MiRNAs Expression Profiling in Raw264.7 Macrophages after Nfatc1-Knockdown Elucidates Potential Pathways Involved in Osteoclasts Differentiation

Differentiation of macrophages toward osteoclasts is crucial for bone homeostasis but can be detrimental in disease states, including osteoporosis and cancer. Therefore, understanding the osteoclast differentiation process and the underlying regulatory mechanisms may facilitate the identification of new therapeutic targets. Hereby, we tried to reveal new miRNAs potentially involved in the regulation of early steps of osteoclastogenesis, with a particular focus on those possibly correlated with NFATc1 expression, by studying miRNAs profiling. During the first 24 h of osteoclastogenesis, 38 miRNAs were differentially expressed between undifferentiated and RANKL-stimulated RAW264.7 cells, while 10 miRNAs were differentially expressed between RANKL-stimulated cells transfected with negative control or NFATc1-siRNAs. Among others, the expression levels of miR-411, miR-144 and members of miR-29, miR-30, and miR-23 families changed after RANKL stimulation. Moreover, the potential role of miR-124 during osteoclastogenesis was explored by transient cell transfection with anti-miR-124 or miR-124-mimic. Two relatively unknown miRNAs, miR-880-3p and miR-295-3p, were differentially expressed between RANKL-stimulated/wild-type and RANKL-stimulated/NFATc1-silenced cells, suggesting their possible correlation with NFATc1. KEGG enrichment analyses showed that kinase and phosphatase enzymes were among the predicted targets for many of the studied miRNAs. In conclusion, our study provides new data on the potential role and possible targets of new miRNAs during osteoclastogenesis.

Plumbagin, a Biomolecule with (Anti)Osteoclastic Properties

Plumbagin is a plant-derived naphthoquinone that is widely used in traditional Asian medicine due to its anti-inflammatory and anti-microbial properties. Additionally, plumbagin is cytotoxic for cancer cells due to its ability to trigger reactive oxygen species (ROS) formation and subsequent apoptosis. Since it was reported that plumbagin may inhibit the differentiation of bone resorbing osteoclasts in cancer-related models, we wanted to elucidate whether plumbagin interferes with cytokine-induced osteoclastogenesis. Using C57BL/6 mice, we unexpectedly found that plumbagin treatment enhanced osteoclast formation and that this effect was most pronounced when cells were pre-treated for 24 h with plumbagin before subsequent M-CSF/RANKL stimulation. Plumbagin caused a fast induction of NFATc1 signalling and mTOR-dependent activation of p70S6 kinase which resulted in the initiation of protein translation. In line with this finding, we observed an increase in RANK surface expression after Plumbagin stimulation that enhanced the responsiveness for subsequent RANKL treatment. However, in Balb/c mice and Balb/c-derived RAW264.7 macrophages, these findings could not be corroborated and osteoclastogenesis was inhibited. Our results suggest that the effects of plumbagin depend on the model system used and can therefore either trigger or inhibit osteoclast formation.

Osteoclasts Differentiation from Murine RAW 264.7 Cells Stimulated by RANKL: Timing and Behavior

The development of multi-nucleated cells is critical for osteoclasts (OCs) maturation and function. Our objective was to extend knowledge on osteoclastogenesis, focusing on pre-OC fusion timing and behavior. RAW 264.7 cells, which is a murine monocyte-macrophage cell line, provide a valuable and widely used tool for in vitro studies on osteoclastogenesis mechanisms. Cells were treated with the receptor activator of nuclear factor κ-B ligand (RANKL) for 1–4 days and effects on cell morphology, cytoskeletal organization, protein distribution, and OC-specific gene expression examined by TEM, immunofluorescence, and qPCR. Multinucleated cells began to appear at two days of Receptor Activator of Nuclear factor κ-B Ligand (RANKL) stimulation, increasing in number and size in the following days, associated with morphological and cytoskeletal organization changes. Interesting cellular extensions were observed in three days within cells labeled with wheat germ agglutinin (WGA)-Fluorescein isothiocyanate (FITC). The membrane, cytoplasmic, or nuclear distribution of RANK, TRAF6, p-p38, pERK1/2, and NFATc1, respectively, was related to OCs maturation timing. The gene expression for transcription factors regulating osteoclastogenesis (NFATc1, c-fos, RelA, MITF), molecules involved in RANKL-signaling transduction (TRAF6), cytoskeleton regulation (RhoA), fusion (DC-STAMP), migration (MMP9), and OC-specific enzymes (TRAP, CtsK), showed different trends related to OC differentiation timing. Our findings provide an integrated view on the morphological and molecular changes occurring during RANKL stimulation of RAW 264.7 cells, which are important to better understand the OCs’ maturation processes.

miR-155 promotes T reg cell development by safeguarding medullary thymic epithelial cell maturation

During thymocyte development, medullary thymic epithelial cells (mTECs) provide appropriate instructive cues in the thymic microenvironment for not only negative selection but also the generation of regulatory T (T reg) cells. Here, we identify that miR-155, a microRNA whose expression in T reg cells has previously been shown to be crucial for their development and homeostasis, also contributes to thymic T reg (tT reg) cell differentiation by promoting mTEC maturation. Mechanistically, we show that RANKL stimulation induces expression of miR-155 to safeguard the thymic medulla through targeting multiple known and previously uncharacterized molecules within the TGFβ signaling pathway, which is recognized for its role in restricting the maturation and expansion of mTECs. Our work uncovers a miR-155–TGFβ axis in the thymic medulla to determine mTEC maturity and, consequently, the quantity of tT reg cells and suggests that miR-155 ensures proper tT reg cell development in both cell-intrinsic and -extrinsic manners.

Dual Oxidase Maturation Factor 1 Positively Regulates RANKL-Induced Osteoclastogenesis via Activating Reactive Oxygen Species and TRAF6-Mediated Signaling

Receptor activator of NF-κB ligand (RANKL) induces generation of intracellular reactive oxygen species (ROS), which act as second messengers in RANKL-mediated osteoclastogenesis. Dual oxidase maturation factor 1 (Duoxa1) has been associated with the maturation of ROS-generating enzymes including dual oxidases (Duox1 and Duox2). In the progression of osteoclast differentiation, we identified that only Duoxa1 showed an effective change upon RANKL stimulation, but not Duox1, Duox2, and Duoxa2. Therefore, we hypothesized that Duoxa1 could independently act as a second messenger for RANKL stimulation and regulate ROS production during osteoclastogenesis. Duoxa1 gradually increased during RANKL-induced osteoclastogenesis. Using siRNA or retrovirus transduction, we found that Duoxa1 regulated RANKL-stimulated osteoclast formation and bone resorption positively. Furthermore, knockdown of Duoxa1 decreased the RANKL-induced ROS production. During Duoxa1-related control of osteoclastogenesis, activation of tumor necrosis factor receptor-associated factor 6 (TRAF6)-mediated early signaling molecules including MAPKs, Akt, IκB, Btk, Src and PLCγ2 was affected, which sequentially modified the mRNA or protein expression levels of key transcription factors in osteoclast differentiation, such as c-Fos and NFATc1, as well as mRNA expression of osteoclast-specific markers. Overall, our data indicate that Duoxa1 plays a crucial role in osteoclastogenesis via regulating RANKL-induced intracellular ROS production and activating TRAF6-mediated signaling.

FRI0370 DUAL OXIDASE MATURATION FACTOR 1 POSITIVELY REGULATES RANKL-INDUCED OSTEOCLASTOGENESIS VIA ACTIVATING REACTIVE OXYGEN SPECIES PRODUCTION AND TRAF6-MEDIATED SIGNALING

Background:Reactive oxygen species (ROS) are one of the significant factors of chemical or physical cell signaling in a wide variety of cell types including skeletal cells. Receptor activator of NF-βB ligand (RANKL) induces generation of intracellular ROS, which act as second messengers in RANKL-mediated osteoclastogenesis. Dual oxidase maturation factor 1 (Duoxa1) was first identified as aDrosophilaNumb-interacting protein (NIP), and has been associated with the maturation of ROS generating enzymes including dual oxidases (Duox1 and Duox2). In the progression of osteoclast differentiation using mouse bone marrow-derived macrophages (BMMs), we identified that only Duoxa1 level showed an effective change upon RANKL stimulation, but not Duox1, Duox2, and Duoxa2.Objectives:we hypothesized that Duoxa1 could independently act as a second messenger for RANKL stimulation and regulate ROS production during osteoclast differentiation.Methods:Using siRNA or retrovirus transduction and knockdown of Duoxa1 via siRNAResults:Duoxa1 level gradually increased during RANKL-induced osteoclast differentiation. We found that Duoxa1 regulated RANKL-stimulated osteoclast formation and bone resorption positively. knockdown of Duoxa1 via siRNA decreased the RANKL-induced ROS production. During Duoxa1-related control of osteoclastogenesis, activation of tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)-mediated early signaling molecules including MAPKs, Akt, IβB, Btk, and PLC 2 was affected, which sequentially modified the mRNA or protein expression levels of key transcription factors in osteoclastogenesis, such as c-Fos and NFATc1, as well as mRNA expression of osteoclast-specific markers including OSCAR, ATP6v0d2, and CtsK.Conclusion:Overall, our data indicate that Duoxa1 plays a crucial role in osteoclastogenesis via regulating RANKL-induced intracellular ROS production and activating TRAF6-mediated signaling.Disclosure of Interests:None declared

A Sub-Clone of RAW264.7-Cells Form Osteoclast-Like Cells Capable of Bone Resorption Faster than Parental RAW264.7 through Increased De Novo Expression and Nuclear Translocation of NFATc1

The murine macrophage cell line RAW264.7 is extensively used as a progenitor to study osteoclast (OC) differentiation. RAW264.7 is a heterogeneous cell line, containing sub-clones with different abilities to form OCs. The aim of this study was to identify characteristics within the heterogeneous RAW264.7 cells that define sub-clones with an augmented ability to form bone-resorbing OCs (H9), as well as sub-clones representing non-OCs (J8). RAW264.7 sub-clones were isolated by single cell cloning. Selection was based on TRAP/cathepsin K expression in sub-clone cultures without added RANKL. Sub-clones before and after differentiation with RANKL were assayed for multiple OC-characteristics. Sub-clone H9 cells presented a higher expression of OC-markers in cultures without added RANKL compared to the parental RAW264.7. After 6 days of RANKL stimulation, sub-clone H9 cells had equal expression levels of OC-markers with RAW264.7 and formed OCs able to demineralize hydroxyapatite. However, sub-clone H9 cells displayed rapid differentiation of OC already at Day 2 compared to Day 4 from parental RAW264.7, and when cultured on plastic and on bone they were more efficient in resorption. This rapid differentiation was likely due to high initial expression/nuclear translocation of OC master transcription factor, NFATc1. In contrast to H9, J8 cells expressed initially very low levels of OC-markers, and they did not respond to RANKL-stimulation by developing OC-characteristics/OC-marker expression. Hence, H9 is an additional clone suitable for experimental setup requiring rapid differentiation of large numbers of OCs.

Targeting CD26 with Humanized Monoclonal Antibody, As a Novel Approach to Inhibit Human Osteoclast Differentiation and Subsequent Bone Resorption

Abstract 1348 Bone disease is a hallmark of malignancy with osteolytic bone metastasis, including multiple myeloma (MM) and targeting osteoclasts (OCs) to alleviate bone destruction is a component of the standard care for MM. CD26 is a 110-kDa multifunctional membrane-bound glycoprotein, with dipeptidyl peptidase IV (DPPIV) enzyme activity in its extracellular domain and is critical in T-cell activation and several tumor developments, including malignant lymphoma. However, little is known about the role of CD26 in regulating bone remodeling. In this study, we show that CD26 is expressed on normal human osteoclasts and moreover, intensely expressed on activated human osteoclasts with osteolytic bone metastasis, including MM. We explore the function of CD26 in osteoclastgenesis (OCG) and investigate the effects of humanized anti-CD26 monoclonal antibody (huCD26mAb), which has shown promising clinical activity in T-cell lymphoma, on human OC differentiation, maturation and function. We further define the molecular targets of CD26 signaling cascade in OCG and explore the therapeutic potential of huCD26mAb for treating osteolytic bone metastasis. Human bone marrow mononuclear cells (BMMs) were cultured with human M-CSF (25ng/ml) plus sRANKL (50ng/ml) in the absence or presence of huCD26mAb for the indicated times. Then, M-CSF and sRANKL stimulate CD26 expressions during OCG, in a dose-dependent manner. The expression of CD26 up-regulates mitogen-activated protein kinase14 (p38MAPK) phosphorylation. P38MAPK phosphorylation also occurs downstream of RANK signaling in OCs and stimulates its downstream activation of microphthalmia-associated transcription factor (mi/Mitf), which plays an important role in OC function. Importantly, huCD26mAb decreased the number of multinucleated OCs (>3 nuclei) by tartrate-resistant phosphates (TRAP)/CD26 staining and the secretion of TRAP-5b and type 1 collagen; specific mature OC markers. It decreased the size of OCs and the number of nuclei per OC, with significantly defective bone resorption activity, as evidenced by diminished pit formation on fluoresceinated calcium phosphate-coated plates. In contrast, huCD26mAb added after 4- or 7- days' BMM cultures with M-CSF plus sRANKL did not have significant effects on mature osteoclast formation and function. Given these dual roles of CD26 in OCG, we next examined the effects of huCD26mAb on the phosphorylation of p38MAPK in OC precursor cells and mature OCs. At first, in the absence of huCD26mAb, similar amounts of p38MAPK and MKK3/6 (a molecule that is upstream of p38MAPK) were present in OC precursor cells and OCs. In response to RANKL, MKK3/6-p38MAPK was phosphorylated within 15 minutes in OC precursor cells and reached a maximal level within 30 minutes, and was maintained up to 60 minutes. Moreover, mi/Mitf was subsequently rapidly activated and persisted for 24hours. In the presence of huCD26mAb, when huCD26mAb bound to CD26 on OC precursor cells, only the MKK3/6-p38MAPK pathway was specifically rapidly inactivated, as shown by the persistent decrease in the phosphorylation of p38MAPK, together with MKK3/6, starting within 15 minutes of RANKL stimulation. Subsequent mi/Mitf phosphorylation was also persistently inhibited. In contrast, MKK3/6-p38MAPK was not phosphorylated at all in mature OCs after RANKL stimulation, regardless of the absence or presence of huCD26mAb. These results suggest that huCD26mAb suppressed RANKL induced p38MAPK phosphorylation in OC precursor cells, but not in OCs. The activation of other MAPKs including ERK and SAPK/JNK, or NFκB were rapidly induced in response to RANKL both in OC precursor cells and OCs, regardless of the absence or presence of huCD26mAb. Moreover, p38MAPK inhibitor also strongly inhibited OC formation and function through the suppression of p38 MAPK phosphorylation and subsequent mi/Mitf activation in OC precursor cells, but not in OCs. In conclusion, these data demonstrate that targeting CD26 on OC precursor cells with huCD26mAb suppressed human osteoclast differentiation, via the inhibition of MKK3/6-p38MAPK-mi/Mitf phosphorylation pathway and impaired subsequent mature osteoclast formation and function. Our results strongly suggest that targeting OCs with huCD26mAb has a promising alternative therapeutic potential for the treatment of osteolytic bone metastasis, including MM, to reduce the occurrence of total skeletal-related events. Disclosures: No relevant conflicts of interest to declare.

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

Abstract 1835 Blocking 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.