Aims. Receptor activator of nuclear factor-κB ligand (RANKL) is a key molecule that is expressed in bone stromal cells and is associated with metastasis and poor prognosis in many cancers. However, cancer cells that directly express RANKL have yet to be unveiled. The current study sought to evaluate how a single subunit of G protein, guanine nucleotide-binding protein G(q) subunit alpha (GNAQ), transforms cancer cells into RANKL-expressing cancer cells. Methods. We investigated the specific role of GNAQ using GNAQ wild-type cell lines (non-small-cell lung cancer cell lines; A549 cell lines), GNAQ knockdown cell lines, and patient-derived cancer cells. We evaluated GNAQ, RANKL, macrophage colony-stimulating factor (M-CSF), nuclear transcription factor-κB (NF-κB), inhibitor of NF-κB (IκB), and protein kinase B (Akt) signalling in the GNAQ wild-type and the GNAQ-knockdown cells. Osteoclastogenesis was also evaluated in both cell lines. Results. In the GNAQ-knockdown cells, RANKL expression was significantly upregulated (p < 0.001). The expression levels of M-CSF were also significantly increased in the GNAQ-knockdown cells compared with control cells (p < 0.001). GNAQ knockdown cells were highly sensitive to tumour necrosis factor alpha (TNF-α) and showed significant activation of the NF-κB pathway. The expression levels of RANKL were markedly increased in GNAQ mutant compared with GNAQ wild-type in patient-derived tumour tissues. Conclusion. The present study reveals that the alterations of GNAQ activate NF-κB pathway in cancers, which increase RANKL and M-CSF expression and induce osteoclastogenesis in cancers. Cite this article:Bone Joint Res. 2020;9(1):29–35

Introduction: The biological processes underlying osteolysis in aseptic loosening are not completely understood, but are believed to include factors such as hydrostatic pressure and wear debris. Characterisation of the pseudosynovial membrane from failed implants has revealed numerous cell types with well characterised roles in osteoclastogenesis and bone resorption. More recent work has demonstrated the presence of immunomodulatory cells, including T cells. IL-17 is a T cell product that is believed to be capable of inducing bone resorption. The aims of our study were to characterise the effects of IL-17 on the expression of RANKL and OPG by synovial fibroblasts and to evaluate its role in supporting osteoclastogenesis in vitro.

Materials and Methods: Synovial fibroblasts (SFB) were isolated from tissue obtained at joint replacement surgery. SFB were expanded in culture and used in experiments between passage 4 and 5. Human SFB, and for comparison the human osteosarcoma cell line MG63, were treated with IL-17 (5 and 50ng/ml) for up to 48 hours. The expression and production of RANKL and OPG at 6, 24 and 48hours was assessed by RT-PCR, quantiative real-time PCR, Northern blot and Western blot analyses. To investigate osteoclastogenesis, peripheral blood mononuclear cells (PBMCs) were cultured with IL-17 (5 and 50ng/ml) either alone or with M-CSF (25 ng/ml). After 14–21 days, cultures were fixed and stained for tartrate-resistant acid phosphatase (TRAP) and multinucleated, TRAP positive cells counted. Experiments were repeated on ivory slices and resorption evaluated.

Results: RT-PCR and QT-PCR analysis demonstrated that RANKL mRNA levels in SFB (4 of 5 patients) are enhanced by IL-17 in a biphasic manner. RANKL expression was elevated at 6 hours, returned to near control values at 24 hours before demonstrating increased levels at 48 hours. The expression of RANKL in MG63 cells was enhanced by IL-17 (5ng/ml) at 6 and 24 hours, and by IL-17 (50ng/ml) at 48 hours. The expression of OPG by SFB was upregulated by IL-17 (5 and 50ng/ml) at 6, 24 and 48 hours. The elevated expression of OPG in MG63 cells by IL-17 was time dependent, and this elevated expression was confirmed by Western blot. In cultures of PBMCs, IL-17 alone increased the numbers of TRAP+ve multinucleate cells dose-dependently. Similar levels of TRAP+ve cells were observed in the cultures treated with RANKL and M-CSF, but numbers of multinucleated cells were further increased when M-CSF was supplemented with IL-17. Resorption of ivory wafers was also observed in cultures treated with IL-17.

Conclusions: These results suggest that IL-17 induced osteoclast formation could contribute to the bone loss associated with a wide range of pathological states involving osteolysis and aseptic loosening.

Osteoporosis is one of the most common diseases in modern aging society. Receptor activator of nuclear factor-κB ligand (RANKL) plus macrophage colony stimulating factor (M-CSF)-mediated osteoclastogenesis has been recently implicated in the pathogenesis of this disease. Among other causes, the anticoagulant drug heparin is a notable inducer of secondary osteoporosis, although the molecular pathway underlying this process, particularly in human model, has not been clarified yet. Recently, we reported the differentiation of two subtypes of osteoclasts starting from human peripheral blood CD14-positive monocytes (Monocytes), respectively fusion regulatory protein-1 (FRP-1/CD98)-mediated osteoclasts and RANKL+M-CSF-mediated osteoclasts. We, therefore, investigated in details effects of heparin on differentiation and activation using a simple system of human osteoclastogenesis.

When Monocytes were cultured with osteoclastogenesis-relating factors and a high dose of heparin, heparin suppressed osteoclastogenesis in both pathways. However, a proper quantity of heparin enhanced tartrate-resistant acid phosphatase-positive multinucleated giant cell formation. There were significant differences in fusion indices between control osteoclasts and osteoclasts stimulated by moderate concentrations of heparin in two systems (P< 0.05). As a result of osteoclastic activity, FRP-1-mediated osteoclasts treated with a proper quantity of heparin formed larger pits on Ca plates. Moreover, lacunae on dentin surfaces induced by FRP-1-mediated osteoclasts were enhanced with moderate concentration of heparin. In contrast, heparin did not increase pit-formation area on Ca plates and on dentin surfaces by RANKL+M-CSF-mediated osteoclasts. Evaluating the relation between the concentration of heparin and the osteolytic areas on Ca plates, Pearson’s correlation coefficient of the FRP-1 and the RANKL+M-CSF were −0.973 (P< 0.05) and −0.695 (P=0.19), respectively.

In present study, although moderate doses of heparin stimulated differentiation in both systems, in osteoclastic activity, heparin promoted only to the FRP-1 system, not to RANKL+M-CSF system. Our results suggested FRP-1-induced osteoclastogenesis mainly contributes to development of heparin osteoporosis and also that the onset mechanism after long-term administration of heparin may be affected by the characteristic bone resorption ability of FRP-1osteoclasts.

Introduction and Aims: To investigate the expression of Vascular Endothelial Growth Factor (VEGF) and its receptors in bone metastases from primary breast tumors and further characterise its effects on osteoclasts in vitro.

Method and Results: Seventeen specimens of breast cancer metastases to bone were immunohistochemically stained for VEGF, its receptors VEGFR1 and 2, and the macrophage marker CD68. This demonstrated that breast cancer metastases express VEGF strongly and that surrounding osteoclasts express both VEGFR1 (12 of 14 specimens) and VEGFR2 (14 of 14 specimens).

To investigate osteoclastogenesis in vitro, Peripheral Blood Mononuclear Cells (PBMC) were isolated from healthy volunteers and cultured under stimulation by cytokines. Tartrate Resistant Acid Phophatase (TRAP) positive multinucleated cells were counted in duplicate per treatment and experiments repeated three times. VEGF and RANKL together induced differentiation of multinucleated TRAP-positive cells in similar numbers (22±4.7[SE]) per field of view to M-CSF and RANKL (27.3±7.2[SE]). Stimulation with PlGF (a specific ligand for VEGFR1) and RANKL induced osteoclastogenesis, but VEGF-D (a specific ligand for VEGFR2) with RANKL had little effect.

RAW 264.7 cells (mouse monocyte cell line) differentiated into osteoclast-like cells after stimulation with VEGF and RANKL similar to M-CSF and RANKL. Culture under the same conditions on ivory disks was performed and resorption of ivory by osteoclasts from both PBMC and RAW cells was identified.

Conclusion: VEGF, the angiogenic cytokine, is expressed highly by many solid tumors often correlating with poor prognosis. We have shown that VEGF induces monocytes to differentiate into osteoclast-like cells in the presence of RANKL and this seems to be mediated by VEGFR1. VEGF may therefore play a role in physiological bone resorption and in pathological situations, such as tumor osteolysis and consequently VEGF signalling may be a therapeutic target for osteoclast inhibition.

Background

Aseptic loosening remains the primary reason for failure of orthopaedic implants. Therefore a prime focus of Orthopaedic research is to improve osteointegration and outcomes of joint replacements. The topography of a material surface has been shown to alter cell adhesion, proliferation and growth. The use of nanotopography to promote cell adhesion and bone formation is hoped to improve osteointegration and outcomes of implants. We have previously shown that 15nm high features are bioactive. The arrangement of nanofeatures has been shown to be of importance and block-copolymer separation allows nanopillars to be anodised into the titania layer, providing a compromise of control of order and height of nanopillars. Osteoblast/osteoclast stem cell co-cultures are believed to give the most accurate representation of the in vivo environment, allowing assessment of bone remodelling related to biomaterials.

Aims: Compelling evidences suggest that increased production of osteoclastogenic cytokines by activated T cells plays a relevant role in the bone loss induced by estrogen (E) deficiency in the mouse. However, little information is available on the role of T cells in post-menopausal bone loss in humans.

Methods: To investigate this issue we have assessed the production of cytokines involved in osteoclasts (OCs) formation (RANKL, TNFα and OPG), in vitro Ocs formation in pre and postmenopausal women, the latter with or without osteoporosis. We also evaluate OC precursors in peripheral blood and the ability of peripheral blood mononuclear cells to produce TNFα in both basal and stimulated condition by flow cytometry in these subjects.

Results: Our data demonstrate that E enhances the production of the pro-osteoclastogenetic cytokines TNF alpha and RANKL and increases the number of circulating OCs precursors. Furthermore we show that T cells and monocytes from women with osteoporosis exhibit a higher production of TNF α then those from the other two groups.

Conclusions: Our findings suggest that E deficiency stimulates OCs formation both by increasing the production of TNF and RANKL and increasing the number of OCs precursors. Women with postmenopausal osteoporosis have a higher T cells activity than healthy postmenopausal subjects, T cells thus contribute to the bone loss induced by E deficiency in humans as they do in the mouse.

Summary

We demonstrate that osteoclast-like cells of GCT result from the spontaneous fusion and differentiation of CD14+ cells of the monoblastic lineage by an autocrine mechanism mediated by RANKL, rather than induced by stromal cells. This process is further enhanced by the simultaneous impairment of the negative feed-back regulation of osteoclastogenesis by interferon β.

During bone development and repair, angiogenesis, osteogenesis and bone remodeling (resorption) are closely associated processes with some common mediators involved. BMPs, VEGF and other cytokines are released from bone during bone resorption. Recent study showed that VEGF caused a dose- and time-dependent increase in bone resorption in vitro and in vivo, and BMP-2 markedly enhanced osteoclast differentiation induced by sRANKL and M-CSF in mouse osteoclast culture system. The aim of this study was to further examine the effects of VEGF and BMP-2 on osteoclastogenesis using in vitro human osteoclast culture system. Mononuclear cells were isolated by Lympo-Prep density gradient centrifugation from bone marrow washouts in bone samples from patients undergone total hip replacement. Mononuclear cells were plated at a density of 1 x 106/cm2 in a T-75 flask with aMEM and 15% FCS. The first medium change was made at day 7, when the floating cells were collected from the withdrawn media by centrifugation, and plated in a separate flask. The non-adherent cells in the 2nd flask were harvested again 24 hours later in a similar fashion. The non-adherent cells were then cultured in 24-well plates or calcium phosphate (Ca-P) coated plates, with osteoclast-inducing media (OC media) containing sRANKL 30 ng/ml and M-CSF 30 ng/ml, media were changed every 4 days. After 4 days culture in OC media, rhBMP-2 (3, 30, 300 ng/ml) and VEGF (25 ng/ml) were added respectively or in combination to the cell culture, and the culture was kept for total 16 days. The number of TRAP positive multinuclear cells in each well and the resorptive pit areas on the Ca-P coated plates were calculated and compared. Osteoclastic cell phenotype was defined by expressing tartrate resistant acid phosphatase (TRAP), vitronectin receptor (VNR) and resorptive pit assay. By day 12–14, osteoclastic cells were found in all the experimental groups, they were positive for TRAP and VNR. The number of TRAP+ multinuclear cells were significantly reduced (p< 0.05, t-test) when rhBMP-2 (30 and 300 ng/ml) were present, and this was further reduced (p< 0.01) when rhVEGF was added together with rhBMP-2, comparing to the culture with OC media alone. Extensive lacunar resorption pits in the Ca-P coated plates were found in the culture treated with OC media and OC media with rhVEGF (25 ng/ml). The resorption pit areas were, however, significantly reduced when rhBMP-2 was added at 30 and 300 ng/ml with or without rhVEGF (25 ng/ml, p< 0.05, t-test). The presence of low concentration of rhBMP-2 (3 ng/ml) with VEGF had no effect on osteoclast number or the areas of resorption pit formation. In contrary to previous findings in the mouse osteoclast culture system, the present study had shown that the presence of rhBMP-2 at 30 and 300 ng/ml had strongly inhibited osteoclast differentiation and bone resorptive capability in the human osteoclast culture system, and the inhibition was further enhanced by the presence of rhVEGF. This study implies that VEGF and BMP-2 may be important, yet to be defined regulators, for osteoclastogenesis.

Aims

Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the zinc-dependent matrix metalloproteinases (MMP) and A disintegrin and metalloproteinases (ADAM) involved in extracellular matrix modulation. The present study aims to develop the TIMPs as biologics for osteoclast-related disorders.

Aims

LY3023414 is a novel oral phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dual inhibitor designed for advanced cancers, for which a phase II clinical study was completed in March 2020; however, little is known about its effect on bone modelling/remodelling. In this study, we aimed to explore the function of LY3023414 in bone modelling/remodelling.

Objectives

Many in vitro studies have investigated the mechanism by which mechanical signals are transduced into biological signals that regulate bone homeostasis via periodontal ligament fibroblasts during orthodontic treatment, but the results have not been systematically reviewed. This review aims to do this, considering the parameters of various in vitro mechanical loading approaches and their effects on osteogenic and osteoclastogenic properties of periodontal ligament fibroblasts.

Summary Statement. Obovatol inhibits receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis and prevents inflammatory bone loss in mice. Introduction. Adult skeletal mass and integrity are maintained by balancing osteoclast-mediated bone resorption and osteoblast-induced bone formation during bone remodeling. Abnormal increases in osteoclastic bone resorption can lead to excessive bone destruction as observed in osteoporosis, rheumatoid arthritis, and metastatic cancers Therefore, Modulation of osteoclast formation and function is a promising strategy for the treatment of bone-destructive diseases. To search for compounds that inhibit osteoclast formation, we tested the effect of obovatol, a natural product isolated from the medicinal plant Magnolia obovata, on osteoclastogenesis and inflammatory bone loss. Methods. Osteoclastogenesis was assessed using bone marrow-derived macrophages. RANKL signaling was assessed by immunoblotting and apoptosis by cell death ELISA assay. Actin ring staining and resorption pit assay was performed. Bone morphometric parameters were determined using a microcomputed tomography system. Results. We found that obovatol strongly inhibited osteoclast formation from bone marrow-derived macrophages in a dose-dependent manner without cytotoxicity. Obovatol significantly suppressed RANKL-induced activation of NF-κB, JNK, and ERK signaling pathways. Obovatol also inhibited RANKL-induced expression of the genes c-Fos and nuclear factor of activated T cells c1, which are transcription factors important for osteoclastogenesis. In addition to osteoclast differentiation, obovatol blocked cytoskeletal organization and abrogated the bone resorbing activity of mature osteoclast. Obovatol also accelerated osteoclast apoptosis through the induction of caspase-3 activation. Consistent with its in vitro anti-resorptive effect, obovatol prevented bone loss induced by lipopolysaccharide (LPS) in vivo. Conclusion. Our findings demonstrate that obovatol, a natural compound isolated from Magnolia obovata, suppresses the differentiation, function, and survival of osteoclasts. Furthermore, obovatol protected against LPS-induced bone loss in vivo. Therefore, we suggest that obovatol may have therapeutic potential for the treatment of bone-destructive diseases characterised by increased osteoclast number and/or activity