling.