Osteoclast stimulatory transmembrane protein and dendritic cell–specific transmembrane protein cooperatively modulate cell–cell fusion to form osteoclasts and foreign body giant cells

Cell–cell fusion is a dynamic phenomenon promoting cytoskeletal reorganization and phenotypic changes. To characterize factors essential for fusion of macrophage lineage cells, we identified the multitransmembrane protein, osteoclast stimulatory transmembrane protein (OC-STAMP), and analyzed its function. OC-STAMP–deficient mice exhibited a complete lack of cell–cell fusion of osteoclasts and foreign body giant cells (FBGCs), both of which are macrophage-lineage multinuclear cells, although expression of dendritic cell specific transmembrane protein (DC-STAMP), which is also essential for osteoclast/FBGC fusion, was normal. Crossing OC-STAMP–overexpressing transgenic mice with OC-STAMP–deficient mice restored inhibited osteoclast and FBGC cell–cell fusion seen in OC-STAMP–deficient mice. Thus, fusogenic mechanisms in macrophage-lineage cells are regulated via OC-STAMP and DC-STAMP.     

The small molecule harmine regulates NFATc1 and Id2 expression in osteoclast progenitor cells

Small molecule compounds that potently affect osteoclastogenesis could be useful as chemical probes for elucidating the mechanisms of various biological phenomena and as effective therapeutic strategies against bone resorption. An osteoclast progenitor cell-based high-throughput screening system was designed to target activation of NFAT, which is a key event for osteoclastogenesis. Orphan ligand library screening using this system identified the β-carboline derivative harmine, which is a highly potent inhibitor of dual-specificity tyrosine-phosphorylation regulated kinase 1A (DYRK1A), to be an NFAT regulator in osteoclasts. RAW264.7 cells highly expressed DYRK1A protein, and in vitro phosphorylation assay demonstrated that harmine directly inhibited the DYRK1A-mediated phosphorylation (in-activation) of NFATc1. Harmine promoted the dephosphorylation (activation) of NFATc1 in RAW264.7 cells within 24h, and it significantly increased the expression of NFATc1 in RAW264.7 cells and mouse primary bone marrow macrophages (BMMs) both in the presence and absence of RANKL stimulation. Although harmine promoted NFATc1 expression and stimulated target genes for osteoclastogenesis, cell-cell fusion and the formation of TRAP-positive multinucleated osteoclasts from RAW264.7 cells and BMMs was significantly inhibited by harmine treatment. Meanwhile, harmine remarkably promoted the expression of inhibitor of DNA binding/differentiation-2 (Id2), which is a negative regulator for osteoclastogenesis, in RAW264.7 cells and BMMs. An Id2-null-mutant showed slightly increased osteoclast formation from BMMs, and the harmine-mediated inhibition of osteoclast formation was abolished in the BMMs of Id2-null-mutant mice. These results suggest that harmine is a potent activator of NFATc1 that interferes with the function of DYRK1A in osteoclast precursors and also up-regulates Id2 protein, which may dominantly inhibit expression pathways associated with cell-cell fusion, thereby leading to the disruption of the fusion events mediating osteoclastogenesis. The small molecule harmine is therefore expected to provide an experimental tool for investigating signaling cascades in osteoclastogenesis, especially those centered on DYRK1A-mediated NFATc1 and Id2 regulation.     

Role of DC-STAMP in cellular fusion of osteoclasts and macrophage giant cells

Osteoclasts are the only cells that can resorb bone matrix physiologically and maintain the bone content. Osteoclasts are derived from macrophage/monocyte lineage cells; stimulation by macrophage colony stimulating factor and receptor activator of NFκB ligand induces osteoclastogenesis. During osteoclastogenesis, preosteoclasts fuse to form multinuclear mature osteoclasts. Cellular fusion is a unique phenomenon and enables fertilization, myotube formation, and efficient bone resorption in vertebrates. To date, several molecules have been reported to be fusion related in osteoclasts, namely CD44, CD47, ADAM12, MCP-1, and CD9, although the molecules which regulate osteoclast cellular fusion remain unclear. Here, we show that the seven-transmembrane-region receptor dendritic cell-specific transmembrane protein (DC-STAMP) is required for cell–cell fusion of osteoclasts and foreign body giant cells.     

E-cadherin is important for cell differentiation during osteoclastogenesis

E-cadherin, a protein responsible for intercellular adhesion between epithelial cells, is also expressed in the monocyte/macrophage lineage. In this study we have explored the involvement of E-cadherin during receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclast differentiation. Osteoclastogenesis involves a period of precursor expansion followed by multiple fusion events to generate a multinuclear osteoclast that is capable of bone resorption. We asked whether E-cadherin participated in early precursor interactions and recognition or was a component of the osteoclast fusion machinery. Here, we show that endogenous E-cadherin expression is the highest during early stages of osteoclast differentiation, with surface expression visible on small precursor cells (fewer than four nuclei per cell) in both RAW 264.7 cells and primary macrophages. Blocking E-cadherin function with neutralizing antibodies prior to the onset of fusion delayed the expression of TRAP, Cathepsin K, DC-STAMP and NFATc1 and significantly diminished multinucleated osteoclast formation. Conversely, E-cadherin-GFP overexpressing macrophages displayed earlier NFATc1 nuclear translocation along with faster formation of multinucleated osteoclasts compared to control macrophages. Through live imaging we identified that disrupting E-cadherin function prolonged the proliferative phase of the precursor population while concomitantly decreasing the proportion of migrating precursors. The lamellipodium and polarized membrane extensions appeared to be the principal sites of fusion, indicating precursor migration was a critical factor contributing to osteoclast fusion. These findings demonstrate that E-cadherin-mediated cell–cell contacts can modulate osteoclast-specific gene expression and prompt differentiating osteoclast precursors toward migratory and fusion activities.     

Sialylation of cell surface glycoconjugates is essential for osteoclastogenesis

Sialic acid, which is located at the end of the carbohydrate moiety of cell surface glycoconjugates, is involved in many biologic responses, such as intercellular reactions and virus-cell fusion, especially in hematopoietic cells. Here we provide experimental evidence that the sialic acid of cell surface glycoconjugates has a role in osteoclast differentiation. Lectin histochemical study demonstrated the existence of both alpha (2,3)-linked-sialic acid and alpha (2,6)-linked-sialic acid in mouse bone marrow-derived macrophages and in the RAW264.7 macrophage cell line, which are osteoclast precursors. Flow cytometric analysis of surface lectin staining revealed the kinetics of these sialic acids during osteoclastogenesis: alpha (2,3)-linked-sialic acid was abundantly expressed throughout osteoclastogenesis, whereas alpha (2,6)-linked-sialic acid levels declined at the terminal stage of osteoclast differentiation. To investigate the role of sialic acid in osteoclast differentiation, we performed an osteoclastogenesis assay with or without exogenous sialidase treatment. Desialylated cells formed TRAP-positive mononuclear cells, but did not become multinuclear cells despite the normal expression of osteoclast markers such as cathepsin K, integrin beta3, and nuclear factor-ATc1. Flow cytometric analysis also demonstrated that exogenous sialidase effectively removed alpha (2,6)-linked-sialic acid, but only slightly changed the alpha (2,3)-linked-sialic acid content, suggesting that alpha (2,6)-linked-sialic acid might be involved in osteoclast differentiation. Findings from knockdown analysis using small interfering RNA oligonucleotides against alpha 2,6-sialyltransferase support this idea: alpha (2,6)-linked-sialic acid-deficient cells markedly inhibit the formation of multinuclear osteoclasts. Our findings suggest that alpha (2,6)-linked-sialic acid of cell surface glycoconjugates has a role in osteoclast differentiation, possibly via its role in the cell-cell fusion process.     

Osteoclastogenesis is Influenced by Modulation of Gap Junctional Communication with Antiarrhythmic Peptides

Osteoclasts are formed by the fusion of mononuclear precursor cells of the monocyte–macrophage lineage. Among several putative mechanisms, gap-junctional intercellular communication (GJC) has been proposed to have a role in osteoclast fusion and bone resorption. We examined the role of GJC in osteoclastogenesis and in vitro bone resorption with mouse bone marrow hematopoietic stem cells and RAW 264.7 cells. Blocking of gap junctions with 18-α-glycyrrhetinic acid (18GA) led to inhibition of osteoclastogenesis and in vitro bone resorption. Similarly, the GJC inhibitor GAP27 inhibited osteoclast formation. GJC modulation with the antiarrhythmic peptides (AAPs) led to increased amounts of multinuclear RAW 264.7 osteoclasts as well as increased number of nuclei per multinuclear cell. In the culture of bone marrow hematopoietic stem cells in the presence of bone marrow stromal cells AAP reduced the number of osteoclasts, and coculture of MC3T3-E1 preosteoblasts with RAW 264.7 macrophages prevented the action of AAPs to promote osteoclastogenesis. The present data indicate that AAPs modulate the fusion of the pure culture of cells of the monocyte–macrophage lineage. However, the fusion is influenced by GJC in cells of the osteoblast lineage.     

Lentivirus-mediated RNA interference of DC-STAMP expression inhibits the fusion and resorptive activity of human osteoclasts

Cell–cell fusion is a critical step in osteoclast development, because only after osteoclasts become multinucleated can they efficiently resorb bone. Although recent studies suggest that dendritic cell-specific transmembrane protein (DC-STAMP) may play a key role in the process of fusion of mouse osteoclasts, little information has been available on the role of DC-STAMP in human osteoclasts. In this study, we screened and identified an in vitro-transcribed short-hairpin RNA targeting human DC-STAMP from four candidates and generated a lentivirus vector. Subsequent experiments indicated that this lentiviral transgenic system could effectively transfer into target human osteoclasts, at more than 80 % gene transfer efficiency at multiplicity of infection of 15, and significantly and specifically inhibited DC-STAMP expression at both mRNA and protein levels. We also found that DC-STAMP inhibition by RNAi consequently suppressed fusion and bone resorption of human osteoclasts. In conclusion, these data indicated the lentivirus-mediated RNAi was capable of efficiently suppressing DC-STAMP expression in primary human osteoclasts and inhibiting osteoclastogenesis, demonstrating an essential role of DC-STAMP in the differentiation of human osteoclasts.     

Regulation of RANKL‐induced osteoclastogenesis by RING finger protein RNF114

Normal bone remodeling is a continuous process orchestrated by bone‐resorbing osteoclasts and bone‐forming osteoblasts, which an imbalance in bone remodeling results in metabolic bone diseases. RANKL, a member of the TNF cytokine family, functions as a key stimulator for osteoclast differentiation and maturation. Here, we report that RNF114, previously identified as a psoriasis susceptibility gene, plays a regulatory role in the RANKL/RANK/TRAF6 signaling pathway that mediates osteoclastogenesis. Our results demonstrated that RNF114 expression was significantly down‐regulated in mouse osteoclast precursor cells undergoing RANKL‐induced osteoclast differentiation. RNF114 knockout did not affect development or viability of the subpopulation of bone marrow macrophages capable of differentiating into osteoclasts in culture. However, in the presence of RANKL, RNF114 knockout bone marrow macrophages exhibited enhanced cell proliferation and augmented osteoclast differentiation, as shown by an increased expression of mature osteoclast markers, increased osteoclastic TRAP activity and bone resorption. Conversely, ectopic expression of RNF114 inhibited CTSK expression, TRAP activity, and bone resorption in RANKL‐treated pre‐osteoclasts. RNF114 also suppressed RANKL‐activated NFATc1 expression and NFAT‐regulated promoter activity. RNF114 suppressed TRAF6‐, but not TAK1/TAB2‐mediated NF‐κB activation downstream of RANKL/RANK. In particular, TRAF6 protein levels were down‐regulated by RNF114, possibly via K48‐mediated proteasome‐dependent degradation. These data suggested that RNF114's inhibitory effect on RANKL‐stimulated osteoclastogenesis was mediated by blocking RANK/TRAF6/NF‐κB signal transduction. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:159–166, 2018.     

Detection of osteoclastic cell-cell fusion through retroviral vector packaging

Cell-cell fusion generates multinucleated cells such as osteoclasts in bone, myotubes in muscle, and trophoblasts in placenta. Molecular details governing these fusion processes are still largely unknown. As a step toward identification of fusogenic genes, we tested the concept that retroviral vectors can be packaged as a result of cell-cell fusion. First, we introduced replication-deficient retroviral vectors expressing mCAT-1, which mediates fusogenic interaction with the retroviral envelope protein Env, into Chinese hamster ovary (CHO) cells to generate vector cells. Plasmids expressing virion proteins Gag, Pol, and Env were introduced into a separate culture of CHO cells to generate packaging cells. Co-culturing vector and packaging cells resulted in production of infectious retroviruses carrying the mCAT-1 gene as a consequence of cell-cell fusion. Second, we introduced a retroviral vector into primary osteoclast precursors and co-cultured them with established osteoclast precursor RAW264.7 cells, which turned out to harbor packaging activity. Packaged retroviral vector was detected in culture supernatants only where the osteoclast differentiation factor receptor activator for NF-kappaB ligand (RANKL) induced fusion between these two cell types. These data suggest that retrovirus production can occur as a result of cell-cell fusion. This provides a novel approach for isolating and characterizing fusogenic genes using retroviral expression vectors.     

NF-kappaB p50 and p52 expression is not required for RANK-expressing osteoclast progenitor formation but is essential for RANK- and cytokine-mediated osteoclastogenesis.

Expression of RANKL by stromal cells and of RANK and both NF-kappaB p50 and p52 by osteoclast precursors is essential for osteoclast formation. To examine further the role of RANKL, RANK, and NF-KB signaling in this process, we used NF-kappaB p50-/- ;p52-/- double knockout (dKO) and wild-type (WT) mice. Osteoclasts formed in cocultures of WT osteoblasts with splenocytes from WT mice but not from dKO mice, a finding unchanged by addition of RANKL and macrophage colony-stimulating factor (M-CSF). NF-kappaB dKO splenocytes formed more colony-forming unit granulocyte macrophage (CFU-GM) colonies than WT cells, but no osteoclasts were formed from dKO CFU-GM colonies. RANKL increased the number of CFU-GM colonies twofold in WT cultures but not in dKO cultures. Fluorescence-activated cell sorting (FACS) analysis of splenocytes from NF-kappaB dKO mice revealed a two-to threefold increase in the percentage of CD11b (Mac-1) and RANK double-positive cells compared with WT controls. Treatment of NF-kappaB dKO splenocytes with interleukin (IL)-1, TNF-alpha, M-CSF, GM-CSF, and IL-6 plus soluble IL-6 receptor did not rescue the osteoclast defect. No increase in apoptosis was observed in cells of the osteoclast lineage in NF-kappaB dKO or p50-/-;p52+/- (3/4KO) mice. Thus, NF-kappaB p50 and p52 expression is not required for formation of RANK-expressing osteoclast progenitors but is essential for RANK-expressing osteoclast precursors to differentiate into TRAP+ osteoclasts in response to RANKL and other osteoclastogenic cytokines.     

Specific RANK Cytoplasmic Motifs Drive Osteoclastogenesis

Upon receptor activator of NF-κB ligand (RANKL) binding, RANK promotes osteoclast formation through the recruitment of tumor necrosis factor (TNF) receptor-associated factors (TRAFs). In vitro assays identified two RANK intracellular motifs that bind TRAFs: PVQEET^560–565 (Motif 2) and PVQEQG^604–609 (Motif 3), which potently mediate osteoclast formation in vitro. To validate the in vitro findings, we have generated knock-in (KI) mice harboring inactivating mutations in RANK Motifs 2 and 3. Homozygous KI (RANK^KI/KI) mice are born at the predicted Mendelian frequency and normal in tooth eruption. However, RANK^KI/KI mice exhibit significantly more trabecular bone mass than age- and sex-matched heterozygous KI (RANK^+/KI) and wild-type (RANK^+/+) counterparts. Bone marrow macrophages (BMMs) from RANK^KI/KI mice do not form osteoclasts when they are stimulated with macrophage colony-stimulating factor (M-CSF) and RANKL in vitro. RANKL is able to activate the NF-κB, ERK, p38, and JNK pathways in RANK^KI/KI BMMs, but it cannot stimulate c-Fos or NFATc1 in the RANK^KI/KI cells. Previously, we showed that RANK signaling plays an important role in Porphyromonas gingivalis (Pg)-mediated osteoclast formation by committing BMMs into the osteoclast lineage. Here, we show that RANKL-primed RANK^KI/KI BMMs are unable to differentiate into osteoclasts in response to Pg stimulation, indicating that the two RANK motifs are required for Pg-induced osteoclastogenesis. Mechanistically, RANK Motifs 2 and 3 facilitate Pg-induced osteoclastogenesis by stimulating c-Fos and NFATc1 expression during the RANKL pretreatment phase as well as rendering c-Fos and NFATc1 genes responsive to subsequent Pg stimulation. Cell-penetrating peptides (CPPs) conjugated with RANK segments containing Motif 2 or 3 block RANKL- and Pg-mediated osteoclastogenesis. The CPP conjugates abrogate RANKL-stimulated c-Fos and NFATc1 expression but do not affect RANKL-induced activation of NF-κB, ERK, p38, JNK, or Akt signaling pathway. Taken together, our current findings demonstrate that RANK Motifs 2 and 3 play pivotal roles in osteoclast formation in vivo and mediate Pg-induced osteoclastogenesis in vitro.