Biomedical subjects

Jean-Claude Scimeca

Publications and source records attributed to Jean-Claude Scimeca.

4 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2004

Molecular cloning and localization of a PMCA P-type calcium ATPase from the coral Stylophora pistillata.

Plasma-membrane calcium pumps (PMCAs) are responsible for the expulsion of Ca(2+) from the cytosol of all eukaryotic cells and are one of the major transport systems involved in long-term regulation of resting intracellular Ca(2+) concentration. An important feature of stony corals, one of the major groups of calcifying animals, is the continuous export of large quantities of Ca(2+) for skeletogenesis. Here, we report the cloning and functional expression of the stpPMCA gene from the coral Stylophora pistillata, and whose features resemble those of the plasma-membrane Ca(2+)-ATPase family of mammalian cells. This is the first known example of a Ca(2+)-ATPase from the phylum Cnidaria, and thus, the most phylogenetically distant PMCA sequence in the animal kingdom described to date. We demonstrate that the localization of stpPMCA within calicoblastic cells is fully coherent with its role in calcification. We also show that the coral Ca(2+) pump is more closely related to vertebrate PMCAs than to Caenorhabditis elegans PMCAs. The cloning of evolutionarily conserved genes from cnidarian species repeatedly shows that these genes encode similar functional domains. Moreover, this high level of gene conservation further validates the use of cnidarian model systems for studying processes shared by Eumetazoans.

Amino Acid Sequence↗

PubMed2003

Novel mutations in the TCIRG1 gene encoding the a3 subunit of the vacuolar proton pump in patients affected by infantile malignant osteopetrosis.

Fifty percent of the infantile malignant osteopetrosis (IMO) cases reported in the literature present mutations in the TCIRG1 gene encoding the 116-kDa osteoclast specific subunit of the vacuolar proton ATPase (ATP6I). In this study, we identified four novel mutations in a series of six IMO patients. All of these mutations correspond to single nucleotide changes and affect splice acceptor or donor sites, resulting in aberrant transcription products. We report also a missense mutation, G405R, previously described in several Costa Rican patients. This independent finding suggests that the highly conserved residue at amino acid 405 plays a critical role in the a3 subunit function. Finally, the results of this study were used to provide a prenatal diagnosis to one of the families.

Chromosomes, Human, Pair 11↗