Human osteoclast-poor osteopetrosis with hypogammaglobulinemia due to TNFRSF11A (RANK) mutations

Autosomal-Recessive Osteopetrosis (ARO) comprises a heterogeneous group of bone diseases for which mutations in five genes are known as causative. Most ARO are classified as osteoclast-rich, but recently a subset of osteoclast-poor ARO has been recognized as due to a defect in TNFSF11 (also called RANKL or TRANCE, coding for the RANKL protein), a master gene driving osteoclast differentiation along the RANKL-RANK axis. RANKL and RANK (coded for by the TNFRSF11A gene) also play a role in the immune system, which raises the possibility that defects in this pathway might cause osteopetrosis with immunodeficiency. From a large series of ARO patients we selected a Turkish consanguineous family with two siblings affected by ARO and hypogammaglobulinemia with no defects in known osteopetrosis genes. Sequencing of genes involved in the RANKL downstream pathway identified a homozygous mutation in the TNFRSF11A gene in both siblings. Their monocytes failed to differentiate in vitro into osteoclasts upon exposure to M-CSF and RANKL, in keeping with an osteoclast-intrinsic defect. Immunological analysis showed that their hypogammaglobulinemia was associated with impairment in immunoglobulin-secreting B cells. Investigation of other patients revealed a defect in both TNFRSF11A alleles in six additional, unrelated families. Our results indicate that TNFRSF11A mutations can cause a clinical condition in which severe ARO is associated with an immunoglobulin-production defect.     

Connection between B lymphocyte and osteoclast differentiation pathways

Osteoclasts differentiate from the hemopoietic monocyte/macrophage cell lineage in bone marrow through cell-cell interactions between osteoclast progenitors and stromal/osteoblastic cells. Here we show another osteoclast differentiation pathway closely connected with B lymphocyte differentiation. Recently the TNF family molecule osteoclast differentiation factor/receptor activator of NF-kappaB ligand (ODF/RANKL) was identified as a key membrane-associated factor regulating osteoclast differentiation. We demonstrate that B-lymphoid lineage cells are a major source of endogenous ODF/RANKL in bone marrow and support osteoclast differentiation in vitro. In addition, B-lymphoid lineage cells in earlier developmental stages may hold a potential to differentiate into osteoclasts when stimulated with M-CSF and soluble ODF/RANKL in vitro. B-lymphoid lineage cells may participate in osteoclastogenesis in two ways: they 1) express ODF/RANKL to support osteoclast differentiation, and 2) serve themselves as osteoclast progenitors. Consistent with these observations in vitro, a decrease in osteoclasts is associated with a decrease in B-lymphoid cells in klotho mutant mice (KL(-/-)), a mouse model for human aging that exhibits reduced turnover during bone metabolism, rather than a decrease in the differentiation potential of osteoclast progenitors. Taken together, B-lymphoid lineage cells may affect the pathophysiology of bone disorders through regulating osteoclastogenesis.     

PTEN regulates RANKL- and osteopontin-stimulated signal transduction during osteoclast differentiation and cell motility

PTEN (also known as MMAC-1 or TEP-1) is a frequently mutated tumor suppressor gene in human cancer. PTEN functions have been identified in the regulation of cell survival, growth, adhesion, migration, and invasiveness. Here, we characterize the diverse signaling networks modulated by PTEN in osteoclast precursors stimulated by RANKL and osteopontin (OPN). RANKL dose-dependently stimulated transient activation of Akt before activation of PTEN, consistent with a role for PTEN in decreasing Akt activity. PTEN overexpression blocked RANKL-activated Akt stimulated survival and osteopontin-stimulated cell migration while a dominant-negative PTEN increased the actions of RANKL and OPN. PTEN overexpression suppressed RANKL-mediated osteoclast differentiation and OPN-stimulated cell migration. The PTEN dominant-negative constitutively induced osteoclast differentiation and cell migration. Our data demonstrate multiple roles for PTEN in RANKL-induced osteoclast differentiation and OPN-stimulated cell migration in RAW 264.7 osteoclast precursors.     

Estrogen inhibits RANKL-stimulated osteoclastic differentiation of human monocytes through estrogen and RANKL-regulated interaction of estrogen receptor-α with BCAR1 and Traf6

The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at ∼ 1 nM reduced RANKL-dependent osteoclast differentiation by 40-50%. Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 h. In CD14+ cells apoptosis was rare and was not augmented by RANKL or by 17-β-estradiol. Estrogen receptor-α (ERα) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ERα. However, ERα was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ERα in the presence of estrogen, was abundant. Immunoprecipitation showed rapid (∼ 5 min) estrogen-dependent formation of ERα-BCAR1 complexes, which were increased by RANKL co-treatment. The RANKL-signaling intermediate Traf6, which regulates NF-κB activity, precipitated with this complex. Reduction of NF-κB nuclear localization occurred within 30 min of RANKL stimulation, and estradiol inhibited the phosphorylation of IκB in response to RANKL. Inhibition by estradiol was abolished by siRNA knockdown of BCAR1. We conclude that estrogen directly, but only partially, curtails human osteoclast formation. This effect requires BCAR1 and involves a non-genomic interaction with ERα.     

Osteoporosis regulation by salubrinal through eIF2α mediated differentiation of osteoclast and osteoblast

Nuclear factor-κB (NF-κB) ligand (RANKL) was shown to induce osteoclast differentiation by increasing the expression of c-Fos, NFATc1 and TRAP. Salubrinal treatment to bone marrow macrophage (BMM) cells, however, significantly blocked NFATc1 expression and osteoclast differentiation by RANKL. Overexpression of NFATc1 further confirmed that NFATc1 is a key factor affected by salubrinal in osteoclast differentiation by RANKL. Unexpectedly, NFATc1 and c-Fos mRNA expressions were not affected by salubrinal, implicating that NFATc1 expression is regulated at a translational stage. In support of this, salubrinal increased the phosphorylation of a translation factor eIF2α, decreasing the global protein synthesis including NFATc1. In contrast, a phosphorylation mutant plasmid pLenti-eIF2α-S51A restored RANKL-induced NFATc1 expression and osteoclast differentiation even in the presence of salubrinal. Furthermore, knockdown of ATF4 significantly reduced salubrinal-induced osteoblast differentiation as evidenced by decreased calcium accumulation and lowered expressions of the osteoblast differentiation markers, alkaline phosphatase and RANKL in MC3T3-E1 osteoblast cells. Salubrinal treatment to co-cultured BMM and MC3T3-E1 cells also showed reduction of osteoclast differentiation. Finally, salubrinal efficiently blocked osteoporosis in mice model treated with RANKL as evidenced by elevated bone mineral density (BMD) and other osteoporosis factors. Collectively, our data indicate that salubrinal could affect the differentiation of both osteoblast and osteoclast, and be developed as an excellent anti-osteoporosis drug. In addition, modulation of ATF4 and NFATc1 expressions through eIF2α phosphorylation could be a valuable target for the treatment of osteoporosis.     

Siglec-15, a member of the sialic acid-binding lectin, is a novel regulator for osteoclast differentiation

Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells derived from monocyte/macrophage-lineage precursors and are critically responsible for bone resorption. In giant cell tumor of bone (GCT), numerous TRAP-positive multinucleated giant cells emerge and severe osteolytic bone destruction occurs, implying that the emerged giant cells are biologically similar to osteoclasts. To identify novel genes involved in osteoclastogenesis, we searched genes whose expression pattern was significantly different in GCT from normal and other bone tumor tissues. By screening a human gene expression database, we identified sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) as one of the genes markedly overexpressed in GCT. The mRNA expression level of Siglec-15 increased in association with osteoclast differentiation in cultures of mouse primary unfractionated bone marrow cells (UBMC), RAW264.7 cells of the mouse macrophage cell line and human osteoclast precursors (OCP). Treatment with polyclonal antibody to mouse Siglec-15 markedly inhibited osteoclast differentiation in primary mouse bone marrow monocyte/macrophage (BMM) cells stimulated with receptor activator of nuclear factor κB ligand (RANKL) or tumor necrosis factor (TNF)-α. The antibody also inhibited osteoclast differentiation in cultures of mouse UBMC and RAW264.7 cells stimulated with active vitamin D₃ and RANKL, respectively. Finally, treatment with polyclonal antibody to human Siglec-15 inhibited RANKL-induced TRAP-positive multinuclear cell formation in a human OCP culture. These results suggest that Siglec-15 plays an important role in osteoclast differentiation.     

MCP-5 suppresses osteoclast differentiation through Ccr5 upregulation

Human monocyte chemoattractant protein-1 (MCP-1) in mice has two orthologs, MCP-1 and MCP-5. MCP-1, which is highly expressed in osteoclasts rather than in osteoclast precursor cells, is an important factor in osteoclast differentiation. However, the roles of MCP-5 in osteoclasts are completely unknown. In this study, contrary to MCP-1, MCP-5 was downregulated during receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation and was considered an inhibitory factor in osteoclast differentiation. The inhibitory role of MCP-5 in osteoclast differentiation was closely related to the increase in Ccr5 expression and the inhibition of IκB degradation by RANKL. Transgenic mice expressing MCP-5 controlled by Mx-1 promoter exhibited an increased bone mass because of a decrease in osteoclasts. This result strongly supported that MCP-5 negatively regulated osteoclast differentiation. MCP-5 also prevented severe bone loss caused by RANKL.     

Defective osteoclastogenesis by IKKbeta-null precursors is a result of receptor activator of NF-kappaB ligand (RANKL)-induced JNK-dependent apoptosis and impaired differentiation

It has been reported previously that inhibitory kappaB kinase (IKK) supports osteoclastogenesis through NF-kappaB-mediated prevention of apoptosis. This finding suggests that the ligand for receptor activator of NF-kappaB (RANKL), the master osteoclastogenic cytokine, induces apoptosis of osteoclast precursors (OCPs) in the absence of IKKbeta/NF-kappaB competency. To validate this hypothesis, we sought to determine the pro-apoptotic signaling factors induced by RANKL in IKKbeta-null osteoclast OCPs and to rescue osteoclast differentiation in the absence of IKKbeta through their inhibition. To accomplish this, we generated mice that lack IKKbeta in multiple hematopoietic lineages, including OCPs. We found that these mice possess both in vitro and in vivo defects in osteoclast generation, in concurrence with previous reports, and that this defect is a result of susceptibility to RANKL-mediated apoptosis as a result of gain-of-function of JNK activation. We demonstrate that differentiation of OCPs depends on IKKbeta because reduced IKKbeta mRNA expression correlates with impaired induction of osteoclast differentiation markers in response to RANKL stimulation. We further show that fine-tuned inhibition of JNK activation in these cells inhibits RANKL-induced apoptosis and restores the ability of IKKbeta-null OCPs to become mature osteoclasts. Our data highlight the pro-osteoclastogenic and anti-apoptotic roles of IKKbeta in OCPs and identify a pro-apoptotic mechanism activated within the RANK signalosome.     

Direct inhibition of human RANK+ osteoclast precursors identifies a homeostatic function of IL-1beta

IL-1β is a key mediator of bone resorption in inflammatory settings, such as rheumatoid arthritis (RA). IL-1β promotes osteoclastogenesis by inducing RANKL expression on stromal cells and synergizing with RANKL to promote later stages of osteoclast differentiation. Because IL-1Rs share a cytosolic Toll-IL-1R domain and common intracellular signaling molecules with TLRs that can directly inhibit early steps of human osteoclast differentiation, we tested whether IL-1β also has suppressive properties on osteoclastogenesis in primary human peripheral blood monocytes and RA synovial macrophages. Early addition of IL-1β, prior to or together with RANKL, strongly inhibited human osteoclastogenesis as assessed by generation of TRAP(+) multinucleated cells. IL-1β acted directly on human osteoclast precursors (OCPs) to strongly suppress expression of RANK, of the costimulatory triggering receptor expressed on myeloid cells 2 receptor, and of the B cell linker adaptor important for transmitting RANK-induced signals. Thus, IL-1β rendered early-stage human OCPs refractory to RANK stimulation. Similar inhibitory effects of IL-1β were observed using RA synovial macrophages. One mechanism of RANK inhibition was IL-1β-induced proteolytic shedding of the M-CSF receptor c-Fms that is required for RANK expression. These results identify a homeostatic function of IL-1β in suppressing early OCPs that contrasts with its well-established role in promoting later stages of osteoclast differentiation. Thus, the rate of IL-1-driven bone destruction in inflammatory diseases, such as RA, can be restrained by its direct inhibitory effects on early OCPs to limit the extent of inflammatory osteolysis.     

Clinical development of anti-RANKL therapy

The receptor activator of nuclear factor-kappaB ligand (RANKL), its cognate receptor RANK, and its natural decoy receptor osteoprotegerin have been identified as the final effector molecules of osteoclastic bone resorption. This has provided an ideal target for therapeutic interventions in metabolic bone disease. As described in previous reviews in this supplement, RANKL signaling is required for osteoclast differentiation, activation, and survival. Furthermore, in vivo inhibition of RANKL leads to immediate osteoclast apoptosis, and there are no in vivo models of bone resorption that are refractory to RANKL inhibition. Thus, the only step remaining in the development of a clinical intervention is the generation of a safe, effective, and specific drug that can inhibit RANKL in humans. Here we review the clinical development of denosumab (formerly known as AMG 162), which is a fully human mAb directed against RANKL. This discussion includes the breadth of 21 human studies that have led to the current phase 3 clinical trials seeking approval for use of this agent to treat postmenopausal women with low bone mineral density (osteoporosis) and patients with metastatic lytic bone lesions (multiple myeloma, and prostate and breast cancer).     

Cloning and characterization of osteoclast precursors from the raw264.7 cell line

Summary Osteoclasts are bone-resorbing cells that differentiate from macrophage precursors in response to receptor activator of NF-κB ligand (RANKL). In vitro models of osteoclast differentiation are principally based on primary cell cultures, which are poorly suited to molecular and transgene studies because of the limitations associated with the use of primary macrophage. RAW264.7 is a transfectable macrophage cell line with the capacity to form osteoclast-like cells. In the present study, we have identified osteoclast precursors among clones of RAW264.7 cells. RAW264.7 cell were cloned by limiting dilution and induced to osteoclast differentiation by treatment with recombinant RANKL. Individual RAW264.7 cell clones formed tartrate resistant acid phosphatase (TRAP)-positive multinuclear cells to various degrees with RANKL treatment. All clones tested expressed the RANKL receptor RANK. Each of the clones expressed the osteoclast marker genes TRAP and cathepsin-K mRNA with RANKL treatment. However, we noted that only select clones were able to form large, well-spread, TRAP-positive multinuclear cells. Clones capable of forming large TRAP-positive multinuclear cells also expressed β₃ integrin and calcitonin receptor mRNAs and were capable of resorbing a mineralized matrix. All clones tested activated NF-κB with RANKL treatment. cDNA expression profiling of osteoclast precursor RAW264.7 cell clones demonstrates appropriate expression of a large number of genes before and after osteoclastic differentiation. These osteoclast precursor RAW264.7 cell clones provide a valuable model for dissecting the cellular and molecular regulation of osteoclast differentiation and activation.     

Roles of stromal cell RANKL, OPG, and M-CSF expression in biphasic TGF-beta regulation of osteoclast differentiation

To better understand the complex roles of transforming growth factor-beta (TGF-beta) in bone metabolism, we examined the impact of a range of TGF-beta concentrations on osteoclast differentiation. In co-cultures of support cells and spleen or marrow osteoclast precursors, low TGF-beta concentrations stimulated while high concentrations inhibited differentiation. We investigated the influences of TGF-beta on macrophage colony stimulating factor (M-CSF), receptor activator of NF-kappaB ligand (RANKL), and osteoprotegerin (OPG) expression and found a dose dependent inhibition of M-CSF expression. RANKL expression was elevated at low TGF-beta concentrations with a less dramatic increase in OPG. Addition of OPG blocked differentiation at the stimulatory TGF-beta dose. Thus, low TGF-beta concentrations elevated the RANKL/OPG ratio while high concentrations did not, supporting that, at low TGF-beta concentrations, there is sufficient M-CSF and a high RANKL/OPG ratio to stimulate differentiation. At high TGF-beta concentrations, the RANKL/OPG ratio and M-CSF expression were both repressed and there was no differentiation. We examined whether TGF-beta-mediated repression of osteoclasts differentiation is due to these changes by adding M-CSF and/or RANKL and did not observe any impact on differentiation repression. We studied direct TGF-beta impacts on osteoclast precursors by culturing spleen or marrow cells with M-CSF and RANKL. TGF-beta treatment dose-dependently stimulated osteoclast differentiation. These data indicate that low TGF-beta levels stimulate osteoclast differentiation by impacting the RANKL/OPG ratio while high TGF-beta levels repress osteoclast differentiation by multiple avenues including mechanisms independent of the RANKL/OPG ratio or M-CSF expression regulation.     

Induction of chemokines and chemokine receptors CCR2b and CCR4 in authentic human osteoclasts differentiated with RANKL and osteoclast like cells differentiated by MCP-1 and RANTES

Chemokines MCP-1 and RANTES are induced when authentic bone resorbing human osteoclasts differentiate from monocyte precursors in vitro. In addition, MCP-1 and RANTES can stimulate the differentiation of cells with the visual appearance of osteoclasts, being multinuclear and positive for tartrate resistance acid phosphatase (TRAP +). We show here that MIP1alpha is also potently induced by RANKL during human osteoclast differentiation and that this chemokine also induces the formation of TRAP + multinucleated cells in the absence of RANKL. MIP1alpha was able to overcome the potent inhibition of GM-CSF on osteoclast differentiation, permitting the cells to pass through to TRAP + multinuclear cells, however these were unable to form resorption pits. Chemokine receptors CCR2b and CCR4 were potently induced by RANKL (12.6- and 49-fold, P = 4.0 x 10(-7) and 4.0 x 10(-8), respectively), while CCR1 and CCR5 were not regulated. Chemokine treatment in the absence of RANKL also induced MCP-1, RANTES and MIP1alpha. Unexpectedly, treatment with MCP-1 in the absence of RANKL resulted in 458-fold induction of CCR4 (P = 1.0 x 10(-10)), while RANTES treatment resulted in twofold repression (P = 1.0 x 10(-4)). Since CCR2b and CCR4 are MCP-1 receptors, these data support the existence of an MCP-1 autocrine loop in human osteoclasts differentiated using RANKL.     

Vasohibin-1 promotes osteoclast differentiation in periodontal disease by stimulating the expression of RANKL in gingival fibroblasts

Vasohibin-1 (VASH1) is a key inhibitor of vascular endothelial growth factor-induced angiogenesis. Although the involvement of VASH1 in various pathological processes has been extensively studied, its role in periodontal disease (PD) remains unclear. We aimed to investigate the role of VASH1 in PD by focusing on osteoclastogenesis regulation. We investigated VASH1 expression in PD by analyzing data from the online Gene Expression Omnibus (GEO) database and using a mouse ligature-induced periodontitis model. The effects of VASH1 on osteoclast differentiation and osteoclastogenesis-supporting cells were assessed in mouse bone marrow-derived macrophages (BMMs) and human gingival fibroblasts (GFs). To identify the stimulant of VASH1, we used culture broth from Porphyromonas gingivalis (Pg), a periopathogen. The GEO database and mouse periodontitis model revealed that VASH1 expression was upregulated in periodontitis-affected gingival tissues, which was further supported by immunohistochemistry and qRT-PCR analyses. VASH1 expression was significantly stimulated in GFs after treatment with the Pg broth. Direct treatment with recombinant VASH1 protein did not stimulate osteoclast differentiation in BMMs but did contribute to osteoclast differentiation by inducing RANKL expression in GFs through a paracrine mechanism. Small interfering RNA-mediated silencing of VASH1 in GFs abrogated RANKL-mediated osteoclast differentiation in BMMs. Additionally, VASH1-activated RANKL expression in GFs was significantly suppressed by MK-2206, a selective inhibitor of AKT. These results suggest that Pg-induced VASH1 may be associated with RANKL expression in GFs in a paracrine manner, contributing to osteoclastogenesis via an AKT-dependent mechanism during PD progression.