Effect of Flightless protein on skin architecture, cellular responses and Epidermolysis Bullosa.

Abstract

Wound healing is an area of largely unmet medical need with patients often relying on wound management practice rather than specific therapies. Recent research in our laboratory has identified a cytoskeletal protein Flightless (Flii) as a negative regulator of wound healing. This highly conserved protein is important in development and has a unique structure allowing it to act as a multifunctional protein. Flii expression increases in response to wounding, inhibiting cellular migration and proliferation while its deficiency improves wound healing. The aim of this study was to investigate the effect of differential Flii expression on skin architecture, cellular responses during wound healing, adhesionmediated cell signaling and skin blistering associated with the genetic skin disorder Epidermolysis Bullosa (EB). Chapter 3 of this thesis describes the effect of differential Flii expression on skin architecture and formation of hemidesmosomes which anchor the skin layers. Using primary fibroblasts and keratinocytes with varying Flii expression this study investigated the effect of Flii expression on cellular adhesion, spreading and migration on different extracellular matrix substrates. The results presented in Chapter 3 also describe the effect of Flii neutralising antibodies on primary keratinocyte proliferation illustrating improved proliferation in response to decreased Flii expression. In Chapter 4 an incisional wound healing model was used to investigate the effect of differential Flii expression on different components of hemidesmosomes. Flightless was shown to regulate hemidesmosome formation through its effects on integrin-mediated cellular adhesion and migration. Using immunoprecipitation studies, Flii association with structural and signaling proteins present at the dermal-epidermal junction was investigated. Flii was found to form a cytoskeletal complex with talin, vinculin and paxillin suggesting its role in downstream signaling. The association of Flii with paxillin was further investigated in Chapter 5 where the effect of Flii over-expression on fibroblast adhesion and formation of adhesion structures was examined. Flii over-expression inhibited paxillin activation and the turnover of adhesion structures through down regulation of signaling proteins involved in cell adhesion signaling pathways. Chapter 6 of this thesis summarises the effect of Flii in skin blistering by utilizing both human samples and two mouse models of Epidermolysis Bullosa. Flii expression is significantly increased in response to skin blistering and its effects on integrin mediated cellular adhesion, migration and type VII collagen expression make Flii a negative contributor to blister formation. Decreasing Flii expression genetically or using neutralizing antibodies reduces skin blistering, improves cellular adhesion and decreases TGF-β mediated collagen contraction. In summary, Flii adversely affects skin strength and blister formation. Using a multidi-mensional approach of both in vitro and in vivo methodologies, human tissue and animal models this thesis reveals several novel findings and contributes to better understanding the involvement of Flii in both maintaining skin homeostasis and regulating wound repair. Flii is a novel target for development of mechanistic based therapy for improved wound healing. Findings presented in this thesis may open doors to significant changes in clinical practice and contribute to better therapeutic design by which would healing of blisters in patients with Epidermolysis Bullosa might be improved.