Mast Cells Trigger Disturbed Bone Healing in Osteoporotic Mice

Mast cells are important tissue-resident sensor and effector immune cells but also play a major role in osteoporosis development. Mast cells are increased in numbers in the bone marrow of postmenopausal osteoporotic patients, and mast cell–deficient mice are protected from ovariectomy (OVX)-induced bone loss. In this study, we showed that mast cell–deficient Mcpt5-Cre R-DTA mice were protected from OVX-induced disturbed fracture healing, indicating a critical role for mast cells in the pathomechanisms of impaired bone repair under estrogen-deficient conditions. We revealed that mast cells trigger the fracture-induced inflammatory response by releasing inflammatory mediators, including interleukin-6, midkine (Mdk), and C-X-C motif chemokine ligand 10 (CXCL10), and promote neutrophil infiltration into the fracture site in OVX mice. Furthermore, mast cells were responsible for reduced osteoblast and increased osteoclast activities in OVX mice callus, as well as increased receptor activator of NF-κB ligand serum levels in OVX mice. Additional in vitro studies with human cells showed that mast cells stimulate osteoclastogenesis by releasing the osteoclastogenic mediators Mdk and CXCL10 in an estrogen-dependent manner, which was mediated via the estrogen receptor alpha on mast cells. In conclusion, mast cells negatively affect the healing of bone fractures under estrogen-deficient conditions. Hence, targeting mast cells might provide a therapeutic strategy to improve disturbed bone repair in postmenopausal osteoporosis. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Palmitic Acid and DGAT1 Deficiency Enhance Osteoclastogenesis,while Oleic Acid‐Induced Triglyceride Formation Prevents It

Both obesity and diabetes mellitus are associated with alterations in lipid metabolism as well as a change in bone homeostasis and osteoclastogenesis. We hypothesized that increased fatty acid levels affect bone health by altering precursor cell differentiation and osteoclast activation. Here we show that palmitic acid (PA, 16:0) enhances receptor activator of NF‐κB ligand (RANKL)‐stimulated osteoclastogenesis and is sufficient to induce osteoclast differentiation even in the absence of RANKL. TNFα expression is crucial for PA‐induced osteoclastogenesis, as shown by increased TNFα mRNA levels in PA‐treated cells and abrogation of PA‐stimulated osteoclastogenesis by TNFα neutralizing antibodies. In contrast, oleic acid (OA, 18:1) does not enhance osteoclast differentiation, leads to increased intracellular triglyceride accumulation, and inhibits PA‐induced osteoclastogenesis. Adenovirus‐mediated expression of diacylglycerol acyl transferase 1 (DGAT1), a gene involved in triglyceride synthesis, also inhibits PA‐induced osteoclastogenesis, suggesting a protective role of DGAT1 for bone health. Accordingly, Dgat1 knockout mice have larger bone marrow‐derived osteoclasts and decreased bone mass indices. In line with these findings, mice on a high‐fat PA‐enriched diet have a greater reduction in bone mass and structure than mice on a high‐fat OA‐enriched diet. Thus, we propose that TNFα mediates saturated fatty acid‐induced osteoclastogenesis that can be prevented by DGAT activation or supplementation with OA. © 2014 American Society for Bone and Mineral Research.     

Formulated siRNAs targeting Rankl prevent osteolysis and enhance chemotherapeutic response in osteosarcoma models

The development of osteosarcoma, the most common malignant primary bone tumor is characterized by a vicious cycle established between tumor proliferation and paratumor osteolysis. This osteolysis is mainly regulated by the receptor activator of nuclear factor κB ligand (RANKL). Preclinical studies have demonstrated that Rankl blockade by soluble receptors is an effective strategy to prevent osteolytic lesions leading to osteosarcoma inhibition. A new therapeutic option could be to directly inhibit Rankl expression by small interfering RNAs (Rkl‐siRNAs) and combine these molecules with chemotherapy to counteract the osteosarcoma development more efficiently. An efficient siRNA sequence directed against both mouse and rat mRNAs coding Rankl was first validated in vitro and tested in two models of osteosarcoma: a syngenic osteolytic POS‐1 model induced in immunocompetent mice and a xenograft osteocondensant model of rat OSRGA in athymic mice. Intratumor injections of Rankl‐directed siRNAs in combination with the cationic liposome RPR209120/DOPE reduced the local and systemic Rankl production and protected bone from paratumor osteolysis. Although Rkl‐siRNAs alone had no effect on tumor development in both osteosarcoma models, it significantly blocked tumor progression when combined with ifosfamide compared with chemotherapy alone. Our results indicate that siRNAs could be delivered using cationic liposomes and thereby could inhibit Rankl production in a specific manner in osteosarcoma models. Moreover, the Rankl inhibition mediated by RNA interference strategy improves the therapeutic response of primary osteosarcoma to chemotherapy. © 2011 American Society for Bone and Mineral Research     

RANKL subcellular trafficking and regulatory mechanisms in osteocytes

The receptor activator of the NF‐κB ligand (RANKL) is the central player in the regulation of osteoclastogenesis, and the quantity of RANKL presented to osteoclast precursors is an important factor determining the magnitude of osteoclast formation. Because osteoblastic cells are thought to be a major source of RANKL, the regulatory mechanisms of RANKL subcellular trafficking have been studied in osteoblastic cells. However, recent reports showed that osteocytes are a major source of RANKL presentation to osteoclast precursors, prompting a need to reinvestigate RANKL subcellular trafficking in osteocytes. Investigation of molecular mechanisms in detail needs well‐designed in vitro experimental systems. Thus, we developed a novel co‐culture system of osteoclast precursors and osteocytes embedded in collagen gel. Experiments using this model revealed that osteocytic RANKL is provided as a membrane‐bound form to osteoclast precursors through osteocyte dendritic processes and that the contribution of soluble RANKL to the osteoclastogenesis supported by osteocytes is minor. Moreover, the regulation of RANKL subcellular trafficking, such as OPG‐mediated transport of newly synthesized RANKL molecules to lysosomal storage compartments, and the release of RANKL to the cell surface upon stimulation with RANK are confirmed to be functional in osteocytes. These results provide a novel understanding of the regulation of osteoclastogenesis.     

MiR‐503 Regulates Osteoclastogenesis via Targeting RANK

MicroRNAs (miRNAs) play important roles in osteoclastogenesis and bone resorption. However, no study has investigated the role of miRNA in postmenopausal osteoporosis. Here, we report that miR‐503 was markedly reduced in circulating progenitors of osteoclasts–CD14⁺ peripheral blood mononuclear cells (PBMCs) from postmenopausal osteoporosis patients compared with those from postmenopausal healthy women. Overexpression of miR‐503 in CD14⁺ PBMCs inhibited receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis. Conversely, silencing of miR‐503 in CD14⁺ PBMCs promoted osteoclastogenesis. RANK, which is activated by the binding of RANKL and inducing osteoclast differentiation, was confirmed to be a target of miR‐503. In vivo, silencing of miR‐503 using a specific antagomir in ovariectomy (OVX) mice increased RANK protein expression, promoted bone resorption, and decreased bone mass, whereas overexpression of miR‐503 with agomir inhibited bone resorption and prevented bone loss in OVX mice. Thus, our study revealed that miR‐503 plays an important role in the pathogenesis of postmenopausal osteoporosis and contributes to a new therapeutic way for osteoporosis. © 2014 American Society for Bone and Mineral Research.     

Insulin-like growth factor-binding protein-2 is required for osteoclast differentiation

Global deletion of the Igfbp2 gene results in the suppression of bone turnover. To investigate the role of insulin‐like growth factor‐binding protein‐2 (IGFBP‐2) in regulating osteoclast differentiation, we cultured Igfbp2^?/? bone marrow cells and found a reduction in the number of osteoclasts and impaired resorption. Addition of full‐length IGFBP‐2 restored osteoclast differentiation, fusion, and resorption. To determine the molecular domains of IGFBP‐2 that were required for this effect to be manifest, Igfbp2^?/? bone marrow cells were transfected with constructs in which the heparin‐binding (HBD) or the IGF‐binding domains of IGFBP‐2 were mutated. We found that both domains were necessary for osteoclastogenesis because expression of the mutated forms of either domain failed to support the formation of functionally mature osteoclasts. To discern the mechanism by which IGFBP‐2 regulates osteoclast formation, PTEN abundance and phosphorylation status as well as AKT responsiveness to IGF‐I were analyzed. Igfbp2^?/? cells had elevated levels of PTEN and phospho‐PTEN compared with controls. Expression of wild‐type IGFBP‐2 reduced the level of PTEN to that of wild‐type cells. Cells expressing the IGF‐binding mutant showed suppression of PTEN and phospho‐PTEN equivalent to the wild‐type protein, whereas those expressing the IGFBP‐2 HBD mutant showed no PTEN suppression. When the ability of IGF‐I to stimulate AKT activation, measured by Thr³⁰⁸ and Ser⁴⁷³ phosphorylation, was analyzed, stimulation of Ser⁴⁷³ in response to IGF‐I in preosteoclasts required the presence of intact IGFBP‐2. This effect was duplicated by the addition of a CK2 inhibitor that prevents the phosphorylation of PTEN. In contrast, in fully differentiated osteoclasts, stimulation of Thr³⁰⁸ phosphorylation required the presence of intact IGFBP‐2. We conclude that IGFBP‐2 is an important regulator of osteoclastogenesis and that both the heparin‐ and the IGF‐binding domains of IGFBP‐2 are essential for the formation of fully differentiated and functional osteoclasts. © 2012 American Society for Bone and Mineral Research     

The Calcineurin Inhibitor Tacrolimus as a New Therapy in Severe Cherubism

Cherubism is a rare genetic disorder characterized by extensive growth of a bilateral granuloma of the jaws, resulting in facial disfigurement. Cherubism is caused by gain‐of‐function mutations in the SH3BP2 gene, leading to overactivation of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1)‐dependent osteoclastogenesis. Recent findings in human and mouse cherubism have suggested that calcineurin inhibitors might be drug candidates in cherubism medical treatment. A 4‐year‐old boy with aggressive cherubism was treated with the calcineurin inhibitor tacrolimus for 1 year, and clinical, radiological, and molecular data were obtained. Immunohistologic analysis was performed to compare preoperative and postoperative NFATc1 staining and tartrate resistant acid phosphatase (TRAP) activity. Real‐time PCR was performed to analyze the relative expression levels of OPG and RANKL. After tacrolimus therapy, the patient showed significant clinical improvement, including stabilization of jaw size and intraosseous osteogenesis. Immunohistologic analyses on granuloma showed that tacrolimus caused a significant reduction in the number of TRAP‐positive osteoclasts and NFATc1 nuclear staining in multinucleated giant cells. Molecular analysis showed that tacrolimus treatment also resulted in increased OPG expression. We present the first case of effective medical therapy in cherubism. Tacrolimus enhanced bone formation by stimulating osteogenesis and inhibiting osteoclastogenesis. © 2014 American Society for Bone and Mineral Research.