Intercellular communication via gap junction channels between chondrocytes and bone cells

Abstract

Cell-to-cell communication between bone, cartilage and the synovial membrane is not fully understood and it is only attributed to the diffusion of substances through the extracellular space or synovial fluid. In this study, we found for the first time that primary bone cells (BCs) including osteocytes, synovial cells (SCs) and chondrocytes (CHs) are able to establish cellular contacts and to couple through gap junction (GJ) channels with connexin43 (Cx43) being dominant. Transwell co-culture and identification by mass spectrometry revealed the exchange of essential amino acids, peptides and proteins including calnexin, calreticulin or CD44 antigen between contacting SCs, BCs and CHs. These results reveal that CHs, SCs and BCs are able to establish intercellular connections and to communicate through GJ channels, which provide a selective signalling route by the direct exchange of potent signalling molecules and metabolites.

Keywords: Articular chondrocyte; Bone cells; Cartilage; Cellular communication; Gap junctions; Joint; Osteoarthritis; Synovial cells; connexin43.

Fig.1.. Human primary chondrocytes (CHs), synoviocytes (SCs) and bone cells (BCs) can establish functional GJ channels.

(A) Hematoxylin-eosin staining was used to study the cartilage and subchondral bone at the convergence area between both tissues in samples from healthy donors and patients with OA. Cartilage from OA patients shows destruction of ECM with structural changes in the deepest zone, which borders the subchondral bone. The tidemark (green) and the cement line (black) indicate the calcified cartilage and the merge with subchondral bone. (B) The images represent the joint under healthy or rheumatic disease such as OA or RA. Inflammation of synovial tissue (green), degeneration of articular cartilage (blue) and alteration of subchondral bone are typical of these disorders. (C) Evaluation of gap junctional communication between bone cells and chondrocytes. Gap junctional currents (I_j) elicited by a bipolar protocol (see text for details) from heterologous pair of bone cell and chondrocytes (upper left panel) and bone cell pair. Lower panel, fluorescent labelling to differentiate between CHs and BCs. (D) Average of junctional conductance measured from the pairs of CHs (13.4±2.1 nS, n=11), BCs (13.7±5.6 nS, n=5) and BC-CHs (12.3±2.0 nS, n=15). Statistical analysis revealed no statistical difference between groups (P=0.930). (E) Anti-Cx43 immunoblotting to study the levels of Cx43 in primary CHs, SCs and BCs in monolayer and underwent a total of 6 – 8 passages (1:2) in T-75 culture flask.

Fig. 2.. Establishment of cell contacts allows the direct transfer of free amino acids and proteins.

(A) Transwell co-culture system used to study the transfer of amino acids and peptides/proteins through cell-cell contacts and GJ channels. Primary cells in monolayer underwent 6 passages (1:2) in 100-mm dish for stable isotopic labeling. The diagram shows the workflow used for the identification of labelled amino acids and peptides in receiver cells (cells previously grown in non-labelled medium). Immunohistochemistry to detect Cx43 was performed on transwell membranes with positive spots in cells and through the pore of the membrane (cellular extensions). (B) Analyses and quantification of L-lysine (¹³C₆) detected on donors (CHs), receivers and controls (BCs and SCs). Data are shown as mean ± SEM, n = 2– 5; Student’s t test; p < 0.01 (receivers versus control). (C) The graph represents an example of the mass spectrum of a peptide (SSGVSEIRHTADR) identified to HSPB90 protein. The spectrum shows the m/z (Da) of the ions plotted against their intensities. Each peak shows a component of unique m/z in the sample. Heights of the peaks connote the relative abundance of the components. Green boxes show the peaks corresponding to light (1414,6559 Da) and heavy (1434,7572 Da) forms when two L-arginine (¹³C₆- ¹⁵N₄) have been added to the peptide. The diagram on the right represents the number of heavy peptides identified during the co-culture system and LC-MS/MS analysis for 2 independent experiments. Venn diagram is shown on the right. The identified peptides/proteins are listed in Table 1, 2 and 3.