Osteoclast Development Research Articles

The Notch1 signaling pathway directly modulates the human RANKL-induced osteoclastogenesis

Notch signaling is an evolutionary conserved pathway with a key role in tissue homeostasis, differentiation and proliferation. It was reported that Notch1 receptor negatively regulates mouse osteoclast development and formation by inhibiting the expression of macrophage colony-stimulating factor in mesenchymal cells. Nonetheless, the involvement of Notch1 pathway in the generation of human osteoclasts is still controversial. Here, we report that the constitutive activation of Notch1 signaling induced a differentiation block in human mononuclear CD14+ cells directly isolated from peripheral blood mononuclear cells (PBMCs) upon in vitro stimulation to osteoclasts. Additionally, using a combined approach of single-cell RNA sequencing (scRNA-Seq) simultaneously with a panel of 31 oligo-conjugated antibodies against cell surface markers (AbSeq assay) as well as unsupervised learning methods, we detected four different cell stages of human RANKL-induced osteoclastogenesis after 5 days in which Notch1 signaling enforces the cell expansion of specific subsets. These cell populations were characterized by distinct gene expression and immunophenotypic profiles and active Notch1, JAK/STAT and WNT signaling pathways. Furthermore, cell–cell communication analyses revealed extrinsic modulators of osteoclast progenitors including the IL7/IL7R and WNT5a/RYK axes. Interestingly, we also report that Interleukin-7 receptor (IL7R) was a downstream effector of Notch1 pathway and that Notch1 and IL7R interplay promoted cell expansion of human RANKL-induced osteoclast progenitors. Taken together, these findings underline a novel cell pattern of human osteoclastogenesis, outlining the key role of Notch1 and IL-7R signaling pathways.

Koumine inhibits RANKL-induced ubiquitination and NF-κB activation to prevent ovariectomy and aging-induced bone loss.

Osteoporosis (OP) is a bone remodeling disease characterized by an imbalance between bone resorption and formation. Osteoclasts are the primary therapeutic targets for treating bone destruction. Koumine (KM), the most bioactive component in Gelsemium alkaloids, exhibits antitumor, immunosuppressive, anti-inflammatory, and analgesic properties. However, the effects of bone loss have not been well studied. This study conducted in vitro and in vivo verification experiments on KM. The results showed that KM inhibited bone resorption and tartrate-resistant acid phosphatase positive (TRAP+) osteoclasts development by mature osteoclasts in a dose-dependent manner. Moreover, KM prevented OVX-induced OP in vivo and potentially inhibited ubiquitination, a process closely related to various biological activities, including protein interaction, transcription, and transmembrane signal transduction regulation, especially within the nuclear factor-κB (NF-κB) pathway. Previous studies have demonstrated that several proteins ubiquitination promotes osteoclastogenesis, our study indicated that KM inhibits early NF-κB activation and receptor activator of NF-κB ligandinduced ubiquitination, a critical factor in osteoclast differentiation. In conclusion, our research suggests that KM holds potential as an effective therapeutic agent for OP.

Urolithin A attenuates osteoclast differentiation and compensates for ovariectomy-induced bone loss in mice by inhibiting PI3K/AKT/mTOR signaling pathway

Backgrounds and purposesOsteoporosis (OP) is a debilitating illness distinguished by a reduction in bone density and microstructure, which leads to fragility, fracture risk, and increased mortality, particularly in the elderly and postmenopausal women. Growing evidence suggests that various plant derivatives could be a potential therapy for osteoporosis. Ellagic acid is a phenolic found naturally in extracts from plants such as pomegranate, blackberry and berry. In earlier research, ellagic acid (EA) was reported to have certain benefits on osteoporosis, however, EA was limited by its weak bioactivity. Urolithin A (UA), an ellagic acid metabolite present in the intestinal bacteria, which had a greater bioavailability. However, its therapeutic effects and mechanisms in osteoporosis remain to be explored. Study designIn this work, we explored about how UA affected the molecular processes of osteoclasts differentiation in vitro. We also performed the ovariectomy-induced osteoporotic mice model in order to evaluate how UA impacted the bone loss of osteoporosis. Methods1. TRAP staining analysis to examine the impact of UA on osteoclast differentiation. 2. RT-PCR for evaluating UA's impact on the expression of genes relevant to osteoclasts. Bone resorption experiment and scan by electron microscope in order to determine the impact of UA on osteoclast function. 3.Using western blotting, investigate the impact of UA on the osteoclast differentiation-related signaling pathway. 4. Developed an osteoporosis mouse model for evaluating the preventative impact of UA on bone loss after an ovariectomy. Results1. UA reduced osteoclast development in a dosage manner without cytotoxicity. 2. UA suppresses osteoclast-related gene expression as well as the function of osteoclast bone resorption. 3. UA regulates osteoclast development by decreasing PI3K/AKT/mTOR signaling pathway phosphorylation as well as Nfatc1 transcription and translation. 4. Animal study demonstrated that UA reduced bone loss in ovariectomy-induced osteoporotic mice. ConclusionUA reduced the development and bone resorption function of osteoclast, was shown as inhibiting expression of osteoclast-associated specific genes as well as the PI3K/AKT/mTOR signaling pathway and decreasing Nfatc1 expression. In vivo, UA prevented bone loss in ovariectomy-induced osteoporotic mice. Finally, UA shown tremendous promise in the prevention and treatment of osteoporosis.

Vascular Pericyte-Derived Exosomes Inhibit Bone Resorption via Traf3.

Blood vessels distribute cells, oxygen, and nutrients throughout the body to support tissue growth and balance. Pericytes and endothelial cells form the inner wall of blood vessels, crucial for organ development and tissue homeostasis by producing paracrine signaling molecules. In the skeletal system, pericyte-derived vascular factors along with angiogenic factors released by bone cells regulate angiogenesis and bone formation. Although the involvement of angiogenic factors and skeletal blood vessels in bone homeostasis is relatively clear, the role of pericytes and the underlying mechanisms remain unknown. Here, our objective was to elucidate the significance of pericytes in regulating osteoclast differentiation. We used tissue staining to detect the coverage of pericytes and osteoclasts in femoral tissues of osteoporotic mice and mice of different ages, analyzing their correlation. We developed mice with conditionally deleted pericytes, observing changes in bone mass and osteoclast activity using micro-computer tomography and tissue staining to detect the regulatory effect of pericytes on osteoclasts. Pericytes-derived exosomes (PC-EVs) were collected and co-cultured with monocytes that induce osteoclast differentiation to detect the effect of the former on the exosomes. Finally, the specific mechanism of PC-EVs regulating osteoclast differentiation was verified using RNA sequencing and Western blotting. Our study indicates a significant correlation between pericytes and age-related bone resorption. Conditional deletion of pericytes activated bone resorption and led to osteopenia in vivo. We discovered that PC-EVs inhibited the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, which is mediated by tumor necrosis factor receptor-associated factor 3 (Traf3), negatively regulating osteoclast development and bone resorption. Silencing Traf3 in PC-EVs canceled their inhibitory effect on osteoclast differentiation. Our study provides a novel perspective into the regulatory role of pericytes on bone resorption and may provide potential strategies for developing novel anti-bone resorption therapies.

The JNK signaling pathway against titanium-particle-induced osteoclastogenesis and bone resorption in vivo.

The c-Jun N-terminal kinases (JNK) signaling pathway may be involved in the regulation of osteoclast development. The purpose of this investigation was to investigate whether SB600125, a JNK inhibitor, could attenuate titanium-particle-induced inflammatory osteolysis in vivo. A total of 45 mice were randomly divided into a Sham group, a Titanium group, and a Titanium + JNK inhibitor group, 15 mice per group. After establishing an air pouch bone graft model, we injected phosphate-buffered saline (PBS), titanium particles, or titanium particles + JNK inhibitor into the air pouch of the three groups. The pouch membranes containing bone implants were taken for morphological and molecular analysis 14 days after the mice were sacrificed. General morphological structure observation results, Hematoxylin and Eosin (H&E)-Stained Sections, anti-tartaric acid phosphatase (TRAP) staining, and the transmission electron microscope showed that SB600125, by inhibiting the expression of JNK, attenuated titanium particle-induced inflammatory osteolysis (p<0.05). Immunohistochemical appearance results and reverse transcription-polymerase chain reaction (RT-PCR) results showed SB600125 reduced expression of IL-6, and TNF-α in osteolytic sites stimulated with wear debris (p<0.05). The Western blot results showed the expression of the p-JNK protein in the titanium particle + SB600125 group was significantly reduced compared to the titanium particle stimulation group (p<0.05). Interfering with the JNK signaling pathway may be beneficial in reducing osteolysis, providing a therapeutic target for preventing and treating aseptic loosening caused by debris-induced inflammatory osteolysis.

The Role of XBP1 in bone metabolism.

Bone is a dynamic organ that, once formed, undergoes a constant remodeling process that includes bone resorption and synthesis. Osteoclasts and osteoblasts are primarily responsible for controlling this process. X-box binding protein 1 (XBP1), a transcription factor, affects the metabolism of bones in various ways. In recent years, numerous studies have revealed that XBP1 plays a vital role in bone metabolism, including osteoclast and osteoblast development, as well as in regulating immune cell differentiation that affects the immune microenvironment of bone remodeling. In this review, we highlight the regulatory mechanisms of XBP1 on osteoclasts and osteoblasts, how XBP1 affects the immune microenvironment of bone remodeling by influencing the differentiation of immune cells, and predict the possible future research directions of XBP1 to provide new insights for the treatment of bone-related metabolic diseases.

Early tissue and healing responses after maxillary sinus augmentation using horizontal platelet rich fibrin bone blocks

BackgroundThe effects of horizontal platelet-rich fibrin (H-PRF) bone block on the healing and immune response during sinus augmentation have not been fully investigated histologically at early time points.MethodsEighteenth male New Zealand white rabbits underwent bilateral sinus augmentation and were divided into two groups: deproteinized bovine bone mineral (DBBM) alone and H-PRF + DBBM (H-PRF bone block) group. Maxilla samples were collected at 3, 7 and 14 days post sinus augmentation procedures and analyzed using histological staining for the number of inflammatory cells, new blood vessels and evidence for early osteoclast bone turnover/remodeling. Furthermore, the effects of H-PRF bone blocks on the migration of osteoblasts and THP-1 macrophages were evaluated using a Transwell assay in vitro.ResultsA higher number of immune cells were found in the H-PRF bone block group at 3 and 7 days post-surgery when compared to the DBBM alone group,most notably in the regions close to the mucosal lining and bone plates. Furthermore, a significantly greater number of new blood vessel formations and early signs of osteoclast development were found in the H-PRF bone block group at 14 days. The in vitro transwell assay further confirmed that culture medium from H-PRF bone block markedly promote the migration of osteoblasts and THP-1 macrophages.ConclusionsThe findings from this study have shown that H-PRF bone block is capable of increasing early immune cell infiltration leading to the acceleration of neovascularization and speeding the process of bone metabolism in vivo following maxillary sinus grafting with DBBM.

Clinical masks of marble disease

Marble disease, or osteopetrosis (OPT), is rare in the practice of a rheumatologist, internist or pediatrician. This group of hereditary diseases is based on a defect in the formation, development and functioning of osteoclasts (OCL), which leads to disruption of the processes of resorption and remodeling of bone tissue. Disturbance of resorption leads to increased density and changes in the quality of the bones, as a result of which they become more fragile. As a rule, the disease manifests with pathological fractures. In recent decades, 70% of patients with OPT have been found to have mutations in at least 10 genes that lead to impaired functioning of the OCL. Depending on the variant of inheritance, autosomal dominant, autosomal recessive and intermediate types of OPT are distinguished. Autosomal dominant OPT has a benign course that can be asymptomatic or characterized by multiple bone fractures and other spinal anomalies. The disease usually manifests in adulthood or adolescence. Life expectancy in patients of this group does not differ from that in the general population. Malignant, or infantile, OPT is associated with an autosomal recessive inheritance pattern. Its clinical manifestations are observed from the moment of birth, without treatment, patients die within the first decade of life. In such patients, in addition to the skeletal pathology, there is involvement of the hematopoietic system, compression of the cranial nerves and their function disturbance.The article presents a clinical case of autosomal dominant OPT diagnosed in adulthood (at the age of 38), when the patient referred to the doctor for the first time. Differential diagnosis with ankylosing spondylitis and paraneoplastic spondyloarthritis was performed.

Orientin suppresses osteoclastogenesis and ameliorates ovariectomy-induced osteoporosis via suppressing ROS production.

The aberrant differentiation of osteoclasts is a key feature of the pathogenesis of osteoporosis, which has a devastating impact on human health. While the effects of Orientin (Ori) on osteoporosis, particularly on RANKL-stimulated osteoclast production and activation, remain still unclear, Ori has been found to display several biological activities, including antioxidant and anti-inflammatory. In this work, we investigated the possible pathways through which Ori suppressed RANKL-induced osteoclast development and showed for the first time that it does so. The macrophages from the bone marrow (BMMs) were cultivated and then treated with Ori after being stimulated with RANKL. Then, TRAP-positive multinucleated cells were counted, and F-actin ring analysis was used to assess Ori's impact on mature osteoclast development. In addition, dihydroethidium (DHE) staining was used to evaluate the impact of Ori on RANKL-induced reactive oxygen species (ROS). In addition, we performed western blotting and quantitative RT-PCR analysis to investigate probable causes of these downregulation effects. We discovered that Ori inhibits the creation of osteoclasts, the gene and protein expressions unique to osteoclasts, and the ROS production. By activating Nrf2 and other ROS-scavenging enzymes, Ori reduces intracellular ROS levels. The expression of the main transcription factor of osteoclast development, c-Fos, was downregulated together with NFATc1, CTSK, and NFATc2, thanks to Ori's inhibition of RANKL-induced NF-κB. Consistent with its in vitro antiosteoclastogenic action, Ori therapy in the ovariectomized (OVX) rat model was also able to restore bone mass and improve microarchitecture in the distal femurs. Together, our results demonstrate that Ori is a flavonoid molecule with therapeutic promise for bone illnesses associated with osteoclasts, such as osteoporosis.

Semaphorin 4D Promotes Osteoclast Formation but Inhibits Osteoblast Formation: Implication in Bisphosphonate-Related Osteonecrosis of the Jaw

Introduction: Bisphosphonates are widely used for the treatment of osteoporosis, which could cause osteonecrosis of the jaw (also known as bisphosphonate-related osteonecrosis of the jaw [BRONJ]). Currently, there is no effective treatment for BRONJ. Here, we investigated the role of human recombinant semaphorin 4D (Sema4D) in BRONJ in vitro. Methods: MG-63 and RAW264.7 cells were used to determine the effects of Sema4D on BRONJ. Osteoclast and osteoblast were differentiated by treatment with 50 ng/mL RANKL for 7 days. In vitro BRONJ model was induced by treatment with ZOL (2.5 μ<sc>m</sc>). The development of osteoclasts and osteoblasts was evaluated using ALP activity and ARS staining. qRT-PCR was used to measure the genes relative expression involved in the development of osteoclasts and osteoblasts. In addition, ZOL decreased TRAP-positive area; TRAP protein and mRNA expression were determined using Western blot and qTR-PCR. Results: ZOL treatment remarkedly suppressed Sema4D expression in RAW264.7 cells. Moreover, ZOL reduced TRAP-positive area and TRAP protein and mRNA expression. In parallel, genes involved in osteoclast formation were reduced by ZOL treatment. In contrast, osteoclast apoptosis was increased by ZOL treatment. Recombinant human Sema4D significantly abolished these effects of ZOL. In addition, ALP activity was reduced by recombinant human Sema4D. Discussions: Genes involved in osteoblast formation were decreased by recombinant human Sema4D in a dose-dependent manner. We demonstrated that ZOL treatment inhibited Sema4D expression in RAW264.7 cells. Conclusion: Recombinant human Sema4D treatment can effectively alleviate ZOL-induced inhibition of osteoclast formation and apoptosis and promote osteoblast formation.

Study on the mechanism of NLRP3 effect on the skeleton of de-ovalized mice

Postmenopausal osteoporosis caused by estrogen deficiency affects millions of women worldwide. By influencing both osteoblast and osteoclast development, NOD-like receptor thermoprotein structural domain-associated protein 3 (NLRP3) is a key player in the etiology of osteoporosis (OP). The purpose of this research was to look into the mechanism of action of NLRP3 in osteoporosis caused by a lack of estrogen, highlighting that NLRP3 induces osteoblast pyroptosis and thus inflammatory responses in de-ovulated mice, thereby inhibiting osteogenic differentiation and participating in the development of osteoporosis. In de-ovulated mice, we found an enhanced inflammatory response and suppression of osteogenic activity. In vitro experiments, we found a significant increase in markers of cell pyroptosis and inflammatory responses and a significant decrease in markers of osteogenic differentiation in osteoblasts from de-ovulated mice. However, knockdown of the NLRP3 gene inhibited this cell pyroptosis and improved osteogenic differentiation of osteoblasts. Our findings indicate a potential therapeutic potential for the treatment of estrogen deficiency-induced osteoporosis by demonstrating the critical role that NLRP3 inflammatory vesicles and their downstream-mediated cellular pyroptosis play in bone differentiation.

TRAF-STOP alleviates osteoclastogenesis in periodontitis.

The enhanced osteoclastogenesis contributes to alveolar bone resorption in periodontitis, which increases the risk of tooth loss. To reduce bone destruction, the inhibition of osteoclast development is proposed as a feasible treatment. CD40L-CD40-TRAF6 signal transduction plays a crucial role in inflammation, but how it regulates osteoclast activity in periodontitis has not been elucidated. In this study, we showed the potential role of CD40L-CD40-TRAF6 signaling in periodontitis. CD40L obviously promoted osteoclast formation and bone resorption capacity in vitro. Mechanistically, we found that osteoclastogenesis was enhanced by the overexpression of NFATc1 and NF-κB activation. Importantly, osteoclast activity was effectively suppressed by TRAF-STOP, a small molecular inhibitor of TRAF6. Furthermore, local injection of TRAF-STOP-loaded injectable PLGA-PEG-PLGA hydrogel could alleviate ligation-induced periodontitis in vivo. Taken together, TRAF-STOP shows promising clinical efficacy in periodontitis through alleviating osteoclastogenesis.

Schnurri-3 inhibition suppresses bone and joint damage in models of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads to systemic and articular bone loss by activating bone resorption and suppressing bone formation. Despite current therapeutic agents, inflammation-induced bone loss in RA continues to be a significant clinical problem due to joint deformity and lack of articular and systemic bone repair. Here, we identify the suppressor of bone formation, Schnurri-3 (SHN3), as a potential target to prevent bone loss in RA. SHN3 expression in osteoblast-lineage cells is induced by proinflammatory cytokines. Germline deletion or conditional deletion of Shn3 in osteoblasts limits articular bone erosion and systemic bone loss in mouse models of RA. Similarly, silencing of SHN3 expression in these RA models using systemic delivery of a bone-targeting recombinant adenoassociated virus protects against inflammation-induced bone loss. In osteoblasts, TNF activates SHN3 via ERK MAPK-mediated phosphorylation and, in turn, phosphorylated SHN3 inhibits WNT/β-catenin signaling and up-regulates RANKL expression. Accordingly, knock-in of a mutation in Shn3 that fails to bind ERK MAPK promotes bone formation in mice overexpressing human TNF due to augmented WNT/β-catenin signaling. Remarkably, Shn3-deficient osteoblasts are not only resistant to TNF-induced suppression of osteogenesis, but also down-regulate osteoclast development. Collectively, these findings demonstrate SHN3 inhibition as a promising approach to limit bone loss and promote bone repair in RA.

LOX overexpression programming mediates the osteoclast mechanism of low peak bone mass in female offspring rats caused by pregnant dexamethasone exposure

BackgroundOsteoporosis is a degenerative disease characterized by reduced bone mass, with low peak bone mass being the predominant manifestation during development and having an intrauterine origin. Pregnant women at risk of preterm delivery are commonly treated with dexamethasone to promote fetal lung development. However, pregnant dexamethasone exposure (PDE) can lead to reduced peak bone mass and susceptibility to osteoporosis in offspring. In this study, we aimed to investigate the mechanism of PDE-induced low peak bone mass in female offspring from the perspective of altered osteoclast developmental programming.Methods0.2 mg/kg.d dexamethasone was injected subcutaneously into rats on gestation days (GDs) 9–20. Some pregnant rats were killed at GD20 to remove fetal rat long bones, the rest were delivered naturally, and some adult offspring rats were given ice water swimming stimulation for two weeks.ResultsThe results showed that the fetal rat osteoclast development was inhibited in the PDE group compared with the control group. In contrast, the adult rat osteoclast function was hyperactivation with reduced peak bone mass. We further found that the promoter region methylation levels of lysyl oxidase (LOX) were decreased, the expression was increased, and the production of reactive oxygen species (ROS) was raised in PDE offspring rat long bone before and after birth. Combined in vivo and in vitro experiments, we confirmed that intrauterine dexamethasone promoted the expression and binding of the glucocorticoid receptor (GR) and estrogen receptor β (ERβ) in osteoclasts and mediated the decrease of LOX methylation level and increase of expression through upregulation of 10–11 translocator protein 3 (Tet3).ConclusionsTaken together, we confirm that dexamethasone causes osteoclast LOX hypomethylation and high expression through the GR/ERβ/Tet3 pathway, leading to elevated ROS production and that this intrauterine epigenetic programming effect can be carried over to postnatal mediating hyperactivation in osteoclast and reduced peak bone mass in adult offspring. This study provides an experimental basis for elucidating the mechanism of osteoclast-mediated intrauterine programming of low peak bone mass in female offspring of PDE and for exploring its early targets for prevention and treatment.4bkEv6r2XLhG_tYaB2veA5Video

RhoA promotes osteoclastogenesis and regulates bone remodeling through mTOR-NFATc1 signaling

BackgroundThe cytoskeletal architecture of osteoclasts (OCs) and bone resorption activity must be appropriately controlled for proper bone remodeling, which is associated with osteoporosis. The RhoA protein of GTPase plays a regulatory role in cytoskeletal components and contributes to osteoclast adhesion, podosome positioning, and differentiation. Although osteoclast investigations have traditionally been performed by in vitro analysis, however, the results have been inconsistent, and the significance of RhoA in bone physiology and pathology is still unknown.MethodsWe generated RhoA knockout mice by specifically deleting RhoA in the osteoclast lineage to understand more about RhoA’s involvement in bone remodeling. The function of RhoA in osteoclast differentiation and bone resorption and the mechanisms were assessed using bone marrow macrophages (BMMs) in vitro. The ovariectomized (OVX) mouse model was adopted to examine the pathological effect of RhoA in bone loss.ResultsConditional deletion of RhoA in the osteoclast lineage causes a severe osteopetrosis phenotype, which is attributable to a bone resorption suppression. Further mechanistic studies suggest that RhoA deficiency suppresses Akt-mTOR-NFATc1 signaling during osteoclast differentiation. Additionally, RhoA activation is consistently related to the significant enhancement the osteoclast activity, which culminates in the development of an osteoporotic bone phenotype. Furthermore, in mice, the absence of RhoA in osteoclast precursors prevented occurring OVX-induced bone loss.ConclusionRhoA promoted osteoclast development via the Akt-mTOR-NFATc1 signaling pathway, resulting a osteoporosis phenotype, and that manipulating RhoA activity might be a therapeutic strategy for osteoporotic bone loss.

The role played by ailanthone in inhibiting bone metastasis of breast cancer by regulating tumor-bone microenvironment through the RANKL-dependent pathway.

Introduction: Bone metastasis of breast cancer (BC) is a process in which the disruption of the bone homeostatic microenvironment leads to an increase in osteoclast differentiation. Ailanthus altissima shows an inhibitory effect on osteoclast differentiation. Ailanthone (AIL) refers to a natural compound isolated from Ailanthus altissima, a Chinese herbal medicine, and has effective anti-tumor activity in numerous cell lines. Its impact on bone metastases for BC is yet unclear. Methods: We measured the effect of AIL on MDA-MB-231 cells by wound healing experiments, Transwell and colony formation experiment. Using the Tartrate-resistant Acid Phosphatase (TRAP) staining tests, filamentous (F-actin) staining and bone resorption test to detect the effect of AIL on the osteoclast cell differentiation of the Bone Marrow-derived Macrophages (BMMs), activated by the MDA-MB-231 cell Conditioned Medium (MDA-MB-231 CM) and the Receptor Activator of Nuclear factor-κB Ligand (RANKL),and to explore its possibility Mechanisms. In vivo experiments verified the effect of AIL on bone destruction in breast cancer bone metastasis model mice. Results: In vitro, AIL significantly decrease the proliferation, migration and infiltration abilities of MDA-MB-231 cells at a safe concentration, and also reduced the expression of genes and proteins involved in osteoclast formation in MDA-MB-231 cells. Osteoclast cell differentiation of the BMMs, activated by MDA-MB-231 CM and RANKL, were suppressed by AIL in the concentration-dependent manner. Additionally, it inhibits osteoclast-specific gene and protein expression. It was noted that AIL inhibited the expression of the osteoclast differentiation-related cytokines RANKL and interleukin-1β (IL-1β) that were secreted by the MDA-MB-231 cells after upregulating the Forkhead box protein 3 (FOXP3) expression. Furthermore, AIL also inhibits the expression of the Mitogen-Activated Protein Kinase (MAPK), Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), and Nuclear factor-κB Ligand (NF-κB) signaling pathways, which then suppresses the MDA-MB-231CM-induced development of Osteoclasts. Conclusion: Our study shows that AIL blocks osteoclast differentiation in the bone metastasis microenvironment by inhibiting cytokines secreted by BC cells, which may be a potential agent for the treatment of BC and its secondary bone metastasis.

New Insights into the Physiology of Iron Transport: An Interdisciplinary Approach

The TransCure project entitled 'Iron Transporters DMT1 and FPN1' took an interdisciplinary approach combining structural biology, chemistry and physiology to gain new insights into iron transport. Proteins studied included Divalent Metal Transporter 1 (DMT1, SLC11A2), enabling the import of Fe2+ into the cytoplasm, and the iron efflux transporter Ferroportin (FPN1, SLC40A1). The physiology and pathophysiology, and the mechanisms underlying iron transport in the gut, across the placenta and in bone were investigated. Small molecule high-throughput screening was used to identify improved modulators of DMT1. The characterization of DMT1 inhibitors have provided first detailed insights into the pharmacology of a human iron transport protein. In placental physiology, the identification of the expressional and functional alterations and underlying mechanisms in trophoblast cells clarified the association between placental iron transport by DMT1/FPN1 and gestational diabetes mellitus. In bone, iron metabolism was found to differ between cells of the monocyte/ macrophage lineages, including osteoclasts. Osteoclast development and activity depended on exogenous iron, the expression of high levels of the transferrin receptor (TFR) and low levels of FPN1 suggesting the expression of an “iron storage” phenotype by these cells. The principles and main findings of the TransCure studies on transmembrane iron transport physiology are summarized in this review.

From Stem to Sternum: The Role of Shp2 in the Skeleton.

Src homology-2 domain-containing phosphatase 2 (SHP2) is a ubiquitously expressed phosphatase that is vital for skeletal development and maintenance of chondrocytes, osteoblasts, and osteoclasts. Study of SHP2 function in small animal models has led to insights in phenotypes observed in SHP2-mutant human disease, such as Noonan syndrome. In recent years, allosteric SHP2 inhibitors have been developed to specifically target the protein in neoplastic processes. These inhibitors are highly specific and have great potential for disease modulation in cancer and other pathologies, including bone disorders. In this review, we discuss the importance of SHP2 and related signaling pathways (e.g., Ras/MEK/ERK, JAK/STAT, PI3K/Akt) in skeletal development. We review rodent models of pathologic processes caused by germline mutations that activate SHP2 enzymatic activity, with a focus on the skeletal phenotype seen in these patients. Finally, we discuss SHP2 inhibitors in development and their potential for disease modulation in these genetic diseases, particularly as it relates to the skeleton.

B7-H3 Trike Enhances Killing of Myeloid Derived Suppressor Cells in Multiple Myeloma

Multiple myeloma (MM) is sensitive to Natural Killer (NK) cell-mediated killing but develops methods to attenuate NK cell function. One method to enhance NK cell specificity against myeloma is antibody dependent cellular cytotoxicity through the CD16 receptor. We developed a tri-specific killer engager (TriKE®) with a camelid single-domain antibody (sdAb) fragment against CD16 (on NK cells) as well as a linker region containing IL-15, to enhance NK cell survival. Delivery of IL-15 by TriKE supports NK cell proliferation while avoiding off-target effects on T cells. The second sdAb targets B7-H3 (CD276), a tumor antigen that is expressed on myeloma and is associated with decreased progression free survival. B7-H3 is differentially expressed on myeloma tumor lines MM1S, RPMI-8226, U266, and H929 but all four MM lines are significantly killed by peripheral blood NK cells in the presence of 3nM B7-H3 TriKE compared to NK cells alone. Bone marrow aspirates from 5 MM patients (3 newly diagnosed and 2 relapsed) were analyzed by flow cytometry and showed 58-96% expression of B7-H3 on CD138+ plasma cells. The bone marrow biopsy aspirates also showed monocytic myeloid derived suppressor cells (Mo-MDSC, CD14+, CD11b+) that expressed B7-H3 (38-87%) as well as the damage associated molecular pattern (DAMP) S100A8/9. In comparison, CD14+ cells from the peripheral blood of healthy donors expressed very low levels of B7-H3 or S100A8/9 (<5%). Mo-MDSC are important to the pathogenesis of MM because they support MM growth and contribute to suppression of NK in the tumor microenvironment. B7-H3 is highly expressed on mature osteoclasts and MDSC can develop into osteoclasts in mouse models of MM and breast cancer. We saw that Mo-MDSC expressing B7-H3 also co-expressed RANK, a receptor necessary for signaling osteoclastic development. We developed Mo-MDSC from CD33+ myeloid cells from healthy donors using IL-6 and GM-CSF or co-culture with MM cell lines at 1:1000 ratio. Both types of MDSC suppressed NK proliferation. Peripheral blood derived MDSC expressed >80% B7-H3 and RANK. We separated B7-H3 positive and negative populations by flow cytometry and attempted to develop osteoclasts with M-CSF and RANK-L over 14 days. Tartrate resistant acid phosphatase (TRAP) staining was performed but both groups were negative and exhibited no phenotypic changes by light microscopy. We then isolated CD14+ cells from the bone marrow of an MM patient who was in complete remission (CR) after autologous stem cell transplant but had a distinct population of B7-H3+, RANK+, S100A8/9+ Mo-MDSC in her surveillance bone marrow aspirate. After fourteen days of culture with M-CSF and RANK-L, TRAP staining was positive, and cells had grown larger, consistent with osteoclastic differentiation (Figure 1A). We tested the ability of B7-H3 TriKE to eliminate MDSC by NK cells, therefore diminishing there immune suppression. Cytotoxicity assays were conducted over 48 hours with NK cells with or without B7-H3 TriKE. Killing was significantly enhanced by B7-H3 TriKE (Figure 1B). Since MDSC are known to suppress NK cell function, we assessed metabolic fitness in NK cells with MDSC at 1:1 ratio for 48 hours with or without 3nM B7-H3 TriKE and found that B7-H3 TriKE improved both the oxygen consumption and extracellular acidification rates. These data show that B7-H3 TriKE enhances NK cell killing of MM through direct targeting and eliminates suppressive Mo-MDSC. B7-H3+ Mo-MDSC can differentiate into osteoclasts and persist even in patients who are in CR, possibly contributing to relapse. B7-H3 TriKE may be particularly useful for patients with bone lesions or as consolidative therapy. Commercial manufacturing of B7-H3 TriKE (GTB-5550) has begun, and a phase I trial is expected in late 2023. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mitochondrial fatty acid β-oxidation is important for normal osteoclast formation in growing female mice.

Skeletal remodeling is an energy demanding process that is linked to nutrient availability and the levels of metabolic hormones. While recent studies have examined the metabolic requirements of bone formation by osteoblasts, much less is known about the energetic requirements of bone resorption by osteoclasts. The abundance of mitochondria in mature osteoclasts suggests that the production of an acidified micro-environment conducive to the ionization of hydroxyapatite, secretion of matrix-degrading enzymes, and motility during resorption requires significant energetic capacity. To investigate the contribution of mitochondrial long chain fatty acid β-oxidation to osteoclast development, we disrupted the expression of carnitine palmitoyltransferase-2 (Cpt2) in myeloid-lineage cells. Fatty acid oxidation increases dramatically in bone marrow cultures stimulated with RANKL and M-CSF and microCT analysis revealed that the genetic inhibition of long chain fatty acid oxidation in osteoclasts significantly increases trabecular bone volume in female mice secondary to reduced osteoclast numbers. In line with these data, osteoclast precursors isolated from Cpt2 mutants exhibit reduced capacity to form large-multinucleated osteoclasts, which was not rescued by exogenous glucose or pyruvate, and signs of an energetic stress response. Together, our data demonstrate that mitochondrial long chain fatty acid oxidation by the osteoclast is required for normal bone resorption as its inhibition produces an intrinsic defect in osteoclast formation.