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099 E-Cadherin is dispensable for epidermal localization of Langerhans cells
Epidermal Structure and Function | ABSTRACTS 094
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Prostaglandin E2 regulates osteoclastogenesis through the induction of RANKL in middle ear cholesteatoma R Imai1,2, T Sato2, M Terao1, H Murota1, H Inohara2 and I Katayama1 1 Dermatology, Osaka University Graduate School of Medicine, Suita, Japan and 2 Otorhinolaryngology, Head and Neck Surgery, Osaka University Graduate School of Medicine, Suita, Japan Middle ear cholesteatoma is structurally similar to epidermal cyst composed of both stratified squamous epithelium and surrounding fibrosis tissue. In spite of benign tumor, cholesteatoma destructs adjacent bones and causes various complications such as hearing loss, vertigo, facial nerve palsy and brain abscess. Although mechanism of bone destruction by cholestatoma has been explored for many years, it has not been cleared. We succeeded in generating a model mouse for cholesteatoma, epidermal cyst like tissue by injecting both of ear pinna-derived fibroblasts and keratinocytes on calvarial bone. This model cyst destroys the adjacent calvarial bone and induced osteoclasts. Also RANKL was expressed by fibroblasts surrounding cysts. We focused on Prostaglandin E2, which were reported to promote RANKL expression. The PGE2 concentration of extraction from the model cysts was higher than that from control mouse skin by ELISA. The expression of PGE Synthase was detected in the cells of model cyst by immunohistochemistory. In vitro culture system, we confirmed that PGE2 significantly upregulated RANKL expression in the primary fibroblasts from mouse ear pinna. The upregulation of RANKL expression is inhibited by EP3 antagonist. EP3 is known for one of the PGE2 receptor. In clinical samples, the PGE2 concentration of extraction from cholesteatoma was higher than that from control skin. Quantitative real time PCR revealed that RANKL mRNA expression in cholesteatoma was higher than that in skin. The number of osteoclasts on the surface of bone adjacent to cholesteatoma was significantly increased, compared with cholesteatoma. These data suggested that PGE2 regulated osteoclast differentiation through the induction of RANKL expression in cholesteatoma. Furthermore, these were implicated that cyclooxygenase-2 inhibitors or EP3 antagonist have possibility of new therapeutic strategies for cholesteatoma-induced bone destruction.
PRC2 complex is required for human epidermal differentiation and proliferation I Asamaowei, V Botchkarev, NV Botchkareva and AN Mardaryev University of Bradford, Bradford, United Kingdom Polycomb repressive complex 2 (PRC2) catalyses the methylation of ‘Lys-27’ of histone H3 leading to transcriptional repression of target genes through its catalytic subunit Enhancer of zeste homolog 1/2 (EZH1/2). PRC2 functions as a critical regulator of stem cell identity and their differentiation in embryonic and adult tissues. In mouse skin, PRC2 prevents premature differentiation of epidermal progenitor cells and their ectopic differentiation to Merkle cells. The role of subunits of PRC2 complex in human skin homeostasis remains unclear. Here, we show that in human skin EZH2 is predominantly expressed in the epithelial cells. In the epidermis, its expression is increased in differentiating cells of the suprabasal layer. Indeed, EZH2 expression and H3K27 histone methyltransferase activity is markedly up-regulated in Ca2+-induced differentiation in primary human keratinocytes. Interestingly, knockdown of subunits of PRC2 complex - EZH1/2 and EED using siRNA and small molecule inhibitors GSK126 and UNC1999 up-regulated expression of KRT1, LOR and FLG genes and suppressed cell proliferation in primary human keratinocytes. These results suggest that PRC2 complex plays a critical role in the control of human epidermal progenitor cells proliferation and differentiation.
Novel amino acid duplication in collagen XVII causes mild junctional epidermolysis bullosa by altering the coiled-coil structure and impairing collagen XVII trimerization and maturation J Kroeger1, S Hofmann2, J Leppert1, C Has1 and C Franzke1 1 Department of Dermatology, Medical Center, University of Freiburg, Freiburg, Germany and 2 Center for Dermatology, Allergy and Dermatosurgery, HELIOS University Hospital Wuppertal, University Witten/ Herdecke, Wuppertal, Germany The function and stability of collagens depend on accurate triple helix formation of three distinct polypeptide chains. Disruption of this triple-helical structure can result in connectivetissue disorders. Triple helix formation is thought to depend on three-stranded coiled-coil oligomerization sites. However, only little is known about the physiological relevance of these coiled-coil structures. Transmembrane collagen XVII, also known as 180kDa bullous pemphigoid antigen provides mechanical stability through the anchorage of epithelial cells to the basement membrane. Mutations in the collagen XVII gene, COL17A1, cause junctional epidermolysis bullosa (JEB), characterized by chronic trauma-induced skin blistering. Here we exploited a novel naturally occurring COL17A1 mutation, leading to an in-frame lysine duplication within the coiled-coil structure of the NC16A domain of collagen XVII, which resulted in a mild phenotype of JEB due to reduced membrane-anchored collagen XVII molecules. This mutation causes structural changes in the mutant molecule and interferes with its maturation. The destabilized coiled-coil structure of the mutant collagen XVII unmasks a furin cleavage site that results in excessive and non-physiological ectodomain shedding during its maturation. Furthermore, it decreases its triple-helical stability due to defective coiled-coil oligomerization, which makes it highly susceptible to proteolytic degradation. As a consequence of altered maturation and decreased trimer stability, reduced collagen XVII is incorporated into the cell membrane, resulting in compromised dermalepidermal adhesion. Taken together, using this genetic model, we provide the first proof that alteration of the coiled-coil structure destabilizes oligomerization and impairs physiological shedding of collagen XVII in vivo.
Loss of loricrin exacerbates psoriasis-like skin inflammation through altered dendritic cell homeostasis T Ogawa, Y Ishitsuka, Y Nakamura, R Watanabe, N Okiyama, Y Fujisawa and M Fujimoto Dermatology, University of Tsukuba, Tsukuba, Japan Psoriasis is a chronic inflammatory skin disease associated with multiple predisposing factors, and one of susceptible loci known as PSORS4 is on human chromosome 1, in which epidermal differentiation complex (EDC) is located. An EDC gene loricrin (Lor) accounts for a major protein mass of cornified cell envelope (CE), the vital structure for epidermal integrity. In contrast to competent epidermal permeability barrier, Lor knockout (LKO) mice had reduced epidermal mechanical stability of the epidermis resulting from impaired crosslinking of filaggrin and keratin 1/10 in their CEs. Since mechanical stress/trauma can contribute to psoriasis induction and exacerbation, we evaluated the impact of the loss LOR in a psoriasis-like disease model. Reflecting mechanical weakness, application of mechanical stress onto the ear skin resulted in enhanced and sustained keratin 6 (K6) protein expression in LKO epidermis as determined by immunohistochemistry (IHC). LKO mice had significantly increased expression levels of cytokines that initiate psoriatic inflammatory response, including Tnf, Il1b and Ifn1a on quantitative reverse transcription PCR (RT-qPCR). Topical application of imiquimod (Aldara) cream on LKO mice resulted in a severe inflammatory phenotype; ear skin had augmented swelling, and back skin had enhanced erythema and hyperkeratosis accompanied by increased transcriptional expression levels of K6a. Since Ifn-a produced by plasmacytoid dendritic cell (pDC) is known to play a pivotal role in this model, pDC marker expression was analyzed in a steady state. Unchallenged LKO mice had significantly enhanced expression of Bst2 in the ear skin as determined by RT-qPCR and IHC. Taken together, these results indicate that reduced mechanical stability in the epidermis can exacerbate psoriasis-like skin inflammation through the alteration of DC homeostasis.
iRHOM2 regulation of loricrin in the epidermal barrier L McGinty, DP Kelsell and D Blaydon Centre for Cell Biology and Cutaneous research, Queen Mary University of London, London, United Kingdom iRHOM2 is an inactive rhomboid protease, which has been shown to facilitate barrier formation and transglutaminase activity through ADAM17 interaction. Gain-of-function mutations in iRHOM2 underlie the autosomal-dominant syndrome Tylosis with oesophageal cancer (TOC), with patients possessing an altered epithelial barrier. Here, through analysing differences in the expression intensity and localisation of epidermal barrier proteins in TOC tissue and irhom2 -/- mice, we identify a role for iRHOM2 in the regulation of loricrin (LOR) in the epidermal barrier. Immunofluorescent (IF) analysis of LOR in TOC palm epidermis shows increased levels of basal nuclear LOR localisation compared to controls or interfollicular TOC epidermis. In contrast, in irhom2 -/- mice, we found lower levels of lor expression compared to WT mice, with neither exhibiting nuclear lor. TOC keratinocytes grown normally in monolayer alongside control keratinocyte cell lines exhibited LOR expression throughout the cell nucleus and cytoplasm, with higher nuclear LOR in TOC cells. When grown in the presence of the small-molecule ADAM17 inhibitor, TMI-005, LOR was found to localise heavily at the nuclear periphery as opposed to the nucleus in all cell lines. TOC keratinocytes grown in a 3D model also display nuclear LOR in the basal cell layer, mimicking TOC palm. Furthermore, when grown in the presence of TMI-005, nuclear LOR is absent in these models, indicating that inhibition of ADAM17 activity reduces epithelial stress. In addition to our cutaneous findings, we observe nuclear LOR in TOC oesophageal epithelium, which like the palm, is also hyperproliferative. We demonstrate the importance of the iRHOM2/ADAM17 signalling axis in the regulation and localisation of LOR. Modulation of this pathway signifies a relation between iRHOM2/ADAM17 and a possible stress-related nuclear translocation mechanism for LOR in hyperproliferative tissue.
E-Cadherin is dispensable for epidermal localization of Langerhans cells B Dorn1, B Raghavan1, S Kunz2, S Martin2 and T Jakob1 1 Experimental Dermatology and Allergy Research Group, Department of Dermatology and Allergy, Justus-Liebig University Giessen, University Medical Center Giessen (UKGM), Giessen, Germany and 2 Allergy Research Group, Department of Dermatology, Medical Center - University of Freiburg, Freiburg, Germany Langerhans cells (LC) are potent antigen presenting cells localized in the epidermis. LC express high levels of the adhesion molecule E-Cadherin (E-cad) which has been suggested to be responsible for adhesion to keratinocytes and thus LC localization to the epidermis, supported by three lines of evidence: formation of adherens junctions between E-cad expressing LC like dendritic cells and keratinocytes, down regulation of E-cad during activation, maturation and emigration of LC, and requirement of TGF-b for E-cad expression in dendritic cells (DC) and lack of epidermal LC in TGF-b null mutants. Since most of this evidence is indirect, we here address the question whether E-cad expression in LC is required for their epidermal localization in vivo. For this we used mice with a selective deficiency for E-Cad in CD11c+ cells generated by Cre/LoxP mediated recombination. In E-cadfl/flCD11c Cre+ mice E-cad expression was absent in LC as shown by flow cytometry. Surprisingly, lack of E-cad in LC had little effect on epidermal localization of LC as demonstrated by almost normal LC numbers in epidermal sheets and whole mount skin in Cre+ mice as compared to Cre- littermates. No differences were observed with regard to activation status of LC, in vivo LC migration to regional lymph nodes and TNCB induced contact hypersensitivity. In contrast, in vitro significantly more LC could be isolated from the epidermis of Cre+ mice than that of Crecontrols, which may indicate that E-cad is involved but not essential for adhesion of LC to epidermal keratinocytes. In conclusion our findings reveal that despite the lack of E-cad LC are still largely present in the epidermis, suggesting that E-Cadherin is dispensable for localization of LC within the epidermal layers of the skin.
www.jidonline.org S209
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Prostaglandin E2 regulates osteoclastogenesis through the induction of RANKL in middle ear cholesteatoma R Imai1,2, T Sato2, M Terao1, H Murota1, H Inohara2 and I Katayama1 1 Dermatology, Osaka University Graduate School of Medicine, Suita, Japan and 2 Otorhinolaryngology, Head and Neck Surgery, Osaka University Graduate School of Medicine, Suita, Japan Middle ear cholesteatoma is structurally similar to epidermal cyst composed of both stratified squamous epithelium and surrounding fibrosis tissue. In spite of benign tumor, cholesteatoma destructs adjacent bones and causes various complications such as hearing loss, vertigo, facial nerve palsy and brain abscess. Although mechanism of bone destruction by cholestatoma has been explored for many years, it has not been cleared. We succeeded in generating a model mouse for cholesteatoma, epidermal cyst like tissue by injecting both of ear pinna-derived fibroblasts and keratinocytes on calvarial bone. This model cyst destroys the adjacent calvarial bone and induced osteoclasts. Also RANKL was expressed by fibroblasts surrounding cysts. We focused on Prostaglandin E2, which were reported to promote RANKL expression. The PGE2 concentration of extraction from the model cysts was higher than that from control mouse skin by ELISA. The expression of PGE Synthase was detected in the cells of model cyst by immunohistochemistory. In vitro culture system, we confirmed that PGE2 significantly upregulated RANKL expression in the primary fibroblasts from mouse ear pinna. The upregulation of RANKL expression is inhibited by EP3 antagonist. EP3 is known for one of the PGE2 receptor. In clinical samples, the PGE2 concentration of extraction from cholesteatoma was higher than that from control skin. Quantitative real time PCR revealed that RANKL mRNA expression in cholesteatoma was higher than that in skin. The number of osteoclasts on the surface of bone adjacent to cholesteatoma was significantly increased, compared with cholesteatoma. These data suggested that PGE2 regulated osteoclast differentiation through the induction of RANKL expression in cholesteatoma. Furthermore, these were implicated that cyclooxygenase-2 inhibitors or EP3 antagonist have possibility of new therapeutic strategies for cholesteatoma-induced bone destruction.
PRC2 complex is required for human epidermal differentiation and proliferation I Asamaowei, V Botchkarev, NV Botchkareva and AN Mardaryev University of Bradford, Bradford, United Kingdom Polycomb repressive complex 2 (PRC2) catalyses the methylation of ‘Lys-27’ of histone H3 leading to transcriptional repression of target genes through its catalytic subunit Enhancer of zeste homolog 1/2 (EZH1/2). PRC2 functions as a critical regulator of stem cell identity and their differentiation in embryonic and adult tissues. In mouse skin, PRC2 prevents premature differentiation of epidermal progenitor cells and their ectopic differentiation to Merkle cells. The role of subunits of PRC2 complex in human skin homeostasis remains unclear. Here, we show that in human skin EZH2 is predominantly expressed in the epithelial cells. In the epidermis, its expression is increased in differentiating cells of the suprabasal layer. Indeed, EZH2 expression and H3K27 histone methyltransferase activity is markedly up-regulated in Ca2+-induced differentiation in primary human keratinocytes. Interestingly, knockdown of subunits of PRC2 complex - EZH1/2 and EED using siRNA and small molecule inhibitors GSK126 and UNC1999 up-regulated expression of KRT1, LOR and FLG genes and suppressed cell proliferation in primary human keratinocytes. These results suggest that PRC2 complex plays a critical role in the control of human epidermal progenitor cells proliferation and differentiation.
Novel amino acid duplication in collagen XVII causes mild junctional epidermolysis bullosa by altering the coiled-coil structure and impairing collagen XVII trimerization and maturation J Kroeger1, S Hofmann2, J Leppert1, C Has1 and C Franzke1 1 Department of Dermatology, Medical Center, University of Freiburg, Freiburg, Germany and 2 Center for Dermatology, Allergy and Dermatosurgery, HELIOS University Hospital Wuppertal, University Witten/ Herdecke, Wuppertal, Germany The function and stability of collagens depend on accurate triple helix formation of three distinct polypeptide chains. Disruption of this triple-helical structure can result in connectivetissue disorders. Triple helix formation is thought to depend on three-stranded coiled-coil oligomerization sites. However, only little is known about the physiological relevance of these coiled-coil structures. Transmembrane collagen XVII, also known as 180kDa bullous pemphigoid antigen provides mechanical stability through the anchorage of epithelial cells to the basement membrane. Mutations in the collagen XVII gene, COL17A1, cause junctional epidermolysis bullosa (JEB), characterized by chronic trauma-induced skin blistering. Here we exploited a novel naturally occurring COL17A1 mutation, leading to an in-frame lysine duplication within the coiled-coil structure of the NC16A domain of collagen XVII, which resulted in a mild phenotype of JEB due to reduced membrane-anchored collagen XVII molecules. This mutation causes structural changes in the mutant molecule and interferes with its maturation. The destabilized coiled-coil structure of the mutant collagen XVII unmasks a furin cleavage site that results in excessive and non-physiological ectodomain shedding during its maturation. Furthermore, it decreases its triple-helical stability due to defective coiled-coil oligomerization, which makes it highly susceptible to proteolytic degradation. As a consequence of altered maturation and decreased trimer stability, reduced collagen XVII is incorporated into the cell membrane, resulting in compromised dermalepidermal adhesion. Taken together, using this genetic model, we provide the first proof that alteration of the coiled-coil structure destabilizes oligomerization and impairs physiological shedding of collagen XVII in vivo.
Loss of loricrin exacerbates psoriasis-like skin inflammation through altered dendritic cell homeostasis T Ogawa, Y Ishitsuka, Y Nakamura, R Watanabe, N Okiyama, Y Fujisawa and M Fujimoto Dermatology, University of Tsukuba, Tsukuba, Japan Psoriasis is a chronic inflammatory skin disease associated with multiple predisposing factors, and one of susceptible loci known as PSORS4 is on human chromosome 1, in which epidermal differentiation complex (EDC) is located. An EDC gene loricrin (Lor) accounts for a major protein mass of cornified cell envelope (CE), the vital structure for epidermal integrity. In contrast to competent epidermal permeability barrier, Lor knockout (LKO) mice had reduced epidermal mechanical stability of the epidermis resulting from impaired crosslinking of filaggrin and keratin 1/10 in their CEs. Since mechanical stress/trauma can contribute to psoriasis induction and exacerbation, we evaluated the impact of the loss LOR in a psoriasis-like disease model. Reflecting mechanical weakness, application of mechanical stress onto the ear skin resulted in enhanced and sustained keratin 6 (K6) protein expression in LKO epidermis as determined by immunohistochemistry (IHC). LKO mice had significantly increased expression levels of cytokines that initiate psoriatic inflammatory response, including Tnf, Il1b and Ifn1a on quantitative reverse transcription PCR (RT-qPCR). Topical application of imiquimod (Aldara) cream on LKO mice resulted in a severe inflammatory phenotype; ear skin had augmented swelling, and back skin had enhanced erythema and hyperkeratosis accompanied by increased transcriptional expression levels of K6a. Since Ifn-a produced by plasmacytoid dendritic cell (pDC) is known to play a pivotal role in this model, pDC marker expression was analyzed in a steady state. Unchallenged LKO mice had significantly enhanced expression of Bst2 in the ear skin as determined by RT-qPCR and IHC. Taken together, these results indicate that reduced mechanical stability in the epidermis can exacerbate psoriasis-like skin inflammation through the alteration of DC homeostasis.
iRHOM2 regulation of loricrin in the epidermal barrier L McGinty, DP Kelsell and D Blaydon Centre for Cell Biology and Cutaneous research, Queen Mary University of London, London, United Kingdom iRHOM2 is an inactive rhomboid protease, which has been shown to facilitate barrier formation and transglutaminase activity through ADAM17 interaction. Gain-of-function mutations in iRHOM2 underlie the autosomal-dominant syndrome Tylosis with oesophageal cancer (TOC), with patients possessing an altered epithelial barrier. Here, through analysing differences in the expression intensity and localisation of epidermal barrier proteins in TOC tissue and irhom2 -/- mice, we identify a role for iRHOM2 in the regulation of loricrin (LOR) in the epidermal barrier. Immunofluorescent (IF) analysis of LOR in TOC palm epidermis shows increased levels of basal nuclear LOR localisation compared to controls or interfollicular TOC epidermis. In contrast, in irhom2 -/- mice, we found lower levels of lor expression compared to WT mice, with neither exhibiting nuclear lor. TOC keratinocytes grown normally in monolayer alongside control keratinocyte cell lines exhibited LOR expression throughout the cell nucleus and cytoplasm, with higher nuclear LOR in TOC cells. When grown in the presence of the small-molecule ADAM17 inhibitor, TMI-005, LOR was found to localise heavily at the nuclear periphery as opposed to the nucleus in all cell lines. TOC keratinocytes grown in a 3D model also display nuclear LOR in the basal cell layer, mimicking TOC palm. Furthermore, when grown in the presence of TMI-005, nuclear LOR is absent in these models, indicating that inhibition of ADAM17 activity reduces epithelial stress. In addition to our cutaneous findings, we observe nuclear LOR in TOC oesophageal epithelium, which like the palm, is also hyperproliferative. We demonstrate the importance of the iRHOM2/ADAM17 signalling axis in the regulation and localisation of LOR. Modulation of this pathway signifies a relation between iRHOM2/ADAM17 and a possible stress-related nuclear translocation mechanism for LOR in hyperproliferative tissue.
E-Cadherin is dispensable for epidermal localization of Langerhans cells B Dorn1, B Raghavan1, S Kunz2, S Martin2 and T Jakob1 1 Experimental Dermatology and Allergy Research Group, Department of Dermatology and Allergy, Justus-Liebig University Giessen, University Medical Center Giessen (UKGM), Giessen, Germany and 2 Allergy Research Group, Department of Dermatology, Medical Center - University of Freiburg, Freiburg, Germany Langerhans cells (LC) are potent antigen presenting cells localized in the epidermis. LC express high levels of the adhesion molecule E-Cadherin (E-cad) which has been suggested to be responsible for adhesion to keratinocytes and thus LC localization to the epidermis, supported by three lines of evidence: formation of adherens junctions between E-cad expressing LC like dendritic cells and keratinocytes, down regulation of E-cad during activation, maturation and emigration of LC, and requirement of TGF-b for E-cad expression in dendritic cells (DC) and lack of epidermal LC in TGF-b null mutants. Since most of this evidence is indirect, we here address the question whether E-cad expression in LC is required for their epidermal localization in vivo. For this we used mice with a selective deficiency for E-Cad in CD11c+ cells generated by Cre/LoxP mediated recombination. In E-cadfl/flCD11c Cre+ mice E-cad expression was absent in LC as shown by flow cytometry. Surprisingly, lack of E-cad in LC had little effect on epidermal localization of LC as demonstrated by almost normal LC numbers in epidermal sheets and whole mount skin in Cre+ mice as compared to Cre- littermates. No differences were observed with regard to activation status of LC, in vivo LC migration to regional lymph nodes and TNCB induced contact hypersensitivity. In contrast, in vitro significantly more LC could be isolated from the epidermis of Cre+ mice than that of Crecontrols, which may indicate that E-cad is involved but not essential for adhesion of LC to epidermal keratinocytes. In conclusion our findings reveal that despite the lack of E-cad LC are still largely present in the epidermis, suggesting that E-Cadherin is dispensable for localization of LC within the epidermal layers of the skin.
www.jidonline.org S209