Osteoclastogenesis In Bone Marrow Monocytes Research Articles

Irpetones A and B, Anti-Osteoclastic Heptaketides from a Marine Mesophotic Zone Ircinia Sponge-Associated Fungus Irpex sp. NBUF088.

Chemical investigation of Irpex sp. NBUF088, associated with an Ircinia sp. sponge located at an 84 m deep mesophotic zone, led to the discovery of two new heptaketides, named irpetones A (1) and B (2). Their structures were identified by analysis of spectroscopic data and quantum-chemical calculations. Compound 1 exhibited inhibition against the receptor activator of NF-κB ligand-induced osteoclastogenesis in bone marrow monocytes with an IC50 of 6.3 ± 0.2 μM, causing no notable cytotoxicity. It was also determined that 1 inhibited the phosphorylation of ERK1/2-JNK1/2-p38 MAPKs and the nuclear translocation of NF-κB, consequently suppressing the activation of MAPK and NF-κB signaling pathways induced by the NF-κB ligand.

Casticin promotes osteogenic differentiation of bone marrow stromal cells and improves osteoporosis in rats by regulating nuclear factor-κB/mitogen-activated protein kinase.

Osteoporosis has influenced millions of people, especially postmenopausal women, which has become a big burden to the whole world. Although the diverse roles of casticin (CAS) on different diseases were identified, whether it was implicated with osteoporosis was unknown. A rat model of osteoporosis was established through dexamethasone (DEX) treatment and a cell model reflecting the osteogenic and osteoclast induction was constructed in bone marrow stromal cells (BMSCs). The calcification at the late stage of induction was measured via Alizarin Red S staining. Western blot was applied to evaluate the levels of proteins. Hematoxylin and eosin staining revealed that the number of bone trabecular in DEX-induced osteoporosis rats was decreased, while increased doses of CAS treatment elevated the number of bone trabecular. CAS treatment alleviated DEX-induced osteoporosis in rats. Moreover, we found that CAS inhibited the nuclear factor-κB/mitogen-activated protein kinase (NF-κB/MAPK) pathway. In addition, CAS promoted osteogenic differentiation of BMSCs and reduced osteoclastogenesis of bone marrow monocytes. Finally, CAS was observed to retard the receptor activator of NFκ-B ligand-induced NF-κB/MAPK pathway. CAS promoted osteogenic differentiation of BMSCs and improved osteoporosis in rats by regulating the NF-κB/MAPK pathway. This might shed a light into using CAS as a drug treating osteoporosis in the future.

Fgf9 Negatively Regulates Bone Mass by Inhibiting Osteogenesis and Promoting Osteoclastogenesis Via MAPK and PI3K/AKT Signaling.

Fibroblast growth factor 9 (Fgf9) is a well-known factor that regulates bone development; however, its function in bone homeostasis is still unknown. Previously, we identified a point mutation in the FGF9 gene (p.Ser99Asn, S99N) and generated an isogeneic knock-in mouse model, which revealed that this loss-of-function mutation impaired early joint formation and was responsible for human multiple synostosis syndrome 3 (SYNS3). Moreover, newborn and adult S99N mutant mice exhibited significantly increased bone mass, suggesting that Fgf9 also participated in bone homeostasis. Histomorphology, tomography, and serological analysis of homozygous newborns and heterozygous adults showed that the Fgf9S99N mutation immensely increased bone mass and bone formation in perinatal and adult bones and decreased osteoclastogenesis in adult bone. An in vitro differentiation assay further revealed that the S99N mutation enhanced bone formation by promoting osteogenesis and mineralization of bone marrow mesenchymal stem cells (BMSCs) and attenuating osteoclastogenesis of bone marrow monocytes (BMMs). Considering the loss-of-function effect of the S99N mutation, we hypothesized that Fgf9 itself inhibits osteogenesis and promotes osteoclastogenesis. An in vitro differentiation assay revealed that Fgf9 prominently inhibited BMSC osteogenic differentiation and mineralization and showed for the first time that Fgf9 promoted osteoclastogenesis by enhancing preosteoclast aggregation and cell-cell fusion. Furthermore, specific inhibitors and in vitro differentiation assays were used and showed that Fgf9 inhibited BMSC osteogenesis mainly via the MEK/ERK pathway and partially via the PI3K/AKT pathway. Fgf9 also promoted osteoclastogenesis as a potential costimulatory factor with macrophage colony-stimating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) by coactivating the MAPK and PI3K/AKT signaling pathways. Taken together, our study demonstrated that Fgf9 is a negative regulator of bone homeostasis by regulating osteogenesis and osteoclastogenesis and provides a potential therapeutic target for bone degenerative diseases. © 2020 American Society for Bone and Mineral Research (ASBMR).

Neobavaisoflavone inhibits osteoclastogenesis through blocking RANKL signalling-mediated TRAF6 and c-Src recruitment and NF-κB, MAPK and Akt pathways.

Psoralea corylifolia (P corylifolia) has been popularly applied in traditional Chinese medicine decoction for treating osteoporosis and promoting fracture healing since centuries ago. However, the bioactive natural components remain unknown. In this study, applying comprehensive two‐dimensional cell membrane chromatographic/C18 column/time‐of‐flight mass spectrometry (2D CMC/C18 column/TOFMS) system, neobavaisoflavone (NBIF), for the first time, was identified for the bioaffinity with RAW 264.7 cells membranes from the extracts of P corylifolia. Here, we revealed that NBIF inhibited RANKL‐mediated osteoclastogenesis in bone marrow monocytes (BMMCs) and RAW264.7 cells dose dependently at the early stage. Moreover, NBIF inhibited osteoclasts function demonstrated by actin ring formation assay and pit‐formation assay. With regard to the underlying molecular mechanism, co‐immunoprecipitation showed that both the interactions of RANK with TRAF6 and with c‐Src were disrupted. In addition, NBIF inhibited the phosphorylation of P50, P65, IκB in NF‐κB pathway, ERK, JNK, P38 in MAPKs pathway, AKT in Akt pathway, accompanied with a blockade of calcium oscillation and inactivation of nuclear translocation of nuclear factor of activated T cells cytoplasmic 1 (NFATc1). In vivo, NBIF inhibited osteoclastogenesis, promoted osteogenesis and ameliorated bone loss in ovariectomized mice. In summary, P corylifolia‐derived NBIF inhibited RANKL‐mediated osteoclastogenesis by suppressing the recruitment of TRAF6 and c‐Src to RANK, inactivating NF‐κB, MAPKs, and Akt signalling pathways and inhibiting calcium oscillation and NFATc1 translocation. NBIF might serve as a promising candidate for the treatment of osteoclast‐associated osteopenic diseases.

Diterpenoids from the aerial parts of Flueggea acicularis and their activity against RANKL-induced osteoclastogenesis.

Compounds 1-11 were isolated from the aerial parts of Flueggea acicularis Croizz Webster, including three new rearranged clesistanthane diterpenoids fluacinoids A-C (1-3) and five new norditerpenoid fluacinoids D-H (4-8). The new compounds were identified from spectroscopic data combined with single crystal X-ray diffraction analysis, modified Mosher's methods, and ECD data analyses. All the isolated compounds were evaluated for their activities on RANKL-induced osteoclastogenesis in bone marrow monocytes (BMMs). Compound 6 showed the most potent inhibition against osteoclast differentiation (IC50, 0.7μM) and decreased the expression level of osteoclast-related genes. Moreover, compound 6 prompted the apoptosis of osteoclasts. Compound 6 also suppressed RANKL-induced NF-κB activation. This study reveals that norditerpenoids may be resource for anti-osteoporosis agents.

Blockade of adrenergic β-receptor activation through local delivery of propranolol from a 3D collagen/polyvinyl alcohol/hydroxyapatite scaffold promotes bone repair in vivo.

ObjectivesActivation of the sympathetic system and adrenergic β‐receptors following traumatic bone defects negatively impairs bone regeneration. Whether preventing β‐receptor activation could potentially improve bone defect repair is unknown. In this study, we investigated the effect of systematic administration and local delivery of propranolol through composite scaffolds on bone healing.Materials and methodsCollagen/PVA/propranolol/hydroxyapatite（CPPH）composite scaffolds were fabricated with 3D printing technique and characterized by scanning electron microscope (SEM). Micro‐CT analysis and bone formation histology were performed to detect new bone formation. Osteogenic differentiation of bone marrow stromal cells (BMSCs) and osteoclastogenesis of bone marrow monocytes cultured with scaffolds extract were performed for further verification.ResultsIntraperitoneal injection of propranolol did not significantly improve bone repair, as indicated by micro‐CT analysis and bone formation histology. However, CPPH scaffolds exhibited sustained release of propranolol in vitro and significantly enhanced bone regeneration compared with vehicle collagen/PVA/hydroxyapatite (CPH) scaffolds in vivo. Moreover, in vitro experiments indicated the scaffolds containing propranolol promoted the osteogenic differentiation and migration of rat BMSCs and inhibited osteoclastogenesis by preventing β‐receptor activation.ConclusionsThis study demonstrates that local adrenergic β‐receptor blockade can effectively enhance the treatment of bone defects by stimulating osteogenic differentiation, inhibiting osteoclastogenesis and enhancing BMSCs migration.

Inhibition of osteoclastogenesis by stem cell-derived extracellular matrix through modulation of intracellular reactive oxygen species

Decellularized extracellular matrix (ECM) derived from stem cells has been shown as a promising biomaterial for bone regeneration because of the promotion effect on osteogenesis in mesenchymal stem cells (MSCs). However, bone regeneration is also influenced by bone resorption and little is known about the effect of cell-derived ECM on osteoclast differentiation. In this study, ECM was deposited by MSCs and, after decellularization, the effect of ECM on osteoclastogenesis of bone marrow monocytes (BMMs) was investigated in comparison to standard tissue culture polystyrene. Our results showed that cell-derived ECM improved BMM proliferation but potently inhibited osteoclast differentiation, evidenced by down-regulation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells, areas of actin rings, and osteoclast-specific gene expression. ECM-mediated attenuation of intracellular reactive oxygen species (ROS) was suggested to play a rival role in the inhibition of osteoclastogenesis, because exogenous hydrogen peroxide supplementation partially rescued the ECM-inhibited osteoclastogenesis. Furthermore, rather than collagen type I, fibronectin in the ECM contributed to ECM-mediated anti-osteoclastogenesis. In conclusion, stem cell-derived decellularized ECM significantly suppressed osteoclastogenesis via the attenuation of intracellular ROS. The anti-osteoclastogenic property of cell-derived ECM may benefit its clinical use for modulating bone remodeling and promoting bone tissue engineering. Statement of SignificanceDecellularized extracellular matrix (ECM) derived from stem cells has been shown as a promising biomaterial for bone regeneration; however, bone remodeling is influenced by bone resorption and little is known about the effect of cell-derived ECM on osteoclast differentiation. Cell-derived ECM improved BMM proliferation but potently inhibited osteoclast differentiation. ECM-mediated attenuation of intracellular reactive oxygen species was suggested to play a rival role in osteoclastogenesis. Fibronectin in cell-derived ECM also contributed to ECM-mediated anti-osteoclastogenesis. The anti-osteoclastogenic property of cell-derived ECM may benefit clinically for modulating bone remodeling and promoting bone tissue engineering.

Melatonin at pharmacological concentrations suppresses osteoclastogenesis via the attenuation of intracellular ROS.

Osteoporosis is a bone disorder linked to impaired bone formation and excessive bone resorption. Melatonin has been suggested to treat osteoporosis due to its beneficial actions on osteoblast differentiation. However, the direct effects of melatonin on osteoclastogenesis in bone marrow monocytes (BMMs) remain unknown. This study was to investigate whether melatonin at either physiological or pharmacological concentrations could affect osteoclast differentiation. Primary BMMs were isolated from the femurs and tibias of C57BL/6 mice and were induced toward multinucleated osteoclasts, in the presence of melatonin at either physiological (0.01 to 10nM) or pharmacological (1 to 100μM) concentrations. Tartrate-resistant acid phosphatase (TRAP) staining was used to label multinucleated osteoclasts and the levels of osteoclast-specific genes were evaluated. To further explore the underlying mechanisms, the roles of silent information regulator type 1 (SIRT1) and reactive oxygen species (ROS) were evaluated. We found that melatonin at pharmacological concentrations, rather than at physiological concentrations, significantly inhibited osteoclast formation in a dose-dependent manner. The number of TRAP-positive cells and the gene expression of osteoclast-specific markers were significantly downregulated in melatonin-treated BMMs. The melatonin-mediated repression of osteoclast differentiation involved the inhibition of the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. The treatment with SIRT1 inhibitors did not affect osteoclast differentiation but, when supplemented with exogenous hydrogen peroxide, a partial rescue of melatonin-suppressed osteoclastogenesis was observed. Melatonin at pharmacological doses directly inhibited osteoclastogenesis of BMMs by a ROS-mediated but not a SIRT1-independent pathway.

HLA–B27 Alters the Response to Tumor Necrosis Factor α and Promotes Osteoclastogenesis in Bone Marrow Monocytes From HLA–B27–Transgenic Rats

To determine whether HLA-B27 expression alters the response of bone marrow monocytes from HLA-B27/human β2 -microglobulin-transgenic (B27-Tg) rats to tumor necrosis factor α (TNFα) and, if so, whether this affects the cells involved in bone homeostasis. Bone marrow monocytes were treated with RANKL or with TNFα to promote osteoclast formation. Osteoclasts were quantified by counting. Gene expression was measured using quantitative polymerase chain reaction analysis, and protein was detected by enzyme-linked immunosorbent assay, immunoblotting, or immunofluorescence. Effects of endogenously produced cytokines on osteoclast formation were determined with neutralizing antibodies. TNFα treatment enhanced osteoclast formation 2.5-fold in HLA-B27-expressing cells as compared to wild-type or to HLA-B7/human β2 -microglobulin-expressing monocytes. TNFα induced ∼4-fold up-regulation of HLA-B27, which was associated with the accumulation of misfolded heavy chains, binding of the endoplasmic reticulum (ER) chaperone BiP, and activation of an ER stress response, which was not seen with HLA-B7. No differences were seen with RANKL-induced osteoclastogenesis. Enhanced interleukin-1α (IL-1α) production from ER-stressed bone marrow monocytes from B27-Tg rats was found to be necessary and sufficient for enhanced osteoclast formation. However, bone marrow monocytes from B27-Tg rats also produced more interferon-β (IFNβ), which attenuated the effect of IL-1α on osteoclast formation. HLA-B27-induced ER stress alters the response of bone marrow monocytes from B27-Tg rats to TNFα, which is associated with enhanced production of IL-1α and IFNβ, cytokines that exhibit opposing effects on osteoclast formation. The altered response of cells expressing HLA-B27 to proinflammatory cytokines suggests that this class I major histocompatibility complex allele may contribute to the pathogenesis of spondyloarthritis and its unique phenotype through downstream effects involving alterations in bone homeostasis.