ELMO1 Regulates RANKL-Stimulated Differentiation and Bone Resorption of Osteoclasts.
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ABSTRACT:
SUBMITTER: Liang X
PROVIDER: S-EPMC8350380 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Osteoclasts are large multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage-derived precursors that are thought to undergo apoptosis once resorption is complete. Here, by intravital imaging, we reveal that RANKL-stimulated osteoclasts have an alternative cell fate in which they fission into daughter cells called osteomorphs. Inhibiting RANKL blocked this cellular recycling and resulted in osteomorph accumulation. Single-cell RNA sequencing showed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and express a number of non-canonical osteoclast genes that are associated with structural and functional bone phenotypes when deleted in mice. Furthermore, genetic variation in human orthologs of osteomorph genes causes monogenic skeletal disorders and associates with bone mineral density, a polygenetic skeletal trait. Thus, osteoclasts recycle via osteomorphs, a cell type involved in the regulation of bone resorption that may be targeted for the treatment of skeletal diseases.
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Project description:Vav1 is a Rho/Rac guanine nucleotide exchange factor primarily expressed in hematopoietic cells. In this study, we investigated the potential role of Vav1 in osteoclast (OC) differentiation by comparing the ability of bone marrow mononuclear cells (BMMCs) obtained from Vav1-deficient (Vav1-/-) and wild-type (WT) mice to differentiate into mature OCs upon stimulation with macrophage colony stimulating factor and receptor activator of nuclear kappa B ligand in vitro. Our results suggested that Vav1 deficiency promoted the differentiation of BMMCs into OCs, as indicated by the increased expression of tartrate-resistant acid phosphatase, cathepsin K, and calcitonin receptor. Therefore, Vav1 may play a negative role in OC differentiation. This hypothesis was supported by the observation of more OCs in the femurs of Vav1-/- mice than in WT mice. Furthermore, the bone status of Vav1-/- mice was analyzed in situ and the femurs of Vav1-/- mice appeared abnormal, with poor bone density and fewer number of trabeculae. In addition, Vav1-deficient OCs showed stronger adhesion to vitronectin, an ?v?3 integrin ligand important in bone resorption. Thus, Vav1 may inhibit OC differentiation and protect against bone resorption. [BMB Reports 2019; 52(11): 659-664].
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Project description:The development of multi-nucleated cells is critical for osteoclasts (OCs) maturation and function. Our objective was to extend knowledge on osteoclastogenesis, focusing on pre-OC fusion timing and behavior. RAW 264.7 cells, which is a murine monocyte-macrophage cell line, provide a valuable and widely used tool for in vitro studies on osteoclastogenesis mechanisms. Cells were treated with the receptor activator of nuclear factor κ-B ligand (RANKL) for 1-4 days and effects on cell morphology, cytoskeletal organization, protein distribution, and OC-specific gene expression examined by TEM, immunofluorescence, and qPCR. Multinucleated cells began to appear at two days of Receptor Activator of Nuclear factor κ-B Ligand (RANKL) stimulation, increasing in number and size in the following days, associated with morphological and cytoskeletal organization changes. Interesting cellular extensions were observed in three days within cells labeled with wheat germ agglutinin (WGA)-Fluorescein isothiocyanate (FITC). The membrane, cytoplasmic, or nuclear distribution of RANK, TRAF6, p-p38, pERK1/2, and NFATc1, respectively, was related to OCs maturation timing. The gene expression for transcription factors regulating osteoclastogenesis (NFATc1, c-fos, RelA, MITF), molecules involved in RANKL-signaling transduction (TRAF6), cytoskeleton regulation (RhoA), fusion (DC-STAMP), migration (MMP9), and OC-specific enzymes (TRAP, CtsK), showed different trends related to OC differentiation timing. Our findings provide an integrated view on the morphological and molecular changes occurring during RANKL stimulation of RAW 264.7 cells, which are important to better understand the OCs' maturation processes.
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Project description:Osteopontin (OPN) is a non-collagenous extracellular sialylated glycoprotein located in bone. It is believed to be one of the key components in osteoclast attachment to bone during resorption. In this study, we characterized OPN and other glycoproteins found in the resorption lacunae to confirm the role of osteoclasts in OPN secretion using electron microscopy and mass spectrometry. Additionally, we examined the glycan epitopes of resorption pits and the effects of different glycan epitopes on the differentiation and function of osteoclasts. Osteoarthritic femoral heads were examined by immunohistochemistry to reveal the presence of OPN in areas of increased bone metabolism in vivo. Our results demonstrate that human osteoclasts secrete OPN into resorption lacunae on native human bone and on carbonated hydroxyapatite devoid of natural OPN. OPN is associated with an elevated bone turnover in osteoarthritic bone under experimental conditions. Our data further confirm that osteoclasts secrete OPN into the resorption pit where it may function as a chemokine for subsequent bone formation. We show that ?2,3- and ?2,6-linked sialic acids have a role in the process of osteoclast differentiation. OPN is one of the proteins that has both of the above sialic residues, hence we propose that de-sialylation can effect osteoclast differentiation in bone.
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Project description:The LIM homeobox 2 (Lhx2) transcription factor Lhx2 has a variety of functions, including neural induction, morphogenesis, and hematopoiesis. Here we show the involvement of Lhx2 in osteoclast differentiation. Lhx2 was strongly expressed in osteoclast precursor cells but its expression was significantly reduced during receptor activator of nuclear factor-?B ligand (RANKL)-mediated osteoclastogenesis. Overexpression of Lhx2 in bone marrow-derived monocyte/macrophage lineage cells (BMMs), which are osteoclast precursor cells, attenuated RANKL-induced osteoclast differentiation by inhibiting the induction of nuclear factor of activated T cells c1 (NFATc1). Interestingly, interaction of Lhx2 proteins with c-Fos attenuated the DNA-binding ability of c-Fos and thereby inhibited the transactivation of NFATc1. Furthermore, Lhx2 conditional knockout mice exhibited an osteoporotic bone phenotype, which was related with increased osteoclast formation in vivo. Taken together, our results suggest that Lhx2 acts as a negative regulator of osteoclast formation in vitro and in vivo. The anti-osteoclastogenic effect of Lhx2 may be useful for developing a therapeutic strategy for bone disease.
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Project description:Osteoclasts are multinuclear giant cells that have unique ability to degrade bone. The search for new medicines that modulate the formation and function of osteoclasts is a potential approach for treating osteoclast-related bone diseases. Piceatannol (PIC) is a natural organic polyphenolic stilbene compound found in diverse plants with a strong antioxidant and anti-inflammatory effect. However, the effect of PIC on bone health has not been scrutinized systematically. In this study, we used RAW264.7, an osteoclast lineage of cells of murine macrophages, to investigate the effects and the underlying mechanisms of PIC on osteoclasts. Here, we demonstrated that PIC treatment ranging from 0 to 40 µM strongly inhibited osteoclast formation and bone resorption in a dose-dependent manner. Furthermore, the inhibitory effect of PIC was accompanied by the decrease of osteoclast-specific genes. At the molecular level, PIC suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK1/2), NF-?B p65, I?B? and AKT. Besides, PIC promoted the apoptosis of mature osteoclasts by inducing caspase-3 expression. In conclusion, our results suggested that PIC inhibited RANKL-induced osteoclastogenesis and bone resorption by suppressing MAPK, NF-?B and AKT signalling pathways and promoted caspase3-mediated apoptosis of mature osteoclasts, which might contribute to the treatment of bone diseases characterized by excessive bone resorption.
| S-EPMC6599799 | biostudies-literature
Project description:Osteoclasts resorb bone by attaching on the bone matrix and forming a sealing zone. In Src-deficient mice, osteoclasts cannot form the actin ring, a characteristic actin structure that seals the resorbed area, and resorb hardly any bone as a result. However, the molecular mechanism underlying the role of Src in the regulation and organization of the actin ring is still unclear. We identified an actin-regulatory protein, protein phosphatase 1 regulatory subunit 18 (PPP1r18), as an Src-binding protein in an Src-, Yes-, and Fyn-deficient fibroblast (SYF) cell line overexpressing a constitutively active form of Src. PPP1r18 was localized in the nucleus and actin ring. PPP1r18 overexpression in osteoclasts inhibited terminal differentiation, actin ring formation, and bone-resorbing activity. A mutation of the protein phosphatase 1 (PP1)-binding domain of PPP1r18 rescued these phenotypes. In contrast, PPP1r18 knockdown promoted terminal differentiation and actin ring formation. In summary, we showed that PPP1r18 likely plays a role in podosome organization and bone resorption.
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Project description:Butyrophilins, which are members of the extended B7 family of immunoregulators structurally related to the B7 family, have diverse functions on immune cells as co-stimulatory and co-inhibitory molecules. Despite recent advances in the understanding on butyrophilins' role on adaptive immune cells during infectious or autoimmune diseases, nothing is known about their role in bone homeostasis. Here, we analyzed the role of one specific butyrophilin, namely Btn2a2, as we have recently shown that Btn2a2 is expressed on the monocyte/macrophage lineage that also gives rise to bone degrading osteoclasts. We found that expression of Btn2a2 on monocytes and pre-osteoclasts is upregulated by the receptor activator of nuclear factor κ-B ligand (RANKL), an essential protein required for osteoclast formation. Interestingly, in Btn2a2-deficient osteoclasts, typical osteoclast marker genes (Nfatc1, cathepsin K, TRAP, and RANK) were downregulated following RANKL stimulation. In vitro osteoclast assays resulted in decreased TRAP positive osteoclast numbers in Btn2a2-deficient cells. However, Btn2a2-deficient osteoclasts revealed abnormal fusion processes shown by their increased size. In vivo steady state µCT and histological analysis of bone architecture in complete Btn2a2-deficient mice showed differences in bone parameters further highlighting the fine-tuning effect of BTN2a2. Moreover, in rheumatoid arthritis patients and experimental arthritis, we detected significantly decreased serum levels of the secreted soluble Btn2a2 protein. Taken together, we identified the involvement of the immunomodulatory molecule Btn2a2 in osteoclast differentiation with potential future implications in basic and translational osteoimmunology.
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Project description:Cholesteatoma starts as a retraction of the tympanic membrane and expands into the middle ear, eroding the surrounding bone and causing hearing loss and other serious complications such as brain abscess and meningitis. Currently, the only effective treatment is complete surgical removal, but the recurrence rate is relatively high. In rheumatoid arthritis (RA), osteoclasts are known to be responsible for bone erosion and undergo differentiation and activation by receptor activator of NF-?B ligand (RANKL), which is secreted by synovial fibroblasts, T cells, and B cells. On the other hand, the mechanism of bone erosion in cholesteatoma is still controversial. In this study, we found that a significantly larger number of osteoclasts were observed on the eroded bone adjacent to cholesteatomas than in unaffected areas, and that fibroblasts in the cholesteatoma perimatrix expressed RANKL. We also investigated upstream transcription factors of RANKL using RNA sequencing results obtained via Ingenuity Pathways Analysis, a tool that identifies relevant targets in molecular biology systems. The concentrations of four candidate factors, namely interleukin-1?, interleukin-6, tumor necrosis factor ?, and prostaglandin E2, were increased in cholesteatomas compared with normal skin. Furthermore, interleukin-1? was expressed in infiltrating inflammatory cells in the cholesteatoma perimatrix. This is the first report demonstrating that a larger-than-normal number of osteoclasts are present in cholesteatoma, and that the disease involves upregulation of factors related to osteoclast activation. Our study elucidates the molecular basis underlying bone erosion in cholesteatoma.
| S-EPMC6797677 | biostudies-literature
Project description:BackgroundResolvin E1 (RvE1) derived from the ω-3 polyunsaturated fatty acid eicosapentaenoic acid is known to be a potent pro-resolving lipid mediator that prevents chronic inflammation and osteoclastogenesis. We investigated the inhibitory effects of RvE1 on osteoclastogenesis and bone resorption to clarify its therapeutic potential for rheumatoid arthritis (RA).MethodsReceptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation was assessed with tartrate-resistant acid phosphatase staining. RANKL-induced bone resorption was assessed by the measurement of pit formation using calcium phosphate-labeled fluorescent polyanionic molecules in RAW264.7 cells as osteoclast precursors. The effects of RvE1 on the RANKL-induced mRNA expression of osteoclast-specific genes and transcriptional factors such as c-fos and nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells were measured by quantitative real-time PCR. The distribution of NFATc1 induced by RANKL was evaluated by immunofluorescence staining in RAW264.7 cells. To analyze the mechanism of the inhibitory effect of RvE1 on osteoclastogenesis, we measured IL-17-induced RANKL mRNA expression in MC3T3-E1 osteoblast cells treated with RvE1 using quantitative real-time PCR and determined the level of prostaglandin E2 (PGE2) production by enzyme-linked immunosorbent assay.ResultsRvE1 significantly suppressed RANKL-induced osteoclast differentiation and bone resorption. RvE1 inhibited the RANKL-induced mRNA expression of osteoclast-specific genes along with the transcription factors NFATc1 and c-fos. Moreover, NFATc1 translocation from the cytoplasm to the nucleus of RAW264.7 cells was suppressed following RvE1 treatment. RvE1 also inhibited IL-17-induced RANKL mRNA expression and PGE2 production in MC3T3-E1 cells.ConclusionRvE1 inhibited osteoclastogenesis and bone resorption by suppressing RANKL-induced NFATc1 and c-fos expression in osteoclasts and IL-17-induced RANKL expression through the autocrine action of PGE2 in osteoblasts. Our data suggest RvE1 as a new therapeutic target of RA.
| S-EPMC5871721 | biostudies-literature