Next we investigated the transfer

Next, we investigated the transfer of peptides and proteins between contacting cells by nano-LC-MS/MS analysis using the fractions corresponding to molecules higher than 3 kDa. The MS data analysis revealed the direct transfer of several labelled peptides between receivers and donors. Labelled peptides were not identified in controls (non-contacting cells, seeFig. 213C) and/or L-arginine (13C-15N). The peptides were identified and selected using fragmentation spectra (Fig. 2C). The short time frame of the assays together with the restricted number of cells in the transwell co-culture system limited the number of identified heavy peptides and proteins transferred to receivers by MS. In addition, only proteins with a threshold >95% confidence (>1.3 Unused Score) were considered for protein identification in these assays. Regardless, the identified heavy peptides in receivers, and the corresponding proteins are listed inTable 1, Table 2, Table 3. As expected, the same heavy peptides were identified in labelled cells (CHs, donors).
Several groups have found evidence of cellular communication between different tissues in the joint, which include paracrine regulation, secreted mediators travelling through extracellular space and microcrack channels or secretion of molecules to the extracellular space or synovial fluid. However, this is the first experimental evidence of intercellular communication via gap junction membrane channels. In this study, we have demonstrated that cells found in the bone, cartilage and synovium have the ability to physically interact and can communicate directly with another via contact between membrane-bound cell surface molecules and GJ channels formed by Cx43. Through GJ channels, cells can exchange ions, small molecules, second messengers, miRNAs and other compounds such as glucose, Caspase-1, human recombinant proteinase and metabolites[23]. In addition, our results showed that contacting cells are able to exchange peptides and proteins probably through mechanisms that require the formation of connections between membranes or through extracellular vesicles[24]. These membrane vesicles, which include exosomes, are secreted by cells and participate in intercellular communication increasing the complexity of cell signalling by transfer proteins, peptides, lipids, nucleic acids and other components. Cx43 is the major protein subunit that co-assembles to form GJs and HCs in cartilage, bone and synovium[4]. Alterations in cellular communication through GJs and HCs are associated with disorders that affect cartilage structure, synovial membrane and bone remodelling[25]. Our results demonstrate that these cells when in contact, may rapidly form intercellular communication channels. An example of cell-cell contacts in vivo in joint tissues is CHs in the deepest region of cartilage and the surrounding (contact surface). These CHs may establish cellular contacts through projections or tunnelling nanotubes with subchondral BCs, and these connections could participate in cartilage-bone intercellular communication and biochemical activities. In the same way, CHs in the growth plate are in contact with BCs and these connections may be implicated in ossification processes and the longitudinal growth of long bones. In addition, synovial fibroblast invasion across cartilage[26]could also establish cell-cell contacts with chondrocytes allowing GJ plaque formation, and may be a contributing factor to cartilage and bone destruction in different arthritis conditions. On the other hand, the intercellular transfer of peptides and proteins between contacting cells that are too large to permeate the gap junctional pore may occur through mechanisms involving endocytosis of two membranes, exosomes, microvesicles or membrane nanotubules. Interestingly, Cx43 has been involved in fusion and internalization events between EVs and Cx43-expressing cells[27,28]. Furthermore, intercellular communication through nanotubes allows for the transfer of membrane proteins and cytoplasmic organelles, including mitochondria[29], between several cell types. Nanotubes are dynamic structures that form de novo within a few minutes, yet the dimension of nanotubes represent a very high resistance pathway between cells. In spite of that, a novel function of tunnelling nanotubes is the long-distance electrical coupling of nanotube-connected cells[30]. This function was reported to depend on the presence of connexin channels interposed in the nanotube connections[30]. The electrical and chemical coupling provided by GJ channels and nanotubes allow the synchronization of distant cells and have been suggested to play important roles during biomechanical perturbation and healing by modulation of synthetic activity, phenotype and signalling pathways. Further studies will be required to describe these connections in vivo and to investigate their role in disease progression.