Osteoclast Precursor Cells Research Articles

Role of Dynamic Actin Cytoskeleton Remodeling in Foxp3+ Regulatory TCell Development and Function: Implications for Osteoclastogenesis.

In T cells, processes such as migration and immunological synapse formation are accompanied by the dynamic reorganization of the actin cytoskeleton, which has been suggested to be mediated by regulators of RhoGTPases and by F-actin bundlers. SWAP-70 controls F-actin dynamics in various immune cells, but its role in T cell development and function has remained incompletely understood. CD4+ regulatory T (Treg) cells expressing the transcription factor Foxp3 employ diverse mechanisms to suppress innate and adaptive immunity, which is critical for maintaining immune homeostasis and self-tolerance. Here, we propose Swap-70 as a novel member of the Foxp3-dependent canonical Treg cell signature. We show that Swap-70-/- mice have increased numbers of Foxp3+ Treg cells with an effector/memory-like phenotype that exhibit impaired suppressor function in vitro, but maintain overall immune homeostasis in vivo. Upon formation of an immunological synapse with antigen presenting cells in vitro, cytosolic SWAP-70 protein is selectively recruited to the interface in Treg cells. In this context, Swap-70-/- Treg cells fail to downregulate CD80/CD86 on osteoclast precursor cells by trans-endocytosis and to efficiently suppress osteoclastogenesis and osteoclast function. These data provide first evidence for a crucial role of SWAP-70 in Treg cell biology and further highlight the important non-immune function of Foxp3+ Treg cells in bone homeostasis mediated through direct SWAP-70-dependent mechanisms.

Effect of cadmium on Rho GTPases signal transduction during osteoclast differentiation.

Osteoclasts are the key target cells for cadmium (Cd)-induced bone metabolism diseases, while Rho GTPases play an important role in osteoclast differentiation and bone resorption. To identify new therapeutic targets of Cd-induced bone diseases; we evaluated signal transduction through Rho GTPases during osteoclast differentiation under the influence of Cd. In osteoclastic precursor cells, 10nM Cd induced pseudopodia stretching, promoted cell migration, upregulated the levels of Cdc42, and RhoQ mRNAs and downstream Rho-associated coiled-coil kinase 1 (ROCK1) and ROCK2 proteins, and downregulated the actin-related protein 2/3 (ARP2/3) levels. Cd at 2 and 5μM shortened the pseudopodia, inhibited cell migration, and decreased ROCK1, ROCK2, and ARP2/3 protein levels; Cd at 5μM also reduced the mRNA expression levels of Rac1, Rac2, and RhoU mRNAs and decreased the level of phosphorylated (p)-cofilin. In osteoclasts, 10nM Cd induced the formation of sealing zones, slightly upregulated Cdc42 mRNA levels and ROCK2 and ARP2/3 protein levels and significantly reduced p-cofilin levels. Cd at 2μM and 5μM Cd blocked the fusion of precursor cells; and 5μM Cd downregulated the expression levels of RhoB, Rac1, Rac3, and RhoU mRNAs, and ROCK1, p-cofilin and ARP2/3 protein levels, significantly. In vivo, Cd (at 5 or 25 mg/L) increased the levels of key proteins RhoA, Rac1/2/3, Cdc42, and RhoU and their mRNAs in bone marrow cells. In summary, the results suggested that Cd affected the differentiation process of osteoclast and altered the expression of several Rho GTPases, which might be crucial targets of Cd during the differentiation of osteoclasts.

Interleukin-17 promotes osteoclastogenesis and periodontal damage via autophagy in vitro and in vivo

ObjectiveBecause of its potent pro-inflammatory properties, interleukin-17 (IL-17) contributes to the pathogenesis of various inflammatory diseases. This study explored the effects of IL-17 on osteoclastogenesis in an osteoclast monoculture and osteoblast-osteoclast co-culture system, as tools to investigate the molecular mechanisms underlying the interactions between osteoclastogenesis and autophagy. MethodsVarious ratios of calvarial osteoblasts (OB) and osteoclast precursor cells (mouse macrophage cell line RAW264.7, hereinafter referred to as OC) were tested. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the optimum osteoblasts:osteoclasts ratio. IL-17 was added to the co-culture system to test its effects on multinucleated osteoclast formation and osteoclast-related proteins. We assessed the effects of IL-17 on receptor activator of nuclear factor-kappa B ligand (RANKL) expression in osteoblasts, and determined if IL-17 alone could modulate osteoclast formation in an osteoclast monoculture. Administration of exogenous RANKL combined with IL-17 was employed to stimulate RAW264.7 cells osteoclastogenesis and to determine production of osteoclasts and autophagy-related proteins. We knocked down Beclin1 expression in RAW264.7 cells and examined the expression of autophagy-related and osteoclast-related proteins in RAW264.7 cells and the co-culture system, and the TAK1-binding protein 3 (TAB3)/ extracellular signal regulated kinase (ERK) pathway. ResultsA ratio of 20 OB : 1 OC yielded the highest rate of osteoclast formation. Low IL-17 concentrations increased osteoclastogenesis in co-cultures significantly, but high levels of IL-17 had the opposite effect. IL-17 alone could not induce formation of TRAP+ multinucleated cells in RAW264.7 cells. Low IL-17 concentrations promoted osteoclast differentiation and autophagy in RAW264.7 cells induced by exogenous RANKL, but high IL-17 concentrations inhibited this process. Knockdown of Beclin1 reversed the enhanced effects of 0.1 ng/mL IL-17 on osteoclastogenesis and autophagy in RAW264.7 cells. The TAB3/ERK pathway was also blocked after autophagy inhibition. ConclusionIn the co-culture model used in this study, a ratio of 20 OB:1 OC proved to be the optimal ratio to facilitate osteoclast formation. IL-17 regulated RANKL-induced osteoclastogenesis via autophagy. The Beclin1/TAB3/ERK pathway was involved in osteoclast autophagy.

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.

Characterization, pharmacokinetics, and pharmacodynamics of anti-Siglec-15 antibody and its potency for treating osteoporosis and as follow-up treatment after parathyroid hormone use.

Recent studies have established the idea that Siglec-15 is involved in osteoclast differentiation and/or function, and it is anticipated that therapies suppressing Siglec-15 function can be used to treat bone diseases such as osteoporosis. We have produced rat monoclonal anti-Siglec-15 antibody (32A1) and successively generated humanized monoclonal anti-Siglec-15 antibody (DS-1501a) from 32A1. Studies on the biological properties of DS-1501a showed its specific binding affinity to Siglec-15 and strong activity to inhibit osteoclastogenesis. 32A1 inhibited multinucleation of osteoclasts and bone resorption (pit formation) in cultured mouse bone marrow cells. 32A1 also inhibited pit formation in cultured human osteoclast precursor cells. Maximum serum concentration and serum exposure of DS-1501a in rats were increased in a dose-dependent manner after single subcutaneous or intravenous administration. Furthermore, single administration of DS-1501a significantly suppressed bone resorption markers with minimal effects on bone formation markers and suppressed the decrease in bone mineral density (BMD) of the lumbar vertebrae in ovariectomized (OVX) rats. In histological analysis, the osteoclasts distant from the chondro-osseous junction of the tibia tended to be flattened, shrunken, and functionally impaired in 32A1-treated rats, while alkaline phosphatase-positive osteoblasts were observed throughout the metaphyseal trabeculae. In addition, we compared the efficacy of 32A1 with that of alendronate (ALN) as follow-up medicine after treatment with parathyroid hormone (PTH) using mature established osteoporosis rats. The beneficial effect of PTH on bone turnover disappeared 8weeks after discontinuing the treatment. The administration of 32A1 once every 4weeks for 8weeks suppressed bone resorption and bone formation when the treatment was switched from PTH to 32A1, leading to the maintenance of BMD and bone strength. Unlike with ALN, the onset of suppression of bone resorption with 32A1 was rapid, while the suppression of bone formation was mild. The improvement of bone mass, beneficial bone turnover balance, and suppression of osteoclast differentiation/multinucleation achieved by 32A1 were supported by histomorphometry. Notably, the effects of 32A1 on bone strength, not only structural (extrinsic) but also material (intrinsic) properties, were significantly greater than those of ALN. Since the effect of 32A1 on BMD was moderate, its effect on bone strength could not be fully explained by the increase in BMD. The beneficial balance of bone turnover caused by 32A1 might, at least in part, be responsible for the improvement in bone quality. This is the first report describing the effects of anti-Siglec-15 antibody in OVX rats; the findings suggest that this antibody could be an excellent candidate for treating osteoporosis, especially in continuation therapy after PTH treatment, due to its rapid action and unprecedented beneficial effects on bone quality.

Novel animal model of soft tissue tumor due to aberrant hedgehog signaling activation in pericyte lineage.

Pericytes are pluripotent cells that enclose the endothelium of small blood vessels in the whole body. These cells are thought to play a limited role in vascular development and blood pressure regulation; however, current evidence from numerous studies suggests several significant biologic aspects of pericytes in animals. One viewpoint is that pericytes are also known as potential cellular origin of multiple soft tissue tumors. Experimental evidence of the cellular origin of pericytic tumors is still insufficient, however, and their molecular pathogenesis is poorly understood. Here, we used a conditional constitutively active Smoothened allele (Rosa-SmoM2) and Cre recombinase mice to activate hedgehog (Hh) signaling, exclusively in the monocyte/macrophage and osteoclast lineage (LysMcre) or in RANK expressing cells (RANKcre) that are recognized as osteoclast precursor cells. Mice conditionally expressing SmoM2 with LysMcre displayed no significant skeletal phenotype; surprisingly, however, RANKcre; Rosa-SmoM2 mice frequently developed progressive soft tissue tumors in regions of the leg. Genetic lineage tracing analysis uncovered a new domain of RANKcre-expressing cells in the skeletal muscle interstitial cells that display markers consistent with vascular pericytes. Neoplasms arising from these cells showed increased expression of Matrix metalloproteinases (MMPs) that are molecular indicators of malignancy. Moreover, the tumors displayed strong bone invasive potency associated with osteoclastic bone resorption. Thus, these findings provide a novel insight into tumor pathology: Hh signal activated-pericytes can be a potential cellular origin of multiple soft tissue tumors.

Ethanolic extract of Pyrrosia lingua (Thunb.) Farw. ameliorates OVX-induced bone loss and RANKL-induced osteoclastogenesis

Pyrrosia lingua (Thunb.) Farw is a common plant that has been widely used as a traditional herbal medicine in China and Korea to treat patients suffering from pain, vaginal bleeding and urolithiasis. However, the pharmacological effects of P. lingua on bone remain unknown. We investigated the anti-osteoporotic effects of an ethanolic extract of P. lingua (EEPL). We found that EEPL suppressed osteoclast differentiation by directly acting on osteoclast precursor cells. EEPL suppressed the expression of receptor activator of nuclear factor-κB ligand (RANKL)-induced nuclear factor of activated T cells 1, a major transcription factor for osteoclastogenesis, by inhibiting RANKL-induced expression of aryl hydrocarbon receptor/c-Fos, and activation of nuclear factor-κB and mitogen-activated protein kinases. Moreover, administration of EEPL inhibited trabecular bone loss and weight gain in ovariectomized mice. Furthermore, we identified phytochemicals in EEPL that are known to exert anti-osteoclastogenic or anti-osteoporotic effects using ultra-high-performance liquid chromatography–tandem mass-spectrometry analysis. Overall, the results of this study suggest that EEPL is effective therapeutic candidate that can be used to prevent or treat postmenopausal osteoporosis.

Opa1-mediated mitochondrial dynamics is important for osteoclast differentiation.

Opatic atrophy 1 (Opa1) is a mitochondrial GTPase that regulates mitochondrial fusion and maintenance of cristae architecture. Osteoclasts are mitochondrial rich-cells. However, the role of Opa1 in osteoclasts remains unclear. Here, we demonstrate that Opa1- deficient osteoclast precursor cells do not undergo efficient osteoclast differentiation and exhibit abnormal cristae morphology. Thus, Opa1 is a key factor in osteoclast differentiation through regulation of mitochondrial dynamics.

The Cytoplasmic Dynein Associated Protein NDE1 Regulates Osteoclastogenesis by Modulating M-CSF and RANKL Signaling Pathways.

Cytoskeleton organization and lysosome secretion play an essential role in osteoclastogenesis and bone resorption. The cytoplasmic dynein is a molecular motor complex that regulates microtubule dynamics and transportation of cargos/organelles, including lysosomes along the microtubules. LIS1, NDE1, and NDEL1 belong to an evolutionary conserved pathway that regulates dynein functions. Disruption of the cytoplasmic dynein complex and deletion of LIS1 in osteoclast precursors arrest osteoclastogenesis. Nonetheless, the role of NDE1 and NDEL1 in osteoclast biology remains elusive. In this study, we found that knocking-down Nde1 expression by lentiviral transduction of specific shRNAs markedly inhibited osteoclastogenesis in vitro by attenuating the proliferation, survival, and differentiation of osteoclast precursor cells via suppression of signaling pathways downstream of M-CSF and RANKL as well as osteoclast differentiation transcription factor NFATc1. To dissect how NDEL1 regulates osteoclasts and bone homeostasis, we generated Ndel1 conditional knockout mice in myeloid osteoclast precursors (Ndel1ΔlysM) by crossing Ndel1-floxed mice with LysM-Cre mice on C57BL/6J background. The Ndel1ΔlysM mice developed normally. The µCT analysis of distal femurs and in vitro osteoclast differentiation and functional assays in cultures unveiled the similar bone mass in both trabecular and cortical bone compartments as well as intact osteoclastogenesis, cytoskeleton organization, and bone resorption in Ndel1ΔlysM mice and cultures. Therefore, our results reveal a novel role of NDE1 in regulation of osteoclastogenesis and demonstrate that NDEL1 is dispensable for osteoclast differentiation and function.

Tumor necrosis factor-α promotes osteoclast differentiation via sialylation in mice

To explore the mechanism through which tumor necrosis factor-α (TNF-α) promotes osteoclast differentiation. Bilateral knee joint samples were collected from 4-month-old wild-type mice and TNF-α transgenic mice for CT scan analysis, TRAP staining and sialic acid staining analysis. The osteoclast precursor (RAW264.7) cells were cultured for 3 days in induction medium in the presence of vehicle, TNF-α, or TNF-α and sialidase, and were then examined with RT-qPCR, TRAP staining, and sialic acid immunofluorescence co-localization staining. Bone marrow-derived macrophages isolated from the wild-type mice and TNF-α transgenic mouse and cultured in induction medium with or without the addition of sialidase, and TRAP and sialic acid staining was performed after 3 days of cell culture. TRAP staining showed that the number of osteoclasts increased significantly in TNF-α transgenic mice as compared with the wild-type mice (P < 0.0001), and micro-CT analysis revealed significant reductions of BV/TV, Tb.N, and Tb.Th in TNF-α transgenic mice (P < 0.001). The osteoclasts in TNF-α transgenic mice also showed a significantly increased expression of sialic acid (P=0.004). In the cell experiment, RAW264.7 cells cultured with TNF-α showed a significantly higher expression of sialic acid (P < 0.0001) and a greater osteoclast formation rate (P=0.0007) than the the control cells, while the addition of sialidase significantly reduced sialic acid expression, osteoclast formation rate and TRAP mRNA level in TNF-α-treated cells (P < 0.0001). Similarly, in the bone marrow-derived macrophages, sialic acid expression and osteoclast formation rate were significantly increased by incubation with TNF-α (P < 0.0001), but the increments were obviously reduced by addition of sialidase in the medium (P < 0.0001). TNF-α can promote the differentiation and activity of osteoclasts by increasing the sialylation level in the osteoclasts.

In vitro and in vivo detection of tunneling nanotubes in normal and pathological osteoclastogenesis involving osteoclast fusion

AbstractOsteoclasts are multinucleated cells formed through specific recognition and fusion of mononuclear osteoclast precursors derived from hematopoietic stem cells. Detailed cellular events concerning cell fusion in osteoclast differentiation remain ambiguous. Tunneling nanotubes (TNTs), actin-based membrane structures, play an important role in intercellular communication between cells. We have previously reported the presence of TNTs in the fusion process of osteoclastogenesis. Here we analyzed morphological details of TNTs using scanning electron microscopy. The osteoclast precursor cell line RAW-D was stimulated to form osteoclast-like cells, and morphological details in the appearance of TNTs were extensively analyzed. Osteoclast-like cells could be classified into three types; early osteoclast precursors, late osteoclast precursors, and multinucleated osteoclast-like cells based on the morphological characteristics. TNTs were frequently observed among these three types of cells. TNTs could be classified into thin, medium, and thick TNTs based on the diameter and length. The shapes of TNTs were dynamically changed from thin to thick. Among them, medium TNTs were often observed between two remote cells, in which side branches attached to the culture substrates and beaded bulge-like structures were often observed. Cell-cell interaction through TNTs contributed to cell migration and rapid transport of information between cells. TNTs were shown to be involved in cell-cell fusion between osteoclast precursors and multinucleated osteoclast-like cells, in which movement of membrane vesicles and nuclei was observed. Formation of TNTs was also confirmed in primary cultures of osteoclasts. Furthermore, we have successfully detected TNTs formed between osteoclasts observed in the bone destruction sites of arthritic rats. Thus, formation of TNTs may be important for the differentiation of osteoclasts both in vitro and in vivo. TNTs could be one target cellular structure for the regulation of osteoclast differentiation and function in bone diseases.

Anti-Siglec-15 antibody suppresses bone resorption by inhibiting osteoclast multinucleation without attenuating bone formation

Anti-resorptive drugs are widely used for the treatment of osteoporosis, but excessive inhibition of osteoclastogenesis can suppress bone turnover and cause the deterioration of bone quality. Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is a transmembrane protein expressed on osteoclast precursor cells and mature osteoclasts. Siglec-15 regulates proteins containing immunoreceptor tyrosine-based activation motif (ITAM) domains, which then induce nuclear factor of activated T-cells 1 (NFATc1), a master transcription factor of osteoclast differentiation. Anti-Siglec-15 antibody modulates ITAM signaling in osteoclast precursors and inhibits the maturation of osteoclasts in vitro. However, in situ pharmacological effects, particularly during postmenopausal osteoporosis, remain unclear. Here, we demonstrated that anti-Siglec-15 antibody treatment protected against ovariectomy-induced bone loss by specifically inhibiting the generation of multinucleated osteoclasts in vivo. Moreover, treatment with anti-Siglec-15 antibody maintained bone formation to a greater extent than with risedronate, the first-line treatment for osteoporosis. Intravital imaging revealed that anti-Siglec-15 antibody treatment did not cause a reduction in osteoclast motility, whereas osteoclast motility declined following risedronate treatment. We evaluated osteoclast activity using a pH-sensing probe and found that the bone resorptive ability of osteoclasts was lower following anti-Siglec-15 antibody treatment compared to after risedronate treatment. Our findings suggest that anti-Siglec-15 treatment may have potential as an anti-resorptive therapy for osteoporosis, which substantially inhibits the activity of osteoclasts while maintaining physiological bone coupling.

Titanium surface with nanospikes tunes macrophage polarization to produce inhibitory factors for osteoclastogenesis through nanotopographic cues

Definitive prevention of inflammatory osteolysis around peri-implant bone tissue remains unestablished. M1 macrophages play a key role in the host defense against inflammatory osteolysis, and their polarization depends on cell shape. Macrophage polarization is controlled by environmental stimuli, particularly physicochemical cues and hence titanium nanosurface might tune macrophage polarization and function. This study determined whether titanium nanosurfaces with anisotropically patterned nanospikes regulates macrophage polarization for inhibiting osteoclast differentiation of osteoclast precursors. Alkaline-etching treatment with different protocols created two types of titanium nanosurfaces that had anisotropically patterned nanospikes with high or low distribution density, together with superhydrophilicity and the presence of hydroxyl groups. J774A.1 cells (mouse macrophage-like cell line), cultured on both titanium nanosurfaces, exhibited truly circulated shapes and highly expressed M1, but less M2, markers, without loss of viability. M1-like polarization of macrophages on both titanium nanosurfaces was independent of protein-mediated ligand stimulation or titanium surface hydrophilic or chemical status. In contrast, other smooth or micro-roughened titanium surfaces with little or no nanospikes did not activate macrophages under any culture conditions. Macrophage culture supernatants on both titanium nanosurfaces inhibited osteoclast differentiation of RAW264.7 cells (mouse osteoclast precursor cell line), even when co-incubated with osteoclast differentiation factors. The inhibitory effects on osteoclast differentiation tended to be higher in macrophages cultured on titanium nanosurfaces with denser nanospikes. These results showed that titanium nanosurfaces with anisotropically patterned nanospikes tune macrophage polarization for inhibiting osteoclast differentiation of osteoclast precursors, with nanotopographic cues rather than other physicochemical properties. Statement of significancePeri-implant inflammatory osteolysis is one of the serious issues for dental and orthopedic implants. Macrophage polarization and function are key for prevention of peri-implant inflammatory osteolysis. Macrophage polarization can be regulated by the biomaterial's surface physicochemical properties such as hydrophilicity or topography. However, there was no titanium surface modification to prevent inflammatory osteolysis through immunomodulation. The present study showed for the first time that the titanium nanosurfaces with anisotropically patterned nanospikes, created by the simple alkali-etching treatment polarized macrophages into M1-like type producing the inhibitory factor on osteoclast differentiation. This phenomenon attributed to nanotopographic cues, but not hydrophilicity on the titanium nanosurfaces. This nanotechnology might pave the way to develop the smart implant surface preventing peri-implant inflammatory osteolysis through immunomodulation.

Effects of Bisphenol A and Its Alternatives, Bisphenol F and Tetramethyl Bisphenol F on Osteoclast Differentiation.

Bisphenol A (BPA) is a typical environmental endocrine disruptor that exhibits estrogen-mimicking, hormone-like properties and can cause the collapse of bone homeostasis by an imbalance between osteoblasts and osteoclasts. Various BPA substitutes, structurally similar to BPA, have been used to manufacture ‘BPA-free’ products; however, the regulatory role of BPA alternatives in osteoclast differentiation still remains unelucidated. This study aimed to investigate the effects of these chemicals on osteoclast differentiation using the mouse osteoclast precursor cell line RAW 264.7. Results confirmed that both BPA and its alternatives, bisphenol F and tetramethyl bisphenol F (TMBPF), were nontoxic to RAW 264.7 cells. In particular, tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell staining and activity calculation assays revealed that TMBPF enhanced osteoclast differentiation upon stimulation of the receptor activator of nuclear factor-kappa B ligand (RANKL). Additionally, TMBPF activated the mRNA expression of osteoclast-related target genes, such as the nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), tartrate-resistant acid phosphatase (TRAP), and cathepsin K (CtsK). Western blotting analysis indicated activation of the mitogen-activated protein kinase signaling pathway, including phosphorylation of c-Jun N-terminal kinase and p38. Together, the results suggest that TMBPF enhances osteoclast differentiation, and it is critical for bone homeostasis and skeletal health.

IRF2 enhances RANKL-induced osteoclast differentiation via regulating NF-κB/NFATc1 signaling

Interferon regulatory factors (IRFs) play roles in various biological processes including cytokine signaling, cell growth regulation and hematopoietic development. Although it has been reported that several IRFs are involved in bone metabolism, the role of IRF2 in bone cells has not been elucidated. Here, we investigated the involvement of IRF2 in RANKL-induced osteoclast differentiation. IRF2 overexpression in osteoclast pre-cursor cells enhanced osteoclast differentiation by regulating the expression of NFATc1, a master regulator of osteoclasto-genesis. Conversely, IRF2 knockdown inhibited osteoclast differentiation and decreased the NFATc1 expression. Moreover, IRF2 increased the translocation of NF-κB subunit p65 to the nucleus in response to RANKL and subsequently induced the expression of NFATc1. IRF2 plays an important role in RANKL-induced osteoclast differentiation by regulating NF-κB/NFATc1 signaling pathway. Taken together, we demonstrated the molecular mechanism of IRF2 in osteoclast differentiation, and provide a molecular basis for potential therapeutic targets for the treatment of bone diseases characterized by excessive bone resorption.

Although Anatomically Micrometers Apart: Human Periodontal Ligament Cells Are Slightly More Active in Bone Remodeling Than Alveolar Bone Derived Cells

The periodontal ligament (PDL) and the alveolar bone are part of the periodontium, a complex structure that supports the teeth. The alveolar bone is continuously remodeled and is greatly affected by several complex oral events, like tooth extraction, orthodontic movement, and periodontitis. Until now, the role of PDL cells in terms of osteogenesis and osteoclastogenesis has been widely studied, whereas surprisingly little is known about the bone remodeling capacity of alveolar bone. Therefore, the purpose of this study was to compare the biological character of human alveolar bone cells and PDL cells in terms of osteogenesis and osteoclastogenesis in vitro. Paired samples of PDL cells and alveolar bone cells from seven patients with compromised general and oral health were collected and cultured. Bone A (early outgrowth) and bone B (late outgrowth) were included. PDL, bone A, bone B cell cultures all had a fibroblast appearance with similar expression pattern of six mesenchymal markers. These cultures were subjected to osteogenesis and osteoclastogenesis assays. For osteoclastogenesis assays, the cells were co-cultured with peripheral blood mononuclear cells, a source for osteoclast precursor cells. The total duration of the experiments was 21 days. Osteogenesis was slightly favored for PDL compared to bone A and B as shown by stronger Alizarin red staining and higher expression of RUNX2 and Collagen I at day 7 and for ALP at day 21. PDL induced approximately two times more osteoclasts than alveolar bone cells. In line with these findings was the higher expression of cell fusion marker DC-STAMP in PDL-PBMC co-cultures compared to bone B at day 21. In conclusion, alveolar bone contains remodeling activity, but to a different extent compared to PDL cells. We showed that human alveolar bone cells can be used as an in vitro model to study bone remodeling.

Replacement Process of Carbonate Apatite by Alveolar Bone in a Rat Extraction Socket.

The objective of this study was to investigate a bone graft substitute containing carbonate apatite (CO3Ap) to analyze bone replacement and the state of bone formation in vitro and in vivo compared with autogenous bone (AB) or control. An osteoclast precursor cell line was cultured with AB or CO3Ap, and morphological analysis using scanning electron microscopy and a tartrate-resistant acid phosphatase activity assay were performed. The right maxillary first and second molars of Wistar rats were extracted and compensated by AB or CO3Ap granules. Following implantation, the bone formation state was evaluated after 3, 5, 7, 14, and 28 days of surgery by micro-computed tomography and immunohistostaining. The osteoclast-like cell morphology was typical with many cell protrusions in the AB and CO3Ap groups. Additionally, the number of osteoclast-like cells formed in the culture increased in each group; however, there was no significant difference between the AB and CO3Ap groups. Five days after tooth extraction, osteoclasts were observed near CO3Ap. The bone thickness in the CO3Ap group was significantly increased than that in the control group and the bone formation in the CO3Ap group increased by the same level as that in the AB group. CO3Ap is gradually absorbed by osteoclasts in the extraction socket and is easily replaced by alveolar bone. The process of bone replacement by osteoclasts is similar to that of autologous bone. By observing the process of bone replacement in more detail, it may be possible to gain a better understanding of the bone formation and control the amount of bone after surgery.

Water Extract of Mentha arvensis L. Attenuates Estrogen Deficiency-Induced Bone Loss by Inhibiting Osteoclast Differentiation.

Mentha arvensis L., is an aromatic herb that belongs to the Lamiaceae family and is widely used in medicinal applications, essential oil applications, and food flavoring. The extract of M. arvensis has been reported to exert sedative-hypnotic, anti-inflammatory, anti-fungal, and anti-bacterial effects. However, its effects on bone metabolism have not yet been studied. Here, we investigated the effects of the water extract of M. arvensis (WEMA) on osteoclast formation in vitro and bone loss in an ovariectomized mouse model. We found that WEMA inhibited osteoclast differentiation by directly acting on osteoclast precursor cells. WEMA inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced the expression of cellular oncogene fos (c-Fos) and nuclear factor of activated T cells c1 (NFATc1), crucial transcription factors for osteoclast differentiation, by suppressing RANKL-induced activation of early signaling pathways such as those of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB). In addition, oral administration of WEMA suppressed ovariectomy-induced trabecular bone loss in mice. We additionally identified phytochemicals in WEMA that are known to have anti-osteoclastogenic or anti-osteoporotic properties. Collectively, these results suggest that WEMA is a promising herbal candidate that can be used to prevent or treat postmenopausal osteoporosis.

CD14, A toll-like receptor co-factor, influences osteoclast differentiation and activity, but does not alter osteoblastic potential of bone-marrow preursors in mice

Purpose: Changes in subchondral bone structure occur in osteoarthritis (OA), and are visible by imaging as subchondral sclerosis on radiographs, and bone marrow lesions (BMLs) by MRI. These changes are the result of active bone remodeling that occurs in OA. BMLs in particular are correlated with pain in OA patients. We previously demonstrated that bone remodeling was significantly reduced after joint injury in mice deficient in CD14. CD14 enhances Toll-like receptor (TLR) mediated inflammation by acting as a co-receptor for multiple TLRs, increasing TLR sensitivity to damage associated molecular patterns (DAMPS) to activate proinflammatory signaling pathways. Soluble CD14, TLRs, and DAMPs (TLR ligands) are all elevated in OA patients, and sCD14 has been implicated as a biomarker of symptom and structural severity in OA. Increasing evidence suggestions CD14/TLR signaling influences bone remodeling as well, through activation of osteoclast precursor cells. Here, we investigate the potential mechanism by which CD14 influences bone remodeling through in vitro analysis of osteoclastogenic and osteoblastic potential and function. Methods: Bone marrow (BM) hematopoietic and mesenchymal precursors were isolated from long bones of 12-week-old male wild-type C57BL/6 (WT) and CD14-deficient (CD14-/-) mice. Briefly, BM was flushed from the femurs and tibia, and cells were cultured for 24 hours to separate non-adherent from adherent population. For osteoclastic differentiation, the non-adherent cells were seeded at a density of 26,000 cells per cm2 and were cultured in presence of monocyte/macrophage colony-stimulating factor (M-CSF; 35 ng/ml) and receptor activator of nuclear factor kβ ligand (RANKL; 100 ng/ml) for 9 days. Osteoblastic differentiation was accomplished by seeding the adherent BM cells at 30,000 cells per cm2 and culturing in the presence of 10 mM β-glycerophosphate ,50 μM ascorbic acid, and 100 nM Dexamethasone for up to 19 days. Osteoclasts were identified as multinucleated cells with positive TRAP staining. The resorptive ability of osteoclasts was assayed by quantifying resorption pits formed on bone slices. For osteoblast differentiation, Alizarin red was used to detect mineralized bone matrix. At the completion of each assay RNA was isolated from cultures and Real-time quantitative PCR analysis was performed to detect changes in mRNA levels of osteoclast (i.e. calcitonin receptor/CTR and cathepsin K/Ctsk) and osteoblast (i.e. osteopontin/OPN and osteocalcin/BLAP) differentiation markers. Results: Cells from CD14-/- mice demonstrated decreased percentages of TRAP positive cells and multi-nucleated cells (p value = 0.01; Fig. 1 A-D) and reduced Ctsk mRNA (p value = 0.03; Fig. 1 E&F), compared to WT controls. CD14-/- cells showed a 50% reduction in resorption pits and area compared to WT (Fig. 1 G&I). No significant differences were observed between strains in osteoblastic potential measured by mineralization and gene expression of osteoblastic markers (Fig. 2). Conclusions: CD14-/- BM precursor cells have reduced capacity to differentiate into functional osteoclasts, while osteoblastic potential is retained. This suggests that the reduced capacity of CD14-deficient osteoclastic cells may lead to the reduced bone remodeling in response to joint injury seen in the DMM model, by disrupting the balance between resorption and formation. Future experiments will determine the influence of DAMPs on osteoclast differentiation and activity, and the density of osteoclasts in SCB after DMM injury. These experiments will lead to a better understanding of the role of CD14 and TLR pathways on bone remodeling in response to joint injury and OA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

CX3CL1/fractalkine regulates the differentiation of human peripheral blood monocytes and monocyte-derived dendritic cells into osteoclasts

Osteoclast differentiation is promoted under inflammatory conditions and osteoclasts play a major role in bone destruction in rheumatoid arthritis (RA). Chemokine (C-X3-C motif) ligand 1 (CX3CL1), also known as fractalkine, functions as a chemoattractant and adhesion molecule, and is involved in the pathogenesis of RA. The blockade of CX3CL1 inhibits the migration of macrophages and osteoclast precursor cells into the inflamed synovium. In the present study, we investigated the direct stimulatory effects of CX3CL1 on osteoclast differentiation from human peripheral blood monocytes and monocyte-derived dendritic cells. A stimulation with CX3CL1 significantly promoted osteoclast differentiation from CD16- monocytes and also monocyte-derived dendritic cells induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). On the other hand, CD16+ monocytes treated with M-CSF and RANKL did not differentiate into osteoclasts, even with CX3CL1. Calcium resorption was significantly increased by monocyte-derived osteoclasts, but not by dendritic cell-derived osteoclasts, following the addition of CX3CL1. The present results suggest that CX3CL1 directly regulates osteoclast differentiation. CX3CL1 may play important roles in the pathogenesis of RA, not only through the accumulation of inflammatory cells, but also through osteoclastogenesis.