Suppressors of cytokine signaling-1 and -3 regulate osteoclastogenesis in the presence of inflammatory cytokines

Bone metabolism and the immune system have a correlative relationship, and both are controlled by various common cytokines, such as IFNs and ILs, produced in the bone microenvironments. The suppressor of cytokine signaling-1 (SOCS1) and SOCS3 are negative regulators of such cytokines. Although SOCSs are shown to be induced during osteoclast differentiation, their physiological roles in osteoclast differentiation and function have not been clarified. Thus, we examined the roles of SOCS1 and SOCS3 in osteoclastogenesis using SOCS1- and SOCS3-deficient mice. IFN-gamma-mediated inhibition of osteoclast differentiation from bone marrow-derived monocytes (BMMs) was strongly enhanced in SOCS1-deficient BMMs, but was diminished in SOCS1-overexpressing BMMs. Moreover, LPS-induced osteoclastogenesis and bone destruction in vivo were suppressed in SOCS1(+/-) mice compared with those in wild-type mice, suggesting that SOCS1 antagonizes the inhibitory effect of IFN-gamma on osteoclastogenesis. SOCS3 did not alter the inhibitory effect of IFNs in osteoclastogenesis in both gain and loss of functional assays; however, the suppressive effect of IL-6 on osteoclast differentiation was greater in SOCS3-deficient BMMs than in wild-type BMMs in vitro. In addition, IL-6 significantly prevented LPS-induced bone destruction in SOCS3-deficient mice, although it failed in wild-type mice in vivo. In SOCS3-deficient BMMs, expression levels of TNF-receptor-associated factor-6 and IkappaB were drastically reduced and receptor activator of the NF-kappaB ligand-induced IkappaB phosphorylation was severely impaired in the presence of IL-6. These data suggest that both SOCS1 and SOCS3 regulate osteoclastogenesis by blocking the inhibitory effect of inflammatory cytokines on receptor activator of the NF-kappaB ligand-mediated osteoclast differentiation signals. Selective suppression of SOCS1 and SOCS3 in osteoclast precursors may be a possible therapeutic strategy for inflammatory bone destruction.     

Isorhamnetin attenuates osteoarthritis by inhibiting osteoclastogenesis and protecting chondrocytes through modulating reactive oxygen species homeostasis

Increasing evidence indicates that osteoarthritis (OA) is a musculoskeletal disease affecting the whole joint, including both cartilage and subchondral bone. Reactive oxygen species (ROS) have been demonstrated to be one of the important destructive factors during early‐stage OA development. The objective of this study was to investigate isorhamnetin (Iso) treatment on osteoclast formation and chondrocyte protection to attenuate OA by modulating ROS. Receptor activator of nuclear factor‐kappa B ligand (RANKL) was used to establish the osteoclast differentiation model in bone marrow macrophages (BMMs) in vivo. H₂O₂ was used to induce ROS, which could further cause chondrocyte apoptosis. We demonstrated that Iso suppressed RANKL‐induced ROS generation, which could mediate osteoclastogenesis. Moreover, we found that Iso inhibited osteoclast formation and function by suppressing the expression of osteoclastogenesis‐related genes and proteins. We proved that Iso inhibited RANKL‐induced activation of mitogen‐activated protein kinase activation of mitogen‐activated protein kinase (MAPK), nuclear factor‐kappa B (NF‐κB) and AKT signalling pathways in BMMs. In addition, Iso inhibited ROS‐induced chondrocyte apoptosis by regulating apoptosis‐related proteins. Moreover, Iso was administered to an anterior cruciate ligament transection (ACLT)‐induced OA mouse model. The results indicated that Iso exerted beneficial effects on inhibiting excessive osteoclast activity and chondrocyte apoptosis, which further remedied cartilage damage. Overall, our data showed that Iso is an effective candidate for treating OA.     

Receptor Activator of NF-κB (RANK) Cytoplasmic IVVY535–538 Motif Plays an Essential Role in Tumor Necrosis Factor-α (TNF)-mediated Osteoclastogenesis

Tumor necrosis factor-α (TNF) enhances osteoclast formation and activity leading to bone loss in various pathological conditions, but its precise role in osteoclastogenesis remains controversial. Although several groups showed that TNF can promote osteoclastogenesis independently of the receptor activator of NF-κB (RANK) ligand (RANKL), others demonstrated that TNF-mediated osteoclastogenesis needs permissive levels of RANKL. Here, we independently reveal that although TNF cannot stimulate osteoclastogenesis on bone slices, it can induce the formation of functional osteoclasts on bone slices in the presence of permissive levels of RANKL or from bone marrow macrophages (BMMs) pretreated by RANKL. TNF can still promote the formation of functional osteoclasts 2 days after transient RANKL pretreatment. These data have confirmed that TNF-mediated osteoclastogenesis requires priming of BMMs by RANKL. Moreover, we investigated the molecular mechanism underlying the dependence of TNF-mediated osteoclastogenesis on RANKL. RANK, the receptor for RANKL, contains an IVVY^535–538 motif that has been shown to play a vital role in osteoclastogenesis by committing BMMs to the osteoclast lineage. We show that TNF-induced osteoclastogenesis depends on RANKL to commit BMMs to the osteoclast lineage and RANKL regulates the lineage commitment through the IVVY motif. Mechanistically, the IVVY motif controls the lineage commitment by reprogramming osteoclast genes into an inducible state in which they can be activated by TNF. Our findings not only provide important mechanistic insights into the action of RANKL in TNF-mediated osteoclastogenesis but also establish that the IVVY motif may serve as an attractive therapeutic target for bone loss in various bone disorders.     

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.     

Downregulated fat mass and obesity-associated protein inhibits bone resorption and osteoclastogenesis by nuclear factor-kappa B inactivation

During osteoporosis, fat mass and obesity-associated protein (FTO) promotes the shift of bone marrow mesenchymal stem cells to adipocytes and represses osteoblast activity. However, the role and mechanisms of FTO on osteoclast formation and bone resorption remain unknown. In this study, we investigated the effect of FTO on RAW264.7 cells and bone marrow monocytes (BMMs)-derived osteoclasts in vitro and observed the influence of FTO on ovariectomized (OVX) mice model to mimic postmenopausal osteoporosis in vivo. Results found that FTO was up-regulated in BMMs from OVX mice. Double immunofluorescence assay showed co-localization of FTO with tartrate-resistant acid phosphatase (TRAP) in femurs of OVX mice. FTO overexpression enhanced TRAP-positive osteoclasts and F-actin ring formation in RAW264.7 cells upon RANKL stimulation. The expression of osteoclast differentiation-related genes, including nuclear factor of activated T cells c1 (NFATc1) and c-FOS, was upregulated in BMMs and RAW264.7 cells after FTO overexpression. FTO overexpression induced the phosphorylation and nuclear translocation of factor-kappa B (NF-κB) p65 in BMMs and RAW264.7 cells exposed to RANKL. ChIP and dual-luciferase assays revealed that FTO overexpression contributed to RANKL-induced binding of NF-κB to NFATc1 promoter. Rescue experiments suggested that FTO overexpression-mediated osteoclast differentiation was suppressed after intervention with a NF-κB inhibitor pyrrolidine dithiocarbamate. Further in vivo evidence revealed that FTO knockdown increased bone trabecula and bone mineral density, inhibited bone resorption and osteoclastogenesis in osteoporotic mice. Collectively, our research demonstrates that downregulated FTO inhibits bone resorption and osteoclastogenesis through NF-κB inactivation, which provides a novel reference for osteoporosis treatment.     

Bone microenvironment specific roles of ITAM adapter signaling during bone remodeling induced by acute estrogen-deficiency

Immunoreceptor tyrosine-based activation motif (ITAM) signaling mediated by DAP12 or Fcepsilon receptor Igamma chain (FcRgamma) have been shown to be critical for osteoclast differentiation and maturation under normal physiological conditions. Their function in pathological conditions is unknown. We studied the role of ITAM signaling during rapid bone remodeling induced by acute estrogen-deficiency in wild-type (WT), DAP12-deficient (DAP12-/-), FcRgamma-deficient (FcRgamma-/-) and double-deficient (DAP12-/-FcRgamma-/-) mice. Six weeks after ovariectomy (OVX), DAP12-/-FcRgamma-/- mice showed resistance to lumbar vertebral body (LVB) trabecular bone loss, while WT, DAP12-/- and FcRgamma-/- mice had significant LVB bone loss. In contrast, all ITAM adapter-deficient mice responded to OVX with bone loss in both femur and tibia of approximately 40%, relative to basal bone volumes. Only WT mice developed significant cortical bone loss after OVX. In vitro studies showed microenvironmental changes induced by OVX are indispensable for enhanced osteoclast formation and function. Cytokine changes, including TGFbeta and TNFalpha, were able to induce osteoclastogenesis independent of RANKL in BMMs from WT but not DAP12-/- and DAP12-/-FcRgamma-/- mice. FSH stimulated RANKL-induced osteoclast differentiation from BMMs in WT, but not DAP12-/- and DAP12-/-FcRgamma-/- mice. Our study demonstrates that although ITAM adapter signaling is critical for normal bone remodeling, estrogen-deficiency induces an ITAM adapter-independent bypass mechanism allowing for enhanced osteoclastogenesis and activation in specific bony microenvironments.     

Pectolinarigenin prevents bone loss in ovariectomized mice and inhibits RANKL-induced osteoclastogenesis via blocking activation of MAPK and NFATc1 signaling

Osteoporosis (OP) is a metabolic disease caused by multiple factors, which is characterized by a reduction of bone mass per unit volume and destruction of bone microstructure. Aberrant osteoclast function is the main cause of OP, therefore, regulating the differentiation and function of osteoclast is one of the treatment strategies for OP. Pectolinarigenin (PEC) is a medicinal implant isolated from Fragrant Eupatorium. Our experimental data showed that PEC was able to inhibit receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in vitro, by tartrate-resistant acid phosphatase (TRAcP) staining, Fibrous actin ring formation, and hydroxyapatite resorption assays. In terms of mechanism, PEC inhibited the expression of the osteoclastogenesis-related gene, including cathepsin K (Ctsk), matrix metalloproteinase 9 (Mmp9), and TRAcP (Acp5). Western blot analysis demonstrated that PEC could significantly block the activation of RANKL-induced mitogen-activated protein kinase signaling cascades and was able to suppress the protein expression of nuclear factor of activated T-cells and c-Fos. Meanwhile, the intracellular reactive oxygen species levels were also reduced by PEC in a concentration-dependent manner. Further, PEC could prevent the ovariectomy-induced bone loss in vivo. Summarizing all, our data suggested that PEC inhibits osteoclast formation and function and RANKL signaling pathways, and thus could potentially be used in the treatment the osteoclast-related bone loss diseases.     

AZD8835 inhibits osteoclastogenesis and periodontitis-induced alveolar bone loss in rats

Chronic periodontitis (CP) is one of the most common oral diseases, which is characterized by the loss of connective tissue and alveolar bone in adults. AZD8835, a novel dual phosphoinositide-3-kinase (PI3K) inhibitor, is currently in phase 1 clinical evaluation to treat breast cancer. However, whether AZD8835 has any effect on teeth and alveolar bone health remains unclear. In the current study, we aimed to investigate the potential effect of AZD8835 in treating CP in vitro and in vivo. We found that AZD8835 could inhibit osteoclast differentiation, bone resorption, and downregulate the expression of osteoclast marker genes, such as tartrate-resistant acid phosphatase (Trap), cathepsin K (Ctsk), V-ATPase d2 (Atp6v0d2), and calcitonin receptor (Ctr). In addition, AZD8835 suppressed osteoclastogenesis by inhibiting receptor activator of nuclear factor kappa B ligand (RANKL)-induced PI3K/protein kinase B (AKT), extracellular signal-regulated kinase, and nuclear factor-κB signaling in BMMs. In vivo, AZD8835 greatly ameliorated alveolar bone (ABL) loss in rats with CP. Meanwhile, histological examination showed fewer osteoclasts in the treatment group. In conclusion, these results indicated that AZD8835 is a promising agent to reduce ABL in CP.     

Bone Morphogenetic Proteins Signal Via SMAD and Mitogen-activated Protein (MAP) Kinase Pathways at Distinct Times during Osteoclastogenesis

To investigate the role of bone morphogenetic protein (BMP) signaling in osteoclastogenesis in vivo, we eliminated BMPRII in osteoclasts by creating a BMPRII^fl/fl;lysM-Cre mouse strain. Conditional knock-out (cKO) mice are osteopetrotic when compared with WT controls due to a decrease in osteoclast activity. Bone marrow macrophages (BMMs) isolated from cKO mice are severely inhibited in their capacity to differentiate into mature osteoclasts in the presence of M-CSF and receptor activator of NF-κB (RANK) ligand. We also show that BMP noncanonical (MAPK) and canonical (SMAD) pathways are utilized at different stages of osteoclast differentiation. BMP2 induces p38 phosphorylation in pre-fusion osteoclasts and increases SMAD phosphorylation around osteoclast precursor fusion. Phosphorylation of MAPKs was decreased in differentiated BMMs from cKO animals. Treating BMMs with the SMAD inhibitor dorsomorphin confirms the requirement for the canonical pathway around the time of fusion. These results demonstrate the requirement for BMP signaling in osteoclasts for proper bone homeostasis and also explore the complex signaling mechanisms employed by BMP signaling during osteoclast differentiation.     

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.     

SH3BP2 Gain-Of-Function Mutation Exacerbates Inflammation and Bone Loss in a Murine Collagen-Induced Arthritis Model

Objective

SH3BP2 is a signaling adapter protein which regulates immune and skeletal systems. Gain-of-function mutations in SH3BP2 cause cherubism, characterized by jawbone destruction. This study was aimed to examine the role of SH3BP2 in inflammatory bone loss using a collagen-induced arthritis (CIA) model.

Methods

CIA was induced in wild-type (Sh3bp2^+/+) and heterozygous P416R SH3BP2 cherubism mutant knock-in (Sh3bp2^KI/+) mice, an SH3BP2 gain-of-function model. Severity of the arthritis was determined by assessing the paw swelling and histological analyses of the joints. Micro-CT analysis was used to determine the levels of bone loss. Inflammation and osteoclastogenesis in the joints were evaluated by quantitating the gene expression of inflammatory cytokines and osteoclast markers. Furthermore, involvement of the T- and B-cell responses was determined by draining lymph node cell culture and measurement of the serum anti-mouse type II collagen antibody levels, respectively. Finally, roles of the SH3BP2 mutation in macrophage activation and osteoclastogenesis were determined by evaluating the TNF-α production levels and osteoclast formation in bone marrow-derived M-CSF-dependent macrophage (BMM) cultures.

Results

Sh3bp2^KI/+ mice exhibited more severe inflammation and bone loss, accompanying an increased number of osteoclasts. The mRNA levels for TNF-α and osteoclast marker genes were higher in the joints of Sh3bp2^KI/+ mice. Lymph node cell culture showed that lymphocyte proliferation and IFN-γ and IL-17 production were comparable between Sh3bp2^+/+ and Sh3bp2^KI/+ cells. Serum anti-type II collagen antibody levels were comparable between Sh3bp2^+/+ and Sh3bp2^KI/+ mice. In vitro experiments showed that TNF-α production in Sh3bp2^KI/+ BMMs is elevated compared with Sh3bp2^+/+ BMMs and that RANKL-induced osteoclastogenesis is enhanced in Sh3bp2^KI/+ BMMs associated with increased NFATc1 nuclear localization.

Conclusion

Gain-of-function of SH3BP2 augments inflammation and bone loss in the CIA model through increased macrophage activation and osteoclast formation. Therefore, modulation of the SH3BP2 expression may have therapeutic potential for the treatment of rheumatoid arthritis.     

Cytisine attenuates bone loss of ovariectomy mouse by preventing RANKL‐induced osteoclastogenesis

Postmenopausal Osteoporosis (PMOP) is oestrogen withdrawal characterized of much production and activation by osteoclast in the elderly female. Cytisine is a quinolizidine alkaloid that comes from seeds or other plants of the Leguminosae (Fabaceae) family. Cytisine has been shown several potential pharmacological functions. However, its effects on PMOP remain unknown. This study designed to explore whether Cytisine is able to suppress RANKL‐induced osteoclastogenesis and prevent the bone loss induced by oestrogen deficiency in ovariectomized (OVX) mice. In this study, we investigated the effect of Cytisine on RAW 264.7 cells and bone marrow monocytes (BMMs) derived osteoclast culture system in vitro and observed the effect of Cytisine on ovariectomized (OVX) mice model to imitate postmenopausal osteoporosis in vivo. We found that Cytisine inhibited F‐actin ring formation and tartrate‐resistant acid phosphatase (TRAP) staining in dose‐dependent ways, as well as bone resorption by pit formation assays. For molecular mechanism, Cytisine suppressed RANK‐related trigger RANKL by phosphorylation JNK/ERK/p38‐MAPK, IκBα/p65‐NF‐κB, and PI3K/AKT axis and significantly inhibited these signalling pathways. However, the suppression of PI3K‐AKT‐NFATc1 axis was rescued by AKT activator SC79. Meanwhile, Cytisine inhibited RANKL‐induced RANK‐TRAF6 association and RANKL‐related gene and protein markers such as NFATc1, Cathepsin K, MMP‐9 and TRAP. Our study indicated that Cytisine could suppress bone loss in OVX mouse through inhibited osteoclastogenesis. All data provide the evidence that Cytisine may be a promising agent in the treatment of osteoclast‐related diseases such as osteoporosis.