R. Gago-Fuentes

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.

The C-terminal domain of connexin43 modulates cartilage structure via chondrocyte phenotypic changes

Chondrocytes in cartilage and bone cells population express connexin43 (Cx43) and gap junction intercellular communication (GJIC) is essential to synchronize cells for coordinated electrical, mechanical, metabolic and chemical communication in both tissues. Reduced Cx43 connectivity decreases chondrocyte differentiation and defective Cx43 causes skeletal defects. The carboxy terminal domain (CTD) of Cx43 is located in the cytoplasmic side and is key for protein functions. Here we demonstrated that chondrocytes from the CTD-deficient mice, K258stop/Cx43KO and K258stop/K258stop, have reduced GJIC, increased rates of proliferation and reduced expression of collagen type II and proteoglycans. We observed that CTD-truncated mice were significantly smaller in size. Together these results demonstrated that the deletion of the CTD negatively impacts cartilage structure and normal chondrocyte phenotype. These findings suggest that the proteolytic cleavage of the CTD under pathological conditions, such as under the activation of metalloproteinases during tissue injury or inflammation, may account for the deleterious effects of Cx43 in cartilage and bone disorders such as osteoarthritis.

Normal development of mice lacking PAXX, the paralogue of XRCC4 and XLF

DNA repair consists of several cellular pathways which recognize and repair damaged DNA. The classical nonhomologous DNA end‐joining (NHEJ) pathway repairs double‐strand breaks in DNA. It is required for maturation of both B and T lymphocytes by supporting V(D)J recombination as well as B‐cell differentiation during class switch recombination (CSR). Inactivation of NHEJ factors Ku70, Ku80, XRCC4, DNA ligase 4, DNA‐PKcs, and Artemis impairs V(D)J recombination and blocks lymphocyte development. Paralogue of XRCC4 and XLF (PAXX) is an accessory NHEJ factor that has a significant impact on the repair of DNA lesions induced by ionizing radiation in human, murine, and chicken cells. However, the role of PAXX during development is poorly understood. To determine the physiological role of PAXX, we deleted part of the Paxx promoter and the first two exons in mice. Further, we compared Paxx‐knockout mice with wild‐type (WT) and NHEJ‐deficient controls including Ku80‐ and Dna‐pkcs‐null and severe combined immunodeficiency mice. Surprisingly, Paxx‐deficient mice were not distinguishable from the WT littermates; they were the same weight and size, fertility status, had normal spleen, thymus, and bone marrow. Paxx‐deficient mice had the same number of chromosomal and chromatid breaks as WT mice. Moreover, Paxx‐deficient primary B lymphocytes had the same level of CSR as lymphocytes isolated from WT mice. We concluded that PAXX is dispensable for normal mouse development.

Robust DNA repair in PAXX‐deficient mammalian cells

To ensure genome stability, mammalian cells employ several DNA repair pathways. Nonhomologous DNA end joining (NHEJ) is the DNA repair process that fixes double‐strand breaks throughout the cell cycle. NHEJ is involved in the development of B and T lymphocytes through its function in V(D)J recombination and class switch recombination (CSR). NHEJ consists of several core and accessory factors, including Ku70, Ku80, XRCC4, DNA ligase 4, DNA‐PKcs, Artemis, and XLF. Paralog of XRCC4 and XLF (PAXX) is the recently described accessory NHEJ factor that structurally resembles XRCC4 and XLF and interacts with Ku70/Ku80. To determine the physiological role of PAXX in mammalian cells, we purchased and characterized a set of custom‐generated and commercially available NHEJ‐deficient human haploid HAP1 cells, PAXXΔ, XRCC4Δ, and XLFΔ. In our studies, HAP1 PAXXΔ cells demonstrated modest sensitivity to DNA damage, which was comparable to wild‐type controls. By contrast, XRCC4Δ and XLFΔ HAP1 cells possessed significant DNA repair defects measured as sensitivity to double‐strand break inducing agents and chromosomal breaks. To investigate the role of PAXX in CSR, we generated and characterized Paxx−/− and Aid−/− murine lymphoid CH12F3 cells. CSR to IgA was nearly at wild‐type levels in the Paxx−/− cells and completely ablated in the absence of activation‐induced cytidine deaminase (AID). In addition, Paxx−/− CH12F3 cells were hypersensitive to zeocin when compared to wild‐type controls. We concluded that Paxx‐deficient mammalian cells maintain robust NHEJ and CSR.

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.

FRI0031 Targeting Sialic Acid-Modified Receptors as A Potential Therapy for Osteoarthritis

Background Osteoarthritis (OA) is one of the most common diseases worldwide. Its prevalence and severity increase with age, but treatments only provide symptomatic relief. The articular cartilage consists of a collagen-proteoglycan matrix containing highly glycosylated proteins synthesized by chondrocytes. A notable shift from glycoproteins containing a-2,6-linked sialic acids to those containing a-2,3-linked sialic acids has been associated with progressive cartilage degeneration and with the onset of disorders such as rheumatoid arthritis (RA) and OA1–3. However, the pathophysiology of a-2,3-sialylation in cartilage has not yet been elucidated. Objectives Lectins recognize specific terminal aspects of glycan chains and the Maackia amurensis seed lectin (MASL) is a plant lectin that can bind to sialylated glycoproteins. Here, we study the effects of the lectin MASL on chondrocytes and cartilage integrity from healthy donors, OA patients and animal models of arthritis. Methods Cell viability, cell adhesion and growth were performed using commercial kits. Reactive oxygen species (ROS) levels were measured by DCFH-DA and by Flow Cytometry. Gene expression was analyzed by quantitative real-time PCR. Staining methods were used to study cartilage integrity. Oligomycin and LPS were used to induce cartilage degeneration in vitro and in vivo (human cartilage and mouse model, Mus musculus BALC/c). Results he expression of the a-2,3-sialylated transmembrane mucin receptor podoplanin (PDPN) is induced in cartilage from osteoarthritic patients. Co-immunofluorescence technique showed that MASL can be used to target PDPN. Nanomolar concentrations of MASL protected primary chondrocytes and prevented cartilage breakdown in human tissue from OA patients ex vivo and in an animal model of OA initiated by ROS, inflammatory cytokines, and metalloproteinases. Besides, the increased levels of the α-2,3 sialyltransferase isoforms and the corresponding increase in the levels of a-2,3-sialylated glycoproteins in osteoarthritic chondrocytes may shed mechanistic light on the pathophysiology of OA. These findings based on various experimental models of arthritis reveal that specific lectins that target a-2,3-sialylated transmembrane receptors, such as PDPN on chondrocytes, may effectively inhibit cartilage destruction in the face of various arthritic insults. We also provide a three-dimensional molecular model for such an interaction. Conclusions The ability of MASL to target a-2,3-sialylated glycoproteins, such as PDPN, and to protect chondrocytes from insults leading to cartilage degradation might offer further possibilities for therapeutic interventions and novel arthritis treatments that may include the regulation of sialylation during acute disease stages. References Toegel, S. et al. Arthritis Res Ther 15, R147. (2013). Toegel, S. et al. Osteoarthritis Cartilage 18, 240–248. (2010). Toegel, S. et al. In Vitro Cell Dev Biol Anim 45, 351–360. (2009). Disclosure of Interest None declared

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

OP0250 The Regulatory Role of the C-Terminal Domain of Connexin 43 in Articular Cartilage

Background Gap junction (GJ) channels are composed by a family of proteins called connexins. The ability to synchronize groups of cells for coordinated electrical, mechanical, metabolic and chemical communication make these proteins essential for tissue function. Mutations in connexin-encoding genes lead to developmental of a wide variety of diseases. The inhibition of GJIC in micromass culture of chondrocytes reduces chondrocytes differentiation and defective Cx43 functions cause skeletal defects. Recent results from our group have convincingly demonstrated the involvement of the overexpression of Cx43 in OA pathogenesis. Cx43 has multiple GJ-independent functions that affect signalling pathways, cell growth, and cell proliferation. The carboxy terminal domain (CTD) of Cx43 is located in the cytoplasmic side and is key for protein functions. Objectives The aim of this study was to investigate the role of the CTD of Cx43 in cartilage structure and function. Methods Global knockout of Cx43 is embryonic lethal and homozygous K258stop animals, which Cx43 lacks the last 125 amino acid residues of the CTD, died shortly after birth. Cartilage and primary chondrocytes of 8 wild type, Cx43/KO, 6 K258stop/Cx43, 6 K258stop/KO and 6 K258stop/K258stop (21-day gestational section age mice) were subjected to the study. Mouse genotyping was achieved by PCR using DNA extracted from ear tissue and western-blot. Entire knee joints were stored in formalin-fixed paraffin-embedded sections (decalcified before embedding and sectioning) and analysed by conventional staining methods and immunohistochemistry (IHC) assays. Chondrocytes from cartilage were isolated using an inverted microscope and tissue was digested using trypsin and collagenase. Primary chondrocytes were grown to 80-90% confluence. Proliferation assay was performed by counting cells with using automated cell counter. For IHC cells were seed onto coverslips. The following are the antibodies used in this study: anti-osteoponin, anti-fibronectin, anti-collagen type II, anti-Cx43, anti-PCNA and Ki67. Scrape loading assay was used to examine GJIC Results Analysis of cartilage sections staining with Safranin O/Fast Green, Hematoxylin-eosin eosin and Toluidine blue did not show significant differences between different phenotypes. However the K258stop/KO cartilage was thinner than wild type and Cx43/KO. Besides cartilage from K258stop/KO showed higher rate of positive cells for PCNA. IHC experiments revealed that K258stop/KO chondrocytes in primary culture contain less levels of collagen type II. K258stop/K258stop was found to have 1.5-fold increase in cell proliferation in comparison with wild type or Cx43/KO. Scrape loading assays suggest that the deletion of CTD slightly reduce GJIC. Conclusions We have used a genetically modified murine model to directly characterize, for the first time, the role of the CTD of Cx43 on cartilage structure. Our results strongly support the notion that the CTD of Cx43 plays an important role in chondrocyte phenotype. It is well know that through its CTD, Cx43 serves as scaffolding proteins that associates with structural and signaling molecules leading to regulation of intracellular signaling, independently of channel activity. This study illustrates that a complete isolation of Cx43 from its CTD may have a negative impact on cartilage structure and chondrocyte functions within the tissue. Disclosure of Interest None declared