Increased Osteoclast Formation Research Articles

Unraveling ferroptosis in osteogenic lineages: implications for dysregulated bone remodeling during periodontitis progression

Periodontitis is a highly prevalent disease characterized by inflammation and destruction of tooth-supporting tissues that leads to tooth loss in extreme situations. Elucidating the underlying mechanisms of periodontitis pathogenesis and progression will establish the groundwork for developing effective treatment strategies. Recently, evidence concerning the role of ferroptosis in periodontitis progression has emerged. Osteogenic lineage cells are key regulators of bone remodeling. Osteogenic cell death, as observed in experimental periodontitis models, disrupts the balance between bone resorption and bone formation. However, whether the osteogenic lineage undergoes ferroptosis during periodontitis and the corresponding effect on periodontitis progression remain elusive. Here, we investigated cell-specific ferroptosis within the alveolar bone in a murine periodontitis model. Through immunofluorescence double staining and immunohistochemistry, we identified ferroptotic osteocytes and osteoblasts in inflammatory alveolar bone. Next, in vivo administration of erastin or liproxstatin-1 was conducted to either induce or inhibit ferroptosis, respectively. Severe bone resorption and inflammation, accompanied by increased osteoclast formation and impaired osteogenic potential were detected following ferroptosis activation. Subsequently, we carried out in vitro experiments on osteocytes and further verified that ferroptosis enhanced the osteocytic expression of RANKL and IL-6. These findings suggest that ferroptosis occurring within the osteogenic lineage acts as a catalyst in the progression of periodontitis by stimulating osteoclastogenesis through the secretion of inflammatory cytokines and inhibiting osteoblastic function, providing insights into ferroptosis-induced alterations in microenvironment-based intercellular communication. Ferroptosis is a promising target for controlling inflammation and preventing bone resorption in periodontitis.

Glycogen Synthase Kinase-3 Beta (GSK3β) as a Potential Drug Target in Regulating Osteoclastogenesis: An Updated Review on Current Evidence.

Glycogen synthase kinase 3-beta (GSK3β) is a highly conserved protein kinase originally involved in glucose metabolism, insulin activity, and energy homeostasis. Recent scientific evidence demonstrated the significant role of GSK3β in regulating bone remodelling through involvement in multiple signalling networks. Specifically, the inhibition of GSK3β enhances the conversion of osteoclast progenitors into mature osteoclasts. GSK3β is recognised as a pivotal regulator for the receptor activator of nuclear factor-kappa B (RANK)/receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG), phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT), nuclear factor-kappa B (NF-κB), nuclear factor-erythroid 2-related factor 2 (NRF2)/Kelch-like ECH-associated protein 1 (KEAP1), canonical Wnt/beta (β)-catenin, and protein kinase C (PKC) signalling pathways during osteoclastogenesis. Conversely, the inhibition of GSK3β has been shown to prevent bone loss in animal models with complex physiology, suggesting that the role of GSK3β may be more significant in bone formation than bone resorption. Divergent findings have been reported regarding the efficacy of GSK3β inhibitors as bone-protecting agents. Some studies demonstrated that GSK3β inhibitors reduced osteoclast formation, while one study indicated an increase in osteoclast formation in RANKL-stimulated bone marrow macrophages (BMMs). Given the discrepancies observed in the accumulated evidence, further research is warranted, particularly regarding the use of GSK3β silencing or overexpression models. Such efforts will provide valuable insights into the direct impact of GSK3β on osteoclastogenesis and bone resorption.

Loss-of-function OGFRL1 variants identified in autosomal recessive cherubism families.

Cherubism (OMIM 118400) is a rare craniofacial disorder in children characterized by destructive jawbone expansion due to the growth of inflammatory fibrous lesions. Our previous studies have shown that gain-of-function mutations in SH3 domain-binding protein 2 (SH3BP2) are responsible for cherubism and that a knock-in mouse model for cherubism recapitulates the features of cherubism, such as increased osteoclast formation and jawbone destruction. To date, SH3BP2 is the only gene identified to be responsible for cherubism. Since not all patients clinically diagnosed with cherubism had mutations in SH3BP2, we hypothesized that there may be novel cherubism genes and that these genes may play a role in jawbone homeostasis. Here, using whole exome sequencing, we identified homozygous loss-of-function variants in the opioid growth factor receptor like 1 (OGFRL1) gene in 2 independent autosomal recessive cherubism families from Syria and India. The newly identified pathogenic homozygous variants were not reported in any variant databases, suggesting that OGFRL1 is a novel gene responsible for cherubism. Single cell analysis of mouse jawbone tissue revealed that Ogfrl1 is highly expressed in myeloid lineage cells. We generated OGFRL1 knockout mice and mice carrying the Syrian frameshift mutation to understand the in vivo role of OGFRL1. However, neither mouse model recapitulated human cherubism or the phenotypes exhibited by SH3BP2 cherubism mice under physiological and periodontitis conditions. Unlike bone marrow-derived M-CSF-dependent macrophages (BMMs) carrying the SH3BP2 cherubism mutation, BMMs lacking OGFRL1 or carrying the Syrian mutation showed no difference in TNF-ɑ mRNA induction by LPS or TNF-ɑ compared to WT BMMs. Osteoclast formation induced by RANKL was also comparable. These results suggest that the loss-of-function effects of OGFRL1 in humans differ from those in mice and highlight the fact that mice are not always an ideal model for studying rare craniofacial bone disorders.

Murine IRF8 Mutation Offers New Insight into Osteoclast and Root Resorption

Interferon regulatory factor 8 (IRF8), a transcription factor expressed in immune cells, functions as a negative regulator of osteoclasts and helps maintain dental and skeletal homeostasis. Previously, we reported that a novel mutation in the IRF8 gene increases susceptibility to multiple idiopathic cervical root resorption (MICRR), a form of tooth root resorption mediated by increased osteoclast activity. The IRF8 G388S variant in the highly conserved C-terminal motif is predicted to alter the protein structure, likely impairing IRF8 function. To investigate the molecular basis of MICRR and IRF8 function in osteoclastogenesis, we generated Irf8 knock-in (KI) mice using CRISPR/Cas9 technique modeling the human IRF8 G388S mutation. The heterozygous (Het) and homozygous (Homo) Irf8 KI mice showed no gross morphological defects, and the development of hematopoietic cells was unaffected and similar to wild-type (WT) mice. The Irf8 KI Het and Homo mice showed no difference in macrophage gene signatures important for antimicrobial defenses and inflammatory cytokine production. Consistent with the phenotype observed in MICRR patients, Irf8 KI Het and Homo mice demonstrated significantly increased osteoclast formation and resorption activity in vivo and in vitro when compared to WT mice. The oral ligature–inserted Het and Homo mice displayed significantly increased root resorption and osteoclast-mediated alveolar bone loss compared to WT mice. The increased osteoclastogenesis noted in KI mice is due to the inability of IRF8 G388S mutation to inhibit NFATc1-dependent transcriptional activation and downstream osteoclast specific transcripts, as well as its impact on autophagy-related pathways of osteoclast differentiation. This translational study delineates the IRF8 domain important for osteoclast function and provides novel insights into the IRF8 mutation associated with MICRR. IRF8 G388S mutation mainly affects osteoclastogenesis while sparing immune cell development and function. These insights extend beyond oral health and significantly advance our understanding of skeletal disorders mediated by increased osteoclast activity and IRF8’s role in osteoclastogenesis.

In Vitro Analysis of Bone Remodeling Alterations in Hemophilia: Influence of Coagulation Factors on Bone Cells

Background. Today, one of the main comorbidities affecting the quality of life of the haemophilia patient is bone pathology. Hemophilia A (HA) is associated with reduced bone mass and mineral density (BMD). Due to the rarity of the disease and the heterogeneity among the studies, pathogenesis of bone loss is still under investigation and both processes of bone resorption and bone formation could be hypothesized to be altered. FVIII deficiency could alter BMD directly affecting bone cells or indirectly by decreasing thrombin generation. The reduction of BMD seems due to perturbations of the Receptor Activator of Nuclear factor-B RANK Ligand (RANKL) and osteoprotegerin (OPG) pathways. In haemophilia alterations of bone remodeling, osteoclasts seem to play a major role. Osteoclasts are multinucleated cells, which derive from the CD14+ monocyte/macrophage lineage. Until recently, the identity of osteoclast progenitors has not been well defined, but evidences report that CD16−CD14+ rather than CD16+CD14+ monocytes were prone to differentiate into osteoclasts (Komano et al. Arthritis Res Ther. 2006). Objectives. To dissect the mechanism of bone loss in hemophilia, we studied the effects of coagulation factors, such as factor VIII, von Willebrand factor (VWF), activated factor X (FXa) and thrombin, on both osteoclasts and osteoblasts biology and we characterized the osteoclastogenic potential of HA patients' osteoclast precursors. Patients/Methods: Peripheral Blood Mononuclear Cells (PBMC) isolated from healthy donors were induced to differentiate into osteoclasts and treated with plasma derived VWF/FVIII complex, human rVWF, human full length rFVIII, activated FX and thrombin. Osteoclastogenesis and expression analysis of RANK, TRAF6 (TNF receptor-associated factor 6), TCIRG1 (T Cell Immune Regulator 1) and osteoclasts protease CTSK (cathepsin K) expression were assessed. Moreover, in vitro assays assessed the osteoclastogenic potential of PBMC isolated from different hemophilic A patients. FACS analyses of osteoclast precursors isolated from patients were performed. Osteoblasts differentiation, mineralization and genes expression (Alkaline Phosphatase and COL1A2) were performed in the presence of aforementioned coagulation factors. Results. We showed a significant reduction of mature osteoclasts after treatment of HD-PBMC with FVIII, VWF, FVIII/VWF, FXa and thrombin. VWF appears to play a major role to regulate osteoclast differentation from healthy donor-derived PBMC. Indeed, it inhibits by itself ~45% the osteoclastogenesis comparable to OPG, and even more if is complexed with FVIII (53% inhibition). About 50% and 70% reduced levels of osteoclast differentiation were also revealed following treatment with FXa and thrombin, respectively. Interestingly, PBMC from HA patients showed increased ability to form mature osteoclasts compared to those obtained from healthy controls. Osteoclast precursors (CD16−CD14+CD11b+) are significantly higher in HA patients than age and sex matched controls. Moreover, transcriptional analysis revealed increased RANK, TRAF6, CTSK and TCIRG1 genes expression in adult hemophilia patient's osteoclasts compared to matched controls. FVIII and VWF treatments led also to a statistically significant reduction of ALP positivity in control osteoblasts; opposite effect was shown following thrombin treatment. Conclusions. All these data support that bone loss observed in haemophilia patients could be related to increased osteoclast formation and activity and that coagulation factors directly impact on bone cells. These results may have important implications in the clinical management of hemophilic patients to prevent bleeding as well as to preserve bone health.

Jagged1 Acts as an RBP-J Target and Feedback Suppresses TNF-Mediated Inflammatory Osteoclastogenesis.

TNF plays a crucial role in inflammation and bone resorption in various inflammatory diseases, including rheumatoid arthritis (RA). However, its direct ability to drive macrophages to differentiate into osteoclasts is limited. Although RBP-J is recognized as a key inhibitor of TNF-mediated osteoclastogenesis, the precise mechanisms that restrain TNF-induced differentiation of macrophages into osteoclasts are not fully elucidated. In this study, we identified that the Notch ligand Jagged1 is a previously unrecognized RBP-J target. The expression of Jagged1 is significantly induced by TNF mainly through RBP-J. The TNF-induced Jagged1 in turn functions as a feedback inhibitory regulator of TNF-mediated osteoclastogenesis. This feedback inhibition of osteoclastogenesis by Jagged1 does not exist in RANKL-induced mouse osteoclast differentiation, as RANKL does not induce Jagged1 expression. The Jagged1 level in peripheral blood monocytes/osteoclast precursors is decreased in RA compared with the nonerosive inflammatory disease systemic lupus erythematosus, suggesting a mechanism that contributes to increased osteoclast formation in RA. Moreover, recombinant Jagged1 suppresses human inflammatory osteoclastogenesis. Our findings identify Jagged1 as an RBP-J direct target that links TNF and Notch signaling pathways and restrains TNF-mediated osteoclastogenesis. Given that Jagged1 has no effect on TNF-induced expression of inflammatory genes, its use may present a new complementary therapeutic approach to mitigate inflammatory bone loss with little impact on the immune response in disease conditions.

Concomitant induction of SLIT3 and microRNA-218–2 in macrophages by toll-like receptor 4 activation limits osteoclast commitment

BackgroundToll-like receptor 4 (TLR4) conducts a highly regulated inflammatory process by limiting the extent of inflammation to avoid toxicity and tissue damage, even in bone tissues. Thus, it is plausible that strategies for the maintenance of normal bone-immunity to prevent undesirable bone damage by TLR4 activation can exist, but direct evidence is still lacking.MethodsOsteoclast precursors (OCPs) obtained from WT or Slit3-deficient mice were differentiated into osteoclast (OC) with macrophage colony-stimulating factor (M-CSF), RANK ligand (RANKL) and lipopolysaccharide (LPS) by determining the number of TRAP-positive multinuclear cells (TRAP+ MNCs). To determine the alteration of OCPs population, fluorescence-activated cell sorting (FACS) was conducted in bone marrow cells in mice after LPS injection. The severity of bone loss in LPS injected WT or Slit3-deficient mice was evaluated by micro-CT analysis.ResultWe demonstrate that TLR4 activation by LPS inhibits OC commitment by inducing the concomitant expression of miR-218–2-3p and its host gene, Slit3, in mouse OCPs. TLR4 activation by LPS induced SLIT3 and its receptor ROBO1 in BMMs, and this SLIT3-ROBO1 axis hinders RANKL-induced OC differentiation by switching the protein levels of C/EBP-β isoforms. A deficiency of SLIT3 resulted in increased RANKL-induced OC differentiation, and the elevated expression of OC marker genes including Pu.1, Nfatc1, and Ctsk. Notably, Slit3-deficient mice showed expanded OCP populations in the bone marrow. We also found that miR-218–2 was concomitantly induced with SLIT3 expression after LPS treatment, and that this miRNA directly suppressed Tnfrsf11a (RANK) expression at both gene and protein levels, linking it to a decrease in OC differentiation. An endogenous miR-218–2 block rescued the expression of RANK and subsequent OC formation in LPS-stimulated OCPs. Aligned with these results, SLIT3-deficient mice displayed increased OC formation and reduced bone density after LPS challenge.ConclusionOur findings suggest that the TLR4-dependent concomitant induction of Slit3 and miR-218–2 targets RANK in OCPs to restrain OC commitment, thereby avoiding an uncoordinated loss of bone through inflammatory processes. These observations provide a mechanistic explanation for the role of TLR4 in controlling the commitment phase of OC differentiation.CNR6qWg43XLt4z6Q5VLfxQVideo

Abnormal expression pattern of lncRNA H19 participates in multiple myeloma bone disease by unbalancing osteogenesis and osteolysis

Accumulating genetic and epigenetic alterations in multiple myeloma (MM) have been demonstrated to be closely associated with osteolytic bone disease, generally characterized as increased osteoclast formation and decreased osteoblast activity. Previously, serum long non-coding RNA (lncRNA) H19 has been proved to be a biomarker for the diagnosis of MM. Whereas, its role in MM-associated bone homeostasis remains largely elusive. A cohort of 42 MM patients and 40 healthy volunteers were enrolled for evaluating differential expressions of H19 and its downstream effectors. The proliferative capacity of MM cells was monitored by CCK-8 assay. Alkaline phosphatase (ALP) staining and activity detection, either with Alizarin red staining (ARS) were employed to assess osteoblast formation. Osteoblast- or osteoclast-associated gene were detected using qRT-PCR and western blot analysis. Bioinformatics analysis, RNA pull-down, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP) were subjected to verify H19/miR-532-3p/E2F7/EZH2 axis, which was accounted for epigenetic suppression of PTEN. The functional role of H19 on MM development through unbalancing osteolysis and osteogenesis was also confirmed in the murine MM model. Upregulation of serum H19 was observed in MM patients, suggesting its positive correlation with the poor prognosis of MM patients. Loss of H19 dramatically weakened cell proliferation of MM cells, promoted osteoblastic differentiation, and impaired osteoclast activity. While reinforced H19 exhibited the opposite effects. Akt/mTOR signaling plays an indispensable role in H19-mediated osteoblast formation and osteoclastgenesis. Mechanistically, H19 served as a sponge for miR-532-3p to upregulate E2F7, a transcriptional activator of EZH2, thereby accounting for modulating epigenetic suppression of PTEN. The in vivo experiments further validated that H19 exerted important impacts on tumor growth through breaking the balance between osteogenesis and osteolysis via Akt/mTOR signaling. Collectively, increased enrichment of H19 in MM cells exhibits an essential role in MM development by disturbing bone homeostasis.

DNA damage-inducible transcript 3 deficiency promotes bone resorption in murine periodontitis models.

Periodontitis is a multifactorial inflammatory disease that leads to the destruction of supporting structures of the teeth. DNA damage-inducible transcript 3 (DDIT3) plays crucial roles in cell survival and differentiation. DDIT3 regulates bone mass and osteoclastogenesis in femur. However, the role of DDIT3 in periodontitis has not been elucidated. This research aimed to explore the role and mechanisms of DDIT3 in periodontitis. DDIT3 gene knockout (KO) mice were generated using a CRISPR/Cas9 system. Experimental periodontitis models were established to explore the role of DDIT3 in periodontitis. The expression of DDIT3 in periodontal tissues was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). The alveolar bone phenotypes were observed by micro-CT and stereomicroscopy. The inflammation levels and osteoclast activity were examined by histological staining, immunostaining, and qRT-PCR. Bone marrow-derived macrophages (BMMs) were isolated to confirm the effects of DDIT3 on osteoclast formation and function in vitro. The increased expression of DDIT3 in murine inflamed periodontal tissues was detected. DDIT3 knockout aggravated alveolar bone loss and enhanced expression levels of inflammatory cytokines in murine periodontitis models. Increased osteoclast formation and higher expression levels of osteoclast-specific markers were observed in the inflamed periodontal tissues of KO mice. In vitro, DDIT3 deficiency promoted the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts and the bone resorption activity of mature osteoclasts. Our results demonstrate that DDIT3 deletion aggravated alveolar bone loss in experimental periodontitis through enhanced inflammatory reactions and osteoclastogenesis. The anti-inflammation and the inhibition of bone loss by DDIT3 in murine periodontitis provides a potential novel therapeutic strategy for periodontitis.

SOCS2 regulates alveolar bone loss in Aggregatibacter actinomycetemcomitans-induced periodontal disease.

The role of suppressor of cytokine signaling 2 (SOCS2) in Aggregatibacter actinomycetemcomitans (Aa)-induced alveolar bone loss is unknown; thus, it was investigated in this study. Alveolar bone loss was induced by infecting C57BL/6 wild-type (WT) and Socs2-knockout (Socs2-/-) mice with Aa. Bone parameters, bone loss, bone cell counts, the expression of bone remodeling markers, and cytokine profile were evaluated by microtomography, histology, qPCR, and/or ELISA. Bone marrow cells (BMC) from WT and Socs2-/- mice were differentiated in osteoblasts or osteoclasts for analysis of the expression of specific markers. Socs2-/- mice intrinsically exhibited irregular phenotypes in the maxillary bone and an increased number of osteoclasts. Upon Aa infection, SOCS2 deficiency resulted in the increased alveolar bone loss, despite decreased proinflammatory cytokine production, in comparison to the WT mice. In vitro, SOCS2 deficiency resulted in the increased osteoclasts formation, decreased expression of bone remodeling markers, and proinflammatory cytokines after Aa-LPS stimulus. Collectively, data suggest that SOCS2 is a regulator of Aa-induced alveolar bone loss by controlling the differentiation and activity of bone cells, and proinflammatory cytokines availability in the periodontal microenvironment and an important target for new therapeutic strategies. Thus, it can be helpful in preventing alveolar bone loss in periodontal inflammatory conditions.

HBP/O-GlcNAcylation Metabolic Axis Regulates Bone Resorption Outcome

Osteoclasts play a key role in the regulation of bone mass and are highly active metabolically. Here we show that a metabolic reprogramming toward the hexosamine biosynthetic pathway (HBP) is required not only for osteoclast differentiation but also to determine the bone resorption mode during physiological and pathological bone remodeling. We found that pharmacological inhibition of O-GlcNAc transferase (OGT) significantly reduced protein O-GlcNAcylation and osteoclast differentiation. Accordingly, genetic deletion of OGT also inhibited osteoclast formation and downregulated critical markers related to osteoclasts differentiation and function (NFATc1, αvintegrin, cathepsin K). Indeed, cells treated with OSMI-1, an OGT inhibitor, also reduced nuclear translocation of NFATc1. Furthermore, the addition of exogenous N-acetylglucosamine (GlcNAc) strongly increased osteoclast formation and demineralization ability. Strikingly, our data show for the first time that O-GlcNAcylation facilitates an aggressive trench resorption mode in human cells. The incubation of osteoclasts with exogenous GlcNAc increases the percentage of erosion by trench while having no effect on pit resorption mode. Through time-lapse recording, we documented that osteoclasts making trenches moving across the bone surface are sensitive to GlcNAcylation. Finally, osteoclast-specific Ogt-deficient mice show increased bone density and reduced inflammation-induced bone loss during apical periodontitis model. We show that osteoclast-specific Ogt-deficient mice are less susceptible to develop bacterial-induced periapical lesion. Consistent with this, Ogt-deleted mice showed a decreased number of tartrate-resistant acid phosphatase-positive cells lining the apical periodontitis site. In summary, here we describe a hitherto undiscovered role of the HBP/O-GlcNAcylation axis tuning resorption mode and dictating bone resorption outcome.

Fibronectin-LILRB4/gp49B interaction negatively regulates osteoclastogenesis through inhibition of RANKL-induced TRAF6/TAK1/NF-kB/MAPK signaling.

Dysregulation of osteoclasts, the multinucleated cells responsible for bone resorption, contributes to several degenerative bone disorders. Previously, we showed that blocking the leukocyte immunoglobulin (Ig)-like receptor B4 (LILRB4), a kind of inhibitory receptor that plays an important role in immune regulation, promotes osteoclast differentiation in vitro. Here, we explored whether gp49B, the murine ortholog of LILRB4, regulates osteoclastogenesis in vivo, and whether fibronectin (FN), a ligand of LILRB4/gp49B, certainly contributes to LILRB4/gp49B-mediated osteoclastogenesis. In comparison with wild-type mice, gp49B deficiency mice exhibited a loss of trabecular bone number and an increase in osteoclast formation. Gp49B knockout improved the bone resorptive capacity of osteoclasts derived from murine Raw264.7 cells by increasing osteoclast formation. We further revealed that gp49B deficiency increased the receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced signaling transduction by increasing the phosphorylation of transforming growth factor (TGF)-activated kinase 1 (TAK1), NF-κB and mitogen-activated protein kinases (MAPKs). Furthermore, the N-terminal 30 kDa proteolytic fragments of FN promoted gp49B-mediated inhibition of osteoclastogenesis by increasing Src homology-2-containing tyrosine phosphatase 1 (SHP-1) phosphorylation and tumor necrosis factor receptor-associated factor 6 (TRAF6)-SHP-1 association. In summary, the FN-LILRB4/gp49B interaction negatively regulates RANKL-induced TRAF6/TAK1/NF-κB/MAPK signaling in osteoclastogenesis.

Aberrant activation of TGF-β1 induces high bone turnover via Rho GTPases-mediated cytoskeletal remodeling in Camurati-Engelmann disease.

In the adult skeleton, the bone remodeling process involves a dynamic coordination between osteoblasts and osteoclasts, which is disrupted in diseases with high bone turnover rates and dysregulated transforming growth factor beta 1 (TGF-β1). However, little is known about how TGF-β1 signaling mediates bone resorption. Here, we described a pedigree with a heterozygous variant in TGF-β1 (R218C) that resulted in aberrant activation of TGF-β1 through an activating mechanism that caused Camurati-Engelmann disease (CED). We showed that CED patients have high levels of active Rho GTPases and the migration-related proteins Integrin β1 and Integrin β3 in their peripheral blood. HEK293T cells transfected with a plasmid encoding this mutant expressed high levels of TGF-β1 and active Rho GTPases. Furthermore, activation of Rho by TGF-β1 increased osteoclast formation and bone resorption, with increased migration of pre-osteoclasts, as well as cytoskeletal remodeling of pre-osteoclasts and mature osteoclasts. Importantly, pharmacological inhibition of Rho GTPases effectively rescued hyperactive TGF-β1-induced osteoclastogenesis in vitro. Overall, we propose that Rho GTPases mediate TGF-β1-induced osteoclastogenesis and suggest that Rho-TGF-β1 crosstalk is associated with high bone turnover in CED.

Age-Related Low Bone Mineral Density in C57BL/6 Mice Is Reflective of Aberrant Bone Morphogenetic Protein-2 Signaling Observed in Human Patients Diagnosed with Osteoporosis.

Osteoporosis (OP) is a bone disorder characterized by decreased bone mineral density (BMD). Bone Morphogenetic Protein-2 (BMP-2) injections are used to promote bone formation in OP patients. However, patients are unresponsive to BMP-2 while displaying an upregulation of BMP Receptor Type 1a (BMPRIa) and protein kinase CK2α (CK2α). A synthetically produced peptide named casein kinase 2.3 (CK2.3) utilizes the BMP-signaling pathway as it enhances osteogenesis of primary osteoblasts isolated from OP patients, whereas BMP-2 does not. Although shown in OP patients, there is currently no reliable mouse model to study BMP-2 and CK2.3 signaling. In this publication, we show that BMPRIa was required for CK2.3-mediated osteogenesis in C2C12 cells with a CRISPR-Cas9-mediated gene knockout for BMPRIa. We utilized the C57BL/6 (B6) mouse strain as an aging-model to study aberrant BMP-2 signaling, demonstrating that, like OP patients, in 15 and 20-month mice, BMP-2 did not increase bone growth and displayed upregulated BMPRIa and CK2α protein expression. Furthermore, CK2.3 enhanced osteogenesis and decreased osteoclastogenesis in all age groups, whereas BMP-2 only increased mineralization in 6-month mice while increasing osteoclast formation in all age groups. These data demonstrated that aging B6 mice were a reliable model and mimicked data obtained from OP patients.

Hyponatremia elicits gene expression changes driving osteoclast differentiation and functions

Growing evidence indicates that chronic hyponatremia represents a significant risk for bone loss, osteoporosis, and fractures in our aging population. Our prior studies on a rat model of the syndrome of inappropriate antidiuretic hormone secretion indicated that chronic hyponatremia causes osteoporosis by increasing osteoclastic bone resorption, thereby liberating stored sodium from bone. Moreover, studies in RAW264.7 pre-osteoclastic cells showed increased osteoclast formation and resorptive activity in response to low extracellular fluid sodium ion concentration (low [Na+]). These studies implicated a direct stimulatory effect of low [Na+] rather than the low osmolality on cultured osteoclastic cells. In the present cellular studies, we explored gene expression changes triggered by low [Na+] using RNA sequencing and gene ontology analysis. Results were confirmed by mouse whole genome microarray, and quantitative RT-PCR. Findings confirmed gene expression changes supporting osteoclast growth and differentiation through stimulation of receptor activator of nuclear factor kappa-B ligand (RANKL), and PI3K/Akt pathways, and revealed additional pathways. New findings on low [Na+]-induced upregulation of lysosomal genes, mitochondrial energy production, MMP-9 expression, and osteoclast motility have supported the significance of osteoclast transcriptomic responses. Functional assays demonstrated that RANL and low [Na+] independently enhance osteoclast functions. Understanding the molecular mechanisms of hyponatremia-induced osteoporosis provides the basis for future studies identifying sodium-sensing mechanisms in osteoclasts, and potentially other bone cells, and developing strategies for treatment of bone fragility in the vulnerable aging population most affected by both chronic hyponatremia and osteoporosis. Issue sectionsSignaling Pathways; Parathyroid, Bone, and Mineral Metabolism.

IFT80 negatively regulates osteoclast differentiation via association with Cbl-b to disrupt TRAF6 stabilization and activation

Excess bone loss due to increased osteoclastogenesis is a significant clinical problem. Intraflagellar transport (IFT) proteins have been reported to regulate cell growth and differentiation. The role of IFT80, an IFT complex B protein, in osteoclasts (OCs) is completely unknown. Here, we demonstrate that deletion of IFT80 in the myeloid lineage led to increased OC formation and activity accompanied by severe bone loss in mice. IFT80 regulated OC formation by associating with Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b) to promote protein stabilization and proteasomal degradation of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6). IFT80 knockdown resulted in increased ubiquitination of Cbl-b and higher TRAF6 levels, thereby hyperactivating the receptor activator of nuclear factor-κβ (NF-κβ) ligand (RANKL) signaling axis and increased OC formation. Ectopic overexpression of IFT80 rescued osteolysis in a calvarial model of bone loss. We have thus identified a negative function of IFT80 in OCs.

Monocyte Chemotactic Protein-1 (MCP1) Accumulation in Human Osteoclast Precursor Cultures.

In vitro osteoclast methods require constant treatment with macrophage colony stimulating factor (M-CSF) to support precursor survival and addition of the differentiation agent receptor activator of NF-κB ligand (RANKL). Constant exposure to granulocyte macrophage colony stimulating factor (GM-CSF) suppresses human osteoclast formation in vitro. Addition of the chemokine monocyte chemotactic protein-1 (MCP1) to such cultures dramatically increases osteoclast formation and overcomes GM-CSF mediated suppression. We investigated the effect of M-CSF, GM-CSF and the combination of M-CSF and GM-CSF treatment on the expression of chemokines in human CD14+ cells in culture. Of assayed chemokines, MCP1 was the most abundant in terms of mRNA transcript and protein in M-CSF treated cultures and was suppressed by GM-CSF. MCP1 protein accumulated up to 50 ng/mL in culture medium, greatly exceeding other assayed chemokines. C-C chemokine receptor-2 (CCR2) is the receptor for MCP1: the formation of osteoclast-like cells was inhibited by constant exposure to the CCR2 antagonist RS102895, in part by decreasing expression of RANK, the receptor for RANKL.

Notch2 Blockade Mitigates Methotrexate Chemotherapy-Induced Bone Loss and Marrow Adiposity.

Childhood cancer methotrexate (MTX) chemotherapy often causes bone growth impairments, bone loss, and increased risks of fractures during or after treatment, for which the pathobiology is unclear and there is a lack of specific treatment. Our time course analyses of long bones from rats receiving intensive MTX treatment (mimicking a clinical protocol) found decreased trabecular bone volume, increased osteoclast formation and activity, increased adipogenesis in the expense of osteogenesis from the bone marrow stromal cells at days 6 and 9 following the first of five daily MTX doses. For exploring potential mechanisms, PCR array expression of 91 key factors regulating bone homeostasis was screened with the bone samples, which revealed MTX treatment-induced upregulation of Notch receptor NOTCH2, activation of which is known to be critical in skeletal development and bone homeostasis. Consistently, increased Notch2 activation in bones of MTX-treated rats was confirmed, accompanied by increased expression of Notch2 intracellular domain protein and Notch target genes HEY1, HES1 and HEYL. To confirm the roles of Notch2 signalling, a neutralising anti-Notch2 antibody or a control IgG was administered to rats during MTX treatment. Microcomputed tomography analyses demonstrated that trabecular bone volume was preserved by MTX+anti-Notch2 antibody treatment. Anti-Notch2 antibody treatment ameliorated MTX treatment-induced increases in osteoclast density and NFATc1 and RANKL expression, and attenuated MTX-induced bone marrow adiposity via regulating Wnt/β-catenin signalling and PPARγ expression. Thus, Notch2 signalling plays an important role in mediating MTX treatment-induced bone loss and bone marrow adiposity, and targeting Notch2 could be a potential therapeutic option.

Metabolic Bone Disease of Prematurity.

Metabolic Bone Disease of Prematurity.

The Programmed Death-1 Pathway Counter-Regulates Inflammation-Induced Osteoclast Activity in Clinical and Experimental Settings.

ObjectiveThe programmed death-1 (PD-1) pathway is essential for maintaining self-tolerance and plays an important role in autoimmunity, including rheumatoid arthritis (RA). Here, we investigated how membrane-bound and soluble (s)PD-1 influence bone homeostasis during chronic inflammation, exemplified in RA.MethodsBone mineral density and bone microstructure were examined in PD-1 and PD-L1 knockout (KO) mice and compared with wild-type (WT) mice. Receptor activator of nuclear factor kappa-B ligand (RANKL) was measured in serum, and the expression examined on activated bone marrow cells. Osteoclast formation was examined in cells from murine spleen and bone marrow and from human synovial fluid cells. sPD-1 was measured in chronic and early (e)RA patients and correlated to markers of disease activity and radiographic scores.ResultsPD-1 and PD-L1 KO mice showed signs of osteoporosis. This was supported by a significantly reduced trabecular bone volume fraction and deteriorated microstructure, as well as increased osteoclast formation and an increased RANKL/OPG ratio. The recombinant form of sPD-1 decreased osteoclast formation in vitro, but was closely associated with disease activity markers in eRA patients. Sustained elevated sPD-1 levels indicated ongoing inflammation and were associated with increased radiographic progression.ConclusionThe PD-1 pathway is closely associated with bone homeostasis, and lacking members of this pathway causes a deteriorated bone structure. The immunological balance in the microenvironment determines how the PD-1 pathway regulates osteoclast formation. In eRA patients, sPD-1 may serve as a biomarker, reflecting residual but clinically silent disease activity and radiographic progression.