Human Articular Chondrocytes Express Multiple Gap Junction Proteins

• DOI: 10.1016/j.ajpath.2012.12.018
• Publication Date: 2013-02-13T08:47:19Z
• Authors: Maria D. Mayan; Paula Carpintero-...; Raquel Gago-Fuentes; Oskar Martinez-de...; Hong-Zhang Wang; Virginijus Valiunas; Peter Brink; Francisco J. Blanco
• Keywords: Adult; Aged, 80 and over; Cartilage, Articular; Cell Nucleus; 0301 basic medicine; Gap Junctions; Middle Aged; Connexins; Pathology and Forensic Medicine; 03 medical and health sciences; Chondrocytes; Gene Expression Regulation; Connexin 43; Proliferating Cell Nuclear Antigen; Osteoarthritis; Humans; Cells, Cultured; Aged

Osteoarthritis (OA) is the most common joint disease and involves progressive degeneration of articular cartilage. The aim this study was to investigate if chondrocytes from human cartilage express gap junction proteins called connexins (Cxs). We show that in tissue Cx43, Cx45, Cx32, Cx46. also find primary adults retain capacity form functional voltage-dependent junctions. Immunohistochemistry experiments OA patients revealed significantly elevated levels Cx43 Cx45 superficial zone down through next approximately 1000 μm tissue. These zones corresponded with regions damaged had high proliferative cell nuclear antigen. An increased number Cxs may help explain proliferation cells clusters finally lead homeostasis loss. Conversely, reflect adjacent are able interact directly by junctions as compared hemichannels on single normal Our data provide strong evidence have a loss usual ordered distribution reductions accompanied correct Cx localization nondamaged areas. surfaces articulating bones covered cartilage, which required for smooth painless movement skeleton. condition characterized degradation matrix components leads mobility function chronic pain. disorder Western populations, its incidence increases age. molecular mechanisms regulating pathogenesis progression OA, however, poorly understood, no proven disease-modifying therapy currently available. In adult remain resting nonproliferating state, but display moderate metabolic activity ability maintain surrounding matrix. Chondrocytes isolated inside their lacunae communication between middle deeper layers occurs diffusion, although it has been reported cultured animal located both Cx43,1Donahue H.J. Guilak F. Vander Molen M.A. McLeod K.J. Rubin C.T. Grande D.A. Brink P.R. mature junctions.J Bone Miner Res. 1995; 10: 1359-1364Crossref PubMed Scopus (66) Google Scholar, 2Knight M.M. McGlashan S.R. Garcia M. Jensen C.G. Poole C.A. Articular connexin 43 P2 receptors — putative mechanoreceptor complex involving cilium?.J Anat. 2009; 214: 275-283Crossref (108) 3Schwab W. Hofer A. Kasper Immunohistochemical rats mice.Histochem J. 1998; 30: 413-419Crossref (44) 4D'Andrea P. Vittur Propagation intercellular Ca2+ waves mechanically stimulated chondrocytes.FEBS Lett. 1997; 400: 58-64Abstract Full Text PDF (61) 5Jones S.J. Gray C. Sakamaki H. Arora Boyde Gourdie R. Green size bone rat calvaria.Anat Embryol (Berl). 1993; 187: 343-352Crossref (115) 6Stains J.P. Civitelli Gap skeletal development function.Biochim Biophys Acta. 2005; 1719: 69-81Crossref (123) Scholar protein forms (GJ) channels implicated cell-to-cell communication. Maintenance homeostatic balance controlled extracellular networks. GJs membrane often assemble large rafts identified plaques at surface. Vertebrate GJ composed encoded (Cx) gene family consist connect cytoplasm cells.7Sohl G. Willecke K. family.Cardiovasc 2004; 62: 228-232Crossref (788) addition role providing pathway direct communication, involved release exchange small molecules matrix.8Burra S. Jiang J.X. Regulation cellular hemichannels.Int J Biochem Mol Biol. 2011; 2: 119-128PubMed Cells can interchange molecules, such metabolites (eg, ATP), nutrients glucose), second messengers IP3, Ca2+, or AMPc,), even synthetic oligonucleotides weight 2 4 kDa. ultimately regulate transcription cell. Historically speaking, were first described those responsible electrical synapses central nervous system, they subsequently found tissues. Actually, functioning differentiation.9Laird D.W. Life cycle health disease.Biochem 2006; 394: 527-543Crossref (648) There 21 expressed humans, each play unique roles not interchangeable.10Plum Hallas Magin T. Dombrowski Hagendorff Schumacher B. Wolpert Kim Lamers W.H. Evert Meda Traub O. Unique shared functions different mice.Curr 2000; 1083-1091Abstract (244) For example, liver, serve heart conduct signals. multiple impairment expression several diseases.9Laird alterations, changes occur related channels. Correct cell-matrix cell-cell essential structural integrity any addition, many other types cell, including catenins claudins, activate pathways. levels, assembly characteristic variety Cx-dysregulated diseases.11Talhouk R.S. Zeinieh M.P. Mikati El-Sabban M.E. junctional hypoxia-ischemia-induced neuronal injury.Prog Neurobiol. 2008; 84: 57-76Crossref (43) 12Wang C.M. Lincoln Cook J.E. Becker D.L. Abnormal underlies delayed wound healing diabetic skin.Diabetes. 2007; 56: 2809-2817Crossref (139) 13Fernandez-Cobo Gingalewski De Maio Expression kidneys lungs injected bacterial lipopolysaccharide.Shock. 97-102Crossref 14Toubas J, Beck S, Pageaud AL, Huby AC, Mael-Ainin M, Dussaule JC, Chatziantoniou C, Chadjichristos CE: Alteration an early signal kidney disease. Am Physiol Renal 301:F24–F32Google 15De Vuyst E, Boengler K, Antoons G, Sipido KR, Schulz R, Leybaert L: Pharmacological modulation connexin-formed cardiac pathophysiology. Br Pharmacol 163:469–483Google 16Green C.R. Nicholson L.F. Interrupting inflammatory diseases do answer?.Cell Biol Int. 32: 1578-1583Crossref (16) work contain results presented here demonstrate Cx46, Cx32 all Primary Remarkably, we higher lost mid deep zones. Human knee femoral head donors obtained after surgery. All signed informed consent Institutional Ethics Committee (Galicia, Spain) approved study. Cartilage healthy persons who suffered hip fracture following same procedure. Histological samples (from subjects radiological diagnoses) graded using modified Mankin score17Pascual Garrido Hakimiyan A.A. Rappoport L. Oegema T.R. Wimmer Chubinskaya Anti-apoptotic treatments prevent acute trauma ankle cartilage.Osteoarthritis Cartilage. 17: 1244-1251Abstract (51) (mean age, 70.6 years; range, 60 89 score, 0 13 points). A score corresponds lower (patients age-matched subjects) divided into groups: i) 1 indicates normal/healthy, ii) 3 grade iii) 5 mild iv) 6 indicated grades (6 7) (8 13) severe OA. Samples RNA isolation immediately frozen liquid nitrogen stored −80°C. previously described.18Ruiz-Romero Carreira V. Rego I. Remeseiro Lopez-Armada M.J. Blanco F.J. Proteomic analysis osteoarthritic reveals stress glycolysis.Proteomics. 8: 495-507Crossref (93) seeded onto 100 162 cm2 flasks assays. Dulbecco's Eagle's medium (Life Technologies Ltd, Madrid, supplemented μg/mL Primocin (Nucliber, 15% fetal calf serum Ltd). grown 90% confluence. Cultured recovered trypsinization Frozen digested digestion buffer (200 mmol/L Tris-HCl, 200 NaCl, 5% SDS, sodium citrate) containing proteinase TRIzol Reagent (Invitrogen, Paisley, UK) added sample according manufacturer's instructions. Whole treated DNase (RNase-free DNase; Invitrogen, instructions ensure total DNA sample. situ Cryomold Standard Tissue-Tek OCT Compound (Sakura, Japan) isopentanol (BDH, Poole, sections serially sectioned (4 μm) −20°C Cryostat (Leica CM1510). Tissue fixed acetone (BDH) 10 minutes 4°C, dried room temperature, washed PBS 0.1% Tween 20, pH7.6 (PBST). Before staining, endogenous peroxidase inhibited H2O2 methanol one time PBST. antibody applied hour temperature. After three washes PBST, incubated peroxidase-labeled polymer conjugated goat anti-mouse/rabbit Ig (Dako, Glostrup, Denmark) hour. developed substrate chromogen solution prepared freshly 3,3 diaminobenzidine tetrahydrochloride H2O2, (Dako). Sections then distilled water counterstained Gill's hematoxylin, gradually dehydrated alcohols, mounted xylene DePeX (SERVA, Heidelberg, Germany). chamber slides PBST before performing procedure described. Negative controls (omitting antibody) performed test specificity antibody. To morphology verify disease, stained H&E, Safranin O, Fast Green, Masson's trichrome, Alcian Blue-PAS, Sirius red polarization method, toluidine blue. imaged Olympus BX61 microscope DP71 digital camera (Olympus Biosystems, Hamburg, antibodies procured followed: (ab66020) Abcam (Cambridge, UK), anti-Cx46 (sc-365394) Santa Cruz Biotechnology (Santa Cruz, CA), anti-Cx45 (MAB3101), anti–collagen-II (MAB1330) Millipore Iberica (Madrid, Spain), anti-Cx43 (610062) BD Transduction Laboratories anti-proliferating antigen (PCNA) (NA03) Calbiochem Spain). Calibration quantification images AnalySISD software version 5.0 genes ion transport analyzed electronic Northerns (E-Northerns). E-Northerns retrieved sequences dbEST (Database Expressed Sequence Tags) division GenBank identification BLAST (National Center Information, Bethesda, MD). search analyses reference query database, limiting matches unequivocally either best-characterized libraries pools 5000 sequence clones cartilage.19Kumar Connor J.R. Dodds R.A. Halsey Van Horn Mao Sathe Mui Agarwal Badger A.M. Lee J.C. Gowen Lark M.W. Identification initial characterization sequenced tags (ESTs) cDNA libraries.Osteoarthritis 2001; 9: 641-653Abstract (52) Putative differences detected transport. transcripts corresponding p21 p27 regard transmembrane transport, overexpression GRID2 (glutamate receptor) GRIA2 albeit relatively low levels. DPP10 (a known regulator Kv4 channels) Several channel representative CDKN1A (p21), CDKN1B (p27), DPP10, GRIA4, GRID2, GABARAP, GABARAPL2, GABRG2. Cell lysates confluent monolayer cells. Equal amounts resolved 10% SDS-PAGE transferred nitrocellulose (Bio-Rad, membranes.18Ruiz-Romero Experiments pairs. dual voltage-clamp method whole-cell and/or perforated patch recording used control potential measure currents described.20Valiunas Gemel Beyer E.C. formed coexpressed connexin40 connexin43.Am Heart Circ Physiol. 281: H1675-1689PubMed 21Valiunas Cardiac quantitative selectivity.Circ 2002; 91: 104-111Crossref (128) Dye transfer investigated pairs Lucifer yellow (Molecular Probes; Ltd).21Valiunas GraphPad Prism (La Jolla, CA). Statistical groups assessed Student's t-test Kruskal-Wallis Dunn's Multiple Comparison test. Significant represented P < 0.05 0.01. Data means ± SEM. Despite limitations chondrocyte cultures, these served useful model synthesize collagen type 2. With time, become more fibroblastic down-regulate synthesis. hypothesis maintained original phenotype culture our assays, overall structure assayed immunohistochemistry (IHC) (Figure 1A). Quantitative real-time RT-PCR very mRNA 1B). (intron-exon junctions; transcripts; Cx43p) processed (exon-exon; mRNA; Cx43m) blot showed comparison line expressing 1C). membrane, IHC coupled hematoxylin counterstaining monoclonal 1D). mainly localized cytoplasm, could detect cytoplasms positive spots edge cytoplasm. electrically channels, conducted whole-cell/perforated currents. displayed Figure 2A current recordings typical behavior pattern Cx43. When joints, only 2B). mRNA, did observe experiments. Analysis gap-junction-permeable dye communicate 2C). Quantification PCR demonstrated within expresses (both transcript) 3A). differs 1B), case tissue, transcript (Cx43p). rule out alternative splicing, primer (Table 1) against Tag database. examined antibody, observed 3, B C). population immunopositive mid-layers similar (at 100% 80% population, respectively). margin might correspond connections lacunae. per edges varied (mean, 6.8).Table 1Sequences Primers Used Gene 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 products bp.m, p, transcript. Open table new tab bp. m, patients, nucleus around 3D). followed signals Biosystems) 3E) staining (approximately 40 times than healthy) layer well mid-region μm. zones, intermediate (where less damaged), lacuna 20% 30% population. when signals, instead injury D). damage severity rated (see Materials Methods). area surface predisposed be later 3B). or, bigger High already stage (grade Overall, 8 36 25.1) 3F). It important note cytoplasmic PCNA 4A). contrast, areas, covering ∼1000 Compared donors, 30-fold 4-fold higher, respectively 3E 4B). intermediate-deep (ie, distribution) Very proliferating exclusively cartilage22Holloway Kayser Bader Bentley Knight Increased presence elongated processes 12: 17-24Abstract (36) 4C).22Holloway chondrocytes, Cx46 5). especially top 5A). petite cells, diffuse areas 5, B), what possibly incorporated 5C). 5D). beneath. Nevertheless, some whereas others negative Unlike distinctive patterns This demonstrates altered gating. electrophysiological techniques Studies structures look like pearl necklaces Within immersed because there usually lacuna, therefore hemichannels. tubular canals (termed canaculi tracks) tissue,23Gonzalez Fragoso-Soriano R.J. Kouri J.B. interconnecting tracks projections – transmission electron microscopy, atomic force two-photon excitation microscopy studies.Microsc Res Tech. 70: 1072-1078Crossref (9) together unpublished group suggests possible being formed. Direct cell–cell paired suggested.24Chi S.S. Rattner Matyas Communication cartilage.J 205: 363-370Crossref (53) Single allow matrix, wide mammalian lines.25Saez Schalper KA, Retamal MA, Orellana JA, Shoji KF, Bennett MV: permeabilization via living dying Exp 316:2377–2389Google ATP, glutamate, NAD+ space,26Stout C.E. Costantin J.L. Naus C.C. Charles A.C. Intercellular calcium signaling astrocytes ATP hemichannels.J Chem. 277: 10482-10488Crossref (738) 27Bruzzone Guida Zocchi E. Franco Flora Connexin hemi mediate Ca2+-regulated NAD