Investigating the role of UBR5, a ubiquitin-protein ligase of the N-end rule pathway, in skeletal tissue homeostasis
Kubiak, Malgorzata Maria
Using a conditional Ubr5 mutant (Ubr5mt) mouse the Ditzel lab identified a novel role for the ubiquitin-protein ligase UBR5 in maintaining articular cartilage (AC) homeostasis, suppressing osteoarthritis (OA) and controlling heterotopic tendon ossification (HO), potentially through influencing the Hedgehog signalling pathway. Histological observations revealed aberrant chondrocyte behaviour in both the AC and ossifying tendons. Interestingly, application of the Hedgehog pathway inhibitor cyclopamine enhanced the Ubr5mt OA and HO phenotypes. Based on these observations I hypothesised that UBR5 utilises Hh signalling to control AC and tendon homeostasis through influencing AC- or tendon-resident stem/progenitor cell function. I planned to address this hypothesis by designing an in vitro cell-based model to investigate the chondrogenic potential of UBR5-deficient stem/progenitor cells and by investigating the Hh signalling expression pattern in tendons collected from normal and Ubr5mt animals. To address the molecular and cellular defects on Ubr5mt cells I established an in vitro system to culture mesenchymal stem cells (MSCs) and promote their differentiation into chondrocytes. First, I optimised the experimental conditions for the extraction and culture of bone marrow derived cells from C57BL/6 mice. The identity of the isolated cells was confirmed by functional chondrogenesis assays and expression of specific MSCs markers. Once I was confident about the isolation and culture techniques I aimed to culture cells derived from conditionally Ubr5mt animals. Unfortunately, these Cre-recombinase bearing MSCs proved difficult to culture, which severely hampered experimentation and made the proposed experimental approach unfeasible. To investigate the Hh signalling expression and cellular pattern in tendons affected by HO, I compared the presence of various markers in patellar tendons collected from normal and Ubr5mt mice. It turned out that the level of IHH and PTCH1, proteins involved in Hh signalling pathway, was much higher in the latter. Overall, I have identified differential expression of Hedgehog pathway components in control and Ubr5mt tendons as well as successfully isolated and cultured MSC. These findings and achievement have contributed to a manuscript and will form the basis of a grant proposal.