Background Neurons, hepatocytes, cardiocytes, chondrocytes and almost all cell types express connexins and form gap junction (GJ) channels that are critical for cellular function and tissue homeostasis. GJ channels provide a selective signalling route by the direct exchange of potent signalling molecules such as cAMP, second messengers, electrical signals, ions and several molecules that regulate cell survival, growth and metabolism. GJ channels also play a key nutritional role by the direct exchange of amino acids, glucose and several metabolites. We have recently reported that articular chondrocytes in tissue contain long cytoplasmic arms that physically connect two distant cells and cell-to-cell communication occurs through GJ channels. Cx43 protein is overexpressed in several diseases including in the articular cartilage and synovial membrane from patients with osteoarthritis (OA) and rheumatoid arthritis. An increase in Cx43 protein levels is known to alter gene expression, cell signalling, growth and cell proliferation.
Objectives The goal of this research work was to investigate if bone cells (subchondral bone, SB), synovial cells (SC) and chondrocytes (CH) are able to establish GJs among them and to investigate the consequence of that coupling in disorders that affect these tissues such as OA.
Methods Human cartilage, synovial membrane and subchondral bone were obtained from adult donors after joint surgery. The cells were grown to 80-90% confluence. The Electrophysiological techniques dual voltage-clamp methods, whole-cell/perforated patch experiment and InSitu Porator™ were used to detect the transfer of glucose and lucifer yellow. To confirm the exchange of amino acids, peptides and proteins between contacting cells, transwell co-culture system (3.0μm pore) and SILACã labelling combined with mass spectrometry (MS) were performed. Amino acids were derivatized and analyzed using EZ:faastTM kit and ESI*/LC/MS-Orbitrap. Transjuctional peptides and proteins were isolated and identified by SDS-PAGE, in gel digestion and MALDI/TOF-TOF.
Results Dual voltage-clamp and whole-cell/perforated patch methods demonstrated that primary SB and SC are able to establish functional GJ with CH, being Cx43 properties dominant. Dye injection experiments confirmed that SB, SC and CH exchange via GJ lucifer yellow and the fluorescent glucose derivative (2-NBDG). Transwell co-culture system demonstrated the transference of at least 5 pmol/ml of [13C6]-L-lisine and 3 pmol/mL of [13C6, 15N4]-L-arginine between contacting SB, SC and CH. MALDI/TO-TOF analysis revealed the exchange of peptides and proteins between contacting cells including calnexin, calreticulin or CD44 antigen.
Conclusions These findings suggest that Cx43-mediated intercellular communication between cells located in the subchondral bone, synovial membrane and cartilage may contribute to the cellular signalling and homeostasis of the joint and may have protective effect in the injured tissues and hence warrants further investigation. So far, the results presented here demonstrated for the first time that SB, SC and CH are able to physically interact and directly communicate by GJ channels.
Disclosure of Interest None declared
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