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.
Rheumatology Division, Cartilage Biology Research Group, INIBIC-Hospital Universitario A Coruña, A Coruña, Spain.
Maria D
MD
Mayan
Rheumatology Division, Cartilage Biology Research Group, INIBIC-Hospital Universitario A Coruña, A Coruña, Spain.
Raquel
R
Gago-Fuentes
Rheumatology Division, Cartilage Biology Research Group, INIBIC-Hospital Universitario A Coruña, A Coruña, Spain.
Paula
P
Carpintero-Fernandez
Rheumatology Division, ProteoRed/ISCIII, Proteomics Group, INIBIC-Hospital Universitario A Coruña, A Coruña, Spain.
Patricia
P
Fernandez-Puente
Rheumatology Division, CIBER-BBN/ISCIII, INIBIC-Hospital Universitario A Coruña, A Coruña, Spain.
Purificacion
P
Filgueira-Fernandez
Rheumatology Division, CIBER-BBN/ISCIII, INIBIC-Hospital Universitario A Coruña, A Coruña, Spain.
Noa
N
Goyanes
Department of Physiology and Biophysics, State University of New York, Stony Brook, New York, USA.
Virginijus
V
Valiunas
Department of Physiology and Biophysics, State University of New York, Stony Brook, New York, USA.
Peter R
PR
Brink
Department of Molecular Biology, Medical Center Drive, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey, USA.
Gary S
GS
Goldberg
Rheumatology Division, Cartilage Biology Research Group, INIBIC-Hospital Universitario A Coruña, A Coruña, Spain Rheumatology Division, ProteoRed/ISCIII, Proteomics Group, INIBIC-Hospital Universitario A Coruña, A Coruña, Spain Rheumatology Division, CIBER-BBN/ISCIII, INIBIC-Hospital Universitario A Coruña, A Coruña, Spain.
Francisco J
FJ
Blanco
D000601:Amino Acids, Essential / Q000378:metabolism; D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism* / Q000648:ultrastructure; D002450:Cell Communication; D019902:Chondrocytes / Q000378:metabolism* / Q000648:ultrastructure; D017630:Connexins / Q000378:metabolism* / Q000648:ultrastructure; D017629:Gap Junctions / Q000378:metabolism* / Q000648:ultrastructure; D005947:Glucose / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007719:Knee Joint; D008855:Microscopy, Electron, Scanning; D013552:Swine
D000601:Amino Acids, Essential; D017630:Connexins; D005947:Glucose
Arthritis; Chondrocytes; Osteoarthritis