Proinflammatory Modulation of the Surface and Cytokine Phenotype of Monocytes in Patients With Acute Charcot Foot

Despite increased information on the importance of an inappropriate inflammatory response in the acute Charcot process, there has been no previous attempt to define the specific pathways that mediate its pathogenesis. Here, the role played by monocytes was analyzed

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

International audienceHistone 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 promotes STAT3 activation, VEGF production, and invasion in osteosarcoma cell lines

<p>Abstract</p> <p>Background</p> <p>We have previously demonstrated that both canine and human OSA cell lines, as well as 8 fresh canine OSA tumor samples, exhibit constitutive phosphorylation of STAT3, and that this correlates with enhanced expression of matrix metalloproteinase-2 (MMP2). While multiple signal transduction pathways can result in phosphorylation of STAT3, stimulation of the cytokine receptor gp130 through either IL-6 or Oncostatin M (OSM) is the most common mechanism through which STAT3 is activated. The purpose of this study was to evaluate the role of IL-6 and OSM stimulation on both canine and human OSA cell lines to begin to determine the role of these cytokines in the biology of OSA.</p> <p>Methods</p> <p>RT-PCR and Western blotting were used to interrogate the consequences of OSM and IL-6 stimulation of OSA cell lines. OSA cells were stimulated with OSM and/or hepatocyte growth factor (HGF) and the effects on MMP2 activity (gel zymography), proliferation (CyQUANT), invasion (Matrigel transwell assay), and VEGF production (Western blotting, ELISA) were assessed. The small molecule STAT3 inhibitor LLL3 was used to investigate the impact of STAT3 inhibition following OSM stimulation of OSA cells.</p> <p>Results</p> <p>Our data demonstrate that the OSM receptor (OSMR), but not IL-6 or its receptor, is expressed by all human and canine OSA cell lines and canine OSA tumor samples; additionally, OSM expression was noted in all tumor samples. Treatment of OSA cell lines with OSM induced phosphorylation of STAT3, Src, and JAK2. OSM stimulation also resulted in a dose dependent increase in MMP2 activity and VEGF expression that was markedly reduced following treatment with the small molecule STAT3 inhibitor LLL3. Lastly, OSM stimulation of OSA cell lines enhanced invasion through Matrigel, particularly in the presence of rhHGF. In contrast, both OSM and HGF stimulation of OSA cell lines did not alter their proliferative capacity.</p> <p>Conclusions</p> <p>These data indicate OSM stimulation of human and canine OSA cells induces STAT3 activation, thereby enhancing the expression/activation of MMP2 and VEGF, ultimately promoting invasive behavior and tumor angiogenesis. As such, OSM and its receptor may represent a novel target for therapeutic intervention in OSA.</p

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

RANKL/RANK/OPG: new therapeutic targets in bone tumours and associated osteolysis

The emergence of the molecular triad osteoprotegerin (OPG)/Receptor Activator of NF-kB (RANK)/RANK Ligand (RANKL) has helped elucidate a key signalling pathway between stromal cells and osteoclasts. The interaction between RANK and RANKL plays a critical role in promoting osteoclast differentiation and activation leading to bone resorption. OPG is a soluble decoy receptor for RANKL that blocks osteoclast formation by inhibiting RANKL binding to RANK. The OPG/RANK/RANKL system has been shown to be abnormally regulated in several malignant osteolytic pathologies such as multiple myeloma [MM, where enhanced RANKL expression (directly by tumour cells or indirectly by stromal bone cells or T-lymphocytes)] plays an important role in associated bone destruction. By contrast, production of its endogenous counteracting decoy receptor OPG is either inhibited or too low to compensate for the increase in RANKL production. Therefore, targeting the OPG/RANK/RANKL axis may offer a novel therapeutic approach to malignant osteolytic pathologies. In animal models, OPG or soluble RANK was shown both to control hypercalcaemia of malignancy and the establishment and progression of osteolytic metastases caused by various malignant tumours. To this day, only one phase I study has been performed using a recombinant OPG construct that suppressed bone resorption in patients with multiple myeloma or breast carcinoma with radiologically confirmed bone lesions. RANK-Fc also exhibits promising therapeutic effects, as revealed in animal models of prostate cancer and multiple myeloma. If the animal results translate to similar clinical benefits in humans, using RANK-Fc or OPG may yield novel and potent strategies for treating patients with established or imminent malignant bone diseases and where standard therapeutic regimens have failed

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

Sensitization of osteosarcoma cells to apoptosis by oncostatin M depends on STAT5 and p53

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

DU145 human prostate cancer cells express functional receptor activator of NF kappa B: New insights in the prostate cancer bone metastasis process

Prostate cancer metastases to bone are observed in around 80% of prostate cancer patients and represent the most critical complication of advanced prostate cancer, frequently resulting in significant morbidity and mortality. As the underlying mechanisms are not fully characterized, understanding the biological mechanisms that govern prostate cancer metastases to bone at the molecular level should lead to the determination of new potential therapeutic targets. Receptor activator of NFκB ligand (RANKL)/RANK/Osteoprotegerin (OPG) are the key regulators of bone metabolism both in normal and pathological condition, including prostate cancer bone metastases. In the present study, we demonstrated that human prostate cancer cell lines, DU145 and PC3 express biologically functional RANK. Indeed, soluble human RANKL (shRANKL, 100 ng/ml) treatment induced ERK 1/2, p38 and IκB phosphorylations in these cells. shRANKL administration also promoted DU145 and PC3 prostate cancer cell invasion in vitro. Whereas human OPG (hOPG) administration alone (100 ng/ml) had no marked effect, combined association of both agents abolished the RANKL-induced DU145 cell invasion. As RANKL had no direct effect on DU145 cell proliferation, the observed effects were indeed related to RANKL-induced cell migration. DU145 human prostate cancer cells promoted osteoclastogenesis of osteoclast precursors generated from mouse bone marrow. Moreover, DU145 cells produced soluble factor(s) that up-regulate the proliferation of MC3T3-E1 pre-osteoblasts through the activation of the ERK 1/2 and STAT3 signal transduction pathways. This stimulation of pre-osteoblast proliferation resulted in an increased local RANKL expression that can activate both osteoclasts/osteoclast precursors and prostate cancer cells, thus facilitating prostate cancer metastasis development in bone. We confirm that RANKL is a factor that facilitates metastasis to bone by acting as an activator of both osteoclasts and RANK-positive prostate cancer cells in our model. Furthermore, the present study provides the evidence that blocking RANKL–RANK interaction offer new therapeutic approach not only at the level of bone resorbing cells, but also by interfering with RANK-positive prostate cancer cells in the prostate cancer bone metastasis development