Osteoclast Progenitors Research Articles

Modulation of osteoclastogenesis through adrenomedullin receptors on osteoclast precursors: initiation of differentiation by asymmetric cell division

Adrenomedullin (ADM), a member of the calcitonin family of peptides, is a potent vasodilator and was shown to have the ability to modulate bone metabolism. We have previously found a unique cell surface antigen (Kat1 antigen) expressed in rat osteoclasts, which is involved in the functional regulation of the calcitonin receptor (CTR). Cross-linking of cell surface Kat1 antigen with anti-Kat1 antigen monoclonal antibody (mAbKat1) stimulated osteoclast formation only under conditions suppressed by calcitonin. Here, we found that ADM provoked a significant stimulation in osteoclastogenesis only in the presence of calcitonin; a similar biological effect was seen with mAbKat1 in the bone marrow culture system. This stimulatory effect on osteoclastogenesis mediated by ADM was abolished by the addition of mAbKat1. 125I-labeled rat ADM (125I-ADM)-binding experiments involving micro-autoradiographic studies demonstrated that mononuclear precursors of osteoclasts abundantly expressed ADM receptors, and the specific binding of 125I-ADM was markedly inhibited by the addition of mAbKat1, suggesting a close relationship between the Kat1 antigen and the functional ADM receptors expressed on cells in the osteoclast lineage. ADM receptors were also detected in the osteoclast progenitor cells in the late mitotic phase, in which only one daughter cell of the dividing cell express ADM receptors, suggesting the semiconservative cell division of the osteoclast progenitors in the initiation of osteoclastogenesis. Messenger RNAs for the receptor activity-modifying-protein 1 (RAMP1) and calcitonin receptor-like receptor (CRLR) were expressed in cells in the osteoclast lineage; however, the expression of RAMP2 or RAMP3 was not detected in these cells. It is suggested that the Kat1 antigen is involved in the functional ADM receptor distinct from the general ADM receptor, consisting of CRLR and RAMP2 or RAMP3. Modulation of osteoclastogenesis through functional ADM receptors abundantly expressed on mononuclear osteoclast precursors is supposed to be important in the fine regulation of osteoclast differentiation in a specific osteotrophic hormonal condition with a high level of calcitonin in blood.Adrenomedullin (ADM) significantly stimulates osteoclastogenesis only in the presence of calcitonin. Functional ADM receptor partly composed of osteoclast-specific cell surface Kat1 antigen was detected in cells in the osteoclast-lineage. Expression of ADM receptor was firstly detected in proliferating osteoclast progenitors performing asymmetric cell division and is then expressed in mononuclear osteoclast precursors. Specific regulation of ADM receptors expressed on mononuclear osteoclast precursors could provide an alternative way to modulate bone remodeling therapeutically.

Mechanism of alveolar bone destruction in periodontitis - Periodontal bacteria and inflammation.

Periodontal disease is an inflammatory disease caused by periodontopathogenic bacteria, which eventually leads to bone tissue (alveolar bone) destruction as inflammation persists. Periodontal tissues have an immune system against the invasion of these bacteria, however, due to the persistent infection by periodontopathogenic bacteria, the host innate and acquired immunity is impaired, and tissue destruction, including bone tissue destruction, occurs. Osteoclasts are essential for bone destruction. Osteoclast progenitor cells derived from hematopoietic stem cells differentiate into osteoclasts. In addition, bone loss occurs when bone resorption by osteoclasts exceeds bone formation by osteoblasts. In inflammatory bone disease, inflammatory cytokines act on osteoblasts and receptor activator of nuclear factor-κB ligand (RANKL)-producing cells, resulting in osteoclast differentiation and activation. In addition to this mechanism, pathogenic factors of periodontal bacteria and mechanical stress activate osteoclasts and destruct alveolar bone in periodontitis. In this review, we focused on the mechanism of osteoclast activation in periodontitis and provide an overview based on the latest findings.

Novel population of human monocyte and osteoclast progenitors from pluripotent stem cells and peripheral blood

AbstractOsteoclasts are multinuclear cells of monocytic lineage, with the ability to resorb bone. Studies in mouse have identified bone marrow clonal progenitors able to generate mature osteoclast cells (OCs) in vitro and in vivo. These osteoclast progenitors (OCPs) can also generate macrophages and dendritic cells. Interestingly, cells with equivalent potential can be detected in periphery. In humans, cells with OCP activity have been identified in bone marrow and periphery; however, their characterization has not been as extensive. We have developed reproducible methods to derive, from human pluripotent stem cells, a population containing monocyte progenitors able to generate functional OCs. Within this population, we have identified cells with monocyte and osteoclast progenitor activity based on CD11b and CD14 expression. A population double positive for CD11b and CD14 contains cells with expected osteoclastic potential. However, the double negative (DN) population, containing most of the hematopoietic progenitor activity, also presents a very high osteoclastic potential. These progenitor cells can also be differentiated to macrophage and dendritic cells. Further dissection within the DN population identified cells bearing the phenotype CD15−CD115+ as the population with highest monocytic progenitor and osteoclastic potential. When similar methodology was used to identify OCPs from human peripheral blood, we confirmed a published OCP population with the phenotype CD11b+CD14+. In addition, we identified a second population (CD14−CD11bloCD115+) with high monocytic progenitor activity that was also able to form osteoclast like cells, similar to the 2 populations identified from pluripotent stem cells.

Osteoblasts-derived exosomes regulate osteoclast differentiation through miR-503-3p/Hpse axis

MicroRNAs (miRNAs) are involved in bone remodeling by regulating the balance of bone formation and resorption. Increasing evidence has confirmed that the communication between osteoclast and osteoblast through secreting exosomes and transferring miRNAs. It has been reported that mineralized osteoblasts release exosomes containing more miR-503-3p. However, the roles and molecular mechanisms of osteoblast exosomes-derived miR-503-3p in osteoclast differentiation remain elusive. Here, we isolated exosomes from the supernatant of osteoblasts and identified the exosome characterization through transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot assay. In addition, we found that exosomes and miR-503-3p secreted by osteoblasts inhibited the differentiation of osteoclast progenitor cells. Meanwhile, we found that Hpse (heparanase gene) was a target gene of miR-503-3p and miR-503-3p inhibited the osteoclast differentiation through downregulating the expression of Hpse. In summary, our results demonstrated the roles and the mechanism of osteoblast-derived exosomes inhibited the osteoclast differentiation via miR-503-3p/Hpse axis.

Abstract 3154: Host and neoplastic cell Axl inhibition impair prostate and breast cancer bone metastasis

Abstract Prostate and breast cancers are the most prevalent and the second leading causes of cancer-deaths for men and women in the United States. Cancer survival rates generally worsen significantly with advanced and disseminated disease with nearly 90% of all cancer deaths resulting from metastasis. For breast and prostate cancers, bone is the most common site of metastasis. A key signaling pathway involved in metastatic dissemination of tumor cells is the receptor tyrosine kinase Axl. Axl belongs to the Tyro3, Axl, and MerTK (TAM) subfamily, is expressed in a variety of tumor types, including breast and prostate, and is associated with poor prognosis, metastasis, and outcome. The goal of the present study was to characterize the role of Axl signaling in prostate and breast cancer cell dissemination to bone. This was accomplished using Axl knockdown neoplastic cells, a selective small molecule Axl inhibitor (BGB324), and osteoclast progenitor cells in vitro, as well as intracardiac injection of Axl knockdown tumor cells in vivo. The results showed that Axl inhibition significantly decreases tumor cell migration and invasion in vitro, and suppression of Axl signaling in osteoclast precursor cells reduces the formation of mature osteoclasts. In vivo, Axl knockdown in prostate and breast cancer cells significantly suppressed the formation of neoplastic bone lesions. Taken together our findings indicate that therapeutic targeting of Axl may impair tumor metastasis to the bones through neoplastic and host cell signaling axes. Citation Format: Mai Tanaka, Dietmar W. Siemann. Host and neoplastic cell Axl inhibition impair prostate and breast cancer bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3154.

Neoplastic Monocyte-Derived Osteoclasts Are Highly Proliferative, but Sub-Functional in MPN-Associated Myelofibrosis

Background: Myeloproliferative neoplasms (MPN) are clonal disorders characterized by the proliferation of myeloid progenitors. A defining hallmark of MPN-associated myelofibrosis (MF) is the deposition of fibrous tissue inside the bone marrow (BM) stroma, arising either as a primary process (PMF), or secondary to the essential thromocythemia (post-ET MF) or polycythemia vera. In at least 1/3 of cases, BM fibrosis is accompanied by the osteosclerotic thickening of the bone trabeculae. It was generally held that these changes are a reaction of mesenchymal cells to the stimuli from the malignant clone. However, the recent discovery of neoplastic monocyte-derived fibrocytes capable of directly inducing BM fibrosis in situ and in vivo has led us to hypothesize that other tissue remodeling cells of the myeloid lineage may play similar roles in the pathogenesis of MPN-associated MF. Osteoclasts (OC) are giant multinuclear cells formed by the fusion of multiple monocytic progenitors. Apart from being exclusively bone-resorbing cells, OCs have a critical role in regulating bone formation by the mesenchymal cell-derived osteoblasts (OB) and maintaining the hematopoietic BM microenvironment. The aim of this study was to examine the status of these cells in MF patients and explore the effects that the neoplastic OC progenitors exert on the development of MPN-associated osteosclerosis.Methods: In order to quantitate bone remodeling cells, trephine biopsies were obtained from patients with PMF (n = 32) and post-ET MF (n = 27), as well as healthy controls (n = 3). A tyramide signal amplification-based fluorescence IHC approach was employed using TRAP and cathepsin K as OC-specific and CD56and osterix as OB-specific markers. In addition, CD14+ low-density peripheral blood cells from the representative MF patients and healthy controls were cultured in the presence of M-CSF and RANKL. After 3 weeks mature OCs were visualized using fluorescently labeled F-actin and TRAP with DAPI as the nuclear counterstain, and the JAK2V617F allele frequency was assessed via RT-PCR. For the OC functional assay, cells were plated on a mineralized surface, which was subsequently stained using a modified von Kossa method. To assess the OC fusion capacity, two populations of progenitors labeled with different lipophilic probes were mixed in a 1:1 ratio and the colocalized signal was quantified for each mature cell. All imaging was conducted using an automated multispectral or a dual spinning disk confocal system.Results: Based on the multiplex BM imaging analysis, we observed a significant increase in the number of OCs in MF patients compared to healthy controls (median: 21.8 vs. 7.0 per 100 mm of bone perimeter; p < 0.0001) that was followed by a substantial increase in the total number of OBs and the density of trabecular bone. After inducing differentiation of JAK2+ monocytes from MF patients into mature TRAP+ OCs in vitro, these cells consistently showed a high mutant allele frequency. Compared to OCs cultured from healthy monocytes, MF patients' OCs exhibited a 40.5% decrease in the median number of nuclei per cell (p= 0.024) and a tendency to incompletely develop F-actin rings. Simultaneously, there was no change in the mean TRAP positivity between the two groups. Finally, MF OCs were significantly less apt at resorbing mineral-coated areas in contrast to healthy cells (median: 21.5% vs. 7.3%; p= 0.016), and there was a 3-fold decrease in the proportion of fused mature OCs from MF monocytes compared to healthy ones (p= 0.008).Conclusions: The development of OCs is highly skewed towards proliferation in both primary and secondary MPN-associated MF. We were able to demonstrate that neoplastic monocytic progenitors retain their aberrant genetic constitution even after differentiating into mature OCs. However, fusion of such progenitors seems to be profoundly impaired, and MF OCs are unable to fully acquire the phenotypical features associated with efficient bone resorption, particularly multinucleation and the development of actin-rich structures. At least to a certain extent, this process happens independently of the non-malignant stroma, although it seems to be impacting the OBs as well. Taken together, we show evidence that even though MF OCs are hyperproliferative, their function is intrinsically supressed due to the inherited neoplastic burden, which in turn contributes to the osteosclerotic dysplasia of the MPN-affected BM. DisclosuresVerstovsek:Blueprint Medicines Corp: Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Galena BioPharma: Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Celgene: Research Funding; Roche: Research Funding; Pfizer: Research Funding; Lilly Oncology: Research Funding; Astrazeneca: Research Funding; Incyte: Research Funding; Promedior: Research Funding; Genentech: Research Funding; Galena BioPharma: Research Funding; Pfizer: Research Funding; Gilead: Research Funding; NS Pharma: Research Funding; Seattle Genetics: Research Funding; Astrazeneca: Research Funding; Lilly Oncology: Research Funding; Roche: Research Funding; CTI BioPharma Corp: Research Funding; NS Pharma: Research Funding; Blueprint Medicines Corp: Research Funding; Gilead: Research Funding; CTI BioPharma Corp: Research Funding; Celgene: Research Funding; Promedior: Research Funding.

Melphalan flufenamide inhibits osteoclastogenesis by suppressing proliferation of monocytes

Myeloma bone disease is a major complication in multiple myeloma affecting quality of life and survival. It is characterized by increased activity of osteoclasts, bone resorbing cells. Myeloma microenvironment promotes excessive osteoclastogenesis, a process of production of osteoclasts from their precursors, monocytes. The effects of two anti-myeloma drugs, melphalan flufenamide (melflufen) and melphalan, on the activity and proliferation of osteoclasts and their progenitors, monocytes, were assessed in this study. In line with previous research, differentiation of monocytes was associated with increased expression of genes encoding DNA damage repair proteins. Hence monocytes were more sensitive to DNA damage-causing alkylating agents than their differentiated progeny, osteoclasts. In addition, differentiated progeny of monocytes showed increased gene expression of immune checkpoint ligands which may potentially create an immunosuppressive microenvironment. Melflufen was ten-fold more active than melphalan in inhibiting proliferation of osteoclast progenitors. Furthermore, melflufen was also superior to melphalan in inhibition of osteoclastogenesis and bone resorption. These results demonstrate that melflufen may exert beneficial effects in patients with multiple myeloma such as reducing bone resorption and immunosuppressive milieu by inhibiting osteoclastogenesis.

Mitochondrial Sirt3 contributes to the bone loss caused by aging or estrogen deficiency.

Altered mitochondria activity in osteoblasts and osteoclasts has been implicated in the loss of bone mass associated with aging and estrogen deficiency — the 2 most common causes of osteoporosis. However, the mechanisms that control mitochondrial metabolism in bone cells during health or disease remain unknown. The mitochondrial deacetylase sirtuin-3 (Sirt3) has been earlier implicated in age-related diseases. Here, we show that deletion of Sirt3 had no effect on the skeleton of young mice but attenuated the age-related loss of bone mass in both sexes. This effect was associated with impaired bone resorption. Osteoclast progenitors from aged Sirt3-null mice were able to differentiate into osteoclasts, though the differentiated cells exhibited impaired polykaryon formation and resorptive activity, as well as decreased oxidative phosphorylation and mitophagy. The Sirt3 inhibitor LC-0296 recapitulated the effects of Sirt3 deletion in osteoclast formation and mitochondrial function, and its administration to aging mice increased bone mass. Deletion of Sirt3 also attenuated the increase in bone resorption and loss of bone mass caused by estrogen deficiency. These findings suggest that Sirt3 inhibition and the resulting impairment of osteoclast mitochondrial function could be a novel therapeutic intervention for the 2 most important causes of osteoporosis.

CD13 is a critical regulator of cell\u2013cell fusion in osteoclastogenesis

The transmembrane aminopeptidase CD13 is highly expressed in cells of the myeloid lineage, regulates dynamin-dependent receptor endocytosis and recycling and is a necessary component of actin cytoskeletal organization. Here, we show that CD13-deficient mice present a low bone density phenotype with increased numbers of osteoclasts per bone surface, but display a normal distribution of osteoclast progenitor populations in the bone marrow and periphery. In addition, the bone formation and mineral apposition rates are similar between genotypes, indicating a defect in osteoclast-specific function in vivo. Lack of CD13 led to exaggerated in vitro osteoclastogenesis as indicated by significantly enhanced fusion of bone marrow-derived multinucleated osteoclasts in the presence of M-CSF and RANKL, resulting in abnormally large cells containing remarkably high numbers of nuclei. Mechanistically, while expression levels of the fusion-regulatory proteins dynamin and DC-STAMP1 must be downregulated for fusion to proceed, these are aberrantly sustained at high levels even in CD13-deficient mature multi-nucleated osteoclasts. Further, the stability of fusion-promoting proteins is maintained in the absence of CD13, implicating CD13 in protein turnover mechanisms. Together, we conclude that CD13 may regulate cell–cell fusion by controlling the expression and localization of key fusion regulatory proteins that are critical for osteoclast fusion.

Overexpression of miRNA-22-3p attenuates osteoporosis by targeting MAPK14.

Osteoporosis (OP) results from an imbalance between bone formation, which is regulated by osteoblasts, and bone resorption, which is mediated by osteoclasts. MicroRNA-22-3p (miR-22-3p) expression is decreased during the process of osteoclast differentiation and p38α mitogen-activated protein kinase (MAPK)14 promotes the proliferation and differentiation of osteoclast progenitors. However, whether miR-22-3p could target MAPK14 to regulate the progression of OP remains unknown, which was the aim of the present study. CD14+ PBMCs were used for the establishment of osteoclastic differentiation in vitro. In the present study, reverse transcription quantitative PCR was used to determine the mRNA expression of MAPK14, tartrate resistant acid phosphatase (TRAP), nuclear factor of activated T-cells (NFATC1) and cathepsin K (CTSK). Western blotting was applied to determine the protein expression of MAPK14, TRAP, NFATC1, CTSK, p-p65 and p65. Dual luciferase reporter assay was applied to confirm the relation between miR-22-3p and MAPK14. Cell Counting Kit-8 assay and flow cytometry assays were used to determine the cell proliferation and cell apoptosis, respectively. The results demonstrated that miR-22-3p expression was lower while MAPK14 expression was higher in the serum from patients with OP compared with healthy volunteers. Furthermore, miR-22-3p expression was negatively correlated with MAPK14 expression in patients with OP. In addition, miR-22-3p expression was decreased and MAPK14 expression was increased during the progression of CD14+peripheral blood mononuclear cells (PBMCs) osteoclastic differentiation in a time-dependent manner. Furthermore, miR-22-3p inhibited the proliferation and differentiation and promoted the apoptosis of CD14+PBMCs by targeting MAPK14. In summary, the findings from the present study suggested that miR-22-3p may serve a potential therapeutic role in patients with OP.

Human osteoclastogenesis in Epstein-Barr virus-induced erosive arthritis in humanized NOD/Shi-scid/IL-2Rγnull mice.

Many human viruses, including Epstein-Barr virus (EBV), do not infect mice, which is challenging for biomedical research. We have previously reported that EBV infection induces erosive arthritis, which histologically resembles rheumatoid arthritis, in humanized NOD/Shi-scid/IL-2Rγnull (hu-NOG) mice; however, the underlying mechanisms are not known. Osteoclast-like multinucleated cells were observed during bone erosion in this mouse model, and therefore, we aimed to determine whether the human or mouse immune system activated bone erosion and analyzed the characteristics and origin of the multinucleated cells in hu-NOG mice. Sections of the mice knee joint tissues were immunostained with anti-human antibodies against certain osteoclast markers, including cathepsin K and matrix metalloproteinase-9 (MMP-9). Multinucleated cells observed during bone erosion stained positively for human cathepsin K and MMP-9. These results indicate that human osteoclasts primarily induce erosive arthritis during EBV infections. Human osteoclast development from hematopoietic stem cells transplanted in hu-NOG mice remains unclear. To confirm their differentiation potential into human osteoclasts, we cultured bone marrow cells of EBV-infected hu-NOG mice and analyzed their characteristics. Multinucleated cells cultured from the bone marrow cells stained positive for human cathepsin K and human MMP-9, indicating that bone marrow cells of hu-NOG mice could differentiate from human osteoclast progenitor cells into human osteoclasts. These results indicate that the human immune response to EBV infection may induce human osteoclast activation and cause erosive arthritis in this mouse model. Moreover, this study is the first, to our knowledge, to demonstrate human osteoclastogenesis in humanized mice. We consider that this model is useful for studying associations of EBV infections with rheumatoid arthritis and human bone metabolism.

Inhibition of the Axl pathway impairs breast and prostate cancer metastasis to the bones and bone remodeling

Approximately 90% of cancer-related deaths result from cancer metastasis. In prostate and breast cancers, bone is the most common site of cancer cell dissemination. Key steps in the metastatic cascade are promoted through upregulation of critical cell signaling pathways in neoplastic cells. The present study assessed the role of the receptor tyrosine kinase Axl in prostate and breast cancer cell metastasis to bones using (i) Axl knockdown neoplastic cells and osteoclast progenitor cells in vitro, (ii) intracardiac injection of Axl knockdown tumor cells in vivo, and (iii) selective Axl inhibitor BGB324. Axl inhibition in neoplastic cells significantly decreased their metastatic potential, and suppression of Axl signaling in osteoclast precursor cells also reduced the formation of mature osteoclasts. In vivo, Axl knockdown in prostate and breast cancer cells significantly suppressed the formation and progression of bone metastases. Hence, therapeutic targeting of Axl may impair tumor metastasis to the bones through neoplastic and host cell signaling axes.

EphA2 Is a Clinically Relevant Target for Breast Cancer Bone Metastatic Disease.

ABSTRACTEphA2 receptor tyrosine kinase (RTK) is highly expressed in breast tumor cells across multiple molecular subtypes and correlates with poor patient prognosis. In this study, the potential role of EphA2 in this clinically relevant phenomenon is investigated as metastasis of breast cancer to bone is a major cause of morbidity and mortality in patients. It was found that the EphA2 function in breast cancer cells promotes osteoclast activation and the development of osteolytic bone disease. Blocking EphA2 function molecularly and pharmacologically in breast tumors reduced the number and size of bone lesions and the degree of osteolytic disease in intratibial and intracardiac mouse models, which correlated with a significant decrease in the number of osteoclasts at the tumor–bone interface. EphA2 loss of function in tumor cells impaired osteoclast progenitor differentiation in coculture, which is mediated, at least in part, by reduced expression of IL‐6. EPHA2 transcript levels are enriched in human breast cancer bone metastatic lesions relative to visceral metastatic sites; EphA2 protein expression was detected in breast tumor cells in bone metastases in patient samples, supporting the clinical relevance of the study's findings. These data provide a strong rationale for the development and application of molecularly targeted therapies against EphA2 for the treatment of breast cancer bone metastatic disease. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

A novel modified RANKL variant can prevent osteoporosis by acting as a vaccine and an inhibitor.

BackgroundThe discovery of receptor activator of nuclear factor‐ĸB ligand (RANKL) as the final effector in the pathogenesis of osteoporosis has led to a better understanding of bone remodeling. When RANKL binds to its receptor (RANK), osteoclastic differentiation and activation are initiated. Herein, we propose a strategy using a novel RANKL variant as a competitive inhibitor for RANKL. The RANKL variant activates LGR4 signaling, which competitively regulates RANK and acts as an immunogen that induces anti‐RANKL antibody production.MethodsWe modified the RANK‐binding site on RANKL using minimal amino acid changes in the RANKL complex and its counterpart receptor RANK and tried to evaluate the inhibitory effects on osteoclastogenesis.ResultsThe novel RANKL variant did not bind RANK in osteoclast progenitor cells, but activated LGR4 through the GSK3‐β signaling pathway, thereby suppressing activated T cell cytoplasmic nuclear factor calcineurin‐dependent 1 (NFATc1) expression and activity during osteoclastogenesis. Our RANKL variant generated high levels of RANKL‐specific antibodies, blocked osteoclastogenesis, and inhibited osteoporosis in ovariectomized mouse models. Generated anti‐RANKL antibodies showed a high inhibitory effect on osteoclastogenesis in vivo and in vitro.ConclusionsWe observed that the novel RANKL indeed blocks RANKL via LGR4 signaling and generates anti‐RANKL antibodies, demonstrating an innovative strategy in the development of general immunotherapy.

Skin Chronological Aging Drives Age-Related Bone Loss via Secretion of Cystatin-A

Although clinical evidence has indicated an association between skin atrophy and bone loss during ageing, their causal relationship and the underlying mechanisms are unknown. Here we show that premature skin ageing drives age-related bone loss in mice. We further identify that cystatin-A (Csta), a keratinocyte-enriched secreted factor, mediates the effect of skin on bone. Keratinocyte-derived Csta binds the receptor for activated C-kinase 1 (Rack1) in osteoblast and osteoclast progenitors, thus promoting their proliferation, but inhibiting osteoclast differentiation. Csta secretion decreases with skin ageing in mice and humans, thereby causing senile osteoporosis by decreasing osteoblasts and osteoclasts numbers differentially. In contrast, topical application of calcipotriol stimulates Csta production in the epidermis and alleviates osteoporosis. These results uncover a mode of endocrine regulation of bone metabolism in the skin, identify Csta as an epidermally derived hormone linking skin ageing to bone loss, and calcipotriol as a promising topical drug for senile osteoporosis.

Hdac3 deletion in myeloid progenitor cells enhances bone healing in females and limits osteoclast fusion via Pmepa1

Previous studies examining the role of the histone deacetylase Hdac3 within myeloid cells demonstrated that Hdac3 promotes M2 activation and tissue healing in inflammatory conditions. Since myeloid lineage cells are required for proper bone formation and regeneration, in this study we examined the functions of Hdac3 during bone healing. Conditional deletion of Hdac3 within myeloid progenitors accelerates healing of cortical bone defects. Moreover, reduced osteoclast numbers within the defect site are correlated with Hdac3 suppression. Ex vivo osteoclastogenesis assays further demonstrate that Hdac3 deficiency limits osteoclastogenesis, the number of nuclei per cell and bone resorption, suggesting a defect in cell fusion. High throughput RNA sequencing identified the transmembrane protein Pmepa1 as a differentially expressed gene within osteoclast progenitor cells. Knockdown of Pmepa1 partially restores defects in osteoclastogenesis induced by Hdac3 deficiency. These results show that Hdac3 is required for optimal bone healing and osteoclast fusion, potentially via its regulation of Pmepa1 expression.

Chronic Systemic Infection of Mice with Leishmania infantum Leads to Increased Bone Mass.

Vector-borne infections of humans with the protozoan parasite Leishmania (L.) infantum can cause a systemic and potentially lethal disease termed visceral leishmaniasis. In the corresponding mouse model, an intravenous infection with L. infantum leads to the persistence of parasites in various organs, including bone marrow (BM). Considering the anatomical proximity between the BM and the cortical bone, we investigated whether a chronic infection with L. infantum affected bone homeostasis. Unexpectedly, chronic infection with L. infantum caused an increase in bone mass in mice. In vivo, an increased number of osteoblasts and osteocytes and a decreased maturation of osteoclasts characterized the phenotype. Confocal laser scanning fluorescence microscopy confirmed the infection of BM macrophages but also revealed the presence of parasites in osteoclasts. In vitro, mature osteoclasts took up L. infantum parasites. However, infection of osteoclast progenitors abolished their differentiation and function. In addition, secretory products of infected BM-derived macrophages inhibited the maturation of osteoclasts. Both in vitro and in vivo, infected macrophages and osteoclasts showed an enhanced expression of the anti-osteoclastogenic chemokine CCL5 (RANTES). Neutralization of CCL5 prevented the inhibition of osteoclast generation seen in the presence of culture supernatants from L. infantum-infected macrophages. Altogether, our study shows that chronic infection with Leishmania increases bone mass by inducing bone formation and impairing osteoclast differentiation and function. © 2022 American Society for Bone and Mineral Research (ASBMR).

CXCL9 Predicts the Risk of Osteoporotic Hip Fracture in a Prospective Cohort of Chinese Men-A Matched Case-Control Study.

Recent experimental work has identified CXCL9 as a promoter for the differentiation of osteoclast progenitors into osteoclasts, with resultant bone resorption. However, no human study has validated an association between this chemokine and osteoporosis or fracture risk. We conducted a matched case-control study nested in the prospective, population-based Singapore Chinese Health Study. Fifty-five men and 119 women with incident hip fractures, occurring median 6.2 years after blood collection, were matched individually to controls by age at recruitment, sex, and duration of blood storage. Serum chemokines, CXCL9 and CXCL10, were measured using immunoassays. Multivariable conditional logistic regression models that included age at blood collection, body mass index, smoking, and diabetes as covariates were used to estimate odds ratios (OR) and 95% confidence intervals (CI) for association with hip fracture risk. Predictive utility of chemokine for hip fracture risk was examined by comparing area under receiver operating characteristic curves (AUC) between prognostic models with and without the chemokine. Increasing CXCL9 levels were associated with increasing hip fracture risk in men but not in women (pinteraction =0.002); comparing extreme quartiles, the OR (95% CI) in the highest quartile was 10.35 (1.90-56.39) in men (ptrend =0.002) but 1.46 (0.59-3.60) in women (ptrend =0.32). Adding CXCL9 to a prognostic model that already incorporated age and other risk factors improved the AUC (95% CI) from 0.65 (0.55-0.76) to 0.74 (0.65-0.83) for the predictive utility of hip fractures in men but not in women. Conversely, the association between CXCL10 and hip fracture risk was not statistically significant in either sex. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Dendritic cells development into osteoclast-type APCs by 4T1 breast tumor T cells milieu boost bone consumption.

Bone metastases occur in 70% of patients with advanced breast cancer, causing severe morbidity and increased mortality due to osteolytic lesions driven by osteoclasts (OCs) inside the bone marrow (BM) microenvironment. A reciprocal vicious cycle between bone remodeling system and the tumor itself is established by the release of growth factors stored in the mineralized matrix, which in turn feed the tumor, changing tumor behavior and growth. However, BM is not a passive host microenvironment for circulating tumor cells, but instead can be actively modified by the primary tumor before metastatic spread occurs. Indeed, we have shown that T cells specific for the 4T1 mammary carcinoma cell line, are characteristically RANKL+ IL-17F+ CD4+ T cells. Those cells arrive in the BM before metastatic cells and set the pre-metastatic niche. In the absence of T cell derived RANKL, there is no pre-metastatic osteolytic disease and bone metastases do not take place. Recently, dendritic cells (DCs), the main T cell partner at the beginning of the immune response, came into the spotlight as a potential source of OCs progenitors under inflammatory conditions. Regarding bone metastasis, nothing is currently known about DCs plasticity or even its partnership with tumor induced T cells for BM pre-metastatic niche formation. Here, we show that splenic CD11c+ DCs stimulated with 4T1 conditioned media (CM) efficiently differentiated into mature and activated multinucleated giant cells (DC-OC) expressing TRAP and IL-23 cytokine. More important, 4T1 CM derived DC-OCs build a positive loop which amplifies the osteolytic phenomena by maintaining the RANKL+ Th17 T cells and by its own osteoclastic activity. In conclusion, our results indicate that differentiation of OCs from DCs may be achievable in the bone pre osteolytic disease context representing an alternative OC differentiation pathway. Besides being induced by high levels of T cells pro osteoclastogenic cytokines, especially by RANKL, DC-OC keep a positive feedback loop towards osteolysis, maintaining the pro-osteoclastogenic T cell phenotype in the BM.

Tooth extraction in mice administered zoledronate increases inflammatory cytokine levels and promotes osteonecrosis of the jaw.

Osteonecrosis of the jaw (ONJ) occurring after invasive dental treatment often adversely affects patients' activities of daily living. Long-term administration of strong anti-bone resorptive agents such as bisphosphonates prior to invasive dental treatment is considered an ONJ risk factor; however, pathological mechanisms underlying ONJ development remain unclear. We developed an ONJ mouse model in which a tooth is extracted during treatment with the bisphosphonate zoledronate. We observed induction of apoptosis in osteocytes, resulting in formation of empty lacunae in jaw bones at sites of tooth extraction but not in other bones of the same mice. We also observed elevated levels of inflammatory cytokines such as TNFα, IL-6 and IL-1 in jaw bone at the extraction site relative to other sites in zoledronate-treated mice. We also report that treatment in vitro with either zoledronate or an extract from Porphyromonas gingivalis, an oral bacteria, promotes expression of inflammatory cytokines in osteoclast progenitor cells. We demonstrate that gene-targeting of either TNFα, IL-6 or IL-1 or treatment with etanercept, a TNFα inhibitor, or a neutralizing antibody against IL-6 can antagonize ONJ development caused by combined tooth extraction and zoledronate treatment. Taken together, the cytokine storm induced by invasive dental treatment under bisphosphonate treatment promotes ONJ development due to elevated levels of inflammatory cytokine-producing cells. Our work identifies novel targets potentially useful to prevent ONJ.