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

Author

Fanny Burel-Vandenbos, Mari C. Mañas-Torres, David Momier, Cristina Blanco-Elices, Marianne Goracci, Francisco J Vazquez-Perez, Modesto T. López-López, Pavel Kuzhir, Luis Álvarez de Cienfuegos, Ismael Rodríguez, Cristina Gila-Vilchez, Miguel Alaminos, Arnaud Borderie, Jean-Claude Scimeca, Universidad de Granada (UGR), ibs.GRANADA Instituto de Investigación Biosanitaria [Granada, Spain], Institut de Physique de Nice (INPHYNI), Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), Institut de Biologie Valrose (IBV), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Université Côte d'Azur (UCA), University of Córdoba [Córdoba], COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), SCIMECA, Jean-Claude, Universidad de Granada = University of Granada (UGR), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), and Universidad de Córdoba = University of Córdoba [Córdoba]

Subjects
Biocompatible Materials, 02 engineering and technology, [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, 01 natural sciences, Mice, Tissue engineering, Materials Testing, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], General Materials Science, Magnetite Nanoparticles, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Mice, Inbred BALB C, [SDV.MHEP.RSOA] Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, Molecular Structure, Hybrid hydrogels, Hydrogels, Self-assembly, self-assembly, 021001 nanoscience & nanotechnology, 3. Good health, [SDV.MHEP.RSOA]Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, tissue engineering, Self-healing hydrogels, Magnetic nanoparticles, Regenerative medicine, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], 0210 nano-technology, Research Article, biomaterials, magnetic nanoparticles, Materials science, Biocompatibility, Macromolecular Substances, Injections, Subcutaneous, Supramolecular chemistry, regenerative medicine, Nanotechnology, [SDV.CAN]Life Sciences [q-bio]/Cancer, macromolecular substances, 010402 general chemistry, complex mixtures, Biomaterials, [SDV.CAN] Life Sciences [q-bio]/Cancer, In vivo, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], hybrid hydrogels, Animals, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, Cell Proliferation, Osteoblasts, technology, industry, and agriculture, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, 0104 chemical sciences, [SDV.IB.BIO] Life Sciences [q-bio]/Bioengineering/Biomaterials, peptides, Peptides, Ex vivo
Abstract

This study was supported by project FIS2017-85954-R funded by MCIN/AEI/10.13039/501100011033/FEDER "Una manera de hacer Europa", Spain, grants FIS PI20/0317 and ICI19/00024 (BIOCLEFT) (MINECO, Instituto de Salud Carlos III, Spain, cofinanced by FEDER funds, European Union), grant PE-0395-2019 (Consejeri ' a de Salud y Familias, Junta de Andalucia ', Spain), and project PPJIB2020.07 (Universidad de Granada, Spain). M.C.M.-T. acknowledges grant PRE2018-083773 funded by MCIN/AEI/10.13039/501100011033 and FSE "El FSE invierte en tu futuro", Spain. C.G.-V. acknowledges grant FPU17/00491 funded by MCIN/AEI/10.13039/501100011033 and FSE "El FSE invierte en tu futuro", Spain. P.K., D.M., and J.-C.S. acknowledge the French Agence Nationale de la Recherche, Project Future Investments UCA JEDI no. ANR-15-IDEX-01 (project RheoGels) for financial support. Funding for open access charge: Universidad de Granada/CBUA., 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 threedimensional (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., Instituto de Salud Carlos III FIS PI20/0317 ICI19/00024, European Commission, FSE "El FSE invierte en tu futuro", Spain, French National Research Agency (ANR) ANR-15-IDEX-01, Universidad de Granada/CBUA, FIS2017-85954-R MCIN/AEI/10.13039/501100011033/FEDER PE-0395-2019 PPJIB2020.07 PRE2018-083773 MCIN/AEI/10.13039/501100011033 FPU17/00491

Published
2021
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2. Proteomic analysis identified LBP and CD14 as key proteins in blood/biphasic calcium phosphate microparticle interactions

Author

Jean-Marie Guigonis, Jean-Michel Bouler, Lun Jing, Solène Rota, Michel Samson, Sébastien Schaub, Jean-Claude Scimeca, David Momier, Nathalie Rochet, Patricia Lagadec, Florian Olivier, Transporteurs et Imagerie, Radiothérapie en Oncologie et Mécanismes biologiques des Altérations du Tissu Osseux (TIRO-MATOs - UMR E4320), Service Hospitalier Frédéric Joliot (SHFJ), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-UMR E4320 (TIRO-MATOs), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA), Institut de Biologie Valrose (IBV), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Interfaces, Confinement, Matériaux et Nanostructures ( ICMN), Université d'Orléans (UO)-Centre National de la Recherche Scientifique (CNRS), Chimie Et Interdisciplinarité : Synthèse, Analyse, Modélisation (CEISAM), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Nantes université - UFR des Sciences et des Techniques (Nantes univ - UFR ST), Nantes Université - pôle Sciences et technologie, Nantes Université (Nantes Univ)-Nantes Université (Nantes Univ)-Nantes Université - pôle Sciences et technologie, Nantes Université (Nantes Univ)-Nantes Université (Nantes Univ), Laboratoire de Biologie du Développement de Villefranche sur mer (LBDV), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de la Mer de Villefranche (IMEV), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut de recherche en santé, environnement et travail (Irset), Université d'Angers (UA)-Université de Rennes (UR)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), Université de Nantes (UN)-Université de Nantes (UN)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), UMR E4320 (TIRO-MATOs), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Service Hospitalier Frédéric Joliot (SHFJ), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Centre National de la Recherche Scientifique (CNRS)-Université d'Orléans (UO), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Institut de la Mer de Villefranche (IMEV), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Institut National de la Santé et de la Recherche Médicale (INSERM)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Université d'Angers (UA), and Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)

Subjects
Calcium Phosphates, Proteomics, 0206 medical engineering, Biomedical Engineering, [SDV.CAN]Life Sciences [q-bio]/Cancer, 02 engineering and technology, Bone healing, Fibrinogen, Biochemistry, Monocytes, Stiffness, Biomaterials, Blood cell, Mice, Downregulation and upregulation, Osteogenesis, Tandem Mass Spectrometry, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], medicine, Animals, Humans, TLR4, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], LC-MS/MS, [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Tissue Scaffolds, Chemistry, Biomaterial, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Medicine, 021001 nanoscience & nanotechnology, 020601 biomedical engineering, Blood proteins, Cell biology, medicine.anatomical_structure, [SDV.MHEP.RSOA]Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, Hydroxyapatites, Signal transduction, 0210 nano-technology, LBP/CD14, Biotechnology, medicine.drug, Chromatography, Liquid
Abstract

International audience; 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.

Published
2021
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3. Differential Binding of Poly(ADP-Ribose) Polymerase-1 and JunD/Fra2 Accounts for RANKL-Induced Tcirg1 Gene Expression During Osteoclastogenesis

Author

David Momier, Jean-Claude Scimeca, Guillaume E. Beranger, Michel Samson, Georges F. Carle, Jean-Marie Guigonis, Génétique, physiopathologie et ingénierie du tissu osseux (GéPITOS), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS), Génétique et signalisation moléculaire (GSM), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre Hospitalier Universitaire de Nice (CHU Nice)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Immunite anti-tumorale et chimiotactisme. Adenocarcinomes et métastases, COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Signalisation et Réponses aux Agents Infectieux et Chimiques (SeRAIC), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Institut de Biologie Valrose (IBV), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Université Nice Sophia Antipolis (1965 - 2019) (UNS), Université de Rennes (UR), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), and SCIMECA, Jean-Claude

Subjects
Proto-Oncogene Proteins c-jun, Endocrinology, Diabetes and Metabolism, Poly (ADP-Ribose) Polymerase-1, Osteoclasts, Fos-Related Antigen-2, MESH: Poly (ADP-Ribose) Polymerase-1, [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], MESH: Base Sequence, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Mice, 0302 clinical medicine, MESH: Osteoclasts, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Gene expression, MESH: Animals, Orthopedics and Sports Medicine, MESH: Models, Genetic, Promoter Regions, Genetic, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.MHEP.RSOA] Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, 0303 health sciences, Cell Differentiation, MESH: RANK Ligand, MESH: Gene Expression Regulation, MESH: Promoter Regions (Genetics), Cross-Linking Reagents, [SDV.MHEP.RSOA]Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, RANKL, 030220 oncology & carcinogenesis, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Poly(ADP-ribose) Polymerases, Dimerization, Protein Binding, musculoskeletal diseases, MESH: Cell Differentiation, Gene isoform, Vacuolar Proton-Translocating ATPases, Ultraviolet Rays, Recombinant Fusion Proteins, MESH: Cross-Linking Reagents, Poly ADP ribose polymerase, Molecular Sequence Data, MESH: Vacuolar Proton-Translocating ATPases, [SDV.CAN]Life Sciences [q-bio]/Cancer, MESH: Fos-Related Antigen-2, Biology, 03 medical and health sciences, [SDV.CAN] Life Sciences [q-bio]/Cancer, Downregulation and upregulation, MESH: Mice, Inbred C57BL, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], MESH: Promoter Regions, Genetic, MESH: Recombinant Fusion Proteins, Animals, MESH: Protein Binding, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, RNA, Messenger, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Binding site, [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, MESH: Mice, MESH: RNA, Messenger, 030304 developmental biology, Binding Sites, MESH: Molecular Sequence Data, Base Sequence, Models, Genetic, MESH: Proto-Oncogene Proteins c-jun, Activator (genetics), RANK Ligand, MESH: Poly(ADP-ribose) Polymerases, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Promoter, Molecular biology, Mice, Inbred C57BL, [SDV.IB.BIO] Life Sciences [q-bio]/Bioengineering/Biomaterials, Gene Expression Regulation, MESH: Binding Sites, MESH: Dimerization, biology.protein, MESH: Ultraviolet Rays
Abstract

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.

Published
2007
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4. Effect of G-CSF on the osteoinductive property of a BCP/blood clot composite

Author

Paul, Adrien J., David Momier, Florian Boukhechba, Jean-François Michiels, Patricia Lagadec, Nathalie Rochet, Institut de Biologie Valrose (IBV), Université Nice Sophia Antipolis (... - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA)

Subjects
Blood Cells, Bone Regeneration, Biocompatible Materials, Prostheses and Implants, Hematopoietic Stem Cells, Rats, Mice, Inbred C57BL, Mice, Bone Substitutes, Granulocyte Colony-Stimulating Factor, Materials Testing, Animals, Humans, Hydroxyapatites, [SDV.BDD]Life Sciences [q-bio]/Development Biology, Granulocytes
Abstract

International audience; We previously reported that blood clot combined with biphasic calcium phosphate microparticles constitute a biomaterial (BRB) that can repair a bone critical defect in rat and induces subcutaneous bone formation in mice. The granulocyte colony-stimulating factor (G-CSF) is the agent most commonly used in human to enrich blood with hematopoietic stem and progenitor cells (HSPCs) as well as granulocytes (GCs). Moreover, recent data also suggest that it can mobilize mesenchymal stem cells (MSCs). Here, we asked whether the osteoinductive properties of the BRB could be further enhanced by G-CSF, either by replacing normal blood by G-CSF-mobilized blood (BRBe) or by treating the recipient animals with G-CSF. The experiments performed in C57BL/6 mice showed that G-CSF induces a marked increase of circulating HPCs and GCs, but not of MSCs. BRBe prepared with G-CSF-enriched blood induced a slight but significant decrease of subcutaneous bone formation compared to BRB prepared with normal blood. Additional injection of G-CSF to the recipient mice had no significant effect on the bone formation induced by BRB or BRBe. Altogether these results indicate that, in this model of ectopic implantation, cell mobilization induced by G-CSF has a negative effect on the osteoinductive property of this blood/BCP composite.

Published
2015

5. Preventive cancer stem cell-based vaccination reduces liver metastasis development in a rat colon carcinoma syngeneic model

Author

Patrick Baqué, Michel Samson, Maria Pedroso de Lima, Valérie Pierrefite-Carle, Vincent Casanova, Georges F. Carle, Agnès Loubat, Sónia Duarte, Jean-Marie Guigonis, Marie-Christine Saint-Paul, Pascal Staccini, David Momier, Mécanismes biologiques des Altérations du Tissu Osseux (MATOs), UMR E4320 (TIRO-MATOs), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Department of Life Sciences, University of Coimbra [Portugal] (UC), Université Nice Sophia Antipolis - Faculté de Médecine (UNS UFR Médecine), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA), Transporteurs et Imagerie, Radiothérapie en Oncologie et Mécanismes biologiques des Altérations du Tissu Osseux (TIRO-MATOs - UMR E4320), Service Hospitalier Frédéric Joliot (SHFJ), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-UMR E4320 (TIRO-MATOs), LabSTIC Santé, COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Nice Sophia Antipolis (1965 - 2019) (UNS), Pôle Digestif, Hôpital Archet 2 [Nice] (CHU), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Service Hospitalier Frédéric Joliot (SHFJ), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS)

Subjects
Male, Colorectal cancer, Carcinogenicity Tests, Population, Aldehyde dehydrogenase, Cancer Vaccines, Aldehyde Dehydrogenase 1 Family, Metastasis, 03 medical and health sciences, 0302 clinical medicine, Cancer stem cell, Heat shock protein, Cell Line, Tumor, medicine, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, education, 030304 developmental biology, 0303 health sciences, education.field_of_study, biology, Liver Neoplasms, Retinal Dehydrogenase, Cell Biology, medicine.disease, 3. Good health, Rats, Vaccination, Disease Models, Animal, 030220 oncology & carcinogenesis, Cancer cell, Immunology, Colonic Neoplasms, biology.protein, Cancer research, Neoplastic Stem Cells, Molecular Medicine, Developmental Biology
Abstract

Cancer stem cells (CSCs) represent a minor population of self-renewing cancer cells that fuel tumor growth. As CSCs are generally spared by conventional treatments, this population is likely to be responsible for relapses that are observed in most cancers. In this work, we analyzed the preventive efficiency of a CSC-based vaccine on the development of liver metastasis from colon cancer in a syngeneic rat model. We isolated a CSC-enriched population from the rat PROb colon carcinoma cell line on the basis of the expression of the aldehyde dehydrogenase-1 (ALDH1) marker. Comparative analysis of vaccines containing lysates of PROb or ALDHhigh cells by mass spectrometry identifies four proteins specifically expressed in the CSC subpopulation. The expression of two of them (heat shock protein 27-kDa and aldose reductase) is already known to be associated with treatment resistance and poor prognosis in colon cancer. Preventive intraperitoneal administration of vaccines was then performed before the intrahepatic injection of PROb cancer cells. While no significant difference in tumor occurrence was observed between control and PROb-vaccinated groups, 50% of the CSC-based vaccinated animals became resistant to tumor development. In addition, CSC-based vaccination induced a 99.5% reduction in tumor volume compared to the control group. To our knowledge, this study constitutes the first work analyzing the potential of a CSC-based vaccination to prevent liver metastasis development. Our data demonstrate that a CSC-based vaccine reduces efficiently both tumor volume and occurrence in a rat colon carcinoma syngeneic model.

Published
2013
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6. Isolation of head and neck squamous carcinoma cancer stem-like cells in a syngeneic mouse model and analysis of hypoxia effect

Author

Henrique Faneca, Maria C. Pedroso de Lima, Valérie Pierrefite-Carle, Georges F. Carle, Sónia Duarte, Agnès Loubat, David Momier, Majlinda Topi, Mécanismes biologiques des Altérations du Tissu Osseux (MATOs), UMR E4320 (TIRO-MATOs), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Department of Life Sciences, University of Coimbra [Portugal] (UC), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), Programme EGIDE / PESSOA, Université Nice Sophia Antipolis (1965 - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (1965 - 2019) (UNS)

Subjects
Cancer Research, Pathology, MESH: Tumor Burden, Cell, MESH: Cell Hypoxia, Gene Expression, MESH: Spheroids, Cellular, MESH: Flow Cytometry, Mice, 0302 clinical medicine, MESH: Animals, Mice, Inbred C3H, 0303 health sciences, education.field_of_study, MESH: Carcinoma, Squamous Cell, General Medicine, Cell cycle, Flow Cytometry, Cell Hypoxia, Tumor Burden, 3. Good health, Isoenzymes, medicine.anatomical_structure, Oncology, Head and Neck Neoplasms, 030220 oncology & carcinogenesis, Carcinoma, Squamous Cell, Neoplastic Stem Cells, MESH: Isoenzymes, Female, MESH: Transplantation, Isogeneic, medicine.medical_specialty, MESH: Cell Line, Tumor, MESH: Gene Expression, Population, MESH: Retinal Dehydrogenase, Biology, Aldehyde Dehydrogenase 1 Family, 03 medical and health sciences, Cancer stem cell, Cell Line, Tumor, Spheroids, Cellular, MESH: Cell Proliferation, medicine, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, education, MESH: Mice, Inbred C3H, MESH: Mice, Cell Proliferation, 030304 developmental biology, Cell growth, Retinal Dehydrogenase, medicine.disease, Head and neck squamous-cell carcinoma, MESH: Neoplastic Stem Cells, Squamous carcinoma, MESH: Head and Neck Neoplasms, Transplantation, Isogeneic, Cell culture, MESH: Female, Neoplasm Transplantation, MESH: Neoplasm Transplantation
Abstract

The incidence of oral tumors is increasing around the world and despite recent advances in early detection and diagnosis, current treatments are still unsatisfactory. Recent data suggest that tumor persistence and recurrence could be due to the presence of a rare cell population called cancer stem cells (CSCs), which are generally spared by traditional treatments. Therefore, identification and characterization of CSCs are extremely important to develop novel and effective treatment strategies for cancer. The aim of this study was to identify and isolate CSCs in an established murine head and neck squamous cell carcinoma (HNSCC) cell line and to investigate the influence of hypoxic conditions on the isolated cell popul-ation. Using the expression of the aldehyde dehydrogenase 1 (ALDH1) enzymatic activity, which is now recognized as a CSC marker in various tumors, we isolated a cell population expressing high levels of ALDH1 (ALDH1high) representing 1±0.6% in the murine SCC-VII cell line. These cells were injected subcutaneously in syngeneic animals to evaluate their tumorigenic properties. For the lowest injected cell dose (250 injected cells), tumor occurrence and median tumor size were higher in ALDH1high injected mice than in ALDH1low injected mice. Following an in vivo passage and culture in serum-free medium, the percentage of ALDH1high cells increased by 3‑fold in SCC-VII CSCs (oral spheres) compared to the SCC-VII cell line. This percentage was further increased when oral spheres were cultured under hypoxic conditions. In conclusion, this study reports for the first time the isolation of HNSCC CSCs in a syngeneic mouse model and the use of hypoxia as a method to further enrich the ALDH1high cell population.

Published
2012
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7. Monocytes differentiation upon treatment with a peptide corresponding to the C-terminus of activated T cell-expressed Tirc7 protein

Author

Danielle Quincey, Heidy Schmid-Antomarchi, David Momier, Guillaume E. Beranger, Caroline C. Mouline, Georges F. Carle, Jean-Claude Scimeca, Florian Boukhechba, Nathalie Rochet, Génétique, physiopathologie et ingénierie du tissu osseux (GéPITOS), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Valrose (IBV), COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Université Côte d'Azur (UCA), Graftys SARL, COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Régulations des réactions immunitaires et inflammatoires, COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Nice Sophia-Antipolis (UNSA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Mécanismes biologiques des Altérations du Tissu Osseux (MATOs), UMR E4320 (TIRO-MATOs), Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), and COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA)

Subjects
musculoskeletal diseases, MESH: Cell Differentiation, Vacuolar Proton-Translocating ATPases, Physiology, T cell, [SDV]Life Sciences [q-bio], Clinical Biochemistry, MESH: Vacuolar Proton-Translocating ATPases, Osteoclasts, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, MESH: Monocytes, Monocytes, Mice, 03 medical and health sciences, Paracrine signalling, 0302 clinical medicine, Bone Marrow, Osteoclast, MESH: Osteoclasts, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], medicine, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Animals, Autocrine signalling, [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, MESH: Mice, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, MESH: Peptides, Cell Differentiation, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, Dendritic cell, Molecular biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], medicine.anatomical_structure, [SDV.MHEP.RSOA]Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, RANKL, Cell culture, biology.protein, MESH: Bone Marrow, Bone marrow, Peptides, 030215 immunology
Abstract

International audience; 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. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

Published
2012
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8. Tumor microenvironment modifications induced by soluble VEGF receptor expression in a rat liver metastasis model

Author

Adolfo Gavelli, Samuel Bertin, Georges F. Carle, Valérie Pierrefite-Carle, Tala Mohsen-Kanson, Fabienne Anjuère, Patrick Baqué, David Momier, Institut de signalisation, biologie du développement et cancer (ISBDC), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), Génétique, physiopathologie et ingénierie du tissu osseux (GéPITOS), Biologie et physiopathologie cutanées : expression génique, signalisation et thérapie, Université Nice Sophia Antipolis (... - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Male, Cancer Research, medicine.medical_specialty, Cell Survival, medicine.medical_treatment, Dendritic cell differentiation, Biology, Transfection, Umbilical vein, Metastasis, 03 medical and health sciences, chemistry.chemical_compound, Mice, 0302 clinical medicine, Internal medicine, Cell Line, Tumor, medicine, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Myeloid Cells, 030304 developmental biology, 0303 health sciences, Tumor microenvironment, Vascular Endothelial Growth Factor Receptor-1, Neovascularization, Pathologic, Growth factor, Liver Neoplasms, Endothelial Cells, Rats, Inbred Strains, Dendritic cell, medicine.disease, Rats, Tumor Burden, Vascular endothelial growth factor, Gene Expression Regulation, Neoplastic, Disease Models, Animal, Endocrinology, Oncology, chemistry, 030220 oncology & carcinogenesis, Culture Media, Conditioned, embryonic structures, Colonic Neoplasms, Cancer research, Myeloid-derived Suppressor Cell, Cytokines, Chemokines
Abstract

International audience; Vascular endothelial growth factor is a potent pro-angiogenic growth factor which is also known to alter tumor microenvironment by inhibiting dendritic cell differentiation and promoting accumulation of myeloid-derived suppressor cells. In the present study, we analyzed the modifications induced by intratumoral expression of sFLT-1, a soluble VEGF receptor, in a rat metastatic colon carcinoma model. We generated colon cancer cell lines stably expressing sFLT-1 or a mock construct. Human umbilical vein endothelial cells cultured with conditioned medium from sFLT-1-expressing tumor cells exhibit a significantly decreased survival, demonstrating the functionality of the secreted sFLT-1. Invivo, sFLT-1 expression induced a 30% decrease in microvessel density in 15-day old experimental liver metastasis from colon carcinoma. Tumor growth was inhibited by 63% and 52% in left and right liver lobes respectively within 25days. In these tumors, sFLT-1 expression was associated with a decreased myeloid cell infiltration and a modification in the expression of several cytokines/chemokines. Altogether, these results suggest that VEGF trapping by sFLT-1 intratumoral expression results in reduced vascularization, tumor growth inhibition and modification of immune tumor microenvironment.

Published
2010
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9. Poly(adp-ribose) polymerase-1 regulates Tracp gene promoter activity during RANKL-induced osteoclastogenesis

Author

Jean-Claude Scimeca, Nathalie Rochet, Georges F. Carle, Guillaume E. Beranger, David Momier, Génétique, physiopathologie et ingénierie du tissu osseux (GéPITOS), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Génétique et signalisation moléculaire (GSM), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre Hospitalier Universitaire de Nice (CHU Nice)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), Institut de Biologie Valrose (IBV), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), SCIMECA, Jean-Claude, and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA)

Subjects
Transcription, Genetic, Endocrinology, Diabetes and Metabolism, Osteoclasts, Electrophoretic Mobility Shift Assay, [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Mice, 0302 clinical medicine, MESH: Osteoclasts, Gene expression, MESH: RNA, Small Interfering, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Orthopedics and Sports Medicine, MESH: Animals, RNA, Small Interfering, Promoter Regions, Genetic, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Tartrate-resistant acid phosphatase, Regulation of gene expression, 0303 health sciences, [SDV.MHEP.RSOA] Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, Mice, Inbred C3H, biology, MESH: Gene Expression Regulation, Enzymologic, Transfection, MESH: RANK Ligand, Isoenzymes, [SDV.MHEP.RSOA]Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, RANKL, 030220 oncology & carcinogenesis, MESH: Isoenzymes, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Poly(ADP-ribose) Polymerases, musculoskeletal diseases, Acid Phosphatase, [SDV.CAN]Life Sciences [q-bio]/Cancer, Poly(ADP-ribose) Polymerase Inhibitors, Gene Expression Regulation, Enzymologic, Cell Line, MESH: Poly(ADP-ribose) Polymerase Inhibitors, 03 medical and health sciences, MESH: Acid Phosphatase, [SDV.CAN] Life Sciences [q-bio]/Cancer, MESH: Mice, Inbred C57BL, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], MESH: Promoter Regions, Genetic, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, RNA, Messenger, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH: Tartrate-Resistant Acid Phosphatase, MESH: Mice, Inbred C3H, [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, Gene, MESH: Mice, 030304 developmental biology, MESH: RNA, Messenger, Messenger RNA, Tartrate-Resistant Acid Phosphatase, MESH: Transcription, Genetic, MESH: Poly(ADP-ribose) Polymerases, RANK Ligand, Promoter, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Molecular biology, MESH: Cell Line, [SDV.IB.BIO] Life Sciences [q-bio]/Bioengineering/Biomaterials, Mice, Inbred C57BL, MESH: Electrophoretic Mobility Shift Assay, biology.protein
Abstract

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.

Published
2007
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10. RANKL Treatment Releases the Negative Regulation of the Poly(ADP-Ribose) Polymerase-1 on Tcirg1 Gene Expression During Osteoclastogenesis

Author

Michel Samson, Guillaume E. Beranger, Georges F. Carle, David Momier, Nathalie Rochet, Jean-Marie Guigonis, Danielle Quincey, Jean-Claude Scimeca, Génétique, physiopathologie et ingénierie du tissu osseux (GéPITOS), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Génétique et signalisation moléculaire (GSM), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre Hospitalier Universitaire de Nice (CHU Nice)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Signalisation et Réponses aux Agents Infectieux et Chimiques (SeRAIC), Université de Rennes (UR), Institut de Biologie Valrose (IBV), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), SCIMECA, Jean-Claude, Immunite anti-tumorale et chimiotactisme. Adenocarcinomes et métastases, COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Carle, Georges, Université Nice Sophia Antipolis (... - 2019) (UNS), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR50-Université Nice Sophia Antipolis (... - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)

Subjects
Transcription, Genetic, Endocrinology, Diabetes and Metabolism, Osteoclasts, MESH: Amino Acid Sequence, [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], MESH: Base Sequence, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Mice, 0302 clinical medicine, Transcription (biology), MESH: Osteoclasts, Gene expression, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Orthopedics and Sports Medicine, MESH: Animals, Nuclear protein, Promoter Regions, Genetic, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], 0303 health sciences, [SDV.MHEP.RSOA] Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, General transcription factor, Transfection, MESH: RANK Ligand, MESH: Gene Expression Regulation, [SDV.MHEP.RSOA]Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, RANKL, 030220 oncology & carcinogenesis, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Poly(ADP-ribose) Polymerases, Gene isoform, musculoskeletal diseases, MESH: Cell Nucleus, Vacuolar Proton-Translocating ATPases, Molecular Sequence Data, MESH: Vacuolar Proton-Translocating ATPases, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, Cell Line, 03 medical and health sciences, [SDV.CAN] Life Sciences [q-bio]/Cancer, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], MESH: Promoter Regions, Genetic, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, RNA, Messenger, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, MESH: Mice, 030304 developmental biology, MESH: RNA, Messenger, Cell Nucleus, MESH: Molecular Sequence Data, Base Sequence, MESH: Transcription, Genetic, RANK Ligand, MESH: Poly(ADP-ribose) Polymerases, Promoter, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Molecular biology, MESH: Cell Line, [SDV.IB.BIO] Life Sciences [q-bio]/Bioengineering/Biomaterials, Gene Expression Regulation, biology.protein
Abstract

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 ∼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.

Published
2006
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