UNDERSTANDING THE ROLE OF SFRP1 AND RSPONDIN2 IN REGULATING KELOID PATHOGENESIS THROUGH THE CANONICAL WNT SIGNALLING PATHWAY

Abstract

Keloids are exuberant and autonomous scar tissue which grossly extends beyond the clinical borders of the original wound. They uniquely occur in humans and there is no satisfactory treatment to date. There is currently no suitable animal model for keloid/scar research, and much is focused on understanding the complex molecular and cellular biology of keloid pathogenesis as a strategy to find appropriate therapy for this disfiguring disorder. The canonical Wnt signalling was recently implicated with keloid pathogenesis but it remains to be elucidated how this pathway is directly up-regulated at the protein level. A microarray study conducted within our laboratory concurred with a recent publication that keloid fibroblasts (KFs) have a much lower mRNA expression of secreted Fzd-related protein 1 (SFRP1)- a known Wnt signalling inhibitor, compared to normal skin fibroblasts (NFs). The work in this thesis confirmed that KFs indeed secrete much less SFRP1 protein compared to NFs in the conditioned media. It was therefore hypothesized that significant loss of SFRP1 gave rise to keloid pathogenesis through the up-regulation of Wnt signalling. To test this hypothesis, the overexpression of KFs with SFRP1 was studied and investigated in terms of cellular growth, migration, protein expression of extra-cellular matrix (ECM) components and degree of contraction in fibroblast populated collagen lattice (FPCL). Concurrently, a proteomic study performed in our laboratory detected Rspondin2 (Rspo2) in the conditioned media of KFs. Rspo is a relatively new group of secreted proteins known to be a co-activator of Wnt signalling. This novel class of proteins was recently found: 1) to be implicated in epithelial-mesenchymal interaction of the skin, 2) to promote angiogenesis and 3) to be non-tumorigenic despite long term administration in mice. The above three points stated were all previously reported to be implicated or associated with keloids or their pathogenesis. Here, it was hypothesized that Rspo2 might have a role in keloid formation through the Wnt signalling pathway. The approach for this part of the project was to study and compare the expression levels of Rspo2 in keloids and normal skin; with a focus of understanding the function of this protein in terms of epithelial-mesenchymal interactions. Finally, quercetin was tested if it had inhibitory effects on the Wnt signalling pathway in KFs. Quercetin, a strong dietary antioxidant, was found to inhibit Wnt signalling in some cancer cells, but was also previously reported to mitigate KFs growth and increased contraction via other signalling pathways. If it is found to act on the Wnt/ß-catenin signalling in KFs, it would strongly suggest that quercetin is a suitable candidate for keloid treatment as a multi-target inhibitor. In summary, this thesis aims to show that Wnt signalling is one of the main modulators of keloid pathogenesis and that the upregulation of this canonical pathway in keloids is supported by both a negligible expression of SFRP1 and a higher expression of Rspo2 proteins. It is hoped that by identifying some of the key players of Wnt signalling in keloids, suitable therapeutic intervention can be identified to treat this fibrotic condition.