Afsari, Farinaz.

2018-01-31T11:44:31Z

2018-01-31T11:44:31Z

2005

The mechanotransduction pathway in chondrocytes is facilitated by a αSßI mechanoreceptor at the cell surface and involves tyrosine phosphorylation of paxillin, focal adhesion kinase and ß catenin. The availability of ß-catenin at the cell membrane, in the cytoplasm and in the nucleus plays a key part in the process of mesenchymal condensation during chondrogenesis, which is regulated by Wnt signalling and interaction with other signalling pathways. GSK3ß (glycogen synthase kinase 3ß), is a key mediator in the Wnt pathway and is involved in regulating ß catenin cytoplasmic and nuclear distribution. Wnt binds to its receptor Frizzled (Fz) and subsequent canonical signalling leads to inhibition of GSK3ß, and cytoplasmic accumulation of ß catenin. This, in turn, promotes ß catenin binding to LEF/TCF transcription factors and induction of target gene expression. In the absence of a Wnt signal, GSK3p, as a part of an axin and APC (adenomatous polyposis coli) complex, phosphorylates ß catenin and induces its degradation via the ubiquitin/ proteosome pathway.

This thesis has set out to investigate whether Wnt pathway components are expressed in human chondrocyte cell lines and to explore whether the Wnt pathway plays any role in mechanotransduction pathway in chondrocytes.

Using RT-PCR, cloning, immunofluorescence and confocal microscopy it was for the first time demonstrated that the Wnt signalling components,Wnt-l, Fz-2, Fzrp and ß catenin were expressed in human chondrocyte cell lines. Using confocal microscopy, 7 fibronectin and CD44 were identified in association with chondrocyte Wnt-Fz complexes, suggesting that they may be coreceptors necessary for transducing Wnt signals intracellularly. A kinase assay demonstrated that GSK3ß activity is increased following 40 minute ofmechanical stimulation and that a phosphoinositol-3 OHkinase (PI3K) inhibitor decreased the activity ofthis kinase. A Wnt agonist, Lithium, on the other hand, increased the GSK3ß activity following 20 and 60 minute of mechanical stimulation. Western blotting suggested that in this study the formation of GSK3ß/ß catenin complexes were induced in the presence of Lithium and mechanical stimulation delayed this process. However, this evidence of the complex formation ofGSK3ß with ß catenin in Western blots was not supported by preliminary analysis of densitometric data and further investigation is required to confirm these findings.

The results indicate that Wnt signalling components are expressed in chondrocyte cell lines and may be involved in mechanical signalling in these cells. However, the induction ofGSK3ß activity following mechanical stimulation was mediated by a PI3K dependent pathway rather than a Wnt pathway. This, in turn, may influence the stabilisation of GSK3ß/ßcatenin complex following recruitment of activated protein kinase B (PKB)by PI3K and phosphorylation of GSK3ß. These, in turn, control the cytoplasmic/nuclear distribution of ß-catenin which affects the regulation of downstream target genes such as CD44, fibronectin and some metalloproteinases. CD44 and fibronectin are essential components of cartilage, which are involved in matrix assembly and the maintenance of cartilage integrity.

http://hdl.handle.net/1842/28121

The University of Edinburgh

Annexe Thesis Digitisation Project 2017 Block 16

Already catalogued

Role of Wnt signalling pathway in mechanotransduction pathway in SV-40 immortalised human chondrocyte cell lines

Thesis or Dissertation

PhD Doctor of Philosophy