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102 EZH2 is required for human hair follicle growth and epidermal differentiation
ABSTRACTS | Epidermal Structure and Function 101
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Not only ectodomain shedding but also C-terminal cleavage induces neoepitopes on proteolyzed collagen XVII E Toyonaga, W Nishie, K Izumi, H Ujiie and H Shimizu Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan Collagen XVII (COL17/BP180) is a hemidesmosmal type II transmembrane protein between the epidermis and dermis which can be targeted by autoantibodies in autoimmune blistering disorders, including linear IgA bullous dermatosis (LAD). The ectodomain of COL17 is known to be physiologically or pathologically cleaved within the juxtamembranous noncollagenous NC16A region, which results in the 120kD (LAD-1) and the further C-terminally processed 97kD (LABD-97) ectodomains. Significantly, LAD autoantibodies preferentially target LAD-1 and LABD-97 but not full-length COL17, indicating proteolysis within the NC16A domain induces neoepitopes on the cleaved COL17 ectodomains. However, little is known about how neoepitopes are produced on processed COL17, especially in relation to C-terminal cleavage. This study shows that not only ectodomain shedding but also cleavage of the Cterminus is associated with development of conformational neoepitopes on proteolyzed COL17. We first produced a novel monoclonal antibody (mAb) that specifically reacts with LAD-1 and LABD-97, but not with full-length COL17. The epitope of the mAb was mapped on the 15th collagenous domain of the COL17 ectodomain, indicating comformational neoepitopes are induced in this region after ectodomain shedding. To investigate the roles of C-terminal cleavage in the development of neoepitopes, we produced a recombinant COL17 (COL17-DC; Met1-Arg1174) with a C-terminus 323 amino acids shorter than the full length COL17 (Met1-Pro1497). Interestingly, the mAb reacted with COL17-DC, suggesting cleaving only the C-terminal of COL17 also induces structural changes and exposes comformational neoepitopes on the 15th collagenous domain. Notably, the LAD autoantibodies also reacted with COL17-DC, indicating C-terminal processing of COL17 is associated with the autoimmunity of LAD. The present study shows that proteolysis of COL17 induces dynamic structural changes on proteolyzed COL17 and conformational neoepitopes, which are targeted by LAD autoantibodies.
EZH2 is required for human hair follicle growth and epidermal differentiation I Asamaowei1, N Farjo2, V Botchkarev1, NV Botchkareva1 and AN Mardaryev1 1 University of Bradfoprd, Bradford, United Kingdom and 2 Farjo Hair Institute, Manchester, United Kingdom Enhancer of zeste homolog 2 (Ezh2) is a catalytic subunit of the polycomb repressive complex 2 which methylates ‘Lys-27’ of histone H3 leading to transcriptional repression of target genes. Ezh2 functions as a critical regulator of stem cell identity and their differentiation in embryonic and adult tissues. In mouse skin, Ezh2 prevents premature differentiation of epidermal progenitor cells, and is required for proliferative activity of hair follicle stem cells. The role of EZH2 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 is markedly up-regulated in Ca2+-induced differentiation in primary human keratinocytes. In anagen hair follicles, Ezh2 is expressed in Keratin15/Keratin 19/CD34-positive progenitor cells in the outer root sheath, as well as in differentiating cells of the inner root sheath and precortex, whilst no Ezh2 expression was detected in the dermal papilla and connective tissue sheath. Interestingly, EZH2 knockdown using siRNA together with a small molecule inhibitor GSK126 up-regulated expression of KER1 and LOR genes in primary human keratinocytes. Inhibition of Ezh2 with a selective small molecule, 3-Deazaneplanocin A, caused a dosedependent retardation of hair growth ex vivo. The latter was associated with decreased proliferation and increased apoptosis in the outer root sheath. These results suggest that Ezh2 plays a critical role in the control of human epithelial progenitor cells proliferation and differentiation.
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Sphingomyelin concentrates into lipid microdomains only in non-senescent cells and enhances keratinocyte migration A Mound1, C Warnon1, V lozanova1, C Guere2, K Vie2, D Tyteca3, C Lambert de Rouvroit1, F Debacq-Chainiaux1 and Y Poumay1 1 University of Namur, Namur, Belgium, 2 Laboratoires Clarins, Paris, France and 3 De Duve Institute, UCL, Brussels, Belgium Epidermal barrier against environment threats requires homeostasis involving keratinocytes proliferation, migration and differentiation. This turnover is affected by ageing as it slows down dramatically. Keratinocytes contain membrane lipid microdomains which are involved in cell control and communication. Thus, this study aims to understand the changes of lipid microdomains during senescence of human keratinocytes and how this could affect cell signaling and cell fate. In keratinocytes, replicative senescence (RS) appears after a dozen of population doublings and is characterized by an increase in the senescence-associated bgalactosidase activity and a slowdown of proliferative and migration abilities. Alteration of keratinocyte lipid microdomains activates several signaling pathways. However, despite unaltered expression of receptors and downstream signaling intermediates, senescent keratinocytes exhibit altered signaling suggesting changes in membrane properties. Specific lipid domains were analyzed by vital imaging in human keratinocytes throughout their proliferative lifespan, using lysenin and theta toxin fragments fused to mCherry (lysenin*, theta*). These probes were demonstrated as specific, sensitive and quantitative for sphingomyelin (SM) and cholesterol localization. SM-enriched lipid domains are detected in pre-senescent keratinocytes, but gradually disappear with population doublings (RS). Domains are longer maintained in keratinocytes treated with the Y27632 ROCK inhibitor delaying senescence and are stable in immortalized hTERT keratinocytes. Moreover, keratinocyte migration is stimulated by Y27632, but is decreased after SM and cholesterol depletion, similarly to observations performed during RS. Thus, while altered lipid microdomains are known to affect keratinocyte signaling, proliferation, and migration, this study indicates that their properties are modified during RS in this cell type.
Understanding sensitive skin: a role of inflammation, mast cells, and skin barrier NE Uzunbajakava1, R Richters2, D Falcone2, P Caspers3, G Puppels3, P van Erp2 and PC van de Kerkhof2 1 Philips Research, Eindhoven, Netherlands, 2 Department of Dermatology, Radboud University Medical Center, Nijmegen, Netherlands and 3 RiverD International B.V., Rotterdam, Netherlands Sensitive skin (SS) has high prevalence in the Western world. Potential mechanisms include impaired skin barrier, increased vascular- and neuro-reactivity, increased TRP receptor activation and involvement of immune system. Atopic subjects report to have SS more frequently. Yet, no agreement exists in the field. The aim of this study was to gain new insights on pathomechanisms of SS by identifying key objective physiological markers. Subjects were recruited based on a multifactorial questionnaire; those with atopy (atopic dermatitis, asthma and allergic rhinoconjunctivitis) were observed separately. Skin response to mechanical, chemical and fractional non-ablative provocations was evaluated up to 72 hours using: TEWL; spectrophotometer; corneometer; immunohistochemistry (cycling keratinocytes, mast cells, T-cells, dendritic- and Langerhans cells, endothelium, differentiation markers and others) and confocal Raman spectroscopy. High prevalence of SS was confirmed (41%) in response to multiple provocations; atopy group reported SS more frequently. Objective signs in the no atopy group were: flare after tapestripping; lower a* value 30 min both after tapestripping and fractional non-ablative laser; aberrant adhesion of corneocytes; spongiosis; and significantly lower number of mast cells. Stratum corneum thickness, water- and NMF content were not different for SS compared to NSS group. Yet a trend towards lower ceramides was observed in the cheek. Compared to AD, SS group showed higher ceramides in the forearm. We confirm existence of ‘generalized’ SS, which is not a subclinical form of atopic dermatitis. Treatments solely based on hydration and ceramides supplementation do not reflect the identified properties. Future investigations should address the role of TRP channels; immune response, inter-corneocyte adhesion, and development of specific noninvasive tools for in vivo skin barrier analysis, in order to facilitate treatments.
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The effect of oxygen on keratinocyte differentiation R Koh, I Szevere´nyi, P Benny, P Nguyen and EB Lane Epithelial Biology Laboratory, Institute of Medical Biology, Singapore, Singapore A major function of the epidermis is maintaining good barrier integrity. The barrier is maintained by a constant renewal process where basal keratinocytes terminally differentiate to form corneocytes on the outermost layer of the epidermis. The structural and biochemical changes occurring during keratinocyte differentiation has been well studied. However the specific mechanisms triggering differentiation is less clear. Oxygen has been implicated as a driver of differentiation in other tissues such as the haematopoietic system. Oxygen concentration can be a powerful metabolic signal, and the proximity of the epidermis to atmospheric oxygen makes it a likely target for oxygen-induced differentiation. Immunohistochemistry staining of human epidermis with the oxygen-sensitive transcription factor Hif-2a revealed stabilization of the protein in the basal and lower spinous layers but not in the upper layers of the epidermis, suggesting that an oxygen gradient exists within the epidermis. Using 3-dimensional organotypic models constructed in varying oxygen levels, we show that lowering the culture oxygen results in decreased stratification, organization and expression of key differentiation markers of the epidermis.
S178 Journal of Investigative Dermatology (2016), Volume 136
Glutathione (GSH) distribution by quantitative MALDI imaging in reconstructed human skin upon activation of GSH biosynthesis by Nrf2 pathway activator R Legouffe1, D Bonnel1, C Jones2, N Cavusoglu2, G Le´reaux2, J Eilstein2, A Heron1, J Stauber1 and J Soeur2 1 ImaBiotech, Loos, France and 2 L’ore´al, Paris, France Skin is particularly exposed to oxidative stress, either from environmental insults such as sunlight or pollution or as a consequence of specific impairments in antioxidant status resulting from pathologies or aging. Traditionally, antioxidant products are exogenously provided to neutralize pro-oxidant species. However, another approach based on stimulation of endogenous antioxidant defense pathways is more original. Resveratrol (RSV) was reported to activate the Nrf2 pathway at nontoxic doses, from 20 mM up to 100mM, in primary culture of NHKs or in full-thickness reconstructed human skin. In parallel, a significant increase in glutathione (GSH) content, assessed by LC/MS analysis, was observed in both models. Following the development of a dedicated protocol for the reconstructed skin sectioning, a perfect visualization of the Stratum corneum, Epidermis was obtained on 10mm sections of some frozen samples. Thus MALDI-FTICR imaging at 20mm of spatial resolution was used to investigate the GSH distribution and relative quantification in both Stratum corneum and Epidermis of RSV treated reconstructed human skins at 20 and 100mM compared to a nontreated condition. GSH amount was confirmed to be significatively increased in the reconstructed tissues following a Resveratrol treatment at 20 mM and 100mM.
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Not only ectodomain shedding but also C-terminal cleavage induces neoepitopes on proteolyzed collagen XVII E Toyonaga, W Nishie, K Izumi, H Ujiie and H Shimizu Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan Collagen XVII (COL17/BP180) is a hemidesmosmal type II transmembrane protein between the epidermis and dermis which can be targeted by autoantibodies in autoimmune blistering disorders, including linear IgA bullous dermatosis (LAD). The ectodomain of COL17 is known to be physiologically or pathologically cleaved within the juxtamembranous noncollagenous NC16A region, which results in the 120kD (LAD-1) and the further C-terminally processed 97kD (LABD-97) ectodomains. Significantly, LAD autoantibodies preferentially target LAD-1 and LABD-97 but not full-length COL17, indicating proteolysis within the NC16A domain induces neoepitopes on the cleaved COL17 ectodomains. However, little is known about how neoepitopes are produced on processed COL17, especially in relation to C-terminal cleavage. This study shows that not only ectodomain shedding but also cleavage of the Cterminus is associated with development of conformational neoepitopes on proteolyzed COL17. We first produced a novel monoclonal antibody (mAb) that specifically reacts with LAD-1 and LABD-97, but not with full-length COL17. The epitope of the mAb was mapped on the 15th collagenous domain of the COL17 ectodomain, indicating comformational neoepitopes are induced in this region after ectodomain shedding. To investigate the roles of C-terminal cleavage in the development of neoepitopes, we produced a recombinant COL17 (COL17-DC; Met1-Arg1174) with a C-terminus 323 amino acids shorter than the full length COL17 (Met1-Pro1497). Interestingly, the mAb reacted with COL17-DC, suggesting cleaving only the C-terminal of COL17 also induces structural changes and exposes comformational neoepitopes on the 15th collagenous domain. Notably, the LAD autoantibodies also reacted with COL17-DC, indicating C-terminal processing of COL17 is associated with the autoimmunity of LAD. The present study shows that proteolysis of COL17 induces dynamic structural changes on proteolyzed COL17 and conformational neoepitopes, which are targeted by LAD autoantibodies.
EZH2 is required for human hair follicle growth and epidermal differentiation I Asamaowei1, N Farjo2, V Botchkarev1, NV Botchkareva1 and AN Mardaryev1 1 University of Bradfoprd, Bradford, United Kingdom and 2 Farjo Hair Institute, Manchester, United Kingdom Enhancer of zeste homolog 2 (Ezh2) is a catalytic subunit of the polycomb repressive complex 2 which methylates ‘Lys-27’ of histone H3 leading to transcriptional repression of target genes. Ezh2 functions as a critical regulator of stem cell identity and their differentiation in embryonic and adult tissues. In mouse skin, Ezh2 prevents premature differentiation of epidermal progenitor cells, and is required for proliferative activity of hair follicle stem cells. The role of EZH2 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 is markedly up-regulated in Ca2+-induced differentiation in primary human keratinocytes. In anagen hair follicles, Ezh2 is expressed in Keratin15/Keratin 19/CD34-positive progenitor cells in the outer root sheath, as well as in differentiating cells of the inner root sheath and precortex, whilst no Ezh2 expression was detected in the dermal papilla and connective tissue sheath. Interestingly, EZH2 knockdown using siRNA together with a small molecule inhibitor GSK126 up-regulated expression of KER1 and LOR genes in primary human keratinocytes. Inhibition of Ezh2 with a selective small molecule, 3-Deazaneplanocin A, caused a dosedependent retardation of hair growth ex vivo. The latter was associated with decreased proliferation and increased apoptosis in the outer root sheath. These results suggest that Ezh2 plays a critical role in the control of human epithelial progenitor cells proliferation and differentiation.
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Sphingomyelin concentrates into lipid microdomains only in non-senescent cells and enhances keratinocyte migration A Mound1, C Warnon1, V lozanova1, C Guere2, K Vie2, D Tyteca3, C Lambert de Rouvroit1, F Debacq-Chainiaux1 and Y Poumay1 1 University of Namur, Namur, Belgium, 2 Laboratoires Clarins, Paris, France and 3 De Duve Institute, UCL, Brussels, Belgium Epidermal barrier against environment threats requires homeostasis involving keratinocytes proliferation, migration and differentiation. This turnover is affected by ageing as it slows down dramatically. Keratinocytes contain membrane lipid microdomains which are involved in cell control and communication. Thus, this study aims to understand the changes of lipid microdomains during senescence of human keratinocytes and how this could affect cell signaling and cell fate. In keratinocytes, replicative senescence (RS) appears after a dozen of population doublings and is characterized by an increase in the senescence-associated bgalactosidase activity and a slowdown of proliferative and migration abilities. Alteration of keratinocyte lipid microdomains activates several signaling pathways. However, despite unaltered expression of receptors and downstream signaling intermediates, senescent keratinocytes exhibit altered signaling suggesting changes in membrane properties. Specific lipid domains were analyzed by vital imaging in human keratinocytes throughout their proliferative lifespan, using lysenin and theta toxin fragments fused to mCherry (lysenin*, theta*). These probes were demonstrated as specific, sensitive and quantitative for sphingomyelin (SM) and cholesterol localization. SM-enriched lipid domains are detected in pre-senescent keratinocytes, but gradually disappear with population doublings (RS). Domains are longer maintained in keratinocytes treated with the Y27632 ROCK inhibitor delaying senescence and are stable in immortalized hTERT keratinocytes. Moreover, keratinocyte migration is stimulated by Y27632, but is decreased after SM and cholesterol depletion, similarly to observations performed during RS. Thus, while altered lipid microdomains are known to affect keratinocyte signaling, proliferation, and migration, this study indicates that their properties are modified during RS in this cell type.
Understanding sensitive skin: a role of inflammation, mast cells, and skin barrier NE Uzunbajakava1, R Richters2, D Falcone2, P Caspers3, G Puppels3, P van Erp2 and PC van de Kerkhof2 1 Philips Research, Eindhoven, Netherlands, 2 Department of Dermatology, Radboud University Medical Center, Nijmegen, Netherlands and 3 RiverD International B.V., Rotterdam, Netherlands Sensitive skin (SS) has high prevalence in the Western world. Potential mechanisms include impaired skin barrier, increased vascular- and neuro-reactivity, increased TRP receptor activation and involvement of immune system. Atopic subjects report to have SS more frequently. Yet, no agreement exists in the field. The aim of this study was to gain new insights on pathomechanisms of SS by identifying key objective physiological markers. Subjects were recruited based on a multifactorial questionnaire; those with atopy (atopic dermatitis, asthma and allergic rhinoconjunctivitis) were observed separately. Skin response to mechanical, chemical and fractional non-ablative provocations was evaluated up to 72 hours using: TEWL; spectrophotometer; corneometer; immunohistochemistry (cycling keratinocytes, mast cells, T-cells, dendritic- and Langerhans cells, endothelium, differentiation markers and others) and confocal Raman spectroscopy. High prevalence of SS was confirmed (41%) in response to multiple provocations; atopy group reported SS more frequently. Objective signs in the no atopy group were: flare after tapestripping; lower a* value 30 min both after tapestripping and fractional non-ablative laser; aberrant adhesion of corneocytes; spongiosis; and significantly lower number of mast cells. Stratum corneum thickness, water- and NMF content were not different for SS compared to NSS group. Yet a trend towards lower ceramides was observed in the cheek. Compared to AD, SS group showed higher ceramides in the forearm. We confirm existence of ‘generalized’ SS, which is not a subclinical form of atopic dermatitis. Treatments solely based on hydration and ceramides supplementation do not reflect the identified properties. Future investigations should address the role of TRP channels; immune response, inter-corneocyte adhesion, and development of specific noninvasive tools for in vivo skin barrier analysis, in order to facilitate treatments.
105
106
The effect of oxygen on keratinocyte differentiation R Koh, I Szevere´nyi, P Benny, P Nguyen and EB Lane Epithelial Biology Laboratory, Institute of Medical Biology, Singapore, Singapore A major function of the epidermis is maintaining good barrier integrity. The barrier is maintained by a constant renewal process where basal keratinocytes terminally differentiate to form corneocytes on the outermost layer of the epidermis. The structural and biochemical changes occurring during keratinocyte differentiation has been well studied. However the specific mechanisms triggering differentiation is less clear. Oxygen has been implicated as a driver of differentiation in other tissues such as the haematopoietic system. Oxygen concentration can be a powerful metabolic signal, and the proximity of the epidermis to atmospheric oxygen makes it a likely target for oxygen-induced differentiation. Immunohistochemistry staining of human epidermis with the oxygen-sensitive transcription factor Hif-2a revealed stabilization of the protein in the basal and lower spinous layers but not in the upper layers of the epidermis, suggesting that an oxygen gradient exists within the epidermis. Using 3-dimensional organotypic models constructed in varying oxygen levels, we show that lowering the culture oxygen results in decreased stratification, organization and expression of key differentiation markers of the epidermis.
S178 Journal of Investigative Dermatology (2016), Volume 136
Glutathione (GSH) distribution by quantitative MALDI imaging in reconstructed human skin upon activation of GSH biosynthesis by Nrf2 pathway activator R Legouffe1, D Bonnel1, C Jones2, N Cavusoglu2, G Le´reaux2, J Eilstein2, A Heron1, J Stauber1 and J Soeur2 1 ImaBiotech, Loos, France and 2 L’ore´al, Paris, France Skin is particularly exposed to oxidative stress, either from environmental insults such as sunlight or pollution or as a consequence of specific impairments in antioxidant status resulting from pathologies or aging. Traditionally, antioxidant products are exogenously provided to neutralize pro-oxidant species. However, another approach based on stimulation of endogenous antioxidant defense pathways is more original. Resveratrol (RSV) was reported to activate the Nrf2 pathway at nontoxic doses, from 20 mM up to 100mM, in primary culture of NHKs or in full-thickness reconstructed human skin. In parallel, a significant increase in glutathione (GSH) content, assessed by LC/MS analysis, was observed in both models. Following the development of a dedicated protocol for the reconstructed skin sectioning, a perfect visualization of the Stratum corneum, Epidermis was obtained on 10mm sections of some frozen samples. Thus MALDI-FTICR imaging at 20mm of spatial resolution was used to investigate the GSH distribution and relative quantification in both Stratum corneum and Epidermis of RSV treated reconstructed human skins at 20 and 100mM compared to a nontreated condition. GSH amount was confirmed to be significatively increased in the reconstructed tissues following a Resveratrol treatment at 20 mM and 100mM.