Staphylococcus aureus osteomyelitis leads to extensive bone destruction. Osteoclasts are bone resorbing cells that are often increased in bone infected with S. aureus. The cytokine RANKL is essential ...for osteoclast formation under physiological conditions but in vitro evidence suggests that inflammatory cytokines may by-pass the requirement for RANKL. The goal of this study was to determine whether RANKL-dependent osteoclast formation is essential for the bone loss that occurs in a murine model of S. aureus osteomyelitis. To this end, humanized-RANKL mice were infected by direct inoculation of S. aureus into a unicortical defect in the femur. Mice were treated with vehicle or denosumab, a human monoclonal antibody that inhibits RANKL, both before and during a 14-day infection period. The severe cortical bone destruction caused by infection was completely prevented by denosumab administration even though the bacterial burden in the femur was not affected. Osteoclasts were abundant near the inoculation site in vehicle-treated mice but absent in denosumab-treated mice. In situ hybridization demonstrated that S. aureus infection potently stimulated RANKL expression in bone marrow stromal cells. The extensive reactive bone formation that occurs in this osteomyelitis model was also reduced by denosumab administration. Lastly, there was a notable lack of osteoblasts near the infection site suggesting that the normal coupling of bone formation to bone resorption was disrupted by S. aureus infection. These results demonstrate that RANKL-mediated osteoclast formation is required for the bone loss that occurs in S. aureus infection and suggest that disruption of the coupling of bone formation to bone resorption may also contribute to bone loss in this condition.
•Blocking RANKL prevented bone loss in S. aureus osteomyelitis in mice.•S. aureus infection stimulated profound RANKL production by stromal cells.•Bone formation appeared to be uncoupled to bone resorption at the infection site.
Nuclear Factor Kappa-Β (NF-kB) is recognized as one of the main inflammatory pathways in the Autoimmune Disease (AD) Rheumatoid Arthritis (RA), which exhibits high levels of inflammatory cytokines ...such as IL-1, TNFa and IL-6 linked to bone erosion and disease progression. NF-kB is also the most studied pathophysiological mechanism in RA, however, over the last few decades, a more recently discovered Receptor Activator of Nuclear Factor Kappa-Β Ligand (RANKL), also linked to NF-kB activation and bone erosion, has been the topic of interest for research in the area of AD management. As the non-discriminative long term suppression of the NF-kB pathway by pharmacological agents in the management of RA has been linked with a number of side effects and with the discovery of the RANKL mechanism, which may present a more targeted approach to the management of the AD, there has been renewed interest in research on the potential impact of nutritional interventions influencing the NF-kB pathway, RANKL as well as RA disease outcomes.
Existing research highlights the potential utility of nutrients such as Omega 3 and Vitamin D, which may lower NF-kB activation in RA. There is, however, a gap in the knowledge of the effects of nutritional interventions on pathophysiological mechanisms contributing to RA and a more robust systematic analysis of whether nutrients or specific vitamins can have an effect on the NF-kB and RANKL main drivers of pathology in RA.
Findings from this study suggest the potential of Vitamin D supplementation in lowering the levels of RANKL and related markers/cytokines such as Th17 cell levels, OPG/RANKL ratio and CXCL10 pathway, which may present as a viable nutrition intervention for the management of RA.
The methodology of this review involved a Systematic Search of the Literature with a Critical Appraisal of papers. It incorporated three tranche searches of 1. review, 2. animal/in vitro and 3. intervention peer reviewed research published in the last 10 years, resulting in a total of 119 papers. Results provide an overview of the NF-kB pathway, a detailed mechanistic examination of the Receptor Activator of Nuclear Factor Kappa-Β Ligand (RANKL) which is linked to bone erosion, and finally a review of nutritional interventions relating to this mechanism of pathophysiology. The accepted papers were critically appraised using SIGN50 for human studies and the ARRIVE guidelines for animal studies; the narrative was and the extracted information coded into key themes.
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•N-cyclopropyl-4-((sulfonylpiperazin-1-yl)methyl)benzamide derivatives were synthesized for potential anti-osteoclast activity.•Derivative 5b has IC50 value of 0.64 µM for osteoclast ...differentiation.•Derivative 5b attenuated F-actin ring formation and bone resorption activity.•Derivative 5b suppresses osteoclast-specific marker gene and protein expressions.
Both cyclopropyl amide and piperazine sulfonamide functional groups are known for their various biological properties used for drug development. Herein, we synthesized nine new derivatives with different substituent groups incorporating these moieties and screened them for their anti-osteoclast differentiation activity. After analyzing the structure–activity relationship (SAR), the inhibitory effect against osteoclastogenesis was determined to be dependent on the lipophilicity of the compound. Derivative 5b emerged as the most effective dose-dependent inhibitor after TRAP staining with an IC50 of 0.64 µM against RANKL-induced osteoclast cells. 5b was also able to suppress F-acting ring formation and bone resorption activity of osteoclasts in vitro. Finally, well-acknowledged gene and protein osteoclast-specific marker expression levels were decreased after 5b administration on primary murine osteoclast cells.
Bone‐related diseases, such as osteoporosis and rheumatoid arthritis, affect hundreds of millions of people worldwide and pose a tremendous burden to health care. By deepening our understanding of ...the molecular mechanisms of bone metabolism and bone turnover, it became possible over the past years to devise new and promising strategies for treating such diseases. In particular, three tumor necrosis factor (TNF) family molecules, the receptor activator of NF‐κB (RANK), its ligand RANKL, and the decoy receptor of RANKL, osteoprotegerin (OPG), have attracted the attention of scientists and pharmaceutical companies alike. Genetic experiments revolving around these molecules established their pivotal role as central regulators of osteoclast development and osteoclast function. RANK‐RANKL signaling not only activates a variety of downstream signaling pathways required for osteoclast development, but crosstalk with other signaling pathways also fine‐tunes bone homeostasis both in normal physiology and disease. In addition, RANKL and RANK have essential roles in lymph node formation, establishment of the thymic microenvironment, and development of a lactating mammary gland during pregnancy. Consequently, novel drugs specifically targeting RANK, RANKL, and their signaling pathways in osteoclasts are expected to revolutionize the treatment of various ailments associated with bone loss, such as arthritis, periodontal disease, cancer metastases, and osteoporosis.
In orthodontic treatment, tooth movement occurs based on bone remodeling in response to orthodontic forces. Interleukin-17A (IL-17) is a proinflammatory cytokine that is primarily secreted by Th 17 ...cells. In the present study, we investigated the mechanisms by which transforming growth factor β-activated kinase 1 (TAK1) inhibits osteoclast differentiation and the role of IL-17 in the intracellular signaling pathways IκB kinase α (IKKα) and nuclear factor-κB (NF-κB) p65, which are downstream of mitogen-activated protein kinases (MAPKs) (p38 and c-Jun N-terminal kinases (JNK)). We confirmed that IL-17 dose-dependently suppressed tartrate-resistant acid phosphatase (TRAP) activity in RAW264.7 cells in the presence of receptor for activation of NF-κB ligand (RANKL). We also found that a treatment with takinib, a potent and selective TAK1 inhibitor, significantly inhibited RANKL-stimulated TRAP activity in RAW264.7 cells. Furthermore, we demonstrated that IL-17 reduced the phosphorylation of TAK1, IKKα, and NF-κB p65. Collectively, these results suggest that IL-17 suppressed RANKL-induced osteoclast differentiation by inhibiting the canonical NF-κB pathway via TAK1. Therefore, IL-17 may be one of the factors suppressing RANKL-induced osteoclast differentiation. In the future, we would like to contribute to the development of reagents that locally control osteoclast differentiation by combining with nanocapsules that selectively deliver IL-17 to osteoclast progenitor cells.
Abstract The maintenance of bone homeostasis is largely dependent upon cellular communication between osteoclasts and osteoblasts. Microvesicles (MVs) have received a good deal of attention and are ...increasingly considered as mediators of intercellular communication due to their capacity to merge with and transfer a repertoire of bioactive molecular content (cargo) to recipient cells, triggering a variety of biologic responses. Here, we demonstrated that MVs shed from osteoblasts contain RANKL protein and can transfer it to osteoclast precursors through receptor ligand (RANKL–RANK), leading to stimulation of RANKL–RANK signaling to facilitate osteoclast formation. Such MV-mediated intercellular communication between osteoblasts and osteoclasts may represent a novel mechanism of bone modeling and remodeling. It may be worthwhile to further explore MVs as tools to modify the biological responses of bone cells or develop an alternative drug to treat bone diseases.
Osteoimmunology peeks into the interaction of bone and the immune system, which has largely proved to be a multiplex reaction. Osteocytes have been shown to regulate bone resorption through the ...expression of RANKL in physiologic and pathologic conditions. TNF-α, a product of the immune system, is an important cytokine regulating bone resorption in inflammatory conditions either directly or by increasing RANKL and M-CSF expressions by osteoblasts and stromal cells. The effect of TNF-α on a wide range of cell types has been documented; however, the direct effect of TNF-α on osteocytes has not been established yet. In this study, primary osteocytes were isolated by cell sorting from neonatal calvaria of Dmp1-Topaz mice, which express the green fluorescent protein under the influence of dentin matrix protein 1 promoter. The results show that osteocytes have a significantly higher RANKL mRNA expression when cultured with TNF-α. A co-culture system of osteocytes and TNF receptors I and II deficient osteoclast precursors treated with TNF-α show a significant increase in TRAP-positive cells while cultures without TNF-α failed to show TRAP-positive cells. Additionally,
experiments of TNF-α injected to mouse calvaria show an increase in TRAP-positive cell number in the suture mesenchyme and an increase in the percentage of RANKL-positive osteocytes compared to PBS-injected calvaria. Osteocytes cultured with TNF-α show up-regulation of MAPKs phosphorylation measured by western blot, and adding MAPKs inhibitors to osteocytes cultured with TNF-α significantly decreases RANKL mRNA expression compared to osteocytes cultured with TNF-α alone. We also found that TNF-α activates the NF-κB pathway in osteocytes measured as a function of p65 subunit nuclear translocation. TNF-α directly affects osteocyte RANKL expression and increases osteoclastogenesis; our results demonstrate that osteocytes guard an important role in inflammatory bone resorption mediated by TNF-α.
Osteoporosis is a major public health concern, which requires novel therapeutic strategies to prevent or mitigate bone loss. Natural compounds have attracted attention as potential therapeutic agents ...due to their safety and efficacy. In this study, we investigated the regulatory activities of boeravinone B (BOB), a natural rotenoid isolated from the medicinal plant
Boerhavia diffusa
, on the differentiation of osteoclasts and mesenchymal stem cells (MSCs), the two main cell components responsible for bone remodeling. We found that BOB inhibited osteoclast differentiation and function, as determined by TRAP staining and pit formation assay, with no significant cytotoxicity. Furthermore, our results showing that BOB ameliorates ovariectomy-induced bone loss demonstrated that BOB is also effective
in
vivo
. BOB exerted its inhibitory effects on osteoclastogenesis by downregulating the RANKL/RANK signaling pathways, including NF-κB, MAPK, and PI3K/Akt, resulting in the suppression of osteoclast-specific gene expression. Further experiments revealed that, at least phenomenologically, BOB promotes osteoblast differentiation of bone marrow-derived MSCs but inhibits their differentiation into adipocytes. In conclusion, our study demonstrates that BOB inhibits osteoclastogenesis and promotes osteoblastogenesis
in vitro
by regulating various signaling pathways. These findings suggest that BOB has potential value as a novel therapeutic agent for the prevention and treatment of osteoporosis.
Osteoporosis is a major public health concern, which requires novel therapeutic strategies to prevent or mitigate bone loss. Natural compounds have attracted attention as potential therapeutic agents ...due to their safety and efficacy. In this study, we investigated the regulatory activities of boeravinone B (BOB), a natural rotenoid isolated from the medicinal plant Boerhavia diffusa, on the differentiation of osteoclasts and mesenchymal stem cells (MSCs), the two main cell components responsible for bone remodeling. We found that BOB inhibited osteoclast differentiation and function, as determined by TRAP staining and pit formation assay, with no significant cytotoxicity. Furthermore, our results showing that BOB ameliorates ovariectomyinduced bone loss demonstrated that BOB is also effective in vivo. BOB exerted its inhibitory effects on osteoclastogenesis by downregulating the RANKL/RANK signaling pathways, including NF-κB, MAPK, and PI3K/Akt, resulting in the suppression of osteoclast-specific gene expression. Further experiments revealed that, at least phenomenologically, BOB promotes osteoblast differentiation of bone marrow-derived MSCs but inhibits their differentiation into adipocytes. In conclusion, our study demonstrates that BOB inhibits osteoclastogenesis and promotes osteoblastogenesis in vitro by regulating various signaling pathways. These findings suggest that BOB has potential value as a novel therapeutic agent for the prevention and treatment of osteoporosis. BMB Reports 2023; 56(10): 545-550
RANKL/RANK/OPG system consists of three essential signaling molecules: i) the receptor activator of nuclear factor (NF)-kB-ligand (RANKL), ii) the receptor activator of NF-kB (RANK), and iii) the ...soluble decoy receptor osteoprotegerin (OPG). Although this system is critical for the regulation of osteoclast differentiation/activation and calcium release from the skeleton, different studies have elucidated its specific role in mammary gland physiology and hormone-driven epithelial proliferation during pregnancy. Of note, several data suggest that progesterone induces mammary RANKL expression in mice and humans. In turn, RANKL controls cell proliferation in breast epithelium under physiological conditions typically associated with higher serum progesterone levels, such as luteal phase of the menstrual cycle and pregnancy. Hence, RANKL/RANK system can be regarded as a major downstream mediator of progesterone-driven mammary epithelial cells proliferation, potentially contributing to breast cancer initiation and progression. Expression of RANKL, RANK, and OPG has been detected in breast cancer cell lines and in human primary breast cancers. To date, dysregulation of RANKL/RANK/OPG system at the skeletal level has been widely documented in the context of metastatic bone disease. In fact, RANKL inhibition through the RANKL-blocking human monoclonal antibody denosumab represents a well-established therapeutic option to prevent skeletal-related events in metastatic bone disease and adjuvant therapy-induced bone loss in breast cancer. On the other hand, the exact role of OPG in breast tumorigenesis is still unclear. This review focuses on molecular mechanisms linking RANKL/RANK/OPG system to mammary tumorigenesis, highlighting pre-clinical and clinical evidence for the potential efficacy of RANKL inhibition as a prevention strategy and adjuvant therapy in breast cancer settings.
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