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

Abstract

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. 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. The surfaces of articulating bones are covered by articular cartilage, which is required for the smooth and painless movement of the skeleton. Osteoarthritis (OA) is a condition that is characterized by the progressive degradation of matrix components that leads to a loss of joint mobility and function accompanied by chronic pain. OA is the most common joint disorder in Western populations, and its incidence increases with age. The molecular mechanisms regulating the pathogenesis and progression of OA, however, are poorly understood, and no proven disease-modifying therapy is currently available. In adult cartilage, the chondrocytes remain resting in a nonproliferating state, but display moderate metabolic activity and the ability to maintain the surrounding matrix. Chondrocytes are isolated inside their lacunae and the communication between chondrocytes in the superficial zone and chondrocytes in the middle and deeper layers occurs through diffusion, although it has been reported that cultured animal primary chondrocytes and the chondrocytes located in the superficial zone of adult cartilage both express Cx43,1Donahue H.J. Guilak F. Vander Molen M.A. McLeod K.J. Rubin C.T. Grande D.A. Brink P.R. Chondrocytes isolated from mature articular cartilage retain the capacity to form functional gap junctions.J Bone Miner Res. 1995; 10: 1359-1364Crossref PubMed Scopus (63) Google Scholar, 2Knight M.M. McGlashan S.R. Garcia M. Jensen C.G. Poole C.A. Articular chondrocytes express connexin 43 hemichannels and P2 receptors — a putative mechanoreceptor complex involving the primary cilium?.J Anat. 2009; 214: 275-283Crossref PubMed Scopus (104) Google Scholar, 3Schwab W. Hofer A. Kasper M. Immunohistochemical distribution of connexin 43 in the cartilage of rats and mice.Histochem J. 1998; 30: 413-419Crossref PubMed Scopus (42) Google Scholar, 4D’Andrea P. Vittur F. Propagation of intercellular Ca2+ waves in mechanically stimulated articular chondrocytes.FEBS Lett. 1997; 400: 58-64Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 5Jones S.J. Gray C. Sakamaki H. Arora M. Boyde A. Gourdie R. Green C. The incidence and size of gap junctions between the bone cells in rat calvaria.Anat Embryol (Berl). 1993; 187: 343-352Crossref PubMed Scopus (113) Google Scholar, 6Stains J.P. Civitelli R. Gap junctions in skeletal development and function.Biochim Biophys Acta. 2005; 1719: 69-81Crossref PubMed Scopus (121) Google Scholar a protein that forms gap junction (GJ) channels and is implicated in cell-to-cell communication. Maintenance of the homeostatic tissue balance is controlled by communication between the extracellular and intercellular networks. GJs are membrane channels that often assemble as large membrane rafts and are identified as plaques at the membrane surface. Vertebrate GJ channels are composed of proteins encoded by the connexin (Cx) gene family and consist of intercellular channels that directly connect the cytoplasm of adjacent cells.7Sohl G. Willecke K. Gap junctions and the connexin protein family.Cardiovasc Res. 2004; 62: 228-232Crossref PubMed Scopus (759) Google Scholar In addition to their role in providing a pathway for direct intercellular communication, hemichannels are also involved in the release and exchange of small molecules between cells and the surrounding matrix.8Burra S. Jiang J.X. Regulation of cellular function by connexin hemichannels.Int J Biochem Mol Biol. 2011; 2: 119-128PubMed Google Scholar Cells can interchange small molecules, such as metabolites (eg, ATP), nutrients (eg, glucose), second messengers (eg, IP3, Ca2+, or AMPc,), and even synthetic oligonucleotides with a molecular weight of approximately 2 to 4 kDa. These small molecules can ultimately regulate transcription in the cell. Historically speaking, GJs were first described as those responsible for the electrical synapses in the central nervous system, however, they were subsequently found in most tissues. Actually, GJ intercellular communication is required for normal cellular functioning and for tissue development and differentiation.9Laird D.W. Life cycle of connexins in health and disease.Biochem J. 2006; 394: 527-543Crossref PubMed Scopus (619) Google Scholar There are 21 expressed Cxs in humans, and each connexin may play unique roles that are not interchangeable.10Plum A. Hallas G. Magin T. Dombrowski F. Hagendorff A. Schumacher B. Wolpert C. Kim J. Lamers W.H. Evert M. Meda P. Traub O. Willecke K. Unique and shared functions of different connexins in mice.Curr Biol. 2000; 10: 1083-1091Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar For example, in the liver, GJs serve metabolic functions and in the heart they conduct electrical signals. Cxs have been implicated in multiple cellular functions and the impairment or loss of Cx expression has been implicated in the pathogenesis of several diseases.9Laird D.W. Life cycle of connexins in health and disease.Biochem J. 2006; 394: 527-543Crossref PubMed Scopus (619) Google Scholar OA is characterized by multiple molecular alterations, and most changes that occur in OA cartilage are directly related to functions controlled by Cx channels. Correct cell-matrix and cell-cell communication is essential to maintain the structural integrity and function of any tissue. In addition, GJ proteins interact with many other types of proteins in the cell, including catenins and claudins, and activate multiple molecular pathways. Actually, changes in Cx protein levels, assembly state, or localization are characteristic of a variety of Cx-dysregulated diseases.11Talhouk R.S. Zeinieh M.P. Mikati M.A. El-Sabban M.E. Gap junctional intercellular communication in hypoxia-ischemia-induced neuronal injury.Prog Neurobiol. 2008; 84: 57-76Crossref PubMed Scopus (43) Google Scholar, 12Wang C.M. Lincoln J. Cook J.E. Becker D.L. Abnormal connexin expression underlies delayed wound healing in diabetic skin.Diabetes. 2007; 56: 2809-2817Crossref PubMed Scopus (131) Google Scholar, 13Fernandez-Cobo M. Gingalewski C. De Maio A. Expression of the connexin 43 gene is increased in the kidneys and the lungs of rats injected with bacterial lipopolysaccharide.Shock. 1998; 10: 97-102Crossref PubMed Scopus (43) Google Scholar, 14Toubas J, Beck S, Pageaud AL, Huby AC, Mael-Ainin M, Dussaule JC, Chatziantoniou C, Chadjichristos CE: Alteration of connexin expression is an early signal for chronic kidney disease. Am J Physiol Renal Physiol 301:F24–F32Google Scholar, 15De Vuyst E, Boengler K, Antoons G, Sipido KR, Schulz R, Leybaert L: Pharmacological modulation of connexin-formed channels in cardiac pathophysiology. Br J Pharmacol 163:469–483Google Scholar, 16Green C.R. Nicholson L.F. Interrupting the inflammatory cycle in chronic diseases — do gap junctions provide the answer?.Cell Biol Int. 2008; 32: 1578-1583Crossref PubMed Scopus (16) Google Scholar The aim of this work was to investigate if adult human chondrocytes express Cx proteins and contain functional gap junctions. The results presented here demonstrate that normal human adult articular chondrocytes express Cx43, Cx45, Cx46, and Cx32 in all zones of adult cartilage. Primary chondrocytes retain the capacity to form functional Cx43 gap junctions. Remarkably, we have found that cartilage from OA patients has higher levels of Cx43 and Cx45 protein in the damaged proliferative zones and that Cx43 localization is lost in the mid and deep nondamaged zones. Human knee and femoral head articular cartilage from adult donors was obtained after joint surgery. All patients signed the informed consent form and the Institutional Ethics Committee (Galicia, Spain) approved the study. Cartilage from healthy persons who suffered a knee or hip fracture was obtained following the same procedure. Histological samples (from healthy and OA subjects with radiological diagnoses) were graded using a modified Mankin score17Pascual Garrido C. Hakimiyan A.A. Rappoport L. Oegema T.R. Wimmer M.A. Chubinskaya S. Anti-apoptotic treatments prevent cartilage degradation after acute trauma to human ankle cartilage.Osteoarthritis Cartilage. 2009; 17: 1244-1251Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar (mean age, 70.6 years; range, 60 to 89 years; Mankin score, 0 to 13 points). A higher Mankin score corresponds to a lower structural integrity of the tissue. The samples (patients and age-matched healthy subjects) were divided into 4 groups: i) 0 to 1 indicates normal/healthy, ii) 2 to 3 indicates grade 1 early OA, iii) 4 to 5 indicates grade 2 mild OA, and iv) 6 to 13 indicated grades 3 (6 to 7) and 4 (8 to 13) severe OA. Samples for RNA isolation were immediately frozen in liquid nitrogen and stored at −80°C. Primary chondrocytes were isolated as previously described.18Ruiz-Romero C. Carreira V. Rego I. Remeseiro S. Lopez-Armada M.J. Blanco F.J. Proteomic analysis of human osteoarthritic chondrocytes reveals protein changes in stress and glycolysis.Proteomics. 2008; 8: 495-507Crossref PubMed Scopus (91) Google Scholar Cells were seeded onto 100 or 162 cm2 flasks for gene expression assays. Chondrocytes were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies Ltd, Madrid, Spain) supplemented with 100 μg/mL Primocin (Nucliber, Madrid, Spain) and 15% fetal calf serum (Life Technologies Ltd). Cells were grown to approximately 90% confluence. Cultured primary chondrocytes were recovered by trypsinization and stored at −80°C. Frozen articular cartilage samples were digested in digestion buffer (200 mmol/L Tris-HCl, 200 mmol/L NaCl, 5% SDS, and 100 mmol/L sodium citrate) containing proteinase K. TRIzol Reagent (Invitrogen, Paisley, UK) was added to the sample according to the manufacturer’s instructions. Whole RNA was treated with DNase (RNase-free DNase; Invitrogen, Madrid, Spain) according to the manufacturer’s instructions to ensure total degradation of the DNA in the sample. In situ cartilage was frozen immediately in a Cryomold Standard using Tissue-Tek OCT Compound (Sakura, Japan) and isopentanol (BDH, Poole, UK) in liquid nitrogen and stored at −80°C. Cartilage sections were serially sectioned (4 μm) at −20°C in a Cryostat (Leica CM1510). Tissue sections were fixed with acetone (BDH) for 10 minutes at 4°C, dried at room temperature, and washed for 10 minutes with PBS with 0.1% Tween 20, pH7.6 (PBST). Before staining, endogenous peroxidase was inhibited by H2O2 and methanol for 10 minutes and was washed one time with PBST. Primary antibody was applied 1 hour at room temperature. After three washes of 10 minutes with PBST, sections were incubated with a peroxidase-labeled polymer conjugated to goat anti-mouse/rabbit Ig (Dako, Glostrup, Denmark) for 1 hour. After three washes with PBST, peroxidase activity was developed using a substrate chromogen solution prepared freshly and containing 3,3 diaminobenzidine tetrahydrochloride and H2O2, (Dako). Sections were then washed in distilled water and counterstained with Gill’s hematoxylin, gradually dehydrated with graded alcohols, and mounted in xylene with DePeX (SERVA, Heidelberg, Germany). Cultured cells were seeded onto chamber slides and fixed with acetone for 10 minutes at 4°C, dried at room temperature, and washed for 10 minutes with PBST before performing the procedure previously described. Negative controls (omitting primary antibody) were performed to test the specificity of the antibody. To study cartilage morphology and verify the grade of the disease, cartilage sections were stained with H&E, Safranin O, Fast Green, Masson’s trichrome, Alcian Blue-PAS, Sirius red polarization method, and toluidine blue. The slides were imaged using an Olympus BX61 microscope and a DP71 digital camera (Olympus Biosystems, Hamburg, Germany). The antibodies were procured as followed: Cx32 antibody (ab66020) from Abcam (Cambridge, UK), anti-Cx46 (sc-365394) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-Cx45 (MAB3101), and anti–collagen-II (MAB1330) from Millipore Iberica (Madrid, Spain), anti-Cx43 (610062) from BD Transduction Laboratories (Madrid, Spain), and anti-proliferating cell nuclear antigen (PCNA) (NA03) from Calbiochem (Madrid, Spain). Calibration and quantification of the images was performed with AnalySISD software version 5.0 (Olympus Biosystems, Hamburg, Germany). Expression of different genes related to cell cycle and ion transport in cartilage was analyzed by electronic Northerns (E-Northerns). E-Northerns retrieved expression data from DNA sequences stored in the dbEST (Database of Expressed Sequence Tags) division of GenBank through identification using the BLAST software (National Center for Biotechnology Information, Bethesda, MD). To search for genes expressed in normal or OA cartilage, BLAST analyses were performed using reference sequences of query genes in the GenBank dbEST database, limiting the results to matches unequivocally identified as either normal or OA cartilage. The best-characterized libraries were obtained from pools of RNA with approximately 5000 sequence clones from normal and OA cartilage.19Kumar S. Connor J.R. Dodds R.A. Halsey W. Van Horn M. Mao J. Sathe G. Mui P. Agarwal P. Badger A.M. Lee J.C. Gowen M. Lark M.W. Identification and initial characterization of 5000 expressed sequenced tags (ESTs) each from adult human normal and osteoarthritic cartilage cDNA libraries.Osteoarthritis Cartilage. 2001; 9: 641-653Abstract Full Text PDF PubMed Scopus (51) Google Scholar Putative differences in gene expression between normal and osteoarthritic cartilage were detected for several genes related to cell cycle and ion transport. The number of transcripts corresponding to p21 and p27 were significantly elevated in normal cartilage compared to OA. In regard to ion and transmembrane transport, overexpression of GRID2 (glutamate receptor) was detected in normal cartilage. GRIA2 was also detected in normal cartilage, albeit at relatively low levels. DPP10 (a known regulator of Kv4 channels) was detected in normal cartilage but not in OA. Several cell cycle and ion channel genes are expressed in normal and OA. The most representative differences found between normal and OA cartilage corresponded to CDKN1A (p21), CDKN1B (p27), DPP10, GRIA4, GRID2, GABARAP, GABARAPL2, and GABRG2. Cell lysates were prepared from confluent monolayer cells. Equal amounts of cellular protein were resolved by 10% SDS-PAGE and transferred to nitrocellulose (Bio-Rad, Madrid, Spain) membranes.18Ruiz-Romero C. Carreira V. Rego I. Remeseiro S. Lopez-Armada M.J. Blanco F.J. Proteomic analysis of human osteoarthritic chondrocytes reveals protein changes in stress and glycolysis.Proteomics. 2008; 8: 495-507Crossref PubMed Scopus (91) Google Scholar Experiments were performed on cell pairs. A dual voltage-clamp method and whole-cell and/or perforated patch recording were used to control the membrane potential of both cells and to measure currents as previously described.20Valiunas V. Gemel J. Brink P.R. Beyer E.C. Gap junction channels formed by coexpressed connexin40 and connexin43.Am J Physiol Heart Circ Physiol. 2001; 281: H1675-1689PubMed Google Scholar, 21Valiunas V. Beyer E.C. Brink P.R. Cardiac gap junction channels show quantitative differences in selectivity.Circ Res. 2002; 91: 104-111Crossref PubMed Scopus (128) Google Scholar Dye transfer through gap junctions was investigated using cell pairs and Lucifer yellow (Molecular Probes; Life Technologies Ltd).21Valiunas V. Beyer E.C. Brink P.R. Cardiac gap junction channels show quantitative differences in selectivity.Circ Res. 2002; 91: 104-111Crossref PubMed Scopus (128) Google Scholar The data were analyzed using the GraphPad Prism software version 5 (La Jolla, CA). Statistical differences between sample groups were assessed using the Student’s t-test and Kruskal-Wallis test with Dunn’s Multiple Comparison test. Significant differences are represented as P < 0.05 and P < 0.01. Data are represented as the means ± SEM. Despite the limitations of monolayer chondrocyte cultures, these cells have served as a useful model for the study of cartilage. Chondrocytes synthesize high levels of collagen type 2. With time, cultured chondrocytes become more fibroblastic and down-regulate type 2 collagen synthesis. To test the hypothesis that these cells maintained their original phenotype in culture at the time of our assays, collagen type 2 levels and overall cellular structure were assayed by immunohistochemistry (IHC) (Figure 1A). Quantitative gene expression analysis using real-time RT-PCR revealed that human primary chondrocytes express very high levels of Cx43 mRNA (Figure 1B). We analyzed both primary transcription (intron-exon junctions; primary transcripts; Cx43p) and the processed RNA (exon-exon; mRNA; Cx43m) (Figure 1B). Western blot analysis also showed high levels of Cx43 protein in comparison with a cell line expressing Cx43 (Figure 1C). To test if Cx43 is found in the membrane, we used IHC coupled with Gill’s hematoxylin counterstaining using a monoclonal anti-Cx43 antibody (Figure 1D). Cx43 was mainly localized in the cytoplasm, but we could detect Cx43 between the cytoplasms of 2 adjacent cells. We also detected multiple positive spots at the edge of the cytoplasm. To test if human primary chondrocytes are electrically coupled to each other by voltage-dependent GJ channels, we conducted a dual voltage-clamp method and whole-cell/perforated patch recording to control the membrane potential of both cell pairs and to measure currents. The results displayed in Figure 2A show the junctional current recordings corresponding to the typical voltage-dependent behavior pattern described for GJ channels containing Cx43. When we investigated the expression of different Cxs using primary chondrocytes from healthy joints, we only detected the expression of Cx43 and Cx45 (Figure 2B). We could detect low levels of Cx45 mRNA, although we did not observe any corresponding Cx45 protein signal in our IHC experiments. Analysis of the typical gap-junction-permeable dye Lucifer yellow revealed that primary chondrocytes communicate and transfer molecules through Cx channels (Figure 2C). RNA was isolated from frozen sections of cartilage tissue. Quantification using real-time PCR demonstrated that chondrocytes within cartilage expresses Cx43 mRNA (both mRNA and primary transcript) (Figure 3A). The pattern of expression differs from cultured chondrocytes (Figure 1B), however, as in the case of tissue, we could detect both mRNA and primary transcript (Cx43p). To rule out alternative splicing, the primer sequences (Table 1) were compared against the GenBank Expressed Sequence Tag database. When we examined protein localization by IHC using an anti-Cx43 antibody, we observed multiple positive Cx43 spots in all layers of the healthy cartilage, including the deep zone (Figure 3, B and C). The population of immunopositive chondrocytes in the superficial and mid-layers of healthy cartilage was similar to that of the deeper layers (at 90% to 100% and 80% to 90% of the cell population, respectively). We found that Cx43 is localized at the margin of cells. We also detected positive spots that might correspond to connections between chondrocytes located in the same lacunae. In case of healthy cartilage, the number of positive spots per cell that were located in the edges of the lacunae varied between 4 and 10 (mean, 6.8).Table 1Sequences of the Primers Used for Quantitative Gene Expression AnalysisGeneTargetPrimer sequenceGJA1Cx43m5′-CAATCACTTGGCGTGACTTC-3′5′-AACGAAAGGCAGACTGCTCA-3′Cx43p5′-TTGCAATCTGTGATCCTTGAA-3′5′-AACGAAAGGCAGACTGCTCA-3′GJC1Cx45m5′-ATCTGGAAAAATTGCAATCAAAA-3′5′-CTGTAAGGACGATCCGGAAG-3′Cx45p5′-TGGGTAACCGAAGTTCTGGA-3′5′-TCCAGGATCCAGGTAAAAACC-3′GJA3Cx46m5′-GCCGGCCAGTACTTTCTGTA-3′5′-CCTGCTTGAGCTTCTTCCAG-3′Cx46p5′-ATGCCTGTCCTGTGGAGAAG-3′5′-GAAGATGAACAGCACGGTCA-3′GJB2Cx26m5′-CTTCCTCCCGACGCAGAG-3′5′-GCTCATCTCCCCACACCTC-3′GJA5Cx40m5′-AAGTCCAGGGAGGAGGAAAA-3′5′-TCGTATCACACCGGAAATCA-3′GJB1Cx32m5′-TGCAGACATTCTCTGGGAAA-3′5′-TCTCATCACCCCACACACTC-3′HPRT-15′-TTGAGTTTGGAAACATCTGGAG-3′5′-GCCCAAAGGGAACTGATAGTC-3′The PCR products size is approximately 200 bp.m, mRNA; p, primary transcript. Open table in a new tab The PCR products size is approximately 200 bp. m, mRNA; p, primary transcript. In the case of cartilage from OA patients, Cx43 was found in both the nucleus and the cytoplasm, with positive spots around the edges of the lacunae (Figure 3D). Analysis of Cx43 using IHC followed by quantification of positive signals using the AnalySISD software version 5.0 (Olympus Biosystems) (Figure 3E) revealed that cartilage from OA donors showed very high levels of Cx43 positive staining (approximately 40 times more than healthy) in the 80% to 90% of cells in the superficial layer as well as in 100% of the cells in the mid-region down through 1000 μm. The protein localization was the same in both the superficial and damaged zones, however, in the intermediate and deeper zones (where the tissue was less damaged), we detected positive spots in the edges of the lacuna in only 20% to 30% of the cell population. In the deeper zones, when the cells did show positive signals, the Cx43 was mainly localized in the cytoplasm instead of the membrane (Figure 3D). Cartilage from OA donors showed a different grade of injury (Figure 3, B and D). Cartilage damage severity was rated (see Materials and Methods). The area around the articular surface and down to 100 to 1000 μm is predisposed to be damaged in early and later grades of the disease (Figure 3B). These zones either showed severe damage or, in the case of patients with grade 1 disease, bigger lacunae. High levels of positive staining were already detected in the first stage of OA disease (grade 1) (Figure 3D). Overall, in case of OA cartilage, the number of positive spots per cell that were located in the edges of the lacunae varied between 8 and 36 (mean, 25.1) (Figure 3F). It is important to note that we observed low levels of cytoplasmic PCNA in cartilage from healthy donors (Figure 4A). In contrast, cartilage from OA patients showed high levels of nuclear PCNA in the damaged areas, covering the superficial layer and down through ∼1000 μm (Figure 4A). Compared to tissue from healthy donors, the levels of Cx43 and PCNA in OA tissue were 30-fold and 4-fold higher, respectively (Figure 3E and 4B). In contrast, the less damaged intermediate-deep zone cartilage showed a PCNA staining pattern similar to healthy cartilage (ie, lower levels and a more cytoplasmic PCNA distribution) (Figure 4A). Very high levels of Cx43 were detected in proliferating cells that are exclusively found in the superficial layer of OA cartilage22Holloway I. Kayser M. Lee D.A. Bader D.L. Bentley G. Knight M.M. Increased presence of cells with multiple elongated processes in osteoarthritic femoral head cartilage.Osteoarthritis Cartilage. 2004; 12: 17-24Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar (Figure 4C).22Holloway I. Kayser M. Lee D.A. Bader D.L. Bentley G. Knight M.M. Increased presence of cells with multiple elongated processes in osteoarthritic femoral head cartilage.Osteoarthritis Cartilage. 2004; 12: 17-24Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar In cultured chondrocytes, we detected Cx45 mRNA, but no corresponding protein (Figure 2B). IHC of cartilage from healthy joints, however, showed positive signals for Cx45, Cx32, and Cx46 in all zones (Figure 5). Cx45 protein was detected in all healthy cartilage zones, especially in the superficial layer through the top 1000 μm (Figure 5A). Cx45 formed petite positive spots around the margin of the cells, but it was diffuse in the cytoplasm (Figure 5A). In contrast, Cx45 in OA cartilage showed significantly more positive staining in the superficial and damaged areas (Figure 5, A and B), similar to what was previously observed for Cx43 and PCNA (Figure 3E and 4B). In case of OA joints, Cx45 localized in positive spots around the margin of the cells and in the nucleus but was diffuse in the cytoplasm (Figure 5A). In the case of Cx32 and Cx46, these proteins localized around the edges of the cell and were possibly incorporated into the membrane (Figure 5C). In cartilage from patients, Cx32 was mainly found in the damaged zones and in the intermediate cartilage (Figure 5D). Cx46, however, was mainly detected in the superficial zone and in the 1000 μm of tissue immediately beneath. Nevertheless, some OA patients showed higher levels of Cx32 and/or Cx46, whereas others were negative for both Cxs (Figure 5D). Unlike Cx43 and Cx45, we could not find distinctive patterns for either Cx32 or Cx46. This study demonstrates for the first time that normal human adult articular chondrocytes express several GJ proteins that are altered in OA. We have also demonstrated that human articular chondrocytes from adults retain the capacity to form functional GJs with voltage-dependent gating. The characteristic junctional current pattern obtained using electrophysiological techniques demonstrate that adult chondrocytes form functional GJs composed by Cx43. Studies conducted in tissue demonstrate that Cxs are well localized around the cell and form structures that look like pearl necklaces around the edge of the lacunae. Within tissue, chondrocytes are immersed in lacunae and because there is usually one cell per lacuna, most of these positive spots could therefore correspond to hemichannels. The potential presence of tubular canals (termed canaculi tracks) in the tissue,23Gonzalez S. Fragoso-Soriano R.J. Kouri J.B. Chondrocytes interconnecting tracks and cytoplasmic projections observed within the superficial zone of normal human articular cartilage – a transmission electron microscopy, atomic force microscopy, and two-photon excitation microscopy studies.Microsc Res Tech. 2007; 70: 1072-1078Crossref PubMed Scopus (9) Google Scholar however, together with unpublished results from our group suggests that it is also possible that GJs are being formed. Direct cell–cell communication between paired chondrocytes have already been suggested.24Chi S.S. Rattner J.B. Matyas J.R. Communication between paired chondrocytes in the superficial zone of articular cartilage.J Anat. 2004; 205: 363-370Crossref PubMed Scopus (50) Google Scholar Single hemichannels allow cells to interchange components with the matrix, and Cx43 has been reported to form functional hemichannels in a wide variety of mammalian cell lines.25Saez JC, Schalper KA, Retamal MA, Orellana JA, Shoji KF, Bennett MV: Cell membrane permeabilization via connexin hemichannels in living and dying cells. Exp Cell Res 316:2377–2389Google Scholar Cx43 hemichannels were involved in the release of ATP, glutamate, or NAD+ in the extracellular space,26Stout C.E. Costantin J.L. Naus C.C. Charles A.C. Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels.J Biol Chem. 2002; 277: 10482-10488Crossref PubMed Scopus (723) Google Scholar, 27Bruzzone S. Guida L. Zocchi E. Franco L. De Flora A. Connexin 43 hemi channels mediate Ca2+-regulated transmembrane NAD