P. Carpintero-Fernandez

Human Articular Chondrocytes Express Multiple Gap Junction Proteins: Differential Expression of Connexins in Normal and Osteoarthritic Cartilage

Osteoarthritis (OA) is the most common joint disease and involves progressive degeneration of articular cartilage. The aim of this study was to investigate if chondrocytes from human articular cartilage express gap junction proteins called connexins (Cxs). We show that human chondrocytes in tissue express Cx43, Cx45, Cx32, and Cx46. We also find that primary chondrocytes from adults retain the capacity to form functional voltage-dependent gap junctions. Immunohistochemistry experiments in cartilage from OA patients revealed significantly elevated levels of Cx43 and Cx45 in the superficial zone and down through the next approximately 1000 μm of tissue. These zones corresponded with regions damaged in OA that also had high levels of proliferative cell nuclear antigen. An increased number of Cxs may help explain the increased proliferation of cells in clusters that finally lead to tissue homeostasis loss. Conversely, high levels of Cxs in OA cartilage reflect the increased number of adjacent cells in clusters that are able to interact directly by gap junctions as compared with hemichannels on single cells in normal cartilage. Our data provide strong evidence that OA patients have a loss of the usual ordered distribution of Cxs in the damaged zones and that the reductions in Cx43 levels are accompanied by the loss of correct Cx localization in the nondamaged areas.

Articular chondrocyte network mediated by gap junctions: role in metabolic cartilage homeostasis

Objective This study investigated whether chondrocytes within the cartilage matrix have the capacity to communicate through intercellular connections mediated by voltage-gated gap junction (GJ) channels. Methods Frozen cartilage samples were used for immunofluorescence and immunohistochemistry assays. Samples were embedded in cacodylate buffer before dehydration for scanning electron microscopy. Co-immunoprecipitation experiments and mass spectrometry (MS) were performed to identify proteins that interact with the C-terminal end of Cx43. GJ communication was studied through in situ electroporation, electrophysiology and dye injection experiments. A transwell layered culture system and MS were used to identify and quantify transferred amino acids. Results Microscopic images revealed the presence of multiple cellular projections connecting chondrocytes within the matrix. These projections were between 5 and 150 µm in length. MS data analysis indicated that the C-terminus of Cx43 interacts with several cytoskeletal proteins implicated in Cx trafficking and GJ assembly, including α-tubulin and β-tubulin, actin, and vinculin. Electrophysiology experiments demonstrated that 12-mer oligonucleotides could be transferred between chondrocytes within 12 min after injection. Glucose was homogeneously distributed within 22 and 35 min. No transfer was detected when glucose was electroporated into A549 cells, which have no GJs. Transwell layered culture systems coupled with MS analysis revealed connexins can mediate the transfer of L-lysine and L-arginine between chondrocytes. Conclusions This study reveals that intercellular connections between chondrocytes contain GJs that play a key role in cell–cell communication and a metabolic function by exchange of nutrients including glucose and essential amino acids. A three-dimensional cellular network mediated through GJs might mediate metabolic and physiological homeostasis to maintain cartilage tissue.

Proteomic Analysis of Connexin 43 Reveals Novel Interactors Related to Osteoarthritis

We have previously reported that articular chondrocytes in tissue contain long cytoplasmic arms that physically connect two distant cells. Cell-to-cell communication occurs through connexin channels termed Gap Junction (GJ) channels, which achieve direct cellular communication by allowing the intercellular exchange of ions, small RNAs, nutrients, and second messengers. The Cx43 protein is overexpressed in several human diseases and inflammation processes and in articular cartilage from patients with osteoarthritis (OA). An increase in the level of Cx43 is known to alter gene expression, cell signaling, growth, and cell proliferation. The interaction of proteins with the C-terminal tail of connexin 43 (Cx43) directly modulates GJ-dependent and -independent functions. Here, we describe the isolation of Cx43 complexes using mild extraction conditions and immunoaffinity purification. Cx43 complexes were extracted from human primary articular chondrocytes isolated from healthy donors and patients with OA. The proteomic content of the native complexes was determined using LC-MS/MS, and protein associations with Cx43 were validated using Western blot and immunolocalization experiments. We identified >100 Cx43-associated proteins including previously uncharacterized proteins related to nucleolar functions, RNA transport, and translation. We also identified several proteins involved in human diseases, cartilage structure, and OA as novel functional Cx43 interactors, which emphasized the importance of Cx43 in the normal physiology and structural and functional integrity of chondrocytes and articular cartilage. Gene Ontology (GO) terms of the proteins identified in the OA samples showed an enrichment of Cx43-interactors related to cell adhesion, calmodulin binding, the nucleolus, and the cytoskeleton in OA samples compared with healthy samples. However, the mitochondrial proteins SOD2 and ATP5J2 were identified only in samples from healthy donors. The identification of Cx43 interactors will provide clues to the functions of Cx43 in human cells and its roles in the development of several diseases, including OA.

Intercellular communication via gap junction channels between chondrocytes and bone cells

Cell-to-cell communication between bone, cartilage and the synovial membrane is not fully understood and it is only attributed to the diffusion of substances through the extracellular space or synovial fluid. In this study, we found for the first time that primary bone cells (BCs) including osteocytes, synovial cells (SCs) and chondrocytes (CHs) are able to establish cellular contacts and to couple through gap junction (GJ) channels with connexin43 (Cx43) being dominant. Transwell co-culture and identification by mass spectrometry revealed the exchange of essential amino acids, peptides and proteins including calnexin, calreticulin or CD44 antigen between contacting SCs, BCs and CHs. These results reveal that CHs, SCs and BCs are able to establish intercellular connections and to communicate through GJ channels, which provide a selective signalling route by the direct exchange of potent signalling molecules and metabolites.

OP0308 CELL –To-Cell Communication VIA GAP Junctions Between Cartilage, Synovial Membrane and Subchondral Bone: Implications for Joint Homeostasis

Background Neurons, hepatocytes, cardiocytes, chondrocytes and almost all cell types express connexins and form gap junction (GJ) channels that are critical for cellular function and tissue homeostasis. GJ channels provide a selective signalling route by the direct exchange of potent signalling molecules such as cAMP, second messengers, electrical signals, ions and several molecules that regulate cell survival, growth and metabolism. GJ channels also play a key nutritional role by the direct exchange of amino acids, glucose and several metabolites. We have recently reported that articular chondrocytes in tissue contain long cytoplasmic arms that physically connect two distant cells and cell-to-cell communication occurs through GJ channels. Cx43 protein is overexpressed in several diseases including in the articular cartilage and synovial membrane from patients with osteoarthritis (OA) and rheumatoid arthritis. An increase in Cx43 protein levels is known to alter gene expression, cell signalling, growth and cell proliferation. Objectives The goal of this research work was to investigate if bone cells (subchondral bone, SB), synovial cells (SC) and chondrocytes (CH) are able to establish GJs among them and to investigate the consequence of that coupling in disorders that affect these tissues such as OA. Methods Human cartilage, synovial membrane and subchondral bone were obtained from adult donors after joint surgery. The cells were grown to 80-90% confluence. The Electrophysiological techniques dual voltage-clamp methods, whole-cell/perforated patch experiment and InSitu Porator™ were used to detect the transfer of glucose and lucifer yellow. To confirm the exchange of amino acids, peptides and proteins between contacting cells, transwell co-culture system (3.0μm pore) and SILACã labelling combined with mass spectrometry (MS) were performed. Amino acids were derivatized and analyzed using EZ:faastTM kit and ESI*/LC/MS-Orbitrap. Transjuctional peptides and proteins were isolated and identified by SDS-PAGE, in gel digestion and MALDI/TOF-TOF. Results Dual voltage-clamp and whole-cell/perforated patch methods demonstrated that primary SB and SC are able to establish functional GJ with CH, being Cx43 properties dominant. Dye injection experiments confirmed that SB, SC and CH exchange via GJ lucifer yellow and the fluorescent glucose derivative (2-NBDG). Transwell co-culture system demonstrated the transference of at least 5 pmol/ml of [13C6]-L-lisine and 3 pmol/mL of [13C6, 15N4]-L-arginine between contacting SB, SC and CH. MALDI/TO-TOF analysis revealed the exchange of peptides and proteins between contacting cells including calnexin, calreticulin or CD44 antigen. Conclusions These findings suggest that Cx43-mediated intercellular communication between cells located in the subchondral bone, synovial membrane and cartilage may contribute to the cellular signalling and homeostasis of the joint and may have protective effect in the injured tissues and hence warrants further investigation. So far, the results presented here demonstrated for the first time that SB, SC and CH are able to physically interact and directly communicate by GJ channels. Disclosure of Interest None declared

AB0861 Lectins That Target Sialic Acid Modified Receptors May Hold Therapeutic Potential for Degenerative Arthritis

Background Glycosylated proteins are essential components of the extracellular matrix (ECM) of cartilage and contribute to the maintenance of its function. A shift from a-2,6- to a-2,3-linked sialic acids of glycoproteins modifies the binding ability of proteins to substrates influencing cellular anchoring and affecting signal transduction. Intriguingly, the predominance of a-2,3-sialylation of chondrocytes glycoproteins was associated with the pathophysiology of rheumatic diseases including rheumatoid arthritis (RA) and osteoarthritis (OA). A highly O-glycolysated protein with α-2,3-sialic acid, involved in the induction of inflammation and tissue repair, is the transmembrane mucin receptor named Podoplanin (PDPN). Objectives The present study aimed to assess the effect of specifically targets a-2-3-sialic acid residues with a lectin-based drug (MASL) on chodrocyte dedifferentiation and cartilage breakdown processes. Methods For immunofluorescence and immunohistochemistry assays, in situ cartilage was fixed and frozen immediately using Tissue-Tek O.C.T. and isopentanol in liquid nitrogen. Primary cells in monolayer culture were fixed with formaldehyde for optical microscopy assays. 4mm cartilage punches were prepared from cartilage explants that were cut in the operating room immediately after surgery and cultured in DMEM with 0.1% FCS. Chondrocytes were isolated from articular cartilage and cultured in DMEM with 15% FCS. Cell viability was evaluated by the colorimetric MTT assay. Cell adhesion and grown was assess with fibrinogen-coated well plates and Wound Healing Assay Kit. Reactive oxygen species levels were measured by DCFH-DA and by Flow Cytometry. RNA was isolated with TRIZOL® Reagent and analyzed by Real-Time RT-PCR. Results The treatment of chondrocytes with 400 and 720 nM of MASL did not affect cell viability, adhesion or growth. To mimic pathological conditions, cells and cartilage explants were treated with 5 μg/ml oligomycin. Treatment of chondrocytes with oligomycin did not affect cell viability but increased ROS levels over 10 fold and MMP3, IL-6 and COX2 mRNA levels over 3-10 folds. The treatment of cells with MASL effectively protected chondrocytes from ROS production when incubated in the presence of oligomycin. Moreover, oligomycin induced the expression of inflammatory cytokines including IL-6 and COX2, and this induction was reverted by treatment with nanomolar concentrations of MASL. 5 μg/ml of oligomycin for 7 days decreased safranin uptake and disrupted the ECM structure of cartilage punches as evidenced by ulceration increasing lacunae space. However, the presence of 400 nM of MASL prevented the cartilage destruction and inhibited COX2 and MMP3 induction by oligomycin treatment. Immunohistochemistry assays revealed that OA cartilage contained significantly higher levels of PDPN protein in comparison with cartilage from healthy donors. Conclusions This study demonstrates that physiologically relevant concentrations of MASL protect chondrocytes from detrimental effects of ROS, inflammatory cytokines and MMPs and preserve chondrocyte phenotype and articular cartilage structure under pathological conditions. Disclosure of Interest None declared