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Articular chondrocyte network mediated by gap junctions: role in metabolic cartilage homeostasis.

ABSTRACT: 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.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.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 ?-tubulin and ?-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.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.

SUBMITTER: Mayan MD

PROVIDER: S-EPMC5500216 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Articular chondrocyte network mediated by gap junctions: role in metabolic cartilage homeostasis.

Mayan Maria D MD Gago-Fuentes Raquel R Carpintero-Fernandez Paula P Fernandez-Puente Patricia P Filgueira-Fernandez Purificacion P Goyanes Noa N Valiunas Virginijus V Brink Peter R PR Goldberg Gary S GS Blanco Francisco J FJ

Annals of the rheumatic diseases 20131113 1

<h4>Objective</h4>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.<h4>Methods</h4>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 identi ...[more]

PMID: 24225059

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Project description:The degradation of proteoglycan was examined in cultured slices of pig articular cartilage. Pig leucocyte catabolin (10 ng/ml) was used to stimulate the chondrocytes and induce a 4-fold increase in the rate of proteoglycan loss from the matrix for 4 days. Material in the medium of both control and depleted cultures was mostly a degradation product of the aggregating proteoglycan. It was recovered as a very large molecule slightly smaller than the monomers extracted with 4M-guanidinium chloride and lacked a functional hyaluronate binding region. The size and charge were consistent with a very limited cleavage or conformational change of the core protein near the hyaluronate binding region releasing the C-terminal portion of the molecule intact from the aggregate. The 'clipped' monomer diffuses very rapidly through the matrix into the medium. The amount of proteoglycan extracted with 4M-guanidinium chloride decreased during culture from both the controls and depleted cartilage, and the average size of the molecules initially remained the same. However, the proportion of molecules with a smaller average size increased with time and was predominant in explants that had lost more than 70% of their proteoglycan. All of this material was able to form aggregates when mixed with hyaluronate, and glycosaminoglycans were the same size and charge as normal, indicating either that the core protein had been cleaved in many places or that larger molecules were preferentially released. A large proportion of the easily extracted and non-extractable proteoglycan remained in the partially depleted cartilage and the molecules were the same size and charge as those found in the controls. There was no evidence of detectable glycosidase activity and only very limited sulphatase activity. A similar rate of breakdown and final distribution pattern was found for newly synthesized proteoglycan. Increased amounts of latent neutral metalloproteinases and acid proteinase activities were present in the medium of depleted cartilage. These were not thought to be involved in the breakdown of proteoglycan. Increased release of proteoglycan ceased within 24h of removal of the catabolin, indicating that the effect was reversible and persisted only while the stimulus was present.

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Articular chondrocyte network mediated by gap junctions: role in metabolic cartilage homeostasis.

Publications

Articular chondrocyte network mediated by gap junctions: role in metabolic cartilage homeostasis.

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