Affect Osteoclast Formation Research Articles

Atsttrin regulates osteoblastogenesis and osteoclastogenesis through the TNFR pathway

Osteoporosis is a systemic metabolic bone disorder for which inflammatory cytokines play an important role. To develop new osteoporosis treatments, strategies for improving the microenvironment for osteoblast and osteoclast balance are needed. Tumor necrosis factor-α (TNF-α) plays an important role in the initiation and development of osteoporosis. Atsttrin is an engineered protein derived from the growth factor, progranulin (PGRN). The present study investigates whether Atsttrin affects osteoclast formation and osteoblast formation. Here we show Atsttrin inhibits TNF-α-induced osteoclastogenesis and inflammation. Further mechanistic investigation indicates Atsttrin inhibits TNF-α-induced osteoclastogenesis through the TNFR1 signaling pathway. Moreover, Atsttrin rescues TNF-α-mediated inhibition of osteoblastogenesis via the TNFR1 pathway. Importantly, the present study indicates that while Atsttrin cannot directly induce osteoblastogenesis, it can significantly enhance osteoblastogenesis through TNFR2-Akt-Erk1/2 signaling. These results suggest that Atsttrin treatment could potentially be a strategy for maintaining proper bone homeostasis by regulating the osteoclast/osteoblast balance. Additionally, these results provide new insights for other bone metabolism-related diseases.

LSD1 knockdown confers protection against osteoclast formation by reducing histone 3 lysine 9 monomethylation and dimethylation in ITGB3 promoter

ITGB3, an osteoclast marker, is involved in osteoclast formation. Nevertheless, its related mechanism remains poorly characterized. Herein, this study examines the mechanisms affecting osteoclast formation with the involvement of ITGB3. Osteoclast formation was induced with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand (RANKL), followed by measurement of the mRNA and protein expression of ITGB3 and LSD1. After gain- and loss-of-function assays, cell viability and the expression of osteoclast marker genes (NFATc1, ACP5, and CTSK) were assessed, and osteoclast formation was evaluated with TRAP staining. ChIP assays were used to examine histone 3 lysine 9 (H3K9) monomethylation (H3K9me1) and H3K9 dimethylation (H3K9me2) modifications and LSD1 protein enrichment in the ITGB3 promoter. During osteoclast formation, ITGB3 and LSD1 were gradually augmented. Knockdown of LSD1 or ITGB3 curbed cell viability, the expression of osteoclast marker genes, and osteoclast formation. Moreover, overexpression of ITGB3 nullified the suppressive impact of LSD1 knockdown on osteoclast formation. Mechanistically, LSD1 promoted ITGB3 expression by reducing H3K9 levels in the ITGB3 promoter. LSD1 enhanced ITGB3 expression by decreasing H3K9me1 and H3K9me2 levels in ITGB3 promoter to boost osteoclast formation.

Staphylococci planktonic and biofilm environments differentially affect osteoclast formation

IntroductionThe pathophysiology of chronic implant-related bone infections is characterized by an increase in osteoclast numbers and enhanced bone resorption. Biofilms are a major reason for chronicity of such infections as the biofilm matrix protects bacteria against antibiotics and impairs the function of immune cells. Macrophages are osteoclast precursor cells and therefore linked to inflammation and bone destruction.Objective and methodInvestigations on the impact of biofilms on the ability of macrophages to form osteoclasts are yet missing and we, therefore, analyzed the effect of Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) planktonic and biofilm environments on osteoclastogenesis using RAW 264.7 cells and conditioned media (CM).ResultsPriming with the osteoclastogenic cytokine RANKL before CM addition enabled the cells to differentiate into osteoclasts. This effect was highest in SE planktonic or SA biofilm CM. Simultaneous stimulation with CM and RANKL, however, suppressed osteoclast formation and resulted in formation of inflammation-associated multinucleated giant cells (MGCs) which was most pronounced in SE planktonic CM.ConclusionOur data indicate that the biofilm environment and its high lactate levels are not actively promoting osteoclastogenesis. Hence, the inflammatory immune response against planktonic bacterial factors through Toll-like receptors seems to be the central cause for the pathological osteoclast formation. Therefore, immune stimulation or approaches that aim at biofilm disruption need to consider that this might result in enhanced inflammation-mediated bone destruction.

The Effect of Sclerostin and Monoclonal Sclerostin Antibody Romosozumab on Osteogenesis and Osteoclastogenesis Mediated by Periodontal Ligament Fibroblasts.

Sclerostin is a bone formation inhibitor produced by osteocytes. Although sclerostin is mainly expressed in osteocytes, it was also reported in periodontal ligament (PDL) fibroblasts, which are cells that play a role in both osteogenesis and osteoclastogenesis. Here, we assess the role of sclerostin and its clinically used inhibitor, romosozumab, in both processes. For osteogenesis assays, human PDL fibroblasts were cultured under control or mineralizing conditions with increasing concentrations of sclerostin or romosozumab. For analyzing osteogenic capacity and alkaline phosphatase (ALP) activity, alizarin red staining for mineral deposition and qPCR of osteogenic markers were performed. Osteoclast formation was investigated in the presence of sclerostin or romosozumab and, in PDLs, in the presence of fibroblasts co-cultured with peripheral blood mononuclear cells (PBMCs). PDL-PBMC co-cultures stimulated with sclerostin did not affect osteoclast formation. In contrast, the addition of romosozumab slightly reduced the osteoclast formation in PDL-PBMC co-cultures at high concentrations. Neither sclerostin nor romosozumab affected the osteogenic capacity of PDL fibroblasts. qPCR analysis showed that the mineralization medium upregulated the relative expression of osteogenic markers, but this expression was barely affected when romosozumab was added to the cultures. In order to account for the limited effects of sclerostin or romosozumab, we finally compared the expression of SOST and its receptors LRP-4, -5, and -6 to the expression in osteocyte rich-bone. The expression of SOST, LRP-4, and LRP-5 was higher in osteocytes compared to in PDL cells. The limited interaction of sclerostin or romosozumab with PDL fibroblasts may relate to the primary biological function of the periodontal ligament: to primarily resist bone formation and bone degradation to the benefit of an intact ligament that is indented by every chew movement.

Dehydromiltirone inhibits osteoclast differentiation in RAW264.7 and bone marrow macrophages by modulating MAPK and NF-κB activity.

Background: Osteoporosis is a type of systematic metabolic bone disease caused by the decrease in osteogenic activity or excessive resorption of bone with the relative enhancement of osteoclast function. As osteoporosis seriously affects the quality of patients’ life, effective drugs are needed to treat this disease. Based on the combination of network pharmacology and cellular studies, this study aimed to investigate the probable mechanism of Dehydromiltirone (DHT) in the treatment of osteoporosis. Method: The targets of DHT in osteoporosis were searched using the PharmGKB, OMIM, and Genecard platforms. The PPI core targets, and the GO and KEGG enrichment analysis results were obtained using Cytoscape software, and the David and Metascape databases, respectively. The network pharmacology results were also verified via in vitro cellular experiments. Results: Through network pharmacology and docking analysis, we found DHT was involved in peptide tyrosine phosphorylation, cell surface receptor tyrosine kinase signaling pathways, and MAPK signaling pathways. According to the molecular docking results, the binding of DHT to MAPK14 was more stable than other proteins, which suggests that DHT may affect osteoclast formation through the MAPK signaling pathway. Moreover, DHT was found to inhibit the expression of osteoclast-associated genes, including NFATc1, CTSK, c-Fos, Acp5, and MMP9; as well as the phosphorylation of P38, ERK, and JNK of the MAPK signaling pathway; and the degradation of IκB-α of NF-κB signaling pathway. Conclusion: DHT exhibited an anti-osteoclastogenesis effect by reducing the expression of related genes, ultimately inhibiting bone resorption in vitro.

Osteoclast formation from mouse bone marrow cells on micro/nano-scale patterned surfaces

ObjectivesOsteoclasts can sense the surface topography of materials. However, it is difficult to identify the structural factors that affect osteoclast formation and its function. Furthermore, we hypothesized that the type of osteoclast precursor cells also affects osteoclastogenesis in the materials. In this study, we investigated the effects of defined micro/nanoscale patterns on osteoclastogenesis from bone marrow cells (BMCs). MethodsVarious cyclo-olefin polymer (COP) patterns were prepared using nanoimprinting. The effects of shape, size, and height of the patterns, and the wettability of the patterned surfaces on osteoclastogenesis from BMCs were evaluated in vitro. ResultsOsteoclast formation was promoted on pillars (diameter, 1 μm or 500 nm; height, 500 nm). Notably, osteoclastogenesis from BMCs was better promoted on hydrophobic pillars than on hydrophilic pillars. In contrast, decreased osteoclast formation was observed on the nanopillars (diameter, 100 nm; height, 200 nm). ConclusionsWe demonstrated the promotion of osteoclast formation from BMCs on hydrophobic pillars with diameters of 1 μm and 500 nm. Some cellular behaviors in the patterns were dependent on the type of osteoclast precursor cells. The designed patterns are useful for designing the surface of dental implants or bone replacement materials with a controllable balance between osteoblast and osteoclast activities.

ThPOK Inhibits Osteoclast Formation Via NFATc1 Transcription and Function.

ABSTRACTBoth LRF (Zbtb7a) and ThPOK (Zbtb7b) belong to the POK (BTB/POZ and Kruppel) family of transcription repressors that participate in development, differentiation, and oncogenesis. Although LRF mediates osteoclast differentiation by regulating NFATc1 expression, the principal established function of ThPOK is transcriptional control of T‐cell lineage commitment. Whether ThPOK affects osteoclast formation or function is not known. We find that marrow macrophage ThPOK expression diminishes with exposure to receptor activator of NF‐kB ligand (RANKL), but ThPOK deficiency does not affect osteoclast differentiation. On the other hand, enhanced ThPOK, in macrophages, substantially impairs osteoclastogenesis. Excess ThPOK binds the NFATc1 promoter and suppresses its transcription, suggesting a mechanism for its osteoclast inhibitory effect. Despite suppression of osteoclastogenesis by excess ThPOK being associated with diminished NFATc1, osteoclast formation is not rescued by NFATc1 overexpression. Thus, ThPOK appears to inhibit NFATc1 transcription and its osteoclastogenic capacity, while its depletion has no effect on the bone‐resorptive cell. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

ThPOK inhibits osteoclast formation via NFATc1 transcription and function

Both LRF (Zbtb7a) and ThPOK (Zbtb7b) belong to the POK (BTB/POZ and Kruppel) family of transcription repressors which participate in development, differentiation and oncogenesis. While LRF mediates osteoclast differentiation by regulating NFATc1 expression, the principal established function of ThPOK is transcriptional control of T-cell lineage commitment. Whether ThPOK affects osteoclast formation or function, is not known.We find that marrow macrophage ThPOK expression diminishes with exposure to RANKL but ThPOK deficiency does not affect osteoclast differentiation. On the other hand, enhanced ThPOK, in macrophages, substantially impairs osteoclastogenesis. Excess ThPOK binds the NFATc1 promoter and suppresses its transcription suggesting a mechanism for its osteoclast inhibitory effect. Despite suppression of osteoclastogenesis by excess ThPOK being associated with diminished NFATc1, osteoclast formation is not rescued by NFATc1 overexpression. Thus, ThPOK appears to inhibit NFATc1 transcription and its osteoclastogenic capacity while its depletion has no effect on the bone resorptive cell. This article is protected by copyright. All rights reserved.

Enamel matrix derivative does not affect osteoclast formation or bone resorption in cultures of mouse bone marrow macrophages or human monocytes

Objective Enamel matrix derivative (EMD) is widely used under the brand name Emdogain® to promote periodontal regeneration in surgical treatment of periodontitis and peri-implantitis. The molecular mechanisms are unclear, but it has been proposed that EMD has stimulatory effects on the root cementum and periodontal ligament cells. Since dental implants lack these structures, we hypothesized that EMD-induced bone gain involve interactions with osteoclast precursor cells, with consequent inhibitory effect on osteoclast formation and/or activity. The aim was to evaluate this hypothesis. Material and methods Primary mouse bone marrow macrophages (BMMs) and human peripheral blood monocytes were cultured in the presence of receptor activator nuclear factor-κB ligand (RANKL) to stimulate osteoclast formation. A purified Emdogain® fraction was added to the cell cultures and the effect on number and size of newly formed osteoclasts were evaluated. In cultures on natural bone slices, bioanalytical methods were used to assay osteoclast number and bone resorption. Results EMD had a negative effect on osteoclastogenesis in mouse cultures on plastic surface, whereas addition of EMD to osteoclast precursor cells on bone substrate did not affect osteoclast formation or bone resorption. Conclusions The results on natural bone matrix contradict a direct effect of EMD on osteoclast precursor cells.

GPR120 Inhibits RANKL-Induced Osteoclast Formation and Resorption by Attenuating Reactive Oxygen Species Production in RAW264.7 Murine Macrophages.

Osteoclasts are large, multinucleated cells that are responsible for the resorption of bone. Bone degenerative diseases, such as osteoporosis, are characterized by overactive osteoclasts. Receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) binding to its receptor on osteoclast precursors will trigger osteoclast formation and resorption. The production of reactive oxygen species (ROS) is known to play a crucial role in RANKL-induced osteoclast formation and resorption. G-protein coupled receptor 120 (GPR120) signalling has been shown to affect osteoclast formation, but the exact mechanisms of action require further investigation. RAW264.7 murine macrophages were seeded into culture plates and exposed to the GPR120 agonist, TUG-891, at varying concentrations (20–100 µM) and RANKL to induce osteoclast formation. TUG-891 was shown to inhibit osteoclast formation and resorption without affecting cell viability in RAW264.7 macrophages. TUG-891 further decreased ROS production when compared to RANKL only cells. Antioxidant proteins, Nrf2, HO-1 and NQO1 were shown to be upregulated while the ROS inducing protein, Nox1, was downregulated by TUG-891. Gene silencing revealed that TUG-891 exerted its effects specifically through GPR120. This study reveals that GPR120 signalling may inhibit osteoclast formation and resorption through inhibition on ROS production.

Tumor necrosis factor-α enhances the expression of vascular endothelial growth factor in a mouse orthodontic tooth movement model

Background/purposeTooth movement that is achieved using orthodontic mechanical principles relies on bone resorption which takes place on the compression side via osteoclasts. Tumor necrosis factor-α (TNF-α) has been known to affect osteoclast formation in orthodontic tooth movement (OTM). Vascular endothelial growth factor (VEGF), which is one of the mediators of angiogenesis, also plays an important role in OTM by inducing vascular permeability and chemotaxis of osteoclast precursors. Therefore, the purpose of this research was to evaluate the effect of TNF-α on VEGF expression during OTM. Materials and methodsIn order to demonstrate the effect of TNF-α on VEGF expression during OTM, a nickel titanium closed coil spring was fixed to the upper left first molar and the alveolar bone beneath the upper incisors of both wild type (WT) and TNF receptors (TNFRs) deficient mice resulting in a mesial movement of the molar for 12 days. The maxilla was removed for histological analysis and real-time RCR analysis of VEGF expression. ResultsImmunohistochemical analysis revealed that there were fewer VEGF-positive cells in the periodontal membrane on the mesial side of the distobuccal root in TNFRs-deficient mice than that in WT mice during the OTM for 12 days. Furthermore, expression of VEGF mRNA is lower level in TNFRs-deficient mice than that in WT mice. ConclusionOur results indicate that TNF-α plays an important role in VEGF expression during tooth movement.

Apremilast inhibits inflammatory osteoclastogenesis.

Psoriatic arthritis (PsA) is associated with bone erosion and inflammation-induced bone loss, which are mediated by osteoclasts (OC) and modulated by inflammatory cytokines. Apremilast (APR) (a selective phosphodiesterase 4 inhibitor) is efficacious in PsA and acts by inhibiting cytokine production. However, there are no direct data informing whether and how APR affects osteoclast formation in humans. Osteoclastogenic cytokine production by activated human peripheral blood mononuclear cells (PBMCs) was measured in the presence and absence of APR. Effects of APR on osteoclast differentiation were tested (i) in co-cultures of activated PBMCs and human CD14+ blood monocytes as well as (ii) in CD14+ blood monocytes stimulated with activated-PBMCs supernatant, TNF or IL-17A. Bone resorption was measured on OsteoAssay plates. Effects of APR on ex vivo osteoclast differentiation were compared in PsA, pre-PsA and psoriasis patients, as well as in healthy controls. APR significantly impaired the expression of key osteoclastogenic cytokines in activated PBMCs. Furthermore, APR dose-dependently and significantly inhibited activated PBMC-driven osteoclast differentiation and ex vivo osteoclast differentiation of PBMCs derived from PsA and pre-PsA patients, but not from psoriasis patients or healthy controls. TNF and IL-17A-enhanced osteoclastogenesis and osteolytic activity of CD14+ blood monocytes from PsA patients was also significantly inhibited by APR. Finally, APR inhibited expression of the key osteoclast fusion protein dendritic cell-specific transmembrane protein. Phosphodiesterase 4 targeting by APR not only inhibits osteoclastogenic cytokine production, but also directly suppresses inflammation-driven osteoclastogenesis. These data provide initial evidence that APR has the potential to provide a direct bone protective effect in PsA.

Use of Therapeutic Pathogen Recognition Receptor Ligands for Osteo-Immunomodulation.

Therapeutic pathogen recognition receptor (PRR) ligands are reaching clinical practice following their ability to skew the immune response in a specific direction. We investigated the effects of various therapeutic PRR ligands on bone cell differentiation and inflammation. Following stimulation, alkaline phosphatase (ALP) activity (Day 10), osteocalcin, osteonectin expression (Day 14), and calcium deposition (Day 21) were quantified in bone marrow-derived human mesenchymal stem cells (hMSCs). The osteoclastogenic response was determined by measuring tartrate-resistant acid phosphate (TRAP) activity in human monocytes. TNF-α, IL-6, IL-8, and IL-10 expressions were measured by enzyme-linked immunosorbent assay as an indicator of the ligands’ inflammatory properties. We found that nucleic acid-based ligands Poly(I:C) and CpG ODN C increased early ALP activity in hMSCs by 4-fold without affecting osteoclast formation. These ligands did not enhance expression of the other, late osteogenic markers. MPLA, Curdlan, and Pam3CSK4 did not affect osteogenic differentiation, but inhibited TRAP activity in monocytes, which was associated with increased expression of all measured cytokines. Nucleic acid-based ligands are identified as the most promising osteo-immunomodulators, as they favor early osteogenic differentiation without inducing an exaggerated immune-cell mediated response or interfering in osteoclastogenesis and thus can be potentially harnessed for multifunctional coatings for bone biomaterials.

Inhibitory Effect of Probenecid on Osteoclast Formation via JNK, ROS and COX-2.

Probenecid is a representative drug used in the treatment of gout. A recent study showed that probenecid effectively inhibits oxidative stress in neural cells. In the present study, we investigated whether probenecid can affect osteoclast formation through the inhibition of reactive oxygen species (ROS) formation in RAW264.7 cells. Lipopolysaccharide (LPS)-induced ROS levels were dose-dependently reduced by probenecid. Fluorescence microscopy analysis clearly showed that probenecid inhibits the generation of ROS. Western blot analysis indicated that probenecid affects two downstream signaling molecules of ROS, cyclooxygenase 2 (COX-2) and c-Jun N-terminal kinase (JNK). These results indicate that probenecid inhibits ROS generation and exerts antiosteoclastogenic activity by inhibiting the COX-2 and JNK pathways. These results suggest that probenecid could potentially be used as a therapeutic agent to prevent bone resorption.

Effect of calcium phosphate phases affecting the crosstalk between osteoblasts and osteoclasts in vitro.

Previous studies have reported that octacalcium phosphate (OCP) enhances osteoblast differentiation and osteoclast formation during the hydrolysis process to hydroxyapatite (HA). However, the crystal phases that affect the crosstalk between osteoclasts and osteoblasts are unknown, which should determine the bone substitute material's property of OCP. The present study was designed to investigate whether the chemical composition and crystal structure of calcium phosphates affect osteoclast formation and the osteoclast-osteoblast crosstalk. Biodegradable β-tricalcium phosphate (β-TCP) was used as the control material. Osteoclasts were cultured on HA/OCP or HA/TCP disks and their cellular responses were assessed. Both OCP and β-TCP had a similar ability to create multinucleated osteoclasts. However, OCP promoted the expression of complement component 3a (C3a), a positive coupling factor, in osteoclasts, whereas β-TCP enhanced that of EphrinB2 (EfnB2) and collagen triple helix repeat containing 1 (Cthrc1). During osteoclast culture, phosphate ions were released from the crystals, and OCP-HA conversion was advanced in HA/OCP mixtures and OCP. X-ray diffraction analysis revealed no remarkable changes in the crystal structures of HA/TCP mixtures and β-TCP before and after osteoclast culture. These results indicate that the distinct chemical environment induced by the calcium phosphate phases affects the crosstalk between osteoclasts and osteoblasts. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1001-1013, 2019.

Loss of cyclin-dependent kinase 1 impairs bone formation, but does not affect the bone-anabolic effects of parathyroid hormone

Bone mass is maintained by a balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Although recent genetic studies have uncovered various mechanisms that regulate osteoblast differentiation, the molecular basis of osteoblast proliferation remains unclear. Here, using an osteoblast-specific loss-of-function mouse model, we demonstrate that cyclin-dependent kinase 1 (Cdk1) regulates osteoblast proliferation and differentiation. Quantitative RT-PCR analyses revealed that Cdk1 is highly expressed in bone and is down-regulated upon osteoblast differentiation. We also noted that Cdk1 is dispensable for the bone-anabolic effects of parathyroid hormone (PTH). Cdk1 deletion in osteoblasts led to osteoporosis in adult mice due to low bone formation, but did not affect osteoclast formation in vivo Cdk1 overexpression in osteoblasts promoted proliferation, and conversely, Cdk1 knockdown inhibited osteoblast proliferation and promoted differentiation. Of note, we provide direct evidence that PTH's bone-anabolic effects occur without enhancing osteoblast proliferation in vivo Furthermore, we found that Cdk1 expression in osteoblasts is essential for bone fracture repair. These findings may help reduce the risk of nonunion after bone fracture and identify patients at higher risk for nonresponse to PTH treatment. Collectively, our results indicate that Cdk1 is essential for osteoblast proliferation and that it functions as a molecular switch that shifts osteoblast proliferation to maturation. We therefore conclude that Cdk1 plays an important role in bone formation.

The effect of Activin-A on periodontal ligament fibroblasts-mediated osteoclast formation in healthy donors and in patients with fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a genetic disease characterized by heterotopic ossification (HO). The disease is caused by a mutation in the activin receptor type 1 (ACVR1) gene that enhances this receptor's responsiveness to Activin‐A. Binding of Activin‐A to the mutated ACVR1 receptor induces osteogenic differentiation. Whether Activin‐A also affects osteoclast formation in FOP is not known. Therefore we investigated its effect on the osteoclastogenesis‐inducing potential of periodontal ligament fibroblasts (PLF) from teeth of healthy controls and patients with FOP. We used western blot analysis of phosphorylated SMAD3 (pSMAD3) and quantitative polymerase chain reaction to assess the effect of Activin‐A on the PLF. PLF‐induced osteoclast formation and gene expression were studied by coculturing control and FOP PLF with CD14‐positive osteoclast precursor cells from healthy donors. Osteoclast formation was also assessed in control CD14 cultures stimulated by macrophage colony‐stimulating factor (M‐CSF) and receptor activator of nuclear factor kappa‐B ligand (RANK‐L). Although Activin‐A increased activation of the pSMAD3 pathway in both control and FOP PLF, it increased ACVR1, FK binding protein 12 (FKBP12), an inhibitor of DNA binding 1 protein (ID‐1) expression only in FOP PLF. Activin‐A inhibited PLF mediated osteoclast formation albeit only significantly when induced by FOP PLF. In these cocultures, it reduced M‐CSF and dendritic cell‐specific transmembrane protein (DC‐STAMP) expression. Activin‐A also inhibited osteoclast formation in M‐CSF and RANK‐L mediated monocultures of CD14+ cells by inhibiting their proliferation.This study brings new insight on the role of Activin A in osteoclast formation, which may further add to understanding FOP pathophysiology; in addition to the known Activin‐A‐mediated HO, this study shows that Activin‐A may also inhibit osteoclast formation, thereby further promoting HO formation.

Reduced miR-144-3p expression in serum and bone mediates osteoporosis pathogenesis by targeting RANK.

Osteoblasts and osteoclasts are responsible for the formation and resorption of bone, respectively. An imbalance between these two processes results in a disease called osteoporosis, in which a decreased level of bone strength increases the risk of a bone fracture. MicroRNAs (miRNAs) are small non-coding RNA molecules of 18-25 nucleotides that have been previously shown to control bone metabolism by regulating osteoblast and osteoclast differentiation. In this study, we detected the expression pattern of 10 miRNAs in serum samples from patients with osteoporosis, and identified the altered expression of 6 miRNAs by comparison with patients without osteoporosis. We selected miR-144-3p for further investigation, and showed that it regulates osteoclastogenesis by targeting RANK, and that it is conserved amongst vertebrates. Disrupted expression of miR-144-3p in CD14+ peripheral blood mononuclear cells changed TRAP activity and the osteoclast-specific genes TRAP, cathepsin K (CTSK), and NFATC. TRAP staining, CCK-8, and flow cytometry analyses revealed that miR-144-3p also affects osteoclast formation, proliferation, and apoptosis. Together, these results indicate that miR-144-3p critically mediates bone homeostasis, and thus, represents a promising novel therapeutic candidate for the treatment of this disease.

Interleukin-7 Induces Osteoclast Formation via STAT5, Independent of Receptor Activator of NF-kappaB Ligand

Interleukin-7 (IL-7), which is required for the development and survival of T cells in the thymus and periphery, plays a role in joint destruction. However, it remains unclear how IL-7 affects osteoclast formation. Thus, we investigated the mechanism by which IL-7 induced osteoclast formation through IL-7 receptor α (IL-7Rα) in osteoclast precursors. We cultured peripheral blood mononuclear cells or synovial fluid mononuclear cells with IL-7 in the presence or absence of an appropriate inhibitor to analyze osteoclast formation. We also constructed IL-7Rα-expressing RAW264.7 cells to uncover the mechanism(s) by which IL-7 induced osteoclast formation differed from that of receptor activator of nuclear factor κB ligand (RANKL). We found that IL-7 induced osteoclast formation of human monocytes from peripheral blood or synovial fluid in a RANKL-independent and a signal transducer and activator of transcription 5 (STAT5)-dependent manner. IL-7-induced osteoclasts had unique characteristics, such as small, multinucleated tartrate-resistant acid phosphatase positive cells and no alterations even when RANKL was added after IL-7 pretreatment. RAW264.7 cells, if overexpressing IL-7Rα, also were able to differentiate into osteoclasts by IL-7 through a STAT5 signaling pathway. Furthermore, IL-7-induced osteoclast formation was repressed by inhibitors of the IL-7R signaling molecules Janus kinase and STAT5. Our findings demonstrate that IL-7 is a truly osteoclastogenic factor, which may induce osteoclast formation via activation of STAT5, independent of RANKL. We also suggest the possibility that an IL-7R pathway blocker could alleviate joint damage by inhibiting osteoclast formation, especially in inflammatory conditions.

β-Carotene suppresses osteoclastogenesis and bone resorption by suppressing NF-κB signaling pathway

Aimsβ-Carotene is a natural anti-oxidant, which has been used for treatment of cancer and cardiovascular diseases. Recently, the ameliorating function of β-carotene in osteoporosis has been implicated. However, the precise mechanism of β-carotene in prevention and treatment of osteoporosis is largely unknown. In the present study, we aimed to elucidate how β-carotene affects osteoclast formation and bone resorption. Main methodsBone marrow-derived monocytes/-macrophages (BMM) were exposed to 0.05, 0.1, 0.2, 0.4 and 0.6μM β-carotene, followed by evaluation of cell viability, lactate dehydrogenase (LDH) release, receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis and resorption pits formation. Key factors in nuclear factor kappa B (NF-ĸB) and mitogen-activated protein kinases (MAPK) pathways were evaluated with western blot after BMM cells were exposed to RANKL and β-carotene. The effects of β-carotene in nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), c-Fos and cathepsin K (CTSK) expression were also evaluated. Key findingsβ-Carotene significantly inhibited BMM viability and promoted LDH release at concentrations of 0.4 and 0.6μM. A decrease in RANKL-induced osteoclastogenesis and resorption was also observed after β-carotene treatment. β-Carotene attenuated the NF-ĸB pathway activation by RANKL, with no effect on MAPK pathway. β-Carotene suppressed the upregulation of NFATc1 and c-Fos by RANKL. SignificanceWe clarified the anti-osteoclastogenic role of β-carotene, which is mediated by NF-κB signaling.