Journal of Pathology J Pathol 2014; 232: 541–552 Published online 5 February 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.4323 ORIGINAL PAPER Flightless I over-expression impairs skin barrier development, function and recovery following skin blistering Zlatko Kopecki, 1,2 * Gink N Yang, 1 Ruth M Arkell, 3 Jessica E Jackson, 1 Elizabeth Melville, 1 Hiroaki Iwata, 4 Ralf J Ludwig, 4 Detlef Zillikens, 4 Dedee F Murrell 5 and Allison J Cowin 1,2 1 Centre for Regenerative Medicine, Mawson Institute, University of South Australia, Adelaide, Australia 2 Women’s and Children’s Health Research Institute, Adelaide, Australia 3 Research School of Biological Sciences, Australian National University, Canberra, Australia 4 Department of Dermatology, University of Lubeck, Germany 5 Department of Dermatology, St George Hospital, University of New South Wales, Sydney, Australia *Correspondence to: Z Kopecki, Centre for Regenerative Medicine, Mawson Institute, University of South Australia. e-mail: zlatko.kopecki@unisa.edu.au Abstract Development of an intact epidermis is critical for maintaining the integrity of the skin. Patients with epidermolysis bullosa (EB) experience multiple erosions, which breach the epidermal barrier and lead to increased microbial colocalization of wounds, infections and sepsis. The cytoskeletal protein Flightless I (Flii) is a known regulator of both development and wound healing. Using Flii +/- , WT and Flii Tg/Tg mice, we investigated the effect of altering Flii levels in embryos and adult mice on the development of the epidermal barrier and, consequently, how this affects the integrity of the skin in EB. Flii over-expression resulted in delayed formation of the epidermal barrier in embryos and decreased expression of tight junction (TJ) proteins Claudin-1 and ZO-2. Increased intercellular space and transepidermal water loss was observed in Flii Tg/Tg adult mouse skin, while Flii Tg/Tg keratinocytes showed altered TJ protein localization and reduced transepithelial resistance. Flii is increased in the blistered skin of patients with EB, and over-expression of Flii in experimental EBA showed impaired Claudin-1 and -4 TJ protein expression and delayed recovery of functional barrier post-blistering. Immunoprecipitation confirmed Flii associated with TJ proteins and in vivo actin assays showed that the effect of Flii on actin polymerization underpinned the impaired barrier function observed in Flii Tg/Tg mice. These results therefore demonstrate an important role for Flii in the development and regulation of the epidermal barrier, which may contribute to the impaired healing and skin fragility of EB patients. Copyright  2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: Flii; epidermolysis bullosa; skin barrier; epidermis Received 18 September 2013; Revised 10 December 2013; Accepted 20 December 2013 No conflicts of interest were declared. Introduction An intact epidermis is critical for maintaining the integrity and function of healthy skin. This physi- cal barrier consists of the stratum corneum, cell–cell tight junctions (TJ) and associated cytoskeletal proteins [1]. Patients with epidermolysis bullosa (EB) expe- rience recurrent skin blistering, leading to disruption or loss of the protective barrier, increased microbial wound colocalization and the development of chronic infected wounds [2,3]. Accordingly, sepsis is the lead- ing cause of death in infants with EB [4]. During development and epidermal wound healing, TJ proteins are expressed well before stratum corneum formation [5,6]. Similarly to wound healing, blistering results in TJ protein redistribution in marginal cells [7,8] and up- regulation in the hyperproliferative zone surrounding the blister. An initial protective barrier is first formed by cells re-epithelializing the blister wound, well before stratum corneum formation [8–10]. Establishment of an intact barrier contributes to skin strength, while the quick recovery of the barrier post-blistering is critical for blister healing. As the complex response of epi- dermal cells to barrier disruption may exacerbate EB, treatments aimed at aiding barrier re-establishment or dampening the epidermal stress response may improve the healing observed in EB patients. TJs are intercellular junctions which form the para- cellular barrier and consist of occludin, claudins (Cldns) and junctional adhesion molecules linked to the actin cytoskeleton through scaffolding plaque pro- teins; zonula occludens (ZO)-1, -2, -3, MUPP-1 and cingulin [11]. Different studies have shown the impor- tance of Cldns and ZO-1 and -2 in the forma- tion of TJ strands and paracellular barrier functions [12,13]. Protein–protein interactions between Cldns and scaffolding plaque proteins provide the link to the actin cytoskeleton allowing the transduction of the Copyright  2013 Pathological Society of Great Britain and Ireland. J Pathol 2014; 232: 541–552 Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com