Urokinase-plasminogen activator as a target to block osteoarthritic cartilage destruction

Description

Osteoarthritis (OA) is one of the major causes of disability globally, which is particularly significant in an ageing population. Despite its importance, there remains no therapies that can alter disease progression. This PhD studentship will drive new understanding a novel target, with implications for the development of therapeutic strategies for OA.

Background

The breakdown of the articular cartilage is central to OA. Cartilage protects the joint from compression, and once lost, causes significant pain. The matrix metalloproteinases (MMPs) drive the loss of the cartilage collagen, but MMPs are difficult to target with drugs, and attempts to do so have so far been unsuccessful1. The serine proteinases are a different family of proteinases which have emerging roles in the in OA, and are more tractable drug targets2.
Urokinase plasminogen activator (uPA) is a serine proteinase usually associated with fibrinolysis (clot breakdown) by activation of plasmin. However, our recent research supports a role for uPA in cartilage breakdown. We observed that uPA expression is elevated at important timepoints during cartilage degradation in vitro, and that treatment with its endogenous inhibitor - plasminogen activator inhibitor-1 (PAI-1) - can block cartilage collagen destruction. Moreover, despite being considered a proteinase with restricted substrate specificity, we have demonstrated that uPA is able to directly activate proMMP-3. Using unbiased bioinformatic analysis, uPA dysregulation was a major signal from multiple OA datasets and correlates with disease gene signatures (p=0.0009)3. Taken together, these in vitro and in silico data support a role for uPA in driving disease, potentially through activation of MMPs. Now, to progress into translational studies, we must demonstrate the importance of uPA in preclinical models of OA.

Hypothesis

‘Urokinase plasminogen activator (uPA) is an essential activator of catabolic MMPs and driver of cartilage destruction in osteoarthritis’
Objectives and Expected Outcomes:

1. Use a combination of transgenic mice and pharmacological inhibition to determine the importance of uPA in OA progression in preclinical models of OA
2. Identify novel substrates of uPA in cartilage using N-terminal proteomics
3. Visualise uPA activity in OA using a novel activity-based probe.

Methodology

Using uPA-deficient and PAI-1-deficient mice (commercially available) we will characterise the importance of this serine proteinase (and its inhibitor) using a murine model of OA (destabilisation of the medial meniscus; DMM – well established at UoL). We will monitor the effect on cartilage damage using histology and neo-epitope immunohistochemistry4, while bone changes will be monitored using micro-computed tomography (CT). Gait analysis and activity will also be determined. Next, pharmacological inhibitors of uPA will be administered in wild-type mice to determine drug feasibility. Using cartilage from uPA-deficient mice (or recombinant proteinase) we will conduct N-terminal proteomics to identify novel cartilage substrates of uPA. Finally, we will visualise uPA activity during disease progression using a fluorescent activity-based probe and investigate its potential as a diagnostic biomarker of destructive OA.

Training, supervision and resources

Dr David Wilkinson is a mid-career researcher with expertise in osteoarthritis and proteolytic enzymes. As a holder of an externally-funded fellowship, he will be available to provide hands-on laboratory support and training. The supervisory team will foster a supportive environment and provide training in molecular biology, in vivo models, histology, fluorescent imaging and proteomics. The project will be undertaken in the Musculoskeletal and Ageing Sciences department, within a wider institute harbouring many areas of complementary expertise. The project will also make use of strong core facilities at UoL in proteomics (centre for proteomic research), microscopy (centre for cell imaging) and preclinical models (centre for preclinical imaging). This project will be suitable for a dedicated student with an interest in translational medical science, addressing a major unmet clinical need.