Identification of a novel regulator of extracellular matrix degradation in chondrocytes

Thesis

There are currently no drugs that modify the progression of the degenerative joint disease osteoarthritis, which is characterised by protease-mediated degradation of the cartilage extracellular matrix. Identification of molecules that regulate protease activity could therefore lead to the development of new therapies for osteoarthritis. The Wann group previously showed that mutation of Intraflagellar transport protein 88 (IFT88) increased degradation of the cartilage matrix component aggrecan by proteases in vitro. As IFT88 is required for the assembly of a cell surface organelle called the primary cilium, the aim of my thesis was to investigate whether the primary cilium is involved in the regulation of protease activity. Based on the Wann group’s finding that mutation of IFT88 impaired Low-density lipoprotein receptor-related protein 1 (LRP-1)-mediated endocytosis of extracellular proteases, I hypothesised that the primary cilium is a region of efficient endocytosis and thus regulates the extracellular activity of proteases.

To address this aim, I measured protease activity when four genes encoding proteins involved in cilia assembly were knocked down with siRNAs in a chondrocyte cell line. Generation of the AGEG aggrecan neoepitope was used as an indicator of protease activity. I showed that protease activity was not affected by knockdown of three of the ciliary genes tested. However, knockdown of the gene encoding Tau-tubulin kinase 2 (TTBK2) increased the activity of the protease A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5). This increase was not associated with changes in ADAMTS-5 RNA levels during my experiments, constitutive activation of cilia-dependent hedgehog signalling, or reduced levels of the physiological ADAMTS-5 inhibitor, Tissue inhibitor of metalloproteinase 3 (TIMP-3). The effect of knockdown of the TTBK2 gene on ADAMTS-5 endocytosis could not be definitively determined.

Together, these results showed that whilst the primary cilium itself may not be required for protease activity regulation, TTBK2 is a novel regulator of ADAMTS-5 activity. Further investigation of the molecular mechanism of ADAMTS-5 regulation by TTBK2 may help to identify new therapeutic targets in osteoarthritis.

Authors: Collins, I

• DOI: 10.5287/ora-j2j5qajkq
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Osteoarthritis; ADAMTS-5; Proteases; Cartilage; Primary cilia
• Subjects: Cilia biology; Extracellular matrix; Cells; Cartilage biology