Desbuquois dysplasia type 1 (DBQD1) is a rare recessive chondrodysplasia caused by mutations in the CANT1 gene. This gene encodes for a calcium activated nucleotidase of the Golgi that hydrolyses UDP, a by-product of glycosylation reactions, to UMP and phosphate. In particular the functional impairment of CANT1 protein leads to quantitative and qualitative defects in glycosaminoglycan (GAG) biosynthesis. Due to the reduced availability of bone and cartilage biopsies from the affected patients, in vivo and in vitro models were generated to deeper investigate the complex molecular basis of DBQD1. The Cant1 knock-out mouse was generated and validated as an animal model of DBQD1. The analysis of its cartilage tissue and chondrocytes has shown that Cant1 gene mutation leads to reduced PGs synthesis and to oversulfation of GAGs, which also showed a reduced hydrodynamic size. Given the complexity of the animal model, studies in vitro models (i.e. cell cultures), were needed to get new insight on pathogenesis of DBQD1. However, despite being a well-defined system, primary chondrocytes easily undergo a de-differentiation process after few passages and long-term culture. To overcome this problem, immortalized chondrocyte cell lines, which are able to grow very easily through several passages, were generated. However, the cells might lose the original phenotype during the immortalization procedure; thus, these cell lines should be deeply characterized before their use. Therefore, the aim of this thesis was to phenotypically characterize immortalized Cant1 knock-out (Cant1- / -) chondrocytes and wild-type chondrocytes in order to validate them as an in vitro model to study CANT1 role in the pathogenesis of DBQD1. The cells have been immortalized using a plasmid expressing the small T antigen and the large T antigen of SV40. The morphological analysis and the study of processes involved in the synthesis and deposition of PGs and type II collagen (i.e. the main constituents of the ECM) have shown that these immortalized cells maintain their differentiated phenotype through time. Moreover, 35S-sulfate metabolic labelling of immortalized cells demonstrated that Cant1 - / - chondrocytes show the same GAGs biosynthesis defect previously observed in Cant1 knock-out mouse model-derived primary chondrocytes, thus preserving their pathological phenotype. In conclusion, immortalized Cant1 - / - chondrocytes were validated as an in vitro model to study DBQD1, providing an additional tool to better understand the PGs biosynthesis defects. The same approach might be extended to other cartilage disorders.
La displasia di Desbuquois di tipo 1 è una rara forma di condrodisplasia a trasmissione autosomica recessiva, causata da mutazioni del gene CANT1. Questo gene codifica per una nucleotidasi calcio dipendente, espressa nel Golgi, responsabile dell’idrolisi dell’UDP, sottoprodotto delle reazioni di glicosilazione dei GAG, a UMP e fosfato. Per questo motivo, alterazioni della funzionalità della proteina CANT1 portano a difetti quantitativi e qualitativi nel processo di sintesi dei glicosaminoglicani (GAG). A causa della ridotta disponibilità di campioni bioptici di tessuto osseo e cartilagineo dai pazienti, per studiare le complesse basi molecolari di questa malattia è stato necessario generare modelli sperimentali in vivo ed in vitro. Gli esperimenti condotti analizzando la cartilagine e i condrociti estratti dal topo Cant1 knock-out, modello murino della DBQD1, hanno dimostrato che la mutazione del gene CANT1 determina una ridotta sintesi dei proteoglicani (PG) e un’oversolfatazione dei GAG, che presentano inoltre dimensioni idrodinamiche ridotte. Data la complessità del modello animale, al fine di delucidare il ruolo della proteina CANT1, è importante ad oggi condurre studi in vitro utilizzando colture cellulari. Le colture primarie di condrociti però, pure essendo un sistema ben definito, presentano lo svantaggio di andare incontro a de-differenziamento dopo alcuni passaggi in coltura. Per ovviare a questo problema, una possibilità è quella di generare ed utilizzare linee cellulari immortalizzate. Tuttavia, durante il processo di immortalizzazione è possibile che le cellule acquisiscano un fenotipo differente da quello delle cellule di origine, motivo per cui è molto importante caratterizzarle. Lo scopo di questa tesi è stato quindi la caratterizzazione fenotipica di condrociti, ottenuti rispettivamente dal modello murino Cant1 knock-out (Cant1-/-) e da animali wild-type, immortalizzati con un plasmide che esprime lo small T antigen e il large T antigen dell’SV40, al fine di validarli come modello in vitro per lo studio di alcuni aspetti della patogenesi della DBQD1. L’analisi morfologica e lo studio della sintesi e deposizione delle principali molecole della matrice extracellulare, proteoglicani e collagene, hanno dimostrato che queste cellule immortalizzate conservano il fenotipo condrocitario. Inoltre, tramite marcatura metabolica con 35S-solfato, è stato osservato che i condrociti immortalizzati Cant1-/- presentano gli stessi difetti della sintesi dei GAG precedentemente osservati nei condrociti Cant1-/- primari, preservandone il fenotipo patologico. In conclusione, i condrociti immortalizzati Cant1-/-, validati come modello in vitro, costituiscono uno strumento aggiuntivo per approfondire ulteriormente i difetti nella biosintesi dei PG, e suggeriscono di poter estendere questo approccio anche allo studio di altre patologie della cartilagine.
CARATTERIZZAZIONE DI CONDROCITI IMMORTALIZZATI KNOCK-OUT PER IL GENE CANT1
LEONE, ALESSANDRA
2020/2021
Abstract
Desbuquois dysplasia type 1 (DBQD1) is a rare recessive chondrodysplasia caused by mutations in the CANT1 gene. This gene encodes for a calcium activated nucleotidase of the Golgi that hydrolyses UDP, a by-product of glycosylation reactions, to UMP and phosphate. In particular the functional impairment of CANT1 protein leads to quantitative and qualitative defects in glycosaminoglycan (GAG) biosynthesis. Due to the reduced availability of bone and cartilage biopsies from the affected patients, in vivo and in vitro models were generated to deeper investigate the complex molecular basis of DBQD1. The Cant1 knock-out mouse was generated and validated as an animal model of DBQD1. The analysis of its cartilage tissue and chondrocytes has shown that Cant1 gene mutation leads to reduced PGs synthesis and to oversulfation of GAGs, which also showed a reduced hydrodynamic size. Given the complexity of the animal model, studies in vitro models (i.e. cell cultures), were needed to get new insight on pathogenesis of DBQD1. However, despite being a well-defined system, primary chondrocytes easily undergo a de-differentiation process after few passages and long-term culture. To overcome this problem, immortalized chondrocyte cell lines, which are able to grow very easily through several passages, were generated. However, the cells might lose the original phenotype during the immortalization procedure; thus, these cell lines should be deeply characterized before their use. Therefore, the aim of this thesis was to phenotypically characterize immortalized Cant1 knock-out (Cant1- / -) chondrocytes and wild-type chondrocytes in order to validate them as an in vitro model to study CANT1 role in the pathogenesis of DBQD1. The cells have been immortalized using a plasmid expressing the small T antigen and the large T antigen of SV40. The morphological analysis and the study of processes involved in the synthesis and deposition of PGs and type II collagen (i.e. the main constituents of the ECM) have shown that these immortalized cells maintain their differentiated phenotype through time. Moreover, 35S-sulfate metabolic labelling of immortalized cells demonstrated that Cant1 - / - chondrocytes show the same GAGs biosynthesis defect previously observed in Cant1 knock-out mouse model-derived primary chondrocytes, thus preserving their pathological phenotype. In conclusion, immortalized Cant1 - / - chondrocytes were validated as an in vitro model to study DBQD1, providing an additional tool to better understand the PGs biosynthesis defects. The same approach might be extended to other cartilage disorders.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/13950