Background Gap junction (GJ) channels are composed by a family of proteins called connexins. The ability to synchronize groups of cells for coordinated electrical, mechanical, metabolic and chemical communication make these proteins essential for tissue function. Mutations in connexin-encoding genes lead to developmental of a wide variety of diseases. The inhibition of GJIC in micromass culture of chondrocytes reduces chondrocytes differentiation and defective Cx43 functions cause skeletal defects.
Recent results from our group have convincingly demonstrated the involvement of the overexpression of Cx43 in OA pathogenesis. Cx43 has multiple GJ-independent functions that affect signalling pathways, cell growth, and cell proliferation. The carboxy terminal domain (CTD) of Cx43 is located in the cytoplasmic side and is key for protein functions.
Objectives The aim of this study was to investigate the role of the CTD of Cx43 in cartilage structure and function.
Methods Global knockout of Cx43 is embryonic lethal and homozygous K258stop animals, which Cx43 lacks the last 125 amino acid residues of the CTD, died shortly after birth. Cartilage and primary chondrocytes of 8 wild type, Cx43/KO, 6 K258stop/Cx43, 6 K258stop/KO and 6 K258stop/K258stop (21-day gestational section age mice) were subjected to the study. Mouse genotyping was achieved by PCR using DNA extracted from ear tissue and western-blot. Entire knee joints were stored in formalin-fixed paraffin-embedded sections (decalcified before embedding and sectioning) and analysed by conventional staining methods and immunohistochemistry (IHC) assays. Chondrocytes from cartilage were isolated using an inverted microscope and tissue was digested using trypsin and collagenase. Primary chondrocytes were grown to 80-90% confluence. Proliferation assay was performed by counting cells with using automated cell counter. For IHC cells were seed onto coverslips. The following are the antibodies used in this study: anti-osteoponin, anti-fibronectin, anti-collagen type II, anti-Cx43, anti-PCNA and Ki67. Scrape loading assay was used to examine GJIC
Results Analysis of cartilage sections staining with Safranin O/Fast Green, Hematoxylin-eosin eosin and Toluidine blue did not show significant differences between different phenotypes. However the K258stop/KO cartilage was thinner than wild type and Cx43/KO. Besides cartilage from K258stop/KO showed higher rate of positive cells for PCNA. IHC experiments revealed that K258stop/KO chondrocytes in primary culture contain less levels of collagen type II. K258stop/K258stop was found to have 1.5-fold increase in cell proliferation in comparison with wild type or Cx43/KO. Scrape loading assays suggest that the deletion of CTD slightly reduce GJIC.
Conclusions We have used a genetically modified murine model to directly characterize, for the first time, the role of the CTD of Cx43 on cartilage structure. Our results strongly support the notion that the CTD of Cx43 plays an important role in chondrocyte phenotype. It is well know that through its CTD, Cx43 serves as scaffolding proteins that associates with structural and signaling molecules leading to regulation of intracellular signaling, independently of channel activity. This study illustrates that a complete isolation of Cx43 from its CTD may have a negative impact on cartilage structure and chondrocyte functions within the tissue.
Disclosure of Interest None declared
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