082 WITHDRAWN 083 iRHOM2-p63 pathway mediates survival and redox balance in keratinocytes P Arcidiacono, CM Webb, D Blaydon, A Chikh and DP Kelsell Centre for Cell Biology & Cutaneous Research, Blizard nstitute Queen Mary University, London, United Kingdom The palmoplantar keratodermas are characterised by different patterns of hyperproliferative thickening of the palms and soles which are often painful. We have described dominant “gain of function” mutations in RHBDF2, the gene encoding inactive rhomboid protein 2 (iRHOM2), associated with the inherited syndrome Tylosis (Palmoplantar keratoderma) with Oesophageal Cancer (TOC). Recently, we have shown that genetic depletion of iRHOM2 leads to a much thinner mammalian footpad plus decreases keratinocyte hyperproliferation and migration. As little is known about the transcriptional regulation of iRHOM2, its promoter sequence was assessed using transcription factor binding site prediction software (genoma- tix.gsf.de). Here, we report that iRHOM2 is a novel target gene of p63 and that both p63 and iRHOM2 can differentially regulate cellular stress associated signalling pathways in kerati- nocytes. In normal keratinocytes, p63 regulates positively iRHOM2 with iRHOM2 then antagonizing p63 expression whilst, in hyperproliferative keratinocytes, there is an auto- regulatory feedback loop occurring between p63 and iRHOM2. Using human TOC kerati- nocytes and irhom2 À/À mice, we demonstrate that p63-iRHOM2 mediated signalling regu- lates cellular functions including inflammation, survival and oxidative defence. For example, we identify survivin, a member of the inhibitor of apoptosis (IAP) gene family, as a novel binding partner of iRHOM2 and as a p63 target gene. In addition to increased cell survival via survivin expression, TOC keratinocytes showed increased level of reactive oxygen species (ROS) compared to control cells. We show a role for iRHOM2 in the epidermal oxidative defence response is via its interaction with cytoglobin (Cygb), a reported p63 target gene. In summary our data supports therapeutic strategies targeting iRHOM2-p63 axis in hyper- proliferative skin disease and dysplasia. 084 The keratin cytoskeleton as a regulator of keratinocyte mechano-sensing A Laly and J Connelly Centre for Cell Biology and Cutaneous Research, Blizard Institute - Barts and The London School of Medicine and Dentistry, London, United Kingdom Keratinocyte mechanical properties are important for skin’s function and depend on internal cytoskeletal structures, such as keratins. This study aimed to determine the role of the keratin cytoskeleton in cellular mechano-sensing. Human keratinocytes were cultured on collagen- coated polyacrylamide (PAAm) gels, with different elastic moduli, 0.5, 7, 40, and 200 kPa. Consistent with previous findings, cell area increased with substrate stiffness. Keratin bundle organisation analysis by confocal microscopy indicated that substrate stiffness did not alter bundle thickness or spacing, but rather the keratin mesh area increased with cell spreading. Interestingly, immunoblotting of keratin cross-linking revealed a specific increase in K14 multimers on the 70 kPa gels. Stiffness-induced cell spreading and keratin organisation also depended on cross-talk with the actin cytoskeleton. Inhibition of contractility with blebbis- tatin or ROCK inhibitor (Y27632) reduced K14 expression and increased cell spreading, while disruption of actin polymerisation with latrunculin B or Src inhibitor PP1 significantly inhibited cell spreading. Further analysis of downstream signal transduction to the epidermal growth factor receptor (EGFR) demonstrated increased activation of phospho-EGFR (Y1068) with increasing substrate stiffness. Finally, to test the role of keratin organisation in substrate rigidity sensing, mouse keratinocytes lacking the gene for the cytolinker plectin (Plec) were cultured on PAAm gels. Compared to wild type (WT) cells, Plec knockout (KO) keratinocytes displayed increased cell area on stiffer gels and higher levels of pEGFR. Together, these findings indicate that matrix stiffness regulates organisation of keratin cytoskeleton via cross- talk with F-actin cytoskeleton. Moreover, the cytolinker plectin mediates downstream mechanotransduction to the EGF signalling pathway. These results provide new insights into keratinocyte mechano-sensing and may have important implications for blistering skin dis- eases in which the keratin cytoskeleton is perturbed. 085 Type XVII collagen suppresses interfollicular epidermal proliferation in neonatal and aged skin, and helps rejuvenate epidermis M Watanabe 1 , K Natsuga 1 , W Nishie 1 , G Donati 4 , Y Fujimura 1 , T Tsukiyama 2 , H Ujiie 1 ,M Ozaki 3 , FM Watt 4 and H Shimizu 1 1 Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan, 2 Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Japan, 3 Department of Biological Response and Regulation, Faculty of Health Sciences, Hokkaido University, Sapporo, Japan and 4 Centre for Stem Cells and Regenerative Medicine, King’s College London, London, United Kingdom In skin, epidermal basement membrane zone (BMZ) serves as a niche for epidermal stem cells and is vital to cell homeostasis regulation. Type XVII collagen (COL17) is a hemidesmosomal protein located at the epidermal BMZ. COL17 deficiency in mice and human exhibits early aged phenotype as gray hair and hair loss. In this context, COL17 has been known to engage in maintaining melanocyte stem cells and hair follicle stem cells. However, the role of COL17 in regulating stem cells in interfollicular epidermis has not been elucidated. Here we show that COL17 controls epidermal proliferation through Wnt signaling. Loss of COL17 in interfollicular epidermis leads to epidermal hyperproliferation due to aberrant Wnt signaling. Furthermore, COL17 distribution in epidermal basal cells alters with aging, and this change also induces epidermal hyperproliferation. Of particular interest is the fact that over- expression of human COL17 in aged mice epidermis restores COL17 distribution and amends epidermal hyperproliferation. These findings demonstrate that COL17 is crucial for main- taining epidermal homeostasis. Our study implicates that faulty epidermal BMZ is involved in uncontrolled cell proliferation, phenocopying aged skin and oncogenesis. 086 Evidence for a palmoplantar-specific role for microtubules H Louis dit Picard, D Blaydon and DP Kelsell Blizard Institute, Queen Mary University of London, London, United Kingdom The palmoplantar epidermis is uniquely adapted to withstand physical stress. Genetic studies of the palmoplantar keratodermas (PPK) is revealing key molecules that regulate this structure including the inactive rhomboid protease iRHOM2 and Aquaporin 5 (AQP5) associated with Tylosis with Oesophageal Cancer (TOC) and diffuse non-epidermolytic PPK (DNEPPK), respectively. Using a yeast-2-hybrid (Y2H) screen with iRHOM2 as bait, we identified the major cytoskeletal stress keratin, K16, as an interacting binding partner of iRHOM2. From further analysis of the Y2H data, we now report BPAG1 (Bullous Pemphigoid Antigen 1 or Dystonin) and MACF1 (Microtubule-Actin Crosslinking Factor 1 or ACF7) as iRHOM2 interacting partners, via their spectrin repeat domain. These are both spectraplakins; giant cytoskeletal linker proteins that can interact with all three cytoskeletal components: actin, microtubules and intermediate filaments. Whilst the physical resilience of the palmoplantar epidermis has been linked predominantly to an abundance of keratin intermediate filaments, we have now identified a potential role for microtubules in this function. Immunostaining revealed a palmoplantar-specific localization pattern for the microtubule plus-end binding protein, EB1, that correlated with the palmoplantar ridges and also the alternating expression patterns for keratins 9 and 16, indicating that microtubule dynamics differ between inter- follicular and palmoplantar skin. We show increased levels of BPAG1 in TOC palm at the epidermal-dermal junction and also an altered localisation in TOC patient-derived kerati- nocytes compared to controls. In addition, MACF1 displays increased membrane localisation in DNEPPK palmoplantar epidermis. MACF1 is an EB1 interacting protein and EB1 also displayedincreased membrane localisation in DNEPPK palmoplantar. Together, our findings provide evidence of a palmoplantar-specific role for microtubules in both normal and disease physiology of the palmoplantar epidermis. 087 Metabolic and stress kinase signaling control skin barrier function: Implications for diabetic skin complications E Wachsmuth 1 , M Awazawa 2 , S Aghdam 1 , F Tellkamp 1 , M Pasparakis 3 , J Bru ¨ ning 2 and CM Niessen 1 1 Dermatology, University Hospital Cologne, Cologne, Germany, 2 University of Cologne, Center for Endocrinology, Diabetes and Preventive Medicine, Cologne, Germany and 3 Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), Cologne, Germany Type 2 diabetes mellitus is prevalent in western population and is associated with skin dis- eases, such as impaired healing and barrier dysfunction. It remains unresolved whether diabetic skin complications are due to metabolic alterations or from impaired insulin action in the skin. A major aim is to define the contribution of cell autonomous and non-cell autonomous insulin/IGF-1 signalling (IIS) in skin barrier function and development of diabetic skin complications. The epidermal barrier in mice is first formed at embryonic Day (E) 16.5 and protects the organism of chemical and physical insults and serves as an immune barrier. We find that loss of epidermal Insulin Receptor (IR) and Insulin like growth factor receptor (IGF-1R) results in an initially severe barrier defect at E17.5, which is much less pronounced in newborn mice, suggesting a compensatory response. On the molecular level, loss of IIS induces a p38 Map kinase stress response that promotes epidermal barrier formation as combined loss of IIS and p38a in the epidermis resulted in severe barrier defects and perinatal lethality. Spike-in Silac on newborn epidermis identified the epidermal differentiation com- plex as one key target of p38 and IIS. Together, our data provide direct evidence that impaired epidermal IIS alters skin barrier function and that activation of p38 signaling partially repairs this defect in stratum corneum function. Importantly, we find that adult dietary (high-fat diet) and genetically induced hyperglycemic and obese mice present with insulin resistance in the skin, exhibit epidermal barrier dysfunction and epidermal atrophy, thereby linking impaired cell autonomous IIS in the epidermis to diabetes associated skin complications. Epidermal Structure and Function | ABSTRACTS www.jidonline.org S207