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Collagen, the most abundant organic compound of vertebrate organisms, is a supramolecular, protein-made polymer. Details of its post-translational maturation largely determine the mechanical properties of connective tissues. Its assembly requires massive, heterogeneous prolyl-4-hydroxylation (P4H), catalyzed by Prolyl-4-hydroxylases (P4HA1-3), providing thermostability to its elemental, triple helical building block. So far, there was no evidence of tissue-specific regulation of P4H, nor of a differential substrate repertoire of P4HAs. Here, the post-translational modifications of collagen extracted from bone, skin, and tendon were compared, revealing lower hydroxylation of most GEP/GDP triplets, together with fewer other residue positions along collagen α chains, in the tendon. This regulation is mostly conserved in two distant homeotherm species, mouse and chicken. The comparison of detailed P4H patterns in both species suggests a dual mechanism of specificity.P4ha2expression is low in tendon and its genetic invalidation in the ATDC5 cellular model of collagen assembly specifically mimics the tendon-related P4H profile. Therefore, P4HA2 has a better ability than other P4HAs to hydroxylate the corresponding residue positions. Its local expression participates in determining the P4H profile, a novel aspect of the tissue specificities of collagen assembly.Data availabilityProteomics data are availableviaProteomeXchange with the identifier PXD039221. Reviewer account details:login: MSV000091002_reviewerpassword: P4HA2tendon.

Supplementary Table 1 from C-Propeptides of Procollagens Iα1 and II that Differentially Accumulate in Enchondromas versus Chondrosarcomas Regulate Tumor Cell Survival and Migration

Supplementary Figure 2 from C-Propeptides of Procollagens Iα1 and II that Differentially Accumulate in Enchondromas versus Chondrosarcomas Regulate Tumor Cell Survival and Migration

Supplementary Figure and Table Legend from C-Propeptides of Procollagens Iα1 and II that Differentially Accumulate in Enchondromas versus Chondrosarcomas Regulate Tumor Cell Survival and Migration

Chondrogenic tumors that exhibit benign or malignant behaviors synthesize variable amounts of cartilage-like extracellular matrix. To define the regulators of these phenotypes, we performed a proteomic comparison of multiple human chondrogenic tumors, which revealed differential accumulation of the C-propeptides of procollagens Iα1 and II (PC1CP and PC2CP) in malignant versus benign tumors, respectively. Expression patterns of PC1CP correlated with levels of tumor vascularization, whereas expression patterns of PC2CP suggested its susceptibility to immobilization within the extracellular matrix. Prompted by these observations, we investigated the functions of recombinant PC1CP and PC2CP in the extracellular matrix in soluble or immobilized states. Each induced β1 integrin–mediated chondrocyte adhesion by distinct domains and efficacies, suggesting that they initiated distinct signaling pathways. Indeed, immobilized PC2CP, but not PC1CP, induced apoptosis of primary chondrocytes and EAhy926 endothelial cells. In contrast, soluble PC1CP, but not PC2CP, induced the migration of EAhy926 cells and increased vascular endothelial growth factor (VEGF) and CXCR4 expression in chondrocytes. Soluble PC2CP also increased VEGF expression, but along with a more pronounced effect on CXCR4 and matrix metalloproteinase 13 expression. Our findings suggest that PC1CP favors angiogenesis and tumor progression, but that PC2CP acts in a more complex manner, exerting antitumor and antiangiogenic properties through apoptosis induction when immobilized, but progression and metastasis when soluble. In summary, the relative levels of PC1CP and PC2CP and their interactions within the extracellular matrix contribute to tumor progression, angiogenesis, and metastasis in chondrogenic tumors. Cancer Res; 70(11); 4739–48. ©2010 AACR.

The proteolytic conversion of soluble procollagens into mature collagen monomers is a critical step to decrease their solubility and trigger collagen fibril formation. In the case of collagens I, II and III, this maturation process is driven by several extracellular metalloproteinases such as BMP-1, tolloid-like proteinases, meprin α, meprin β, ADAMTS-2 and ADAMTS-14 but the extensive characterization of these proteolytic events has been hampered by the lack of recombinant procollagens. We previously reported the production and partial characterization of recombinant homotrimeric proteins derived from procollagen III (mini-procollagens III) and, in this study, we describe how we have extended this previous work to the production of heterotrimeric mini-procollagen I and homotrimeric mini-procollagen II. These mini-procollagens include truncated triple helices and intact C-telopeptide and C-propeptide domains and were produced in suspension in HEK293-F cells with yields ranging from 2.5 mg/L to 10 mg/L after purification. They proved very useful tools to analyze the effect of calcium on the stability of the procollagen C-terminal region and to compare the procollagen C-proteinase activity of BMP-1 on the three major fibrillar procollagens or their ability to interact with various partners such as PCPE-1. Using mass spectrometry to map BMP-1 cleavage sites on the mini-procollagens, we confirmed all previously described sites but also revealed two additional cleavage sites in the α1 chain of procollagens I and II. This result shows that the mini-procollagen toolkit offers a broad range of perspectives to make functional studies but also possibly structural analyses or to develop drug screening assays.

Here we propose a general strategy to label carbohydrates with N-methyl-anthranilic acid at the anomeric position. Through two examples, we demonstrate that the generated glycoprobes are suitable for fluorescence-based binding/competition assays. Our approach is expected to readily generate series of glycoprobes dedicated to screening assays for the discovery of drugs targeting carbohydrate–protein interactions.

Here we propose a general strategy to label carbohydrates with N-methyl-anthranilic acid at the anomeric position. Through two examples, we demonstrate that the generated glycoprobes are suitable for fluorescence-based binding/competition assays. Our approach is expected to readily generate series of glycoprobes dedicated to screening assays for the discovery of drugs targeting carbohydrate-protein interactions.

Fibrillar collagens and proteoglycans (PGs) are quantitatively the major constituents of extracellular matrices (ECM). They carry numerous crucial post-translational modifications (PTMs) that tune the resulting biomechanical properties of the corresponding tissues. The mechanisms determining these PTMs remain largely unknown, notably because available established cell lines do not recapitulate much of the complexity of the machineries involved. ATDC5 cells are a model of chondrogenesis widely used for decades, but it remains described mostly at histological and transcriptional levels. Here, we asked to what extent this model recapitulates the events of ECM synthesis and processing occurring in cartilage. Insulin-stimulated ATDC5 cells exhibit up- or down-regulation of more than one-hundred proteins, including a number of known participants in chondrogenesis and major markers thereof. However, they also lack several ECM components considered of significant, yet more subtle, function in cartilage. Still, they assemble the large PG aggrecan and type II collagen, both carrying most of their in vivo PTMs, into an ECM. Remarkably, collagen crosslinking is fully lysyl oxidase (LOX)-dependent. The ATDC5 model recapitulates critical aspects of the cartilage ECM-processing machinery and should be useful to decipher the mechanisms involved. Proteomics data are available via ProteomeXchange with identifier PXD014121. SIGNIFICANCE: The present work provides the first proteome characterization of the ATDC5 chondrogenesis model, which has been used for decades in the field of cartilage biology. The results demonstrate the up- and down-regulation of more than one hundred proteins. Overall, specific drawbacks of the model are pointed out, that will be important to take into consideration for future studies. However, major cartilage components are massively assembled into an extracellular matrix and carry most of their post-translational modifications occurring in cartilage tissue. Unlike other available established cell lines, the ATDC5 model recapitulates major aspects of cartilage biosynthesis and should be useful in investigating the mechanisms that regulate collagen maturation events.

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Cartilage tissue engineering is making progress, but the competing available strategies still leave room for improvement and consensual overviews regarding the best combinations of scaffolds and cell sources are limited by the capacity to compare them directly. In addition, because most strategies involve autologous cell transfer, once these are optimized, the resulting implants require individual quality control prior to grafting in order to emphasize patient-to-patient differential responsiveness to engineering processes. Here, cartilage substitutes prepared from human mesenchymal stem cells undergoing chondrogenic differentiation within distinct scaffolds were used as pilot samples to investigate the pertinence of a novel method with the aim of characterizing the implants. The limits and advantages of analysing, by label-free liquid chromatography-coupled matrix-assisted laser desorption and ionization (LC-MALDI) mass spectrometry, the secreted proteome released into culture medium by engineered cartilage tissues were investigated and compared with more classically used methods for biomaterial characterization. This method did not require sacrificing the biomaterials and robustly evidenced their chondrogenic statuses. In more detail, the method highlighted differences between batches prepared from distinct donors. It was adapted to distinct scaffolds and allowed a comparison of the influence of individual engineering steps, such as growth factor combinations and oxygen tension. Finally, it evidenced subtle changes between replicate substitutes within a series, thereby distinguishing the least and most accomplished ones. We conclude that relative quantification of secreted proteins through label-free LC-MALDI will be useful, not only to orientate engineering methodologies, but also to ultimately provide non-invasive quality control of engineered tissue substitutes for the repair of cartilage and possibly other connective tissues.

International audience; Few studies on oncogenesis of chondrosarcoma (CS) are available in the literature. Our previously published experimental evidence suggests that while the C-propeptide of procollagen I alpha 1 (PC1CP), a component of cartilage, favors tumor progression, the C-propeptide of procollagen II alpha 1 (PC2CP) exerts antitumor properties. In this study, we analyzed expression of PC1CP and PC2CP by immunohistochemistry in a series of enchondromas and CS. Our retrospective series consisted of 88 cases, including 43 CSs, 34 enchondromas and 11 nontumor samples. Immunohistochemical staining for PC1CP and PC2CP was evaluated in the cytoplasm and in the extracellular matrix (ECM). Diffuse staining for PC1CP in ECM was significantly more frequent in tumor than in nontumor samples (32 % vs. 0 %; p = 0.03), and in CSs than in enchondromas (44 vs. 18 %; p = 0.02). ECM semiquantitative score was higher in tumors than in nontumor samples (p < 0.005) and higher in CSs than in enchondromas (p = 0.05). Staining for PC2CP in ECM was more frequently found in enchondromas than in CSs (59 vs. 33 %; p = 0.02). ECM semiquantitative score was higher in enchondromas than in CSs (p = 0.02). Diffuse staining for PC1CP in combination with absence of staining for PC2CP had 94 % specificity for CS but with a sensitivity of only 35 %. Expression of neither PC1CP nor PC2CP correlated with recurrence-free survival or occurrence of metastases. In conclusion, we show that the expression of PC1CP is higher and that of PC2CP lower in malignant cartilaginous tumors. These results support an oncogenic role of PC1CP and anti-oncogenic property of PC2CP in cartilaginous tumors.

International audience; Proteomics users enjoy the rapid development of LC-MS-based label-free relative quantification methods but in practice these remain restricted to mass spectrometers using electrospray ionization. Here, tools dedicated to ion chromatogram extraction, time alignment, signal normalization and statistical analysis were used to interpret label-free relative difference between primary human chondrocyte secretomes and dilutions thereof, analyzed successively by LC-MALDI. The analysis of secretomes diluted into culture medium demonstrated that abundant proteins could be relatively quantified within 1.5 20-fold changes with satisfactory statistics. In addition, comparison of multiple samples requires analyzing most samples in TOP mode only, saving considerable machine-time usage. The method allowed identification and quantification of most secreted proteins relevant to the chondrocyte phenotype and evidenced their up- or down regulations by TGF131 and patient-to-patient differential expression. Novel targets of TGFB1 were evidenced, such as pro-collagen C-proteinase enhancer protein 1, Metalloproteinase inhibitor 1, Fibulin-3, Tetranectin and Cartilage Intermediate Layer Protein 1, while others match previous findings. Several were verified by Western blot. This whole workflow is non-invasive, compatible with many cell culture protocols, technically straightforward and rapid, particularly regarding mass spectrometer time usage and could make label-free LC-MALDI analysis of low-complexity proteomes a major tool for routine cell culture characterization. Biological significance The present work presents the adaptation of label free relative protein quantification principles to LC-MALDI data to rapidly measure protein fold-changes between samples of relative complexity and its utility to characterize the secreted proteome of human primary chondrocytes. The method was employed to characterize the chondrocyte secretome regulation by TGF beta 1 and is proposed as a routine tool to assess the quality of biomaterials designed for cartilage repair and to quantitatively investigate the influence of environmental factors upon it. (C) 2014 The Authors. Published by Elsevier B.V.

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International audience; BACKGROUND: Osteoarthritis (OA) is a chronic joint disease characterized by a progressive and irreversible degeneration of articular cartilage. Among the environmental risk factors of OA, tobacco consumption features prominently, although, there is a great controversy regarding the role of tobacco smoking in OA development. Among the numerous chemicals present in cigarette smoke, nicotine is one of the most physiologically active molecules.OBJECTIVE: The aim of the study was (i) to measure the impact of nicotine on the proliferation and chondrogenic differentiation of mesenchymal stem cells from the human Wharton's jelly (hWJ-MSCs) into chondrocytes, (ii) to investigate whether the alpha 7 nicotinic acetylcholine receptors (nAChRs) was expressed in hWJ-MSCs and could play a role in the process. The project benefits from the availability of an umbilical cord bank from which hWJ-MSCs were originated.METHODS: The hWJ-MSCs were cultured and used up to passage 5. The proliferation of hWJ-MSCs with 5 mu M nicotine was measured by the MTT assay on the 1st, 2nd, 3rd, and 6th day. Flow cytometry analysis was used to detect cell apoptosis/necrosis by Annexin V/PI double-staining. The chondrogenic differentiation grade of hWJ-MSCs induced by TGF beta 3 was assessed by the Sirius red and Alcian blue staining. The expression of markers genes was followed by quantitative real-time PCR. The expression of nAChRs was followed by RT-PCR. The functional activity of alpha 7 nAChR was evaluated by calcium (Ca2+) influx mediated by nicotine using the Fluo-4 NW Calcium assay.RESULTS: The proliferation of hWJ-MSCs was significantly impaired by nicotine (5 mu M) from the 3rd day of treatment, but nicotine did not significantly induce modifications on the viability of hWJ-MSCs. Alcian blue staining indicated that the amount of proteoglycan was more abundant in control group than in the nicotine group, but no difference was observed on the total collagen amount using Sirius red staining. The mRNA expression of Sox9, type II collagen (Col2a1), aggrecan in control group was higher than in the nicotine group. We found that hWJ-MSCs expressed alpha 7 nAChR. The receptor agonist nicotine caused calcium (Ca2+) influx into hWJ-MSCs suggesting that the calcium ion channel alpha 7 homopolymer could mediate this response.CONCLUSIONS: At the concentration used, nicotine had an adverse effect on the proliferation and chondrogenic differentiation of hWJ-MSCs which was probably impaired through a alpha 7 nAChR mediation.

Chondrogenic tumors that exhibit benign or malignant behaviors synthesize variable amounts of cartilage-like extracellular matrix. To define the regulators of these phenotypes, we performed a proteomic comparison of multiple human chondrogenic tumors, which revealed differential accumulation of the C-propeptides of procollagens Iα1 and II (PC1CP and PC2CP) in malignant versus benign tumors, respectively. Expression patterns of PC1CP correlated with levels of tumor vascularization, whereas expression patterns of PC2CP suggested its susceptibility to immobilization within the extracellular matrix. Prompted by these observations, we investigated the functions of recombinant PC1CP and PC2CP in the extracellular matrix in soluble or immobilized states. Each induced β1 integrin–mediated chondrocyte adhesion by distinct domains and efficacies, suggesting that they initiated distinct signaling pathways. Indeed, immobilized PC2CP, but not PC1CP, induced apoptosis of primary chondrocytes and EAhy926 endothelial cells. In contrast, soluble PC1CP, but not PC2CP, induced the migration of EAhy926 cells and increased vascular endothelial growth factor (VEGF) and CXCR4 expression in chondrocytes. Soluble PC2CP also increased VEGF expression, but along with a more pronounced effect on CXCR4 and matrix metalloproteinase 13 expression. Our findings suggest that PC1CP favors angiogenesis and tumor progression, but that PC2CP acts in a more complex manner, exerting antitumor and antiangiogenic properties through apoptosis induction when immobilized, but progression and metastasis when soluble. In summary, the relative levels of PC1CP and PC2CP and their interactions within the extracellular matrix contribute to tumor progression, angiogenesis, and metastasis in chondrogenic tumors. Cancer Res; 70(11); 4739–48. ©2010 AACR.

Objective To identify regulators of the cartilaginous phenotype, on the basis of their differential expression in human conventional chondrogenic tumors compared with articular cartilage. Methods Differential proteomics analysis revealed matrilin-3 (MATN3) as a candidate regulator of the cartilaginous phenotype. Its capacity to modulate gene expression was investigated in human HCS-2/8 chondrosarcoma cells and transfected chondrocytes, using cell culture fractionation, reverse transcription–polymerase chain reaction, and Western blot analyses. Results Increased expression of the cartilage-specific matrix protein MATN3 was specifically observed in enchondromas and conventional chondrosarcomas. A substantial fraction of MATN3 was found in cytoplasmic structures of tumor cells, as demonstrated by immunohistochemistry. Analyses of intracellular MATN3 revealed that it corresponded to an imperfectly maturated MATN3 polypeptide, both in HCS-2/8 human chondrosarcoma cells and in transfected human chondrocytes. Moderately increased expression of MATN3 resulted in its intracellular retention. Antibody-mediated blockade of soluble, extracellular MATN3 in HCS-2/8 cell cultures resulted in increased expression of MATN3 and the chondrogenic transcription factor SOX9. Conversely, increased ectopic expression of MATN3 resulted in decreased expression of MATN3 and SOX9 in primary chondrocytes, while a mutant MATN3 lacking its first epidermal growth factor (EGF)–like domain failed to down-regulate SOX9. Conclusion Aberrant expression and processing of MATN3 are hallmarks of conventional cartilaginous neoplasms. A particular step in the maturation of MATN3 limits its processing through the secretion machinery, resulting in its intracellular accumulation upon increased expression. Soluble, secreted MATN3, however, down-regulates SOX9 at the messenger RNA and protein levels. The first EGF-like domain of MATN3 is a critical determinant of its regulatory activity toward SOX9. These activities of MATN3 suggest that its increased expression in osteoarthritis might contribute to the degeneration of articular cartilage.

Chromosome dimers form in bacteria by recombination between circular chromosomes. Resolution of dimers is a highly integrated process involving recombination between dif sites catalysed by the XerCD recombinase, cell division and the integrity of the division septum-associated FtsK protein and the presence of dif inside a restricted region of the chromosome terminus, the dif activity zone (DAZ). We analyse here how these phenomena collaborate. We show that (i) both inter- and intrachromosomal recombination between dif sites are activated by their presence inside the DAZ; (ii) the DAZ-specific activation only occurs in conditions supporting the formation of chromosome dimers; (iii) overexpression of FtsK leads to a general increase in dif recombination irrespective of dif location; (iv) overexpression of FtsK does not improve the ability of dif sites inserted outside the DAZ to resolve chromosome dimers. Our results suggest that the formation of an active XerCD-FtsK-dif complex is restricted to when a dimer is present, the features of chromosome organization that determine the DAZ playing a central role in this control.

International audience; Chondrocalcin is among the most highly synthesized polypeptides in cartilage. This protein is released from its parent molecule, type II pro-collagen, after secretion by chondrocytes. A participation of extracellular, isolated chondrocalcin in mineralization was proposed more than 25 years ago, but never demonstrated. Here, exogenous chondrocalcin was found to trigger MMP13 secretion and cartilage destruction ex vivo in human cartilage explants and did so by modulating the expression of interleukin-1 beta in primary chondrocyte cultures in vitro. Chondrocalcin was found internalized by chondrocytes. Uptake was found mediated by a single 18-mer peptide of chondrocalcin, which does not exhibit homology to any known cell-penetrating peptide. The isolated peptide, when artificially linked as a tetramer, inhibited gene expression regulation by chondrocalcin, suggesting a functional link between uptake and gene expression regulation. At the same time, the tetrameric peptide potentiated chondrocalcin uptake by chondrocytes, suggesting a cooperative mechanism of entry. The corresponding peptide from type I pro-collagen supported identical cell-penetration, suggesting that this property may be conserved among C-propeptides of fibrillar pro-collagens. Structural modeling localized this peptide to the tips of procollagen C-propeptide trimers. Our findings shed light on unexpected function and mechanism of action of these highly expressed proteins from vertebrates.

International audience; Cucurbitacins are a class of natural compounds known for their numerous ă potential pharmacological effects. The purpose of this work was to ă compare the cytotoxicity of three cucurbitacins I, D, E on the ă chondrosarcoma SW 1353 cancer cell line and to investigate their ă biotransformation in man. Cucurbitacins I and D showed a very strong ă cytotoxicity, which was higher than that of cytochalasin D, used as a ă drug reference. Almost 100% of the cells were apoptotic as observed by ă DNA fragmentation (TUNEL assay) after 12 h with cucurbitacins I and D (1 ă mu M) and cucurbitacin E (10 mu M). In terms of IC50 values, ă cucurbitacins I and E presented a higher toxicity compared to that of ă cucurbitacin D (MTT assay). Cucurbitacin E was readily hydrolyzed by ă human hepatic microsomes, leading to cucurbitacin I (K-m 22 mu M, V-max ă 571 nmol/mg proteins/min). On the other hand, the three cucurbitacins ă were hydroxylated at a very low extent, but they were sulfated and ă glucuronidated. In terms of V-max/K-m, the cucurbitacin E was the best ă substrate of UDP-glucuronosyltransferases. This study shows that ă cucurbitacins I, D and E present a potent cytotoxic activity toward the ă chondrosarcoma SW 1353 cell line and are metabolized as sulfate and ă glucuronide conjugates. (C) 2012 Elsevier Ireland Ltd. All rights ă reserved.

International audience

The extracellular matrix (ECM) has long been viewed primarily as an organized network of solid-phase ligands for integrin receptors. During degenerative processes, such as osteoarthritis, the ECM undergoes deterioration, resulting in its remodeling and in the release of some of its components. Matrilin-3 (MATN3) is an almost cartilage specific, pericellular protein acting in the assembly of the ECM of chondrocytes. In the past, MATN3 was found required for cartilage homeostasis, but also involved in osteoarthritis-related pro-catabolic functions. Here, to better understand the pathological and physiological functions of MATN3, its concentration as a circulating protein in articular fluids of human osteoarthritic patients was determined and its functions as a recombinant protein produced in human cells were investigated with particular emphasis on the physical state under which it is presented to chondrocytes. MATN3 down-regulated cartilage extracellular matrix (ECM) synthesis and up-regulated catabolism when administered as a soluble protein. When artificially immobilized, however, MATN3 induced chondrocyte adhesion via a α5β1 integrin-dependent mechanism, AKT activation and favored survival and ECM synthesis. Furthermore, MATN3 bound directly to isolated α5β1 integrin in vitro. TGFβ1 stimulation of chondrocytes allowed integration of exogenous MATN3 into their ECM and ECM-integrated MATN3 induced AKT phosphorylation and improved ECM synthesis and accumulation. In conclusion, the integration of MATN3 to the pericellular matrix of chondrocytes critically determines the direction toward which MATN3 regulates cartilage metabolism. These data explain how MATN3 plays either beneficial or detrimental functions in cartilage and highlight the important role played by the physical state of ECM molecules.

Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, plays a key role in the pathogenesis of many inflammatory diseases, including arthritis. Neutralization of this cytokine by anti-TNF-α antibodies has shown its efficacy in rheumatoid arthritis (RA) and is now widely used. Nevertheless, some patients currently treated with anti-TNF-α remain refractory or become nonresponder to these treatments. In this context, there is a need for new or complementary therapeutic strategies. In this study, we investigated in vitro and in vivo anti-inflammatory potentialities of an anti-TNF-α triplex-forming oligonucleotide (TFO), as judged from effects on two rat arthritis models. The inhibitory activity of this TFO on articular cells (synoviocytes and chondrocytes) was verified and compared to that of small interfering RNA (siRNA) in vitro. The use of the anti-TNF-α TFO as a preventive and local treatment in both acute and chronic arthritis models significantly reduced disease development. Furthermore, the TFO efficiently blocked synovitis and cartilage and bone destruction in the joints. The results presented here provide the first evidence that gene targeting by anti-TNF-α TFO modulates arthritis in vivo, thus providing proof-of-concept that it could be used as therapeutic tool for TNF-α-dependent inflammatory disorders.

Articular cartilage consists mainly of extracellular matrix, mostly made of collagens and proteoglycans. These macromolecules have so far impaired the detailed two-dimensional electrophoresis-based proteomic analysis of articular cartilage. Here we describe a method for selective protein extraction from cartilage, which excludes proteoglycans and collagen species, thus allowing direct profiling of the protein content of cartilage by two-dimensional electrophoresis. Consistent electrophoretic patterns of more than 600 protein states were reproducibly obtained after silver staining from 500 mg of human articular cartilage from joints with diverse pathologies. The extraction yield increased when the method was applied to a chondrosarcoma sample, consistent with selective extraction of cellular components. Nearly 200 of the most intensely stained protein spots were analyzed by MALDI-TOF mass spectrometry after trypsin digestion. They represented 127 different proteins with diverse functions. Our method provides a rapid, efficient, and pertinent alternative to previously proposed approaches for proteomic characterization of cartilage phenotypes. It will be useful for detecting protein expression patterns that relate pathophysiological processes of cartilaginous tissues such as osteoarthritis and chondrosarcoma.

Lymphocyte emigration from the blood into most secondary lymphoid organs and chronically inflamed tissues occurs at the level of high endothelial venules (HEV). A unique characteristic of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. We have previously shown that sulfate uptake into HEVEC is mediated by two distinct functional classes of sulfate transporters: Na + -coupled transporters and sulfate/anion exchangers. Here, we report the molecular characterization from human HEVEC of SLC26A11, a novel member of the SLC26 sulfate/anion exchanger family. Functional expression studies in COS-7 and Sf9 insect cells revealed that SLC26A11 is targeted to the cell membrane and exhibits Na + -independent sulfate transport activity, sensitive to the anion exchanger inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). Northern blot analysis showed the highest SLC26A11 transcript levels in placenta, kidney, and brain. The SLC26A11 gene mapped to human chromosome 17q25, very close to the hereditary hearing loss diseases loci DFNA20, DFNA26, and USH1G. RT-PCR analysis of SLC26 sulfate transporters in human HEVEC revealed coexpression of SLC26A11 with SLC26A2/DTDST and lack of SLC26A1/SAT1, SLC26A3/DRA, and SLC26A8/TAT1. Together, our results indicate that SLC26A11 is a novel Na + -independent sulfate transporter that may cooperate with SLC26A2 to mediate DIDS-sensitive sulfate uptake into HEVEC.

Sulfation is a major modification of many molecules in eukaryotes that is dependent on the enzymatic synthesis of an activated sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). While sulfate activation has long been assumed to occur in the cytosol, we show in this study that human PAPS synthetase 1 (PAPSS1), a bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase enzyme sufficient for PAPS synthesis, accumulates in the nucleus of mammalian cells. Nuclear targeting of the enzyme is mediated by its APS kinase domain and requires a catalytically dispensable 21 amino acid sequence at the amino terminus. Human PAPSS1 and Drosophila melanogaster PAPSS localize to the nucleus in yeast and relieve the methionine auxotrophy of ATP sulfurylase- or APS kinase-deficient strains, suggesting that PAPSS1 is fully functional in vivo when targeted to the nucleus. A second PAPS synthetase gene, designated PAPSS2, has recently been described, mutations of which are responsible for abnormal skeletal development in human spondyloepimetaphyseal dysplasia and murine brachymorphism. We found that PAPSS2, which localizes to the cytoplasm when ectopically expressed in mammalian cells, is relocated to the nucleus when coexpressed with PAPSS1. Taken together, these results indicate that a sulfation pathway might exist in the nucleus of eukaryotic cells. -Besset, S., Vincourt, J.-B., Amalric, F., Girard, J.-P. Nuclear localization of PAPS synthetase 1: a sulfate activation pathway in the nucleus of eukaryotic cells.

Endochondral ossification is the mechanism by which long bones of Vertebrates grow. It requires the formation of a primary cartilage pattern by chondrogenesis, which is then replaced by bone. Chondrogenesis relies on the production of a dedicated type II collagen rich extracellular matrix (ECM), which will then mineralize, and many aspects of which remain difficult to characterize. The ATDC5 cell line can engage into a differentiation program resembling the multistep chondrogenic differentiation observed in vivo after insulin stimulation. Although this cell line is widely used, its proteome is barely described. In order to define to what extent this model recapitulates the main aspects of cartilage ECM formation, a time-resolved proteome analysis by LC-MALDI-TOF / TOF of the regulation of the proteome of differentiating ATDC5 cells focused on the ECM and the level of maturation of its main components. These cells synthesize and incorporate into their ECM a wide range of cartilage components, including aggrecan and type II collagen, which carry most of the maturations described in vivo. Most importantly, the accumulation of collagen in the MEC is correlated with maturation events: proteolytic cleavage and triple helix hydroxylation. The C-propeptide of type II collagen (CPII), is a component of joint and growth plate cartilage, and has been described as involved in the mineralization process of the latter. On the other hand, to what extent and how CPII would regulate mineralization remains to be specified. We have shown that CPII exhibit a different interactome as its type I and III procollagens (CPI and CPIII) counterparts, suggesting mechanistic differences between their maturation. These results are consolidated by additional in vivo studies of CPII cleavage sites, confirming the coexistence of two cleavage sites and demonstrating for the first time that CPII trimers contain the four possible combinations of the two cleavage sites. These data suggest that each monomer within the trimer is cleaved independently of the others. Overall, this work highlights CPII cleavage mechanism as distinct from that of CPI and CPIII and more complex than anticipated. Functional characterization of key events of ECM elaboration in ATDC5 cells should allow better dissecting the mechanisms of chondrogenesis and cartilage mineralization., L'ossification endochondrale est le mécanisme par lequel les os longs se développent chez les Vertébrés. Elle nécessite la formation d'un patron cartilagineux primaire par chondrogenèse, qui est ensuite remplacé par l'os. Elle repose sur la production d'une matrice extracellulaire (MEC) riche en collagènes de type II qui va minéraliser et dont de nombreux aspects restent difficiles à caractériser. La lignée cellulaire ATDC5 s'engage dans un programme de différenciation séquentiel semblable à la chondrogenèse après stimulation à l'insuline. Bien que cette lignée soit largement utilisée, son protéome est à peine décrit. Afin de définir à quel point ce modèle récapitule les principaux aspects de la formation de la MEC du cartilage, une analyse temporelle par LC–MALDI–TOF / TOF de la régulation du protéome a été effectuée, axée sur la MEC et la maturation de ses composants. Ces cellules synthétisent et incorporent dans leur MEC un vaste panel de composants cartilagineux, notamment l’agrécane et le collagène de type II qui portent la plupart des maturations décrites in vivo. Surtout, l'accumulation de collagène dans la MEC est corrélée aux événements de maturation : clivages protéolytiques et hydroxylations de la triple hélice. Le C-propeptide du collagène de type II (CPII), est un composant des cartilages articulaire et de croissance, et a été décrit comme impliqué dans le processus de minéralisation, mais dans quelle mesure et par quel mécanisme CPII est impliqué dans la minéralisation restent à être déterminés. Nous avons montré que le CPII ne possède pas le même intéractome que ses homologues des procollagènes de type I et III (CPI et CPIII), suggérant des divergences mécanistiques entre leurs maturations. Ces résultats sont consolidés par des études complémentaires in vivo des sites de clivage de CPII, confirmant la coexistence de 2 sites de clivage et démontrant pour la première fois que les trimères de CPII contiennent les quatre combinaisons possibles des deux sites de clivage. Ces données suggèrent que chaque monomère au sein du trimère est clivé indépendamment des autres. Dans l’ensemble, ces travaux amènent à considérer le mécanisme de clivage de CPII comme distinct de celui de CPI et CPIII et comme plus complexe qu’il n’était anticipé. La caractérisation fonctionnelle des événements clés de l'élaboration de la MEC dans les cellules ATDC5 devrait permettre de mieux disséquer les mécanismes de la chondrogenèse et de la minéralisation.