Effect of glucosamine on c-jun DNA-binding activity in HPCs stimulated with 10 ng/ml IL-1β

<p><b>Copyright information:</b></p><p>Taken from "Glucosamine affects intracellular signalling through inhibition of mitogen-activated protein kinase phosphorylation in human chondrocytes"</p><p>http://arthritis-research.com/content/9/5/R104</p><p>Arthritis Research & Therapy 2007;9(5):R104-R104.</p><p>Published online 9 Oct 2007</p><p>PMCID:PMC2212570.</p><p></p> Cells were pretreated with 2.5 and 10 mmol/l glucosamine (G2.5 and G10, respectively) for 2 hours and then stimulated with IL-1β for 15 minutes. Nuclear extract was prepared as described in Materials and methods. Results are expressed as optical density (OD) measured at 450 nm and represent the mean ± standard error of data obtained in six different experiments. *≤ 0.05. CTL, control; HPC, human primary chondrocyte

Effect of glucosamine MAPK phosphorylation in HPCs stimulated with 10 ng/ml IL-1β

<p><b>Copyright information:</b></p><p>Taken from "Glucosamine affects intracellular signalling through inhibition of mitogen-activated protein kinase phosphorylation in human chondrocytes"</p><p>http://arthritis-research.com/content/9/5/R104</p><p>Arthritis Research & Therapy 2007;9(5):R104-R104.</p><p>Published online 9 Oct 2007</p><p>PMCID:PMC2212570.</p><p></p> Cells were pretreated for 2 hours with 2.5 or 10 mmol/l glucosamine (G2.5 and G10, respectively), and then stimulated with IL-1β for 15 minutes. Whole cell extract was prepared as described in Materials and methods. Proteins were resolved on SDS-PAGE, electrotransferred and immunoblotted. Antibodies to phosphorylated (p)-c-jun amino-terminal kinase (JNK) and total JNK, p-p38 and total p38, and p-extracellular signal-regulated kinase (ERK)1/2 and total ERK1/2 were used to visualize mitogen-activated protein kinase (MAPK) phosphorylation. Representative data from six independent experiments are shown. CTL, control; HPC, human primary chondrocyte

Effect of glucosamine on MMP-3 expression in lbpva55 cell line stimulated with 10 ng/ml IL-1β

<p><b>Copyright information:</b></p><p>Taken from "Glucosamine affects intracellular signalling through inhibition of mitogen-activated protein kinase phosphorylation in human chondrocytes"</p><p>http://arthritis-research.com/content/9/5/R104</p><p>Arthritis Research & Therapy 2007;9(5):R104-R104.</p><p>Published online 9 Oct 2007</p><p>PMCID:PMC2212570.</p><p></p> Cells were pretreated for 2 hours with 2.5 or 10 mmol/l glucosamine (G2.5 and G10, respectively), and then stimulated with IL-1β for 22 hours. mRNA was extracted and analyzed by quantitative real-time PCR, and cell supernatant was analyzed by ELISA. Shown are matrix metalloprotease (MMP)-3 mRNA and MMP-3 protein levels. Quantitative real-time PCR results are expressed in relative arbitrary units (AU) and ELISA results are expressed in ng/ml. Results are expressed as mean ± standard error, obtained in three different experiments. *≤ 0.05. CTL, control

Effect of glucosamine on MMP-1 and MMP-13 expression in HPCs stimulated with 10 ng/ml IL-1β

<p><b>Copyright information:</b></p><p>Taken from "Glucosamine affects intracellular signalling through inhibition of mitogen-activated protein kinase phosphorylation in human chondrocytes"</p><p>http://arthritis-research.com/content/9/5/R104</p><p>Arthritis Research & Therapy 2007;9(5):R104-R104.</p><p>Published online 9 Oct 2007</p><p>PMCID:PMC2212570.</p><p></p> Cells were pretreated for 2 hours with 2.5 and 10 mmol/l glucosamine (G2.5 and G10, respectively), and then stimulated with IL-1β for 22 hours. mRNA was extracted and analyzed by quantitative real-time PCR, and cell supernatant was analyzed by ELISA. Shown are matrix metalloprotease (MMP)-1 and MMP-13 mRNA levels, and MMP-1 and MMP-13 protein amounts. Quantitative real-time PCR results are expressed in relative arbitrary units (AU) and ELISA results are expressed in ng/ml or pg/ml. Results are expressed as mean ± standard error, obtained in six different experiments. *≤ 0.05. CTL, control; HPC, human primary chondrocyte

Effect of glucosamine on MMP-3 expression in HCPs stimulated with 10 ng/ml IL-1β

<p><b>Copyright information:</b></p><p>Taken from "Glucosamine affects intracellular signalling through inhibition of mitogen-activated protein kinase phosphorylation in human chondrocytes"</p><p>http://arthritis-research.com/content/9/5/R104</p><p>Arthritis Research & Therapy 2007;9(5):R104-R104.</p><p>Published online 9 Oct 2007</p><p>PMCID:PMC2212570.</p><p></p> Cells were pretreated for 2 hours with 2.5 or 10 mmol/l glucosamine (G2.5 and G10, respectively), and then stimulated with IL-1β for 22 hours. mRNA was extracted and analyzed by quantitative real-time PCR, and cell supernatant was analyzed by ELISA. Shown are matrix metalloprotease (MMP)-3 mRNA level and MMP-3 protein level. Quantitative real-time PCR results are expressed in relative arbitrary units (AU) and ELISA results are expressed in ng/ml. Results are expressed as mean ± standard error, obtained in six different experiments. *≤ 0.05. CTL, control; HPC, human primary chondrocyte

Effect of glucosamine on MMP-1 and MMP-13 expression in lbpva55 cells stimulated with 10 ng/ml IL-1β

<p><b>Copyright information:</b></p><p>Taken from "Glucosamine affects intracellular signalling through inhibition of mitogen-activated protein kinase phosphorylation in human chondrocytes"</p><p>http://arthritis-research.com/content/9/5/R104</p><p>Arthritis Research & Therapy 2007;9(5):R104-R104.</p><p>Published online 9 Oct 2007</p><p>PMCID:PMC2212570.</p><p></p> Cells were pretreated for 2 hours with 2.5 or 10 mmol/l glucosamine (G2.5 and G10, respectively), and then stimulated with IL-1β for 22 hours. mRNA was extracted and analyzed by quantitative real-time PCR, and cell supernatant was analyzed by ELISA. Shown are matrix metalloprotease (MMP)-1 and MMP-13 mRNA levels, and MMP-1 and MMP-13 protein amounts. Quantitative real-time PCR results are expressed in relative arbitrary units (AU), and ELISA results are expressed in ng/ml or pg/ml. Results are expressed as mean ± standard error, obtained in three different experiments. *≤ 0.05. CTL, control

Metabolic Labeling of Human Bone Marrow Mesenchymal Stem Cells for the Quantitative Analysis of their Chondrogenic Differentiation

Human mesenchymal stem cells (hMSCs), residing in bone marrow as well as in the synovial lining of joints, can be triggered to differentiate toward chondrocytes. Thus, hMSCs harbor great therapeutic potential for the repair of cartilage defects in osteoarthritis (OA) and other articular diseases. However, the molecular mechanisms underlying the chondrogenesis process are still in part unknown. In this work, we applied for the first time the stable isotope labeling by amino acids in cell culture (SILAC) technique for the quantitative analysis of protein modulation during the chondrogenic differentiation process of hMSCs. First, we have standardized the metabolic labeling procedure on MSCs isolated from bone marrow (hBMSCs), and we have assessed the quality of chondrogenesis taking place in these conditions. Then, chondrogenic differentiation was induced on these labeled cells, and a quantitative proteomics approach has been followed to evaluate protein changes between two differentiation days. With this strategy, we could identify 622 different proteins by LC–MALDI-TOF/TOF analysis and find 65 proteins whose abundance was significantly modulated between day 2 and day 14 of chondrogenesis. Immunohistochemistry analyses were performed to verify the changes on a panel of six proteins that play different biological roles in the cell: fibronectin, gelsolin, vimentin, alpha-ATPase, mitochondrial superoxide dismutase, and cyclophilin A. All of these proteins were increased at day 14 compared to day 2 of chondrogenic induction, thus being markers of the enhanced extracellular matrix synthesis, cell adhesion, metabolism, and response to stress processes that take place in the early steps of chondrogenesis. Our strategy has allowed an additional insight into both specific protein function and the mechanisms of chondrogenesis and has provided a panel of protein markers of this differentiation process in hBMSCs

Pierre Marie Auguste Broussonet, Paris, [France], to James Edward Smith

Has edited works of [Pierre Richer de] Belleval

Quantitative Proteomic Profiling of Human Articular Cartilage Degradation in Osteoarthritis

Osteoarthritis (OA) is the most common rheumatic pathology and is characterized primarily by articular cartilage degradation. Despite its high prevalence, there is no effective therapy to slow disease progression or regenerate the damaged tissue. Therefore, new diagnostic and monitoring tests for OA are urgently needed, which would also promote the development of alternative therapeutic strategies. In the present study, we have performed an iTRAQ-based quantitative proteomic analysis of secretomes from healthy human articular cartilage explants, comparing their protein profile to those from unwounded (early disease) and wounded (advanced disease) zones of osteoarthritic tissue. This strategy allowed us to identify a panel of 76 proteins that are distinctively released by the diseased tissue. Clustering analysis allowed the classification of proteins according to their different profile of release from cartilage. Among these proteins, the altered release of osteoprotegerin (decreased in OA) and periostin (increased in OA), both involved in bone remodelling processes, was verified in further analyses. Moreover, periostin was also increased in the synovial fluid of OA patients. Altogether, the present work provides a novel insight into the mechanisms of human cartilage degradation and a number of new cartilage-characteristic proteins with possible biomarker value for early diagnosis and prognosis of OA

Proteomic Analysis of the E3 Ubiquitin-Ligase Hakai Highlights a Role in Plasticity of the Cytoskeleton Dynamics and in the Proteasome System

Carcinoma, the most common type of cancer, arises from epithelial cells. The transition from adenoma to carcinoma is associated with the loss of E-cadherin and, in consequence, the disruption of cell–cell contacts. E-cadherin is a tumor suppressor, and it is down-regulated during epithelial-to-mesenchymal transition (EMT); indeed, its loss is a predictor of poor prognosis. Hakai is an E3 ubiquitin-ligase protein that mediates E-cadherin ubiquitination, endocytosis and finally degradation, leading the alterations of cell–cell contacts. Although E-cadherin is the most established substrate for Hakai activity, other regulated molecular targets for Hakai may be involved in cancer cell plasticity during tumor progression. In this work we employed an iTRAQ approach to explore novel molecular pathways involved in Hakai-driven EMT during tumor progression. Our results show that Hakai may have an important influence on cytoskeleton-related proteins, extracellular exosome-associated proteins, RNA-related proteins and proteins involved in metabolism. Moreover, a profound decreased expression in several proteasome subunits during Hakai-driven EMT was highlighted. Since proteasome inhibitors are becoming increasingly used in cancer treatment, our findings suggest that the E3 ubiquitin-ligase, such as Hakai, may be a better target than proteasome for using novel specific inhibitors in tumor subtypes that follow EMT