Plasma Rhenium and Selenium Concentrations After Repeated Daily Oral Administration of Rhenium(I)-diselenoether in 4T1 Breast Tumor-bearing Mice.

BACKGROUND/AIM: Rhenium(I)-diselenoether (Re-diSe) is a compound combining a rhenium tricarbonyl(I) core with a diselenide ligand. A high dose of 60 mg/kg had a pro-tumor effect in a previous study, in non-immune deficient 4T1 tumor-bearing mice, while doses of 1 and 10 mg/kg did not affect tumor growth, after repeated oral administrations. This study aimed to examine the tumor effects of a lower dose of 0.1 mg/kg with the same experimental design and to assay plasma Re and Se concentrations. MATERIALS AND METHODS: Syngenic BALB/cByJ (JAX) mice were orthotopically inoculated with 4T1 mammary breast cancer cells. Re-diSe was daily administered orally for 23 days at doses of 0.1, 1, and 10 mg/kg, whereas controls received no treatment. Tumor and mice weights were measured at the end of the experiment. Plasma Re and Se concentrations were assayed by an inductively coupled plasma sector field mass spectrometry instrument (ICP-sf-MS). RESULTS: The weight of the tumors did not vary in treated versus non-treated mice. The limit of detection (LOD) of Re was 0.34 nmol/l. Plasma Re concentrations were 14±20 nmol/l at doses of 0.1 mg/kg, and increased at higher doses, up to 792±167 nmol/l at doses of 10 mg/kg. Plasma Se concentrations were significantly increased in mice treated with the dose of 0.1 mg/kg (4,262±1,511 nmol/l) versus controls (1,262±888 nmol/l), but not from 0.1 to 1 mg/kg, nor from 1 to 10 mg/kg. CONCLUSION: The 0.1 mg/kg dose of Re-diSe resulted in detectable plasma Re concentrations and significantly increased plasma Se concentrations. In the future, doses as low as 0.1 mg/kg of Re-diSe will be tested, exploring its potential immune interest as a metronomic schedule of treatment, but in mouse models that readily develop extensive metastatic disease.

Relationship between the oxidative status and the tumor growth in transplanted triple-negative 4T1 breast tumor mice after oral administration of rhenium(I)-diselenoether.

BACKGROUND: Selective inhibitory effects of rhenium(I)-diselenoether (Re-diSe) were observed in cultured breast malignant cells. They were attributed to a decrease in Reactive Oxygen Species (ROS) production. A concomitant decrease in the production of Transforming Growth Factor-beta (TGFß1), Insulin Growth Factor 1 (IGF1), and Vascular Endothelial Growth Factor A (VEGFA) by the malignant cells was also observed. AIM: The study aimed to investigate the anti-tumor effects of Re-diSe on mice bearing 4T1 breast tumors, an experimental model of triple-negative breast cancer, and correlate them with several biomarkers. MATERIAL AND METHODS: 4T1 mammary breast cancer cells were orthotopically inoculated into syngenic BALB/c Jack mice. Different doses of Re-diSe (1, 10, and 60 mg/kg) were administered orally for 23 consecutive days to assess the efficacy and toxicity. The oxidative status was evaluated by assaying Advanced Oxidative Protein Products (AOPP), and by the dinitrophenylhydrazone (DNPH) test in plasma of healthy mice, non-treated tumor-bearing mice (controls), treated tumor-bearing mice, and tumors in all tumor-bearing mice. Tumor necrosis factor (TNFα), VEGFA, VEGFB, TGFß1, Interferon, and selenoprotein P (selenoP) were selected as biomarkers. RESULTS: Doses of 1 and 10 mg/kg did not affect the tumor weights. There was a significant increase in the tumor weights in mice treated with the maximum dose of 60 mg/kg, concomitantly with a significant decrease in AOPP, TNFα, and TGFß1 in the tumors. SelenoP concentrations increased in the plasma but not in the tumors. CONCLUSION: We did not confirm the anti-tumor activity of the Re-diSe compound in this experiment. However, the transplantation of the tumor cells did not induce an expected pro-oxidative status without any increase of the oxidative biomarkers in the plasma of controls compared to healthy mice. This condition could be essential to evaluate the effect of an antioxidant drug. The choice of the experimental model will be primordial to assess the effects of the Re-diSe compound in further studies.

Injectable Magnetic-Responsive Short-Peptide Supramolecular Hydrogels: Ex Vivo and In Vivo Evaluation.

The inclusion of magnetic nanoparticles (MNP) in a hydrogel matrix to produce magnetic hydrogels has broadened the scope of these materials in biomedical research. Embedded MNP offer the possibility to modulate the physical properties of the hydrogel remotely and on demand by applying an external magnetic field. Moreover, they enable permanent changes in the mechanical properties of the hydrogel, as well as alterations in the micro- and macroporosity of its three-dimensional (3D) structure, with the associated potential to induce anisotropy. In this work, the behavior of biocompatible and biodegradable hydrogels made with Fmoc-diphenylalanine (Fmoc-FF) (Fmoc = fluorenylmethoxycarbonyl) and Fmoc-arginine-glycine-aspartic acid (Fmoc-RGD) short peptides to which MNP were incorporated was studied in detail with physicochemical, mechanical, and biological methods. The resulting hybrid hydrogels showed enhance mechanical properties and withstood injection without phase disruption. In mice, the hydrogels showed faster and improved self-healing properties compared to their nonmagnetic counterparts. Thanks to these superior physical properties and stability during culture, they can be used as 3D scaffolds for cell growth. Additionally, magnetic short-peptide hydrogels showed good biocompatibility and the absence of toxicity, which together with their enhanced mechanical stability and excellent injectability make them ideal biomaterials for in vivo biomedical applications with minimally invasive surgery. This study presents a new approach to improving the physical and mechanical properties of supramolecular hydrogels by incorporating MNP, which confer structural reinforcement and stability, remote actuation by magnetic fields, and better injectability. Our approach is a potential catalyst for expanding the biomedical applications of supramolecular short-peptide hydrogels.

Proteomic analysis identified LBP and CD14 as key proteins in blood/biphasic calcium phosphate microparticle interactions.

Immediately upon implantation, scaffolds for bone repair are exposed to the patient's blood. Blood proteins adhere to the biomaterial surface and the protein layer affects both blood cell functions and biomaterial bioactivity. Previously, we reported that 80-200 µm biphasic calcium phosphate (BCP) microparticles embedded in a blood clot, induce ectopic woven bone formation in mice, when 200-500 µm BCP particles induce mainly fibrous tissue. Here, in a LC-MS/MS proteomic study we compared the differentially expressed blood proteins (plasma and blood cell proteins) and the deregulated signaling pathways of these osteogenic and fibrogenic blood composites. We showed that blood/BCP-induced osteogenesis is associated with a higher expression of fibrinogen (FGN) and an upregulation of the Myd88- and NF-κB-dependent TLR4 signaling cascade. We also highlighted the key role of the LBP/CD14 proteins in the TLR4 activation of blood cells by BCP particles. As FGN is an endogenous ligand of TLR4, able to modulate blood composite stiffness, we propose that different FGN concentrations modify the blood clot mechanical properties, which in turn modulate BCP/blood composite osteoactivity through TLR4 signaling. The present findings provide an insight at the protein level, into the mechanisms leading to an efficient bone reconstruction by blood/BCP composites. STATEMENT OF SIGNIFICANCE: Upon implantation, scaffolds for bone repair are exposed to the patient's blood. Blood proteins adhere to bone substitute surface and this protein layer affects both biomaterial bioactivity and bone healing. Therefore, for the best outcome for patients, it is crucial to understand the molecular interactions between blood and bone scaffolds. Biphasic calcium phosphate (BCP) ceramics are considered as the gold standard in bone reconstruction surgery. Here, using proteomic analyses we showed that the osteogenic properties of 80-200 µm BCP particles embedded in a blood clot is associated with a higher expression of fibrinogen. Fibrinogen upregulates the Myd88- and NF-κB-dependent TLR4 pathway in blood cells and, BCP-induced TLR4 activation is mediated by the LBP and CD14 proteins.

Percutaneous vertebroplasty in tumoral spinal fractures with posterior vertebral wall involvement: Feasibility and safety.

PURPOSE: to evaluate the technical feasibility and safety of CT and fluoroscopy guided percutaneous vertebroplasty in the treatment of tumoral vertebral fractures with posterior wall involvement. MATERIALS AND METHODS: Institutional review board approval and informed consent were obtained for this study. Sixty-three consecutive adult patients (35 women, 28 men; mean age+/- standard deviation: 69 years+/- 14) with tumoral spinal fractures that compromised the posterior wall were treated by means of percutaneous vertebroplasty with CT and fluoroscopy guidance. Only local anesthesia was used during these procedures. Postoperative outcome was assessed using the Kostuik index. RESULTS: Sixty-three vertebroplasties were performed on thirty-four thoracic (54%), twenty-six lumbar (41%), and three (5%) cervical vertebrae. The etiologies of the fractures were metastasis in twenty-eight (44%), myeloma in twenty-five (40%) and hemangioma in ten (16%). Almost all fractures (94%) were consolidated after vertebroplasty (score of Kostuik <3) (p < 0.001). No major complications were reported in our series of cases. CONCLUSION: This study suggests that tumoral spinal fractures with posterior vertebral wall involvement can be successfully and safely treated by CT- and fluoroscopy-guided percutaneous vertebroplasty.

Understanding the Progression of Bone Metastases to Identify Novel Therapeutic Targets.

Bone is one of the most preferential target site for cancer metastases, particularly for prostate, breast, kidney, lung and thyroid primary tumours. Indeed, numerous chemical signals and growth factors produced by the bone microenvironment constitute factors promoting cancer cell invasion and aggression. After reviewing the different theories proposed to provide mechanism for metastatic progression, we report on the gene expression profile of bone-seeking cancer cells. We also discuss the cross-talk between the bone microenvironment and invading cells, which impacts on the tumour actions on surrounding bone tissue. Lastly, we detail therapies for bone metastases. Due to poor prognosis for patients, the strategies mainly aim at reducing the impact of skeletal-related events on patients' quality of life. However, recent advances have led to a better understanding of molecular mechanisms underlying bone metastases progression, and therefore of novel therapeutic targets.

Gallium enhances reconstructive properties of a calcium phosphate bone biomaterial.

Calcium phosphate (CaP)-based biomaterials are commonly used in bone reconstructive surgery to replace the damaged tissue, and can also serve as vectors for local drug delivery. Due to its inhibitory action on osteoclasts, the semi-metallic element gallium (Ga) is used for the systemic treatment of disorders associated with accelerated bone resorption. As it was demonstrated that Ga could be incorporated in the structure of CaP biomaterials, we investigated the biological properties of Ga-loaded CaP biomaterials. Culturing bone cells on Ga-CaP, we observed a decrease in osteoclast number and a downregulation of late osteoclastic markers expression, while Ga-CaP upregulated the expression of osteoblastic marker genes involved in the maturation of bone matrix. We next investigated in vivo bone reconstructive properties of different Ga-loaded biomaterials using a murine bone defect healing model. All implanted biomaterials showed a good osseointegration into the surrounding host tissue, accompanied by a successful bone ingrowth and bone marrow reconstruction, as evidenced by histological analysis. Moreover, quantitative micro-computed tomography analysis of implants revealed that Ga enhanced total defect filling. Lastly, we took advantage for the first time of a particular mode of non-linear microscopy (second harmonic generation) to quantify in vivo bone tissue reconstruction within a CaP bone substitute. By doing so, we showed that Ga exerted a positive impact on mature organized collagen synthesis. As a whole, our data support the hypothesis that Ga represents an attractive additive to CaP biomaterials for bone reconstructive surgery. Copyright © 2017 John Wiley & Sons, Ltd.

Gallium, a promising candidate to disrupt the vicious cycle driving osteolytic metastases.

Bone metastases of breast cancer typically lead to a severe osteolysis due to an excessive osteoclastic activity. On the other hand, the semi-metallic element gallium (Ga) displays an inhibitory action on osteoclasts, and therefore on bone resorption, as well as antitumour properties. Thus, we explored in vitro Ga effects on osteoclastogenesis in an aggressive bone metastatic environment based on the culture of pre-osteoclast RAW 264.7 cells with conditioned medium from metastatic breast tumour cells, i.e. the breast tumour cell line model MDA-MB-231 and its bone-seeking clone MDA-231BO. We first observed that Ga dose-dependently inhibited the tumour cells-induced osteoclastic differentiation of RAW 264.7 cells. To mimic a more aggressive environment where pro-tumourigenic factors are released from bone matrix due to osteoclastic resorption, metastatic breast tumour cells were stimulated with TGF-ß, a mayor cytokine in bone metastasis vicious cycle. In these conditions, we observed that Ga still inhibited cancer cells-driven osteoclastogenesis. Lastly, we evidenced that Ga affected directly and strongly the proliferation/viability of both cancer cell lines, as well as the expression of major osteolytic factors in MDA-231BO cells. With the exception of two small scale clinical studies from 1980s, this is the first time that antitumour properties of Ga have been specifically studied in the context of bone metastases. Our data strongly suggest that, through its action against the vicious cycle involving bone cells and tumour cells, Ga represents a relevant and promising candidate for the local treatment of bone metastases in patients with breast cancer.

Adaptive immune response inhibits ectopic mature bone formation induced by BMSCs/BCP/plasma composite in immune-competent mice.

A combination of autologous bone marrow stromal cells (BMSCs) and biomaterials is a strategy largely developed in bone tissue engineering, and subcutaneous implantation in rodents or large animals is often a first step to evaluate the potential of new biomaterials. This study aimed at investigating the influence of the immune status of the recipient animal on BMSCs-induced bone formation. BMSCs prepared from C57BL/6 mice, composed of a mixture of mesenchymal stromal and monocytic cells, were combined with a biomaterial that consisted of biphasic calcium phosphate (BCP) particles and plasma clot. This composite was implanted subcutaneously either in syngenic C57BL/6 immune-competent mice or in T-lymphocyte-deficient Nude (Nude) mice. Using histology, immunohistochemistry, and histomorphometry, we show here that this BMSC/BCP/plasma clot composite implanted in Nude mice induces the formation of mature lamellar bone associated to hematopoietic areas and numerous vessels. Comparatively, implantation in C57BL/6 results in the formation of woven bone without hematopoietic tissue, a lower number of new vessels, and numerous multinucleated giant cells (MNGCs). In situ hybridization, which enabled to follow the fate of the BMSCs, revealed that BMSCs implanted in Nude mice survived longer than BMSCs implanted in C57BL/6 mice. Quantitative expression analysis of 280 genes in the implants indicated that the differences between C57BL/6 and Nude implants corresponded almost exclusively to genes related to the immune response. Gene expression profile in C57BL/6 implants was consistent with a mild chronic inflammation reaction characterized by Th1, Th2, and cytotoxic T-lymphocyte activation. In the implants retrieved from T-deficient Nude mice, Mmp14, Il6st, and Tgfbr3 genes were over-expressed, suggesting their putative role in bone regeneration and hematopoiesis. In conclusion, we show here that the T-mediated inflammatory microenvironment is detrimental to BMSCs-induced bone formation and shortens the survival of implanted cells. Conversely, the lack of T-lymphocyte reaction in T-deficient animals is beneficial to BMSCs-induced mature bone formation. This should be taken into account when evaluating cell/biomaterial composites in these models.

Therapeutic strategies for treating osteolytic bone metastases.

The recent progress in oncologic management of patients with localized cancer or metastatic disease has permitted a significant improvement in life expectancy. Nevertheless, bone metastases and their consequent skeletal-related events (SREs) are still associated with unfavorable prognosis and greatly affect quality of life. Global management of these bone metastases includes traditional local approaches (surgery, radiotherapy, etc.) and systemic administration of chemotherapeutic agents. This review focuses on treatments specific for bone metastases and, in particular, on inhibitors of bone resorption that are effective for preventing and delaying the development of SREs.

Monocytes differentiation upon treatment with a peptide corresponding to the C-terminus of activated T cell-expressed Tirc7 protein.

Atp6v0a3 gene encodes for two alternative products, Tirc7 and a3 proteins, which are differentially expressed in activated T cells and resorbing osteoclasts, respectively. Tirc7 plays a central role in T cell activation, while a3 protein is critical for osteoclast-mediated bone matrix resorption. Based on the large body of evidences documenting the relationships between T cells and osteoclasts, we hypothesized that the extracellular C-terminus of Tirc7 protein could directly interact with osteoclast precursor cells. To address this issue, we performed the molecular cloning of a mouse Atp6v0a3 cDNA segment encoding the last 40 amino acids of Tirc7 protein, and we used this peptide as a ligand added to mouse osteoclast precursor cells. We evidenced that Tirc7-Cter peptide induced the differentiation of RAW264.7 cells into osteoclast-like cells, stimulated an autocrine/paracrine regulatory loop potentially involved in osteoclastic differentiation control, and strongly up-regulated F4/80 protein expression within multinucleated osteoclast-like cells. Using a mouse bone marrow-derived CD11b(+) cell line, or total bone marrow primary cells, we observed that similarly to Rankl, Tirc7-Cter peptide induced the formation of TRACP-positive large multinucleated cells. At last, using mouse primary monocytes purified from total bone marrow, we determined that Tirc7-Cter peptide induced the appearance of small multinucleated cells (3-4 nuclei), devoid of resorbing activity, and which displayed modulations of dendritic cell marker genes expression. In conclusion, we report for the first time on biological effects mediated by a peptide corresponding to the C-terminus of Tirc7 protein, which interfere with monocytic differentiation pathways.

Fate of bone marrow stromal cells in a syngenic model of bone formation.

Bone marrow stromal cells (BMSCs) have been demonstrated to induce bone formation when associated to osteoconductive biomaterials and implanted in vivo. Nevertheless, their role in bone reconstruction is not fully understood and rare studies have been conducted to follow their destiny after implantation in syngenic models. The aim of the present work was to use sensitive and quantitative methods to track donor and recipient cells after implantation of BMSCs in a syngenic model of ectopic bone formation. Using polymerase chain reaction (PCR) amplification of the Sex determining Region Y (Sry) gene and in situ hybridization of the Y chromosome in parallel to histological analysis, we have quantified within the implants the survival of the donor cells and the colonization by the recipient cells. The putative migration of the BMSCs in peripheral organs was also analyzed. We show here that grafted cells do not survive more than 3 weeks after implantation and might migrate in peripheral lymphoid organs. These cells are responsible for the attraction of host cells within the implants, leading to the centripetal colonization of the biomaterial by new bone.

Osteoclastic differentiation of mouse and human monocytes in a plasma clot/biphasic calcium phosphate microparticles composite.

We recently demonstrated that blood clotted around biphasic calcium phosphate (BCP) microparticles constituted a composite biomaterial that could be used for bone defect filling. In addition, we showed that mononuclear cells, i.e. monocytes and lymphocytes, play a central role in the osteogenic effect of this biomaterial. Hypothesizing that osteoclast progenitors could participate to the pro-osteogenic effect of mononuclear cells we observed previously, we focus on this population through the study of mouse monocyte/macrophage cells (RAW264.7 cell line), as well as human pre-osteoclastic cells derived from mononuclear hematopoietic progenitor cells (monocytes-enriched fraction from peripheral blood). Using monocyte-derived osteoclast progenitors cultured within plasma clot/BCP microparticles composite, we aimed in the present report at the elucidation of transcriptional profiles of genes related to osteoclastogenesis and to bone remodelling. For both human and mouse monocytes, real-time PCR experiments demonstrated that plasma clot/BCP scaffold potentiated the expression of marker genes of the osteoclast differentiation such as Nfactc1, Jdp2, Fra2, Tracp and Ctsk. By contrast, Mmp9 was induced in mouse but not in human cells, and Ctr expression was down regulated for both species. In addition, for both mouse and human precursors, osteoclastic differentiation was associated with a strong stimulation of VegfC and Sdf1 genes expression. At last, using field-emission scanning electron microscopy analysis, we observed the interactions between human monocytes and BCP microparticles. As a whole, we demonstrated that plasma clot/BCP microparticles composite provided monocytes with a suitable microenvironment allowing their osteoclastic differentiation, together with the production of pro-angiogenic and chemoattractant factors.

Biphasic calcium phosphate microparticles for bone formation: benefits of combination with blood clot.

Particulate forms of biphasic calcium phosphate (BCP) biomaterials below 500 µm are promising bone substitutes that provide with interconnected open porosity allowing free circulation of fluids and cells. Dispersion of the particles in the surrounding tissues at the time of implantation is a major drawback preventing from an easy use. We have asked whether blood clot could be a convenient natural hydrogel for handling BCP microparticles, and we hypothesized that blood clot might also confer osteoinductive properties to these particles. We show here that blood clotted around BCP microparticles constitutes a cohesive, moldable, and adaptable biomaterial that can be easily implanted in subcutaneous sites but also inserted and maintained in segmental bone defects, conversely to BCP microparticles alone. Moreover, implantation in bony and ectopic sites revealed that this composite biomaterial has osteogenic properties. It is able to repair a 6-mm critical femoral defect in rat and induced woven bone formation after subcutaneous implantation. Parameters such as particle size and loading into the clot are critical for its osteogenic properties. In conclusion, this blood/BCP microparticle composite is a moldable and osteoinductive biomaterial that could be used for bone defect filling in dental and orthopedic surgery.

Gallium modulates osteoclastic bone resorption in vitro without affecting osteoblasts.

BACKGROUND AND PURPOSE: Gallium (Ga) has been shown to be effective in the treatment of disorders associated with accelerated bone loss, including cancer-related hypercalcemia and Paget's disease. These clinical applications suggest that Ga could reduce bone resorption. However, few studies have studied the effects of Ga on osteoclastic resorption. Here, we have explored the effects of Ga on bone cells in vitro. EXPERIMENTAL APPROACH: In different osteoclastic models [osteoclasts isolated from long bones of neonatal rabbits (RBC), murine RAW 264.7 cells and human CD14-positive cells], we have performed resorption activity tests, staining for tartrate resistant acid phosphatase (TRAP), real-time polymerase chain reaction analysis, viability and apoptotic assays. We also evaluated the effect of Ga on osteoblasts in terms of proliferation, viability and activity by using an osteoblastic cell line (MC3T3-E1) and primary mouse osteoblasts. KEY RESULTS: Gallium dose-dependently (0-100 microM) inhibited the in vitro resorption activity of RBC and induced a significant decrease in the expression level of transcripts coding for osteoclastic markers in RAW 264.7 cells. Ga also dramatically reduced the formation of TRAP-positive multinucleated cells. Ga down-regulated in a dose-dependant manner the expression of the transcription factor NFATc1. However, Ga did not affect the viability or activity of primary and MC3T3-E1 osteoblasts. CONCLUSIONS AND IMPLICATIONS: Gallium exhibits a dose-dependent anti-osteoclastic effect by reducing in vitro osteoclastic resorption, differentiation and formation without negatively affecting osteoblasts. We provide evidence that this inhibitory mechanism involves down-regulation of NFATc1 expression, a master regulator of RANK-induced osteoclastic differentiation.

Cloning and use of a coral 36B4 gene to study the differential expression of coral genes between light and dark conditions.

This paper aims to validate reference genes for gene expression studies between light and dark conditions in the scleractinian coral Stylophora pistillata for future gene expression studies of the "light-enhanced calcification" phenomenon. For this purpose, we cloned, sequenced, and characterized a candidate reference gene, the 36B4 gene from the coral S. pistillata, and validated 36B4 and beta-actin as reference genes. To illustrate the future applications of these reference genes, we tested the dark and light expression of two photosynthetic genes (Rubisco and D1 protein of the photosystem II) and two genes encoding proteins involved in calcium transport for coral calcification (a calcium ATPase and a calcium channel). Results show that both photosynthetic genes are enhanced during the light when standardized against 36B4 and beta-actin, whereas the two genes encoding proteins involved in calcium transport are not differentially expressed between light and dark conditions. The characterization of a coral 36B4 and the establishment of such valid reference genes will be useful for future gene expression studies between diverse conditions (aposymbiotic/symbiotic, stress/control, light/dark conditions) in scleractinian corals.

Poly(adp-ribose) polymerase-1 regulates Tracp gene promoter activity during RANKL-induced osteoclastogenesis.

UNLABELLED: The Tracp gene encodes an acid phosphatase strongly upregulated during osteoclastogenesis on RANKL treatment. Using the mouse osteoclastic model RAW264.7, we studied Tracp gene expression, and we identified PARP-1 as a transcriptional repressor negatively regulated by RANKL during osteoclastogenesis. INTRODUCTION: The Tracp gene encodes an acid phosphatase strongly expressed in differentiated osteoclasts. TRACP enzyme has a dual role and is involved in (1) the regulation of the biological activity of the bone matrix phosphoproteins osteopontin and bone sialoprotein and (2) the intracellular collagen degradation. Based on our previous work on Tcirg1 gene expression, and using data available in the literature, we focused on a 200-bp sequence located upstream the Tracp gene transcriptional start to identify binding activities. MATERIALS AND METHODS: We first performed siRNA transfections and RAW264.7 cell treatment with an inhibitor of poly(ADP-ribose) polymerase-1 (PARP-1) activity. After EMSA and supershift experiments, we measured the promoter activity of wildtype and mutant constructs throughout the osteoclastic differentiation. RESULTS: We first showed that depleting PARP-1 mRNA in the pre-osteoclastic cell line RAW264.7 results in an increase of both matrix metalloproteinase 9 and TRACP mRNA expression (3.5- and 2.5-fold, respectively). Moreover, in response to 3-aminobenzamide treatment, we measured a weak stimulation of MMP9 mRNA expression, whereas up to a 2-fold enhancement above the control condition of TRACP mRNA expression was observed. We next identified in the -839/-639 Tracp promoter region a PARP-1 binding site, and supershift experiments showed the interaction of a PARP-1 binding activity with the Tracp promoter sequence -830/-808. Finally, RAW264.7 cell transfection with a promoter construct mutated for this PARP-1 interacting sequence showed the functionality of this site within intact pre-osteoclastic cells. CONCLUSIONS: In this study, we provide evidence that the transcriptional activity of the Tracp gene, in pre-osteoclastic cells, is negatively regulated by the binding of PARP-1 protein to a potential consensus sequence located in its promoter region. Taken together with our previous results related to the control of Tcirg1 gene expression, our data suggest that PARP-1 exerts a pivotal role in the basal repression of genes that are upregulated during RANKL-induced osteoclastogenesis.

Differential binding of poly(ADP-Ribose) polymerase-1 and JunD/Fra2 accounts for RANKL-induced Tcirg1 gene expression during osteoclastogenesis.

UNLABELLED: We studied Tcirg1 gene expression on RANKL-induced osteoclastic differentiation of the mouse model RAW264.7 cells. We identified a mechanism involving PARP-1 inhibition release and JunD/Fra-2 binding, which is responsible for Tcirg1 gene upregulation. INTRODUCTION: The Tcirg1 gene encodes the a3 isoform of the V-ATPase a subunit, which plays a critical role in the resorption activity of the osteoclast. Using serial deletion constructs of the Tcirg1 gene promoter, we performed a transcriptional study to identify factor(s) involved in the regulation of the RANKL-induced gene expression. MATERIALS AND METHODS: The promoter activity of serial-deletion fragments of the Tcirg1 gene promoter was monitored throughout the RAW264.7 cells differentiation process. We next performed sequence analysis, EMSA, UV cross-linking, qPCR, and gel supershift experiments to identify the factor(s) interacting with the promoter. RESULTS: A deletion of the -1297-1244 region led to the disappearance of the RANKL-induced promoter activity. EMSA experiments showed the binding of two factors that undergo differential binding on RANKL treatment. Supershift experiments led us to identify the dimer JunD/Fra-2 as the binding activity associated with the -1297/-1268 Tcirg1 gene promoter sequence in response to RANKL. Moreover, we observed poly(ADP-ribose) polymerase-1 (PARP-1) binding to an adjacent site (-1270/-1256), and this interaction was disrupted after RANKL treatment. CONCLUSIONS: We provide data that identify junD proto-oncogene (JunD) and Fos-related antigen 2 (Fra-2) as the activator protein-1 (AP-1) factors responsible for the RANKL-induced upregulation of the mouse Tcirg1 gene expression. Moreover, we identified another binding site for PARP-1 that might account for the repression of Tcirg1 gene expression in pre-osteoclastic cells.

RANKL treatment releases the negative regulation of the poly(ADP-ribose) polymerase-1 on Tcirg1 gene expression during osteoclastogenesis.

UNLABELLED: The Tcirg1 gene encodes the osteoclast-specific a3 isoform of the V-ATPase a subunit. Using the mouse osteoclastic model RAW264.7 cells, we studied Tcirg1 gene expression, and we identified PARP-1 as a transcriptional repressor negatively regulated by RANKL during osteoclastogenesis. INTRODUCTION: The TCIRG1 gene encodes the a3 isoform of the V-ATPase a subunit, and mutations at this locus account for approximately 60% of infantile malignant osteopetrosis cases. Using RAW264.7 cells as an osteoclastic differentiation model, we undertook a transcriptional study of the mouse Tcirg1 gene focused on the 4-kb region upstream of the transcription starting point. MATERIALS AND METHODS: The promoter activity of serial-deletion fragments of the Tcirg1 gene promoter was monitored throughout the RAW264.7 cell differentiation process. We next performed EMSA, UV cross-linking, affinity purification, mass spectrometry analysis, gel supershift, and siRNA transfection experiments to identify the factor(s) interacting with the promoter. RESULTS: The -3946/+113 region of the mouse Tcirg1 gene displayed a high basal promoter activity, which was enhanced by RANKL treatment of RAW264.7 cells. Constructs deleted up to -1589 retained this response to RANKL. A deletion up to -1402 induced a 3-fold enhancement of the basal activity, whereas RANKL response was not affected. EMSA experiments led us to identify within the -1589/-1402 region, a 10-nucleotide sequence, which bound a nuclear protein present in nondifferentiated RAW264.7 cells. This interaction was lost using nuclear extracts derived from RANKL-treated cells. Affinity purification followed by mass spectrometry analysis and gel supershift assay allowed the identification of poly(ADP-ribose) polymerase-1 (PARP-1) as this transcriptional repressor, whereas Western blot experiments revealed the cleavage of the DNA-binding domain of PARP-1 on RANKL treatment. Finally, both PARP-1 depletion after siRNA transfection and RAW264.7 cell treatment by an inhibitor of PARP-1 activity induced an increase of a3 mRNA expression. CONCLUSIONS: We provide evidence that the basal transcription activity of the Tcirg1 gene is negatively regulated by the binding of PARP-1 protein to its promoter region in mouse pre-osteoclast. On RANKL treatment, PARP-1 protein is cleaved and loses its repression effect, allowing an increase of Tcirg1 gene expression that is critical for osteoclast function.

Ectopic bone formation using an injectable biphasic calcium phosphate/Si-HPMC hydrogel composite loaded with undifferentiated bone marrow stromal cells.

We have used a new synthetic injectable composite constituted of hydroxyapatite/tricalcium phosphate (HA/TCP) particles in suspension in a self-hardening Si-hydroxypropylmethylcellulose (HPMC) hydrogel. The aim of this study was to evaluate in vivo the biocompatibility and the new bone formation efficacy of this scaffold loaded with undifferentiated bone marrow stromal cells (BMSCs). This biomaterial was mixed extemporaneously with BMSCs prepared from C57BL/6 mice, injected in subcutaneous and intramuscular sites and retrieved 4 and 8 weeks after implantation. Dissection of the implants revealed a hard consistency and the absence of a fibrous capsule reflecting a good integration into the host tissues. Histological analysis showed mineralized woven bone in the granule inter-space with numerous active osteoclasts attached to the particles as assessed by the presence of multinucleated cells positively stained for TRAP activity and for the a3 subunit of the V-ATPase. Small vessels were homogenously distributed in the whole implants. Similar results were obtained in SC and IM sites and no bone formation was observed in the control groups when cell-free and particle-free transplants were injected. These results indicate that this injectable biphasic calcium phosphate-hydrogel composite mixed with undifferentiated BMSCs is a new promising osteoinductive bone substitute. It also provides with an original in vivo model of osteoclast differentiation and function.