Inhibition of BET proteins and epigenetic signaling as a potential treatment for osteoporosis.

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.

Oncostatin m, an inflammatory cytokine produced by macrophages, supports intramembranous bone healing in a mouse model of tibia injury.

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.

Selective inhibition of BET bromodomain epigenetic signalling interferes with the bone-associated tumour vicious cycle.

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.

A functional, new short isoform of death receptor 4 in Ewing's sarcoma cell lines may be involved in TRAIL sensitivity/resistance mechanisms.

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.

Adhesion and osteogenic differentiation of human mesenchymal stem cells on titanium nanopores.

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.

Long term oncostatin M treatment induces an osteocyte-like differentiation on osteosarcoma and calvaria cells.

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.

Interleukin-6 inhibits receptor activator of nuclear factor kappaB ligand-induced osteoclastogenesis by diverting cells into the macrophage lineage: key role of Serine727 phosphorylation of signal transducer and activator of transcription 3.

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.

Receptor activator of nuclear factor-kappaB ligand (RANKL) directly modulates the gene expression profile of RANK-positive Saos-2 human osteosarcoma cells.

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.

Downregulation of osteoblast markers and induction of the glial fibrillary acidic protein by oncostatin M in osteosarcoma cells require PKCdelta and STAT3.

UNLABELLED: 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 PKCdelta 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 by 3H 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 (PKCdelta 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 PKCdelta, ERK1/2, and STAT3, these results offer new lines of investigations for neural cell transplantation and osteosarcoma therapy.