Adrenomedullin and truncated peptide adrenomedullin(22-52) affect chondrocyte response to apoptotis in vitro: downregulation of FAS protects chondrocyte from cell death.

Chondrocyte apoptosis may have a pivotal role in the development of osteoarthritis. Interest has increased in the use of anti-apoptotic compounds to protect against osteoarthritis development. In this work, we investigated the effect of adrenomedullin (AM), a 52 amino-acid hormone peptide, and a 31 amino-acid truncated form, AM(22-52), on chondrocyte apoptosis. Bovine articular chondrocytes (BACs) were cultured under hypoxic conditions to mimic cartilage environment and then treated with Fas ligand (Fas-L) to induce apoptosis. The expression of AM and its calcitonin receptor-like receptor (CLR)/receptor activity-modifying protein (RAMP) (receptor/co-receptor) was assessed by immunostaining. We evaluated the effect of AM and AM(22-52) on Fas-L-induced chondrocyte apoptosis. FAS expression was appreciated by RT-qPCR and immunostainings. The expression of hypoxia-inducible factor 1α (HIF-1α), CLR and one co-receptor (RAMP2) was evidenced. With BACs under hypoxia, cyclic adenosine monophosphate production increased dose-dependently with AM stimulation. AM significantly decreased caspase-3 activity (mean 35% decrease; p = 0.03) as a marker of Fas-L-induced apoptosis. Articular chondrocytes treated with AM showed significantly reduced cell death, along with downregulated Fas expression and production, as compared with AM(22-52). AM decreased articular chondrocyte apoptosis by downregulating a Fas receptor. These findings may pave the way for novel therapeutic approaches in osteoarthritis.

'Click'-xylosides as initiators of the biosynthesis of glycosaminoglycans: Comparison of mono-xylosides with xylobiosides.

Different mono-xylosides and their corresponding xylobiosides obtained by a chemo-enzymatic approach featuring various substituents attached to a triazole ring were probed as priming agents for glycosaminoglycan (GAG) biosynthesis in the xylosyltransferase-deficient pgsA-745 Chinese hamster ovary cell line. Xylosides containing a hydrophobic aglycone moiety were the most efficient priming agents. Mono-xylosides induced higher GAG biosynthesis in comparison with their corresponding xylobiosides. The influence of the degree of polymerization of the carbohydrate part on the priming activity was investigated through different experiments. We demonstrated that in case of mono-xylosides, the cellular uptake as well as the affinity and the catalytic efficiency of ß-1,4-galactosyltransferase 7 were higher than for xylobiosides. Altogether, these results indicate that hydrophobicity of the aglycone and degree of polymerization of glycone moiety were critical factors for an optimal priming activity for GAG biosynthesis.

Lumican Inhibits SNAIL-Induced Melanoma Cell Migration Specifically by Blocking MMP-14 Activity.

Lumican, a small leucine rich proteoglycan, inhibits MMP-14 activity and melanoma cell migration in vitro and in vivo. Snail triggers epithelial-mesenchymal transitions endowing epithelial cells with migratory and invasive properties during tumor progression. The aim of this work was to investigate lumican effects on MMP-14 activity and migration of Snail overexpressing B16F1 (Snail-B16F1) melanoma cells and HT-29 colon adenocarcinoma cells. Lumican inhibits the Snail induced MMP-14 activity in B16F1 but not in HT-29 cells. In Snail-B16F1 cells, lumican inhibits migration, growth, and melanoma primary tumor development. A lumican-based strategy targeting Snail-induced MMP-14 activity might be useful for melanoma treatment.

The elastin peptide (VGVAPG)3 induces the 3D reorganisation of PML-NBs and SC35 speckles architecture, and accelerates proliferation of fibroblasts and melanoma cells.

During melanoma tumour growth, cancerous cells are exposed to the immediate surrounding the micro- and macro environment, which is largely modified through the degradation of the extracellular matrix by fibroblast-derived metalloproteinases. Among the degradation products, (VGVAPG)3, an elastin peptide is known to stimulate the proliferation of both fibroblasts and cancerous cells by binding to the elastin-binding receptor and activating the MEK/ERK signal transduction pathway. As this process strongly modifies mRNA synthesis, we investigated its effect on the relative three-dimensional organisation of the major partners of the mRNA splicing machinery: promyelocytic nuclear bodies (PML-NBs ) and splicing component 35 speckles (SC35) of normal fibroblasts and melanoma SK-MEL-28 cells. SC35 and PML-NBs proteins were immunolabeled and imaged by confocal microscopy within these cells cultured with (VGVAPG)3. Three-dimensional reconstruction was performed to elucidate the organisation of PML-NBs and SC35 speckles and their spatial relationship. In G0 cells, SC35 speckles were sequestered in PML-NBs. Shortly after (VGVAPG)3 stimulation, the three-dimensional organisation of PML-NBs and SC35 speckles changed markedly. In particular, SC35 speckles gradually enlarged and adopted a heterogeneous organisation, intermingled with PML-NBs. Conversely, inhibition of the elastin-binding protein or MEK/ERK pathway induced a remarkable early sequestration of condensed SC35 speckles in PML-NBs, the hallmark of splicing inhibition. The 3D architecture of speckles/PML-NBs highlights the modulation in their spatial relationship, the multiple roles of PML-NBs in activation, inhibition and sequestration, and provides the first demonstration of the dependence of PML-NBs and SC35 speckles on the elastin peptide for these functions.

Lumican: a new inhibitor of matrix metalloproteinase-14 activity.

We previously showed that lumican regulates MMP-14 expression. The aim of this study was to compare the effect of lumican and decorin on MMP-14 activity. In contrast to decorin, the glycosylated form of lumican was able to significantly decrease MMP-14 activity in B16F1 melanoma cells. Our results suggest that a direct interaction occurs between lumican and MMP-14. Lumican behaves as a competitive inhibitor which leads to a complete blocking of the activity of MMP-14. It binds to the catalytic domain of MMP-14 with moderate affinity (KD∼275 nM). Lumican may protect collagen against MMP-14 proteolysis, thus influencing cell-matrix interaction in tumor progression.

Revisiting spatial distribution and biochemical composition of calcium-containing crystals in human osteoarthritic articular cartilage.

INTRODUCTION: Calcium-containing (CaC) crystals, including basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP), are associated with destructive forms of osteoarthritis (OA). We assessed their distribution and biochemical and morphologic features in human knee OA cartilage. METHODS: We prospectively included 20 patients who underwent total knee replacement (TKR) for primary OA. CaC crystal characterization and identification involved Fourier-transform infra-red spectrometry and scanning electron microscopy of 8 to 10 cartilage zones of each knee, including medial and lateral femoral condyles and tibial plateaux and the intercondyle zone. Differential expression of genes involved in the mineralization process between cartilage with and without calcification was assessed in samples from 8 different patients by RT-PCR. Immunohistochemistry and histology studies were performed in 6 different patients. RESULTS: Mean (SEM) age and body mass index of patients at the time of TKR was 74.6 (1.7) years and 28.1 (1.6) kg/m², respectively. Preoperative X-rays showed joint calcifications (chondrocalcinosis) in 4 cases only. The medial femoro-tibial compartment was the most severely affected in all cases, and mean (SEM) Kellgren-Lawrence score was 3.8 (0.1). All 20 OA cartilages showed CaC crystals. The mineral content represented 7.7% (8.1%) of the cartilage weight. All patients showed BCP crystals, which were associated with CPP crystals for 8 joints. CaC crystals were present in all knee joint compartments and in a mean of 4.6 (1.7) of the 8 studied areas. Crystal content was similar between superficial and deep layers and between medial and femoral compartments. BCP samples showed spherical structures, typical of biological apatite, and CPP samples showed rod-shaped or cubic structures. The expression of several genes involved in mineralization, including human homolog of progressive ankylosis, plasma-cell-membrane glycoprotein 1 and tissue-nonspecific alkaline phosphatase, was upregulated in OA chondrocytes isolated from CaC crystal-containing cartilages. CONCLUSIONS: CaC crystal deposition is a widespread phenomenon in human OA articular cartilage involving the entire knee cartilage including macroscopically normal and less weight-bearing zones. Cartilage calcification is associated with altered expression of genes involved in the mineralisation process.

Elastin peptides signaling relies on neuraminidase-1-dependent lactosylceramide generation.

The sialidase activity of neuraminidase-1 (Neu-1) is responsible for ERK 1/2 pathway activation following binding of elastin peptide on the elastin receptor complex. In this work, we demonstrate that the receptor and lipid rafts colocalize at the plasma membrane. We also show that the disruption of these microdomains as well as their depletion in glycolipids blocks the receptor signaling. Following elastin peptide treatment, the cellular GM(3) level decreases while lactosylceramide (LacCer) content increases consistently with a GM(3)/LacCer conversion. The use of lactose or Neu-1 siRNA blocks this process suggesting that the elastin receptor complex is responsible for this lipid conversion. Flow cytometry analysis confirms this elastin peptide-driven LacCer generation. Further, the use of a monoclonal anti-GM(3) blocking antibody shows that GM(3) is required for signaling. In conclusion, our data strongly suggest that Neu-1-dependent GM(3)/LacCer conversion is the key event leading to signaling by the elastin receptor complex. As a consequence, we propose that LacCer is an early messenger for this receptor.

Tomography of the cell nucleus using confocal microscopy and medium voltage electron microscopy.

Changes in nuclear structures are widely used by pathologists as diagnostic and prognostic indicators in cancer cells. Recent studies have demonstrated that the cell nucleus is probably the most complex organelle in the cell. It contains the genome and is the site of all related activities such as DNA repair, DNA duplication, RNA synthesis, RNA processing and RNA transport. These activities take place within dynamic three-dimensional compartments. The detailed study of these compartments requires an approach termed "cell tomography" based on 3D imaging using confocal microscopy and electron tomography. In this paper, we will first summarize the most recent findings concerning the organization of the cell nucleus. We will then describe markers used to identify molecules specific for various nuclear compartments and their use in tomography of the cell nucleus by confocal microscopy and electron tomography.

A protocol for studying the kinetics of RNA within cultured cells: application to ribosomal RNA.

This protocol describes a nonisotopic method for high-resolution investigation of the kinetics of RNA within the cell. This involves the incorporation of bromouridine-5'-triphosphate into RNA of living cells by lipofection followed by immunocytological detection of BrRNAs. The use of the same antibody identified either with fluorescence or with gold particles revealed the three-dimensional organization of sites containing labeled RNAs or their precise localization by using confocal and ultrastructural microscopy, respectively. Comparison of three-dimensional reconstruction obtained from the series of optical sections and ultrathin sections was extremely fruitful to describe topological and spatial dynamics of RNAs from their synthesis site inside the nucleus to the cytoplasm. Combined with immunolocalization of proteins involved in different nuclear activities and with highly resolved three-dimensional visualizations of the labelings, this method should also provide a significant contribution to our understanding of the functional, volumic organization of the cell nucleus. The entire protocol can be completed in approximately 10 d.