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Different macrophage depletion strategies have demonstrated a vital role of macrophages in bone healing, but the underlying molecular mechanisms are poorly understood. Here, with the use of a mouse model of tibia injury, we found that the cytokine oncostatin M [OSM or murine (m)OSM] was overexpressed during the initial inflammatory phase and that depletion of macrophages repressed mOSM expression. In Osm(-/-) mice, by micro-computed tomography and histology we observed a significant reduction in the amount of new intramedullar woven bone formed at the injured site, reduced number of Osterix(+) osteoblastic cells, and reduced expression of the osteoblast markers runt-related transcription factor 2 and alkaline phosphatase. In contrast, osteoclasts were normal throughout the healing period. One day after bone injury, Stat3, the main transcription factor activated by mOSM, was found phosphorylated/activated in endosteal osteoblastic cells located at the hedge of the hematoma. Interestingly, we observed reduced activation of Stat3 in Osm(-/-) mice. In addition, mice deficient in the mOSM receptor (Osmr(-/-)) also had reduced bone formation and osteoblast number within the injury site. These results suggest that mOSM, a product of macrophages, sustains intramembranous bone formation by signaling through Osmr and Stat3, acting on the recruitment, proliferation, and/or osteoblast differentiation of endosteal mesenchymal progenitor cells. Because bone resorption is largely unaltered, OSM could represent a new anabolic treatment for unconsolidated bone fractures.

International audience; Histone modifications are important for maintaining the transcription program. BET proteins, an important class of " histone reading proteins " , have recently been described as essential in bone biology. This study presents the therapeutic opportunity of BET protein inhibition in osteoporosis. We find that the pharmacological BET protein inhibitor JQ1 rescues pathologic bone loss in a post-ovariectomy osteoporosis model by increasing the trabecular bone volume and restoring mechanical properties. The BET protein inhibition suppresses osteoclast differentiation and activity as well as the osteoblastogenesis in vitro. Moreover, we show that treated non-resorbing osteoclasts could still activate osteoblast differentiation. In addition, specific inhibition of BRD4 using RNA interference inhibits osteoclast differentiation but strongly activates osteoblast mineralization activity. Mechanistically, JQ1 inhibits expression of the master osteoclast transcription factor NFATc1 and the transcription factor of osteoblast Runx2. These findings strongly support that targeting epigenetic chromatin regulators such as BET proteins may offer a promising alternative for the treatment of bone-related disorders such as osteoporosis.

Previous in vitro studies on primary osteoblastic and osteosarcoma cells (normal and transformed osteoblasts) have shown that oncostatin M (OSM), a member of the interleukin-6 family, possesses cytostatic and pro-apoptotic effects in association with complex and poorly understood activities on osteoblast differentiation. In this study, we use rat osteosarcoma cells transduced with lentiviral particles encoding OSM (lvOSM) to stably produce this cytokine. We show that after several weeks of culture, transduced OSRGA and ROS 17/2.8 cells are growth inhibited and sensitized to apoptosis induced by the kinase inhibitor Staurosporine (Sts). Moreover, this long term OSM treatment induces (i) a decrease in osteoblastic markers, (ii) morphological changes leading to an elongated and/or stellate shape and (iii) an increase in osteocytic markers (sclerostin and/or E11), suggesting an osteocyte-like differentiation. We also show that non transformed rat calvaria cells transduced with lvOSM differentiate into stellate shaped cells expressing sclerostin, E11, Phex and functional hemichannels. Together, these results indicate that osteosarcoma cells stably producing OSM do not develop resistance to this cytokine and thus could be a valuable new tool to study the anti-cancer effect of OSM in vivo. Moreover, OSM-over-expressing osteoblastic cells differentiate into osteocyte-like cells, the major cellular contingent in bone, providing new culture conditions for this cell type which is difficult to obtain in vitro.

RANK, RANK ligand (RANKL) and osteoprotegerin (OPG) are the key regulators of bone metabolism, both in normal and pathological conditions. Previous data have demonstrated that human osteosarcoma biopsies express RANKL as well as OPG, and functional RANK is expressed in a murine osteosarcoma cell line. As RANK expression in human osteosarcoma remains controversial, the aim of the present study was to analyse its expression in vitro in human osteosarcoma cell lines, ex vivo using pathological tissues, and then to determine its functionality in terms of signal transduction pathways modulated by RANKL. RT-PCR analysis and immunohistochemistry experiments revealed that RANK is expressed at both transcriptional and protein levels in MNNG/HOS, Saos-2 and MG-63 human osteosarcoma cell lines, in contrast to the U-2 OS osteosarcoma cell line and human osteoblasts, which were negative. RANK was also expressed in 57% of osteosarcoma biopsies. Furthermore, western blot experiments clearly demonstrated the functionality of RANK. Thus, RANKL significantly induced the phosphorylation of ERK1/2, p38 and IkappaB in RANK-positive osteosarcoma cells. This study is the first report of functional RANK expression in human osteosarcoma cells: this strengthens the involvement of the RANK-RANKL-OPG axis in primary bone tumour biology and identifies novel therapeutic approaches targeting RANK-positive osteosarcoma.

The effects of OSM on proliferation and differentiation of osteosarcoma and nontransformed osteoblasts were analyzed. OSM downregulates osteoblast markers but induces the glial fibrillary acidic protein by the combined activation of PKCδ and STAT3, offering new lines of therapeutic investigations. Introduction: Oncostatin M (OSM) is a multifunctional cytokine of the interleukin-6 family implicated in embryonic development, differentiation, inflammation, and regeneration of various tissues, mainly the liver, bone, and the central nervous and hematopoietic systems. One particularity of OSM relies on its growth inhibitory and pro-differentiating effects on a variety of tumor cell lines such as melanoma, providing arguments for a therapeutic application of OSM. The objective of this study was to analyze the effects of OSM on osteosarcoma cell lines proliferation and differentiation. Materials and Methods: Proliferation was analyzed by3H thymidine incorporation. Differentiation was analyzed by semiquantitative RT-PCR and immunocytochemistry for various markers. Alizarin red S staining was used to evaluate bone nodule formation. Morphological changes were studied by confocal and electron microscopy. Western blotting, kinases inhibitors, and dominant negative STAT3 were used to identified the signaling pathways implicated. Results: OSM inhibits the growth of rat osteosarcoma cell lines as well as normal osteoblasts, in correlation with induction of the cyclin-dependent kinases inhibitor p21WAF1. However, OSM reduces osteoblast markers such as alkaline phosphatase, osteocalcin, and bone sialoprotein, leading to strong inhibition of mineralized nodule formation. This inhibitory effect is restricted to mature osteoblasts and differentiated osteosarcoma because OSM effectively stimulates osteoblast markers and bone nodule formation in early, but not late, bone marrow mesenchymal stem cell (BMSC) cultures. In osteosarcoma cells or BMSC, OSM induces expression of the glial fibrillary acidic protein (GFAP) as well as morphological and ultrastructural changes, for example, elongated shape and bundles of microfilaments in cell processes. Rottlerin (PKCδ inhibitor), and to a lesser degree UO126 (MEK/ERK inhibitor), prevents the loss of osteoblastic markers by OSM, whereas dominant negative STAT3 prevents GFAP induction. Conclusions: These results highlight the particular gene expression profile of OSM-treated osteosarcoma cells and BMSCs, suggesting either a osteocytic or a glial-like phenotype. Together with the implication of PKCδ, ERK1/2, and STAT3, these results offer new lines of investigations for neural cell transplantation and osteosarcoma therapy.

International audience; Receptor activator of nuclear factor kappa-B (RANK) and RANK-ligand are relevant targets for the treatment of polyethylene particle-induced osteolysis. This study assessed the local administration of siRNA, targeting both human RANK and mouse Rank transcripts in a mouse model. Four groups of mice were implanted with polyethylene (PE) particles in the calvaria and treated locally with 2.5, 5 and 10 mg of RANK siRNA or a control siRNA delivered by the cationic liposome DMAPAP/DOPE. The tissues were harvested at day 9 after surgery and evaluated by micro-computed tomography, tartrate-resistant acid phosphatase (TRAP) immunohistochemistry for macrophages and osteoblasts, and gene relative expression of inflammatory and osteolytic markers. 10 mu g of RANK siRNA exerted a protective effect against PE particle-induced osteolysis, decreasing the bone loss and the osteoclastogenesis, demonstrated by the significant increase in the bone volume (P < 0.001) and by the reduction in both the number of TRAP cells and osteoclast activity (P < 0.01). A bone anabolic effect demonstrated by the formation of new trabecular bone was confirmed by the increased immunopositive staining for osteoblast-specific proteins. In addition, 5 and 10 mu g of RANK siRNA downregulated the expression of pro-inflammatory cytokines (P < 0.01) without depletion of macrophages. Our findings show that RANK siRNA delivered locally by a synthetic vector may be an effective approach for reducing osteolysis and may even stimulate bone formation in aseptic loosening of prosthetic implants. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Ultrasmall superparamagnetic iron oxide (USPIO) contrast agents for use in magnetic resonance imaging (MRI) are currently undergoing clinical evaluation. However, the images observed and the kinetic profiles obtained differ from one agent to another. In this study, BD IX rats received an intravenous penis injection of the USPIO contrast agents AMI-HS and AMI-227. A cytologic study of the liver was performed, and the data obtained were compared with those of MRI. Images acquired in light microscopy, transmission electron microscopy, microanalysis and electron diffraction provided data on the cell categories involved in the processing of these contrast agents, the importance and modalities of each category relative to this processing, and the modalities of agent elimination. AMI-HS was rapidly removed from the bloodstream by Kupffer's cells and hepatocytes and then eliminated through bile ducts. AMI-227 remained much longer in the blood compartment since it was processed very slowly by endothelial and Kuppfer's cells in the near absence of hepatocytic participation and thus of elimination by the bile ducts. These results allowed us to base our interpretation of MRI sequences on cytologic observations.

The vicious cycle established between bone-associated tumours and bone resorption is the central problem with therapeutic strategies against primary bone tumours and bone metastasis. Here we report data to support inhibition of BET bromodomain proteins as a promising therapeutic strategy that target simultaneously the three partners of the vicious cycle. Treatment with JQ1, a BET bromodomain inhibitor, reduces cell viability of osteosarcoma cells and inhibits osteoblastic differentiation both in vitro and in vivo. These effects are associated with transcriptional silencing of MYC and RUNX2, resulting from the depletion of BRD4 from their respective loci. Moreover, JQ1 also inhibits osteoclast differentiation by interfering with BRD4-dependent RANKL activation of NFATC1 transcription. Collectively, our data indicate that JQ1 is a potent inhibitor of osteoblast and osteoclast differentiation as well as bone tumour development.

Ewing's sarcoma (ES) is a high-grade neoplasm arising in bones of children and adolescents. Survival rate decreases from greater than 50% to only 20% after 5 years for patients not responding to treatment or presenting metastases at diagnosis. TRAIL, which has strong antitumoral activity, is a promising therapeutic candidate. To address TRAIL sensitivity, 7 human ES cell lines were used. Cell viability experiments [3′[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro-)benzene sulfonic acid hydrate (XTT) assay] showed that 4 of the 7 ES cell lines were resistant to TRAIL. Western blotting and flow cytometry analyses revealed that DR5 was uniformly expressed by all ES cell lines, whereas DR4 levels were higher in sensitive cell lines. In TRAIL-sensitive TC-71 cells, knockdown of TNFRSF10A/DR4 by short hairpin RNA (shRNA) was associated with a loss of sensitivity to TRAIL, in spite of DR5 presence. Interestingly, we identified a new transcript variant that results from an alternative splicing and encodes a 310–amino acid protein which corresponds to the 468 aa of DR4 original isoform but truncated of aa 11 to 168 within the extracellular TRAIL-binding domain. According to modeling studies, the contact of this new DR4 isoform (bDR4) with TRAIL seemed largely preserved. The overexpression of bDR4 in a TRAIL-resistant cell line restored TRAIL sensitivity. TRAIL resensitization was also observed after c-FLIP knockdown by shRNA in two TRAIL-resistant cell lines, as shown by XTT assay and caspase-3 assay. The results presented in this study showed that DR4, both as the complete form or as its new short isoform, is involved in TRAIL sensitivity in ES. Mol Cancer Res; 10(3); 336–46. ©2012 AACR.

International audience; Osteoclasts are bone-resorptive cells that differentiate from hematopoietic precursors upon receptor activator of nuclear factor kappaB ligand (RANKL) activation. Previous studies demonstrated that IL-6 indirectly stimulates osteoclastogenesis through the production of RANKL by osteoblasts. However, few data described the direct effect of IL-6 on osteoclasts. To investigate this effect, we used several models: murine RAW264.7 cells, mouse bone marrow, and human blood monocytes. In the three models used, the addition of IL-6 inhibited RANKL-induced osteoclastogenesis. Furthermore, IL-6 decreased the expression of osteoclast markers and up-modulated macrophage markers. To elucidate this inhibition, signal transducer and activator of transcription (STAT) 3, the main signaling molecule activated by IL-6, was analyzed. Addition of two STAT3 inhibitors completely abolished RANKL-induced osteoclastogenesis, revealing a key role of STAT3. We demonstrated that a basal level of phosphorylated-STAT3 on Serine(727) associated with an absence of phosphorylation on Tyrosine(705) is essential for osteoclastogenesis. Furthermore, a decrease of Serine(727) phosphorylation led to an inhibition of osteoclast differentiation, whereas an increase of Tyrosine(705) phosphorylation upon IL-6 stimulation led to the formation of macrophages instead of osteoclasts. In conclusion, we showed for the first time that IL-6 inhibits RANKL-induced osteoclastogenesis by diverting cells into the macrophage lineage, and demonstrated the functional role of activated-STAT3 and its form of phosphorylation in the control of osteoclastogenesis.

Oncostatin M (OSM), a cytokine of the interleukin-6 family, induces growth arrest and differentiation of osteoblastic cells into glial-like/osteocytic cells. Here, we asked whether OSM regulates apoptosis of normal or transformed (osteosarcoma) osteoblasts. We show that OSM sensitizes cells to apoptosis induced by various death inducers such as staurosporine, ultraviolet or tumor necrosis factor-alpha. Apoptosis is mediated by the mitochondrial pathway, with release of cytochrome c from the mitochondria to the cytosol and activation of caspases-9 and -3. DNA micro-arrays revealed that OSM modulates the expression of Bax, Bad, Bnip3, Bcl-2 and Mcl-1. Pharmacological inhibitors, dominant-negative signal transducer and activator of transcriptions (STATs), stable RNA interference and knockout cells indicated that the transcription factors p53 and STAT5, which are activated by OSM, are implicated in the sensitization to apoptosis, being responsible for Bax induction and Bcl-2 reduction, respectively. These results indicate that, in addition to growth arrest and induced differentiation, OSM also sensitizes normal and transformed osteoblasts to apoptosis by a mechanism implicating (i) activation and nuclear translocation of STAT5 and p53 and (ii) an increased Bax/Bcl-2 ratio. Therefore, association of OSM with kinase inhibitors such as Sts represents new therapeutic opportunities for wild-type p53 osteosarcoma.

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of nuclear factor κB ligand (RANKL), an inducer of osteoclastogenesis via its receptor RANK. We recently demonstrated that OPG also exerts a direct effect in osteoclasts by regulating protease expression. Herein, we showed that OPG-induced pro-matrix metalloproteinase-9 activity was abolished by ras/MAPK inhibitors in purified osteoclasts. OPG induced the phosphorylation of p38 and ERK1/2 in RAW264.7 cells. Only p38 activation was totally abolished by a blocking anti-RANKL antibody or an excess of RANKL. Surface plasmon resonance experiments revealed that RANK, RANKL and OPG are able to form a tertiary complex. These results suggested a potential formation of a tertiary complex RANK–RANKL–OPG on osteoclasts. Thus, OPG is not only a soluble decoy receptor for RANKL but must be also considered as a direct effector of osteoclast functions.

BACKGROUND—Dietary fibres have been proposed as protective agents against colon cancer but results of both epidemiological and experimental studies are inconclusive. AIMS—Hypothesising that protection against colon cancer may be restricted to butyrate producing fibres, we investigated the factors needed for long term stable butyrate production and its relation to susceptibility to colon cancer. METHODS—A two part randomised blinded study in rats, mimicking a prospective study in humans, was performed using a low fibre control diet (CD) and three high fibre diets: starch free wheat bran (WB), type III resistant starch (RS), and short chain fructo-oligosaccharides (FOS). Using a randomised block design, 96 inbred rats were fed for two, 16, 30, or 44 days to determine the period of adaptation to the diets, fermentation profiles, and effects on the colon, including mucosal proliferation on day 44. Subsequently, 36 rats fed the same diets for 44 days were injected with azoxymethane and checked for aberrant crypt foci 30 days later. RESULTS—After fermentation had stabilised (44 days), only RS and FOS produced large amounts of butyrate, with a trophic effect in the large intestine. No difference in mucosal proliferation between the diets was noted at this time. In the subsequent experiment one month later, fewer aberrant crypt foci were present in rats fed high butyrate producing diets (RS, p=0.022; FOS, p=0.043). CONCLUSION—A stable butyrate producing colonic ecosystem related to selected fibres appears to be less conducive to colon carcinogenesis. Keywords: fibre; fermentation; butyrate; colon carcinogenesis; aberrant crypt foci; rat

We recently demonstrated original anti-tumor effects of zoledronic acid (Zol) on osteosarcoma cell lines independently of their p53 and Rb status. The present study investigated the potential Zol-resistance acquired by osteosarcoma cells after prolonged treatment. After 12 weeks of culture in the presence of 1 microm Zol, the effects of high doses of Zol (10-100 microm) were compared between the untreated rat (OSRGA, ROS) and human (MG63, SAOS2) osteosarcoma cells and Zol-pretreated cells in terms of cell proliferation, cell cycle analysis, migration assay and cytoskeleton organization. Long-term treatment with 1 microm Zol reduced the sensitivity of osteosarcoma cells to high concentrations of Zol. Furthermore, the Zol-resistant cells were sensitive to conventional anti-cancer agents demonstrating that this resistance process is independent of the multidrug resistance phenotype. However, as similar experiments performed in the presence of clodronate and pamidronate evidenced that this drug resistance was restricted to the nitrogen-containing bisphosphonates, we then hypothesized that this resistance could be associated with a differential expression of farnesyl diphos-phate synthase (FPPS) also observed in human osteosarcoma samples. The transfection of Zol-resistant cells with FPPS siRNA strongly increased their sensitivity to Zol. This study demonstrates for the first time the induction of metabolic resistance after prolonged Zol treatment of osteosarcoma cells confirming the therapeutic potential of Zol for the treatment of bone malignant pathologies, but points out the importance of the treatment regimen may be important in terms of duration and dose to avoid the development of drug metabolic resistance.

Osteosarcoma is the most frequent primary bone tumor that develops mainly in young adults. Despite recent improvements of the therapeutic advances, the survival rate at 5 years is below 30% for patients with poor response to treatment or with metastasis. The histones modifications are of critical importance in maintaining of the transcription program of both normal and tumor cells. The bromodomain and extra-terminal domain (BET) protein family is an important class of “histone reading protein” capable to recognize the N-acetylation of lysine residues on histone tails. BET bromain proteins have recently been described as regulators of MYC expression in various tumors. In this study, we present the therapeutic opportunity to pharmacologically target the BET bromodomain family in primary bone tumors. The consequence of this pharmacologic inhibition of bromodomains is a wide gene expression alteration, but it is believed to selectively target malignant cells by disrupting transcription at intense activity, most notably MYC and Runx2 in our model. Considering MYC and Runx2 are of particular importance for the oncogenic potential of primary bone tumors, a therapeutic strategy targeting those networks might be extremely relevant and potent. In osteosarcoma tumor cell lines, BET inhibitor reduced cell growth in a dose-dependent manner and induced apoptosis with an increase of sub-G1 fraction and PARP cleavage. These biological events were accompanied by decreased expression and activity of both MYC and Runx-2, and by an expression modulation of their target genes. Moreover, BET inhibitor affects bone remodeling process by disrupting both osteoclast and osteoblast differenciation. In vivo, BET inhibitor (IP; 50mg/kg) significantly inhibits tumor growth by 70% and prolongs survival in both POS-1 syngenic and HOS xenograft models compared to control. Additionnally, these results were accompanied by a decrease of associated bone lesions. These findings demonstrate that dual pharmacologic inhibition of MYC and Runx-2 is achievable through targeting BET bromodomains to treat osteosarcoma. Note: This abstract was not presented at the conference. Citation Format: Francois Lamoureux, Marc Baud'huin, Lidia Rodriguez, Camille Jacques, Martine Berreur, Francoise Rédini, James E. Bradner, Dominique Heymann, Benjamin Ory. Selective BET bromodomains epigenetic signaling inhibition as a therapeutic strategy in primary bone tumors. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B53.

International audience; Titanium implants are widely used in orthopaedic and dental surgery. Surface properties play a major role in cell and tissue interactions. The adhesion and differentiation of mesenchymal stem cells were studied as a function of nanostructures. Titanium surfaces with nanopores 30, 150 and 300 nm in diameter were prepared by physical vapour deposition. PCR arrays indicated that the expression of integrins was modulated by the nanopore size. Human Mesenchymal Stem Cells (hMSCs) exhibited more branched cell morphology on Ti30 than on other surfaces. Ti30 and Ti150 induced osteoblastic differentiation while Ti300 had a limited effect. Overall, nanopores of 30 nm may promote early osteoblastic differentiation and, consequently, rapid osseointegration of titanium implants.

Receptor activator of nuclear factor kappaB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) are the key regulators of bone metabolism. Recent findings demonstrated a crucial role of RANK in several bone-associated tumors. Indeed, we have recently demonstrated functional RANK expression both in a mouse and several human osteosarcoma cell lines. However, RANKL effects on osteosarcoma cells remain to be determined. In this study, we determined RANKL effects on RANK-positive Saos-2 human osteosarcoma cells. cDNA microarray and quantitative RT-PCR analyses clearly demonstrated that RANK-positive osteosarcoma cells were the target of RANKL as well as osteoclasts/osteoclast precursors. Thus, we present for the first time that RANKL can directly and significantly modulate gene expression of RANK-expressing Saos-2 cells. RANKL-modulated genes included genes that were implicated in protein metabolism, nucleic acid metabolism, intracellular transport, cytoskeleton organization and biogenesis, apoptosis and signaling cascade. Our results strengthen the involvement of the RANK/RANKL/OPG axis in osteosarcoma biology and capability to identify novel therapeutic approaches targeting RANK-positive osteosarcomas.