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|Title:||The activation and disarming of Protease-activated receptor 2|
|Abstract:||Osteoarthritis (OA) is a common degenerative disease of articular joints characterised by the enzymatic degradation of cartilage, mediated predominately by the collagenolytic matrix metalloproteinases (MMP)-1 and -13. Various stimuli have been identified as inducers of MMP expression in chondrocytes, including pro-inflammatory cytokines, abnormal mechanical load, and the activation of protease-activated receptor 2 (PAR2) by matriptase. Both PAR2 and matriptase are strongly implicated in OA. PAR2 is activated by cleavage at a distinct site to reveal the canonical tethered ligand, SLIGKV, which can bind and activate the receptor. PAR2 is also able to undergo non-canonical activation and receptor disarming by cleavage at different sites, revealing different tethered ligands or removing the extracellular domain. This project aimed to identify novel PAR2 cleavages by incubated recombinant proteases with a PAR2 peptide sequence. The classical collagenases, MMP-1, -8 and -13, as well as the cysteine proteases cathepsins V, K, L and B, were all identified as novel PAR2 cleaving enzymes. The collagenases cleaved PAR2 at a distinct site a single amino acid to the C-terminus of the canonical site, and the putative tethered ligand, LIGKVD, was identified as a potential biased agonist for PAR2. The cathepsins generally cleaved PAR2 further towards the cell membrane, and cathepsin V was found to disarm against canonical PAR2 activation. Enzyme kinetics were examined for all novel proteases and compared with matriptase, identifying MMP-13 as the most potent collagenase for PAR2 cleavage, with all cathepsins exhibiting broadly similar kinetic profiles to each other. Canonical PAR2 activation was further explored by DNA microarray analysis which identified a pro-inflammatory expression profile. Multiple identified downstream targets had previously been identified as MMP regulators induced by pro-inflammatory cytokine stimulation of chondrocytes, such as activating transcription factor (ATF) 3, and early growth response (EGR) 2. Furthermore, work performed on the mechanical loading of chondrocytes in 3-dimensional hydrogel culture identified the same regulators to be induced by abnormal load, with subsequent MMP-1 expression. Taken together, disparate stimuli were identified as having common downstream elements suggesting overlapping pathways resulting in a common outcome: the induction of MMP expression, and an increased potential for cartilage catabolism.|
|Appears in Collections:||Institute of Genetic Medicine|
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|Falconer, A. 2017.pdf||Thesis||7.61 MB||Adobe PDF||View/Open|
|dspacelicence.pdf||Licence||43.82 kB||Adobe PDF||View/Open|
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