Pit Formation Assay Research Articles

Abstract 4030: In-vivo evaluation of the anti-metastatic efficacy of small molecule Cathepsin L inhibitor KGP94 in a prostate cancer model

Abstract It is estimated that ∼85% of patients with advanced prostate cancer develop skeletal metastases; an occurrence that results in a substantial reduction in the quality of life and a drastic worsening of the prognosis. The development of novel therapeutic strategies that impair the metastatic process and associated skeletal adversities is therefore critical to improving prostate cancer patient survival. Tumor cells rely on proteolytic enzymes for successful establishment of metastases. Cathepsin L (CTSL) is a lysosomal cysteine protease that is up regulated in a wide range of human cancers including prostate cancer. The elevated secretion of CTSL by aggressively metastasizing tumor cells plays a key role in metastatic spread of tumor cells through proteolytic degradation of extracellular matrix and basement membrane components. In addition, tumor secreted cytokines have been shown to increase osteoclastic secretion of CTSL to promote bone resorption. Intervention strategies targeting CTSL may therefore serve a dual purpose by inhibiting both metastatic dissemination as well as bone resorption. The goal of present studies was to evaluate the anti-metastatic and anti-bone resorptive efficacy of CTSL inhibition using the small molecule CTSL inhibitor 3-bromophenyl-3-hydroxyphenyl-ketone thiosemicarbazone (KGP94). KGP94 treatment significantly impaired the metastatic phenotype of highly metastatic prostate cancer PC-3ML cells. Treatment with 25µM KGP94 decreased the migratory and invasive capacities of PC-3ML cells by 78 and 53% respectively. To validate the anti-metastatic efficacy of KGP94 in-vivo, luciferase labelled PC-3ML cells were injected into the left ventricle of male nu/nu mice followed by daily treatment with 20mg/kg KGP94. Metastatic progression was monitored by weekly bioluminescence imaging using firefly luciferase reporter system. KGP94 treatment led to a significant reduction in metastatic tumor burden (65% reduction) and a reduction in metastatic incidence. Histological assessment of bones further confirmed the reduction in metastatic burden upon KGP94 treatment. CTSL inhibition also led to a significant increase in overall survival of tumor bearing mice (p=0.01). In vitro assessment of anti-resorptive function of KGP94 was performed using osteoclastogenesis and pit formation assays. KGP94 treatment of RAW264.7 osteoclast precursor cells led to a significant decrease (72%) in osteoclast formation as demonstrated by a decrease in the number of TRAP+ multinucleate cells. KGP94 also inhibited the bone resorptive activity of osteoclasts as indicated by a significant reduction in the number of resorptive pits formed by osteoclasts on bone slices. In conclusion, our findings suggest that CTSL inhibition using small molecule KGP94 can significantly impair metastatic dissemination of prostate cancer cells and alleviate bone resorption. Citation Format: Dhivya R. Sudhan, Dietmar W. Siemann. In-vivo evaluation of the anti-metastatic efficacy of small molecule Cathepsin L inhibitor KGP94 in a prostate cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4030. doi:10.1158/1538-7445.AM2014-4030

FRI0367 Celastrol Inhibits Osteoclastogenesis and Bone Resorption in Rankl-Induced Raw264.7 via Nf-κB and MAPK Pathways and Attenuates Bone Erosion in Collagen-Induced Arthritis

BackgroundCelastrus have been long used to treat various inflammatory disorders including Rheumatoid arthritis (RA), SLE and Ankylosing Spondylitis in China. Celastrol is one of the main bioactive component of Celastrus....

Mesoporous Silica Nanoparticles/Hydroxyapatite Composite Coated Implants to Locally Inhibit Osteoclastic Activity

In an attempt to improve implant-bone integration and accelerate bone fracture healing from resisting osteoclastic resorption point of view, we have employed a novel procedure to develop a mesoporous silica nanoparticles/hydroxyapatite (MSNs/HA) composite coating onto stainless Kirschner wire substrate. Characterizations of the surface microstructures indicated enlarged specific surface area compared to HA-coated wires as control, thus the MSNs/HA composite coated implants are endowed with abilities to locally deliver biomedical substances and enhance fracture healing. Herein, zoledronic acid (ZOL) as a model drug, different doses of which were immobilized in the mesoporous coating toward decreasing osteoclastic resorption activity. The loading capacities of ZOL increased almost eight-folds to that of pure HA coating, and the introduction of MSNs obviously retarded ZOL release to achieve a more sustained release profile. After certain periods of osteoclast like cells co-culturing with ZOL contained wires, tartrat-resistant acid phosphatases (TRAP) staining of polynucleated cells and a pit formation assay were performed to investigate the ZOL dose-dependent anti-resorption activity. The promoted local effect on osteoclasts will be of clinical benefit to support implant integration and bone repair.

Kaempferol Inhibits IL-1β-Stimulated, RANKL-mediated Osteoclastogenesis via Downregulation of MAPKs, c-Fos, and NFATc1

Kaempferol is one of the most common flavonoid that is present in a variety of vegetables and fruits and has effects on bone metabolism. The present study was performed to define the effects of kaempferol on interleukin (IL)-1β-stimulated receptor activator of NF-κB ligand (RANKL)-mediated osteoclast differentiation. Bone marrow cells were harvested from 6-week-old male imprinting control region mice, and the differentiation of osteoclasts from these cells was evaluated by tartrate-resistant acid phosphatase staining and resorption pit formation assay. Phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated p38, phosphorylated c-Jun amino-terminal kinase, NF-κB (p65), IκBα, c-Fos, and nuclear factor of activated T cells c1 (NFATc1) expressions were examined by Western blotting and quantitative RT-PCR. Kaempferol inhibits IL-1β-stimulated, RANKL-mediated osteoclast differentiation and also inhibits IL-1β-stimulated, RANKL-mediated phosphorylation of ERK 1/2, p38 and JNK MAP kinases, and expressions of c-Fos and NFATc1. These results indicate that kaempferol has an inhibitory role in the bone loss by preventing osteoclast formation and suggest that it might be a novel therapeutic agent for the treatment of inflammatory arthritis by managing bone destruction.

Cadmium induces the differentiation of duck embryonic bone marrow cells into osteoclasts in vitro

This study aimed to determine the in vitro effect of cadmium on the differentiation of duck embryonic bone marrow cells into osteoclasts. Bone marrow cells were harvested from 23-day old Gaoyou duck embryos and were cultured with either 50nmol/L cadmium alone or different cadmium concentrations (0, 5, 10, 20 and 50nmol/L) in combination with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). Tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay with bovine cortical bone slices, and co-staining with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin and Hoechst 33258 were performed to determine the number of TRAP-positive cells and bone resorption activity.Cadmium at a concentration ⩾10 nmol/L in the presence of M-CSFandRANKL significantly increased in a concentration-dependent manner both the number of TRAP-positive cells (35–160%) and bone resorption activity (36–261%) (P<0.05). High cadmium concentrations in the presence of M-CSFandRANKL markedly promoted the formation of filamentous (F)-actin rings in differentiated osteoclasts. In conclusion, cadmium promotes in vitro the differentiation of duck embryonic osteoclasts in the presence of M-CSF and RANKL.

Estrogen Inhibits RANKL-Induced Osteoclastic Differentiation by Increasing the Expression of TRPV5 Channel

The inhibitor effect of estrogen on osteoclasts differentiation is very important in the etiology of estrogen protecting the adult skeleton against bone loss. However, the precise molecular events underlying the effect of estrogen on osteoclasts differentiation are not known. Recent studies implicated an important role of transient receptor potential vanilloid 5 (TRPV5) in osteoclast differentiation and bone resorption. Furthermore, some studies have confirmed that estrogen is involved in the regulation of calcium ion (Ca(2+)) influx in many cells via TRPV5 channel. Therefore, we hypothesize that TRPV5 channel may be implicated in the process of estrogen-inhibited osteoclastogenesis and bone resorption. Western blot, quantitative real-time PCR, tartrate-resistant acid phosphatase (TRAP) staining, and pit formation assay were employed to investigate the role of TRPV5 in estrogen decreasing osteoclast differentiation and bone resorption. We found that the expression of TRPV5 is significantly down-regulated during estrogen deficiency-induced osteoclastogenesis. Furthermore, TRAP staining and pit formation assay showed that the depletion of TRPV5 significantly blocks the inhibitor effects of estrogen on osteoclasts differentiation and bone resorption activity. Further studies confirmed that estrogen regulates the expression of TRPV5 channel via estrogen receptor. Based on these results above, we can draw conclusion that TRPV5 may contribute to the process of estrogen-inhibited osteoclastogenesis and bone resorption activity.

Knockout of TRPV6 Causes Osteopenia in Mice by Increasing Osteoclastic Differentiation and Activity

Background: Calcium ion (Ca²⁺) signals are required for osteoclast differentiation. Previous study showed that transient receptor potential vanilloid 5 (TRPV5) is an essential Ca²⁺ transporter in osteoclastogenesis and bone resorption. TRPV5 and TRPV6 represent two highly homologous members within the transient receptor potential (TRP) superfamily. However, the role of TRPV6 in bone metabolism is still controversial and little is known about the involvement of TRPV6 in receptor activator of nuclear factor κ-B ligand (RANKL)-induced osteoclastogenesis. Methods: In our study, gene knockout mice, RNA interference, western blot, quantitative real-time PCR, tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay, histomorphometry and measurement of serum parameters were employed to investigate the role of TRPV6 in bone homeostasis, osteoclastogenesis and bone resorption. Results: We found that TRPV6 depletion results in noticeable destruction of bone microarchitecture in TRPV6 knockout mice (TRPV6^-/-), suggesting that TRPV6 is a critical regulator in bone homeostasis. Inactivation of Trpv6 had no effect on osteoblastic bone formation. However, quantification of the TRAP staining showed a significantly increased osteoclast number and surface area in the metaphyseal area of femurs bone sections derived from TRPV6^-/- mice. In agreement with our observations from TRPV6^-/- mice, TRPV6 depletion in vitro significantly increased osteoclasts differentiation and bone resorption activity. Conclusion: Based on these results above, we can draw conclusions that TRPV6 plays an essential role in bone metabolism and is a critical regulator in osteoclasts differentiation and bone resorption.

Combination of Tumor Necrosis Factor α and Interleukin‐6 Induces Mouse Osteoclast‐like Cells With Bone Resorption Activity Both In Vitro and In Vivo

To clarify the function of osteoclast-like multinuclear cells differentiated from bone marrow-derived macrophages (BMMs) by a combination of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6), and to investigate the molecular mechanisms underlying the differentiation. BMMs were stimulated by TNFα and/or IL-6. The cells were then compared with conventional osteoclasts differentiated in vitro by RANKL. An in vitro pit formation assay on dentine slices and an in vivo resorption assay of calvarial bones were performed. We also evaluated the activities and expression levels of NF-κB, c-Fos, and NF-ATc1, which are essential to the differentiation of conventional osteoclasts. Small interfering RNA was used to knock down c-Fos. The effects of genetic ablation of STAT-3 and pharmacologic inhibitors of NF-AT, JAK, and ERK were also studied. Osteoclast-like cell differentiation depended on TNFα and IL-6 and was not inhibited by osteoprotegerin. These differentiated cells were associated with both in vitro and in vivo bone resorption activity. TNFα and IL-6 had a synergistic effect on the activity and expression of c-Fos. Knockdown of c-Fos inhibited the expression of NF-ATc1 and the differentiation of osteoclast-like cells. All of these inhibitors blocked differentiation of the cells in vitro, but surprisingly, the conditional knockout of STAT-3 did not. Tofacitinib also inhibited the bone destruction caused by TNFα and IL-6 in vivo. Our results demonstrate that a combination of the inflammatory cytokines TNFα and IL-6 can induce osteoclast-like cells that have in vitro and in vivo bone-resorptive activity.

A Novel Bruton′s Tyrosine Kinase Inhibitor CC-292 In Combination With The Proteasome Inhibitor Carfilzomib Iimpacts Multiple Myeloma Bone Microenviroment With Resultant Anti-Myeloma Activity

A member of the Tec family kinases, Bruton's tyrosine kinase (Btk) modulates B-cell development and activation, and plays an important role in antibody production. Interestingly, Btk and Tec (the other Tec kinase family) regulate osteoclast (OC) differentiation via Receptor Activator of Nuclear Factor κ B (RANK) signaling. Moreover, OCs derived from X-linked agammaglobulinemia (XLA) patients who harbor Btk null mutations have impaired function.Here we show that a potent and specific Btk inhibitor, CC-292 inhibits OC function in multiple myeloma (MM) patients. CC-292 is a highly selective, covalent Btk inhibitor. OC derived from MM patient monocytes were assayed with or without CC-292. Interestingly, OC function was significantly inhibited in the presence of CC-292 (100 nM and 1000 nM) as demonstrated by pit formation assay. However, mRNA expression for TRAP and Cathepsin K, two OC differentiation markers were increased in the presence of CC-292 suggesting that CC-292 inhibits OC function without inhibiting OC differentiation.OC sealing zone contributes to OC bone resorption function. Given the role of c-Src and Proline-rich tyrosine Kinase 2 (Pyk2) signaling in sealing zone formation and OC function we next evaluated CC-292's effect on Pyk2 and c-Src. Pyk2 plays a role in OC activation and localizes to the sealing zone in OC. RANK signaling activates c-Src, which phosphorylates Pyk2. Moreover c-Src controls OC bone resorption by regulating actin organization via cortactin. Interestingly, CC-292 (100 nM) inhibited c-Src total protein, c-Src phosphorylation and Pyk2 phosphorylation. Furthermore, CC-292 (100 nM) inhibited cortactin protein and mRNA expression, and upregulated c-Cbl protein (E3 ubiquitin ligase for c-Src) expression in OC derived from MM patient monocytes with resultant inhibition of OC sealing zone formation. However, at the same low doses (100 nM) CC-292 did not show any direct in vitro effect against MM cell viability.Because carfilzomib, a proteasome inhibitor that binds irreversibly to its target, has potent anti-MM activity and also inhibits OC resorptive activity, we studied CC-292 in combination with carfilzomib. Our data suggests that carfilzomib (1.25 nM) has no impact on OC sealing zone formation but inhibits OC differentiation. CC-292 (100 nM) in combination with carfilzomib (1.25 nM) inhibited not only sealing zone formation but also OC differentiation, resulting in stronger suppression of OC function than carfilzomib alone.The combination of CC-292 (30mg/kg p.o. for 5 days per week for 6 weeks) and carfilzomib (3 mg/kg i.v. x 2 days per week for 4 weeks and 2 mg/kg i.v. x 2 days per week for 2 weeks) significantly inhibited tumor burden and myeloma cell numbers in a diffuse NOD-SCID MM model. The calvarial cells derived from these mice treated with CC-292 alone, carfilzomib alone or the combination showed higher osteocalcin mRNA (osteoblast differentiation marker) expression. A specific bone resorption marker, carboxy-terminal telopeptide collagen crosslinks (CTX) in mouse serum was significantly inhibited in CC-292 and CC-292 in combination with carfilzomib treatment groups in comparison with control mice. Furthermore, 3D microCT reconstructions showed increase in cancellous bone volume in lumbar vertebrae in mice treated with CC-292 or carfilzomib, while the combination treatment resulted in an increase in cancellous bone volume in an additive manner.These data demonstrate that the novel BTK inhibitor CC-292 inhibits OC function through inhibition of OC sealing zone formation. Moreover, CC-292 in combination with carfilzomib augments effects against the bone microenvironment with resultant anti-MM activity. Disclosures:Arastu-Kapur:Onyx Pharmaceuticals, Inc.: Employment. Evans:Celgene Avilomics Research: Employment, Equity Ownership. Singh:Celgene Avilomics Research: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals, Inc.: Employment. Westlin:Celgene Avilomics Research: Employment, Equity Ownership. Raje:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Amgen: Consultancy; Acetylon: Research Funding; Eli Lilly: Research Funding.

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.

Anti-apoptotic Bcl-2 family member Mcl-1 regulates cell viability and bone-resorbing activity of osteoclasts

Myeloid cell leukemia sequence 1 (Mcl-1) is an anti-apoptotic Bcl-2 family protein and an immediate early gene expressed during myeloid leukemia cell line differentiation. We analyzed the expression and function of Mcl-1 in osteoclasts. Mcl-1 protein exhibited a short half-life in osteoclasts caused by its degradation in the ubiquitin-proteasome system. Mcl-1 had no effect on osteoclast differentiation, but its overexpression prolonged osteoclast survival and suppressed the bone-resorbing activity of these cells, as determined by pit formation assay. Conversely, Mcl-1 depletion suppressed osteoclast survival and increased bone resorption. This negative role for Mcl-1 on the bone-resorptive activities of osteoclasts may be caused by the increase in adenosine triphosphate/adenosine diphosphate ratio. Finally, we showed that the local deletion of Mcl-1 by the injection of the Cre adenovirus into the calvaria of Mcl1fl/fl mice significantly affected GST-RANKL-induced bone resorption in vivo. These results demonstrated that Mcl-1 positively regulates cell viability and negatively regulates the bone-resorbing activity of osteoclasts both in vitro and in vivo.

291: Tumor necrosis factor-α aggravates orthodontically root resorption

Objective Tumor necrosis factor (TNF)-α is known as inflammatory cytokine of potent osteoclast activate factor. TNF-α together with interleukin (IL)-1 promotes differentiation of osteoclast from osteoclast precursor and engages in inflammatory bone resorption. Root resorption is one of a complication associated with orthodontic tooth movement, and that is derived from excessive activation of odontoclast. However, the relationship with tooth root resorption has not been revealed. In this study, therefore, the relationship between TNF-α and tooth root resorption was examined. Materials and methods: Twenty patients who had been receiving orthodontic treatment (fifteen-control subjects and five-severe root resorption subjects) participated in this study. Gingival crevicular fluid (GCF) was collected from the patients. To determine the TNF-α level, the eluted GCF was used for Western blot analysis.25 g of orthodontic force was applied to the upper first molars in mouse in vivo. HE staining and immunohistochemical staining with TNF-α was performed. Human periodontal ligament cells (hPDL cells) were subjected to compressive forces (CF) for up to 48 h in vitro. Real time PCR and enzyme-linked immunosorbent assay (ELISA) were performed to evaluate the production of TNF-α.In addition, we investigated that the effect of TNF-α on osteoclast through TRAP staining and pit formation assay. Results: The TNF-α and RANKL levels were significantly higher concentration in the severe root resorption group than in the control group by Western blot analysis.In immunohistochemical staining, TNF-α positive cells were significantly increased in the root resorption area at 7 days. In vitro study, the mRNA expression of TNF-αwas found to increase by CF. Moreover, the number of TRAP positive cells significantly increased in the TNF-α with RANKL group. Furthermore, the area of resorption pits increased marginally in the same group. Conclusions: TNF-α may induce by CF promote odontoclastogenesis and aggravate root resorption.

Influence of osteoprotegerin on differentiation, activation, and apoptosis of Gaoyou duck embryo osteoclasts in vitro

ABSTRACT The aim of this study was to determine the influence of osteoprotegerin (OPG) on the differentiation, activation, and apoptosis of Gaoyou duck embryo osteoclasts cultured in vitro. Bone marrow cells were harvested from 23-d-old Gaoyou duck embryos and cultured in the presence of different concentrations of OPG (group A: no added factors, group B: 30 ng/mL of OPG, and group C: 100 ng/mL of OPG). Tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay, and co-staining with tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin and Hoechst 33258 were all performed to determine the number of TRAP-positive cells, bone resorption activity, and the level of apoptosis, respectively. The number of TRAP-positive cells and the net expansion of pit formations area peaked on d 7 of culture in all 3 groups. The number of osteoclasts and the total volume of pit formations in OPG-treated groups were significantly lower compared with group A (P < 0.05). At each time point, the net expansion of pit formations area correlated with the number of TRAP-positive cells. The OPG inhibited the de novo formation of filamentous (F)-actin rings and promoted the disruption of existing F-actin rings in mature osteoclasts. In addition, OPG induced apoptosis in mature osteoclasts, as demonstrated by morphological changes in the nuclei. In osteoclast precursors, OPG inhibited differentiation and downregulated the formation of F-actin rings. In mature osteoclasts, OPG suppressed activation and enhanced the development of apoptosis, observed as a decrease in the number of TRAP-positive cells, the disruption of F-actin rings and morphological changes of the nuclei.

AB0116 HIF-2alpha dependent rankl induction and osteoclastogenesis is augmented by inflammatory cytokines

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation and consecutive local hypoxia, leading to cartilage and bone destruction. Hypoxia promotes osteoclasts formation in vitro but its...

Inhibitory effects of high glucose/insulin environment on osteoclast formation and resorption in vitro

Patients with type 2 diabetes mellitus (T2DM) exhibit hyperglycemia and hyperinsulinemia and increased risk of fracture at early stage, but they were found to have normal or even enhanced bone mineral density (BMD). This study was aimed to examine the molecular mechanisms governing changes in bone structure and integrity under both hyperglycemic and hyperinsulinemic conditions. Monocytes were isolated from the bone marrow of the C57BL/6 mice, induced to differentiate into osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) and exposed to high glucose (33.6 mmol/L), high insulin (1 μmol/L), or a combination of high glucose/high insulin (33.6 mmol/L glucose and 1 μmol/L insulin). Cells cultured in α-MEM alone served as control. After four days of incubation, the cells were harvested and stained for tartrate resistant acid phosphatase (TRAP). Osteoclast-related genes including RANK, cathepsin K and TRAP were determined by using real-time PCR. The resorptive activity of osteoclasts was measured by using a pit formation assay. Osteoclasts that were derived from monocytes were of multinucleated nature and positive for TRAP, a characteristic marker of osteoclasts. Cell counting showed that the number of osteoclasts was much less in high glucose and high glucose/high insulin groups than in normal glucose and high insulin groups. The expression levels of RANK and cathepsin K were significantly decreased in high glucose, high insulin and high glucose/high insulin groups as compared with normal glucose group, and the TRAP activity was substantially inhibited in high glucose environment. The pit formation assay revealed that the resorptive activity of osteoclasts was obviously decreased in high glucose group and high glucose/high insulin group as compared with normal group. It was concluded that osteoclastogenesis is suppressed under hyperglycemic and hyperinsulinemic conditions, suggesting a disruption of the bone metabolism in diabetic patients.

Collagen-induced arthritis impairs osteoclastogenesis and reactivity to sympathetic neurotransmitter stimuli in bone marrow-derived macrophages from DA rats

Osteoclasts (OC) are key players in bone destruction in rheumatoid arthritis (RA). Recent work showed that catecholaminergic nerve fibers are reduced in RA synovial tissue. Studies on sweat gland innervation revealed that catecholaminergic fibers are capable of phenotypic transition to cholinergic nerves. The sympathetic neurotransmitters norepinephrine (NE) and acetylcholine (ACh) affect osteoclastogenesis oppositely and that is why we wanted to study osteoclastogenesis at different phases of collagen-induced arthritis (CIA) in an altered neurotransmitter microenvironment. The influence of NE and ACh on differentiation and activity of bone marrow macrophage (BMM)-derived osteoclasts from arthritic and control animals were compared at various time-points after arthritis induction. The expression profile for adrenergic and ACh receptors was analyzed on mRNA level. The number of OCs was tendentially lower in arthritic animals. Stimulation with ACh yielded significantly more OCs in controls. NE decreased osteoclastogenesis via β-adrenoceptors and enhanced via α-adrenoceptor stimulation. Cells from arthritic animals were less affected. In form of a trend, osteoclasts from arthritic animals showed decreased activity in cathepsin K activity and in a pit formation assay. The receptor gene expression profile changed in the time course of arthritis. After 20 days past immunization, muscarinic ACh receptors M3 and M5 were significantly upregulated whereas after 40 days adrenoceptors α1D and α2B were significantly downregulated. We conclude that CIA suppresses OC differentiation and activity as well as reactivity to neurotransmitter stimulation but the underlying processes remain to be demonstrated.

Synovial membrane immunohistology in early-untreated rheumatoid arthritis reveals high expression of catabolic bone markers that is modulated by methotrexate

IntroductionWe aimed to investigate the expression and therapeutic modulation of the receptor activator of the NF-κB ligand (RANKL) system in early-untreated rheumatoid arthritis (RA).MethodsIn this study, 15 patients with newly diagnosed RA (median symptom duration 7 months) were started on methotrexate (MTX) 20 mg weekly. Synovial biopsies were obtained by needle arthroscopy at baseline and 8 weeks after initiation of therapy. X-rays of the hands and feet were obtained at baseline and 1 year after diagnosis. Immunohistochemistry was performed to detect RANKL, receptor activator of nuclear factor-κB (RANK) and osteoprotegerin (OPG) in the synovial biopsies. The in vitro effect of MTX was tested on RA-derived primary fibroblasts and the osteoblasts-like osteosarcoma cell line (rtPCR, Western blot and ELISA) and in osteoclasts (tartrate-resistant acid phosphatase staining and dentine pit formation assay).ResultsMTX decreased synovial cellularity as well as RANK expression and the RANKL/OPG ratio. We confirmed this effect by a decrease of the mRNA and protein RANKL/OPG ratio in synovial-derived fibroblasts and osteoblasts-like tumoral cells exposed in vitro to methotrexate. Supernatants from MTX treated osteoblasts-like tumoral cells prevented pre-osteoclast formation in the absence of exogenous RANKL. Furthermore, MTX blocked osteoclastogenesis from peripheral blood mononuclear cells despite the presence of macrophage colony stimulating factor and RANKL, which indicates that MTX directly inhibits osteoclastogenesis.ConclusionsThe synovial membrane of early-untreated RA is characterized by a high RANKL/OPG ratio that can be reversed by methotrexate.

Decoy receptor 3 suppresses RANKL-induced osteoclastogenesis via down-regulating NFATc1 and enhancing cell apoptosis

Decoy receptor 3 (DCR3) has been known to modulate immune functions of monocyte or macrophage. In the present study, we investigated the mechanism and the effect of DCR3 on RANK ligand (RANKL)-induced osteoclastogenesis. We treated cells with DCR3 in RANKL-induced osteoclastogenesis to monitor osteoclast formation by tartrate-resistant acid phosphatase (TRAP) staining. Osteoclast activity was assessed by pit formation assay. The mechanism of inhibition was studied by biochemical analysis such as RT-PCR and immunoblotting. In addition, cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis and apoptosis signalling were evaluated by immunoblotting and using flow cytometry. DCR3 inhibited RANKL-induced TRAP(+) multinucleated cells and inhibited RANKL-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) nuclear translocation in RAW264.7 cells. Also, DCR3 significantly inhibited the bone-resorbing activity of mature osteoclasts. Moreover, DCR3 enhanced RANKL-induced cell apoptosis and enhanced RANKL-induced Fas ligand expression. The mechanisms were mediated via the intrinsic cytochrome c and activated caspase 9 apoptosis pathway. We postulated that the inhibitory activity of DCR3 on osteoclastogenesis occurs via down-regulation of RANKL-induced NFATc1 expression and induction of cell apoptosis. Our results postulated DCR3 as a possible new remedy against inflammatory bone destruction.

CRM1 Inhibition Abrogates Osteoclast Formation and Bone Resorption Via Inhibition of RANKL-Induced NFκB While Sparing Osteoblastogenesis: Further Therapeutic Implication in Multiple Myeloma

AbstractAbstract 1835Blocking CRM1 by novel selective inhibitors of nuclear export (SINE: KPT-185, KPT-251, KPT-276, and KPT-330) induced potent MM cell apoptosis in vitro and in vivo (Abstract #46829). In addition, these compounds inhibited NFkB p65 DNA-binding activity in MM cells. Here, we investigated whether SINEs have effects on bone and whether this is mediated only through anti-MM activity, or additional effects directly on osteoclasts (OC) are in play. We asked whether SINEs could prevent RANKL/M-CSF-induced osteoclastogenesis via blockade of NFkB activation. Mature OC (TRAP+ multinucleated cell) were derived from the CD138-negative cell fraction from MM patient samples (n=4) stimulated with RANKL/M-CSF for 3 weeks, in the presence or absence of KPT-185. KPT-185 significantly blocked formation of TRAP+ multinucleated OC in a dose-dependent manner, further confirmed by reduction of the selective osteoclastic marker TRAP5b in cell culture supernatant. NFkB p65 activity was induced in nuclear extracts of CD14+ OC precursor cells following RANKL stimulation for 30 min. Importantly, KPT-185 and KPT-330 blocked such induction in a dose-dependent manner. When KPT-185 was added 2 weeks following OC differentiation by RANKL/M-CSF, the effects of KPT-185 on osteoclast culture were not as prominent as when drug was added from the onset. Immunofluorescence staining to examine the actin cytoskeleton in OC cultures performed on glass cover slips further confirmed that actin belt formation in mature OCs is required for bone resorption activity. In the presence of KPT-185 or KPT-330, such critical structure was significantly decreased, consistent with diminished mature OC number and reduced TRAP5b. Pit formation assays on dentine slices clearly showed that KPT-185 and KPT-330, as low as 10 nM, inhibited % erosion area when compared with control group (p<0.005). In RANKL-activated preosteoclasts, both compounds further blocked expression levels of NFATC1, the key osteoclast differentiation transcription factor, as well as fusion-related (Atp6v0d2 and DC-STAMP) and adhesion (integrin αv and integrin β3) molecules. We also assessed the effect of SINE on osteogenesis derived from mesenchymal stem cells of normal healthy donors (n=3). Neither KPT-276 nor KPT-330 blocked calcium deposition, an indicator of bone formation in in vitro culture. Moreover, KPT-185 did not alter INA6 MM cell-inhibited calcium deposition of osteoblasts. Thus, SINEs specifically blocked osteoclast formation and bone resorption activity without significantly impacting osteogenesis. This is the first study to demonstrate a novel role of CRM1 regulating osteoclast formation at least in part by blocking NFkB activity triggered in osteoclast precursor cells by RANKL stimulation. Decreased NFkB p65 activity essential for osteoclast differentiation and fusion was associated with suppressed bone resorption. The potent MM cytotoxicity and prolonged host survival (p=0.0004) demonstrated in our disseminated SCID mouse model of human MM (Abstract#46829), coupled with these bone effects, provide the framework for clinical trials targeting CRM1 with SINEs to simultaneously inhibit both tumor progression and bone destruction in MM. Disclosures:Ghobrial:Millennium pharmaceuticals Inc.: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder, Scientific Founder Other.

Adenosine Regulates Bone Metabolism Via A1, A2A and A2B Receptors in Bone Marrow Cells From Normal and Patients with Multiple Myeloma

Abstract 4977 Background:Multiple myeloma is characterized by osteolytic bone lesions, wherein coupled bone remodeling is disrupted with increased osteoclast activation and decreased osteoblast differentiation. We have previously demonstrated that adenosine, acting via A2A receptors, diminishes human and murine osteoclast formation and others have reported that adenosine, acting at A2Breceptors, promotes osteoblast differentiation in murine osteoblast precursors and cell lines. In this study, we examined the effect of adenosine on osteoblast and osteoclast differentiation derived from multiple myeloma (MM) patients. Methods:Human bone marrow was collected from multiple myeloma patients. Bone marrow stromal cells (BMSCs) and bone marrow derived mononuclear (BMMs) cells were isolated and osteoblasts and osteoclastswere cultured, respectively. Adenosine A1 receptor agonist CHA and antagonist Rolofylline, A2A receptor agonist CGS and antagonist ZM, and A2b receptor agonist BAY and antagonist MRS 1754, A3receptor agonist IB-MECA and antagonist MRS 1191; and dipyridamole, a nucleoside transport inhibitor, were added to the culture media. Alkaline phosphatase (ALP) activity assay was used to quantitate the osteoblast differentiation. In vitro osteoblast calcification was determined by alizarin red staining. TRAP+ staining was used to examine the osteoclast differentiation and bone resorption assay was used to study the osteoclast activity. Results:We found that A1R blockade by rolofyllineand A2aR ligation by CGS21680 inhibited differentiation of both normal and MM BMMs into TRAP+ multinucleated cells (IC50= 1nM for A1R, IC50= 10μM for A2AR;p<0. 001, n=3 for both). The inhibition of osteoclast differentiation by Rolofylline was also seen in bone resorption assay (Pit formation assay). The A2A receptor antagonist completely reversed the effects of CGS21680 on osteoclast differentiation. Moreover, enhanced adenosine accumulation in the presence of dipyridamole (0. 5μM) and A2BR activation promoted the differentiation of BMSCs from myeloma patients into osteoblasts shown byArlizarin red staining and ALP activity assay (by 1. 8 ± 0. 41 and 1. 57 ± 0. 26 fold, respectively, p<0. 05, compared with osteogenic media only, n=3 for both). Conclusions:These results indicate that adenosine A2 receptors may be useful targets for the treatment and prevention of MM-induced bone disease. Disclosures:No relevant conflicts of interest to declare.