title: Biochemical evidence for gap junctions and Cx43 expression in immortalized human chondrocyte cell line: a potential model in the study of cell communication in human chondrocytes.
abstract: OBJECTIVE
  The development of chondrocytic cell lines has enabled the investigation of the role of cellular phenotype and mechanisms in articular cartilage biology and physiopathology of several rheumatic diseases. Among them, the T/C-28a2 cell line has become a common tool in cartilage research. Recent results from our group have revealed that primary human chondrocytes in tissue and in monolayer culture contain high levels of connexin 43 (Cx43) and are able to directly communicate through gap junction (GJ) channels. These results challenge the existing thesis of cartilage physiology, that chondrocytes do not have the capacity to physically communicate with each other. Established cell lines offer the advantage of convenience and uniformity; however, the establishment process may cause a disruption of GJ. This study was performed to investigate if T/C-28a2 cells contain Cx43 protein and form functional channels.
  
  
  METHODS
  Cx43 was characterized by RT-qPCR, Western blotting, and immunohistochemistry (IHC). Electrophysiology experiments, Lucifer Yellow (LY) uptake, electroporation in situ and scrape loading assay were performed to test the functionality of GJs.
  
  
  RESULTS
  T/C-28a2 cells express Cx43. Electrophysiology experiments and LY uptake confirmed the capacity of these cells to communicate through GJ channels, although these cells contain significant levels of active c-Src kinase, presumably due to their immortalization with the Simian Virus 40 large T antigen. The results were validated using primary chondrocytes (PC).
  
  
  CONCLUSIONS
  These results reveal that the T/C-28a2 line may provide a useful in vitro model for the study of Cx43 function and cell communication to understand the physiology of chondrocytes and cartilage.
authors: Cartilage Biology Research Group, Rheumatology Division, INIBIC-Hospital Universitario A Coruña, Xubias de Arriba 84, 15006 A Coruña, Spain.
authors: R
authors: R
authors: Gago-Fuentes
authors: Cartilage Biology Research Group, Rheumatology Division, INIBIC-Hospital Universitario A Coruña, Xubias de Arriba 84, 15006 A Coruña, Spain.
authors: P
authors: P
authors: Carpintero-Fernandez
authors: Tissue Engineering Repair and Regeneration Program, The Hospital for Special Surgery, Weill Cornell Medical College, New York, NY, USA.
authors: M B
authors: MB
authors: Goldring
authors: Department of Physiology and Biophysics, State University of New York, Stony Brook, NY, USA.
authors: P R
authors: PR
authors: Brink
authors: Cartilage Biology Research Group, Rheumatology Division, INIBIC-Hospital Universitario A Coruña, Xubias de Arriba 84, 15006 A Coruña, Spain. Electronic address: Ma.Dolores.Mayan.Santos@sergas.es.
authors: M D
authors: MD
authors: Mayan
authors: Cartilage Biology Research Group, Rheumatology Division, INIBIC-Hospital Universitario A Coruña, Xubias de Arriba 84, 15006 A Coruña, Spain; Rheumatology Division, ProteoRed/ISCIII, Proteomics Group, INIBIC-Hospital Universitario A Coruña, Xubias de Arriba 84, 15006 A Coruña, Spain; Rheumatology Division, CIBER-BBN/ISCIII, INIBIC-Hospital Universitario A Coruña, Xubias de Arriba 84, 15006 A Coruña, Spain. Electronic address: fblagar@sergas.es.
authors: F J
authors: FJ
authors: Blanco
mesh_terms: D015153:Blotting, Western; D000081247:CSK Tyrosine-Protein Kinase; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D002450:Cell Communication / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017629:Gap Junctions / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013194:Staining and Labeling / Q000379:methods; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D018031:Connexin 43; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human
keywords: Articular chondrocyte; Cartilage; Cx43; Gap junction; Hemichannels; T/C-28a2