Actin Ring Formation Research Articles

Direct and indirect effects of functionalised fluorescence-labelled nanoparticles on human osteoclast formation and activity

Recently, it was demonstrated that phosphonate-functionalised nanoparticles were successfully taken up by mesenchymal stem cells without influencing their viability and differentiation capacity, suggesting that they may provide a promising basis for the development of nanoparticles for drug delivery or cell labelling. The present study aimed to investigate the effects of these nanoparticles on osteoclast formation and activity as well as on the inflammatory response of osteoclasts and osteoblasts. The intracellular uptake of the particles by human osteoclasts and osteoblasts was demonstrated by confocal laser scanning microscopy, transmission electron microscopy and fluorescence microscopy. The expression of tartrate-resistant acid phosphatase, carboanhydrase II, cathepsin K, calcitonin receptor and osteoclast-specific vacuolar proton pump subunit TCIRG1 as well as actin ring formation were not significantly altered in osteoclasts by particle treatment, as demonstrated by cytochemical staining and immunostaining. Active calcium phosphate resorption by osteoclasts was also not significantly influenced by the particles. The expression and secretion of pro-inflammatory cytokines (IL-6, IL-1β) by osteoclasts and osteoblasts and the expression of osteoclast-regulating genes (M-CSF, OPG, RANKL) in osteoblasts were similarly not significantly affected. In conclusion, phosphonate-functionalised nanoparticles did not affect osteoclast formation and activity either directly or indirectly, suggesting that they could provide a promising tool for the development of particle-based treatments for anti-resorptive therapies.

Gymnasterkoreayne F inhibits osteoclast formation by suppressing NFATc1 and DC-STAMP expression

Osteoclasts are multinucleated cells that have a unique role in bone degradation. Modulation of osteoclast formation and/or its activity is an important approach for the treatment of bone-destructive diseases such as osteoporosis and rheumatoid arthritis. In this study, Gymnasterkoreayne F (GK-F), a natural compound isolated from Gymnaster koraiensis, was found to inhibit osteoclast differentiation from primary bone marrow-derived macrophages (BMMs) in a dose-dependent manner. The inhibition occurred through the suppression of nuclear factor of activated T cells c1 (NFATc1) expression, which then led to the decreased levels of osteoclastogenic markers, including Cathepsin K and tartrate-resistant acid phosphatase (TRAP). In addition, GK-F abolished pre-osteoclast fusion induced by the receptor activator of nuclear factor kappa B ligand (RANKL), lipopolysaccharide (LPS) and TNF-α. Reflecting its inhibitory effects on cell–cell fusion, GK-F attenuated the gene expression of an essential molecule of osteoclast fusion, the dendritic cell-specific transmembrane protein (DC-STAMP). Furthermore, GK-F inhibited the bone resorptive activity of differentiated osteoclasts through its ability to block RANKL-induced actin ring formation. The effect was associated with a significant decrease in the induction of β3 integrin expression, which is an essential regulator of osteoclast cytoskeletal function. Taken together, these results suggest that GK-F might be useful as a therapeutic agent for bone resorption-related diseases.

Histone deacetylase 2 is required for chromatin condensation and subsequent enucleation of cultured mouse fetal erythroblasts

During the final stages of differentiation of mammalian erythroid cells, the chromatin is condensed and enucleated. We previously reported that Rac GTPases and their downstream target, mammalian homolog of Drosophila diaphanous 2 (mDia2), are required for enucleation of in vitro cultured mouse fetal liver erythroblasts. However, it is not clear how chromatin condensation is achieved and whether it is required for enucleation. Mouse fetal liver erythroblasts were purified from embryonic day 14.5 pregnant mice and cultured in erythropoietin-containing medium. Enucleation was determined by flow-cytometry based analysis after treatment with histone deacetylase inhibitors or infection with lentiviral short hairpin RNA. We showed that histone deacetylases play critical roles in chromatin condensation and enucleation in cultured mouse fetal liver erythroblasts. Enzymatic inhibition of histone deacetylases by trichostatin A or valproic acid prior to the start of enucleation blocked chromatin condensation, contractile actin ring formation and enucleation. We further demonstrated that histone deacetylases 1, 2, 3 and 5 are highly expressed in mouse fetal erythroblasts. Short hairpin RNA down-regulation of histone deacetylase 2, but not of the other histone deacetylases, phenotypically mimicked the effect of trichostatin A or valproic acid treatment, causing significant inhibition of chromatin condensation and enucleation. Importantly, knock-down of histone deacetylase 2 did not affect erythroblast proliferation, differentiation, or apoptosis. These results identify histone deacetylase 2 as an important regulator, mediating chromatin condensation and enucleation in the final stages of mammalian erythropoiesis.

Maslinic acid suppresses osteoclastogenesis and prevents ovariectomy-induced bone loss by regulating RANKL-mediated NF-κB and MAPK signaling pathways

Activation of NF-κB and MAPK/activator protein 1 (AP-1) signaling pathways by receptor activator NF-κB ligand (RANKL) is essential for osteoclast activity. Targeting NF-κB and MAPK/AP-1 signaling to modulate osteoclast activity has been a promising strategy for osteoclast-related diseases. In this study we examined the effects of maslinic acid (MA), a pentacyclic triterpene acid that is widely present in dietary plants, on RANKL-induced osteoclastogenesis, osteoclast function, and signaling pathways by in vitro and in vivo assay systems. In mouse bone marrow monocytes (BMMs) and RAW264.7 cells, MA inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner within nongrowth inhibitory concentration, and MA decreased osteoclastogenesis-related marker gene expression, including TRACP, MMP9, c-Src, CTR, and cathepsin K. Specifically, MA suppressed osteoclastogenesis and actin ring formation at early stage. In ovariectomized mice, administration of MA prevented ovariectomy-induced bone loss by inhibiting osteoclast activity. At molecular levels, MA abrogated the phosphorylation of MAPKs and AP-1 activity, inhibited the IκBα phosphorylation and degradation, blocked NF-κB/p65 phosphorylation, nuclear translocation, and DNA-binding activity by downregulating RANK expression and blocking RANK interaction with TRAF6. Together our data demonstrate that MA suppresses RANKL-induced osteoclastogenesis through NF-κB and MAPK/AP-1 signaling pathways and that MA is a promising agent in the treatment of osteoclast-related diseases such as osteoporosis.

Calpain-6, a target molecule of glucocorticoids, regulates osteoclastic bone resorption via cytoskeletal organization and microtubule acetylation

Glucocorticoids (GCs) inhibit the resorptive capacity of the osteoclast by disrupting its cytoskeleton. We find that calpain-6 (Capn6), a unique, nonproteolytic member of its family, is suppressed 12-fold by dexamethasone (DEX) in the bone-degrading cell. While Capn6 abundance parallels commitment of naive bone marrow macrophages (BMMs) to the osteoclast phenotype, its excess or deletion does not affect the cell's differentiation. On the other hand, Capn6 localizes to the sealing zone, and its overexpression promotes osteoclast spreading and large actin ring formation, eventuating in stimulated bone degradation. Conversely, Capn6 knockdown impairs cytoskeletal organization and the cell's resorptive capacity. Capn6 complexes with tubulin, and its absence inhibits microtubule acetylation and stability in the osteoclast. Knockdown of Capn6 also reduces β3-integrin subunit protein, another essential regulator of osteoclast cytoskeletal function. Reflecting Capn6 as a target molecule of GCs, microtubule stability and acetylation, as well as the expression of β3-integrin protein, are similarly suppressed in DEX-treated osteoclasts. Moreover, overexpression of Capn6 rescues GC-mediated disruption of osteoclast cytoskeleton. Thus Capn6 promotes cytoskeletal organization and microtubule stability in osteoclasts, and its inhibition may mediate the resorption-arresting properties of GCs. © 2011 American Society for Bone and Mineral Research.

Cbp recruitment of Csk into lipid rafts is critical to c-Src kinase activity and bone resorption in osteoclasts

A tyrosine kinase, c-Src, that plays an indispensable role in ruffled border formation and bone resorption is constitutively active in osteoclasts. However, to date, the molecular mechanism underlying increased c-Src activity in osteoclasts is unknown. To address this, we first examined the expression levels and subcellular localization of Csk, a negative regulatory kinase for c-Src. We found that the expression level of Csk in osteoclasts was comparable with that of other tissues. However, in osteoclasts, Csk was hardly localized in lipid rafts, where c-Src is highly expressed. Interestingly, expression of Cbp, which recruits Csk into lipid rafts through physical interaction with Csk, was very low in osteoclasts compared with other tissues. To understand the importance of Cbp in osteoclasts, we introduced Cbp into osteoclasts using an adenovirus gene delivery system. Introduction of Cbp stimulated recruitment of Csk into lipid rafts and suppressed c-Src activity in a dose-dependent manner. Furthermore, introduction of Cbp markedly inhibited formation of actin rings and bone-resorbing activity in osteoclasts. In addition, treatment with RANKL and overexpression of TRAF6 or NFAT2 inhibited Cbp expression in the osteoclastogenic cell line RAW264.7 along with osteoclastic differentiation. NFAT2 overexpression also inhibited Cbp expression in spleen macrophages. Collectively, our results indicate that reduction in Cbp expression is responsible for maintaining high c-Src activity in osteoclasts. These findings contribute to an understanding of the unique regulatory system for c-Src in osteoclasts.

Alisol-B, a novel phyto-steroid, suppresses the RANKL-induced osteoclast formation and prevents bone loss in mice

Osteoclasts, bone-resorbing multinucleated cells, are differentiated from hemopoietic progenitors of the monocyte/macrophage lineage. Bone resorption by osteoclasts is considered a potential therapeutic target to the treatment of erosive bone diseases, including osteoporosis, rheumatoid arthritis, and periodontitis. In the present study, we found that alisol-B, a phyto-steroid from Alisma orientale Juzepczuk, exhibited inhibitory effects on osteoclastogenesis both in vitro and in vivo. Although RT-PCR analysis showed that alisol-B did not affect the 1α,25(OH)2D3-induced expressions of RANKL, OPG and M-CSF mRNAs in osteoblasts, addition of alisol-B to co-cultures of mouse bone marrow cells and primary osteoblasts with 10−8M 1α,25(OH)2D3 caused significant inhibition of osteoclastogenesis. We further examined the direct effects of alisol-B on osteoclast precursors. Alisol-B strongly inhibited RANKL-induced osteoclast formation when added during the early stage of cultures, suggesting that alisol-B acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, alisol-B inhibited the phosphorylation of JNK, which are upregulated in response to RANKL in bone marrow macrophages, alisol-B also inhibited RANKL-induced expression of NFATc1 and c-Fos, which are key transcription factors for osteoclastogenesis. In addition, alisol-B suppressed the pit-forming activity and disrupted the actin ring formation of mature osteoclasts. In a hypercalcemic mouse model induced by 2-methylene-19-nor-(20S)-1α,25(OH)2D3 (2MD), an analog of 1α,25(OH)2D3, administration of alisol-B significantly suppressed 2MD-induced hypercalcemia as resulting from the inhibition of osteoclastogenesis. Taken together, these findings suggest that alisol-B may be a potential novel therapeutic molecule for bone disorders by targeting the differentiation of osteoclasts as well as their functions.

Nano-topography sensing by osteoclasts

Bone resorption by osteoclasts depends on the assembly of a specialized, actin-rich adhesive 'sealing zone' that delimits the area designed for degradation. In this study, we show that the level of roughness of the underlying adhesive surface has a profound effect on the formation and stability of the sealing zone and the associated F-actin. As our primary model substrate, we use 'smooth' and 'rough' calcite crystals with average topography values of 12 nm and 530 nm, respectively. We show that the smooth surfaces induce the formation of small and unstable actin rings with a typical lifespan of approximately 8 minutes, whereas the sealing zones formed on the rough calcite surfaces are considerably larger, and remain stable for more than 6 hours. It was further observed that steps or sub-micrometer cracks on the smooth surface stimulate local ring formation, raising the possibility that similar imperfections on bone surfaces may stimulate local osteoclast resorptive activity. The mechanisms whereby the physical properties of the substrate influence osteoclast behavior and their involvement in osteoclast function are discussed.

Integrins, growth factors, and the osteoclast cytoskeleton

The unique ability of the osteoclast to degrade skeletal tissue depends upon formation of a resorptive microenvironment between the osteoclast and the bone surface. Generation of this privileged space is substantially mediated by signals emanating from alphavbeta3 integrin, which transits to its active high-affinity conformation by growth factor-initiated intracellular events targeting the matrix receptor's cytoplasmic domain. The activated liganded integrin stimulates a signaling complex consisting of c-Src, Syk, immunoreceptor tyrosine-based activation motif proteins, Slp-76, Vav3, and members of the Rho family of GTPases. These events contribute to secretory lysososme insertion into the bone-apposed plasma membrane to form the ruffled border that delivers the bone-degrading molecules (HCl and cathepsin K) into the resorptive microenvironment. Integrin/bone recognition also promotes formation of actin rings, which surround the ruffled border, thereby isolating the focus of skeletal degradation from the general extracellular space.

TRP14 attenuates osteoclast differentiation by inhibiting NF‐κB and MAPK signaling

TRP14 is a disulfide reductase containing the CXXC motif which regulates NF‐κB and mitogen‐activated protein kinases (MAPKs) stimulated by TNFα. Given that NF‐κB and MAPK signaling play an important role in osteoclastogenesis, it was examined whether TRP14 can inhibit osteoclast differentiation. Osteoclast differentiation of RAW264.7 macrophages by receptor activator of NF‐κB ligand (RANKL) was enhanced by depletion of TRP14, while it was inhibited by TRP14 overexpression, as revealed by tartrate‐resistant acid phosphatase (TRAP) staining and TRAP activity assay. When RAW264.7 cells was exposed to RANKL, depletion of TRP14 augmented phosphorylation and degradation of IκBα, and activation of c‐Jun NH2‐terminal kinase (JNK) and p38 as well as consequent induction of nuclear factor of activated T cell, cytoplasmic 1 (NFATc1) that is a pivotal determinant of osteoclastogenesis. In addition depletion of TRP14 strongly expanded actin ring formation that is a prerequisite for osteoclast bone resorption, and significantly increased RANKL‐mediated ROS production. Taken together TRP14 attenuates osteoclast differentiation by inhibiting NF‐κB, JNK and p38 activated by RANKL.

Clathrin-dependent endocytosis of membrane-bound RANKL in differentiated osteoclasts

Bone is continuously repaired and remodelled through well-coordinated activity of osteoblasts that form new bone and osteoclasts, which resorb it. Osteoblasts synthesize and secrete two key molecules that are important for osteoclast differentiation, namely the ligand for the receptor of activator of nuclear factor κB (RANKL) and its decoy receptor osteoprotegerin (OPG). Active membrane transport is a typical feature of the resorbing osteoclast during bone resorption. Normally, one resorption cycle takes several hours as observed by monitoring actin ring formation and consequent disappearance in vitro. During these cyclic changes, the cytoskeleton undergoes remarkable dynamic rearrangement. Active cells show a continuous process of exocytosis that plays an essential role in transport of membrane components, soluble molecules and receptor-mediated ligands thus allowing them to communicate with the environment. The processes that govern intracellular transport and trafficking in mature osteoclasts are poorly known. The principal methodological problem that have made these studies difficult is a physiological culture of osteoclasts that permit observing the vesicle apparatus in conditions similar to the in vivo conditions. In the present study we have used a number of morphological approaches to characterize the composition, formation and the endocytic and biosynthetic pathways that play roles in dynamics of differentiation of mature bone resorbing cells using a tri-dimensional system of physiologic coculture.

Chk1 haploinsufficiency results in anemia and defective erythropoiesis.

BackgroundErythropoiesis is a highly regulated and well-characterized developmental process responsible for providing the oxygen transport system of the body. However, few of the mechanisms involved in this process have been elucidated. Checkpoint Kinase 1 (Chk1) is best known for its role in the cell cycle and DNA damage pathways, and it has been shown to play a part in several pathways which when disrupted can lead to anemia.Methodology/Principal FindingsHere, we show that haploinsufficiency of Chk1 results in 30% of mice developing anemia within the first year of life. The anemic Chk1+/− mice exhibit distorted spleen and bone marrow architecture, and abnormal erythroid progenitors. Furthermore, Chk1+/− erythroid progenitors exhibit an increase in spontaneous DNA damage foci and improper contractile actin ring formation resulting in aberrant enucleation during erythropoiesis. A decrease in Chk1 RNA has also been observed in patients with refractory anemia with excess blasts, further supporting a role for Chk1 in clinical anemia.Conclusions/SignificanceClinical trials of Chk1 inhibitors are currently underway to treat cancer, and thus it will be important to track the effects of these drugs on red blood cell development over an extended period. Our results support a role for Chk1 in maintaining the balance between erythroid progenitors and enucleated erythroid cells during differentiation. We show disruptions in Chk1 levels can lead to anemia.

Difference in Osteoclast Responses to Tricalcium Phosphate in Culture Medium Supplemented with Zinc and to Zinc-Containing Tricalcium Phosphate

Difference in osteoclast responses (i.e., apoptosis, actin ring formation) to tricalcium phosphate (TCP) in culture medium supplemented with zinc and to zinc-containing tricalcium phosphate (ZnTCP) was investigated in this study. On the TCP ceramic, an increase in zinc ion in the culture medium within the range between 0.3 and 6.8 ppm significantly induced an increase in osteoclast apoptosis and a decrease in actin ring formation. However, even a high level of magnesium ion up to 100 ppm in the culture medium or up to 6.8mol% in the ceramic was unlikely to influence osteoclast activity. There was almost no significant difference in osteoclast apoptosis and actin ring formation between ZnTCP with 1mol% zinc and magnesiumcontaining tricalcium phosphate with 1.5mol% magnesium ceramic which have the same solubility and the same dissolution rate. It is suggested that only an increase in zinc level outside resorption lacuna has an inhibitory effect on osteoclast resorption and that an increase in zinc level inside resorption lacuna could not influence osteoclast activity.

Palmatine Attenuates Osteoclast Differentiation and Function through Inhibition of Receptor Activator of Nuclear Factor-.KAPPA.B Ligand Expression in Osteoblast Cells

Osteoclasts are the only cell type capable of resorbing mineralized bone, and they act under the control of numerous cytokines produced by supporting cells such as osteoblasts and stromal cells. Among cytokines, receptor activator of nuclear factor-κB ligand (RANKL) was found to be a key osteoclastogenetic molecule that directly binds to its cognate receptor, RANK, on osteoclast precursor cells. In turn, RANKL, which is an essential factor for differentiation and activation of osteoclasts, is one of the major targets of anti-resorptive agents. In this study, we found that palmatine, an isoquinoline alkaloid originally isolated from Coptis chinensis, had an inhibitory effect on osteoclast differentiation and function in vitro. Palmatine inhibited osteoclast formation in the co-culture system with mouse bone marrow cells (BMC) and osteoblasts in the presence of 10 nM 1α,25-(OH)(2)D(3). Palmatine did not affect osteoclast formation induced by RANKL in the BMC cultures. Reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that palmatine significantly inhibited the expression of 1α,25-(OH)(2)D(3)-induced expression of RANKL mRNAs in stromal cells without loss of cell viability. Moreover, palmatine suppressed resorption pit formation by mature osteoclasts on dentin slices and induced disruption of actin ring formation in mature osteoclasts with an impact on cell viability. Taken together, these results suggest that palmatine attenuates osteoclast differentiation through inhibition of RANKL expression in osteoblast cells, and its inhibitory effect on bone resorption is due to its disruptive effect on actin rings in mature osteoclasts. Therefore, palmatine might be an ideal candidate as an anti-resorptive agent for the prevention and treatment of bone disorders such as osteoporosis.

Enucleation.

Abstract SCI-18Late erythroblasts undergo terminal cell cycle exit, chromatin condensation, and extrusion of the pycnotic nucleus via an asymmetric cell division, but the signaling pathways and genes involved in these last steps of erythropoiesis are not known. Last year we showed that enucleation of mouse fetal erythroblasts requires both Rac GTPases and mDia2, a downstream effector of Rho GTPases and a formin protein required for nucleation of unbranched actin filaments. Rac and mDia2 are required for formation of the cortical actin ring that apparently powers the separation between the membrane- enveloped nucleus and forming reticulocyte. In collaboration with Drs. Tzutzuy Ramirez Hernandez and Maki Murata-Hori of the Temasek Life Sciences Laboratory in Singapore we showed by time-lapse microscopy that extrusion of the nucleus begins with microtubule- dependent “pushing” of the nucleus to one pole of the cell. Final budding off of the nucleus happens quickly – requiring only about 5 minutes. By analyzing fractionated erythroblasts at all stages of differentiation we confirmed that the nucleus undergoes gradual condensation before being extruded out of the cytoplasm. To investigate how chromatin condensation affects condensation and enucleation, we treated cultured erythroblasts with trichostatin A, a pan-histone deacetylase inhibitor, and discovered that enucleation is completely blocked. We further demonstrated that HDAC1, HDAC2 and HDAC3 are highly expressed in erythroid cells. Using a retroviral system to express shRNAs in developing erythroblasts, we found that downregulation of HDAC2 partially blocked enucleation. We also focused on the role of HDAC6 on enucleation since previous reports showed that HDAC6 forms a complex with mDia2. We confirmed the interaction between HDAC6 and mDia2 and demonstrated that HDAC6 can deacetylate mDia2. This may activate mDia2 and eventually promote enucleation. Our hypothesis is that HDACs play two distinct roles in enucleation: HDAC1, HDAC2 and HDAC3 (class I HDACs) are necessary for the condensation of the chromatin which is required for enucleation; on the other hand, HDAC6 (class II HDAC) interacts with and deacetylates mDia2 to promote enucleation. DisclosuresLodish:Amgen: Consultancy, Research Funding.

Glucose Ceramide Synthase Inhibitors Inhibit Osteoclast Activation Induced by Myeloma-Derived and De Novo Synthesized Glycosphingolipids.

Abstract 424In Multiple myeloma (MM), a malignancy of plasma cells, autocrine and paracrine networks involving the malignant cells and their microenvironment, especially osteoclasts (OC), play a crucial role in disease pathogenesis. OC activation and bone destruction are common and devastating events in this disease. Tumour-derived glycosphingolipids (GSL) have been shown to modify the tumour microenvironment by promoting tumour growth, angiogenesis, immune evasion and metastasis. The aim of this work was two-fold: 1) to investigate the role of MM-derived GSL in OC development and activation and 2) to define the role of de novo GSL synthesis in OC development. Using MALDI-TOF MS and MS-MS, we first determined the GSL repertoire of primary CD138+ myeloma cells (n=3) and compared it with non-myeloma bone marrow cells (i.e., without CD138+ cells) (n=3), and various myeloma cell lines (n=5). We found that GM3 was the dominant GSL in primary myeloma cells and GM2/GM3 in myeloma cell lines; by contrast, in non-myeloma marrow the non-polar LacCer was the dominant GSL. As GM3 was the dominant GSL in myeloma cells, we tested its effect on osteoclast function. We found that exogenous GM3 synergistically enhanced the ability of M-CSF and RANKL to induce maturation of murine bone marrow OC in vitro. This, as shown by immunoblotting, was associated with increased ERK1/2, p38, JNK phosphorylation and NFATc dephosphorylation, signal transduction and transcriptional events respectively, required for OC differentiation and maturation in response to RANKL. Furthermore, GM3 further enhanced OC maturation in synergy with IGF-1, a growth factor known to promote myeloma growth and OC activation. Next we tested the effect of inhibition of de novo GSL biosynthesis on osteoclastogenesis. We found that the glucose ceramide synthase (GCS) inhibitors NB-DNJ inhibited RANKL- and M-CSF-dependent development of murine as well as human, monocyte-derived OC in a dose dependent manner when added either in the beginning or during OC differentiation cultures. This effect was associated with significantly reduced RANKL- and M-CSF-dependent phosphorylation of ERK, JNK and p38 as well as reduced localisation of NFATc in the nucleus. OC development in response to RANKL-RANK interaction requires movement of RANK into lipid rafts where it interacts with TRAF6, an adaptor crucial for downstream signalling and with cSrc which is required for actin ring formation and OC resorptive activity. Using sucrose gradient membrane fractionation and GM1 as a marker of rafts we found that GCS inhibitors partially disrupt the integrity of lipid rafts in developing osteoclasts and prevent RANKL-induced localisation of TRAF6 and Src in lipid rafts. Next we tested the ability of NB-DNJ to block OC activation caused by a single injection of alpha-galactosylceramide (αGC), an invariant NKT cell ligand known to rapidly activate the innate immune response. We found that in mice receiving αGC, serum C-telopeptide Type I collagen (CTX) levels increased by ∼50% (p<0.01), while in mice co-injected with αGC plus NB-DNJ (daily for 3 days i.p) CTX levels returned to baseline (p<0.01); in mice receiving only NB-DNJ, CTX levels were not significantly different to vehicle controls (p>0.05). Taken together, our data demonstrate a novel role of GSL in promoting OC differentiation and activation. Thus, GCS inhibitors may be of benefit in reducing OC activation and bone destruction in MM by preventing generation of tumour-derived, pre-osteoclastogenic GSL as well as by inhibiting de novo OC GSL synthesis and thus OC activation. Disclosures:No relevant conflicts of interest to declare.

Dual Src/Abl Kinase Targeted Inhibition in Myeloma Microenvironment Promotes Myeloma Cell Apoptosis Both in Vitro and In Vivo.

Abstract 2813Poster Board II-789Osteoclast, in addition to eroding the bone resulting in lytic lesions, enhances plasma cell proliferation and survival via direct cell to cell contact. Src family protein tyrosine kinases (SFKs) and c-Abl kinase play important role downstream of integrin adhesion receptors, and regulate the cytoskeletal organisation, cell motility and gene expression in response to cell adhesion. We hypothesised targeting SFKs and Abl kinase with the small molecule tyrosine kinase inhibitor Dasatinib has potential to reduce adhesion of plasma cells to ECM proteins in the bone marrow and modify the microenvironment by inhibiting osteoclast function, specifically bone resorption. As a result, myeloma cells could be sensitised to drugs with cytotoxic properties such as dexamethasone.Osteoclasts were generated from primary bone marrow mononuclear cells of myeloma and MGUS patients (n=10). Using Immunofluorescence, we found that Dasatinib 100nM but not dexamethasone inhibited osteoclastogenesis and disrupted the actin cytoskeletal organisation with actin clusters formed in the periphery of the cell. There was absence of actin ring formation at sealing zones which is essential for bone resorption. This effect consistently led to impaired osteoclast function, evidenced by fewer resorption pits formed on rabbit dentine slices on toluidine blue staining. Experiments were repeated ≥ 3 times.In plasma cells, the combination of dexamethasone and Dasatinib synergistically (Calcusyn software) inhibited cell proliferation at clinically relevant concentrations and induced apoptosis of human and murine myeloma cell lines alone and in cocultures with human stromal cells ( p<.001). Dasatinib alone at 200 nM concentration does not inhibit plasma cell proliferation with maximal serum concentration achieved in Phase I CML trials being 180nM. Additionally, Dasatinib and Dexamethasone in combination inhibited secretion of IL-6 but not MIL -1 alpha in stromal cell cocultures. Dasatinib but not dexamethasone significantly inhibited adhesion of myeloma cell lines on Fibronectin despite integrin activation with Magnesium EGTA. This effect was mediated through down regulation of both Src and Abl phosphorylation. Both Dasatinib and Dexamethasone inhibited adhesion of PC on stromal cells and osteoclasts.Taken together, our in vitro results suggest that Dasatinib and dexamethasone could be an effective therapeutic combination with Dasatinib impairing adhesion of plasma cells to the bone marrow microenvironment as well as osteoclast function and resultant bone disease thereby sensitising myeloma cells to the cytotoxic effect of dexamethasone. We have also established that the combination of Dasatinib 75mg/kg and dexamethasone 1mg/kg is not toxic to C57BL/KaLwRij mice. The anti-myeloma efficacy of these drugs alone and in combination is being currently studied. The combination of Dasatinib 100 mg OD days 1-28 and Dexamethasone 20mg OD on Day 1-4, 15-18 has resulted in a partial response (EBMT criteria) in 2 multiply relapsed and steroid refractory myeloma patients without significant toxicity. Serum calcium levels fell commensurate with disease response and we are currently performing experiments to analyse the effect of the drug combination on osteoclast function in vivo. These findings warrant exploring this drug combination in steroid resistant myeloma and patients with extensive skeletal disease prospectively in a phase I/II trial. Disclosures:Off Label Use: Dasatinib is not licensed for Myeloma.

Propolis inhibits osteoclast maturation

Propolis, a natural product produced by the honey bee, has been successfully used in medicine as an anti-inflammatory and antimicrobial agent. Traumatic injuries to the teeth, especially avulsion injuries, present a challenging situation for the clinician because of post-treatment complications, such as inflammatory and/or replacement resorption. Agents that reduce osteoclast numbers and activity may be useful in the treatment of traumatic injuries to the teeth. In this study, we evaluated propolis as an anti-resorptive agent. Calcitriol-stimulated mouse marrow cultures, which contain both osteoclasts and osteoblasts, were exposed to the ethanol extracts of propolis or vehicle control and stained for tartrate-resistant acid phosphatase (TRAP)-activity to identify osteoclasts. A significant, dose-dependent reduction in multinuclear TRAP+ cells was demonstrated, although the propolis treatment accommodated cell growth and survival (P < 0.05). Propolis also reduced the formation of actin rings in pure cultures of RAW 264.7 osteoclast-like cells, suggesting that it exerts direct actions on osteoclast maturation. In summary, our data suggest that propolis inhibits late stages of osteoclast maturation including fusion of osteoclasts precursors to form giant cells and formation of actin rings. This supports the hypothesis that it may prove useful as a medicament to reduce resorption associated with traumatic injuries to the teeth.

Inhibitory regulation of osteoclast bone resorption by signal regulatory protein α

Osteoclasts mediate bone resorption, which is critical for bone development, maintenance, and repair. Proper control of osteoclast development and function is important and deregulation of these processes may lead to bone disease, such as osteoporosis. Previous studies have shown that the cytosolic protein tyrosine phosphatase SHP-1 acts as a suppressor of osteoclast differentiation and function, but putative inhibitory receptors that mediate recruitment and activation of SHP-1 in osteoclasts have remained unknown. In the present study, we identify the SHP-1-recruiting inhibitory immunoreceptor signal regulatory protein (SIRP) alpha as a negative regulator of osteoclast activity. SIRPalpha is expressed by osteoclasts, and osteoclasts from mice lacking the SIRPalpha cytoplasmic tail and signaling capacity display enhanced bone resorption in vitro. Consequently, SIRPalpha-mutant mice have a significantly reduced cortical bone mass. Furthermore, osteoclasts from SIRPalpha-mutant mice show an enhanced formation of actin rings, known to be instrumental in bone resorption. SIRPalpha mutation did not significantly affect osteoclast formation, implying that the role of SIRPalpha was limited to the regulation of mature osteoclast function. This identifies SIRPalpha as a bona fide inhibitory receptor that regulates the bone-resorption activity and supports a concept in which osteoclast function is balanced by the signaling activities of activating and inhibitory immunoreceptors.

Endothelial barrier protection by FTY720 under hyperglycemic condition: involvement of focal adhesion kinase, small GTPases, and adherens junction proteins

Recently, sphingosine 1-phosphate (S1P) has been highlighted as an endothelial barrier-stabilizing mediator. FTY720 is a S1P analog originally developed as a novel immunosuppressant. The phosphorylated form of FTY720 binds to S1P receptors to exert S1P-like biological effects, suggesting endothelial barrier promotion by FTY720. To elucidate whether FTY720 induces signaling events related to endothelial barrier enhancement under hyperglycemic conditions, human microvascular endothelial cells (HMVECs) preincubated with hyperglycemic (30 mM) medium were treated with 100 nM FTY720 for 3 h. Immunofluorescent microscopy and coprecipitation study revealed FTY720-induced focal adhesion kinase (FAK)-associated adherens junction (AJ) assembly at cell-cell contacts coincident with formation of a prominent cortical actin ring. FTY720 also induced transmonolayer electrical resistance (TER) augmentation in HMVEC monolayers in both normoglycemic and hyperglycemic conditions, implying endothelial barrier enhancement. Similar to S1P, site-specific FAK tyrosine phosphorylation analysis revealed FTY720-induced FAK [Y576] phosphorylation without phosphorylation of FAK [Y397/Y925]. Furthermore, FTY720 conditioned the phosphorylation profile of FAK [Y397/Y576/Y925] in hyperglycemic medium to the same pattern observed in normoglycemic medium. FTY720 challenge resulted in small GTPase Rac activation under hyperglycemic conditions, whereas increased Rho activity in hyperglycemic medium was restored to the basal level. Rac protein depletion by small interfering RNA (siRNA) technique completely abolished FTY720-induced FAK [Y576] phosphorylation. These findings strongly suggest the barrier protective effect of FTY720 on HMVEC monolayers in hyperglycemic medium via S1P signaling, further implying the possibility of FTY720 as a therapeutic agent of diabetic vascular disorder.