2006 ESDR ABSTRACTS View metadata, citation and similar papers at core.ac.uk brought to you by CORE 001 [003] 004 [022] provided by Elsevier - Publisher Connector Functional Effects Of Pachyonychia Congenita Type I Mutations on Keratinocyte Migration P120-catenin is a New Member of Desmosome to Recruit desmoglein3 (Dsg3) to Desmosomes and the Actin Cytoskeleton by Binding to its Cytoplasmic Membrane-proximal Domain Van Koningsveld R1, Fitchett CJ1, Rugg EL2 and O’Toole EA1 Kanno M, Aoyama Y, Nagai M, Yamamoto Y and Kitajima Y 1Centre for Cutaneous Research, ICMS, Queen Mary, University of London (United Kingdom) Department of dermatology, Gifu University School of Medicine (Japan) 2Department of Dermatology, University of California, Irvine (USA) p120-catenin (p120-ctn) is armadillo family protein, which binds to classical cadherins at Keratins are the largest group of cytoskeletal proteins forming the intermediate fi lament cy- the juxtata membrane domain (JMD) of cytoplasmic domain through its arm repeat domain. toskeleton within epithelial cells. Keratins K6a and K16 are expressed in wound healing, skin Recently several lines of evidence indicate that p120-ctn stabilizes cadherins and regulates cancer and psoriasis and mutations in these genes cause type I pachyonychia congenita (PC1). the cell adhesion positively . We have already shown the association of endogeneous Dsg 1 The aim of this study was to investigate the effect of over-expression of wild-type or mutant K6a and 3 associated with p120ctn by immunoprecipitation study and the localization of p120- (K6aN171del) and K16 (K16R127C) on human keratinocyte (HK) migration, and study the sig- ctn at desmosome by immuno-electron microscopy. To investigate the association manner of nalling pathways involved. Phoenix cells (HEK293 cells modifi ed to express retroviral packag- Dsg3 and p120-ctn, we constructed expression vectors containing Flag tagged mutant forms of ing proteins) were transfected with target genes and the supernatant was used to infect primary mouse Dsg3 with increasing truncations eliminating various cytoplasmic subdomain from the HK. Migration was assessed using the colloidal gold and in vitro scratch assays. Expression of carboxy-terminus, which is ∆ICS:AA1-810, AA1-761,∆IA:AA1-714, 1-641, respectively. These signalling molecules was investigated using Western blotting and differentially expressed genes constructs and HA tagged mouse p120-ctn were co-transfected into HEK293 cell. Extracts using Afymmetrix chip technology. All cells migrated further in the presence of 10nM EGF, were subjected to immunoprecipitation with anti-HA antibody. Recoverd p120-ctn contained however, the K16R127C-transduced HKs displayed maximal hypermotility in response to EGF all Dsg3 mutants but not 1-641. The result suggests that the cytoplasmic membrane-proximal in the colloidal gold assay (p<0.0001). Both K16 and K16R127C-expressing cells displayed hy- domain including IA region (641-714AA) of Dsg3 is the binding site for p120-ctn. To confi rm permotility in the in vitro scratch assay. Maximal p38 phosphorylation in response to EGF was the role of p120-ctn, we generated another mutant form of mouse Dsg3, ∆641-714. We deter- seen in the K16R127C-transduced HKs. The K16R127C-transduced keratinocytes displayed mined the localization of these recombinant proteins in DJM-1 cells by immunofl uorescence. decreased basal expression of cofi lin, an actin-binding protein. Data-mining of the Afymmetrix P120-ctn was found to co-localize with ∆ICS:AA1-810, AA1-761, ∆IA:AA1-714, but neither chip data using Genespring identifi ed altered expression of several genes involved in actin ∆IA:AA1-641 nor AA641-714, immunocytochemicaly. Interestingly, ∆IA:AA1-641 and AA641- remodelling in the transduced cells suggesting that the PC type I mutation, R127C, signifi cantly 714 which lack the cytoplasmic membrane-proximal region, were localized in the cytoplasm alters keratinocyte migration by affecting actin cytoskeletal dynamics. These changes may play but not localized at the plasma membrane. These results suggest that p120-ctn can associate a role in the stress-induced clinical phenotype seen in PC1. not only with classical cadherin, but also with desmosomal cadherin as a new member of desmosomal constituents, and that p120-ctn might be involved in stabilization of Dsg3 at the 002 [011] plasma membrane. Serpin Squamous Cell Carcinoma Antigen-1 Suppresses UV-induced Cell Death In Vivo and In Vitro 005 [048] Hibino T, Katagiri C and Nakanishi J Novel Targets for Cystatin M/E: A New Route in Epidermal Differentiation Shiseido Life Science Research Center (Japan) Cheng T1, Hitomi K2, Ishida-Yamamoto A3, van Vlijmen-Willems IM1, de Jongh GJ1, Bergers M1, Schalkwijk J1 and Zeeuwen PL1 Recently we have demonstrated that serpin squamous cell carcinoma antigen 1 (SCCA1) plays 1Department of Dermatology, Radboud University Nijmegen Medical Centre (Netherlands) a critical role in UV-induced apoptosis in human keratinocytes. This reaction was mediated via 2Department of Applied Molecular Biosciences, Nagoya University (Japan) specifi c suppression of c-Jun N-terminal kinase-1 (JNK1). In the present study, we generated 3Department of Dermatology, Asahikawa Medical College (Japan) transgenic mice that overexpressed SCCA1 driven from the involucrin promoter and examined the effect of UV irradiation. In addition, we established various cell lines, in which SCCA1 was Cystatin M/E is a high affi nity inhibitor of the asparaginyl endopeptidase legumain. Although overexpressed or downregulated. Using these cell lines we further analyzed the roles of SCCA1 cystatin M/E also contains a predicted binding site for papain-like cysteine proteases, no high in cell death and proliferation. affi nity binding for any member of this family has been demonstrated so far. The present study Since SCCA1 was strongly upregulated in the upper epidermis of sun-exposed skin, we gener- aims on fi nding these unknown target proteins of cystatin M/E. Furthermore, we investigated ated involucrin promoter-driven SCCA1 transgenic mice. In order to obtain hairless phenotype, the possible role of cystatin M/E and its physiological target proteases in epidermal differentia- the involucrin-SCCA1 transgenic mice were crossed with HR-1 mice and SCCA1+/+ mice were tion and desquamation. We report that human cathepsin V (CTSV) and human cathepsin L obtained after three passages. Immunostaining of skin sections revealed strong expression of (CTSL) are strongly inhibited by human cystatin M/E. Kinetic studies show that Ki values of SCCA1 in the upper epidermis of the transgenic mice but not in the skin of wild HR-1 mice. cystatin M/E for the interaction with CTSV and CTSL are 0.47 nM and 1.78 nM, respectively. When 200 mJ/cm2 of UV was irradiated twice on the back of mice, the epidermis of wild Site-directed mutagenesis was used to identify the binding sites of cystatin M/E. We found that type mice was seriously damaged, showing spongiosis in the suprabasal layer. In contrast, the legumain and the papain-like cysteine proteases CTSV and CTSL are inhibited by two distinct mutant mice demonstrated remarkable resistance against UV irradiation. We transfected hu- non-overlapping sites. Localization studies in normal human skin show that cystatin M/E co- man SCCA1 cDNA into 3T3/J2 cells and established 20 individual clones that stably express localizes with CTSV and CTSL in the upper layers of the epidermis. Furthermore, we show that SCCA1. SCCA1-expressing clones showed a strong correlation between the expression levels CTSL is the elusive enzyme that processes and activates epidermal transglutaminase 3, an en- and the suppression of UV-induced apoptosis. In SCCA knockdown HaCaT cells using siRNA, zyme responsible for crosslinking during epidermal cornifi cation. Compartmentalization stud- UV-induced apoptosis dramatically increased. Interestingly downregualtion of SCCA1 resulted ies using immunoelectron microscopy revealed that both cystatin M/E and CTSV are found in in marked increase of phospho-c-Jun and UV irradiation further enhanced the levels of c-Jun the extracellular space in the stratum corneum associated with corneodesmosomes, suggesting phosphorylation. Our data suggest that the up-regulation of SCCA1 is enough to suppress UV- an important role for these molecules in epidermal desquamation. The identifi cation of CTSV induced apoptosis and reveal a novel UV protection mechanism in the human skin. and CTSL as novel targets for cystatin M/E, their co-expression in human skin, and the activity of CTSL towards transglutaminase 3, strongly implies an important role for these molecules in the differentiation process of human epidermis. 003 [021] Functional Study of the Dermokine σ, an Intracellular Isoform Encoded by an Epidermal Gene with a Complex Splicing 006 [049] Leclerc E, Jonca N, Toulza E, Gazeilles L, Serre G and Guerrin M The activity of PI3K-Akt-mTOR Survival Pathway in Keratinocytes is Critically Dependent on “Epidermal Differentiation and Rheumatoïd Autoimmunity”, UMR 5165 CNRS-Toulouse III the Integrity of Lipid Rafts in the Membrane University (France) Calay D and Gniadecki R Department of Dermatology, Bispebjerg Hospital, University of Copenhagen (Denmark) In order to better understand the molecular mechanism underlying the cohesion/desquama- tion processes which are essential for the barrier function, we have analyzed the granular Lipid rafts are nanometer-large, cholesterol- and shingolipid-rich plasma membrane microdo- keratinocyte transcriptome.