Suppression of RANKL-dependent heme oxygenase-1 is required for high mobility group box 1 release and osteoclastogenesis

The differentiation of osteoclasts is regulated by several essential cytokines, such as receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor. Recently, high mobility group box 1 (HMGB1), a chromatin protein, also has been identified as one of these osteoclast differentiation cytokines. However, the molecular mechanisms that control HMGB1 release from osteoclast precursor cells are not known. Here, we report that RANKL-induced suppression of heme oxygenase-1 (HO-1), a heme-degrading enzyme, promotes HMGB1 release during osteoclastogenesis. In contrast, induction of HO-1 with hemin or curcumin in bone marrow-derived macrophages or RAW-D murine osteoclast precursor cells inhibited osteoclastogenesis and suppressed HMGB1 release. Since an inhibitor for p38 mitogen-activated protein kinase (MAPK) prevented the RANKL-mediated HO-1 suppression and extracellular release of HMGB1, these effects were p38 MAPK-dependent. Moreover, suppression of HO-1 in RAW-D cells by RNA interference promoted the activation of caspase-3 and HMGB1 release, whereas overexpression of HO-1 inhibited caspase-3 activation as well as HMGB1 release. Furthermore, these effects were regulated by redox conditions since antioxidant N-acetylcysteine abolished the HO-1/HMGB1/caspase-3 axis. These results suggest that RANKL-dependent HO-1 suppression leads to caspase-3 activation and HMGB1 release during osteoclastogenesis.     

Hesperetin suppresses RANKL-induced osteoclastogenesis and ameliorates lipopolysaccharide-induced bone loss

Destructive bone diseases caused by osteolysis are increasing in incidence. They are characterized by an excessive imbalance of osteoclast formation and activation. During osteolysis, the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways are triggered by receptor activator of NF-κB ligand (RANKL), inflammatory factors, and oxidative stress. Previous studies have indicated that the common flavanone glycoside compound hesperetin exhibits anti-inflammatory and antioxidant activity by inhibition of NF-κB and MAPK signaling pathways. However, the direct relationship between hesperetin and osteolysis remain unclear. In the present study, we investigated the effects of hesperetin on lipopolysaccharide (LPS)-induced osteoporosis and elucidated the related mechanisms. Hesperetin effectively suppressed RANKL-induced osteoclastogenesis, osteoclastic bone resorption, and F-actin ring formation in a dose-dependent manner. It also significantly suppressed the expression of osteoclast-specific markers including tartrate-resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, c-Fos, and nuclear factor of activated T-cells cytoplasmic 1. Furthermore, it inhibited osteoclastogenesis by inhibiting activation of NF-κB and MAPK signaling, scavenging reactive oxygen species, and activating the nuclear factor E2 p45-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathway. Consistent with in vitro results, hesperetin effectively ameliorated LPS-induced bone loss, reduced osteoclast numbers, and decreased the RANKL/OPG ratio in vivo. As such, our results suggest that hesperetin may be a great candidate for developing a novel drug for destructive bone diseases such as periodontal disease, tumor bone metastasis, rheumatoid arthritis, and osteoporosis.     

Protection against Ovariectomy-Induced Bone Loss by Tranilast

Background

Tranilast (N-(3′,4′-dimethoxycinnamonyl) anthranilic acid) has been shown to be therapeutically effective, exerting anti-inflammatory and anti-oxidative effects via acting on macrophage. We hypothesized that Tranilast may protect against oxidative stress-induced bone loss via action in osteoclasts (OCs) that shares precursors with macrophage.

Methodology and Principal Findings

To elucidate the role of Tranilast, ovariectomy (OVX)-induced bone loss in vivo and OC differentiation in vitro were evaluated by µCT and tartrate-resistant acid phosphatase staining, respectively. Oral administration of Tranilast protected against OVX-induced bone loss with decreased serum level of reactive oxygen species (ROS) in mice. Tranilast inhibited OC formation in vitro. Decreased osteoclastogenesis by Tranilast was due to a defect of receptor activator of nuclear factor-κB ligand (RANKL) signaling, at least partly via decreased activation of nuclear factor-κB and reduced induction and nuclear translocation of nuclear factor of activated T cells, cytoplasmic 1 (or NFAT2). Tranilast also decreased RANKL-induced a long lasting ROS level as well as TGF-β to inhibit osteoclastogenesis. Reduced ROS caused by Tranilast was due to the induction of ROS scavenging enzymes (peroxiredoxin 1, heme oxygenase-1, and glutathione peroxidase 1) as well as impaired ROS generation.

Conclusions/Significance

Our data suggests the therapeutic potential of Tranilast for amelioration of bone loss and oxidative stress due to loss of ovarian function.     

CRISPR/Cas9 knockout of MTA1 enhanced RANKL-induced osteoclastogenesis in RAW264.7 cells partly via increasing ROS activities

Metastasis-associated protein 1 (MTA1), belonging to metastasis-associated proteins (MTA) family, which are integral parts of nucleosome remodelling and histone deacetylation (NuRD) complexes. However, the effect of MTA1 on osteoclastogenesis is unknown. Currently, the regulation of MTA1 in osteoclastogenesis was reported for the first time. MTA1 knockout cells (KO) were established by CRISPR/Cas9 genome editing. RAW264.7 cells with WT and KO group were stimulated independently by RANKL to differentiate into mature osteoclasts. Further, western blotting and quantitative qRT-PCR were used to explore the effect of MTA1 on the expression of osteoclast-associated genes (including CTSK, MMP9, c-Fos and NFATc1) during osteoclastogenesis. Moreover, the effects of MTA1 on the expression of reactive oxygen species (ROS) in osteoclastogenesis was determined by 2′, 7′ -dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Nuclear translocation of Nrf2 was assessed by immunofluorescence staining and western blotting. Our results indicated that the MTA1 deletion group could differentiate into osteoclasts with larger volume and more TRAP positive. In addition, compared with WT group, KO group cells generated more actin rings. Mechanistically, the loss of MTA1 increased the expression of osteoclast-specific markers, including c-Fos, NFATc1, CTSK and MMP-9. Furthermore, the results of qRT-PCR and western blotting showed that MTA1 deficiency reduced basal Nrf2 expression and inhibited Nrf2-mediated expression of related antioxidant enzymes. Immunofluorescence staining demonstrated that MTA1 deficiency inhibited Nrf2 nuclear translocation. Taken together, the above increased basal and RANKL-induced intracellular ROS levels, leading to enhanced osteoclast formation.     

Interleukin-4 reversibly inhibits osteoclastogenesis via inhibition of NF-kappa B and mitogen-activated protein kinase signaling.

To define the molecular mechanism(s) by which interleukin (IL)-4 reversibly inhibits formation of osteoclasts (OCs) from bone marrow macrophages (BMMs), we examined the capacity of this T cell-derived cytokine to impact signals known to modulate osteoclastogenesis, which include those initiated by macrophage colony-stimulating factor (M-CSF), receptor for activation of NF-kappa B ligand (RANKL), tumor necrosis factor (TNF), and IL-1. We find that although pretreatment of BMMs with IL-4 does not alter M-CSF signaling, it reversibly blocks RANKL-dependent activation of the NF-kappa B, JNK, p38, and ERK signals. IL-4 also selectively inhibits TNF signaling, while enhancing that of IL-1. Contrary to previous reports, we find that MEK inhibitors dose-dependently inhibit OC differentiation. To identify more proximal signals mediating inhibition of OC formation by IL-4, we used mice lacking STAT6 or SHIP1, two adapter proteins that bind the IL-4 receptor. IL-4 fails to inhibit RANKL/M-CSF-induced osteoclastogenesis by BMMs derived from STAT6-, but not SHIP1-, knockout mice. Consistent with this observation, the inhibitory effects of IL-4 on RANKL-induced NF-kappa B and mitogen-activated protein kinase activation are STAT6-dependent. We conclude that IL-4 reversibly arrests osteoclastogenesis in a STAT6-dependent manner by 1) preventing I kappa B phosphorylation and thus NF-kappa B activation, and 2) blockade of the JNK, p38, and ERK mitogen-activated protein kinase pathways.     

Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo

Wear particle‐stimulated inflammatory bone destruction and the consequent aseptic loosening remain the primary causes of artificial prosthesis failure and revision. Previous studies have demonstrated that curcumin has a protective effect on bone disorders and inflammatory diseases and can ameliorate polymethylmethacrylate‐induced osteolysis in vivo. However, the effect on immunomodulation and the definitive mechanism by which curcumin reduces the receptor activators of nuclear factor‐kappa B ligand (RANKL)‐stimulated osteoclast formation and prevents the activation of osteoclastic signalling pathways are unclear. In this work, the immunomodulation effect and anti‐osteoclastogenesis capacities exerted by curcumin on titanium nanoparticle‐stimulated macrophage polarization and on RANKL‐mediated osteoclast activation and differentiation in osteoclastic precursor cells in vitro were investigated. As expected, curcumin inhibited RANKL‐stimulated osteoclast maturation and formation and had an immunomodulatory effect on macrophage polarization in vitro. Furthermore, studies aimed to identify the potential molecular and cellular mechanisms revealed that this protective effect of curcumin on osteoclastogenesis occurred through the amelioration of the activation of Akt/NF‐κB/NFATc1 pathways. Additionally, an in vivo mouse calvarial bone destruction model further confirmed that curcumin ameliorated the severity of titanium nanoparticle‐stimulated bone loss and destruction. Our results conclusively indicated that curcumin, a major biologic component of Curcuma longa with anti‐inflammatory and immunomodulatory properties, may serve as a potential therapeutic agent for osteoclastic diseases.     

Inhibition of RANKL-mediated osteoclast differentiation by selective TRAF6 decoy peptides

RANK and RANKL are essential mediators of osteoclastogenesis. RANK interacts with members of the tumor necrosis factor receptor-associated factor (TRAF) family, of which TRAF6 is the critical signaling molecule. We identified a unique TRAF6-binding motif in RANK, which was subsequently co-crystallized with TRAF6 revealing distinct molecular interactions. A cell-permeable TRAF6 decoy peptide (T6DP) was shown to specifically target TRAF6 and inhibit RANKL-mediated signaling. In this study, we identified a core motif for binding to TRAF6 by generating a series of deletion mutants linked via palmitate as a means to internalize the peptide, thus making a smaller scaffold for intracellular delivery. The core motif of RKIPTEDEY inhibited RANKL-mediated osteoclastogenesis and bone resorption. In contrast, TRAF2/5 decoy peptides appeared to have no affect. Thus, disruption of the RANK-TRAF6 interaction may prove useful as a novel target for the development of a small molecule therapeutic agent for the treatment of bone-related diseases.     

Regulation of receptor activator of NF-kappa B ligand-induced osteoclastogenesis by endogenous interferon-beta (INF-beta ) and suppressors of cytokine signaling (SOCS). The possible counteracting role of SOCSs- in IFN-beta-inhibited osteoclast formation

Bone resorption and the immune system are correlated with each other, and both are controlled by a variety of common cytokines produced in the bone microenvironments. Among these immune mediators, the involvement of type I interferons (IFNs) in osteoclastic bone resorption remains unknown. In this study, we investigated the participation of IFN-beta and suppressors of cytokine signaling (SOCS)-1 and -3 in osteoclastogenesis. Addition of exogenous IFN-beta to osteoclast progenitors (bone-derived monocytes/macrophages) inhibited their differentiation toward osteoclasts induced by the receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor with/without transforming growth factor-beta, where inhibition was associated with down-regulation of the gene expressions of molecules related to osteoclast differentiation. In addition, RANKL induced the expression of IFN-beta; furthermore, neutralizing antibody against type I IFNs accelerated the osteoclast formation, indicating type I IFNs as potential intrinsic inhibitors. On the other hand, RANKL also induced the expression of SOCS-1 and -3, suppressors of the IFN signaling. Pretreatment with RANKL for a sufficient time for the induction of SOCSs attenuated phosphorylation of STAT-1 in response to IFN-beta in osteoclast progenitors, causing a decrease in the binding activity of nuclear extracts toward the interferon-stimulated response element. mRNA levels of STAT-1, STAT-2, and IFN-stimulated gene factor-3gamma, comprising IFN-stimulated gene factor-3, were not altered by RANKL. Thus, although the inhibitory cytokine such as IFN-beta is produced in response to RANKL, the inhibition of osteoclastogenesis may be rescued by the induction of signaling suppressors such as SOCSs.     

Silibinin inhibits osteoclast differentiation mediated by TNF family members

Silibinin is a polyphenolic flavonoid compound isolated from milk thistle (Silybum marianum), with known hepatoprotective, anticarcinogenic, and antioxidant effects. Herein, we show that silibinin inhibits receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis from RAW264.7 cells as well as from bone marrow-derived monocyte/macrophage cells in a dose-dependent manner. Silibinin has no effect on the expression of RANKL or the soluble RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts. However, we demonstrate that silibinin can block the activation of NF-κB, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, and extracellular signal-regulated kinase (ERK) in osteoclast precursors in response to RANKL. Furthermore, silibinin attenuates the induction of nuclear factor of activated T cells (NFAT) c1 and osteoclast-associated receptor (OSCAR) expression during RANKL-induced osteoclastogenesis. We demonstrate that silibinin can inhibit TNF-α-induced osteoclastogenesis as well as the expression of NFATc1 and OSCAR. Taken together, our results indicate that silibinin has the potential to inhibit osteoclast formation by attenuating the downstream signaling cascades associated with RANKL and TNF-α.     

The association of Notch2 and NF-kappaB accelerates RANKL-induced osteoclastogenesis

Notch signaling plays a key role in various cell differentiation processes including bone homeostasis. However, the specific involvement of Notch in regulating osteoclastogenesis is still controversial. In the present study, we show that RANKL induces expression of Jagged1 and Notch2 in bone marrow macrophages during osteoclast differentiation. Suppression of Notch signaling by a selective γ-secretase inhibitor or Notch2 short hairpin RNA suppresses RANKL-induced osteoclastogenesis. In contrast, induction of Notch signaling by Jagged1 or by ectopic expression of intracellular Notch2 enhances NFATc1 promoter activity and expression and promotes osteoclastogenesis. Finally, we found that Notch2 and p65 interact in the nuclei of RANKL-stimulated cells and that both proteins are recruited to the NFATc1 promoter, driving its expression. Taken together, our results show a new molecular cross talk between Notch and NF-κB pathways that is relevant in osteoclastogenesis.     

Tiliroside is a new potential therapeutic drug for osteoporosis in mice

Osteoporosis is a class of metabolic bone disease caused by complexed ramifications. Overactivation of osteoclasts due to a sudden decreased estrogen level plays a pivotal role for postmenopausal women suffering from osteoporosis. Therefore, inhibiting osteoclast formation and function has become a major direction for the treatment of osteoporosis. Tiliroside (Tle) is a salutary dietary glycosidic flavonoid extracted from Oriental Paperbush flower, which has been reported to have an anti-inflammation effect. However, whether Tle affects the osteoclastogenesis and bone resorption remains unknown. Herein, we demonstrate that Tle prevents bone loss in ovariectomy in mice and inhibits osteoclast differentiation and bone resorption stimulated by receptor activator of nuclear factor-κB ligand (RANKL) in vitro. Molecular mechanism studies reveal that Tle reduces RANKL-induced activation of mitogen-activated protein kinase and T-cell nuclear factor 1 pathways, and osteoclastogenesis-related marker gene expression, including cathepsin K (Ctsk), matrix metalloproteinase 9, tartrate-resistant acid phosphatase (Acp5), and Atp6v0d2. Our research indicates that Tle suppresses osteoclastogenesis and bone loss by downregulating the RANKL-mediated signaling protein activation and expression. In addition, Tle inhibits intracellular reactive oxygen species generation which is related to the formation of osteoclasts. Therefore, Tle might serve as a potential drug for osteolytic disease such as osteoporosis.