Osteoclast Bone Resorption Activity Research Articles

MiR-31 controls osteoclast formation and bone resorption by targeting RhoA

IntroductionIncreased activity of osteoclasts is responsible for bone loss and joint destruction in rheumatoid arthritis. For osteoclast development and bone resorption activity, cytoskeletal organization must be properly regulated. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that suppress expression of their target genes. This study was conducted to identify crucial miRNAs to control osteoclasts.MethodsmiRNA expression in the bone marrow-derived macrophages (BMM) with or without receptor activator of nuclear factor κB ligand (RANKL) stimulation was analyzed by miRNA array. To examine the role of specific miRNAs in osteoclast formation, bone resorption activity and actin ring formation, the BMM were retrovirally transduced with miRNA antagomirs. To confirm whether the suppressive effects on osteoclastogenesis by miR-31 inhibition were mediated by targeting RhoA, osteoclast formation was analyzed in the presence of the RhoA inhibitor, exoenzyme C3.ResultsmiR-31 was identified as one of the highly upregulated miRNAs during osteoclast development under RANKL stimulation. Inhibition of miR-31 by specific antagomirs suppressed the RANKL-induced formation of osteoclasts and bone resorption. Phalloidin staining of osteoclasts revealed that actin ring formation at the cell periphery was severely impaired by miR-31 inhibition, and clusters of small ringed podosomes were observed instead. In these osteoclasts, expression of RhoA, one of the miR-31 target genes, was upregulated by miR-31 inhibition in spite of the impaired osteoclastogenesis. Treatment with the RhoA inhibitor, exoenzyme C3, rescued the osteoclastogenesis impaired by miR-31 inhibition.ConclusionsmiR-31 controls cytoskeleton organization in osteoclasts for optimal bone resorption activity by regulating the expression of RhoA.

HIGH SERUM CONCENTRATION OF INTERLEUKINE-6 AND RANK-LIGAND AS RISK FACTORS FOR OSTEOPOROSIS IN ESTROGEN DEFICIENCY POST-MENOPAUSAL WOMEN

Osteoporosis in post-menopausal women is not merely due to deficient estrogenhormone production. The development of osteoporosis is due to increased boneresorption by osteoclasts. The osteoclast’s number and activity is controlled by activatingfactors such as IL-6 and RANK-L. The objective of this study was to determine that highIL-6 and RANK-L serum concentrations are risks for osteoporosis in estrogen deficientpost-menopausal women. The serum concentration of ß-CrossLaps (CTx) was measuredto determine bone resorption rate. This is an observational analytical study using case andcontrol design conducted at Sanglah General Hospital of Denpasar. The sample size wascalculated using the paired case-control study formula. There were 41 osteoporotic and41 non-osteoporotic (control) estrogen deficient post-menopausal women involved in thestudy.Data were analyzed by using the t-paired and McNemar tests. Mean serumconcentration of IL-6 among the osteoporotic women was significantly higher ascompared to that of the controls (3.47±1.75 pg/ml vs 2.51±1.13 pg/ml, p = 0.001). Meanserum concentration of RANK-L among the osteoporotic women was also significantlyhigher as compared to that of the controls (320.66±122.44ng/ml vs 249.94±82.41 ng/ml,p = 0.002). To qualify as risk factors for osteoporosis, the cut-off point for IL-6 was 2.17pg/ml (OR = 4, CI 95%: 1.23-14.24; p = 0.032); the cut-off point for RANK-L was275.165 ng/ml (OR = 8, CI 95%: 1.84-34.79; p = 0.001). Analysis of both high serumconcentration of IL-6 and RANK-L was associated with an odd ratio of 9 (CI 95%: 4,27-18,96, p=0,000). CTx concentration in the osteoporotic women was significantly higherthan in the controls (0.60±0.22ng/ml vs 0.46±0.16ng/ml, p = 0.004).We found that the high IL-6 and RANK-L serum concentrations were risk factorsin estrogen deficient post-menopausal women. CTx being a marker for osteoclastic boneresorption activity, increased in concentration higher in osteoporotic than in nonosteoporoticwomen. The high serum concentrations of IL-6 and RANK-L could be usedas predictors for osteoporosis in estrogen deficient post-menopausal women.

Functions and action mechanisms of flavonoids genistein and icariin in regulating bone remodeling

Increasingly natural products particularly flavonoids are being explored for their therapeutic potentials in reducing bone loss and maintaining bone health. This study has reviewed previous studies on the two better known flavonoids, genistein and icariin, their structures, functions, action mechanisms, relative potency, and potential application in regulating bone remodeling and preventing bone loss. Genistein, an isoflavone abundant in soy, has dual functions on bone cells, able to inhibit bone resorption activity of osteoclasts and stimulate osteogenic differentiation and maturation of bone marrow stromal progenitor cells (BMSCs) and osteoblasts. Genistein is an estrogen receptor (ER)-selective binding phytoestrogen, with a greater affinity to ERβ. Genistein inhibits tyrosine kinases and inhibits DNA topoisomerases I and II, and may act as an antioxidant. Genistein enhances osteoblastic differentiation and maturation by activation of ER, p38MAPK-Runx2, and NO/cGMP pathways, and it inhibits osteoclast formation and bone resorption through inducing osteoclastogenic inhibitor osteoprotegerin (OPG) and blocking NF-κB signaling. Icariin, a prenylated flavonol glycoside isolated from Epimedium herb, stimulates osteogenic differentiation of BMSCs and inhibits bone resorption activity of osteoclasts. Icariin, whose metabolites include icariside I, icariside II, icaritin, and desmethylicaritin, has no estrogenic activity. However, icariin is more potent than genistein in promoting osteogenic differentiation and maturation of osteoblasts. The existence of a prenyl group on C-8 of icariin molecular structure has been suggested to be the reason why icariin is more potent than genistein in osteogenic activity. Thus, the prenylflavonoids may represent a class of flavonoids with a higher osteogenic activity.

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.

C-Fms Signaling Mediates Neurofibromatosis Type-1 Osteoclast Gain-In-Functions

Skeletal abnormalities including osteoporosis and osteopenia occur frequently in both pediatric and adult neurofibromatosis type 1 (NF1) patients. NF1 (Nf1) haploinsufficient osteoclasts and osteoclast progenitors derived from both NF1 patients and Nf1+/− mice exhibit increased differentiation, migration, and bone resorptive capacity in vitro, mediated by hyperactivation of p21Ras in response to limiting concentrations of macrophage-colony stimulating factor (M-CSF). Here, we show that M-CSF binding to its receptor, c-Fms, results in increased c-Fms activation in Nf1+/ − osteoclast progenitors, mediating multiple gain-in-functions through the downstream effectors Erk1/2 and p90RSK. PLX3397, a potent and selective c-Fms inhibitor, attenuated M-CSF mediated Nf1+/− osteoclast migration by 50%, adhesion by 70%, and pit formation by 60%. In vivo, we administered PLX3397 to Nf1 +/− osteoporotic mice induced by ovariectomy (OVX) and evaluated changes in bone mass and skeletal architecture. We found that PLX3397 prevented bone loss in Nf1+/−-OVX mice by reducing osteoclast differentiation and bone resorptive activity in vivo. Collectively, these results implicate the M-CSF/c-Fms signaling axis as a critical pathway underlying the aberrant functioning of Nf1 haploinsufficient osteoclasts and may provide a potential therapeutic target for treating NF1 associated osteoporosis and osteopenia.

Regulation of osteoclast structure and function by FAK family kinases

Osteoclasts are highly specialized cells that resorb bone and contribute to bone remodeling. Diseases such as osteoporosis and osteolytic bone metastasis occur when osteoclast-mediated bone resorption takes place in the absence of concurrent bone synthesis. Considerable effort has been placed on identifying molecules that regulate the bone resorption activity of osteoclasts. To this end, we investigated unique and overlapping functions of members of the FAK family (FAK and Pyk2) in osteoclast functions. With the use of a conditional knockout mouse model, in which FAK is selectively targeted for deletion in osteoclast precursors (FAK(Δmyeloid)), we found that loss of FAK resulted in reduced bone resorption by osteoclasts in vitro, coincident with impaired signaling through the CSF-1R. However, bone architecture appeared normal in FAK(Δmyeloid) mice, suggesting that Pyk2 might functionally compensate for reduced FAK levels in vivo. This was supported by data showing that podosome adhesion structures, which are essential for bone degradation, were significantly more impaired in osteoclasts when FAK and Pyk2 were reduced than when either molecule was depleted individually. We conclude that FAK contributes to cytokine signaling and bone resorption in osteoclasts and partially compensates for the absence of Pyk2 to maintain proper adhesion structures in these cells.

Inhibitory effects of tert‐butylhydroquinone on osteoclast differentiation via up‐regulation of heme oxygenase‐1 and down‐regulation of HMGB1 release and NFATc1 expression

Osteoclasts (OCLs) are multinucleated bone-resorbing cells that are differentiated by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Our recent studies have shown that heme-oxygenase-1 (HO-1), a stress-induced cytoprotective enzyme, plays an important role in OCL differentiation, although the pharmacological significance of this effect remains unknown. In this study, we investigated the effects of tert-butylhydroquinone (tBHQ), a pharmacological HO-1 inducer, on in vitro differentiation of bone marrow-derived macrophages (BMMs) or murine monocytic cell line RAW-D into OCLs. tBHQ inhibited the formation and the bone-resorbing activity of OCLs. Moreover, tBHQ treatment decreased the expression of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator of OCL differentiation, and of OCL markers transcriptionally regulated by NFATc1, such as Src and cathepsin K. In addition, tBHQ impaired phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase, Akt, and inhibitor of nuclear factor kappa B alpha (IκBα). Finally, we show that tBHQ inhibited the release of high mobility group box 1 (HMGB1), a recently identified activator of OCL differentiation. Thus, tBHQ inhibits OCL differentiation through the HO-1/HMGB1 pathways.

Thickened skull, scoliosis and other skeletal findings in Unverricht–Lundborg disease link cystatin B function to bone metabolism

PurposeUnverricht–Lundborg disease (EPM1) is a rare type of inherited progressive myoclonic epilepsy resulting from mutations in the cystatin B gene, CSTB, which encodes a cysteine cathepsin inhibitor. Cystatin B, cathepsin K, and altered osteoclast bone resorption activity are interconnected in vitro. This study evaluated the skeletal characteristics of patients with EPM1. MethodsSixty-six genetically verified EPM1 patients and 50 healthy controls underwent head MRI. Skull dimensions and regional calvarial thickness was measured perpendicular to each calvarial bone from T1-weighted 3-dimensional images using multiple planar reconstruction tools. All clinical X-ray files of EPM1 patients were collected and reviewed by an experienced radiologist. A total of 337 X-ray studies were analyzed, and non-traumatic structural anomalies, dysplasias and deformities were registered. ResultsEPM1 patients exhibited significant thickening in all measured cranial bones compared to healthy controls. The mean skull thickness was 10.0±2.0mm in EPM1 patients and 7.6±1.2mm in healthy controls (p<0.001). The difference was evident in all age groups and was not explained by former phenytoin use. Observed abnormalities in other skeletal structures in EPM1 patients included thoracic scoliosis (35% of EPM1 patients) and lumbar spine scoliosis (35%), large paranasal sinuses (27%), accessory ossicles of the foot, and arachnodactyly (18%). ConclusionsSkull thickening and an increased prevalence of abnormal findings in skeletal radiographs of patients with EPM1 suggest that this condition is connected to defective cystatin B function. These findings further emphasize the role of cystatin B in bone metabolism in humans.

Class IA phosphatidylinositol 3-kinase regulates osteoclastic bone resorption through protein kinase B–mediated vesicle transport

Class IA phosphatidylinositol 3-kinases (PI3Ks) are activated by growth factor receptors and regulate a wide range of cellular processes. In osteoclasts, they are activated downstream of α(v) β(3) integrin and colony-stimulating factor-1 receptor (c-Fms), which are involved in the regulation of bone-resorbing activity. The physiological relevance of the in vitro studies using PI3K inhibitors has been of limited value, because they inhibit all classes of PI3K. Here, we show that the osteoclast-specific deletion of the p85 genes encoding the regulatory subunit of the class IA PI3K results in an osteopetrotic phenotype caused by a defect in the bone-resorbing activity of osteoclasts. Class IA PI3K is required for the ruffled border formation and vesicular transport, but not for the formation of the sealing zone. p85α/β doubly deficient osteoclasts had a defect in macrophage colony-stimulating factor (M-CSF)-induced protein kinase B (Akt) activation and the introduction of constitutively active Akt recovered the bone-resorbing activity. Thus, the class IA PI3K-Akt pathway regulates the cellular machinery crucial for osteoclastic bone resorption, and may provide a molecular basis for therapeutic strategies against bone diseases.

The epoxyketone-based proteasome inhibitors carfilzomib and orally bioavailable oprozomib have anti-resorptive and bone-anabolic activity in addition to anti-myeloma effects

Proteasome inhibitors (PIs), namely bortezomib, have become a cornerstone therapy for multiple myeloma (MM), potently reducing tumor burden and inhibiting pathologic bone destruction. In clinical trials, carfilzomib, a next generation epoxyketone-based irreversible PI, has exhibited potent anti-myeloma efficacy and decreased side effects compared with bortezomib. Carfilzomib and its orally bioavailable analog oprozomib, effectively decreased MM cell viability following continual or transient treatment mimicking in vivo pharmacokinetics. Interactions between myeloma cells and the bone marrow (BM) microenvironment augment the number and activity of bone-resorbing osteoclasts (OCs) while inhibiting bone-forming osteoblasts (OBs), resulting in increased tumor growth and osteolytic lesions. At clinically relevant concentrations, carfilzomib and oprozomib directly inhibited OC formation and bone resorption in vitro, while enhancing osteogenic differentiation and matrix mineralization. Accordingly, carfilzomib and oprozomib increased trabecular bone volume, decreased bone resorption and enhanced bone formation in non-tumor bearing mice. Finally, in mouse models of disseminated MM, the epoxyketone-based PIs decreased murine 5TGM1 and human RPMI-8226 tumor burden and prevented bone loss. These data demonstrate that, in addition to anti-myeloma properties, carfilzomib and oprozomib effectively shift the bone microenvironment from a catabolic to an anabolic state and, similar to bortezomib, may decrease skeletal complications of MM.

Genes associate with abnormal bone cell activity in bone metastasis

Bone is one of the most frequent sites of metastasis in patients with malignancies. Up to 90% of patients with multiple myeloma, and 60% to 75% patients with prostate cancer and breast cancer develop bone metastasis at the later stages of their diseases. Bone metastases are responsible for tremendous morbidity in patients with cancer, including severe bone pain, pathologic fractures, spinal cord and nerve compression syndromes, life-threatening hypercalcemia, and increased mortality. Multiple factors produced by tumor cells or produced by the bone marrow microenvironment in response to tumor cells play important roles in activation of osteoclastic bone resorption and modulation of osteoblastic activity in patients with bone metastasis. In this chapter, we will review the genes that play important roles in bone destruction, tumor growth, and osteoblast activity in bone metastasis and discuss the potential therapies targeting the products of these genes to block both bone destruction and tumor growth.

Serum Calcium-decreasing Factor, Caldecrin, Inhibits Receptor Activator of NF-κB Ligand (RANKL)-mediated Ca2+ Signaling and Actin Ring Formation in Mature Osteoclasts via Suppression of Src Signaling Pathway

Osteoclasts are essential for bone dynamics and calcium homeostasis. Recently, we reported that serum calcium-decreasing factor, caldecrin, which is a secretory-type serine protease isolated from the pancreas, inhibits osteoclast differentiation by suppression of NFATc1 activity regardless of its own protease activity (Hasegawa, H., Kido, S., Tomomura, M., Fujimoto, K., Ohi, M., Kiyomura, M., Kanegae, H., Inaba, A., Sakagami, H., and Tomomura, A. (2010) Serum calcium-decreasing factor, caldecrin, inhibits osteoclast differentiation by suppression of NFATc1 activity. J. Biol. Chem. 285, 25448-25457). Here, we investigated the effects of caldecrin on the function of mature osteoclasts by treatment with receptor activator of NF-κB ligand (RANKL). Caldecrin inhibited the RANKL-stimulated bone resorptive activity of mature osteoclasts. Furthermore, caldecrin inhibited RANKL-mediated sealing actin ring formation, which is associated with RANKL-evoked Ca(2+) entry through transient receptor potential vanilloid channel 4. The inhibitors of phospholipase Cγ, Syk, and c-Src suppressed RANKL-evoked Ca(2+) entry and actin ring formation of mature osteoclasts. Interestingly, caldecrin significantly inhibited RANKL-stimulated phosphorylation of c-Src, Syk, phospholipase Cγ1 and Cγ2, SLP-76, and Pyk2 but not that of ERK, JNK, or Akt. Caldecrin inhibited RANKL-stimulated c-Src kinase activity and c-Src·Syk association. These results suggest that caldecrin inhibits RANKL-stimulated calcium signaling activation and cytoskeletal organization by suppression of the c-Src·Syk pathway, which may in turn reduce the bone resorptive activity of mature osteoclasts. Thus, caldecrin is capable of acting as a negative regulator of osteoclastogenesis and osteoclast function of bone resorption.

Heparin-like Polysaccharides Reduce Osteolytic Bone Destruction and Tumor Growth in a Mouse Model of Breast Cancer Bone Metastasis

TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β-induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight Escherichia coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β-induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate in vitro siRNA screening results toward in vivo therapeutic concepts.

NF-κB signaling and bone resorption

The transcription factor NF-κB is a family of proteins involved in signaling pathways essential for normal cellular functions and development. Deletion of various components of this pathway resulted with abnormal skeletal development. Research in the last decade has established that NF-κB signaling mediates RANK ligand-induced osteoclastogenesis. Consistently, it was shown that inhibition of NF-κB was an effective approach to inhibit osteoclast formation and bone resorptive activity. Identification of the molecular machinery underlying NF-κB activation permitted osteoclast-specific deletion of the major components of this pathway. As a result, it was clear that deletion of members of the proximal IKK kinase complex and the distal NF-κB subunits and downstream regulators affected skeletal development. These studies provided several targets of therapeutic intervention in osteolytic diseases. NF-κB activity has been also described as the centerpiece of inflammatory responses and is considered a potent mediator of inflammatory osteolysis. Indeed, inflammatory insults exacerbate physiologic RANKL-induced NF-κB signals leading to exaggerated responses and to inflammatory osteolysis. These superimposed NF-κB activities appear to underlie several bone pathologies. This review will describe the individual roles of NF-κB molecules in bone resorption and inflammatory osteolysis.

Potent inhibitory effect of Foeniculum vulgare Miller extract on osteoclast differentiation and ovariectomy-induced bone loss

Inhibition of osteoclast differentiation and bone resorption is considered an effective therapeutic approach to the treatment of postmenopausal bone loss. To find natural compounds that may inhibit osteoclastogenesis, we screened herbal extracts on bone marrow cultures. In this study, we found that an aqueous extract of Foeniculum vulgare Miller seed (FvMs) at low concentration, which has traditionally been used as a treatment for a variety of ailments, inhibits the osteoclast differentiation and bone resorptive activity of mature osteoclasts. We further investigated the effects of FvMs on ovariectomy (OVX)-induced bone loss using microcomputed tomography, biomechanical tests and serum marker assays for bone remodeling. Oral administration of FvMs (30 mg or 100 mg/kg/day) for 6 weeks had an intermediary effect on the prevention of femoral bone mineral density (BMD), bone mineral content (BMC), and other parameters compared to OVX controls. In addition, FvMs slightly decreased bone turnover markers that were accelerated by OVX. The bone-protective effects of FvMs may be due to suppression of an OVX-induced increase in bone turnover. Collectively, our findings indicate that FvMs have potential in preventing bone loss in postmenopausal osteoporosis by reducing both osteoclast differentiation and function.

NIP45 negatively regulates RANK ligand induced osteoclast differentiation

Receptor activator of NF-κB ligand (RANKL)-RANK receptor signaling to induce NFATc1 transcription factor is critical for osteoclast differentiation and bone resorption. RANK adaptor proteins, tumor necrosis factor receptor-associated factors (TRAFs) play an essential role in RANKL signaling. Evidence indicates that NIP45 (NFAT interacting protein) binds with TRAFs and NFATc2. We therefore hypothesized that NIP45 regulates RANKL induced osteoclast differentiation. In this study, we demonstrate that RANKL treatment down regulates NIP45 expression in mouse bone marrow derived pre-osteoclast cells. Lentiviral (pGIPZ) mediated shRNA knock-down of NIP45 expression in RANKL stimulated pre-osteoclast cells resulted in increased levels of NFATc1, NFATc2, and TRAF6 but not TRAF2 expression compared to control shRNA transduced cells. Also, NIP45 suppression elevated p-IκB-α levels and NF-κB-luciferase reporter activity. Confocal microscopy demonstrated NIP45 colocalized with TRAF6 in the cytosol of osteoclast progenitor cells. In contrast, RANKL stimulation induced NIP45 nuclear translocation and colocalization with NFATc2 in these cells. Coimmuneprecipitation assay demonstrated NIP45 binding with NFATc2 but not NFATc1. We further show that shRNA knock-down of NIP45 expression in pre-osteoclast cells significantly increased RANKL induced osteoclast differentiation and bone resorption activity. Taken together, our results indicate that RANKL signaling down regulates NIP45 expression and that NIP45 is a negative regulator of osteoclast differentiation.

Paclitaxel inhibits osteoclast formation and bone resorption via influencing mitotic cell cycle arrest and RANKL‐induced activation of NF‐κB and ERK

Pathological bone destruction (osteolysis) is a hallmark of many bone diseases including tumor metastasis to bone, locally osteolytic giant cell tumor (GCT) of bone, and Paget's disease. Paclitaxel is frequently prescribed in the treatment of several malignant tumors where it has been shown to exert beneficial effects on bone lesions. However, the mechanism(s) through which paclitaxel regulates osteoclast formation and function remain ill defined. In the present study, we demonstrate that paclitaxel dose-dependently inhibits receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis in both RAW264.7 cells and mouse bone marrow macrophage (BMM) systems. In addition, paclitaxel treatment reduces the bone resorptive activity of human osteoclasts derived from GCT of bone, and attenuates lipopolysaccharide (LPS)-induced osteolysis in a mouse calvarial model. Complementary cellular and biochemical analyses revealed that paclitaxel induces mitotic arrest of osteoclastic precursor cells. Furthermore, luciferase reporter gene assays and western blot analysis indicate that paclitaxel modulates key RANKL-induced activation pathways that are essential to osteoclast formation including NF-κB and ERK. Collectively, our findings demonstrate a role for paclitaxel in the regulation of osteoclast formation and function and uncover potential mechanism(s) through which paclitaxel alleviates pathological osteolysis.

NAMPT/PBEF Enzymatic Activity Is Indispensable for Myeloma Cell Growth and Osteoclast Activity

Abstract 886Clinical MRI and PET-CT analyses implicated hypermetabolic activity of multiple myeloma (MM) cells in focal lesions with tumor burden, metastasis and survival. Osteoclasts induced by MM are also naturally hypermetabolic suggesting that targeting cellular metabolism may contribute to controlling MM and associated bone disease. The coenzyme nicotinamide adenine dinucleotide (NAD) not only is essential for proper cellular metabolism but also affects activity of NAD-dependent enzymes, such as PARP1 and sirtuins. Nicotinamide phosphoribosyl transferase (NAMPT/PBEF/Visfatin) is a rate-limiting enzyme in NAD biosynthesis from nicotinamide and also functions extracellularly. APO866 (TopoTarget, Switzerland), an inhibitor of NAMPT, depletes cellular NAD particularly in cancer cells. The aims of the study were to evaluate expression of NAMPT (PBEF1) in clinical and experimental MM samples using global gene expression profiling (GEP) and investigate the effect of APO866 on MM cell growth and osteoclastogenesis. Our molecular classification data revealed elevated expression of PBEF1 in the PR, MAF and LB subtypes of newly diagnosed MM patients (n=796) and in MM cell lines (n=41). Based on our GEP70 risk model, PBEF1 expression was higher in high risk of the CD-1, HY, and MS subtypes relative to their low risk counterparts, and in focal lesions at relapse than at diagnosis. PBEF1 expression was 2.5 fold higher in myelomatous versus nonmyelomatous human bones from SCID-hu mice (p<0.0001). In primary MM cell-osteoclast cocultures (n=8) PBEF1 expression was upregulated by >3 folds in cocultured MM cells or osteoclasts compared to baseline MM cells (p<0.02) or control osteoclasts (p<0.04). APO866 dose dependently inhibited growth of 6 MM cell lines at low nanomolar concentrations (IC50 at 0.1–5 nM, 72 hrs) and similarly inhibited growth of MM cells in coculture with stromal cells or osteoclasts. NAD depletion by APO866 (5 nM) in 3 MM cell lines was consistently associated with reduced enzymatic activities of PARP1 by 73 ± 5% (p<0.001) and of SIRT1 by 49 ± 3% (p<0.005). In vivo, SCID-rab mice engrafted with our DAS MM cell line (MF subtype) were intraperitoneally treated with saline or APO866 (10 mice/group, 20 mg/kg, twice weekly) for 3 weeks. At experiment’s end circulating human Ig levels in control and APO866 groups were 43 ± 15 μg/ml and 1 ± 0.6 μg/ml, respectively (p<0.01). APO866 prevented bone loss as assessed by X-rays and measurement of bone mineral density (p<0.05). Histological examination confirmed high infiltration of MM cells in myelomatous bones of control hosts while in APO866-treated hosts the implanted bones reduced numbers of osteoclasts by 53% (p<0.0007). In vitro, at concentration of ≥0.1 nM, APO866 significantly suppressed osteoclast differentiation and bone resorption activity of mature osteoclasts on dentine slices (p<0.007). We conclude that NAMPT/PBEF/Visfatin is a potential target in certain molecularly classified and high risk MM patients, and that MM cells and osteoclasts are highly sensitive to NAD depletion. Disclosures:Barlogie:Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.

Cell Surface CXCR4 and BTK Expression Are Associated in Myeloma Cells and Osteoclast Precursors and Mediate Myeloma Cell Homing and Clonogenicity, and Osteoclastogenesis

Abstract 884Myeloma cells typically grow in bone, recruit osteoclast precursors and induce their differentiation and activity in areas adjacent to tumor foci. Clonal B-lymphocytes and plasma cells harboring lymphocytic phenotypes have a proposed role in sustaining myeloma. Bruton’s tyrosine kinase (BTK) is expressed in hematopoietic cells and is particularly involved in B-lymphocyte function and osteoclastogenesis. The SDF-1/CXCR4 signaling pathway is reportedly involved in homing to bone of myeloma cells and osteoclast precursors. The aims of the study were to investigate the possible association between CXCR4 and BTK signaling in myeloma cells and osteoclast precursors, and the consequences of BTK inhibition on myeloma cell migration and clonogenicity, and osteoclastogenesis. By global gene expression profiling (GEP), BTK expression was moderately higher in clinical myeloma cells (n=559) than normal plasma cells (n=25, p<0.05). BTK was also detected in IL6–dependent cell lines and myeloma cell lines that can passage in vivo (n=7), using GEP, qRT-PCR and Western Blot analyses. Cell surface CXCR4 determined by flow cytometry was variably expressed in myeloma cells (5%–95%) and was significantly correlated with BTK gene expression (r=0.75, p<0.002, n=14). Most myeloma cell lines grown independently in vitro expressed very low levels of BTK; however, even in such cell lines (e.g. CAG), enrichment of myeloma cells expressing cell-surface CXCR4 by FACS analysis resulted in detectable BTK expression. BTK inhibition by shRNA in IL6–dependent INA6 cells inhibited their migration toward SDF-1 by >50% (p<0.006) and diminished their ability to form colonies in a 2-weeks clonogenic assay (p<0.0002). The BTK small molecule inhibitor LFM-A13 (25–50 μM) consistently reduced SDF-1-induced migration of primary myeloma cells and myeloma lines (n=6, p<0.03), and clonogenicity (colonies number and size) of 3 BTK-expressing myeloma lines by >50% (p<0.03). Using kinase immunoprecipitation assay, SDF-1 rapidly induced BTK phosphorylation (activation) in myeloma cells, an effect that was blocked by LFM-A13. In vivo, pretreatment of luciferase-expressing myeloma cells with LFM-A13 lessened their homing to bone following intravenous injection into SCID-rab mice as determined by live-animal imaging. BTK was highly expressed in osteoclast precursors while cell surface CXCR4 was detected in 5% of this cell population. LFM-A13 also reduced osteoclast precursor migration toward SDF-1 and suppressed osteoclast formation and bone resorption activity on dentine slices (p<0.002). In myeloma-bearing SCID-rab mice, LFM-A13 (n=10, 40 mg/kg, twice daily for 3 weeks, i.p.) reduced osteoclast number by 45% (p<0.004), prevented reduction of bone mineral density (BMD, p<0.04) and attenuated myeloma growth at near significant level (p<0.07). These data indicate that CXCR4 and BTK signaling are linked in myeloma cells and osteoclast precursors and that BTK has potential as a targeted myeloma therapy because of its roles in myeloma cell homing and clonogenicity and myeloma-induced osteolysis. Disclosures:Barlogie:Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.

Erk1 Plays Critical Role in Macrophage Development

Abstract 517Extracellular signal-regulated kinase (ERK 1 and 2) are widely expressed and are involved in the regulation of meiosis, mitosis, and postmitotic functions in multiple cell lineages, including T cells, B cells and osteoblasts. Macrophages are capable of differentiating into osteoclasts, which resorb bone. Abnormal osteoclast development and functions underlie certain diseases, especially skeletal defects. Altered ERK1/2 signaling has been found in several genetic diseases with skeletal phenotypes, including Noonan syndrome, polycystic kidney disease and serious developmental disorders such as cardio-facio-cutaneous syndrome. These clinical findings suggest the importance of the ERK MAPK pathway in human skeletal development. In the present study, we examined the consequence of Erk1 and Erk2 disruption in modulating macrophage development in the murine system. We found that deletion of Erk1 reduced macrophage progenitor numbers. Erk1−/− bone marrow mononuclear cells (BMMNCs) had significant reduction in osteoclast formation as compared to wildtype BMMNCs. In addition, Erk1−/− macrophages; the osteoclast progenitors, had a two-three fold reduction in migration and a two-fold reduction in αv ß3 mediated adhesion as compared to WT macrophages as evaluated by transwell and adhesion assay, respectively. These in vitro data demonstrate that Erk1 positively regulates macrophage differentiation into osteoclasts.To evaluate the impact of deficiency of Erk1 in vivo, we examined bone mineral density and trabecular microarchitecture in the distal femoral metaphysis by dual-energy X-ray absorptiometry (DEXA) with a Lunar Piximus densitometer and a high-resolution desktop microcomputed tomography imaging system (μCT-20; Scanco Medical AG, Basserdorf, Switzerland), respectively. Erk1−/− mice displayed elevated bone mineral density and increased trabecular bone formation as compared to WT mice. Histomorphometric analysis indicated that the Erk1−/− femur had significant reduction in osteoclast numbers as determined by tartrate resistant acid phosphatase staining, an osteoclast specific staining, as compared to femur of wildtype and Erk2−/− mice. Most importantly, Erk1−/− plasma had reduced C-terminal telopeptide of type I collagen, indicating less bone resorption in vivo. These data suggest that the impaired macrophage differentiation and osteoclast bone resorptive activity play an important role in increased bone mass in Erk1−/− mice.Finally, to verify that the macrophage-osteoclast lineage is a key cell lineage for the phenotypic changes in vivo in Erk1−/− mice, we performed bone marrow transplantation. WT mice reconstituted long-term with Erk1−/− hematopoietic stem cells demonstrated increased bone mineral density as compared to WT and Erk2−/− stem cell recipients, implicating marrow autonomous, Erk1-dependent macrophage differentiation and osteoclast bioactivity in vivo. Collectively, our in vitro and in vivo data demonstrate isoform-specific Erk function in macrophage while providing rationale for the development of a specific inhibitor for Erk1 that might be used for the treatment of dysplastic and erosive bone diseases. Disclosures:No relevant conflicts of interest to declare.