Novel intracellular signalling regulators of cartilage progenitor cell populations
Cartilage is a viscoelastic tissue that absorbs shocks and facilitates low friction of the joint. Adult articular cartilage is limited in its ability to self-repair. Lesions or gradual wear-and-tear affect cartilage integrity and lead to damage that, when untreated, can ultimately develop to osteoarthritis. Osteoarthritis is a considerable health burden and is a leading cause of disability worldwide. In order to address this burdensome condition, several treatments have been developed. In particular, therapies that allow delivery of bone marrow and its constituent cell populations to the site of cartilage damage to form a regenerative clot have proved promising in repairing joint cartilage defects. In order to improve such regenerative technique, further research is required to provide a deeper understanding of the mechanisms underlying cartilage regeneration. One of the critical players are the cells that originally reside in the cartilage surrounding the damage. How these resident cells contribute to the activity of cells within the repair tissue at the site of cartilage damage is largely undescribed. In this thesis chondrocytes from three different cartilage areas were compared: chondrocytes from 1) the superficial (SZ) and 2) the middle-deep (MDZ) zone of non-weight bearing femoral condyles, and from 3) the osteoarthritic zone (OAZ) of patients undergoing knee replacement. More inflammatory factors and cytokines are present in MSCs co-culture with OA cartilage chips, and with MDZ cartilage chips. To assess how chondrocyte-MSC crosstalk would affect MSCs chondrogenesis, cartilage chips from the three different zones were co-cultured with BMSC pellets. Results indicated that the SZ induce chondrogenic differentiation of BMSCs, whereas MDZ and OAZ have a negative effect, compared to control conditions. The findings suggest that the presence of SZ, which has been reported to reduce with age, is important to direct BMSCs differentiation towards the chondrogenic fate. In order to better understand the molecular mechanism that differentiate SZ from MDZ and OA chondrocytes, DACTs (Dapper antagonist of catenin) were studied. DACT1 and DACT2 are known to be Wnt and TGFβ pathways regulators, but their role in chondrocytes and MSCs has not been described before. Both proteins are present in chondrocytes throughout the osteoarthritic human tissue, including in chondrocytes forming cell clusters. On the non-weight bearing and visually undamaged cartilage, DACT1 and DACT2 expression is localised to the articular surface. In mouse embryos (E.15.5), DACT2 is expressed at the interzone, site of developing synovial joint, indicating that DACT2 is expressed in cells that give rise to the articular joint. Subsequently the expression of DACT1 and DACT2 was analysed in MSCs: both are expressed in synovial and bone marrow-derived MSCs. Following this observation an RNAi knockdown experiment showed that DACT1 knockdown in both chondrocyte and MSCs causes the cells to undergo apoptosis within 24 hours. To understand the pathway regulated by DACT1, next generation sequencing gene expression analysis was performed on BMSCs where DACT1 had been reduced by RNAi. The study showed that loss of DACT1 influences the expression (p<0.05) of genes involved in both TGFβ and Wnt pathways and putative link to relevant cell regulatory pathways (Ingenuity® Pathway Analysis). SMURF2 and other genes involved in protein phosphorylation and degradation are downregulated following DACT1 knockdown. This suggests that DACT1 is an upstream regulator of SMAD protein phosphorylation affecting proliferation and survival of MSCs. The data presented in this thesis has indicated that different types of chondrocytes are present within human articular cartilage and that they cross talk with MSCs differently according to their regional origin. This information offers a new level of complexity to consider to improve regenerative techniques. In addition, this work describes for the first time, the presence and biological relevance of DACT1 and DACT2 in chondrocytes and MSCs. DACT1 is involved in MSCs survival and is downregulated in OA which suggest that this is an important regulatory protein. Further studies of DACT1 could help elucidate mechanisms involved in OA, but also uncover the relevance of cartilage progenitors loss in the development of cartilage degeneration.
cartilage, cell biology, DACT1, mesenchymal stromal cell, regenerative medicine, osteoarthritis, Extracellular vesicles
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