Regulates Osteoclast Differentiation Research Articles

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

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

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

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

Connexin 43 Hemichannels Regulate Osteoblast to Osteocyte Differentiation.

Connexin 43 (Cx43) is the predominant connexin subtype expressed in osteocytes. Osteocytes, accounting for 90%–95% of total bone cells, function as orchestrators coordinating balanced activity between bone-resorbing osteoclasts and bone-forming osteoblasts. In this study, two newly developed osteocytic cell lines, OCY454 and IDG-SW3, were used to determine the role of Cx43 gap junctions and hemichannels (HCs) in the regulation of osteoblast to osteocyte differentiation. We found that the Cx43 level was substantially increased during the differentiation of IDG-SW3 cells and is also much higher than that of OCY454 cells. We knocked down Cx43 expression using the lentiviral CRISPR/Cas9 approach and inhibition of Cx43 HCs using Cx43 (E2) antibody in IDG-SW3 cells. Cx43 knockdown (KD) or Cx43 HC inhibition decreased gene expression for osteoblast and osteocyte markers, including alkaline phosphatase, type I collagen, dentin matrix protein 1, sclerostin, and fibroblast growth factor 23, whereas increasing the osteoclastogenesis indicator and the receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio at early and late differentiation stages. Moreover, mineralization was remarkably attenuated in differentiated Cx43-deficient IDG-SW3 cells compared to ROSA26 control. The conditioned medium collected from fully differentiated IDG-SW3 cells with Cx43 KD promoted osteoclastogenesis of RAW264.7 osteoclast precursors. Our results demonstrated that Cx43 HCs play critical roles in osteoblast to osteocyte differentiation process and regulate osteoclast differentiation via secreted factors.

BHLHE40 promotes osteoclastogenesis and abnormal bone resorption via c-Fos/NFATc1

BackgroundDysregulated osteoclast activity due to altered osteoclast differentiation causes multiple bone diseases. Osteoclasts are multinucleated giant cells derived from hematopoietic stem cells and play a major role in bone absorption. However, the mechanisms underlying the tight regulation of osteoclast differentiation in multiple pathophysiological status remain unknown.ResultsWe showed that Bhlhe40 upregulation is tightly associated with osteoclast differentiation and osteoporosis. Functionally, Bhlhe40 promoted osteoclast differentiation in vitro, and Bhlhe40 deficiency led to increased bone mass and decreased osteoclast differentiation in vivo. Moreover, Bhlhe40 deficient mice resisted estrogen deficiency and aging-induced osteoporosis. Mechanism study showed that the increase in bone mass due to Bhlhe40 deficiency was a cell intrinsic defect in osteoclast differentiation in these mice. BHLHE40 upregulated the gene expression of Fos and Nfatc1 by directly binding to their promoter regions. Notably, inhibition of Fos/Nfatc1 abrogated the enhanced osteoclast differentiation induced by BHLHE40 overexpression.ConclusionsOur research reveals a novel Bhlhe40/c-Fos/Nfatc1 axis involved in regulating osteoclastogenesis and shows that osteoporosis caused by estrogen deficiency and aging can be rescued by regulating Bhlhe40 in mice. This may help in the development of a new strategy for the treatment of osteoporosis.

NOX4 blockade suppresses titanium nanoparticle-induced bone destruction via activation of the Nrf2 signaling pathway

Periprosthetic osteolysis (PPO) triggered by wear particles is the most severe complication of total joint replacement (TJR) surgeries, representing the major cause of implant failure, which is public health concern worldwide. Previous studies have confirmed the specialized role of osteoclast-induced progressive bone destruction in the progression of PPO. Additionally, the reactive oxygen species (ROS) induced by wear particles can promote excessive osteoclastogenesis and bone resorption. Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), a cellular enzyme, is considered to be responsible for the production of ROS and the formation of mature osteoclasts. However, NOX4 involvement in PPO has not yet been elucidated. Therefore, we investigated the mechanism by which NOX4 regulates osteoclast differentiation and the therapeutic effects on titanium nanoparticle-induced bone destruction. We found that NOX4 blockade suppressed osteoclastogenesis and enhanced the scavenging of intracellular ROS. Our rescue experiment revealed that nuclear factor-erythroid 2-related factor 2 (Nrf2) silencing reversed the effects of NOX4 blockade on ROS production and osteoclast differentiation. In addition, we found increased expression levels of NOX4 in PPO tissues, while NOX4 inhibition in vivo exerted protective effects on titanium nanoparticle-induced osteolysis through antiosteoclastic and antioxidant effects. Collectively, these findings suggested that NOX4 blockade suppresses titanium nanoparticle-induced bone destruction via activation of the Nrf2 signaling pathway and that NOX4 blockade may be an attractive therapeutic approach for preventing PPO.Graphical

Long noncoding RNA KCNQ1OT1 inhibits osteoclast differentiation by regulating the miR-128-3p/NFAT5 axis.

Noncoding RNAs play an important role in regulating osteoclast differentiation. We investigated whether and how potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1), a long noncoding RNA, regulates osteoclast differentiation. We found that the expression of KCNQ1OT1 was downregulated in osteoporotic bone tissue. Then transfection of KCNQ1OT1 overexpression vectors or small interfering RNAs showed that the proliferation, migration, and osteoclast differentiation of RAW 264.7 cells were inhibited by KCNQ1OT1 upregulation, while they were promoted by KCNQ1OT1 knockdown. Interestingly, we found and confirmed that miR-128-3p was a target of KCNQ1OT1 using online databases, dual luciferase reporter assays and quantitative real-time polymerase chain reaction, and that it inhibited the expression of miR-128-3p. Moreover, we confirmed that miR-128-3p directly targeted nuclear factor of activated T cell 5 (NFAT5), a protein that combines with osteoprotegerin and thus regulates osteoclastogenesis with the presence of the receptor activator of nuclear factor κB ligand. Furthermore, we demonstrated that both the knockdown of KCNQ1OT1 and the overexpression of miR-128-3p attenuate the expression of NFAT5, while upregulating the osteoclastogenesis markers c-Fos, NFATc1, and Ctsk. The results from overexpression of KCNQ1OT1 and the inhibition of miR-128-3p were contrary to the above. Finally, we found that the inhibition of osteoclast differentiation by KCNQ1OT1 overexpression could be rescued using a miR-128-3p mimic, while the enhancement of migration and osteoclast differentiation by si-NFAT5 could be reversed with a miR-128-3p inhibitor. These results suggested that KCNQ1OT1 regulates the osteoclast differentiation via the miR-128-3p/NFAT5 axis.

γ-Secretase inhibitors suppress IL-20-mediated osteoclastogenesis via Notch signalling and are affected by Notch2 in vitro.

Rheumatoid arthritis (RA) is a chronic immune disease involving the small joints, which often causes irreversible damage. In recent years, elevated interleukin 20 (IL-20) has been observed in synovial fluid, while IL-20 receptor overexpression has been observed in synovial cells. IL-20 is a pleiotropic cytokine that participates in various immune diseases. Further understanding of the relationship between IL-20 and RA can help to identify a potential clinical treatment for RA. This study demonstrated that IL-20 can regulate osteoclast differentiation and function in a dose-dependent manner, while influencing the expression of Notch signalling. Quantitative reverse transcription polymerase chain reaction and western blotting showed that γ-secretase-inhibiting drugs can reverse the effects of IL-20. The effects of Notch2 on IL-20-induced osteoclastogenesis were investigated by immunofluorescence and Notch2 gene silencing via transfection of small interfering RNA; the results showed that Notch2 obviously affected the expression levels of the key protein NFATc1 and downstream osteoclastic proteins. In conclusion, we found that IL-20 regulated the osteoclastogenesis in a dose-dependent manner via Notch signalling, primarily by means of Notch2 activity. This study may help to find new targets for RA treatment.

Modulation of Differentiation and Bone Resorbing Activity of Human (Pre-) Osteoclasts After X-Ray Exposure.

Low-dose radiotherapy (LD-RT) is a local treatment option for patients with chronic degenerative and inflammatory diseases, in particular musculoskeletal diseases. Despite reported analgesic and anti-inflammatory effects, cellular and molecular mechanisms related to osteoimmunological effects are still elusive. Here we test the hypothesis that X-irradiation inhibits the differentiation of precursor osteoclasts into mature osteoclasts (mOC) and their bone resorbing activity. Circulating monocytes from healthy donors were isolated and irradiated after attachment with single or fractionated X-ray doses, comparable to an LD-RT treatment scheme. Then monocytes underwent ex vivo differentiation into OC during cultivation up to 21 days, under conditions mimicking the physiological microenvironment of OC on bone. After irradiation, apoptotic frequencies were low, but the total number of OC precursors and mOC decreased up to the end of the cultivation period. On top, we observed an impairment of terminal differentiation, i.e. a smaller fraction of mOC, reduced resorbing activity on bone, and release of collagen fragments. We further analyzed the effect of X-irradiation on multinucleation, resulting from the fusion of precursor OC, which occurs late during OC differentiation. At 21 days after exposure, the observation of smaller cellular areas and a reduced number of nuclei per mOC suggest an impaired fusion of OC precursors to form mOC. Before, at 14 days, the nuclear translocation of Nuclear Factor Of Activated T Cells 1 (NFATc1), a master regulator of osteoclast differentiation and fusion, was decreased. In first results, obtained in the frame of a longitudinal LD-RT study, we previously reported a pain-relieving effect in patients. However, in a subgroup of patients suffering from Calcaneodynia or Achillodynia, we did not observe a consistent decrease of established blood markers for resorption and formation of bone, or modified T cell subtypes involved in regulating these processes. To assess the relevance of changes in bone metabolism for other diseases treated with LD-RT will be subject of further studies. Taken together, we observed that in vitro X-irradiation of monocytes results in an inhibition of the differentiation into bone-resorbing OC and a concomitant reduction of resorbing activity. The detected reduced NFATc1 signaling could be one underlying mechanism.

The Paget's disease of bone risk gene PML is a negative regulator of osteoclast differentiation and bone resorption.

ABSTRACTPaget's disease of bone (PDB) is characterized by focal increases in bone remodelling. Genome-wide association studies identified a susceptibility locus for PDB tagged by rs5742915, which is located within the PML gene. Here, we have assessed the candidacy of PML as the predisposing gene for PDB at this locus. We found that the PDB-risk allele of rs5742915 was associated with lower PML expression and that PML expression in blood cells from individuals with PDB was lower than in controls. The differentiation, survival and resorptive activity of osteoclasts prepared from Pml−/− mice was increased compared with wild type. Furthermore, the inhibitory effect of IFN-γ on osteoclast formation from Pml−/− was significantly blunted compared with wild type. Bone nodule formation was also increased in osteoblasts from Pml−/− mice when compared with wild type. Although microCT analysis of trabecular bone showed no differences between Pml−/− mice and wild type, bone histomorphometry showed that Pml−/− mice had high bone turnover with increased indices of bone resorption and increased mineral apposition rate. These data indicate that reduced expression of PML predisposes an individual to PDB and identify PML as a novel regulator of bone metabolism. This article has an associated First Person interview with the first author of the paper.

First person – Sachin Wani

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping early-career researchers promote themselves alongside their papers. Sachin Wani is first author on ‘The Paget's disease of bone risk gene PML is a negative regulator of osteoclast differentiation and bone resorption’, published in DMM. Sachin is a research assistant (postdoctoral) in the lab of Prof. Omar Albagha at University of Edinburgh, Edinburgh, UK, where he applies knowledge of biomedical science to the investigation of health, development, biological function and disease.

Rab34 plays a critical role as a bidirectional regulator of osteoclastogenesis.

Accumulating evidence suggests that Rab GTPases representing the largest branch of Ras superfamily have recently emerged as the core factors for the regulation of osteoclastogenesis through modulating vesicular transport amongst specific subcellular compartments. Among these, Rab34 GTPase has been identified to be important for the post-Golgi secretory pathway and for phagocytosis; nevertheless, its specific role in osteoclastogenesis has been completely obscure. Here, upon the in vitro model of osteoclast formation derived from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we reveal that Rab34 regulates osteoclastogenesis bidirectionally. More specifically, Rab34 serves as a negative regulator of osteoclast differentiation by promoting the lysosome-induced proteolysis of two osteoclastogenic surface receptors, c-fms and RANK, via the axis of early endosomes-late endosomes-lysosomes, leading to alleviate the transcriptional activity of two of the master regulator of osteoclast differentiation, c-fos and NFATc-1, eventually attenuating osteoclast differentiation and bone resorption. Besides, Rab34 plays a crucial role in modulating the secretory network of lysosome-related proteases including matrix metalloprotease 9 and Cathepsin K across the ruffled borders of osteoclasts, contributing to the regulation of bone resorption.

Dysregulation of the HOTAIR-miR-152-CAMKIIα Axis in Craniosynostosis Results in Impaired Osteoclast Differentiation.

Craniosynostosis is one of the most common craniofacial deformities demanding surgical treatment in infancy. LncRNA HOTAIR has verified its important role in osteogenesis and osteoarthritis. However, whether HOTAIR plays an essential role in the development of craniosynostosis is still unclear. In this study, we aimed to investigate the molecular role of HOTAIR in the osteoclast function and development of craniosynostosis.For osteoclast differentiation, RAW264.7 cells were induced by 50 ng/ml of RANKL and 10 ng/mL M-CSF, followed by TRAP staining. Cell proliferation and apoptosis were assayed by the CCK-8 kit and Annexin V-FITC apoptosis detection kit, respectively. The expression of HOTAIR was determined in PBMCs by qRT-PCR. Protein levels of all those involved genes were measured by Western blot assay. A luciferase reporter assay was used to determine the miRNA target validation. The HOTAIR expression in PBMCs from children with craniosynostosis was significantly downregulated. The results of cell proliferation and apoptosis assays indicated that silencing of HOTAIR could inhibit osteoclast differentiation and increase cell apoptosis. Moreover, the luciferase reporter assay revealed that the regulatory axis and HOTAIR-miR-152-CAMKIIα were the regulatory mechanisms of HOTAIR in the osteoclast function and development of craniosynostosis.In this study, our data showed that HOTAIR could promote osteoclast differentiation by binding miR-152. Furthermore, the HOTAIR/HOTAIR-miR-152-CAMKIIα axis was found to regulate osteoclast differentiation. These results indicate that the HOTAIR plays a crucial role in the development of osteoclasts.

Transcription Factor Hematopoietically Expressed Homeobox Protein (Hhex) Negatively Regulates Osteoclast Differentiation by Controlling Cyclin-Dependent Kinase Inhibitors.

ABSTRACTWe investigated the role of hematopoietically expressed homeobox protein (Hhex) in osteoclast development. Trimethylation of lysine 27 of histone H3 at the cis‐regulatory element of Hhex was maintained and that of lysine 4 was reduced during receptor activator of nuclear factor κB ligand (RANKL)‐induced osteoclastogenesis, which was associated with a reduction of Hhex expression. Overexpression of Hhex in bone marrow–derived macrophages inhibited, whereas Hhex suppression promoted, RANKL‐induced osteoclastogenesis in vitro. Conditional deletion of Hhex in osteoclast‐lineage cells promoted osteoclastogenesis and reduced cancellous bone volume in mice, confirming the negative regulatory role of Hhex in osteoclast differentiation. Expression of cyclin‐dependent kinase inhibitors such as Cdkn2a and Cdkn1b in osteoclast precursors was negatively regulated by Hhex, and Hhex deletion increased the ratio of cells at the G1 phase of the cell cycle. In conclusion, Hhex is an inhibitor of osteoclast differentiation that is regulated in an epigenetic manner and regulates the cell cycle of osteoclast precursors and the skeletal homeostasis. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Exploring the relationship between systemic lupus erythematosus and osteoporosis based on bioinformatics.

This study aimed to explore the relationship between systemic lupus erythematosus (SLE) and osteoporosis (OP) based on bioinformatics. The expression profiles of SLE and OP gene chips were searched through the GEO database, and the differentially expressed genes (DEGs) were screened out to obtain the intersection. Then, the Funrich software was used to predict the upstream miRNAs of the intersection genes, and the miRNA-mRNA relationship network was constructed. Afterward, the String database and Cytoscape software were used to construct the protein interaction network of the intersection genes to screen out the key genes. Finally, the functions and related pathways of key genes were analyzed by using the DAVID database. ①A total of 140 intersection genes of SLE and OP were obtained; ②There were 217 miRNAs regulating the intersection genes; ③IL-4, FOS, TLR1, TLR6, CD40LG, CCR1 were the key genes in the protein interaction network; ④The DAVID enrichment analysis mainly covered the positive regulation of cytokine production, the regulation of osteoclast differentiation, macrophage activation and other biological processes, involving Toll-like receptor signaling pathway, T cell receptor signaling pathway, Th1, Th2, and Th17 cells Differentiation, IL-17 signaling pathway. SLE and OP still have some highly overlapping differential gene expressions under the background of complex gene networks. The gene functions and signaling pathways involved can simultaneously regulate the two diseases, suggesting that there is a close relationship between the molecular mechanisms of the two diseases, and that it may be a target of drugs that interfere with two diseases at the same time.

Regulation of osteoclast differentiation by β-glucan mediated by dectin-1 recognition and pharmacological application

Regulation of osteoclast differentiation by <i>β</i>-glucan mediated by dectin-1 recognition and pharmacological application

Calcitonin Gene-related Peptide Inhibits Osteoclast Differentiation by Inducing the Negative Regulators &lt;i&gt;MafB&lt;/i&gt; and &lt;i&gt;Bcl6&lt;/i&gt;

Calcitonin gene-related peptide (CGRP), a neuropeptide derived from sensory nerves, suppresses osteoclast differentiation; however, the detailed mechanism is unknown. Therefore, in this study, we investigated the mechanism underlying the CGRP-mediated suppression of osteoclast differentiation using a model in which mouse monocyte/macrophage RAW264.7 cells were induced to differentiate into osteoclasts with recombinant mouse soluble receptor activator of nuclear factor-kappa B ligand (s-RANKL). RAW264.7 cells were cultured with s-RANKL in the presence or absence of CGRP for 24–72 h and then quantitative real-time PCR was performed. The mRNA expression of the positive regulators of osteoclast differentiation [i.e., nuclear factor of activated T-cells 1 (Nfatc1) and B lymphocyte-induced maturation protein-1 (Blimp1)], as well as that of the negative regulators of osteoclast differentiation [i.e., MAF bZIP transcription factor B (MafB), B-cell lymphoma 6 (Bcl6), and interferon regulatory factor-8 (Irf8)], was analyzed. In RAW264.7 cells treated with s-RANKL, CGRP significantly suppressed Nfatc1 and Blimp1 mRNA expression at 24 and 48 h. On the contrary, CGRP significantly increased the mRNA expression of MafB at 24 and 48 h of culture and of Bcl6 at 48 and 72 h of culture. However, CGRP did not affect Irf8 mRNA expression. In s-RANKL-untreated RAW264.7 cells, MafB mRNA expression significantly increased from 2 to 8 h, whereas Bcl6 mRNA expression significantly increased from 4 to 24 h of culture. These results suggest that CGRP inhibits osteoclast differentiation by increasing the mRNA expression of MafB and Bcl6, which are negative regulators of osteoclast differentiation.

Silence of TANK-binding kinase 1 (TBK1) regulates extracellular matrix degradation of chondrocyte in osteoarthritis by janus kinase (JAK)-signal transducer of activators of transcription (STAT) signaling

ABSTRACT TANK-binding kinase 1 (TBK1) was previously reported to be critical for the regulation of osteoclast differentiation. However, its function in osteoarthritis (OA) has not yet been determined. This study aims to reveal the role of TBK1 in the extracellular matrix (ECM) degradation in OA. C57BL/6 J mice were subjected to anterior cruciate ligament transection (ACLT) surgery to establish an OA animal model. ATDC5 cells were treated with IL-1β to construct a cell model of OA. Changes in the expression of TBK1 were analyzed by qRT-PCR, Western blotting, and immunohistochemistry. Safranin O-fast green staining, ELISA, and Western blotting were performed to evaluate the ECM degradation. By searching GSE75181 and GSE6119 datasets, TBK1 was found to be highly expressed in the OA model. Its upregulation was also confirmed in ACLT mice and in a cell model of OA. Silencing of TBK1 reduced cartilage degradation, OARSI score, and serum levels of CTX-II and COMP. Silencing of TBK1attenuated ECM degradation, as ADAMTS-4, MMP3, and MMP13 were downregulated, whilst SOX9, collagen II, and aggrecan were upregulated. Furthermore, TBK1 activates the JAK/STAT signaling pathway. Transfection of cells with the STAT3 overexpression plasmid blocked the beneficial effects of TBK1 silencing. In conclusion, TBK1 is highly expressed in OA. Silencing of TBK1 inhibited ECM degradation.

Protocadherin-7 Regulates Osteoclast Differentiation through Intracellular SET-Binding Domain-Mediated RhoA and Rac1 Activation.

Protocadherin-7 (Pcdh7) is a member of the non-clustered protocadherin δ1 subgroup of the cadherin superfamily. Although the cell-intrinsic role of Pcdh7 in osteoclast differentiation has been demonstrated, the molecular mechanisms of Pcdh7 regulating osteoclast differentiation remain to be determined. Here, we demonstrate that Pcdh7 contributes to osteoclast differentiation by regulating small GTPases, RhoA and Rac1, through its SET oncoprotein binding domain. Pcdh7 is associated with SET along with RhoA and Rac1 during osteoclast differentiation. Pcdh7-deficient (Pcdh7−/−) cells showed abolished RANKL-induced RhoA and Rac1 activation, and impaired osteoclast differentiation. Impaired osteoclast differentiation in Pcdh7−/− cells was restored by retroviral transduction of full-length Pcdh7 but not by a Pcdh7 mutant that lacks SET binding domain. The direct crosslink of the Pcdh7 intracellular region induced the activation of RhoA and Rac1, which was not observed when Pcdh7 lacks the SET binding domain. Additionally, retroviral transduction of the constitutively active form of RhoA and Rac1 completely restored the impaired osteoclast differentiation in Pcdh7−/− cells. Collectively, these results demonstrate that Pcdh7 controls osteoclast differentiation by regulating RhoA and Rac1 activation through the SET binding domain.

Cardiotrophin Like Cytokine Factor 1 (CLCF1) alleviates bone loss in osteoporosis mouse models by suppressing osteoclast differentiation through activating interferon signaling and repressing the nuclear factor-κB signaling pathway.

A growing body of evidence suggests that immune factors that regulate osteoclast differentiation and bone resorption might be promising therapeutic agents for the treatment of osteoporosis. The expression of CLCF1, an immune cell-derived molecule, has been reported to be reduced in patients with postmenopausal osteoporosis. This suggests that it may be involved in bone remodeling. Thus, we explored the functional role of CLCF1 in osteoclastogenesis and bone loss associated with osteoporosis. Surprisingly, the administration of recombinant CLCF1 repressed excessive bone loss in ovariectomized mice and prevented RANKL-induced bone loss in calvarial mouse model. Likewise, the addition of recombinant CLCF1 to RANKL-stimulated monocytes resulted in a significant suppression in the number of differentiated osteoclasts with small resorption areas being observed on dentine slices in vitro. At the same dosage, CLCF1 did not exhibit any detectable negative effects on the differentiation of osteoblasts. Mechanistically, the inhibition of osteoclast differentiation by the CLCF1 treatment appears to be related to the activation of interferon signaling (IFN) and the suppression of the NF-κB signaling pathway. Interestingly, the expression of the main components of IFN-signaling namely, STAT1 and IRF1, was detected in macrophages as early as 1h after stimulation with CLCF1. Consistent with these results, the blockade of STAT1 in macrophages abolished the inhibitory effect of CLCF1 on osteoclast differentiation in vitro. These collective findings point to a novel immunoregulatory function of CLCF1 in bone remodeling and highlight it as a potentially useful therapeutic agent for the treatment of osteoporosis.

In vitro and in vivo detection of tunneling nanotubes in normal and pathological osteoclastogenesis involving osteoclast fusion

AbstractOsteoclasts are multinucleated cells formed through specific recognition and fusion of mononuclear osteoclast precursors derived from hematopoietic stem cells. Detailed cellular events concerning cell fusion in osteoclast differentiation remain ambiguous. Tunneling nanotubes (TNTs), actin-based membrane structures, play an important role in intercellular communication between cells. We have previously reported the presence of TNTs in the fusion process of osteoclastogenesis. Here we analyzed morphological details of TNTs using scanning electron microscopy. The osteoclast precursor cell line RAW-D was stimulated to form osteoclast-like cells, and morphological details in the appearance of TNTs were extensively analyzed. Osteoclast-like cells could be classified into three types; early osteoclast precursors, late osteoclast precursors, and multinucleated osteoclast-like cells based on the morphological characteristics. TNTs were frequently observed among these three types of cells. TNTs could be classified into thin, medium, and thick TNTs based on the diameter and length. The shapes of TNTs were dynamically changed from thin to thick. Among them, medium TNTs were often observed between two remote cells, in which side branches attached to the culture substrates and beaded bulge-like structures were often observed. Cell-cell interaction through TNTs contributed to cell migration and rapid transport of information between cells. TNTs were shown to be involved in cell-cell fusion between osteoclast precursors and multinucleated osteoclast-like cells, in which movement of membrane vesicles and nuclei was observed. Formation of TNTs was also confirmed in primary cultures of osteoclasts. Furthermore, we have successfully detected TNTs formed between osteoclasts observed in the bone destruction sites of arthritic rats. Thus, formation of TNTs may be important for the differentiation of osteoclasts both in vitro and in vivo. TNTs could be one target cellular structure for the regulation of osteoclast differentiation and function in bone diseases.