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Utilisation du modèle cellulaire ATDC5 pour la caractérisation des mécanismes de maturation des procollagènes et de leurs relations avec le processus de minéralisation matricielle

Abstract : Endochondral ossification is the mechanism by which long bones of Vertebrates grow. It requires the formation of a primary cartilage pattern by chondrogenesis, which is then replaced by bone. Chondrogenesis relies on the production of a dedicated type II collagen rich extracellular matrix (ECM), which will then mineralize, and many aspects of which remain difficult to characterize. The ATDC5 cell line can engage into a differentiation program resembling the multistep chondrogenic differentiation observed in vivo after insulin stimulation. Although this cell line is widely used, its proteome is barely described. In order to define to what extent this model recapitulates the main aspects of cartilage ECM formation, a time-resolved proteome analysis by LC-MALDI-TOF / TOF of the regulation of the proteome of differentiating ATDC5 cells focused on the ECM and the level of maturation of its main components. These cells synthesize and incorporate into their ECM a wide range of cartilage components, including aggrecan and type II collagen, which carry most of the maturations described in vivo. Most importantly, the accumulation of collagen in the MEC is correlated with maturation events: proteolytic cleavage and triple helix hydroxylation. The C-propeptide of type II collagen (CPII), is a component of joint and growth plate cartilage, and has been described as involved in the mineralization process of the latter. On the other hand, to what extent and how CPII would regulate mineralization remains to be specified. We have shown that CPII exhibit a different interactome as its type I and III procollagens (CPI and CPIII) counterparts, suggesting mechanistic differences between their maturation. These results are consolidated by additional in vivo studies of CPII cleavage sites, confirming the coexistence of two cleavage sites and demonstrating for the first time that CPII trimers contain the four possible combinations of the two cleavage sites. These data suggest that each monomer within the trimer is cleaved independently of the others. Overall, this work highlights CPII cleavage mechanism as distinct from that of CPI and CPIII and more complex than anticipated. Functional characterization of key events of ECM elaboration in ATDC5 cells should allow better dissecting the mechanisms of chondrogenesis and cartilage mineralization.
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https://hal.univ-lorraine.fr/tel-02746390
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Dafné Wilhelm. Utilisation du modèle cellulaire ATDC5 pour la caractérisation des mécanismes de maturation des procollagènes et de leurs relations avec le processus de minéralisation matricielle. Rhumatologie et système ostéo-articulaire. Université de Lorraine, 2019. Français. ⟨NNT : 2019LORR0286⟩. ⟨tel-02746390⟩

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