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

Abstract

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.

The Importance of Connexin Hemichannels During Chondroprogenitor Cell Differentiation in Hydrogel Versus Microtissue Culture Models

“…13,14 While the first two molecules disappear in the course of chondrogenesis, 13 gap junctions can still be found in mature cartilage. 15 Gap junctions are intercellular channels formed by connexins that allow for rapid exchange of ions, metabolites, and other small-molecule messengers. 16 They are expressed in virtually all tissues and are involved in various cellular functions, including cell growth, differentiation, and death.…”

Proteomic Analysis of Connexin 43 Reveals Novel Interactors Related to Osteoarthritis

“…In addition, caldesmon plays important roles in the migration of nonmuscle cells via regulating the actin-myosin system (82), and caldesmon binding to myosin has been reported to result in the enhancement of axon extension (83). Interestingly, we have found that chondrocytes from OA patients contain longer cellular extensions (more than 200 m in length) when compared with chondrocytes in healthy cartilage (7). Further studies will be necessary to reveal the role of Cx43 in these processes and to discern its function in OA chondrocytes.…”

“…In fact, the eye lens and articular cartilage of adults are avascular organs, and because no blood supply reaches these tissues, it has been reported that cells in these tissues are maintained by the movement of fluids, ions, nutrients (e.g. glucose), and metabolites through a network of GJs (7,54,55). In addition, results from different reports strongly suggest that inhibition of GJIC modifies glucose uptake and that glucose levels modify Cx43 gene expression (56).…”

“…Our group is interested in investigating the physiopathological mechanism that is associated with osteoarthritis (OA). Recent results suggest a direct role of Cx43 in the development of OA that is not necessary related to its channel function (7,23,24). For instance, only a few connexin-interacting proteins have been described to date, and previous studies have used artificial systems, such as yeast two-hybrid screens or Cx43-domain fusion proteins.…”

Connexins and pannexins in the skeleton: gap junctions, hemichannels and more

“…Direct cell-to-cell communication has been shown in the superficial zone of articular chondrocytes [155]. A paradigm challenging study has even shown that chondrocytes in situ may form long cell processes, 5 to 150 microns in length, that permit communication through gap junction channels [156]. Increased presence of gap junction plaques and Cx43 expression have been found in synovial lining cells obtained from the knee of patients with osteoarthritis [157].…”