Increased Osteoclast Differentiation Research Articles

Water extract of Magnolia officinalis cortex inhibits osteoclastogenesis and bone resorption by downregulation of nuclear factor of activated T cells cytoplasmic 1

BackgroundMagnolia officinalis cortex has been traditionally used to treat stomach and intestine diseases in traditional Korean medicine. In this study, we investigated the effect of water extract of M. officinalis cortex (WEMC) on osteoclast differentiation and function. MethodsPhytochemical characterization of WEMC was performed by high-performance liquid chromatography analysis. Osteoclast differentiation of bone marrow-derived macrophages was determined by tartrate-resistant acid phosphatase activity assay. Receptor activator of nuclear factor-κB ligand (RANKL) signaling factors and transcription factors regulating osteoclast differentiation were analyzed by Western blot and real-time polymerase chain reaction. Bone resorption function of mature osteoclasts was examined by using culture plate coated with inorganic crystalline calcium phosphate. Furthermore, the in vivo effect of WEMC on osteoporosis was examined using RANKL-induced bone loss model, characterized by micro-computed tomography and bone metabolism marker analysis. ResultsWEMC inhibited RANKL-induced osteoclast differentiation and the bone resorbing activity of mature osteoclasts. WEMC contains gallic acid and honokiol as active constituents contributing to the inhibitory effect of WEMC on osteoclast differentiation. Further, WEMC suppressed RANKL-induced activation of p38 and nuclear factor-κB pathways and expression of osteoclastogenic transcription factors such as c-Fos for AP-1 and nuclear factor of activated T cells cytoplasmic 1. Ectopic overexpression of a constitutive active form of nuclear factor of activated T cells cytoplasmic 1 rescued the antiosteoclastogenic effect of WEMC. Consistent with the in vitro results, WEMC suppressed RANKL-induced trabecular bone loss in mice. ConclusionWEMC might have a therapeutic potential to treat pathological bone diseases due to increased osteoclast differentiation and function.

GDF15 Promotes Osteoclast Differentiation and High Serum GDF15 Levels Are Associated with Multiple Myeloma Bone Disease

IntroductionGrowth differentiation factor 15 (GDF15) is a multifunctional growth factor of the transforming growth factor beta (TGFbeta) family that plays a complex role in several types of cancers. In multiple myeloma, GDF15 was recently shown to enhance the tumor-initiating and self-renewal potential of the cancer cells (Tanno et al, Blood 2014). Moreover, blood and bone marrow plasma levels of GDF15 are elevated in myeloma patients compared with healthy persons, and high serum levels are associated with a poor prognosis (Corre et al, Cancer Research 2012). GDF15 seems important for bone remodeling during hypoxia (Hino et al, JBMR 2012), and one study proposed GDF15 to increase osteoclast activation in prostate cancer metastasizing to bone (Wakchoure et al, Prostate 2009). Whether GDF15 plays a role in the bone disease of multiple myeloma is not well characterized.AimOur aim was to investigate whether high GDF15 serum levels are associated with multiple myeloma bone disease and to characterize the effect of GDF15 on osteoclast differentiation in vitro.MethodsGDF15 was measured in serum samples obtained at diagnosis from 138 myeloma patients and 58 age and sex-matched healthy controls. The patient serum samples were collected for the Nordic Myeloma Study Group during a randomized phase 3 clinical trial which compared the effect of two different doses of pamidronate on bone. The bone disease was therefore particularly well-characterized in this study (Gimsing et al, Lancet Oncol 2010). Peripheral blood mononuclear cells (PBMC) isolated from buffy coats were cultured in osteoclast medium (a-MEM with human serum (20%), M-CSF (30ng/ml) and RANKL (50ng/ml)) for up to 14 days with or without GDF15. Purchased pre-osteoclasts (Lonza Inc.) were cultured in purchased bullet kit (OC medium with M-CSF (33ng/ml) and RANKL (66ng/ml)) for 7 days with or without GDF15. Cells positive for tartrate resistant acidic phosphatase (TRAP) staining and with more than two nuclei were counted as osteoclasts.ResultsGDF15 was significantly higher in serum obtained from myeloma patients (median 1.08 ng/ml, range 27.91) compared with healthy controls (median 0.46 ng/ml, range 1.66, Independent samples Kruskal-Wallis test p< 0.0001). Moreover, serum GDF15 was elevated in patients with a more advanced osteolytic bone disease (n= 51, median 1.44 ng/ml, range 6.48) as compared to patients without osteolytic lesions (n= 16, median 0.84 ng/ml, range 10.62) at inclusion (p<0.05). The difference between serum GDF15 in patients with limited bone disease at inclusion (n=51, median 1.07 ng/ml, range 6.84) and patients with no bone disease at baseline was not significantly different.In vitro, addition of GDF15 (0-100 ng/ml) to osteoclast precursors or PBMC increased numbers of multi-nucleated TRAP positive cells in a dose dependent manner (n=3, O ng/ml GDF15 mean 25.7, SEM 5.9, 2 ng/ml GDF15 mean 29.7, SEM 4.3, 20 ng/ml GDF15 mean 38.7, SEM 4.9, 50 ng/ml GDF15 mean 53.3, SEM 11.6, 100 ng/ml GDF15 mean 78.7 SEM 7.8). OPG inhibited the pro-osteoclastogenic activity of GDF15 inferring that the effect is mediated by RANKL. Hence, GDF15 increases osteoclast differentiation.ConclusionSerum GDF15 is elevated in myeloma patients with advanced osteolytic bone disease compared to patients with no lesions. GDF15 increases osteoclast differentiation in vitro. Hence, GDF15 could play a role in regulating bone remodeling in myeloma patients. DisclosuresNo relevant conflicts of interest to declare.

Regulation of osteoclastogenesis by osteocytes through growth differentiation factor-15

Osteocytes are the most abundant cells in bone. However, little attention has been paid to their role in bone remodeling. In this study, osteoclast differentiation was significantly enhanced by conditioned media derived from cultures of osteocytic MLO-Y4 cells that were cultured under hypoxic conditions. Using microarray analysis, we identified growth differentiation factor-15 (GDF15) as a pivotal factor secreted from osteocytes under hypoxia. Indeed, treatment with recombinant GDF15 markedly increased osteoclast differentiation in vitro. Further to investigate the importance of GDF15 in vivo, we used a hypoxic murine model that involved ligation of the right femoral artery. The volume of cancellous bone in the proximal tibia of the ligated limb was significantly reduced, together with a significant increase in osteoclast-related parameters. Addition of anti-GDF15 antibody prevented bone loss and osteoclastic activation in the tibiae of mice that had undergone femoral artery ligation. These results suggest that GDF15, which is secreted from osteocytes under hypoxia during bone remodeling, may be a positive regulator of osteoclastic differentiation. The in vivo usefulness of the anti-GDF15 antibody might provide insights for the development of novel therapeutics for bone disorders related to hypoxia or ischemic insults.

Activation of EPAC1/2 is essential for osteoclast formation by modulating NFκB nuclear translocation and actin cytoskeleton rearrangements.

Bisphosphonates inhibit osteoclast differentiation/function via inhibition of Rap1A isoprenylation. As Rap1 is the effector of exchange protein directly activated by cAMP (EPAC) proteins, we determined the role of EPAC in osteoclast differentiation. We examined osteoclast differentiation as the number of primary murine/human bone-marrow precursors that differentiated into multinucleated TRAP-positive cells in the presence of EPAC-selective stimulus (8-pCTP-2'-O-Me-cAMP, 100 μM; 8-pCTP-2'-O-Me-cAMP-AM, 1 μM) or inhibitor brefeldin A (BFA), ESI-05, and ESI-09 (10 μM each). Rap1 activity was assessed, and signaling events, as well as differentiation in EPAC1/2-knockdown RAW264.7 cells, were studied. Direct EPAC1/2 stimulation significantly increased osteoclast differentiation, whereas EPAC1/2 inhibition diminished differentiation (113 ± 6%, P < 0.05, and 42 ± 10%, P < 0.001, of basal, respectively). Rap1 activation was maximal 15 min after RANKL stimulation (147 ± 9% of basal, P < 0.001), whereas silencing of EPAC1/2 diminished activated Rap1 (43 ± 13 and 20 ± 15% of control, P < 0.001) and NFkB nuclear translocation. TRAP-staining revealed no osteoclast differentiation in EPAC1/2-KO cells. Cathepsin K, NFATc1, and osteopontin mRNA expression decreased in EPAC1/2-KO cells when compared to control. RhoA, cdc42, Rac1, and FAK were activated in an EPAC1/2-dependent manner, and there was diminished cytoskeletal assembly in EPAC1/2-KO cells. In summary, EPAC1 and EPAC2 are critical signaling intermediates in osteoclast differentiation that permit RANKL-stimulated NFkB nuclear translocation and actin rearrangements. Targeting this signaling intermediate may diminish bone destruction in inflammatory arthritis.

T cell costimulation molecules CD80/86 inhibit osteoclast differentiation by inducing the IDO/tryptophan pathway.

Bone resorption is seminal for the physiological remodeling of bone during life. However, this process needs to be strictly controlled; excessive bone resorption results in pathologic bone loss, osteoporosis, and fracture. We describe a control mechanism of bone resorption by the adaptive immune system. CD80/86, a pair of molecules expressed by antigen-presenting cells and involved in T cell costimulation, act as negative regulator for the generation of bone-resorbing osteoclasts. CD80/86-deficient mice were osteopenic because of increased osteoclast differentiation. CD80/86-deficient osteoclasts escaped physiological inhibition by CTLA-4 or regulatory T cells. Mechanistically, engagement of CD80/86 by CTLA-4 induced activation of the enzyme indoleamine 2,3-dioxygenase (IDO) in osteoclast precursors, which degraded tryptophan and promoted apoptosis. Concordantly, IDO-deficient mice also showed an osteopenic bone phenotype with higher numbers of osteoclast precursors and osteoclasts. Also, IDO-deficient mononuclear cells escaped the anti-osteoclastogenic effect of CTLA-4. This molecular mechanism was also present in humans because targeting CD80/86 by abatacept, a CTLA-4-immunoglobulin fusion protein, reduced, whereas blockade of CTLA-4 by ipilimumab antibody enhanced, the frequency of peripheral osteoclast precursors and osteoclastogenesis. In summary, these data show an important role of the adaptive immune system, in particular T cell CD80/86 costimulation molecules, in the physiological regulation of bone resorption and preservation of bone mass, as well as affect the understanding of the function of current and future drugs fostering or blocking the effects of CTLA-4 in humans.

Humanized Anti-CD26 Monoclonal Antibody Inhibits Osteoclast Bone Resorption Activity Via Blockade Of P38MAPK Signaling In Multiple Myeloma

BackgroundBone disease is a hallmark of multiple myeloma (MM) due to increased osteoclast (OC)-mediated bone resorption and decreased osteoblast (OB)-mediated bone formation. CD26 is a 110-kDa cell surface glycoprotein with dipeptidyl peptidase IV (DPPIV) activity and has a well-defined role in T cell activation and several tumor developments including malignant lymphoma, whereas its expression in OCs further suggests a role in osteoclastgenesis (OCG). Interestingly, CD26 is associated with increased phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) which also occurs downstream of RANK signaling in OCG and plays an important role in OC function. Our previous studies showed that CD26 is preferentially expressed in normal human OCs, and is also intensely expressed in activated OCs in MM, but not in MM cells themselves. In this study, we investigated the efficacy of humanized anti-CD26 monoclonal antibody (YS110) on OCs derived from MM patient bone marrow (BM) and MM growth within the BM microenvironment. MethodsOCs derived from MM patient BM mononuclear cells were assayed with macrophage-colony stimulating factor (M-CSF) (25ng/ml) plus receptor activator of NF- κB ligand (RANKL) (50ng/ml) with or without YS110 for OC formation and maturation for TRAP/CD26 staining and functional assay with bone resorptive pit formation assay. To assess the mechanisms of action of YS110 against OC differentiation in MM, RANK signaling proteins were examined by immunoblotting. ResultsSignificantly reduced numbers of multinucleated TRAP/CD26 positive mature OCs (>3 nuclei per OC) were observed in YS110-treated versus non-treated MM OC culture. YS110 blocked RANKL/M-CSF induced phosphorylation of p38MAPK and its downstream activation of mi/Mitf in MM-derived OC precursor cells, which otherwise increased OC differentiation. In contrast, YS110 did not show significant inhibition against other MAPKs involved in RANK signaling pathway in OC precursor cells. Importantly, YS110 remarkably diminished bone erosion area formed by mature OCs on dentine slices in pit formation assay. YS110 also consistently induced changes of cell supernatants in a dose-dependent manner in MM OC culture by ELISA. In fact, OC- or MM- related growth factors such as TRAP5b, collagen type 1, APRIL and BAFF which are secreted by OCs were also down-regulated. DPPIV inhibitor had no significant inhibitory effects on OCG in MM. These results suggest that the mechanism of action by YS110 on OCG in MM result from the blockade of RANKL induced p38MAPK-mi/Mitf phosphorylation in MM-derived OC precursor cells. Next, although YS110 did not demonstrate direct inhibition of proliferation of MM cells, to further investigate the role of CD26 in MM cells in the BM microenvironment, co-cultures of MM cell lines (KMS11, KMS12, KMS18, KMS20, KMS21, KMS26, KMS27, KMS28, KMS34, KMM1, U266) with MM-derived CD26-stained OCs were performed. We examined the expression of CD26 in co-cultured MM cells and co-cultured OCs by flow cytometry and immunoblotting. On day 1 of the co-culture, the expression of CD26 was barely detected in MM cells. However, on day 5, the expression level of CD26 was shown to be further increased in OCs. Moreover, interestingly, the expression of CD26 was also significantly up-regulated in MM cells. By immunohistochemistry, co-cultured MM cells were intensely stained positive for CD26 as well as co-cultured OCs. P38MAPK was activated in co-cultured CD26-stained MM cells; it regulates OC differentiation and bone resorption activity in MM. MM cells in the BM microenvironment may induce functional changes which promote tumor development and progression, as well as microenvironmental alternations. Now on going studies are confirming the anti-myeloma efficacy of YS110 on co-cultures of MM cells with OCs. ConclusionsOur data strongly demonstrate that targeting CD26 with YS110 inhibits MM-derived OC differentiation and function through blocking p38MAPK-mi/Mitf cascade of RANK signaling in MM-derived OC precursor cells. These results suggests that CD26 in OCs may be a novel therapeutic target for the treatment of myeloma-assoiated bone disease. It was further confirmed that the direct interaction between MM cells and OCs results in the up-regulation of CD26 expression in MM cells and that targeting CD26-p38MAPK signaling cascade may have a novel therapeutic potential not only in MM-derived OCs but in MM cells. Disclosures:No relevant conflicts of interest to declare.

RANKL enhances the effect of an antagonist of inhibitor of apoptosis proteins (cIAPs) in RANK-positive breast cancer cells

ObjectiveBetween 65% and 75% of patients with metastatic breast cancer will have decreased 5-year survival and increased morbidity due to cancer relapse in bone. At this stage of disease treatment is palliative, but tumor-targeted compounds could add to the benefits of anti-resorptive agents, improving clinical outcome. Inhibitor-of-apoptosis proteins (IAPs) are overexpressed in many tumors and second mitochondria-derived activator of caspases (Smac) mimetics have been designed to antagonize IAPs. In this work we explored the use of AT-406, a Smac mimetic, to target the tumor compartment of bone metastases. MethodsEffect of AT-406 on cancer cells apoptosis, expression of IAPs and osteogenic potential was addressed in vitro using the RANK-positive MDA-MB-231 breast cancer cell line. Effect of AT-406 on osteoclastogenesis was determined by inducing the differentiation of the RAW 264.7 mouse monocytic cell line. Osteoclastogenesis was measured by TRAP staining and TRACP 5b quantification. ResultsAT-406 increased apoptosis in MDA-MB-231 breast cancer cells in vitro, and activation of RANK-pathway improved this effect. RANKL stimuli induced a strong increase in c-IAP2. AT-406 increased osteoclast differentiation and activity, by up-regulating the osteogenic transcription factor Nfatc1, but also increased the apoptosis of mature osteoclasts in the absence of RANKL. ConclusionsOur results indicate that despite the anti-tumoral effect of AT-406, its use in the context of bone metastatic disease needs to be carefully monitored for the induction of increased bone resorption. We also hypothesize that the combination of AT-406 with anti-RANKL directed therapies could have a beneficial effect, especially in RANK-positive tumors.

Nicotine affects bone resorption and suppresses the expression of cathepsin K, MMP-9 and vacuolar-type H(+)-ATPase d2 and actin organization in osteoclasts.

Tobacco smoking is an important risk factor for the development of several cancers, osteoporosis, and inflammatory diseases such as periodontitis. Nicotine is one of the major components of tobacco. In previous study, we showed that nicotine inhibits mineralized nodule formation by osteoblasts, and the culture medium from osteoblasts containing nicotine and lipopolysaccharide increases osteoclast differentiation. However, the direct effect of nicotine on the differentiation and function of osteoclasts is poorly understood. Thus, we examined the direct effects of nicotine on the expression of nicotine receptors and bone resorption-related enzymes, mineral resorption, actin organization, and bone resorption using RAW264.7 cells and bone marrow cells as osteoclast precursors. Cells were cultured with 10−5, 10−4, or 10−3 M nicotine and/or 50 µM α-bungarotoxin (btx), an 7 nicotine receptor antagonist, in differentiation medium containing the soluble RANKL for up 7 days. 1–5, 7, 9, and 10 nicotine receptors were expressed on RAW264.7 cells. The expression of 7 nicotine receptor was increased by the addition of nicotine. Nicotine suppressed the number of tartrate-resistant acid phosphatase positive multinuclear osteoclasts with large nuclei(≥10 nuclei), and decreased the planar area of each cell. Nicotine decreased expression of cathepsin K, MMP-9, and V-ATPase d2. Btx inhibited nicotine effects. Nicotine increased CA II expression although decreased the expression of V-ATPase d2 and the distribution of F-actin. Nicotine suppressed the planar area of resorption pit by osteoclasts, but did not affect mineral resorption. These results suggest that nicotine increased the number of osteoclasts with small nuclei, but suppressed the number of osteoclasts with large nuclei. Moreover, nicotine reduced the planar area of resorption pit by suppressing the number of osteoclasts with large nuclei, V-ATPase d2, cathepsin K and MMP-9 expression and actin organization.

Estrogen Deficiency Induces the Differentiation of IL-17 Secreting Th17 Cells: A New Candidate in the Pathogenesis of Osteoporosis

Th17 cells produce IL-17, and the latter promotes bone loss in collagen-induced arthritis in mice. Blocking IL-17 action in mouse model of rheumatoid arthritis reduces disease symptoms. These observations suggest that Th17 cells may be involved in the pathogenesis of bone loss. However, the role of Th17 cell in estrogen (E2) deficiency-induced bone loss is still not very clear. We investigated the effect of E2 on Th17 differentiation in vivo and IL-17 mediated regulation of osteoclast and osteoblast differentiation. Additionally, effect of IL-17 functional block under E2 deficiency-induced bone loss was studied. In murine bone marrow cells, E2 suppressed IL-17 mediated osteoclast differentiation. IL-17 inhibited formation of mineralized nodules in osteoblasts and this effect was suppressed by E2. E2 treatment to mouse calvarial osteoblasts inhibited the IL-17-induced production of osteoclastogenic cytokines and NF-kB translocation. In ovariectomized mice, there was increase in the number of Th17 cells, transcription factors promoting Th17 cell differentiation and circulating IL-17 levels. These effects were reversed by E2 supplementation. Treatment of neutralizing IL-17 monoclonal antibody to Ovx mice mitigated the E2 deficiency-induced trabecular bone loss and reversed the decreased osteoprotegerin-to-receptor activator of nuclear factor kappa B ligand (RANKL) transcript levels in long bones, increased osteoclast differentiation from the bone marrow precursor cells and decreased osteoblast differentiation from the bone marrow stromal cells. Our findings indicate that E2 deficiency leads to increased differentiation of Th17 cells with attendant up regulation of STAT3, ROR-γt and ROR-α and downregulation of Foxp3 which antagonizes Th17 cell differentiation. Increased IL-17 production in turn induces bone loss by increasing pro-osteoclastogenic cytokines including TNF-α, IL-6 and RANKL from osteoblasts and functional block of IL-17 prevents bone loss. IL-17 thus plays a critical causal role in Ovx-induced bone loss and may be considered a potential therapeutic target in pathogenesis of post menopausal osteoporosis.

Targeted Disruption of Ephrin B1 in Cells of Myeloid Lineage Increases Osteoclast Differentiation and Bone Resorption in Mice

Disruption of ephrin B1 in collagen I producing cells in mice results in severe skull defects and reduced bone formation. Because ephrin B1 is also expressed during osteoclast differentiation and because little is known on the role of ephrin B1 reverse signaling in bone resorption, we examined the bone phenotypes in ephrin B1 conditional knockout mice, and studied the function of ephrin B1 reverse signaling on osteoclast differentiation and resorptive activity. Targeted deletion of ephrin B1 gene in myeloid lineage cells resulted in reduced trabecular bone volume, trabecular number and trabecular thickness caused by increased TRAP positive osteoclasts and bone resorption. Histomorphometric analyses found bone formation parameters were not changed in ephrin B1 knockout mice. Treatment of wild-type precursors with clustered soluble EphB2-Fc inhibited RANKL induced formation of multinucleated osteoclasts, and bone resorption pits. The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation. Similarly, activation of ephrin B1 reverse signaling by EphB2-Fc treatment led to inhibition of TRAP, cathepsin K and NFATc1 mRNA expression in osteoclasts derived from wild-type mice but not conditional knockout mice. Immunoprecipitation with NHERF1 antibody revealed ephrin B1 interacted with NHERF1 in differentiated osteoclasts. Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin. We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin.

Oncostatin M Promotes Mammary Tumor Metastasis to Bone and Osteolytic Bone Degradation

Oncostatin M (OSM) is an interleukin-6 (IL-6) family cytokine that has been implicated in a number of biological processes including inflammation, hematopoiesis, immune responses, development, and bone homeostasis. Recent evidence suggests that OSM may promote breast tumor invasion and metastasis. We investigated the role of OSM in the formation of bone metastases in vivo using the 4T1.2 mouse mammary tumor model in which OSM expression was knocked down using shRNA (4T1.2-OSM). 4T1.2-OSM cells were injected orthotopically into Balb/c mice, resulting in a greater than 97% decrease in spontaneous metastasis to bone compared to control cells. Intratibial injection of these same 4T1.2-OSM cells also dramatically reduced the osteolytic destruction of trabecular bone volume compared to control cells. Furthermore, in a tumor resection model, mice bearing 4T1.2-OSM tumors showed an increase in survival by a median of 10 days. To investigate the specific cellular mechanisms important for OSM-induced osteolytic metastasis to bone, an in vitro model was developed using the RAW 264.7 preosteoclast cell line co-cultured with 4T1.2 mouse mammary tumor cells. Treatment of co-cultures with OSM resulted in a 3-fold induction of osteoclastogenesis using the TRAP assay. We identified several tumor cell-induced factors including vascular endothelial growth factor, IL-6, and a previously uncharacterized OSM-regulated bone metastasis factor, amphiregulin (AREG), which increased osteoclast differentiation by 4.5-fold. In addition, pretreatment of co-cultures with an anti-AREG neutralizing antibody completely reversed OSM-induced osteoclastogenesis. Our results suggest that one mechanism for OSM-induced osteoclast differentiation is via an AREG autocrine loop, resulting in decreased osteoprotegerin secretion by the 4T1.2 cells. These data provide evidence that OSM might be an important therapeutic target for the prevention of breast cancer metastasis to bone.

Regulation of Breast Cancer-induced Bone Lesions by β-Catenin Protein Signaling

Breast cancer patients have an extremely high rate of bone metastases. Morphological analyses of the bones in most of the patients have revealed the mixed bone lesions, comprising both osteolytic and osteoblastic elements. β-Catenin plays a key role in both embryonic skeletogenesis and postnatal bone regeneration. Although this pathway is also involved in many bone malignancy, such as osteosarcoma and prostate cancer-induced bone metastases, its regulation of breast cancer bone metastases remains unknown. Here, we provide evidence that the β-catenin signaling pathway has a significant impact on the bone lesion phenotype. In this study, we established a novel mouse model of mixed bone lesions using intratibial injection of TM40D-MB cells, a breast cancer cell line that is highly metastatic to bone. We found that both upstream and downstream molecules of the β-catenin pathway are up-regulated in TM40D-MB cells compared with non-bone metastatic TM40D cells. TM40D-MB cells also have a higher T cell factor (TCF) reporter activity than TM40D cells. Inactivation of β-catenin in TM40D-MB cells through expression of a dominant negative TCF4 not only increases osteoclast differentiation in a tumor-bone co-culture system and enhances osteolytic bone destruction in mice, but also inhibits osteoblast differentiation. Surprisingly, although tumor cells overexpressing β-catenin did induce a slight increase of osteoblast differentiation in vitro, these cells display a minimal effect on osteoblastic bone formation in mice. These data collectively demonstrate that β-catenin acts as an important determinant in mixed bone lesions, especially in controlling osteoblastic effect within tumor-harboring bone environment.

Inositol Polyphosphate 4-Phosphatase B as a Regulator of Bone Mass in Mice and Humans

Osteoporosis is a multifactorial genetic disease characterized by reduction of bone mass due to dysregulation of osteoclast differentiation or maturation. Herein, we identified a regulator of osteoclastogenesis, the murine homolog of inositol polyphosphate 4-phosphatase type IIα (Inpp4bα). Expression of Inpp4bα is detected from early osteoclast differentiation to activation stage. Targeted expression of native Inpp4bα exvivo repressed whereas phosphatase-inactive Inpp4bα stimulated osteoclast differentiation. Inpp4bα acts on intracellular calcium level that modulates NFATc1 nuclear translocation and activation. Invivo mice deficient in Inpp4b displayed increased osteoclast differentiation rate and potential resulting in decreased bone mass and osteoporosis. Importantly, INPP4B in human was identified as a susceptibility locus for osteoporosis. This study defined Inpp4b as a major modulator of the osteoclast differentiation and as a gene linked to variability of bone mineral density in mice and humans.

IL-23 Is Critical for Induction of Arthritis, Osteoclast Formation, and Maintenance of Bone Mass

The role of IL-23 in the development of arthritis and bone metabolism was studied using systemic IL-23 exposure in adult mice via hydrodynamic delivery of IL-23 minicircle DNA in vivo and in mice genetically deficient in IL-23. Systemic IL-23 exposure induced chronic arthritis, severe bone loss, and myelopoiesis in the bone marrow and spleen, which resulted in increased osteoclast differentiation and systemic bone loss. The effect of IL-23 was partly dependent on CD4(+) T cells, IL-17A, and TNF, but could not be reproduced by overexpression of IL-17A in vivo. A key role in the IL-23-induced arthritis was made by the expansion and activity of myeloid cells. Bone marrow macrophages derived from IL-23p19(-/-) mice showed a slower maturation into osteoclasts with reduced tartrate-resistant acid phosphatase-positive cells and dentine resorption capacity in in vitro osteoclastogenesis assays. This correlated with fewer multinucleated osteoclast-like cells and more trabecular bone volume and number in 26-wk-old male IL-23p19(-/-) mice compared with control animals. Collectively, our data suggest that systemic IL-23 exposure induces the expansion of a myeloid lineage osteoclast precursor, and targeting IL-23 pathway may combat inflammation-driven bone destruction as observed in rheumatoid arthritis and other autoimmune arthritides.

Dendritic Cell-Mediated In Vivo Bone Resorption

Osteoclasts are resident cells of the bone that are primarily involved in the physiological and pathological remodeling of this tissue. Mature osteoclasts are multinucleated giant cells that are generated from the fusion of circulating precursors originating from the monocyte/macrophage lineage. During inflammatory bone conditions in vivo, de novo osteoclastogenesis is observed but it is currently unknown whether, besides increased osteoclast differentiation from undifferentiated precursors, other cell types can generate a multinucleated giant cell phenotype with bone resorbing activity. In this study, an animal model of calvaria-induced aseptic osteolysis was used to analyze possible bone resorption capabilities of dendritic cells (DCs). We determined by FACS analysis and confocal microscopy that injected GFP-labeled immature DCs were readily recruited to the site of osteolysis. Upon recruitment, the cathepsin K-positive DCs were observed in bone-resorbing pits. Additionally, chromosomal painting identified nuclei from female DCs, previously injected into a male recipient, among the nuclei of giant cells at sites of osteolysis. Finally, osteolysis was also observed upon recruitment of CD11c-GFP conventional DCs in Csf1r(-/-) mice, which exhibit a severe depletion of resident osteoclasts and tissue macrophages. Altogether, our analysis indicates that DCs may have an important role in bone resorption associated with various inflammatory diseases.

Cellular chemotaxis induced by wear particles from joint replacements

The destruction of bone around joint replacements (periprosthetic osteolysis) is an adverse biological response associated with the generation of excessive wear particles. Wear debris from the materials used for joint replacements stimulate a chronic inflammatory and foreign body reaction that leads to increased osteoclast differentiation and maturation, and decreased bone formation. Wear debris induces both local and systemic trafficking of inflammatory cells to the site of particle generation. Recent studies have shown that this effect is mediated primarily by chemotactic cytokines (chemokines) including macrophage chemotactic protein-1 (MCP-1, also known as CCL2), macrophage inhibitory protein-1 (MIP-1), Interleukin-8 (IL-8 or CXCL8) and others. These ligands migrate along a concentration gradient to interact with G-protein-linked transmembrane receptors on the cell surface. Chemokines are involved in the innate and adaptive immune responses, angiogenesis, wound healing and tissue repair. In vitro, in vivo and tissue retrieval studies have shown that chemokine-directed systemic trafficking of polymorphonuclear leukocytes and cells of the monocyte/macrophage lineage to wear particles result in the release of pro-inflammatory factors and subsequent bone loss. Modulation of the chemokine ligand-receptor axis is a potential strategy to mitigate the adverse effects of wear particles from joint replacements.

Id1 Represses Osteoclast-Dependent Transcription and Affects Bone Formation and Hematopoiesis

BackgroundThe bone-bone marrow interface is an area of the bone marrow microenvironment in which both bone remodeling cells, osteoblasts and osteoclasts, and hematopoietic cells are anatomically juxtaposed. The close proximity of these cells naturally suggests that they interact with one another, but these interactions are just beginning to be characterized.Methodology/Principal FindingsAn Id1−/− mouse model was used to assess the role of Id1 in the bone marrow microenvironment. Micro-computed tomography and fracture tests showed that Id1−/− mice have reduced bone mass and increased bone fragility, consistent with an osteoporotic phenotype. Osteoclastogenesis and pit formation assays revealed that loss of Id1 increased osteoclast differentiation and resorption activity, both in vivo and in vitro, suggesting a cell autonomous role for Id1 as a negative regulator of osteoclast differentiation. Examination by flow cytometry of the hematopoietic compartment of Id1−/− mice showed an increase in myeloid differentiation. Additionally, we found increased expression of osteoclast genes, TRAP, Oscar, and CTSK in the Id1−/− bone marrow microenvironment. Lastly, transplantation of wild-type bone marrow into Id1−/− mice repressed TRAP, Oscar, and CTSK expression and activity and rescued the hematopoietic and bone phenotype in these mice.Conclusions/SignificanceIn conclusion, we demonstrate an osteoporotic phenotype in Id1−/− mice and a mechanism for Id1 transcriptional control of osteoclast-associated genes. Our results identify Id1 as a principal player responsible for the dynamic cross-talk between bone and bone marrow hematopoietic cells.

Dlx5, a Positive Regulator of Osteoblastogenesis, is Essential for Osteoblast-Osteoclast Coupling

The homeodomain protein Dlx5 is an activator of Runx2 (a key regulator of osteogenesis) and is thought to be an important regulator of bone formation. At present, however, the perinatal lethality of Dlx5-null mice has hampered the elucidation of its function in osteogenesis. Here we provide the first analysis of the effects of Dlx5 inactivation on bone development. Femurs of Dlx5-null mouse embryos at the end of gestation exhibit a reduction in both total and trabecular bone volume associated with increased trabecular separation and reduced trabecular number. These parameters are often associated with pathological conditions characterized by reduced osteoblast activity and increased bone resorption. Dlx5(-/-) osteoblasts in culture display reduced proliferation and differentiation rate and reduction of Runx2, Osx, Osteocalcin and Bone Sialoprotein expression. In addition to impaired osteoblast function, Dlx5(-/-) femurs exhibit significant increases in osteoclast number. As Dlx5 is not expressed by osteoclasts, we suggest that its osteoblastic expression might control osteoblast/osteoclast coupling. Cultured Dlx5(-/-) osteoblasts displayed a higher RANKL/OPG ratio. Furthermore, Dlx5(-/-) osteoblasts induced a higher number of TRAP-positive multinucleated cells in normal spleen cultures with a globally increased resorption activity. These findings suggest that Dlx5 is a central regulator of bone turnover as it activates bone formation directly and bone resorption indirectly.

A dominant function of p38 mitogen-activated protein kinase signaling in receptor activator of nuclear factor-kappaB ligand expression and osteoclastogenesis induction by Aggregatibacter actinomycetemcomitans and Escherichia coli lipopolysaccharide.

Lipopolysaccharide from gram-negative bacteria is one of the microbial-associated molecular patterns that initiate the immune/inflammatory response, leading to the tissue destruction observed in periodontitis. The aim of this study was to evaluate the role of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in lipopolysaccharide-induced receptor activator of nuclear factor-kappaB ligand (RANKL) expression by murine periodontal ligament cells. Expression of RANKL and osteoprotegerin mRNA was studied by reverse transcription-polymerase chain reaction upon stimulation with lipopolysaccharide from Escherichia coli and Aggregatibacter actinomycetemcomitans. The biochemical inhibitor SB203580 was used to evaluate the contribution of the p38 MAPK signaling pathway to lipopolysaccharide-induced RANKL and osteoprotegerin expression. Stable cell lines expressing dominant-negative forms of MAPK kinase (MKK)-3 and MKK6 were generated to confirm the role of the p38 MAPK pathway. An osteoclastogenesis assay using a coculture model of the murine monocytic cell line RAW 264.7 was used to determine if osteoclast differentiation induced by lipopolysaccharide-stimulated periodontal ligament was correlated with RANKL expression. Inhibiting p38 MAPK prior to lipopolysaccharide stimulation resulted in a significant decrease of RANKL mRNA expression. Osteoprotegerin mRNA expression was not affected by lipopolysaccharide or p38 MAPK. Lipopolysaccharide-stimulated periodontal ligament cells increased osteoclast differentiation, an effect that was completely blocked by osteoprotegerin and significantly decreased by inhibition of MKK3 and MKK6, upstream activators of p38 MAPK. Conditioned medium from murine periodontal ligament cultures did not increase osteoclast differentiation, indicating that periodontal ligament cells produced membrane-bound RANKL. Lipopolysaccharide resulted in a significant increase of RANKL in periodontal ligament cells. The p38 MAPK pathway is required for lipopolysaccharide-induced membrane-bound RANKL expression in these cells.

TNFalpha mediates the skeletal effects of thyroid-stimulating hormone.

We have shown recently that by acting on the thyroid-stimulating hormone (TSH) receptor (TSHR), TSH negatively regulates osteoclast differentiation. Both heterozygotic and homozygotic TSHR null mice are osteopenic with evidence of enhanced osteoclast differentiation. Here, we report that the accompanying elevation of TNFalpha, an osteoclastogenic cytokine, causes the increased osteoclast differentiation. This enhancement in TSHR-/- and TSHR+/- mice is abrogated in compound TSHR-/-/TNFalpha-/- and TSHR+/-/TNFalpha+/- mice, respectively. In parallel studies, we find that TSH directly inhibits TNFalpha production, reduces the number of TNFalpha-producing osteoclast precursors, and attenuates the induction of TNFalpha expression by IL-1, TNFalpha, and receptor activator of NF-kappaB ligand. TSH also suppresses osteoclast formation in murine macrophages and RAW-C3 cells. The suppression is more profound in cells that overexpress the TSHR than those transfected with empty vector. The overexpression of ligand-independent, constitutively active TSHR abrogates osteoclast formation even under basal conditions and in the absence of TSH. Finally, IL-1/TNFalpha and receptor activator of NF-kappaB ligand fail to stimulate AP-1 and NF-kappaB binding to DNA in cells transfected with TSHR or constitutively active TSHR. The results suggest that TNFalpha is the critical cytokine mediating the downstream antiresorptive effects of TSH on the skeleton.