Mollugin from Rubea cordifolia suppresses receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis and bone resorbing activity in vitro and prevents lipopolysaccharide-induced bone loss in vivo

Osteopenic diseases, such as osteoporosis, are characterized by progressive and excessive bone resorption mediated by enhanced receptor activator of nuclear factor-κB ligand (RANKL) signaling. Therefore, downregulation of RANKL downstream signals may be a valuable approach for the treatment of bone loss-associated disorders. In this study, we investigated the effects of the naphthohydroquinone mollugin on osteoclastogenesis and its function in vitro and in vivo. Mollugin efficiently suppressed RANKL-induced osteoclast differentiation of bone marrow macrophages (BMMs) and bone resorbing activity of mature osteoclasts by inhibiting RANKL-induced c-Fos and NFATc1 expression. Mollugin reduced the phosphorylation of signaling pathways activated in the early stages of osteoclast differentiation, including the MAP kinase, Akt, and GSK3β and inhibited the expression of different genes associated with osteoclastogenesis, such as OSCAR, TRAP, DC-STAMP, OC-STAMP, integrin αν, integrin β3, cathepsin K, and ICAM-1. Furthermore, mice treated with mollugin showed significant restoration of lipopolysaccharide (LPS)-induced bone loss as indicated by micro-CT and histological analysis of femurs. Consequently, these results suggested that mollugin could be a novel therapeutic candidate for bone loss-associated disorders including osteoporosis, rheumatoid arthritis, and periodontitis.     

Reactivity of rat bone marrow-derived macrophages to neurotransmitter stimulation in the context of collagen II-induced arthritis

IntroductionNumerous observations indicate that rheumatoid arthritis (RA) has a bone marrow component. In parallel, local synovial changes depend on neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze whether collagen II-induced arthritis (CIA) has an impact on number, adhesion, apoptosis, and proliferation of the macrophage subset of bone marrow cells and how alterations in neurotransmitter microenvironment affect these properties.MethodsBone marrow-derived macrophages (BMMs) were isolated from Dark Agouti rats at different stages of CIA, and number, adhesion, caspase 3/7 activity, and proliferation were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA), and vasoactive intestinal peptide (VIP).ResultsOpposed to enhanced CD11b⁺ (cluster of differentiation 11b-positive) and EMR1⁺ (epidermal growth factor-like module-containing mucin-like hormone receptor-like 1-positive) cells, characterizing the macrophage subset, in native bone marrow of rats with acute inflammatory arthritis, we found decreased numbers of CIA macrophages after enrichment and culture in comparison with healthy (control) animals. Adhesion studies revealed significantly reduced attachment to plastic in acute arthritis and collagen type I and fibronectin in chronic arthritis. Additionally, we found a strong reduction in proliferation of BMMs at CIA onset and in the chronic phase of CIA. Apoptosis remained unaffected. Neurotransmitter stimulation profoundly affected proliferation, adhesion, and apoptosis of BMMs from CIA and control rats, depending on disease time point. Cultured BMMs from CIA and control animals expressed neurotransmitter receptors for ACh, VIP and NA, but the expression profile seemed not to be affected by CIA.ConclusionsInduction of CIA distinctly inhibits proliferation of BMMs in low- and non-inflammatory phases and reduces attachment to plastic at the acute inflammatory arthritis stage and adhesion to collagen I and fibronectin at the chronic stage. Influence of neurotransmitter stimulation on adhesion, apoptosis, and proliferation is altered by CIA depending on disease stage. We suggest an altered reactivity of BMMs to neurotransmitter stimulation caused by CIA and maybe also by aging.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0684-4) contains supplementary material, which is available to authorized users.     

NF-kappaB inhibitor dehydroxymethylepoxyquinomicin suppresses osteoclastogenesis and expression of NFATc1 in mouse arthritis without affecting expression of RANKL, osteoprotegerin or macrophage colony-stimulating factor

Inhibition of NF-kappaB is known to be effective in reducing both inflammation and bone destruction in animal models of arthritis. Our previous study demonstrated that a small cell-permeable NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), suppresses expression of proinflammatory cytokines and ameliorates mouse arthritis. It remained unclear, however, whether DHMEQ directly affects osteoclast precursor cells to suppress their differentiation to mature osteoclasts in vivo. The effect of DHMEQ on human osteoclastogenesis also remained elusive. In the present study, we therefore examined the effect of DHMEQ on osteoclastogenesis using a mouse collagen-induced arthritis model, and using culture systems of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis, and of osteoclast precursor cells from peripheral blood of healthy volunteers. DHMEQ significantly suppressed formation of osteoclasts in arthritic joints, and also suppressed expression of NFATc1 along the inner surfaces of bone lacunae and the eroded bone surface, while serum levels of soluble receptor activator of NF-kappaB ligand (RANKL), osteoprotegerin and macrophage colony-stimulating factor were not affected by the treatment. DHMEQ also did not suppress spontaneous expression of RANKL nor of macrophage colony-stimulating factor in culture of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis. These results suggest that DHMEQ suppresses osteoclastogenesis in vivo, through downregulation of NFATc1 expression, without significantly affecting expression of upstream molecules of the RANKL/receptor activator of NF-kappaB/osteoprotegerin cascade, at least in our experimental condition. Furthermore, in the presence of RANKL and macrophage colony-stimulating factor, differentiation and activation of human osteoclasts were also suppressed by DHMEQ, suggesting the possibility of future application of NF-kappaB inhibitors to rheumatoid arthritis therapy.     

NF-κB inhibitor dehydroxymethylepoxyquinomicin suppresses osteoclastogenesis and expression of NFATc1 in mouse arthritis without affecting expression of RANKL, osteoprotegerin or macrophage colony-stimulating factor

Inhibition of NF-κB is known to be effective in reducing both inflammation and bone destruction in animal models of arthritis. Our previous study demonstrated that a small cell-permeable NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), suppresses expression of proinflammatory cytokines and ameliorates mouse arthritis. It remained unclear, however, whether DHMEQ directly affects osteoclast precursor cells to suppress their differentiation to mature osteoclasts in vivo. The effect of DHMEQ on human osteoclastogenesis also remained elusive. In the present study, we therefore examined the effect of DHMEQ on osteoclastogenesis using a mouse collagen-induced arthritis model, and using culture systems of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis, and of osteoclast precursor cells from peripheral blood of healthy volunteers. DHMEQ significantly suppressed formation of osteoclasts in arthritic joints, and also suppressed expression of NFATc1 along the inner surfaces of bone lacunae and the eroded bone surface, while serum levels of soluble receptor activator of NF-κB ligand (RANKL), osteoprotegerin and macrophage colony-stimulating factor were not affected by the treatment. DHMEQ also did not suppress spontaneous expression of RANKL nor of macrophage colony-stimulating factor in culture of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis. These results suggest that DHMEQ suppresses osteoclastogenesis in vivo, through downregulation of NFATc1 expression, without significantly affecting expression of upstream molecules of the RANKL/receptor activator of NF-κB/osteoprotegerin cascade, at least in our experimental condition. Furthermore, in the presence of RANKL and macrophage colony-stimulating factor, differentiation and activation of human osteoclasts were also suppressed by DHMEQ, suggesting the possibility of future application of NF-κB inhibitors to rheumatoid arthritis therapy.     

PPAR-gamma regulates osteoclastogenesis in mice

Osteoclasts are bone-resorbing cells derived from hematopoietic precursors of the monocyte-macrophage lineage. Regulation of osteoclast function is central to the understanding of bone diseases such as osteoporosis, rheumatoid arthritis and osteopetrosis. Although peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has been shown to inhibit osteoblast differentiation, its role, if any, in osteoclasts is unknown. This is a clinically crucial question because PPAR-gamma agonists, "such as thiazolidinediones-" a class of insulin-sensitizing drugs, have been reported to cause a higher rate of fractures in human patients. Here we have uncovered a pro-osteoclastogenic effect of PPAR-gamma by using a Tie2Cre/flox mouse model in which PPAR-gamma is deleted in osteoclasts but not in osteoblasts. These mice develop osteopetrosis characterized by increased bone mass, reduced medullary cavity space and extramedullary hematopoiesis in the spleen. These defects are the result of impaired osteoclast differentiation and compromised receptor activator of nuclear factor-kappaB ligand signaling and can be rescued by bone marrow transplantation. Moreover, ligand activation of PPAR-gamma by rosiglitazone exacerbates osteoclast differentiation in a receptor-dependent manner. Our examination of the underlying mechanisms suggested that PPAR-gamma functions as a direct regulator of c-fos expression, an essential mediator of osteoclastogenesis. Therefore, PPAR-gamma and its ligands have a previously unrecognized role in promoting osteoclast differentiation and bone resorption.     

Inflammatory osteoclastogenesis can be induced by GM-CSF and activated under TNF immunity

In inflammatory arthritis such as RA, osteoclastic activity is severely enhanced. GM-CSF was reportedly elevated in synovial fluid, but is a strong inhibitor of osteoclastogenesis; here lies a contradiction. Our objective was to examine what type of osteoclasts generate and resorb bone with resistance to GM-CSF in an inflammatory joint. Monocyte-derived cells generated in GM-CSF were morphologically and immunophenotypically different from both the conventional DC and macrophage. They could differentiate into osteoclasts in the presence of RANKL + M-CSF, acquiring a stronger osteoclastic activity under TNF treatment. Furthermore, their differentiation was not inhibited by GM-CSF, while monocyte-derived osteoclast differentiation was completely inhibited. The resorption was suppressed by GM-CSF, and the existence of another osteoclastic pathway has been suggested. Our findings indicate another type of osteoclast exists in inflammatory arthritis.     

Jolkinolide B inhibits RANKL-induced osteoclastogenesis by suppressing the activation NF-κB and MAPK signaling pathways

Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. The unique function and ability of osteoclasts to resorb bone makes them critical in both normal bone homeostasis and pathologic bone diseases such as osteoporosis and rheumatoid arthritis. Thus, new compounds that may inhibit osteoclastogenesis and osteoclast function may be of great value in the treatment of osteoclast-related diseases. In the present study, we examined the effect of jolkinolide B (JB), isolated from the root of Euphorbia fischeriana Steud on receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation. We found that JB inhibited RANKL-induced osteoclast differentiation from bone marrow macrophages (BMMs) without cytotoxicity. Furthermore, the expression of osteoclastic marker genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CtsK), and calcitonin receptor (CTR), was significantly inhibited. JB inhibited RANKL-induced activation of NF-κB by suppressing RANKL-mediated IκBα degradation. Moreover, JB inhibited RANKL-induced phosphorylation of mitogen-activated protein kinases (p38, JNK, and ERK). This study thus identifies JB as an inhibitor of osteoclast formation and provides evidence that JB might be an alternative medicine for preventing and treating osteolysis.     

Downregulation of miR-21 gene expression by CRE-Ter to modulate osteoclastogenesis: De Novo mechanism

miR-21 expression stimulates osteoclast cells in the context of osteoclastogenesis. A previous report showed that NFκB-miR-21 pathway could serve as an innovative alternative to devise therapeutics for healing diabetic ulcers. Furthermore, our study demonstrated that a highly water-soluble curcuminoids-rich extract (CRE-Ter) inhibits osteoclastogenesis through NFκB pathway. The interplay between miR-21 and CRE-Ter in osteoclastogenesis has not yet been investigated. In this study, we examined the relation of CRE-Ter and miR-21 gene expression in receptor of the nuclear factor κB (NFκB) ligand (RANKL) - induced murine monocyte/macrophage RAW 264.7 cells, osteoclast cells, in osteoclastogenesis. Effect of CRE-Ter on generation of intracellular reactive oxygen species (ROS) was estimated by dichlorofluorescein diacetate (DCFH-DA). The results reveal that CRE-Ter reduced expression levels of miR-21 gene in osteoclasts. The inhibitory effects of CRE-Ter on in vitro osteoclastogenesis were evaluated by reduction in tartrate-resistant acid phosphatase (TRAP) content, and by reduction in expression levels of an osteoclast-specific gene, cathepsin K. Treatment of the osteoclast cells with CRE-Ter suppressed RANKL-induced NFκB activation including phospho-NFκB-p65, and phospho IκBα proteins. Western blot analysis revealed that NFκB inhibitor up-regulated CRE-Ter-promoted expression of phospho-NFκB-p65. In addition, CRE-Ter dose-dependently inhibited phospho-Akt expression. CRE-Ter also dose-dependently reduced DNA binding activity of NFκB and Akt as revealed by EMSA. ChIP assay revealed binding of NFκB-p65 to miR-21 promoters. In conclusion, our results demonstrate that CRE-Ter downregulates miR-21 gene expression in osteoclasts via a de novo mechanism, NFκB- Akt-miR-21 pathway.     

Stimulation of adenylyl cyclase and induction of brain-derived neurotrophic factor and TrkB mRNA by NKH477, a novel and potent forskolin derivative

The present study was undertaken to examine whether NKH477, a novel and potent water-soluble forskolin derivative, stimulates adenylyl cyclase and regulates brain-derived neurotrophic factor (BDNF) and TrkB expression in the rat brain. Administration of NKH477 at a dose of 1.0 mg/kg, but not 0.1 mg/kg, increased levels of cyclic AMP (cAMP) in a time-dependent manner in frontal cortex and hippocampus. Repeated administration of NKH477 (1.0 mg/kg) for 7 or 14 days also increased levels of cAMP in these two brain regions, indicating that the response does not desensitize with chronic treatment. In addition, administration of NKH477 at the 1 mg/kg dose increased the expression of BDNF and TrkB mRNA in frontal cortex and hippocampus. This effect was observed after single, as well as repeated (7 or 14 days), administration of NKH477. These results demonstrate that NKH477 administration rapidly increases cAMP levels in brain and provides evidence that stimulation of this second messenger system increases the expression of BDNF and TrkB mRNA.     

High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro

Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the proinflammatory cytokine, high mobility group box 1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43^def), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/M-CSF treated bone marrow cells. In bone marrow macrophages (BMMs), toll-like receptor 4 (TLR4) inhibition with LPS-RS, but not receptor for advanced glycation end products (RAGE) inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly affects differentiation by activating TLR4 in BMMs and RAGE in preosteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43^def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.     

Grape-Seed Proanthocyanidin Extract as Suppressors of Bone Destruction in Inflammatory Autoimmune Arthritis

Chronic autoimmune inflammation, which is commonly observed in rheumatoid arthritis (RA), disrupts the delicate balance between bone resorption and formation causing thedestruction of the bone and joints. We undertook this study to verify the effects of natural grape-seed proanthocyanidin extract (GSPE), an antioxidant, on chronic inflammation and bone destruction. GSPE administration ameliorated the arthritic symptoms of collagen-induced arthritis (CIA), which are representative of cartilage and bone destruction. GSPE treatment reduced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and osteoclast activity and increased differentiation of mature osteoblasts. Receptor activator of NFκB ligand expression in fibroblasts from RA patients was abrogated with GSPE treatment. GSPE blocked human peripheral blood mononuclear cell-derived osteoclastogenesis and acted as an antioxidant. GSPE improved the arthritic manifestations of CIA mice by simultaneously suppressing osteoclast differentiation and promoting osteoblast differentiation. Our results suggest that GSPE may be beneficial for the treatment of inflammation-associated bone destruction.     

The Effect of Vascular Endothelial Growth Factor on Osteoclastogenesis in Rheumatoid Arthritis

Vascular endothelial growth factor (VEGF) has angiogenic, inflammatory, and bone-destructive roles in rheumatoid arthritis (RA). We aimed to determine the unique role of VEGF in osteoclastogenesis in RA. VEGF-induced receptor activator of nuclear factor ҡB ligand (RANKL) expression was determined in RA synovial fibroblasts by real-time PCR, luciferase assays, and ELISA. Osteoclastogenesis in peripheral blood monocytes cultured with VEGF was assessed by determining the numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. Synovial fluid RANKL was correlated with VEGF concentration in the RA patients. VEGF stimulated the expression of RANKL in RA synovial fibroblasts. The RANKL promoter activity was upregulated by VEGF in the synovial fibroblasts transfected with RANKL-reporter plasmids. The VEGF-induced RANKL expression was decreased by the inhibition of both VEGF receptors (VEGFR) 1 and 2, Src, protein kinase C (PKC) and p38 MAPK. VEGF induced osteoclast differentiation from monocytes in the absence of RANKL and this was decreased by the inhibition of VEGFR1 and 2, Src, PKC and p38 MAPK. On coculturing with VEGF-prestimulated RA synovial fibroblasts, the monocytes differentiated into osteoclasts, and the osteoclastogenesis decreased by inhibition of Src and PKC pathways. VEGF plays dual roles on osteoclastogenesis in RA: direct induction of osteoclastogenesis from the precursors and stimulation of RANKL production in synovial fibroblasts, which is mediated by Src and PKC pathways. The axis of VEGF and RANKL could be a potential therapeutic target for RA-associated bone destruction.     

RANK ligand, RANK, and OPG expression in type II collagen-induced arthritis mouse

Rheumatoid arthritis (RA) is a systemic disorder characterized by synovial inflammation and subsequent destruction and deformity of synovial joints. The articular lesions start with synovitis, focal erosion of unmineralized cartilage, and then culminate in the destruction of subarticular bone by pannus tissue. Periarticular osteopenia and systemic osteoporosis follow as late complications of RA. Osteoclasts, specialized cells that resorb bone, play a central role in developing these osteolytic lesions. To elucidate the mechanism of osteoclastogenesis and bone destruction in autoimmune arthritis, we investigated the expression of RANK ligand (RANKL), RANK, and osteoprotegerin (OPG) mRNA in a mouse type II collagen-induced arthritis (CIA) model by in situ hybridization. The results indicated that most of the TRAP-positive mono- and multinucleated cells in the inflamed and proliferating synovium and in the pannus were RANK-positive authentic osteoclasts and their precursors. In the inflamed synovium and pannus of the mouse CIA model, synovial fibroblastic cells around these RANK-positive cells were strongly positive for RANKL. Moreover, RANKL-positive osteoblasts on the endosteal bone surface, at a distance from the affected synovial joints, increased significantly in the mouse CIA model prior to periarticular osteopenia and systemic osteoporosis. These data indicated that the RANKL-RANK system plays an important role for osteoclastogenesis in both local and systemic osteolytic lesions in autoimmune arthritis, and can therefore be a good target for therapeutic intervention.     

Talin1 and Rap1 Are Critical for Osteoclast Function

To determine talin1''s role in osteoclasts, we mated TLN1^fl/fl mice with those expressing cathepsin K-Cre (CtsK-TLN1) to delete the gene in mature osteoclasts or with lysozyme M-Cre (LysM-TLN1) mice to delete TLN1 in all osteoclast lineage cells. Absence of TLN1 impairs macrophage colony-stimulating factor (M-CSF)-stimulated inside-out integrin activation and cytoskeleton organization in mature osteoclasts. Talin1-deficient precursors normally express osteoclast differentiation markers when exposed to M-CSF and receptor activator of nuclear factor κB (RANK) ligand but attach to substrate and migrate poorly, arresting their development into mature resorptive cells. In keeping with inhibited resorption, CtsK-TLN1 mice exhibit an ∼5-fold increase in bone mass. Osteoclast-specific deletion of Rap1 (CtsK-Rap1), which promotes talin/β integrin recognition, yields similar osteopetrotic mice. The fact that the osteopetrosis of CtsK-TLN1 and CtsK-Rap1 mice is substantially more severe than that of those lacking αvβ3 is likely due to added failed activation of β1 integrins. In keeping with osteoclast dysfunction, mice in whom talin is deleted late in the course of osteoclastogenesis are substantially protected from ovariectomy-induced osteoporosis and the periarticular osteolysis attending inflammatory arthritis. Thus, talin1 and Rap1 are critical for resorptive function, and their selective inhibition in mature osteoclasts retards pathological bone loss.     

miR-31 controls osteoclast formation and bone resorption by targeting RhoA

Introduction

Increased activity of osteoclasts is responsible for bone loss and joint destruction in rheumatoid arthritis. For osteoclast development and bone resorption activity, cytoskeletal organization must be properly regulated. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that suppress expression of their target genes. This study was conducted to identify crucial miRNAs to control osteoclasts.

Methods

miRNA expression in the bone marrow-derived macrophages (BMM) with or without receptor activator of nuclear factor κB ligand (RANKL) stimulation was analyzed by miRNA array. To examine the role of specific miRNAs in osteoclast formation, bone resorption activity and actin ring formation, the BMM were retrovirally transduced with miRNA antagomirs. To confirm whether the suppressive effects on osteoclastogenesis by miR-31 inhibition were mediated by targeting RhoA, osteoclast formation was analyzed in the presence of the RhoA inhibitor, exoenzyme C3.

Results

miR-31 was identified as one of the highly upregulated miRNAs during osteoclast development under RANKL stimulation. Inhibition of miR-31 by specific antagomirs suppressed the RANKL-induced formation of osteoclasts and bone resorption. Phalloidin staining of osteoclasts revealed that actin ring formation at the cell periphery was severely impaired by miR-31 inhibition, and clusters of small ringed podosomes were observed instead. In these osteoclasts, expression of RhoA, one of the miR-31 target genes, was upregulated by miR-31 inhibition in spite of the impaired osteoclastogenesis. Treatment with the RhoA inhibitor, exoenzyme C3, rescued the osteoclastogenesis impaired by miR-31 inhibition.

Conclusions

miR-31 controls cytoskeleton organization in osteoclasts for optimal bone resorption activity by regulating the expression of RhoA.     

Inhibition of histone deacetylase suppresses osteoclastogenesis and bone destruction by inducing IFN-beta production

Osteoclasts are bone-resorptive multinucleated cells that are differentiated from hemopoietic cell lineages of monocyte/macrophages in the presence of receptor activator of NF-kappaB ligand (RANKL) and M-CSF. Downstream signaling molecules of the receptor of RANKL, RANK, modulate the differentiation and the activation of osteoclasts. We recently found that histone deacetylase inhibitors (HDIs), known as anticancer agents, selectively suppressed osteoclastogenesis in vitro. However, the molecular mechanism underlying inhibitory action of HDIs in osteoclastogenesis and the effect of HDIs on pathological bone destruction are still not remained to be elucidated. In this study, we show that a depsipeptide, FR901228, inhibited osteoclast differentiation by not only suppressing RANKL-induced nuclear translocation of NFATc1 but also increasing the mRNA level of IFN-beta, an inhibitor of osteoclastogenesis. The inhibition of osteoclast formation by FR901228 was abrogated by the addition of IFN-beta-neutralizing Ab. In addition, treatment of adjuvant-induced arthritis in rats revealed that FR901228 inhibited not only disease development in a prophylactic model but also bone destruction in a therapeutic model. Furthermore, immunostaining of the joints of therapeutically treated rats revealed significant production of IFN-beta in synovial cells. Taken together, these data suggest that a HDI inhibits osteoclastogenesis and bone destruction by a novel action to induce the expression of osteoclast inhibitory protein, IFN-beta.     

c-Fms-mediated differentiation and priming of monocyte lineage cells play a central role in autoimmune arthritis

Introduction

Tyrosine kinases are key mediators of multiple signaling pathways implicated in rheumatoid arthritis (RA). We previously demonstrated that imatinib mesylate--a Food and Drug Administration (FDA)-approved, antineoplastic drug that potently inhibits the tyrosine kinases Abl, c-Kit, platelet-derived growth factor receptor (PDGFR), and c-Fms--ameliorates murine autoimmune arthritis. However, which of the imatinib-targeted kinases is the principal culprit in disease pathogenesis remains unknown. Here we examine the role of c-Fms in autoimmune arthritis.

Methods

We tested the therapeutic efficacy of orally administered imatinib or GW2580, a small molecule that specifically inhibits c-Fms, in three mouse models of RA: collagen-induced arthritis (CIA), anti-collagen antibody-induced arthritis (CAIA), and K/BxN serum transfer-induced arthritis (K/BxN). Efficacy was evaluated by visual scoring of arthritis severity, paw thickness measurements, and histological analysis. We assessed the in vivo effects of imatinib and GW2580 on macrophage infiltration of synovial joints in CIA, and their in vitro effects on macrophage and osteoclast differentiation, and on osteoclast-mediated bone resorption. Further, we determined the effects of imatinib and GW2580 on the ability of macrophage colony-stimulating factor (M-CSF; the ligand for c-Fms) to prime bone marrow-derived macrophages to produce tumor necrosis factor (TNF) upon subsequent Fc receptor ligation. Finally, we measured M-CSF levels in synovial fluid from patients with RA, osteoarthritis (OA), or psoriatic arthritis (PsA), and levels of total and phosphorylated c-Fms in synovial tissue from patients with RA.

Results

GW2580 was as efficacious as imatinib in reducing arthritis severity in CIA, CAIA, and K/BxN models of RA. Specific inhibition of c-Fms abrogated (i) infiltration of macrophages into synovial joints of arthritic mice; (ii) differentiation of monocytes into macrophages and osteoclasts; (iii) osteoclast-mediated bone resorption; and (iv) priming of macrophages to produce TNF upon Fc receptor stimulation, an important trigger of synovitis in RA. Expression and activation of c-Fms in RA synovium were high, and levels of M-CSF were higher in RA synovial fluid than in OA or PsA synovial fluid.

Conclusions

These results suggest that c-Fms plays a central role in the pathogenesis of RA by mediating the differentiation and priming of monocyte lineage cells. Therapeutic targeting of c-Fms could provide benefit in RA.     

Retracted: Daidzin inhibits RANKL-induced osteoclastogenesis in vitro and prevents LPS-induced bone loss in vivo

Osteoclasts are multinuclear giant cells responsible for bone resorption in bone loss diseases, including rheumatoid arthritis, periodontitis, and the aseptic loosening of orthopedic implants. Because of injurious side effects with currently available drugs, it is necessary to continue research novel bone-protective therapies. Daidzin, a naturally occurring isoflavone found in leguminous plants, has numerous beneficial pharmacologic effects, including anti-cancer, anti-cholesterol, and anti-angiocardiopathy, promoting osteoblasts differentiation, and even anti-osteoporosis. However, the effect of daidzin on the regulation of osteoclast activity has not yet been investigated. In this study, our study showed that daidzin significantly inhibited receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclast differentiation of bone marrow macrophages and the hydroxyapatite-resorbing activity of mature osteoclasts by inhibiting RANKL-induced NF-kB signaling pathway. In addition, daidzin could inhibit the expression of osteoclast marker genes, including nuclear factor of activated T cells cytoplasmic 1 (NFATc1), cellular oncogene fos (c-Fos), tartrate-resistant acid phosphatase (TRAP), and cathepsin K (CTSK). Consistent with in vitro results, daidzin inhibited lipopolysaccharide-induced bone loss by suppressing the osteoclast differentiation. Together our data demonstrated that daidzin inhibits RANKL-induced osteoclastogenesis through suppressing NF-ĸB signaling pathway and that daidzin is a promising agent in the treatment of osteolytic diseases.