Regulates Osteoclast Formation Research Articles

New mechanistic understanding of osteoclast differentiation and bone resorption mediated by P2X7 receptors and PI3K-Akt-GSK3β signaling.

Osteoporosis is a global health issue characterized by decreased bone mass and microstructural degradation, leading to an increased risk of fractures. This study aims to explore the molecular mechanism by which P2X7 receptors influence osteoclast formation and bone resorption through the PI3K-Akt-GSK3β signaling pathway. An osteoporosis mouse model was generated through ovariectomy (OVX) in normal C57BL/6 and P2X7f/f; LysM-cre mice. Osteoclasts were isolated for transcriptomic analysis, and differentially expressed genes were selected for functional enrichment analysis. Metabolite analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and multivariate statistical analysis and pattern recognition were used to identify differential lipid metabolism markers and their distribution. Bioinformatics analyses were conducted using the Encyclopedia of Genes and Genomes database and the MetaboAnalyst database to assess potential biomarkers and create a metabolic pathway map. Osteoclast precursor cells were used for in vitro cell experiments, evaluating cell viability and proliferation using the Cell Counting Kit 8 (CCK-8) assay. Osteoclast precursor cells were induced to differentiate into osteoclasts using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-beta ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) staining was performed to compare differentiation morphology, size, and quantity between different groups. Western blot analysis was used to assess the expression of differentiation markers, fusion gene markers, and bone resorption ability markers in osteoclasts. Immunofluorescence staining was employed to examine the spatial distribution and quantity of osteoclast cell skeletons, P2X7 protein, and cell nuclei, while pit assay was used to evaluate osteoclast bone resorption ability. Finally, in vivo animal experiments, including micro computed tomography (micro-CT), hematoxylin and eosin (HE) staining, TRAP staining, and immunohistochemistry, were conducted to observe bone tissue morphology, osteoclast differentiation, and the phosphorylation level of the PI3K-Akt-GSK3β signaling pathway. Transcriptomic and metabolomic data collectively reveal that the P2X7 receptor can impact the pathogenesis of osteoporosis through the PI3K-Akt-GSK3β signaling pathway. Subsequent in vitro experiments showed that cells in the Sh-P2X7 + Recilisib group exhibited increased proliferative activity (1.15 versus 0.59), higher absorbance levels (0.68 versus 0.34), and a significant increase in resorption pit area (13.94 versus 3.50). Expression levels of osteoclast differentiation-related proteins MMP-9, CK, and NFATc1 were markedly elevated (MMP-9: 1.72 versus 0.96; CK: 2.54 versus 0.95; NFATc1: 3.05 versus 0.95), along with increased fluorescent intensity of F-actin rings. In contrast, the OE-P2X7 + LY294002 group showed decreased proliferative activity (0.64 versus 1.29), reduced absorbance (0.34 versus 0.82), and a significant decrease in resorption pit area (5.01 versus 14.96), accompanied by weakened expression of MMP-9, CK, and NFATc1 (MMP-9: 1.14 versus 1.79; CK: 1.26 versus 2.75; NFATc1: 1.17 versus 2.90) and decreased F-actin fluorescent intensity. Furthermore, in vivo animal experiments demonstrated that compared with the wild type (WT) + Sham group, mice in the WT + OVX group exhibited significantly increased levels of CTX and NTX in serum (CTX: 587.17 versus 129.33; NTX: 386.00 versus 98.83), a notable decrease in calcium deposition (19.67 versus 53.83), significant reduction in bone density, increased trabecular separation, and lowered bone mineral density (BMD). When compared with the KO + OVX group, mice in the KO + OVX + recilisib group showed a substantial increase in CTX and NTX levels in serum (CTX: 503.50 versus 209.83; NTX: 339.83 versus 127.00), further reduction in calcium deposition (29.67 versus 45.33), as well as decreased bone density, increased trabecular separation, and reduced BMD. P2X7 receptors positively regulate osteoclast formation and bone resorption by activating the PI3K-Akt-GSK3β signaling pathway.

Nuclear Factor-Kappa B Regulation of Osteoclastogenesis and Osteoblastogenesis.

Maintenance of skeletal integrity requires the coordinated activity of multinucleated bone-resorbing osteoclasts and bone-forming osteoblasts. Osteoclasts form resorption lacunae on bone surfaces in response to cytokines by fusion of precursor cells. Osteoblasts are derived from mesenchymal precursors and lay down new bone in resorption lacunae during bone remodeling. Nuclear factorkappa B (NF-κB) signaling regulates osteoclast and osteoblast formation and is activated in osteoclast precursors in response to the essential osteoclastogenic cytokine, receptor activator of NF-κB ligand (RANKL), which can also control osteoblast formation through RANK-RANKL reverse signaling in osteoblast precursors. RANKL and some pro-inflammatory cytokines, including tumor necrosis factor (TNF), activate NF-κB signaling to positively regulate osteoclast formation and functions. However, these cytokines also limit osteoclast and osteoblast formation through NF-κB signaling molecules, including TNF receptor-associated factors (TRAFs). TRAF6 mediates RANKL-induced osteoclast formation through canonical NF-κB signaling. In contrast, TRAF3 limits RANKL- and TNF-induced osteoclast formation, and it restricts transforming growth factor β (TGFβ)-induced inhibition of osteoblast formation in young and adult mice. During aging, neutrophils expressing TGFβ and C-C chemokine receptor type 5 (CCR5) increase in bone marrow of mice in response to increased NF-κB-induced CC motif chemokine ligand 5 (CCL5) expression by mesenchymal progenitor cells and injection of these neutrophils into young mice decreased bone mass. TGFβ causes degradation of TRAF3, resulting in decreased glycogen synthase kinase-3β/β-catenin-mediated osteoblast formation and age-related osteoporosis in mice. The CCR5 inhibitor, maraviroc, prevented accumulation of TGFβ+/CCR5+ neutrophils in bone marrow and increased bone mass by inhibiting bone resorption and increasing bone formation in aged mice. This paper updates current understanding of how NF-κB signaling is involved in the positive and negative regulation of cytokine-mediated osteoclast and osteoblast formation and activation with a focus on the role of TRAF3 signaling, which can be targeted therapeutically to enhance bone mass.

LncRNA XIST regulates osteoclast formation and promotes orthodontically induced inflammatory root resorption through miR-130b-3p/PTEN axis

ABSTRACT The long non-coding RNA (LncRNA) X-inactive specific transcript (XIST) regulates the biological process of osteoclasts and the process of related diseases. This study was attempted to investigate the mechanism of LncRNA XIST acting in osteoclast formation and orthodontic induced inflammatory root resorption (OIIRR). The compression force (CF) -induced cell model and the orthodontic tooth movement (OTM) rat model were designed and established in this study. The expression of LncRNA XIST, miR-130b-3p, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) as well as osteoclast related marker genes and inflammatory factors level were measured in this study. The interaction among LncRNA XIST, microRNA-130b-3p (miR-130b-3p) and PTEN were researched through luciferase activity and western blot assay. Pathological sections were used to analyze root resorption and osteoclast formation. The OTM rat model was successfully constructed, which was characterized by increased tooth spacing and increased root resorption pits. PTEN and LncRNA XIST was overexpressed in OTM group. Mechanism analysis showed that the overexpression of LncRNA XIST enhanced the PTEN level by sponging miR-130b-3p. The overexpression of LncRNA XIST increased the secretion of inflammatory factors and positive osteoclasts number, but inhibited the differentiation of osteoclasts by sponging miR-130b-3p and promoting the level of PTEN. This finding demonstrates that LncRNA XIST regulates osteoclast formation and aggravated OIIRR through miR-130b-3p/PTEN axis, suggesting that LncRNA XIST may be used as potential targets for OIIRR therapy.

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.

Mechanisms by which kidney-tonifying Chinese herbs inhibit osteoclastogenesis: Emphasis on immune cells.

The immune system plays a crucial role in regulating osteoclast formation and function and has significance for the occurrence and development of immune-mediated bone diseases. Kidney-tonifying Chinese herbs, based on the theory of traditional Chinese medicine (TCM) to unify the kidney and strengthen the bone, have been widely used in the prevention and treatment of bone diseases. The common botanical drugs are tonifying kidney-yang and nourishing kidney-yin herbs, which are divided into two parts: one is the compound prescription of TCM, and the other is the single preparation of TCM and its active ingredients. These botanical drugs regulate osteoclastogenesis directly and indirectly by immune cells, however, we have limited information on the differences between the two botanical drugs in osteoimmunology. In this review, the mechanism by which kidney-tonifying Chinese herbs inhibiting osteoclastogenesis was investigated, emphasizing the immune response. The differences in the mechanism of action between tonifying kidney-yang herbs and nourishing kidney-yin herbs were analysed, and the therapeutic value for immune-mediated bone diseases was evaluated.

Zingerone attenuates Ti particle-induced inflammatory osteolysis by suppressing the NF-κB signaling pathway in osteoclasts.

Aseptic loosening caused by inflammatory osteolysis is one of the most frequent and serious long-term complications after total joint arthroplasty (TJA). Development of a new therapeutic drug is required due to the lack of effective therapy and serious adverse effects. This study aimed to explore the pharmacological properties of zingerone (ZO) in attenuating osteoclast-mediated periprosthetic osteolysis and how ZO modulates osteoclastogenesis. The nontoxic concentration of ZO was clarified by the CCK-8 method. Then, we explored the efficacy of ZO on suppressing osteoclast differentiation, F-actin ring formation, bone resorption, and NF-κB luciferase activity in vitro as well as osteoprotection in vivo. Polymerase chain reaction and western blotting were applied to detect the underlying mechanisms involved in osteoclastogenesis. ZO showed an obvious inhibitory effect on osteoclastogenesis and bone resorption in a dose-dependent manner by mainly suppressing the activation of NF-κB signaling pathways. Furthermore, ZO administration successfully attenuated titanium (Ti) particle-stimulated periprosthetic osteolysis and osteoporosis by regulating osteoclast formation. Our findings demonstrated the pharmacological properties of ZO in inhibiting osteoclast formation and function by downregulation of NF-κB signaling activation. As a result, these findings could be expected to provide a novel reagent for regulating inflammatory osteolysis caused by prosthetic loosening.

A rescue diet raises the plasma calcium concentration and ameliorates rheumatoid arthritis in mice: Role of CaSR-mediated inhibition of osteoclastogenesis.

Calcium modulates bone cell recruitment, differentiation, and function by binding to the calcium-sensing receptor (CaSR). However, the function of CaSR induced by high extracellular calcium (Ca2+ e ) in the regulation of osteoclast formation in rheumatoid arthritis (RA) remains unknown. Here, we used TNFα-transgenic (TNFTG ) RA mice and their wildtype (WT) littermates fed a normal or a rescue diet (high calcium, high phosphorus, and high lactose diet, termed rescue diet) to compare their joint bone phenotypes. In comparison to TNFTG mice fed the normal diet, articular bone volume and cartilage area are increased, whereas inflamed area, eroded surface, TRAP+ surface, and osteoclast-related genes expression are decreased in TNFTG mice fed the rescue diet. Besides, TNFTG mice fed the rescue diet were found to exhibit more CaSR+ area and less NFATc1+ /TRAP+ area. Furthermore, at normal Ca2+ e concentrations, osteoclast precursors (OCPs) from TNFTG mice formed more osteoclasts than OCPs from WT mice, but the number of osteoclasts gradually decreased when the Ca2+ e concentration increased. Meanwhile, the expression of CaSR increased responding to a high level of Ca2+ e , whereas the expression of NF-κB/NFATc1 signaling molecules decreased. At last, the knockdown of CaSR blocked the inhibition of osteoclast differentiation attributed to high Ca2+ e . Taken together, our findings indicate that high Ca2+ e inhibits osteoclast differentiation in RA mice partially through the CaSR/NF-κB/NFATc1 pathway.

AZ-628 delays osteoarthritis progression via inhibiting the TNF-α-induced chondrocyte necroptosis and regulating osteoclast formation

As a degenerative disease, the pathogenesis and treatment of osteoarthritis (OA) are still being studied. The prevailing view is that articular cartilage dysfunction plays an essential role in the development of osteoarthritis. Similarly, dynamic bone remodeling dramatically influences the development of osteoarthritis. The inflammatory response is caused by the overexpression of inflammatory factors, among which tumor necrosis factor-α is one of the main causes of OA, and its sources include the secretion of chondrocytes themselves and osteoclast secretion of subchondral bone. Moreover, TNF-α-induced activation of RIP1, RIP3, and MLKL has been shown to play an important role in cell necroptosis and inflammatory responses. In vitro, AZ-628 alleviates chondrocyte inflammation and necroptosis by inhibiting the NF-κB signaling pathway and RIP3 activation instead of RIP1 activation. AZ-628 also reduces osteoclast activity, proliferation and differentiation, and release of inflammatory substances by inhibiting autophagy, MAPK, and NF-κB pathways. Similarly, the in vivo study demonstrated that AZ-628 could inhibit chondrocyte breakdown and lower osteoclast formation and bone resorption, thereby slowing down subchondral bone changes induced by dynamic bone remodeling and reversing the progression of osteoarthritis in mice. The results of this study indicate that AZ-628 could be used to treat OA byinhibiting chondrocyte necroptosis and regulating osteoclast formation.

The role of C5a-C5aR1 axis in bone pathophysiology: A mini-review.

Bone remodeling is a physiological, dynamic process that mainly depends on the functions of 2 cell types: osteoblasts and osteoclasts. Emerging evidence suggests that complement system is crucially involved in the regulation of functions of these cells, especially during inflammatory states. In this context, complement component 5a (C5a), a powerful pro-inflammatory anaphylatoxin that binds the receptor C5aR1, is known to regulate osteoclast formation and osteoblast inflammatory responses, and has thus been proposed as potential therapeutic target for the treatment of inflammatory bone diseases. In this review, we will analyze the role of C5a-C5aR1 axis in bone physiology and pathophysiology, describing its involvement in the pathogenesis of some of the most frequent inflammatory bone diseases such as rheumatoid arthritis, and also in osteoporosis and bone cancer and metastasis. Moreover, we will examine C5aR1-based pharmacological approaches that are available and have been tested so far for the treatment of these conditions. Given the growing interest of the scientific community on osteoimmunology, and the scarcity of data regarding the role of C5a-C5aR1 axis in bone pathophysiology, we will highlight the importance of this axis in mediating the interactions between skeletal and immune systems and its potential use as a therapeutic target.

MiR-134-5p inhibits osteoclastogenesis through a novel miR-134-5p/Itgb1/MAPK pathway

Osteoporosis affects approximately 200 million people and severely affects quality of life, but the exact pathological mechanisms behind this disease remain unclear. Various miRNAs have been shown to play a predominant role in the regulation of osteoclast formation. In this study, we explored the role of miR-134-5p in osteoclastogenesis both in vivo and in vitro. We constructed an ovariectomized (OVX) mouse model and performed microarray analysis using bone tissue from OVX mice and their control counterparts. Quantitative RT-PCR data from bone tissue and bone marrow macrophages (BMMs) confirmed the decreased expression of miR-134-5p in OVX mice observed in microarray analysis. In addition, a decrease in miR-134-5p was also observed during induced osteoclastogenesis of BMMs collected from C57BL/6N mice. Through transfection with miR-134-5p agomirs and antagomirs, we found that miR-134-5p knockdown significantly accelerated osteoclast formation and cell proliferation and inhibited apoptosis. Furthermore, a luciferase reporter assay showed that miR-134-5p directly targets the integrin surface receptor gene Itgb1. Cotransfection with Itgb1 siRNA reversed the effect of the miR-134-5p antagomir in promoting osteoclastogenesis. Moreover, the abundance levels of MAPK pathway proteins phosphorylated-p38 (p-p38) and phosphorylated-ERK (p-ERK) were significantly increased after transfection with the miR-134-5p antagomir but decreased after transfection with the miR-134-5p agomir or Itgb1 siRNA, which indicated a potential relationship between the miR-134-5p/Itgb1 axis and the MAPK pathway. Collectively, these results revealed that miR-134-5p inhibits osteoclast differentiation of BMMs both in vivo and in vitro and that the miR-134-5p/Itgb1/MAPK pathway might be a potential target for osteoporosis therapy.

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.

Role of HSP90α in osteoclast formation and osteoporosis development.

Osteoporosis (OP) is a systemic metabolic bone disease that occurs most frequently in the elderly. The main pathogenesis of OP is excessive proliferation and differentiation of osteoclasts, in which the peroxisome proliferator-activated receptor γ (PPARγ) pathway has a pivotal role. Recently, heat shock protein (HSP)90α has been identified as an important molecular chaperone with PPARγ, which regulates the effect of the PPARγ pathway. The aim of the present study was to investigate the role of HSP90α involved in the regulation of osteoclast formation and the process of osteoporosis. Firstly, the expression of HSP90α in osteoclast differentiation was detected by western blotting in vitro, then the effect of HSP90α inhibition on the formation and differentiation of osteoclasts was examined. Furthermore, the nuclear import of PPARγ was also assessed to confirm the synergistic effect of HSP90α. Finally, the inhibitory effect of HSP90α in vivo was explored, using a mouse model of osteoporosis. As a result, in the process of osteoclast differentiation and proliferation, the expression of HSP90α was upregulated. Inhibition of HSP90α could block the formation and differentiation of osteoclasts, and remit osteoporosis in mice. Regarding the underlying mechanism, inhibition of HSP90α could block the nuclear import of PPARγ to inhibit osteoclast differentiation and proliferation. In conclusion, these data indicated that the inhibition of HSP90α could block osteoclast formation and remit osteoporosis by reducing the nuclear import of PPARγ.

GPR125 positively regulates osteoclastogenesis potentially through AKT-NF-κB and MAPK signaling pathways.

G-protein-coupled receptors (GPCRs) signaling is critical to cell differentiation and activation. However, the function of GPCRs in osteoclast differentiation and activation remains unclear. We found that the G-protein coupled receptor 125 (GPCR 125) gene (Gpr125) gene was highly expressed in osteoclasts through RNA-sequencing technology, qRT-PCR, and Western blot analysis. We characterized the role of GPCR125 in osteoclast differentiation and activation by loss-of-function and gain-of-function methods in osteoclasts. Osteoclasts with lentivirus-mediated GPR125 silencing demonstrated a dramatic reduction in differentiation and impaired bone resorption function. In contrast, overexpression of Gpr125 in osteoclasts increased NFATC1 expression and enhanced osteoclast differentiation and enhanced osteoclast-mediated bone resorption. These results indicated that GPCR125 positively regulates osteoclast formation and function. Following receptor activator of nuclear factor kappa-Β ligand (RANKL) stimulation, the expression levels of MAPK signaling pathway proteins phosphorylated-ERK (p-ERK) and phosphorylated-p38 (p-p38) were significantly decreased in the Gpr125 knockdown (sh-GPR125) group compared to its control group. We also found that phosphorylated AKT (p-AKT) expression was downregulated, as well as nuclear factor kappa-B (NF-κB) signaling pathway protein phosphorylated-IKB alpha (p-IKBα). Our results demonstrated that GPCR125 positively regulates osteoclasts via RANKL-stimulated MAPK and AKT-NF-κB signaling pathways, and GPCR125 could potentially be utilized as a novel therapeutic target in bone related diseases including osteoporosis.

MiRNA-7062-5p Promoting Bone Resorption After Bone Metastasis of Colorectal Cancer Through Inhibiting GPR65

Bone metastasis is positively associated with a poor prognosis in patients with colorectal cancer (CRC). CRC always leads to osteolytic change, which is regulated by aberrant activation of osteoclasts. MicroRNAs are remarkedly involved in metastasis of CRC; however, their role in bone metastasis of CRC is still unclear. The aim of this study is to find key microRNAs that are critical to bone resorption in bone metastasis of CRC. In this study, bone metastasis model was established through intratibially injecting CT-26 cells or MC-38 cells. Tartrate-resistant acid phosphatase (TRAP) staining was performed to explore the osteoclastogenesis of primary early osteoclast precursors (OCPs) after stimulation by CT-26 conditioned medium (CM). Then, microarray assay was performed to find differentially expressed miRNAs and mRNAs. The target gene of miRNA was confirmed by dual-luciferase analysis. The effect of miRNA, its target gene on osteoclastogenesis, and involved pathways were explored by Western blot, immunofluorescence analysis, and TRAP staining. Finally, the effect of miRNA on bone resorption in vivo was observed. miRNA-7062-5p was upregulated in early OCPs cultured in CT-26 CM or MC-38 CM. GPR65 was proven to be the target gene of miRNA-7062-5p. Overexpression of GPR65 can rescue the osteoclastogenesis caused by miRNA-7062-5p through activation of AMPK pathway. Local injection of miRNA-7062-5p inhibitors efficiently improved the bone resorption. Our study found the role of miRNA-7062-5p in regulating osteoclast formation, and our findings provided a potential therapeutic target in treatment of bone metastasis of CRC.

FAM19A5l Affects Mustard Oil-Induced Peripheral Nociception in Zebrafish.

Family with sequence similarity 19 (chemokine (C-C motif)-like) member A5 (FAM19A5) is a chemokine-like secretory protein recently identified as involved in the regulation of osteoclast formation, post-injury neointima formation, and depression. Although roles for FAM19A5 have been described in nervous system development and psychiatric disorders, its role in the nervous system remains poorly understood. Here, we analyzed the evolutionary history of FAM19A genes in vertebrates and identified FAM19A5l, a paralogous zebrafish gene originating from a common ancestral FAM19A5 gene. Further, zebrafish FAM19A5l is expressed in trigeminal and dorsal root ganglion neurons as well as distinct neuronal subsets of the central nervous system. Interestingly, FAM19A5l+ trigeminal neurons are nociceptive neurons that localized with TRPA1b and TRPV1 and respond to mustard oil treatment. Behavioral analysis further revealed that the nociceptive response to mustard oil decreases in FAM19A5l-knockout zebrafish larvae. In addition, TRPA1b and NGFa mRNA levels are down- and upregulated in FAM19A5l-knockout and -overexpressing transgenic zebrafish, respectively. Together, our data suggest that FAM19A5l plays a role in nociceptive responses to mustard oil by regulating TRPA1b and NGFa expression in zebrafish.

Serum Insufficiency Induces RANKL-Independent Osteoclast Formation during Developing Ischemic ONFH.

Blood supply interruption induces hypoxia and reduces serum provision to cause ischemia-induced osteonecrosis, including avascular osteonecrosis of the femoral head (ONFH). Oxygen deficiency (hypoxia) is known to induce different expression patterns in osteoblasts and osteoclasts, which have been extensively studied. However, the effects of serum insufficiency in nutrients, growth factors, and hormones on osteoblast and osteoclast activity in the damaged area and nearby regions remain poorly understood. In this study, the expression of osteoblast and osteoclast marker proteins was elucidated through in vitro and ex vivo studies. The results indicate that serum insufficiency accelerates the formation of monocyte-derived osteoclasts. The combined effect of serum insufficiency and hypoxia (mimicking ischemia) suppressed the activity of alkaline phosphatase and calcification in osteoblasts after the stimulation of osteogenic growth factors. Serum insufficiency increased the activity of tartrate-resistant acid phosphatase, expression of phosphorylated extracellular signal-regulated kinases, and production of reactive oxygen species in monocyte-derived osteoclasts in the absence of receptor activator of nuclear factor kappa-Β ligand stimulation. The findings indicate that changes in the expression of osteoblast and osteoclast markers in necrotic bone extracts were similar to those observed during an in vitro study. These results also suggest that serum insufficiency may be involved in the regulation of osteoclast formation in patients with ONFH.

Dysregulated miRNAs in bone cells of patients with Gorham-Stout disease.

Gorham-Stout disease (GSD) is a very rare disease characterized by increased bone erosion with angiomatous proliferation. The mechanisms underlying this disorder have not been deeply investigated. Due to its rarity, no guidelines are currently available for treatment and management of GSD. We recently evaluated the cellular alterations of the bone remodeling in patients showing that osteoclast precursors displayed increased ability to differentiate into osteoclasts and that affected osteoclasts resorb bone more actively than control cells. Moreover, osteoblasts isolated from a patient showed a defective ability to form mineralized nodules. In this paper, we investigated the molecular pathways involved in the cellular defects of GSD bone cells. For this study, we recruited nine patients and performed miRNome analysis of bone cells. Between the 178 miRNAs robustly expressed in GSD osteoclasts, significant modulation of three miRNAs (miR-1246, miR-1-3p, and miR-137-3p) involved in the regulation of osteoclast formation and activity or in the angiomatous proliferation was found in patients' cells. Interestingly, miR-1246 was also up-regulated in serum exosomes from patients. Analysis of miRNAs from patient osteoblasts suggested alteration of miR-204a-5p, miR-615-3p and miR-378a-3p regulating osteoblast function and differentiation. The resulting miRNA pattern may help to understand better the mechanisms involved in GSD and to identify new potential therapeutic targets for this rare disease.

Prognostic Value of Bone Formation and Resorption Proteins in Heterotopic Ossification in Critically-Ill Patients. a Single-Centre Study.

IntroductionA potential complication in critically ill patients is the formation of bone in soft tissues, termed heterotopic ossification. The exact pathogenetic mechanisms are still undetermined. Bone morphogenetic proteins induce bone formation, while signalling through the receptor activator of nuclear factor kappa-Β (RANK) and its ligand (RANKL), regulates osteoclast formation, activation, and survival in normal bone modelling and remodelling. Osteoprotegerin protects bone from excessive bone loss by blocking RANKL from binding to RANK.AimThe study aimed to investigate these molecules as potential prognostic biomarkers of heterotopic ossification development in critically ill patients.Materials and MethodsIn this prospective observational study, BMP-2, RANKL, and osteoprotegerin were measured by ELISA in twenty-eight critically-ill, initially non-septic patients, on admission to an ICU, seven days post-admission, and thirty days after ICU discharge.ResultsIn the critically-ill cohort, nine of the twenty-eight patients developed heterotopic ossification up to the 30-day follow-up time-point. The patients who developed heterotopic ossification exhibited significantly reduced BMP-2 and RANKL levels on ICU admission, compared to patients who did not; Osteoprotegerin readings were similar in both groups.ConclusionsCritically-ill patients who will subsequently develop heterotopic ossification, have significantly lower BMP-2 and RANKL levels at the time of ICU admission, suggesting that these proteins may be useful as prognostic markers for this debilitating condition.

The calcium channel TRPV6 is a novel regulator of RANKL-induced osteoclastic differentiation and bone absorption activity through the IGF-PI3K-AKT pathway.

ObjectivesCalcium ion signals are important for osteoclast differentiation. Transient receptor potential vanilloid 6 (TRPV6) is a regulator of bone homeostasis. However, it was unclear whether TRPV6 was involved in osteoclast formation. Therefore, the aim of this study was to evaluate the role of TPRV6 in bone metabolism and to clarify its regulatory role in osteoclasts at the cellular level.Materials and methodsBone structure and histological changes in Trpv6 knockout mice were examined using micro‐computed tomography and histological analyses. To investigate the effects of Trpv6 on osteoclast function, we silenced or overexpressed Trpv6 in osteoclasts via lentivirus transfection, respectively. Osteoclast differentiation and bone resorption viability were measured by tartrate‐resistant acid phosphatase (TRAP) staining and pit formation assays. The expression of osteoclast marker genes, including cathepsin k, DC‐STAMP, Atp6v0d2 and TRAP, was measured by qRT‐PCR. Cell immunofluorescence and Western blotting were applied to explore the mechanisms by which the IGF‐PI3K‐AKT pathway was involved in the regulation of osteoclast formation and bone resorption by Trpv6.ResultsWe found that knockout of Trpv6 induced osteoporosis and enhanced bone resorption in mice, but did not affect bone formation. Further studies showed that Trpv6, which was distributed on the cell membrane of osteoclasts, acted as a negative regulator for osteoclast differentiation and function. Mechanistically, Trpv6 suppressed osteoclastogenesis by decreasing the ratios of phosphoprotein/total protein in the IGF–PI3K–AKT signalling pathway. Blocking of the IGF–PI3K–AKT pathway significantly alleviated the inhibitory effect of Trpv6 on osteoclasts formation.ConclusionsOur study confirmed the important role of Trpv6 in bone metabolism and clarified its regulatory role in osteoclasts at the cellular level. Taken together, this study may inspire a new strategy for the treatment of osteoporosis.

TANK-binding kinase 1 mediates osteoclast differentiation by regulating NF-κB, MAPK and Akt signaling pathways.

TANK-binding kinase 1 (TBK1) belongs to the noncanonical IκB kinase (IKK) family. The ubiquitously expressed protein is well known to play a pivotal role in innate immune response and inflammation. Although excessive inflammatory activities have been shown to affect osteoclast (OC) differentiation and function, direct relevance of TBK1 in bone turnover is not known. In this work, we specifically altered the TBK1 protein level by knocking down or overexpressing it without affecting its homologous protein IKKε expression, and demonstrated the effect of TBK1 on OC differentiation in bone marrow macrophages (BMMs) and RAW264.7 cells upon induction by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL). TBK1 knockdown was found to markedly inhibit the OC differentiation and function, while TBK1 overexpression enhanced OC formation. Downregulation of TBK1 greatly suppressed RANKL-induced gene expression of Mmp9, Atp6v0d2, Acp5, Ctsk andNfatc1 involved in the regulation of OC formation and function in both BMM and RAW264.7 cells. Mechanistic studies indicated that TBK1 affected the NF-κB signaling pathway as well as mitogen-activated protein kinases (MAPKs) and protein kinase B (Akt) activation during OC differentiation. Moreover, the protein level of TNF receptor-associated factor 6 (TRAF6) was increased, and the interaction of TRAF6 with TBK1 was potentiated, by RANKL. Collectively, we provide direct evidence showing that TBK1 effectively mediates OC differentiation and function by regulating NF-κB, MAPKs and Akt signals. A TBK1-targeted therapeutic strategy may be useful for the treatment of bone-related disorders.