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.     

Scutellarein inhibits RANKL-induced osteoclast formation in vitro and prevents LPS-induced bone loss in vivo

Osteoporosis, arthritis, Peget's disease, bone tumor, periprosthetic joint infection, and periprosthetic loosening have a common characteristic of osteolysis, which is characterized by the enhanced osteoclastic bone resorptive function. At present, the treatment target of these diseases is to interfere with osteoclastic formation and function. Scutellarein (Scu), a flavonoids compound, can inhibit the progress of tumor and inflammation. However, the role of Scu in inflammatory osteolysis isn’t elucidated clearly. Our study showed that Scu inhibited bone destruction induced by LPS in vivo and OC morphology and function induced by RANKL in vitro. Mechanistic studies revealed that Scu suppressed osteoclastic marker gene expression by RANKL-induced, such as Ctsk9, Mmp9, Acp5, and Atp6v0d2. In addition, we found that the inhibition effects of osteoclastogenesis and bone resorption function of Scu were mediated via attenuating NF-κB and NFAT signaling pathways. In conclusion, the results showed that Scu may become a potential new drug for the treatment of inflammatory osteolysis.     

EGFR/FAK and c‐Src signalling pathways mediate the internalisation of Staphylococcus aureus by osteoblasts

Internalisation of Staphylococcus aureus in osteoblasts plays a critical role in the persistence and recurrence of osteomyelitis, the mechanisms involved in this process remain largely unknown. In the present study, evidence of internalised S. aureus in osteoblasts was found in long bone of haematogenous osteomyelitis in mice after 2 weeks of infection. Meanwhile, eliminating extracellular S. aureus by gentamicin can partially rescue bone loss, whereas the remaining intracellular S. aureus in osteoblasts may be associated with continuous bone destruction. In osteoblastic MC3T3 cells, intracellular S. aureus was detectable as early as 15 min after infection, and the internalisation rates increased with the extension of infection time. Additionally, S. aureus invasion stimulated the expression of phosphor‐focal adhesion kinase (FAK), phosphor‐epidermal growth factor receptor (EGFR) and phosphor‐c‐Src in a time‐dependent way, and blocking EGFR/FAK or c‐Src signalling significantly reduced the internalisation rate of S. aureus in osteoblasts. Our findings provide new insights into the mechanism of S. aureus internalisation in osteoblast and raise the potential of targeting EGFR/FAK and c‐Src as adjunctive therapeutics for treating chronic S. aureus osteomyelitis.     

Oleanolic acid exerts inhibitory effects on the late stage of osteoclastogenesis and prevents bone loss in osteoprotegerin knockout mice

Postmenopausal women undergo rapid bone loss, which caused by the accelerated osteoclastic bone resorption. Receptor activator of nuclear factor kappa-B ligand (RANKL) plays critical and essential roles on varied stages of osteoclastogenesis. Oleanolic acid (OA), a naturally derived small compound, has been found suppress osteoclastogenesis in early stage of bone marrow macrophages (BMMs). However, whether OA also regulates the late stage of osteoclastogenesis remains unclear. Here, the regulatory effect of OA on the late stage of osteoclastogenesis was investigated in vitro using RANKL-pretreated BMMs and in vivo using osteoprotegerin (OPG) knockout mice. Our in vitro studies demonstrate that OA inhibits the late stage of osteoclastogenesis from RANKL-pretreated BMMs. For in vivo animal investigation, OA attenuates the bone loss phenotypes in OPG-knockout mice by decreasing the densities of osteoclast, which are in consistent with the finding with in vitro osteoclastogenesis. Mechanistic investigations found that OA largely inhibit the activity of c-Fos and Nuclear factor of activated T-cells c1 (NFATc1) with RANKL-pretreated BMMs and OPG-knockout mice. Furthermore, OA suppresses the activities of osteoclast genes, such as Tartrate resistant acid phosphatase (TRAP), CathepsinK (Ctsk), and Matrix metalloproteinase 9 (MMP9). Taken together these findings, they have not only defined an inhibitory effect of OA in the late stage of osteoclastogenesis but have also gained new molecular mechanisms underlying the process of osteoclast formation.     

SH3BP2 is an activator of NFAT activity and osteoclastogenesis

Heterozygous activating mutations in exon 9 of SH3BP2 have been found in most patients with cherubism, an unusual genetic syndrome characterized by excessive remodeling of the mandible and maxilla due to spontaneous and excessive osteoclastic bone resorption. Osteoclasts differentiate after binding of sRANKL to RANK induces a number of downstream signaling effects, including activation of the calcineurin/NFAT (nuclear factor of activated T cells) pathway. Here, we have investigated the functional significance of SH3BP2 protein on osteoclastogenesis in the presence of sRANKL. Our results indicate that SH3BP2 both increases nuclear NFATc1 in sRANKL treated RAW 264.7 preosteoclast cells and enhances expression of tartrate resistant acid phosphatase (TRAP), a specific marker of osteoclast differentiation. Moreover, overexpression of SH3BP2 in RAW 264.7 cells potentiates sRANKL-stimulated phosphorylation of PLCγ1 and 2, thus providing a mechanistic pathway for the rapid translocation of NFATc1 into the nucleus and increased osteoclastogenesis in cherubism.     

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.     

α-Tocotrienol inhibits osteoclastic bone resorption by suppressing RANKL expression and signaling and bone resorbing activity

Vitamin E, an essential nutrient with powerful antioxidant activity, is the mixture of two classes of compounds, tocopherols (TPs) and tocotrienols (TTs). Although TTs exhibit better bone protective activity than α-TP, the underlying mechanism is poorly understood. In this study, we investigated whether α-TT and α-TP can modulate osteoclastic bone resorption. We found that α-TT but not α-TP inhibits osteoclastogenesis in coculture of osteoblasts and bone marrow cells induced by either IL-1 or combined treatment with 1α,25(OH)₂ vitamin D₃ and prostaglandin E₂. In accordance with this, only α-TT inhibited receptor activator of NF-κB ligand (RANKL) expression in osteoblasts. In addition, α-TT but not α-TP inhibited RANKL-induced osteoclast differentiation from precursors by suppression of c-Fos expression, possibly through inhibiting ERK and NF-κB activation. This anti-osteoclastogenic effect was reversed when c-Fos or an active form of NFATc1, a critical downstream of c-Fos during osteoclastogenesis, was overexpressed. Furthermore, only α-TT reduced bone resorbing activity of mature osteoclasts without affecting their survival. Overall, our results demonstrate that α-TT but not α-TP has anti-bone resorptive properties by inhibiting osteoclast differentiation and activation, suggesting that α-TT may have therapeutic value for treating and preventing bone diseases characterized by excessive bone destruction.     

Mangiferin attenuates osteoclastogenesis,bone resorption,and RANKL‐induced activation of NF‐κB and ERK

Osteolytic bone diseases such as osteoporosis have a common pathological feature in which osteoclastic bone resorption outstrips bone synthesis. Osteoclast formation and activation are regulated by receptor activator of nuclear factor κB ligand (RANKL). The induction of RANKL‐signaling pathways occurs following the interaction of RANKL to its cognate receptor, RANK. This specific binding drives the activation of downstream signaling pathways; which ultimately induce the formation and activation of osteoclasts. In this study, we showed that a natural immunomodulator, mangiferin, inhibits osteoclast formation and bone resorption by attenuating RANKL‐induced signaling. Mangiferin diminished the expression of osteoclast marker genes, including cathepsin K, calcitonin receptor, DC‐STAMP, and V‐ATPase d2. Mechanistic studies revealed that mangiferin inhibits RANKL‐induced activation of NF‐κB, concomitant with the inhibition of IκB‐α degradation, and p65 nuclear translocation. In addition, mangiferin also exhibited an inhibitory effect on RANKL‐induced ERK phosphorylation. Collectively, our data demonstrates that mangiferin exhibits anti‐resorptive properties, suggesting the potential application of mangiferin for the treatment and prevention of bone diseases involving excessive osteoclastic bone resorption. J. Cell. Biochem. 112: 89–97, 2011. © 2010 Wiley‐Liss, Inc.     

IL-1β favors osteoclastogenesis via supporting human periodontal ligament fibroblasts

The balance between bone formation and bone resorption in inflammatory diseases is often disturbed. Periodontitis, a chronic inflammation of the tooth gums, leads to unwanted bone loss as a response to inflammatory compounds such as interleukin‐1β (IL‐1β). This excessive bone loss reflects an increased osteoclast formation and activity. Osteoclast formation is a multistep process driven by osteoclastogenesis supporting cells such as periodontal ligament fibroblasts. The inflammatory factors can induce osteoclastogenesis, probably also by affecting the periodontal ligament fibroblast. In this study we investigated how pre‐culture of periodontal ligament fibroblasts with IL‐1β affected osteoclastogenesis. Fibroblasts were pre‐cultured with IL‐1β and/or dexamethasone, a commonly used anti‐inflammatory compound, before being co‐cultured with peripheral blood mononuclear cells (PBMCs). Pre‐culture with IL‐1β (1–100 ng/ml) resulted in an increased number of adhered PBMCs as well as an increased mRNA expression of intercellular adhesion molecule‐1 (ICAM‐1), macrophage colony stimulating factor (M‐CSF) and IL‐1β. Pre‐culture with IL‐1β also caused retraction of fibroblasts and an augmented formation of TRACP⁺ multinucleated cells. Our data suggest that stimulation of fibroblasts with IL‐1β has a long‐lasting effect, leading to a significantly increased osteoclastogenesis. These results provide new insights for understanding excessive bone loss in periodontitis. J. Cell. Biochem. 112: 1890–1897, 2011. © 2011 Wiley‐Liss, Inc.     

Compressive mechanical force augments osteoclastogenesis by bone marrow macrophages through activation of c‐Fms‐mediated signaling

Little is known about the effects of mechanical forces on osteoclastogenesis by bone marrow macrophages (BMMs) in the absence of mechanosensitive cells, including osteoblasts and fibroblasts. In this study, we examined the effects of mechanical force on osteoclastogenesis by applying centrifugal force to BMMs using a horizontal microplate rotor. Our findings, as measured by an in vitro model system, show that tumor necrosis factor (TNF)‐α is capable of inducing osteoclast differentiation from BMMs and bone resorption in the presence of macrophage‐colony stimulating factor (M‐CSF) and is further facilitated by receptor activator of nuclear factor‐kappaB (NF‐κB) ligand (RANKL). Application of force to BMMs accelerated TNF‐α‐induced osteoclastogenesis; this was inhibited either by anti‐TNF‐α or anti‐TNF‐α receptor but not by OPG. TNF‐α also increased c‐Fms expression at both mRNA and protein levels in BMMs. An anti‐c‐Fms antibody completely inhibited osteoclast differentiation and bone resorption induced by TNF‐α but partially blocked osteoclastogenesis stimulated in combination with RANKL. These results suggest that TNF‐α (in the presence of M‐CSF) is capable of inducing osteoclastogenesis from BMMs, and that osteoclastogenesis is significantly stimulated by force application through the activation of c‐Fms‐mediated signaling. Overall, the present study reveals the facilitating effect of mechanical force on osteoclastic differentiation from BMMs without the addition of mechanosensitive cells. J. Cell. Biochem. 111: 1260–1269, 2010. © 2010 Wiley‐Liss, Inc.     

Coaggregation and biofilm formation of Leptospira with Staphylococcus aureus

It is not known how Leptospira react to wound or a cut infected with microbes, such as pathogenic Staphylococcus, or their common habitat on oral or nasal mucosal membranes. In the present study, Staphylococcus aureus MTCC‐737 showed strong co‐aggregation with leptospiral strains (>75%, visual score of + 4) in vitro. All tested strains of Leptospira were able to form biofilm with S. aureus. Scanning electron microscopy analysis revealed intertwined networks of attached cells of L. interrogans and S. aureus, thus providing evidence of a matrix‐like structure. This phenomenon may have implications in Leptospira infection, which occurs via cuts and wounds of the skin.     

Artesunate inhibits RANKL‐induced osteoclastogenesis and bone resorption in vitro and prevents LPS‐induced bone loss in vivo

Osteoclasts are multinuclear giant cells responsible for bone resorption in lytic bone diseases such as osteoporosis, arthritis, periodontitis, and bone tumors. Due to the severe side‐effects caused by the currently available drugs, a continuous search for novel bone‐protective therapies is essential. Artesunate (Art), the water‐soluble derivative of artemisinin has been investigated owing to its anti‐malarial properties. However, its effects in osteoclastogenesis have not yet been reported. In this study, Art was shown to inhibit the nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis, the mRNA expression of osteoclastic‐specific genes, and resorption pit formation in a dose‐dependent manner in primary bone marrow‐derived macrophages cells (BMMs). Furthermore, Art markedly blocked the RANKL‐induced osteoclastogenesis by attenuating the degradation of IκB and phosphorylation of NF‐κB p65. Consistent with the in vitro results, Art inhibited lipopolysaccharide (LPS)‐induced bone resorption by suppressing the osteoclastogenesis. Together our data demonstrated that Art inhibits RANKL‐induced osteoclastogenesis by suppressing the NF‐κB signaling pathway and that it is a promising agent for the treatment of osteolytic diseases.