Background/Purpose: Human adult articular cartilage is composed of a dense extracellular matrix and specialized cells called chondrocytes. The chondrocytes are found to their own lacuna and remain in resting stage refraining form proliferation but displaying a moderate metabolic activity to maintain their surrounding matrix during the whole adult life. We have previously found that human adult chondrocytes express the gap junction (GJ) protein connexin 43 and chondrocytes in tissue have long cytoplasmic arms that physically connect two distant cells. The interaction of proteins with the C-terminal tail of Cx43 directly modulates different cellular activities such as cell growth and proliferation.

Methods: Chondrocytes were isolated by sequential digestion with trypsin-EDTA and Collagenase. Isolated cells from healthy and cartilage from osteoarthritis (OA) patients were maintained for stable short-term cell culture in DMEM supplemented with primocin and 15% FCS. Co-immunoprecipitation (IP) experiments were performed to identify the proteins that interact with the C-Terminal tail of Cx43. In-gel digestion of immunoprecipitated proteins separated by SDS-PAGE were analysed using the nano-liquid chromatography (Nano-LC, Eksigent) coupled to mass spectrometry (MALDI-TOF/TOF, Applied Biosystem). The identification of proteins was performed using ProteinPilot™ 3.0 Software. Samples were evaluated by SDS-PAGE followed by Western blotting with specific antibodies.

Results:

A total of 123 were identified, of which 68 proteins were represented by at least two unique peptides. 118 proteins were specific to the Cx43 IP, not identified in the control IP performed without antibody. Identified interactors show significant enrichment for Gene Ontology (GO) processes directly linked with cytoskeleton dynamics (vimentin, a/b-tubulin or profiling-1), metabolic pathways (enolase, aldolase or GAPDH), nuclear activity (histones, nucleolin and other nucleoar proteins) and translation (several ribosomal and ribosomal-related proteins). IHC experiments showed that chondrocytes from OA patients in cartilage contain higher levels of Cx43 in the nucleus, the cytoplasm and membrane. However Cx43 was only found in the membrane of healthy chondrocytes in tissue. GO terms of proteins identified in OA samples showed an enrichment of Cx43-interactors related with cell adhesion, calmoduling binding, nucleolus or cytoskeleton related proteins in OA samples regards to healthy. However the mitochondrial proteins SOD2 or ATP5J2 were only identified in samples from healthy donors.

Conclusion: Mass-spectrometry results revealed novel functional Cx43 interactors involved in human disease and OA development emphasizing the importance of Cx43-interactions for normal development and physiology. Besides IHC experiments suggest that Cx43 interacts with nuclear and translational components especially in OA cartilage. The interaction of Cx43 with mitochondrial proteins may be involved in ischemic preconditioning as occur in other cell types.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/proteomic-analysis-of-connexin-43-reveals-association-with-proteins-dysfunctional-related-with-osteoarthrosis/