[Show abstract][Hide abstract] ABSTRACT: 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. Increase 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 NFΚB1 (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 extends from corals to vertebrates and uncovers new therapeutics targets for calcified tissue related metabolic pathologies.
[Show abstract][Hide abstract] 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 the present 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 (HSP27 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 intra-hepatic 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.
[Show abstract][Hide abstract] ABSTRACT: We report here for the first time the isolation and characterization of a protein from the organic matrix (OM) of the sclerites
of the alcyonarian, Corallium rubrum. This protein named scleritin is one of the predominant proteins extracted from the EDTA-soluble fraction of the OM. The
entire open reading frame (ORF) was obtained by comparing amino acid sequences from de novo mass spectrometry and Edman degradation with an expressed sequence tag library dataset of C. rubrum. Scleritin is a secreted basic phosphorylated protein which exhibits a short amino acid sequence of 135 amino acids and a
signal peptide of 20 amino acids. From specific antibodies raised against peptide sequences of scleritin, we obtained immunolabeling
of scleroblasts and OM of the sclerites which provides information on the biomineralization pathway in C. rubrum.
Full-text · Article · May 2012 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: We report here for the first time the isolation and characterization of a protein from the organic matrix (OM) of the sclerites of the alcyonarian, Corallium rubrum. This protein named scleritin is one of the predominant proteins extracted from the EDTA-soluble fraction of the OM. The entire open reading frame (ORF) was obtained by comparing amino acid sequences from de novo mass spectrometry and Edman degradation with an expressed sequence tag library dataset of C. rubrum. Scleritin is a secreted basic phosphorylated protein which exhibits a short amino acid sequence of 135 amino acids and a signal peptide of 20 amino acids. From specific antibodies raised against peptide sequences of scleritin, we obtained immunolabeling of scleroblasts and OM of the sclerites which provides information on the biomineralization pathway in C. rubrum.
[Show abstract][Hide abstract] ABSTRACT: Formation and stabilization of postsynaptic glycine receptor (GlyR) clusters result from their association with the polymerized scaffold protein gephyrin. At the cell surface, lateral diffusion and local trapping of GlyR by synaptic gephyrin clusters is one of the main factors controlling their number. However, the mechanisms regulating gephyrin/GlyR cluster sizes are not fully understood. To identify molecular binding partners able to control gephyrin cluster stability, we performed pull-down assays with full-length or truncated gephyrin forms incubated in a rat spinal cord extract, combined with mass spectrometric analysis. We found that heat shock cognate protein 70 (Hsc70), a constitutive member of the heat shock protein 70 (Hsp70) family, selectively binds to the gephyrin G-domain. Immunoelectron microscopy of mouse spinal cord sections showed that Hsc70 could be colocalized with gephyrin at inhibitory synapses. Furthermore, ternary Hsc70-gephyrin-GlyR coclusters were formed following transfection of COS-7 cells. Upon overexpression of Hsc70 in mouse spinal cord neurons, synaptic accumulation of gephyrin was significantly decreased, but GlyR amounts were unaffected. In the same way, Hsc70 inhibition increased gephyrin accumulation at inhibitory synapses without modifying GlyR clustering. Single particle tracking experiments revealed that the increase of gephyrin molecules reduced GlyR diffusion rates without altering GlyR residency at synapses. Our findings demonstrate that Hsc70 regulates gephyrin polymerization independently of its interaction with GlyR. Therefore, gephyrin polymerization and synaptic clustering of GlyR are uncoupled events.
Full-text · Article · Jan 2011 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Cadmium, an environmental stressor due to its toxicity, persistence and accumulation in biota, is widespread in the aquatic environment. Cadmium accumulation kinetics have revealed that Ruditapes decussatus has a high affinity to this metal. Proteomics is an effective tool to evaluate the toxic effects of contaminants. The aim of this study was to investigate the Cd effects in the gill and digestive gland of the sentinel species R. decussatus. Protein expression profiles (PEPs) in the clam tissues exposed to Cd (40 microg l(-1), 21 days) were compared to unexposed ones. Cd induces major changes in tissue-specific protein expression profiles in gill and digestive gland. This tissue dependent response results mainly from differences in Cd accumulation, protein inhibition and/or autophagy. An overall decrease of protein spots was detected in both treated tissues, being higher in gill. Some of the spots more drastically altered after pollutants exposure were excised and nine were identified by micro liquid chromatography tandem mass spectrometry (LC-MS/MS). Proteins identified by homology search in databases included: three proteins (8-fold) up-regulated, one down-regulated, four suppressed and one induced. Cd induces major changes in proteins involved in cytoskeletal structure maintenance (muscle-type actin, adductor muscle actin and beta-tubulin), cell maintenance (Rab GDP) and metabolism (ALDH and MCAD, both identified by de novo sequencing) suggesting potential energetic change. They provide a valuable knowledge of Cd effects at biochemical and molecular levels in the gill and digestive gland of R. decussatus.
No preview · Article · Aug 2009 · Aquatic toxicology (Amsterdam, Netherlands)
[Show abstract][Hide abstract] ABSTRACT: Unmethylated CpG dinucleotides, present in bacterial DNA, are recognized in vertebrates via the Toll-like receptor 9 (TLR9) and are known to act as an anticancer agent by stimulating immune cells to induce a proinflammatory response. Although the effects of CpG-oligodeoxynucleotides (CpG-ODNs) in immune cells have been widely studied, little is known regarding their molecular effects in TLR9-positive tumor cells. To better understand the role of these bacterial motifs in cancer cells, we analyzed proteome modifications induced in TLR9-positive tumor cells in vitro and in vivo after CpG-ODN treatment in a rat colon carcinoma model. Proteomics analysis of tumor cells by two-dimensional gel electrophoresis followed by mass spectrometry identified several proteins modulated by bacterial CpG motifs. Among them, several are related to autophagy including potential autophagic substrates. In addition, we observed an increased glyceraldehyde-3-phosphate dehydrogenase expression, which has been shown to be sufficient to trigger an autophagic process. Autophagy is a self-digestion pathway whereby cytoplasmic material is sequestered by a structure termed the autophagosome for subsequent degradation and recycling. As bacteria are known to trigger autophagy, we assessed whether bacterial CpG motifs might induce autophagy in TLR9-positive tumor cells. We showed that CpG-ODN can induce autophagy in rodent and human tumor cell lines and was TLR9-dependent. In addition, an increase in the number of autophagosomes can also be observed in vivo after CpG motif intratumoral injection. Our findings bring new insights on the effect of bacterial CpG motifs in tumor cells and may be relevant for cancer treatment and more generally for gene therapy approaches in TLR9-positive tissues.
[Show abstract][Hide abstract] ABSTRACT: Two isoforms of human cytoplasmic isocitrate dehydrogenase (IDPc) of close molecular weights and different isoelectric points were identified in human seminal plasma (SP) by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS). These two isoforms were detected in the normospermic men SP and their expressions were markedly altered in patients with testicular seminoma, the most frequent testicular germ cell cancer (TGCC): increase of the more acidic spot and decrease of the more basic one. Since oligospermia has been considered as a high risk pathological condition for developing a testicular cancer, the two IDPc isoforms were analyzed in SP of a group of secretory azoospermic patients. In this group the two spots displayed similar variations of expression to those observed in testicular seminoma. These results propose IDPc as a promising SP biomarker of testicular seminoma. Whether IDPc alteration in secretory azoospermia is predictive of testicular seminoma remains to be elucidated.
No preview · Article · Jun 2008 · Molecular Carcinogenesis
[Show abstract][Hide abstract] ABSTRACT: It is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. Among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. As secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell (hMADS) at an early step of commitment to adipocytes and osteoblasts.
A proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 73 proteins at day 0 and day 3 of adipocyte and osteoblast differentiation. Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly. These proteins were classified into 8 clusters according to their function. Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2. Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome. Furthermore we showed that PAI-1 mRNA was down-regulated in the bone of ovariectomized mice.
Given its regulation during the early events of hMADS cell differentiation and its status in ovariectomized mice, PAI-1 could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis.
Full-text · Article · Feb 2008 · BMC Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: The bacterial cytosine deaminase (CD) gene, associated with the 5-fluorocytosine (5FC) prodrug, is one of the most widely used suicide systems in gene therapy. Introduction of the CD gene within a tumor induces, after 5FC treatment of the animal, a local production of 5-fluorouracil resulting in intratumor chemotherapy. Destruction of the gene-modified tumor is then followed by the triggering of an antitumor immune reaction resulting in the regression of distant wild-type metastasis. The global effects of 5FC on colorectal adenocarcinoma cells expressing the CD gene were analyzed using the proteomic method. Application of 5FC induced apoptosis and 19 proteins showed a significant change in 5FC-treated cells compared with control cells. The up-regulated and down-regulated proteins include cytoskeletal proteins, chaperones, and proteins involved in protein synthesis, the antioxidative network, and detoxification. Most of these proteins are involved in resistance to anticancer drugs and resistance to apoptosis. In addition, we show that the heat shock protein Hsp90beta is phosphorylated on serine 254 upon 5FC treatment. Our results suggest that activation of Hsp90beta by phosphorylation might contribute to tumor regression and tumor immunogenicity. Our findings bring new insights into the mechanism of the anticancer effects induced by CD/5FC treatment.
Preview · Article · Nov 2007 · Molecular Cancer Therapeutics
[Show abstract][Hide abstract] 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.
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.
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.
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.
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.
Full-text · Article · Aug 2007 · Journal of Bone and Mineral Research
[Show abstract][Hide abstract] ABSTRACT: A proteomic approach offers a powerful and complementary tool to genomics. It allows to index and characterize proteins, and, for example, to compare their levels of expression between healthy and pathological states. Proteomic analyses are mainly based on the separation of proteins by two-dimensional gel electrophoresis and their subsequent identification by comparing the data from Mass Spectrometry (SM) analyses to the theoretical ones contained in databases. In mass spectrometry, the detector noise, the electronic and chemical noise, sometimes the small amount of peptides that has to be treated and finally the spectrum reduction noise (due to bad filtering and/or thresholding), can induce Parasitic Mass Peaks (PMP) and/or hide some Useful Mass Peaks (UMP) of low intensities. The immediate consequence is that the presence of the PMP and the absence of the UMP will be detrimental to the protein identification quality. In this article, we propose an original algorithm eliminating the PMP, detecting and amplifying those which are useful. The preprocessing principle uses a multi-scale analysis technique coupled to a fuzzy thresholding (multi-scale fuzzy thresholding), a local amplification of the UMP, and finally an adaptive Base Line Correction. The associated frequencies with the PMP are distributed on all the spectrum pass bandwidth. This leads us to a dyadic tree structure subband decomposition. The algorithm principle consists of dividing the frequential pass bandwidth of each masses spectrum into two subbands, a Low and High Frequency (LFHF) subband, then each subband is in turn divided into two subbands etc. The HF subbands are then thresholded according to the minimization criterion of the Shannon fuzzy entropy, and then amplified locally; the base line is calculated in an adaptive way and subtracted from reconstructed spectrum. To evaluate the quality of this algorithm, we present a comparison of the results obtained by our algorithm, and those obtained by the DataExplorer software. The latter is a reduction software provided within the MALDI-TOF spectrometer software package.
No preview · Article · Jan 2007 · Traitement du Signal
[Show abstract][Hide abstract] 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.
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.
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.
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.
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.
Full-text · Article · Dec 2006 · Journal of Bone and Mineral Research
[Show abstract][Hide abstract] ABSTRACT: To better understand the effects of antiandrogens on the prostate, we investigated the changes in the proteome of rat ventral prostate (VP) following treatment with a well characterized 5alpha-reductase inhibitor, finasteride. Sprague-Dawley rats were treated daily by gavage with finasteride at 0, 1, 5, 25, and 125 mg/kg/day. Changes in plasma hormone levels as well as the weight and histology of sex accessory tissues were determined after 28 days of treatment and showed a dose-related decrease of VP weights together with a marked atrophy of the tissue visible at the macroscopic and microscopic levels. In addition, significant reductions in seminal vesicle and epididymis weights were noted. VP proteins were analyzed by two-dimensional gel electrophoresis: 37 proteins, mainly involved in protein synthesis, processing, and cellular trafficking and in metabolism, detoxification, and oxidative stress, were identified as modulated by finasteride. The prominent feature of this study is the demonstration of finasteride dose-dependent up-regulation of a protein similar to l-amino-acid oxidase 1 (Lao1). An up-regulation of this protein was also observed with the antiandrogen flutamide. Lao1 expression occurred as early as 48 h after antiandrogen administration and persisted throughout the treatment duration. Immunohistochemistry showed that this protein was only detectable in epithelial cells and secretory vesicles. Altogether these data point to a potential use of Lao1 to reveal antiandrogen-induced prostate injury.
[Show abstract][Hide abstract] ABSTRACT: In the analysis of proteomic image (2D Electrophoresis-Gels image), it is necessary to separately detect the proteinic spots before the comparative analysis of the images (matching). The matching is the operation which makes it possible to locate the proteins which, either single, or are modified between an image corresponding to a biological or organic treated sample and an another obtained from a reference sample. The 2DE-G image processing is a crucial stage because it allows to localize pertinently (single, or modified) protein spots candidate for a peptidic identification by mass spectrometry. In the present study, we present a segmentation technique based on two processes, image preprocessing process and spots detection process. The aim of the first is to preprocess the image in order to improve its quatity in term of signal to noise Ratio. It uses the sub-band technique combined with Retinex Method. The protein spots detection algorithm operates by finding the threshold gray level that minimizes the entropy of the fuzziness measure, then uses the watershed transformation.
[Show abstract][Hide abstract] ABSTRACT: Proteomic analysis is done primarily by the use of the two-dimensional electrophoreses (2-DE) technique coupled with the Mass Spectrometry (MS) analysis. The first technique helped by the proteomic imaging leads to the localization of the candidates proteins for mass spectrometry analysis. The comparison between the spectra of masses obtained and those theoretical of DataBase leads to the identification of proteins of interest in term of peptides or amino acids. The presence of parasitic and/or the absence of useful mass peaks distort(s) the result of the identification process. In this article, we propose an original data reduction algorithm with the aim of removing the spectra baseline, then removing parasitic mass peaks and amplifying those useful. The algorithm principle uses the dyadic muli-resolution technique (bio-orthogonal decomposition/reconstruction) coupled to the fuzzy logic thresholding. In order to evaluate the quality of this algorithm, we present a comparison of the results obtained by our algorithm and those obtained using the data reduction software of MALDI-TOF spectrometer (Matrix-Assisted Laser Desorption/ionization).
[Show abstract][Hide abstract] ABSTRACT: The proteomic analysis is a set of process involving modern analytical techniques: -Realization of 2D Electrophoresis-Gels (2DE-G). - Processing of 2DE-G image and location of new or modified proteins. - Peptidic identification by mass spectrometry. - Location in data bases of proteins whose peptidic card correlates with respect to that found. The 2DE-G image processing is a crucial stage because it allows to localize pertinently the protein spots candidate for a peptidic identification by mass spectrometry. One of the major axes of this stage turns around the protein-spots detection. The reliability of this stage depends among others on the quality of the detection tools. In this paper we present a segmentation technique combining both the Top-Hat transformation and the watershed. The segmentation process is an iterative process leading to a simple and effective detection of protein spots. Before this segmentation process, image must be preprocessed in order to improve the image quality in term of signal to noise ratio. Image preprocessing algorithm uses the sub-band technique combined with Retinex Method. The detection algorithm operates by finding the threshold gray level that minimizes the entropy of the fuzziness measure. Key-Words: 2D Electrophoresis-Gels (2DE-G). Protein spots. Detection. Top-Hat Transformation. Watershed Transformation. Sub-band technique. Retinex Method. Entropy of the fuzziness measure.