Osteoclast Development Research Articles

Acod1-mediated inhibition of aerobic glycolysis suppresses osteoclast differentiation and attenuates bone erosion in arthritis

ObjectivesMetabolic changes are crucially involved in osteoclast development and may contribute to bone degradation in rheumatoid arthritis (RA). The enzyme aconitate decarboxylase 1 (Acod1) is known to link the cellular...

Angelicin improves osteoporosis in ovariectomized rats by reducing ROS production in osteoclasts through regulation of the KAT6A/Nrf2 signalling pathway

BackgroundAngelicin, which is found in Psoralea, can help prevent osteoporosis by stopping osteoclast formation, although the precise mechanism remains unclear. MethodsWe evaluated the effect of angelicin on the oxidative stress level of osteoclasts using ovariectomized osteoporosis model rats and RAW264.7 cells. Changes in the bone mass of the femur were investigated using H&E staining and micro-CT. ROS content was investigated by DHE fluorescence labelling. Osteoclast-related genes and proteins were examined for expression using Western blotting, immunohistochemistry, tartrate-resistant acid phosphatase staining, and real-time quantitative PCR. The influence of angelicin on osteoclast development was also evaluated using the MTT assay, double luciferin assay, chromatin immunoprecipitation, immunoprecipitation and KAT6A siRNA transfection. ResultsRats treated with angelicin had considerably higher bone mineral density and fewer osteoclasts. Angelicin prevented RAW264.7 cells from differentiating into osteoclasts in vitro when stimulated by RANKL. Experiments revealed reduced ROS levels and significantly upregulated intracellular KAT6A, HO-1, and Nrf2 following angelicin treatment. The expression of genes unique to osteoclasts, such as MMP9 and NFATc1, was also downregulated. Finally, KAT6A siRNA transfection increased intracellular ROS levels while decreasing KAT6A, Nrf2, and HO-1 protein expression in osteoclasts. However, in the absence of KAT6A siRNA transfection, angelicin greatly counteracted this effect in osteoclasts. ConclusionsAngelicin increased the expression of KAT6A. This enhanced KAT6A expression helps to activate the Nrf2/HO-1 antioxidant stress system and decrease ROS levels in osteoclasts, thus inhibiting oxidative stress levels and osteoclast formation.

Therapeutic potential and recent progression of BTK inhibitors against rheumatoid arthritis.

Rheumatoid arthritis (RA) is a complex chronic inflammatory illness that affects the entire physiology of human body. It has become one of the top causes of disability worldwide. The development and progression of RA involves a complex interplay between an individual's genetic background and various environmental factors. In order to effectively manage RA, a multidisciplinary approach is required, as this disease is complicated and its pathophysiological mechanism is not fully understood yet. In majority of arthritis patients, the presence of abnormal B cells and autoantibodies, primarily anti-citrullinated peptide antibodies and rheumatoid factor affects the progression of RA. Therefore, drugs targeting B cells have now become a hot topic in the treatment of RA which is quite evident from the recent trends seen in the discovery of various B cell receptors (BCRs) targeting agents. Bruton's tyrosine kinase (BTK) is one of these recent targets which play a role in the upstream phase of BCR signalling. BTK is an important enzyme that regulates the survival, proliferation, activation and differentiation of B-lineage cells by preventing BCR activation, FC-receptor signalling and osteoclast development. Several BTK inhibitors have been found to be effective against RA during the invitro and invivo studies conducted using diverse animal models. This review focuses on BTK inhibition mechanism and its possible impact on immune-mediated disease, along with the types of RA currently being investigated, preclinical and clinical studies and future prospective.

The potential calcium content of anchovy (Stolephorus sp.) on mandibular bone growth through osteoprotegerin expression analysis

Abstract Background: Anchovy (Stolephorus sp.) is a commonly used food ingredient due to its high calcium content, which supports craniofacial growth. Calcium stimulates the formation of osteoblasts, which produce osteoprotegerin (OPG). OPG binds to RANKL, blocking RANKL–RANK bonding and limiting osteoclast development. Objective: The objective of this study was to analyze OPG expression in mandibular bones to assess the potential calcium content of anchovies. Methods: Three groups of 27 male Wistar rats were created: control, anchovy, and milk. After 40 days, the rats were decapitated, and their mandibular bones were surgically extracted, decalcified, and prepared for microscopic examination. The results showed a significant difference in OPG expression of rat mandibles between control, anchovy, and milk groups (P < 0.05), as determined by one-way analysis of variance (ANOVA). Tukey’s HSD test revealed a significant difference in the average quantity of rat mandibular OPG expression between the control group and each of the anchovy and milk groups, with probability values of 0.00 and 0.003 (P < 0.05), respectively. However, the average level of OPG expression in anchovy and milk groups did not differ significantly, as indicated by the probability value of 0.064 (P > 0.05). Conclusion: The mandibular development increases after feeding anchovies compared to the control group. Anchovy is not statistically superior to milk in terms of increasing mandibular development. However, as a high-calcium food, anchovy is well-suited to support children’s craniofacial development and growth.

Downregulation of the metalloproteinases ADAM10 or ADAM17 promotes osteoclast differentiation

Bone resorption is driven through osteoclast differentiation by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-Β ligand (RANKL). We noted that a disintegrin and metalloproteinase (ADAM) 10 and ADAM17 are downregulated at the expression level during osteoclast differentiation of the murine monocytic cell line RAW264.7 in response to RANKL. Both proteinases are well known to shed a variety of single-pass transmembrane molecules from the cell surface. We further showed that inhibitors of ADAM10 or ADAM17 promote osteoclastic differentiation and furthermore enhance the surface expression of receptors for RANKL and M-CSF on RAW264.7 cells. Using murine bone marrow-derived monocytic cells (BMDMCs), we demonstrated that a genetic deficiency of ADAM17 or its required regulator iRhom2 leads to increased osteoclast development in response to M-CSF and RANKL stimulation. Moreover, ADAM17-deficient osteoclast precursor cells express increased levels of the receptors for RANKL and M-CSF. Thus, ADAM17 negatively regulates osteoclast differentiation, most likely through shedding of these receptors. To assess the time-dependent contribution of ADAM10, we blocked this proteinase by adding a specific inhibitor on day 0 of BMDMC stimulation with M-CSF or on day 7 of subsequent stimulation with RANKL. Only ADAM10 inhibition beginning on day 7 increased the size of developing osteoclasts indicating that ADAM10 suppresses osteoclast differentiation at a later stage. Finally, we could confirm our findings in human peripheral blood mononuclear cells (PBMCs). Thus, downregulation of either ADAM10 or ADAM17 during osteoclast differentiation may represent a novel regulatory mechanism to enhance their differentiation process. Enhanced bone resorption is a critical issue in osteoporosis and is driven through osteoclast differentiation by specific osteogenic mediators. The present study demonstrated that the metalloproteinases ADAM17 and ADAM10 critically suppress osteoclast development. This was observed for a murine cell line, for isolated murine bone marrow cells and for human blood cells by either preferential inhibition of the proteinases or by gene knockout. As a possible mechanism, we studied the surface expression of critical receptors for osteogenic mediators on developing osteoclasts. Our findings revealed that the suppressive effects of ADAM17 and ADAM10 on osteoclastogenesis can be explained in part by the proteolytic cleavage of surface receptors by ADAM10 and ADAM17, which reduces the sensitivity of these cells to osteogenic mediators. We also observed that osteoclast differentiation was associated with the downregulation of ADAM10 and ADAM17, which reduced their suppressive effects. We therefore propose that this downregulation serves as a feedback loop for enhancing osteoclast development.

Inhibition of caspase-8 cascade restrains the osteoclastogenic fate of bone marrow cells.

Osteoclasts are multinucleated cells of hematopoietic origin, with a pivotal role in bone development and remodeling. Failure in osteoclast differentiation and activation leads to various bone disorders; thus, attention has focused on a search of molecules involved in osteoclast regulatory pathways. Caspase-8 appears to be an interesting candidate for further exploration, due to its potential function in bone development and homeostasis. Mouse bone marrow cells were differentiated into osteoclasts by RANKL stimulation. Increased activation of caspase-8 and its downstream executioner caspases (caspase-3 and caspase-6) was found during osteoclastogenesis. Subsequent inhibition of caspase-8, caspase-3, or caspase-6, respectively, during osteoclast differentiation showed distinct changes in the formation of TRAP-positive multinucleated cells and reduced expression of osteoclast markers including Acp5, Ctsk, Dcstamp, and Mmp9. Analysis of bone matrix resorption confirmed significantly reduced osteoclast function after caspase inhibition. The results clearly showed the role of caspases in the proper development of osteoclasts and contributed new knowledge about non-apoptotic function of caspases.

The “toxic window” of amoxicillin exposure during pregnancy on long bone development in fetal mice

AimsAmoxicillin is a broad-spectrum beta-lactam antibiotic used to treat infectious diseases in pregnant women. Studies have shown that prenatal amoxicillin exposure (PAmE) has developmental toxicity on fetal development. However, the effect of PAmE on long bone development has not been reported. This study aimed to investigate the “toxic window” of PAmE on long bone development and explore its possible mechanism in fetal mice. Materials and methodsPregnant mice were administered amoxicillin by gavage at different stages (gestational day (GD)10–12 and GD16–18), different doses (150 and 300 mg/kg·d) and different courses (single and multiple courses). Fetal femurs were collected at GD18 and bone development related indicators were detected. Key findingsThe results showed that PAmE significantly reduced the length of the femur and primary ossification center of fetal mice, and inhibited the development of fetal growth plate. Meanwhile, PAmE inhibited the development of bone marrow mesenchymal stem cells, osteoclasts and endothelial cells in fetal long bone. Further, we found the fetal long bone developmental toxicity induced by PAmE was most significant at late-pregnancy (GD16–18), high dose (300 mg/kg·d) and multiple-course group. Besides, PAmE inhibited the expression of Wnt/β-catenin signaling pathway in fetal long bone. The β-catenin mRNA expression was significantly positively correlated with the development indexes of fetal long bone. SignificancePAmE has toxic effects on long bone development, and there was an obvious “toxic window” of PAmE on the long bone development in fetal mice. The Wnt/β-catenin signaling pathway may mediate PAmE-induced fetal long bone development inhibition.

From Tumor to Bone: Growth Factor Receptors as Key Players in Cancer Metastasis.

This review article explores the intricate correlation between growth factors and bone metastases, which play a crucial role in the development of several types of malignancies, namely breast, prostate, lung, and renal cancers. The focal point of our discussion is on crucial receptors for growth factors, including Epidermal Growth Factor Receptor (EGFR), Transforming Growth Factor-β (TGFβ), Vascular Endothelial Growth Factor Receptor (VEGFR), and Fibroblast Growth Factor Receptor (FGFR). These receptors, which are essential for cellular activities including growth, differentiation, and survival, have important involvement in the spread of cancer and the interactions between tumors and the bone environment. We discuss the underlying mechanisms of bone metastases, with a specific emphasis on the interaction between growth factor receptors and the bone microenvironment. EGFR signaling specifically enhances the process of osteoclast development and the formation of osteolytic lesions, especially in breast and lung malignancies. TGFβ receptors have a role in both osteolytic and osteoblastic metastases by releasing TGFβ, which attracts cancer cells and promotes bone remodeling. This is a crucial element in the spread of prostate cancer to the bones. The functions of FGFR and VEGFR in the processes of bone formation and tumor angiogenesis, respectively, highlight the complex and diverse nature of these interactions. The review emphasizes the possibility of targeted therapeutics targeting these receptors to interrupt the cycle of tumor development and bone degradation. Therapeutic approaches include focusing on the VEGF/VEGFR, EGF/EGFR, FGF/FGFR, and TGFβ/TGFβR pathways. These include a variety of compounds, such as small molecule inhibitors and monoclonal antibodies, which have shown potential to interfere with tumor-induced alterations in bone. The text discusses clinical trials and preclinical models, offering insights into the effectiveness and constraints of various treatments. Ultimately, this study provides a succinct but thorough summary of the present knowledge and treatment strategies focused on growth factor receptors in bone metastases. This highlights the significance of comprehending the signaling of growth factor receptors in the microenvironment where tumors spread to the bones, as well as the possibility of using targeted therapies to enhance the results for cancer patients with bone metastases. The advancement of treating bone metastases hinges on the development of treatments that specifically target the intricate relationships between malignancies and bone.

Potato protein hydrolysate inhibits RANKL-induced osteoclast development by inhibiting osteoclastogenic genes via the NF-κB/MAPKs signaling pathways.

In recent times, there has been growing attention towards exploring the nutritional and functional aspects of potato protein, along with its diverse applications. In the present study, we examined the anti-osteoclast properties of potato protein hydrolysate (PP902) in vitro. Murine macrophages (RAW264.7) were differentiated into osteoclasts by receptor activator of nuclear factor-κB ligand (RANKL), and PP902 was examined for its inhibitory effect. Initially, treatment with PP902 was found to significantly prevent RANKL-induced morphological changes in macrophage cells, as determined by tartrate-resistant acid phosphatase (TRAP) staining analysis. This notion was further supported by F-actin analysis using a confocal microscope. Furthermore, PP902 treatment effectively and dose-dependently down-regulated the expression of RANKL-induced osteoclastogenic marker genes, including TRAP, CTR, RANK, NFATc1, OC-STAMP, and c-Fos. These inhibitory effects were associated with suppressing NF-κB transcriptional activation and subsequent reduced nuclear translocation. The decrease in NF-κB activity resulted from reduced activation of its upstream kinases, including I-κBα and IKKα. Moreover, PP902 significantly inhibited RANKL-induced p38MAPK and ERK1/2 activities. Nevertheless, PP902 treatment prevents RANKL-induced intracellular reactive oxygen species generation via increased HO-1 activity. The combined antioxidant and anti-inflammatory effects of PP902 resulted in significant suppression of osteoclastogenesis, suggesting its potential as an adjuvant therapy for osteoclast-related diseases.

343 Development of osteoclast derived exosomes for vascular calcification therapy

OBJECTIVES/GOALS: The global incidence of calcific aortic valve disease (CAVD) increased 3.5-fold since 1990. No preventative or therapeutic pharmaceutical therapies exist for CAVD. We will establish the therapeutic potential of osteoclast-derived exosomes though characterization of contents and mechanisms of action to protect against mineralization. METHODS/STUDY POPULATION: Exosomes were purified from conditioned media collected from murine myeloid precursor cells, RAW264.7 (control), and osteoclasts induced to differentiate from RAW264.7 cells (OD). Protein content of exosomes was determined using proteomic analyses. Nucleic acid contents will be identified by sequencing mRNA, miRNA, and DNA. The calcification prevention and reabsorption abilities of control and OD exosomes will be tested using human valvular interstitial cells (VIC) and smooth muscle cell calcification assays and acellular osteologic disc assays, respectively. Comparison between cellular and acellular systems will help identify mechanisms of action, and demonstrate potential therapeutic viability of OD exosomes in preventative vs resorptive treatments. RESULTS/ANTICIPATED RESULTS: OD exosomes, but not control exosomes, prevented calcification in VIC in vitro. OD exosomes contained osteoclast-specific proteins including TRAP, MMP6, cathepsin K, and bone reabsorption factors including V type proton pumps, ATPases, and integrins. These genes are also involved in resorptive activities, and were highly upregulated in OD compared to control exosomes. We anticipate miRNA signatures associated with mineral resorption will also be present. Increased knowledge of exosome cargo will illuminate their mechanism of action and allow future work to engineer increased efficacy. We also anticipate a therapeutic response when OD exosomes are applied after calcification has begun, showing exosomes promote calcium reabsorption. DISCUSSION/SIGNIFICANCE: Establishing therapeutic potential and examining mechanisms of action will pave the way for OD exosomes as a CAVD treatment. Analysis of exosome contents will determine active molecules to be enhanced in future studies. This work will lay a foundation for moving into aortic valve organoid models, which are accepted by the FDA for preclinical trials.

Morusin inhibits breast cancer-induced osteolysis by decreasing phosphatidylinositol 3-kinase (PI3K)-mTOR signalling

Bone metastases caused by breast cancer pose a major challenge to the successful treatment of breast cancer patients. Many researchers have suggested that herbal medicines are extremely effective at preventing and treating cancer-associated osteolysis. Previous studies have revealed that Morusin (MOR) is cytotoxic to many cancer cells ex vivo. Nevertheless, how MOR contributes to osteolysis induced by breast cancer is still unknown, and the potential mechanism of action against osteolysis is worthy of further study. The protective effect and molecular mechanism of MOR in inhibiting breast cancer cell-induced osteolysis were verified by experiments and network pharmacology. Cell function was assessed by cell proliferation, osteoclast (OC) formation, bone resorption, and phalloidin staining. Tumour growth was examined by micro-CT scanning in vivo. To identify potential MOR treatments, the active ingredient–target pathway of breast cancer was screened using network pharmacology and molecular docking approaches. This study is the first to report that MOR can prevent osteolysis induced by breast cancer cells. Specifically, our results revealed that MOR inhibits RANKL-induced osteoclastogenesis and restrains the proliferation, invasion and migration of MDA-MB-231 breast cells through restraining the PI3K/AKT/MTOR signalling pathway. Notably, MOR prevented bone loss caused by breast cancer cell-induced osteolysis in vivo, indicating that MOR inhibited the development of OCs and the resorption of bone, which are essential for cancer cell-associated bone distraction. This study showed that MOR treatment inhibited osteolysis induced by breast cancer in vivo. MOR inhibited OC differentiation and bone resorption ex vivo and in vivo and might be a potential drug candidate for treating breast cancer-induced osteolysis.

Establishment and validation of an efficient method for the 3D culture of osteoclasts in vitro

IntroductionOsteoclasts (OCs) play a crucial role in maintaining bone health. Changes in OC activity are linked to different bone diseases, making them an intriguing focus for research. However, most studies on OCs have relied on 2D cultures, limiting our understanding of their behavior. Yet, there's a lack of knowledge regarding platforms that effectively support osteoclast formation in 3D cultures. MethodsIn our investigation, we explored the capacity of collagen and GelMA hydrogels to facilitate osteoclast development in 3D culture settings. We assessed the osteoclast development by using different hydrogels and cell seeding strategies and optimizing cell seeding density and cytokine concentration. The osteoclast development in 3D cultures was further validated by biochemical assays and immunochemical staining. ResultsOur findings revealed that 0.3 % (w/v) collagen was conducive to osteoclast formation in both 2D and 3D cultures, demonstrated by increased multinucleation and higher TRAP activity compared to 0.6 % collagen and 5 % to 10 % (w/v) GelMA hydrogels. Additionally, we devised a "sandwich" technique using collagen substrates and augmented the initial macrophage seeding density and doubling cytokine concentrations, significantly enhancing the efficiency of OC culture in 3D conditions. Notably, we validated osteoclasts derived from macrophages in our 3D cultures express key osteoclast markers like cathepsin K and TRAP. ConclusionsTo conclude, our study contributes to establishing an effective method for cultivating osteoclasts in 3D environments in vitro. This innovative approach not only promises a more physiologically relevant platform to study osteoclast behavior during bone remodeling but also holds potential for applications in bone tissue engineering. Clinical significanceThis study introduces an efficient method for cultivating osteoclasts in 3D environments in vitro.It offers a more physiologically relevant platform to investigate osteoclast behavior and holds promise to advance research in bone biology and regenerative dentistry.

A novel method to efficiently differentiate human osteoclasts from blood-derived monocytes

BackgroundOsteoclasts are the tissue-specific macrophage population of the bone and unique in their bone-resorbing activity. Hence, they are fundamental for bone physiology in health and disease. However, efficient protocols for the isolation and study of primary human osteoclasts are scarce. In this study, we aimed to establish a protocol, which enables the efficient differentiation of functional human osteoclasts from monocytes.ResultsHuman monocytes were isolated through a double-density gradient from donor blood. Compared to standard differentiation schemes in polystyrene cell culture dishes, the yield of multinuclear osteoclasts was significantly increased upon initial differentiation of monocytes to macrophages in fluorinated ethylene propylene (FEP) Teflon bags. This initial differentiation phase was then followed by the development of terminal osteoclasts by addition of Receptor Activator of NF-κB Ligand (RANKL). High concentrations of RANKL and Macrophage colony-stimulating factor (M-CSF) as well as an intermediate cell density further supported efficient cell differentiation. The generated cells were highly positive for CD45, CD14 as well as the osteoclast markers CD51/ITGAV and Cathepsin K/CTSK, thus identifying them as osteoclasts. The bone resorption of the osteoclasts was significantly increased when the cells were differentiated from macrophages derived from Teflon bags compared to macrophages derived from conventional cell culture plates.ConclusionOur study has established a novel protocol for the isolation of primary human osteoclasts that improves osteoclastogenesis in comparison to the conventionally used cultivation approach.

Mouse mandibular-derived osteoclast progenitors have differences in intrinsic properties compared with femoral-derived progenitors.

Craniofacial osteoclasts are essential for site-specific processes such as alveolar bone resorption, tooth eruption, and orthodontic tooth movement. Much of the current understanding of osteoclast development and function comes from studies using long bone-derived cells. Minimal investigation has been done to explore skeletal site differences. The overall goal of this study was to determine if mandibular- and femoral-derived osteoclasts represent distinct populations. To test this hypothesis, bone marrow cells were initially analyzed from the mandible and femur of 2-month-old mice. It was shown that mandibular-derived osteoclasts have enhanced size (mm2) compared with femoral-derived osteoclasts. Since bone marrow macrophages are a heterogenous population, we additionally selected for monocytes and demonstrated that mandibular-derived monocytes also form osteoclasts with increased size compared with femoral-derived monocytes. Osteoclast precursor populations from both skeletal sites were analyzed by flow cytometry. A newly described Ly6CHigh+ population as well as the Ly6Cint population was increased in the mandibular-derived cells. The difference in differentiation potential between monocyte cultures suggests that the increase in the Ly6CHigh+ population may explain the enhanced differentiation potential in mandibular-derived cells. Monocyte genes such as Pu.1, C/ebp-a, and Prdm1 are increased in expression in mandibular-derived monocytes compared with femoral-derived monocytes. As expected with enhanced differentiation, osteoclast genes including Nfatc1, Dc-stamp, Ctsk, and Rank are upregulated in mandibular-derived osteoclast precursors. Future studies will determine how changes in the environment of the mandible lead to changes in percentages of osteoclast progenitors and their differentiation potential.

De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis

Osteoclasts play a central role in cancer-cell-induced osteolysis, but the molecular mechanisms of osteoclast activation during bone metastasis formation are incompletely understood. By performing RNA sequencing on a mouse breast carcinoma cell line with higher bone-metastatic potential, here we identify the enzyme CYP11A1 strongly upregulated in osteotropic tumor cells. Genetic deletion of Cyp11a1 in tumor cells leads to a decreased number of bone metastases but does not alter primary tumor growth and lungmetastasis formation in mice. The product of CYP11A1 activity, pregnenolone, increases the number and function of mouse and human osteoclasts invitro but does not alter osteoclast-specific gene expression. Instead, tumor-derived pregnenolone strongly enhances the fusion of pre-osteoclasts via prolyl 4-hydroxylase subunit beta (P4HB), identified as a potential interaction partner of pregnenolone. Taken together, our results demonstrate that Cyp11a1-expressing tumor cells produce pregnenolone, which is capable of promoting bone metastasis formation and osteoclast development via P4HB.

Immune Modulation with RANKL Blockade through Denosumab Treatment in Patients with Cancer.

Denosumab is a fully human mAb that binds receptor activator of NFκB ligand (RANKL). It is routinely administered to patients with cancer to reduce the incidence of new bone metastasis. RANK-RANKL interactions regulate bone turnover by controlling osteoclast recruitment, development, and activity. However, these interactions also can regulate immune cells including dendritic cells and medullary thymic epithelial cells. Inhibition of the latter results in reduced thymic negative selection of T cells and could enhance the generation of tumor-specific T cells. We examined whether administering denosumab could modify modulate circulating immune cells in patients with cancer. Blood was collected from 23 patients with prostate cancer and 3 patients with renal cell carcinoma, all of whom had advanced disease and were receiving denosumab, prior to and during denosumab treatment. Using high-dimensional mass cytometry, we found that denosumab treatment by itself induced modest effects on circulating immune cell frequency and activation. We also found minimal changes in the circulating T-cell repertoire and the frequency of new thymic emigrants with denosumab treatment. However, when we stratified patients by whether they were receiving chemotherapy and/or steroids, patients receiving these concomitant treatments showed significantly greater immune modulation, including an increase in the frequency of natural killer cells early and classical monocytes later. We also saw broad induction of CTLA-4 and TIM3 expression in circulating lymphocytes and some monocyte populations. These findings suggest that denosumab treatment by itself has modest immunomodulatory effects, but when combined with conventional cancer treatments, can lead to the induction of immunologic checkpoints. See related Spotlight by Nasrollahi and Davar, p. 383.

Runx2 deletion in hypertrophic chondrocytes impairs osteoclast mediated bone resorption

Deletion of Runx2 gene in proliferating chondrocytes results in complete failure of endochondral ossification and perinatal lethality. We reported recently that mice with Runx2 deletion specifically in hypertrophic chondrocytes (HCs) using the Col10a1-Cre transgene survive and exhibit enlarged growth plates due to decreased HC apoptosis and cartilage resorption. Bulk of chondrogenesis occurs postnatally, however, the role of Runx2 in HCs during postnatal chondrogenesis is unknown. Despite limb dwarfism, adult homozygous (Runx2HC/HC) mice showed a significant increase in length of growth plate and articular cartilage. Consistent with doubling of the hypertrophic zone, collagen type X expression was increased in Runx2HC/HC mice. In sharp contrast, expression of metalloproteinases and aggrecanases were markedly decreased. Impaired cartilage degradation was evident by the retention of significant amount of safranin-O positive cartilage. Histomorphometry and μCT uncovered increased trabecular bone mass with a significant increase in BV/TV ratio, trabecular number, thickness, and a decrease in trabecular space in Runx2HC/HC mice. To identify if this is due to increased bone synthesis, expression of osteoblast differentiation markers was evaluated and found to be comparable amongst littermates. Histomorphometry confirmed similar number of osteoblasts in the littermates. Furthermore, dynamic bone synthesis showed no differences in mineral apposition or bone formation rates. Surprisingly, three-point-bending test revealed Runx2HC/HC bones to be structurally less strong. Interestingly, both the number and surface of osteoclasts were markedly reduced in Runx2HC/HC littermates. Rankl and IL-17a ligands that promote osteoclast differentiation were markedly reduced in Runx2HC/HC mice. Bone marrow cultures were performed to independently establish Runx2 and hypertrophic chondrocytes role in osteoclast development. The culture from the Runx2HC/HC mice formed significantly fewer and smaller osteoclasts. The expression of mature osteoclast markers, Ctsk and Mmp9, were significantly reduced in the cultures from Runx2HC/HC mice. Thus, Runx2 functions extend beyond embryonic development and chondrocyte hypertrophy by regulating cartilage degradation, osteoclast differentiation, and bone resorption during postnatal endochondral ossification.

AAV-based gene editing of type 1 collagen mutation to treat osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a genetic disorder characterized by bone fragility, low bone mass, fractures, and extraskeletal manifestations. Since OI is commonly caused by single-nucleotide mutation(s) in the COL1A1 or COL1A2 genes encodingtype I collagens, we developed a genome-editing strategy to correct a Col1a2 mutation in an OIM mouse model resembling a severe dominant form of human type III OI. Using a recombinant adeno-associated virus (rAAV), we delivered CRISPR-Cas9 to bone-forming osteoblast-lineage cells in the skeleton. Homology-directed repair (HDR)-mediated gene editing efficiency in these cells was improved when CRISPR-Cas9 was coupled with a donor AAV vector containing a promoterless partial mouse Col1a2 complementary DNA sequence. This approach effectively reversed the dysregulation of osteogenic differentiation by a Col1a2 mutation invitro. Furthermore, systemic administration of dual rAAVs in OIM mice lowered bone matrix turnover rates by reducing osteoblast and osteoclast development while improving the cellular network of mechano-sensing osteocytes embedded in the bone matrix. This strategy significantly improved bone architecture/mass/mineralization, skeletal deformities, grip strength, and spontaneous fractures. Our study is the first demonstration that HDR-mediated gene editing via AAV-mediated delivery effectively corrects a collagen mutation in OI osteoblasts and reverses skeletal phenotypes in OIM mice.

An LGR4 agonist activates the GSK‑3β pathway to inhibit RANK‑RANKL signaling during osteoclastogenesis in bone marrow‑derived macrophages.

The binding between receptor‑activated nuclear factor‑κB (RANK) and the RANK ligand (RANKL) during osteoclast development is an important target for drugs that treat osteoporosis. The leucine‑rich repeat‑containing G‑protein‑coupled receptor 4 (LGR4) acts as a negative regulator of RANK‑RANKL that suppresses canonical RANK signaling during osteoclast differentiation. Therefore, LGR4 agonists may be useful in inhibiting osteoclastogenesis and effectively treating osteoporosis. In the present study, bone marrow‑derived macrophages and a mouse model of RANKL‑induced bone loss were used to investigate the effect of mutant RANKL (MT RANKL), which was previously developed based on the crystal structure of the RANKL complex. In the present study, the binding affinity of wild‑type (WT) RANKL and MT RANKL to RANK and LGR4 was determined using microscale thermophoresis analysis, and the effect of the ligands on the AKT‑glycogen synthase kinase‑3β (GSK‑3β)‑nuclear factor of activated T cells, cytoplasmic, calcineurin‑dependent 1 (NFATc1) signaling cascade was investigated using western blotting and confocal microscopy. In addition, the expression of LGR4 and the colocalization of LGR4 with MT RANKL were analyzed in a mouse model of RANKL‑induced bone loss. The results showed that in osteoclast precursor cells, MT RANKL bound with high affinity to LGR4 and increased GSK‑3β phosphorylation independently of AKT, resulting in the inhibition of NFATc1 nuclear translocation. In the mouse model, MT RANKL colocalized with LGR4 and inhibited bone resorption. These results indicated that MT RANKL may inhibit RANKL‑induced osteoclastogenesis through an LGR4‑dependent pathway and this could be exploited to develop new therapies for osteoporosis.

Osteoprotegerin Gene as a Biomarker in the Development of Osteoporosis in Postmenopausal Women.

Osteoporosis is a multifactorial and polygenic disease caused by an imbalance between osteoclastogenesis and osteoblastogenesis, leading to a decrease in bone mineral density and the occurrence of disorders in the microarchitecture and metabolism of bone tissue. In postmenopausal women, there is a significant decrease in the production of estrogens, which play a key role in maintaining proper bone mineral density. Estrogens have an inhibitory effect on the development and activity of osteoclasts by reducing the synthesis of pro-resorption cytokines and stimulating the expression of osteoprotegerin (OPG). Osteoprotegerin is a cytokine that prevents bone loss by inhibiting the process of osteoclastogenesis, reducing bone resorption. The aim of our study was to determine the influence of the rs3102735 (-163A>G), rs3134070 (-245T>G), rs207361 (-950T>C), rs7844539 (6890A>C), and rs2073618 (1181G>C) polymorphisms of the OPG gene on the risk of osteoporosis and osteopenia in postmenopausal Polish women. The study included 802 unrelated women (osteoporosis: n = 317, osteopenia: n = 110, controls: n = 375) at postmenopausal age (54.7 ± 8.6 years). Genetic analysis was performed using real-time PCR. BMD values as well as clinical and bone parameters with the tested polymorphisms were analyzed among the study population. Analysis of the PPARG rs1801282 variants did not show any association with the risk of osteoporosis and osteopenia. However, for the OPG rs207361 polymorphism, we observed a statistically significant association with the risk of osteoporosis, suggesting that the OPG rs207361 variant may be one of the genetic markers associated with the pathogenesis of osteoporosis.