Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.

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 crosslinking, 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.

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.

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.

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 ALDH high 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 CSCbased vaccine reduces efficiently both tumor volume and occurrence in a rat colon carcinoma syngeneic model.

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 preosteoclastic 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.

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.

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.