Increased Osteoclast Differentiation Research Articles

Targeting EZH2 attenuates the ferroptosis-mediated osteoblast–osteoclast imbalance in rheumatoid arthritis

ObjectiveThe enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) can regulate osteogenesis and osteoclastogenesis. This study aimed to further explore the effects of EZH2 modification on ferroptosis and the osteoblast–osteoclast balance in rheumatoid arthritis (RA) in vitro and in vivo. MethodsBone marrow mesenchymal stromal cells were transfected with EZH2 overexpression (oeEZH2) and EZH2 shRNA (shEZH2) plasmids with or without ferrostatin-1 (Fer-1) treatment and subjected to an osteoblast differentiation assay. The cells were then cocultured with bone marrow-derived macrophages and subjected to an osteoclast differentiation assay. Collagen-induced arthritis (CIA) mice were generated and injected with shEZH2 adeno-associated virus (AAV). ResultsOeEZH2 repressed osteoblast differentiation, as reflected by decreased ALP and Alizarin Red S staining and increased OPN, RUNX2, OPG and RANKL; however, shEZH2 had the opposite effects. Besides, oeEZH2 promoted osteoblast ferroptosis, as suggested by increased MDA, Fe2+, ROS, and PTGS2 but decreased GPX4 and SLC7A11; these effects could be attenuated by Fer-1 treatment. In contrast, shEZH2 ameliorated osteoblast ferroptosis. OeEZH2 subsequently increased osteoclast differentiation, as indicated by increased TRAP+ multinucleated cells, NFATC1, CTSK, and c-FOS; however, shEZH2 had the opposite effect, except that it did not regulate CTSK. In CIA mice, shEZH2 AAV decreased the clinical symptom score, histological score of cartilage, and systemic inflammation (TNF-α and IL-6) and repressed bone ferroptosis and restored the osteoblast–osteoclast balance to some extent, as reflected by immunohistochemical staining of related markers. ConclusionTargeting EZH2 attenuates the ferroptosis-mediated osteoblast–osteoclast imbalance in RA, revealing its potential as a treatment target.

LOX, but not LOXL2, promotes bone metastasis formation and bone destruction in triple-negative breast cancer

The primary function of the lysyl oxidase (LOX) family, including LOX and its paralogue LOX-like (LOXL)-2, is to catalyze the covalent crosslinking of collagen and elastin in the extracellular matrix. LOX and LOXL2 are also facilitating breast cancer invasion and metastatic spread to visceral organs (lungs, liver) in vivo. Conversely, the contribution of LOX and LOXL2 to breast cancer bone metastasis remains scant. Here, using gene overexpression or silencing strategies, we investigated the role of LOX and LOXL2 on the formation of metastatic osteolytic lesions in animal models of triple negative breast cancer. In vivo, the extent of radiographic metastatic osteolytic lesions in animals injected with LOX-overexpressing [LOX(+)] tumor cells was 3-fold higher than that observed in animals bearing tumors silenced for LOX [LOX(−)]. By contrast, the extent of osteolytic lesions between LOXL2(+) and LOXL2(−) tumor-bearing animals did not differ, and was comparable to that observed with LOX(−) tumor-bearing animals. In situ, TRAP staining of bone tissue sections from the hind limbs of LOX(+) tumor-bearing animals was substantially increased compared to LOX(−), LOXL2(+) and LOXL2(−)-tumor-bearing animals, which was indicative of enhanced active-osteoclast resorption. In vitro, tumor-secreted LOX increased osteoclast differentiation induced by RANKL, whereas LOXL2 seemed to counteract LOX’s pro-osteoclastic activity. Furthermore, LOX (but not LOXL2) overexpression in tumor cells induced a robust production of IL-6, the latter being a pro-osteoclastic cytokine. Based on these findings, we propose a model in which LOX and IL-6 secreted from tumor cells act in concert to enhance osteoclast-mediated bone resorption that, in turn, promotes metastatic bone destruction in vivo.

The effects of preosteoblast-derived exosomes on macrophages and bone in mice.

The effect of preosteoblast-derived exosomes on bone marrow macrophages (BMMΦ) and calvarial osteoblasts (cOB) was evaluated invitro, and bone formation studies were performed invivo in mice. Preosteoblastic MC3T3-E1 clone 4 (MC4) cell-derived exosomes (MC4exo) were characterized with particle tracking, transmission electron microscopy and western blot analysis to validate size, number, shape and phenotypic exosome markers. Exosomes pre-labelled with PKH67 were incubated with BMMΦ and phagocytosis of exosomes was confirmed. To examine the effect of MC4exo on macrophage polarization, BMMΦ were treated with MC4exo and the expression of pro- and anti-inflammatory cytokines was determined by qPCR. MC4exo treatment upregulated mRNA expression of Cd86, Il1β, Ccl2, Rankl and Nos, and downregulated Cd206, Il10 and Tnfα, suggesting a shift towards pro-inflammatory 'M1-like' macrophage polarization. Combination of RANKL and MC4exo increased osteoclast differentiation of BMMΦ in comparison to RANKL alone as analysed by TRAP staining. MC4exo treatment showed no significant effect on calvarial osteoblast mineralization. For invivo studies, intratibial inoculation of MC4exo (2 × 109 particles in PBS, n = 12) and vehicle control (PBS only, n = 12) was performed in C57Bl/6 mice (8 weeks, male). Micro-CT analyses of the trabecular and cortical bone compartments were assessed at 4 weeks post-injection. Tibial sections were stained for TRAP activity to determine osteoclast presence and immunofluorescence staining was performed to detect osteocalcin (Ocn), osterix (Osx) and F4/80 expression. Intratibial inoculation of MC4exo increased the diaphyseal bone mineral density and trabecular bone volume fraction due to increased trabecular number. This increase in bone was accompanied by a reduction in bone marrow macrophages and osteoclasts at the experimental endpoint. Together, these findings suggest that preosteoblast-derived exosomes enhanced bone formation by influencing macrophage responses.

Bone marrow-derived exosomes promote inflammation and osteoclast differentiation in high-turnover renal osteodystrophy

Introduction Renal osteodystrophy (ROD) is a type of bone metabolic disorder in patients with chronic kidney disease (CKD). Inflammation is associated with bone loss in ROD. However, its precise mechanism has not yet been elucidated. The present study was conducted to investigate whether exosomes (Exos) in bone marrow (BM) are involved in the pathogenesis of high-turnover ROD. Methods Bone mass, osteoclast number, and pro-inflammatory cytokines levels of BM supernatant were detected in adenine-induced ROD rats. The effect of Exos derived from BM (BM-Exos) of ROD (ROD-Exos) on inflammatory genes and osteoclast differentiation of BM-derived macrophages (BMMs) were further examined. Then, exosomal miRNA sequencing was performed and an miRNA-mRNA-pathway network was constructed. Results we found increased osteoclasts and decreased bone mass in ROD rats, as well as inflammatory activation in the BM niche. Furthermore, BMMs from ROD rats displayed overproduction of proinflammatory cytokines and increased osteoclast differentiation, accompanied by nuclear factor κB (NF-κB) signaling activation. Mechanistically, we found that ROD-Exos activates NF-κB signaling to promote the release of proinflammatory cytokines and increase osteoclast differentiation of BMMs. Meanwhile, a total of 24 differentially expressed miRNAs were identified between BM-Exos from ROD and normal control (NC). The miRNA-mRNA-pathway network suggests that rno-miR-9a-5p, rno-miR-133a-3p, rno-miR-30c-5p, rno-miR-206-3p, and rno-miR-17-5p might play pivotal roles in inflammation and osteoclast differentiation. Additionally, we validated that the expression of miR-9a-5p is upregulated in ROD-Exos. Conclusion The BM niche of ROD alters the miRNA cargo of BM-Exos to promote inflammation and osteoclast differentiation of BMMs, at least partially contributing to the pathogenesis of high-turnover ROD.

Deletion of NADPH oxidase 2 in chondrocytes exacerbates ethanol-mediated growth plate disruption in mice without major effects on bone architecture or gene expression.

Excess reactive oxygen species generated by NADPH oxidase 2 (Nox2) in response to ethanol exposure mediate aspects of skeletal toxicity including increased osteoclast differentiation and activity. Because perturbation of chondrocyte differentiation in the growth plate by ethanol could be prevented by dietary antioxidants, we hypothesized that Nox2 in the growth plate was involved in ethanol-associated reductions in longitudinal bone growth. Nox2 conditional knockout mice were generated, where the essential catalytic subunit of Nox2, cytochrome B-245 beta chain (Cybb), is deleted in chondrocytes using a Cre-Lox model with Cre expressed from the collagen 2a1 promoter (Col2a1-Cre). Wild-type and Cre-Lox mice were fed an ethanol Lieber-DeCarli-based diet or pair-fed a control diet for 8 weeks. Ethanol treatment significantly reduced the number of proliferating chondrocytes in the growth plate, enhanced bone marrow adiposity, shortened femurs, reduced body length, reduced cortical bone volume, and decreased mRNA levels of a number of osteoblast and chondrocyte genes. Conditional knockout of Nox2 enzymatic activity in chondrocytes did not consistently prevent any ethanol effects. Rather, knockout mice had fewer proliferating chondrocytes than wild-type mice in both the ethanol- and control-fed animals. Additional analysis of tibia samples from Nox4 knockout mice showed that loss of Nox4 activity also reduced the number of proliferating chondrocytes and altered chondrocyte size in the growth plate. Although Nox enzymatic activity regulates growth plate development, ethanol-associated disruption of the growth plate morphology is independent of ethanol-mediated increases in Nox2 activity.

Accelerated Bone Loss in Transgenic Mice Expressing Constitutively Active TGF-β Receptor Type I.

Transforming growth factor beta (TGF-β) is a key factor mediating the intercellular crosstalk between the hematopoietic stem cells and their microenvironment. Here, we investigated the skeletal phenotype of transgenic mice expressing constitutively active TGF-β receptor type I under the control of Mx1-Cre (Mx1;TβRICA mice). μCT analysis showed decreased cortical thickness, and cancellous bone volume in both femurs and mandibles. Histomorphometric analysis confirmed a decrease in cancellous bone volume due to increased osteoclast number and decreased osteoblast number. Primary osteoblasts showed decreased ALP and mineralization. Constitutive TβRI activation increased osteoclast differentiation. qPCR analysis showed that Tnfsf11/Tnfrsf11b ratio, Ctsk, Sufu, and Csf1 were increased whereas Runx2, Ptch1, and Ptch2 were decreased in Mx1;TβRICA femurs. Interestingly, Gli1, Wnt3a, Sp7, Alpl, Ptch1, Ptch2, and Shh mRNA expression were reduced whereas Tnfsf11/Tnfrsf11b ratio was increased in Mx1;TβRICA mandibles. Similarly, osteoclast-related genes were increased in Mx1;TβRICA osteoclasts whereas osteoblast-related genes were reduced in Mx1;TβRICA osteoblasts. Western blot analysis indicated that SMAD2 and SMAD3 phosphorylation was increased in Mx1;TβRICA osteoblasts, and SMAD3 phosphorylation was increased in Mx1;TβRICA osteoclasts. CTSK was increased while RUNX2 and PTCH1 was decreased in Mx1;TβRICA mice. Microindentation analysis indicated decreased hardness in Mx1;TβRICA mice. Our study indicated that Mx1;TβRICA mice were osteopenic by increasing osteoclast number and decreasing osteoblast number, possibly by suppressing Hedgehog signaling pathways.

EGFL6 promotes bone metastasis of lung adenocarcinoma by increasing cancer cell malignancy and bone resorption.

Lung adenocarcinoma is the most common and aggressive type of lung cancer with the highest incidence of bone metastasis. Epidermal growth factor-like domain multiple 6 (EGFL6) is an exocrine protein, and the expression of EGFL6 is correlated with survival of patient with lung adenocarcinoma. However, the association between EGFL6 expression in lung adenocarcinoma and bone metastasis has not been investigated. In this study, we found that EGFL6 levels in lung adenocarcinoma tissues correlate with bone metastasis and TNM stages in surgical patients. In vitro, overexpression of EGFL6 in lung adenocarcinoma cells promoted their proliferation, migration, and invasion ability compared with control by enhancing EMT process and activating Wnt/β-catenin and PI3K/AKT/mTOR pathways. In the nude mouse model, overexpression of EGFL6 enhanced tumor growth and caused greater bone destruction. Moreover, the exocrine EGFL6 of human lung adenocarcinoma cells increased osteoclast differentiation of bone marrow mononuclear macrophages (BMMs) of mice via the NF-κB and c-Fos/NFATc1 signaling pathways. However, exocrine EGFL6 had no effect on osteoblast differentiation of bone marrow mesenchymal stem cells (BMSCs). In conclusion, high expression of EGFL6 in lung adenocarcinomas is associated with bone metastasis in surgical patients. The underlying mechanism may be the increased metastatic properties of lung adenocarcinoma cells with high EGFL6 level and the enhanced osteoclast differentiation and bone resorption by exocrine EGFL6 from tumors. Therefore, EGFL6 is a potential therapeutic target to reduce the ability of lung adenocarcinomas to grow and metastasize and to preserve bone mass in patients with bone metastases from lung adenocarcinomas.

Geranylgeraniol Application in Human Osteoblasts and Osteoclasts for Reversal of the Effect of Bisphosphonates.

Nitrogen-containing bisphosphonates lead to the depletion of geranylgeranyl pyrophosphate involved in the mevalonate pathway. The effect of geranylgeraniol (GGOH) on human osteoblast and osteoclast activities suppressed by zoledronate was investigated in this study. The effect of GGOH on human osteoblasts and osteoclasts subjected to treatment with zoledronate was analyzed by assessing cell viability, osteoclast differentiation, resorption ability, gene expression, and protein synthesis. Cell viability suppressed by bisphosphonates in osteoblasts and osteoprogenitor cells was restored with GGOH. Osteoclast differentiation was analyzed by vitronectin receptor immunofluorescence staining, and the addition of GGOH to zoledronate significantly increased osteoclast differentiation compared with zoledronate alone. A trend of reversal of osteoclast resorption by GGOH was observed; however, it was not significant in all groups. The expression of ALP, type 1 collagen, and RUNX2 in osteoblasts was recovered by the addition of GGOH. Only CALCR expression in osteoclasts was significantly recovered by GGOH addition in the zoledronate group. Although the activities of osteoblasts and osteoclasts were not entirely restored, the possibility that the topical application of GGOH in MRONJ patients or patients with dental problems and bisphosphonates might lessen the risk of development and recurrence of MRONJ is shown.

ADSC-derived exosomes inhibit cbl-cathepsin K-dependent osteoclast activation in inflammatory arthritis

Abstract Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes joint inflammation and bone destruction due to increased osteoclast differentiation and activity. Adipose-derived stem cells (ADSCs) are used to treat tissue injury because they are easy to harvest and exhibit immunomodulatory and regenerative capacity. Previous studies demonstrated that ADSC-derived exosomes (ADSC-Exo) protect articular cartilage from damage and exert anti-inflammation effects. Thus, we investigated the underlying mechanisms of ADSC-Exo in regulating osteoclast activation in inflammatory arthritis. RANKL is a TNF-family cytokine that binds to the RANK required for mature osteoclasts. However, whether ADSC-Exo is functionally involved in the inhibition of osteoclast formation via regulating RANK-mediated signaling in these pathological conditions is unclear. Our data revealed that ADSC-Exo ameliorated the RA severity and bone loss in vivo and inhibited osteoclast differentiation, RANK downstream signaling, NFATc1, cathepsin K, and cbl-b. Moreover, ADSC-Exo could significantly suppress the population of RANKL-expressing TH17 cells. In brief, ADSC-Exo protects articular cartilage and bone destruction from damage and ameliorates gait abnormality in CIA rats, which may be related to the inhibition of the RANK-signaling pathway in osteoclasts. As it is relatively feasible to obtain human adipose tissue, ADSC-Exo may be a potential therapy for RA. This study could comprehensively understand the immunomodulatory effects of ADSC-Exo on suppressing osteoclastogenesis. This finding is critical for understanding the bone homeostasis modulated by MSC-Exo and provides biological evidence of ADSC-Exo in inflammatory arthritis. Supported by grants from the Ministry of Science and Technology (111-2321-B-418-002, 111-2314-B-418-004), FEMH (2022-C-042 and 2022-C-123), NYCU-FEMH Cooperation grants (111DN12, 111DN22), NTUH-FEMH Cooperation (111-FTN0012).

Effect of Bifidobacterium on osteoclasts: TNF-α/NF-κB inflammatory signal pathway-mediated mechanism.

Osteoporosis is a systemic multifactorial bone disease characterized by low bone quality and density and bone microstructure damage, increasing bone fragility and fracture vulnerability. Increased osteoclast differentiation and activity are important factors contributing to bone loss, which is a common pathological manifestation of bone diseases such as osteoporosis. TNF-a/NF-κB is an inflammatory signaling pathway with a key regulatory role in regulating osteoclast formation, and the classical pathway RANKL/RANK/OPG assists osteoclast formation. Activation of this inflammatory pathway promotes the formation of osteoclasts and accelerates the process of osteoporosis. Recent studies and emerging evidence have consistently demonstrated the potential of probiotics to modulate bone health. Secretions of Bifidobacterium, a genus of probiotic bacteria in the phylum Actinobacteria, such as short-chain fatty acids, equol, and exopolysaccharides, have indicated beneficial effects on bone health. This review discusses the molecular mechanisms of the TNF-a/NF-κB inflammatory pathway in regulating osteoclast formation and describes the secretions produced by Bifidobacterium and their potential effects on bone health through this pathway, opening up new directions for future research.

Circadian Rhythm Disorders Aggravate Periodontitis by Modulating BMAL1

Circadian rhythms regulate the body's homeostasis through the temporal control of tissue-specific circadian rhythm control genes. Circadian rhythm disorders (CRD) affect the expression levels of circadian rhythms-associated genes in brain and muscle aryl hydrocarbon receptor nuclear translocator-like-1(BMAL1), which is thought to contribute to metabolic disorders and an altered immune system. However, the relationship between CRD and the development of periodontitis was poorly reported. Therefore, this study aimed to investigate the role played by BMAL1 in periodontitis. We used a modified multi-platform approach (MMPM) to induce circadian rhythm disturbances in rats to investigate the role of BMAL1 in periodontitis. Our results showed significant downregulation of BMAL1 in the CRD with periodontitis group, significant resorption of alveolar bone, increased osteoclast differentiation, and upregulation of the inflammatory signaling molecule NF-κB. In addition, apoptosis and oxidative stress levels were increased in periodontal tissues. Collectively, our study suggests that BMAL1 is a key regulator in periodontitis exacerbated by CRD and that CRD may lead to the downregulation of BMAL1, thereby exacerbating oxidative stress and apoptosis in periodontal tissues. Our study found that BMAL1 may be associated with the progression of periodontitis and provides a new perspective on the treatment of periodontitis.

HSC70 mediated autophagic degradation of oxidized PRL2 is responsible for osteoclastogenesis and inflammatory bone destruction.

Inflammation leads to systemic osteoporosis or local bone destruction, however, the underlying molecular mechanisms are still poorly understood. In this study, we report that PRL2 is a negative regulator of osteoclastogenesis and bone absorption. Mice with PRL2 deficiency exhibit a decrease in bone volume and an increase in osteoclast numbers. PRL2 negatively regulates RANKL-induced reactive oxygen species production through the activation of RAC1, thus PRL2 deficient osteoclast precursors have both increased osteoclast differentiation ability and bone resorptive capacity. During inflammation, oxidized PRL2 is a selected substrate of HSC70 and conditions of oxidative stress trigger rapid degradation of PRL2 by HSC70 mediated endosomal microautophagy and chaperone-mediated autophagy. Ablation of PRL2 in mouse models of inflammatory bone disease leads to an increase in the number of osteoclasts and exacerbation of bone damage. Moreover, reduced PRL2 protein levels in peripheral myeloid cells are highly correlated with bone destruction in a mouse arthritis model and in human rheumatoid arthritis, while the autophagy inhibitor hydroxychloroquine blocked inflammation-induced PRL2 degradation and bone destruction in vivo. Therefore, our findings identify PRL2 as a new regulator in osteoimmunity, providing a link between inflammation and osteoporosis. As such, PRL2 is a potential therapeutic target for inflammatory bone disease and inhibition of HSC70 mediated autophagic degradation of PRL2 may offer new therapeutic tools for the treatment of inflammatory bone disease.

Effects of extracellular vesicles derived from oral bacteria on osteoclast differentiation and activation

Dysbiosis of the oral microbiota plays an important role in the progression of periodontitis, which is characterized by chronic inflammation and alveolar bone loss, and associated with systemic diseases. Bacterial extracellular vesicles (EVs) contain various bioactive molecules and show diverse effects on host environments depending on the bacterial species. Recently, we reported that EVs derived from Filifactor alocis, a Gram-positive periodontal pathogen, had osteoclastogenic activity. In the present study, we analysed the osteoclastogenic potency and immunostimulatory activity of EVs derived from the Gram-negative periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia, the oral commensal bacterium Streptococcus oralis, and the gut probiotic strain Lactobacillus reuteri. Bacterial EVs were purified by density gradient ultracentrifugation using OptiPrep (iodixanol) reagent. EVs from P. gingivalis, T. forsythia, and S. oralis increased osteoclast differentiation and osteoclstogenic cytokine expression in osteoclast precursors, whereas EVs from L. reuteri did not. EVs from P. gingivalis, T. forsythia, and S. oralis preferentially activated Toll-like receptor 2 (TLR2) rather than TLR4 or TLR9, and induced osteoclastogenesis mainly through TLR2. The osteoclastogenic effects of EVs from P. gingivalis and T. forsythia were reduced by both lipoprotein lipase and polymyxin B, an inhibitor of lipopolysaccharide (LPS), while the osteoclastogenic effects of EVs from S. oralis were reduced by lipoprotein lipase alone. These results demonstrate that EVs from periodontal pathogens and oral commensal have osteoclastogenic activity through TLR2 activation by lipoproteins and/or LPS.

TLR9 Mediates Periodontal Aging by Fostering Senescence and Inflammaging

TLR9 is a critical nucleic acid sensing receptor in mediating periodontitis and periodontitis-associated comorbidities. Emerging evidence implicates TLR9 as a key sensor during aging, although its participation in periodontal aging is unexplored. Here, we investigated whether TLR9-mediated host responses can promote key hallmarks of aging, inflammaging, and senescence, in the course of periodontitis using a multipronged approach comprising clinical and preclinical studies. In a case-control model, we found increased TLR9 gene expression in gingival tissues of older (≥55 y) subjects with periodontitis compared to older healthy subjects as well as those who are younger (<55 y old) with and without the disease. Mechanistically, this finding was supported by an in vivo model in which wild-type (WT) and TLR9–/– mice were followed for 8 to 10 wk (young) and 18 to 22 mo (aged). In this longitudinal model, aged WT mice developed severe alveolar bone resorption when compared to their younger counterpart, whereas aged TLR9–/– animals presented insignificant bone loss when compared to the younger groups. In parallel, a boosted inflammaging milieu exhibiting higher expression of inflammatory/osteoclast mediators (Il-6, Rankl, Cxcl8) and danger signals (S100A8, S100A9) was noted in gingival tissues of aged WT mice compared to the those of aged TLR9–/– mice. Consistently, WT aged mice displayed an increase in prosenescence balance as measured by p16 INK4a /p19 ARF ratio compared to the younger groups and aged TLR9–/– animals. Ex vivo experiments with bone marrow–derived macrophages primed by TLR9 ligand (ODN 1668) further corroborated in vivo and clinical data and showed enhanced inflammatory-senescence circuit followed by increased osteoclast differentiation. Together, these findings reveal first systematic evidence implicating TLR9 as one of the drivers of periodontitis during aging and functioning by boosting a deleterious inflammaging/senescence environment. This finding calls for further investigations to determine whether targeting TLR9 will improve periodontal health in an aging population.

Dynamic changes in O-GlcNAcylation regulate osteoclast differentiation and bone loss via nucleoporin 153

Bone mass is maintained by the balance between osteoclast-induced bone resorption and osteoblast-triggered bone formation. In inflammatory arthritis such as rheumatoid arthritis (RA), however, increased osteoclast differentiation and activity skew this balance resulting in progressive bone loss. O-GlcNAcylation is a posttranslational modification with attachment of a single O-linked β-D-N-acetylglucosamine (O-GlcNAc) residue to serine or threonine residues of target proteins. Although O-GlcNAcylation is one of the most common protein modifications, its role in bone homeostasis has not been systematically investigated. We demonstrate that dynamic changes in O-GlcNAcylation are required for osteoclastogenesis. Increased O-GlcNAcylation promotes osteoclast differentiation during the early stages, whereas its downregulation is required for osteoclast maturation. At the molecular level, O-GlcNAcylation affects several pathways including oxidative phosphorylation and cell-cell fusion. TNFα fosters the dynamic regulation of O-GlcNAcylation to promote osteoclastogenesis in inflammatory arthritis. Targeted pharmaceutical or genetic inhibition of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) arrests osteoclast differentiation during early stages of differentiation and during later maturation, respectively, and ameliorates bone loss in experimental arthritis. Knockdown of NUP153, an O-GlcNAcylation target, has similar effects as OGT inhibition and inhibits osteoclastogenesis. These findings highlight an important role of O-GlcNAcylation in osteoclastogenesis and may offer the potential to therapeutically interfere with pathologic bone resorption.

Conditioned medium of amniotic fluid-derived stromal cells exerts a bone anabolic effect by enhancing progenitor population and angiogenesis.

A cell-free approach utilizing the paracrine effects of mesenchymal stromal cells is receiving attention in regenerative medicine. In the present study, we evaluated the effects of a conditioned medium of amniotic fluid-derived stromal cells (AFSC-CM) on bone metabolism. In mice, intraperitoneal injections of AFSC-CM increased bone mass and enhanced bone turnover. The precursor populations of myeloid and mesenchymal lineages, as well as endothelial cells in bone marrow, were also augmented by AFSC-CM administration. In an in vitro culture experiment, AFSC-CM increased osteoclast differentiation of bone marrow-derived macrophages, but had no significant effect on the osteogenic differentiation of preosteoblasts. However, AFSC-CM administration dramatically accelerated the migration and tube formation of endothelial cells, and a cytokine array showed that AFSC-CM contained many angiogenic factors. These results indicate that AFSC-CM exerts a bone anabolic effect by changing the bone marrow microenvironment, including angiogenesis and precursor expansion. Therefore, ameliorating marrow angiogenesis is a potential therapeutic strategy for bone regeneration, for which AFSCs can be a good cellular source.

Toxic Effects of Indoxyl Sulfate on Osteoclastogenesis and Osteoblastogenesis.

Uremic toxins, such as indoxyl sulfate (IS) and kynurenine, accumulate in the blood in the event of kidney failure and contribute to further bone damage. To maintain the homeostasis of the skeletal system, bone remodeling is a persistent process of bone formation and bone resorption that depends on a dynamic balance of osteoblasts and osteoclasts. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates the toxic effects of uremic toxins. IS is an endogenous AhR ligand and is metabolized from tryptophan. In osteoclastogenesis, IS affects the expression of the osteoclast precursor nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) through AhR signaling. It is possible to increase osteoclast differentiation with short-term and low-dose IS exposure and to decrease differentiation with long-term and/or high-dose IS exposure. Coincidentally, during osteoblastogenesis, through the AhR signaling pathway, IS inhibits the phosphorylation of ERK, and p38 reduces the expression of the transcription factor 2 (Runx2), disturbing osteoblastogenesis. The AhR antagonist resveratrol has a protective effect on the IS/AhR pathway. Therefore, it is necessary to understand the multifaceted role of AhR in CKD, as knowledge of these transcription signals could provide a safe and effective method to prevent and treat CKD mineral bone disease.

Discovering Myeloid Cell Heterogeneity in Mandibular Bone - Cell by Cell Analysis.

The myeloid-derived bone marrow progenitor populations from different anatomical locations are known to have diverse osteoclastogenesis potential. Specifically, myeloid progenitors from the tibia and femur have increased osteoclast differentiation potential compared to myeloid progenitors from the alveolar process. In this study, we explored the differences in the myeloid lineage progenitor cell populations in alveolar (mandibular) bone versus long (femur) bone using flow cytometry and high-throughput single cell RNA sequencing (scRNA-seq) to provide a comprehensive transcriptional landscape. Results indicate that mandibular bone marrow-derived cells exhibit consistent deficits in myeloid differentiation, including significantly fewer myeloid-derived suppressor cell (MDSC)-like populations (CD11b+Ly6C+, CD11b+Ly6G+), as well as macrophages (CD11b+F4/80+). Although significantly fewer in number, MDSCs from mandibular bone exhibited increased immunosuppressive activity compared to MDSCs isolated from long bone. Using flow cytometry panels specific for bone marrow progenitors, analysis of hematopoietic stem cells showed no defects in mandibular bone marrow in LSK (Lin–Sca1+cKit+) cell and LK (Lin–Sca1–cKit+) cell populations. While there was no significant difference in granulocyte progenitors, the granulocyte-monocyte progenitors and monocyte progenitor population were significantly decreased in the mandibular bone marrow. T-lymphocyte subsets were not significantly different between mandibular and femoral bone, except for CD4+CD25+Foxp3+ regulatory T lymphocytes, which were significantly increased in the mandible. In addition, B lymphocytes were significantly increased in mandible. Single cell RNA sequencing from mandible and femur BM revealed distinct differences in transcriptomic profiles in myeloid populations establishing previously unappreciated aspects of mandibular bone marrow populations. These analyses reveal site-specific differences in the myeloid progenitor cellular composition and transcriptional programs providing a deeper appreciation of the complex differences in myeloid cell heterogeneity from different anatomical bone marrow sites.

C-Reactive Protein Promotes Bone Destruction in Human Myeloma By Stimulating Myeloma Cell Production of Osteolytic Cytokines Via the CD32/FcγRII-p38MAPK-Twist Axis

More than 80% of patients with multiple myeloma develop bone destruction that causes pathological fractures, severe bone pain, spinal cord compression, and hypercalcemia. The normally balanced act between bone-resorbing osteoclasts and bone-forming osteoblasts is disrupted in those patients. It is well established that myeloma cells mediate bone destruction by activating osteoclasts. Studies have been shown that myeloma cells produce a number of cytokines such as nuclear factor kappa-B ligand (RANKL), macrophage inflammatory protein (MIP)-1α, and monocyte chemoattractant protein (MCP)-1, which increase osteoclast differentiation and bone resorption activity. Yet the mechanism how myeloma cells are regulated to do so is unknown. Here we show that C-reactive protein (CRP), a protein secreted in elevated amounts by hepatocytes in response to myeloma-derived cytokines, may be responsible for activating myeloma cells to promote osteoclastogenesis and inducing bone destruction in vivo. Using both SCID and SCID-hu models, we found that injection of human CRP into myeloma-bearing, but not myeloma-free, mice accelerated the induction of bone destruction in both murine and human bones, and significantly more osteoclasts were detected in the bone-tumor interface in CRP-injected mice as compared with controls. Mechanistic studies show that CRP binds to surface CD32/FcγRII, activates p38MAPK-twist pathways, and upregulates the secretion of osteolytic cytokines by myeloma cells. Furthermore, clinical studies revealed a highly positive correlation between the level of serum CRP and the number of osteolytic bone lesions in newly diagnosed myeloma patients. These findings establish a novel mechanism by which myeloma cells are activated to promote bone destruction in patients and suggest that CRP may be targeted to prevent or treat myeloma-associated bone disease. DisclosuresYang:Poseida Therapuetics: Research Funding; Cellectis: Research Funding. Orlowski:BioTheryX: Consultancy, Membership on an entity's Board of Directors or advisory committees.

S100 Calcium-Binding Protein P Secreted from Megakaryocytes Promotes Osteoclast Maturation.

Megakaryocytes (MKs) differentiate from hematopoietic stem cells and produce platelets at the final stage of differentiation. MKs directly interact with bone cells during bone remodeling. However, whether MKs are involved in regulating bone metabolism through indirect regulatory effects on bone cells is unclear. Here, we observed increased osteoclast differentiation of bone marrow-derived macrophages (BMMs) cultured in MK-cultured conditioned medium (MK CM), suggesting that this medium contains factors secreted from MKs that affect osteoclastogenesis. To identify the MK-secreted factor, DNA microarray analysis of the human leukemia cell line K562 and MKs was performed, and S100 calcium-binding protein P (S100P) was selected as a candidate gene affecting osteoclast differentiation. S100P was more highly expressed in MKs than in K562 cells, and showed higher levels in MK CM than in K562-cultured conditioned medium. In BMMs cultured in the presence of recombinant human S100P protein, osteoclast differentiation was promoted and marker gene expression was increased. The resorption area was significantly larger in S100P protein-treated osteoclasts, demonstrating enhanced resorption activity. Overall, S100P secreted from MKs promotes osteoclast differentiation and resorption activity, suggesting that MKs indirectly regulate osteoclast differentiation and activity through the paracrine action of S100P.