Investigation of the E3 ubiquitin ligase UBR5; a novel regulator of Hh gene expression

Abstract

The Hedgehog (Hh) signalling pathway is essential for embryogenesis and regulating cellular homeostasis in the adult, however much remains unknown about the molecular mechanisms that control ligand expression. In 2002, Lee et al. demonstrated that conditional mutation of the Drosophila hyperplastic discs gene (hyd) resulted in increased hh levels, suggesting that Hyd can act as a negative regulator of hh gene expression. Based on this evidence, the aim of this project was to investigate the hypothesis that UBR5, the murine homologue of Hyd, acts as a novel regulator of Hh gene expression in mammals. To investigate this hypothesis in vivo, I utilized the developing mouse limb as a model system that is highly sensitive to abnormal Hh expression. Morphological analysis of Ubr5 limb mutant embryos did not reveal an obvious phenotype, however quantitative analysis of Ihh gene expression and its downstream targets at E13.5 demonstrated a significant decrease in levels. In addition, changes in the expression of Runx2 and Msx2 were detected. Therefore, these data indicate that UBR5 can act as a positive regulator of Ihh expression, in addition to regulating other factors involved in chondrogenesis. The role for UBR5 as a positive regulator of Hh expression was also supported by in vitro investigations, demonstrating that UBR5 is required for Shh expression in mouse embryonic stem cells. Morphological analysis of adult Ubr5 limb mutant mice revealed the presence of significantly shorter long bones. These observations support previous reports that interference with IHH during early chondrogenesis can negatively affect long bone growth in the adult. Interestingly, adult Ubr5 limb mutant mice also possess osteophytes, a feature typically observed in osteoarthritis (OA), in addition to sites of ectopic mineralization (EM) near tendons of the knee and ankle. Based on these observations and evidence from the literature, I hypothesize that in addition to the role for UBR5 as a positive regulator of Ihh expression in the bone, UBR5 also plays a role in ligament/tendon development and/or maintenance, whereby its loss results in defective ligaments/tendons that are incapable of stabilizing the joints of the limb, culminating in joint deterioration, as observed in OA, in addition to EM. However, further investigation is required to determine whether this is also related to deregulated Ihh. These experiments suggest that Ubr5 limb mutant mice could provide a novel mouse model in the study of OA and prompt the investigation of the potential role for EDD, the human homologue of UBR5, in OA initiation and progression.