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144 Deciphering the role of the hexosamine pathway in skin homeostasis
Epidermal Structure and Function | ABSTRACTS 143
E-cadherin integrates EGFR signaling and mechanotransduction to control tissue polarization and barrier formation M Ru¨bsam1, A Mertz2, A Kubo3, ER Dufresne2, V Horsley2, W Ziegler5, SA Wickstro¨m4, M Amagai3 and CM Niessen1 1 Dermatology, University of Cologne, Cologne, Germany, 2 Yale University, New Haven, CT, 3 Dermatology, Keio University School of Medicine, Tokyo, Japan, 4 Max Planck Institute for Biology of Ageing, Cologne, Germany and 5 Hannover Medical School, Hannover, Germany Generation of a barrier in multi-layered epithelia like the epidermis requires restricted positioning of functional tight junctions (TJ) to the most suprabasal viable layer. This positioning necessitates tissue-level polarization of junctions and the cytoskeleton through unknown mechanisms. Previously, we showed that epidermal E-cadherin is essential for epidermal TJ barrier function. Using quantitative whole-mount imaging, genetic ablation, and traction force microscopy, we find that ubiquitously localized E-cadherin coordinates tissue polarization of vinculin positive, tension-bearing adherens junction (AJ) and F-actin organization and allows formation of an apical ZO1-EGFR-positive TJ network only in the granular layer 2 (SG2). The SG2 layer itself is highly polarized despite its flattened appearance with a lateral AJ network reaching up to a continuous ZO-1 positive apical tight junctional ring. We find that E-cadherin regulates spatiotemporal positioning of EGFR receptor during epidermal barrier development at embryonic day 16.5 and balances EGFR activity critical for in vitro TJ barrier function. In conclusion, our data link E-cadherin-dependent control of suprabasal EGFR activity and the polarized, tissue-level organization of junctions, tension, and the cytoskeleton to promote in vivo epidermal barrier formation. Our results further reveal a mechanistic role for EGFR at TJs and thereby uncover why EGFR inhibitors compromise skin barrier function in human cancer patients.
144
Deciphering the role of the hexosamine pathway in skin homeostasis K Allmeroth1, M Horn1, Y Hinze2 and MS Denzel1 1 Molecular Genetics of Ageing, Max Planck Institute for Biology of Ageing, Cologne, Germany and 2 Max Planck Insitute for Biology of Ageing, Cologne, Germany As a major structural and functional component of the skin, the extracellular matrix (ECM) plays a critical role in skin homeostasis. ECM components are not only a scaffold surrounding cells, but they exhibit pivotal functions in key cellular events such as migration, proliferation and survival. In addition to proteins like collagen and elastin, glycosaminoglycans (GAGs), such as hyaluronic acid (HA), are major constituents of the ECM. GAGs are long linear disaccharide chains that contain acetylated aminosugars. The hexosamine pathway (HP) generates these aminosugars, such as UDP-N-acetylglucosamine (UDP-GlcNAc), which is a substrate for N-glycosylation and mucin-type O-glycosylation in the endoplasmic reticulum and Golgi apparatus, as well as cytoplasmic O-GlcNAcylation. Interestingly, it was shown previously that HP activation leads to elevated aminosugar levels, improved protein homeostasis and extended lifespan in a nematode model. Thus, the HP might be linked to skin homeostasis in two ways: First, its effect on protein quality control might improve or maintain cellular functions in the skin and should thereby boost all-over robustness. In fact, HP activation significantly increases resistance to oxidative and UVB-induced stress in a murine neuroblastoma cell line. Second, activation of the HP modulates ECM composition and function. This is supported by our observation that HP activation by supplementation with a UDP-GlcNAc precursor, results in increased hyaluronic acid secretion. Therefore, I will further investigate the effect of HP activation on GAG production and matrix composition. Additionally, these matrix changes might affect the stem cell niche and thus stem cell function. Indeed, preliminary data suggest that HP activation promotes self-renewal of hair follicle stem cells in vitro. Furthermore, we have generated transgenic mice allowing for conditional HP activation. Therefore, we will be able to address molecular changes caused by HP activation in the skin, and test their relevance in vivo.
145
Skin barrier impairment downregulates PPARG via IL-1b S Blunder, V Moosbrugger-Martinz, R Gruber, M Schmuth and S Dubrac Dermatology, Innsbruck Medical University, Innsbruck, Austria Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear hormone receptors that comprises PPARa, PPARb/d and PPARg. In skin, PPARs modulate inflammation, lipid synthesis, keratinocyte differentiation and proliferation. Furthermore, activation of all three PPAR isoforms was found to improve cutaneous barrier recovery. In this study we aim (i) to establish easily reproducible models of epidermal barrier impairment using human epidermal equivalents (HEE), (ii) to assess PPAR and cytokine gene expression in these models and (iii) to evaluate potential modulatory effects of keratinocyte-derived cytokines on PPAR expression after skin barrier disruption. First, we show that SDS and acetone successfully impairs epidermal barrier function in HEEs as assessed by transepithelial electrical resistance (TEER). However, SDS but not acetone application renders HEEs permeable to Lucifer yellow fluorescent dye. Nile Red staining does not reveal major changes in the pattern and/or intensity of non-polar lipids. PPARG gene expression levels are decreased 24 hours after SDS- and acetone- mediated barrier impairment. In contrast, PPARD mRNA levels are increased 24 hours after SDS, but not after acetone application. PPARA mRNA levels are not altered with any of the treatments. TNFA mRNA levels are upregulated 6 hours and IL1B mRNA levels are increased 24 hours after both SDS- and acetone- mediated barrier impairment. In line with these results, both IL-1b and TNFa treatment lead to diminished PPARG gene expression levels in HEEs after 24 hours. Yet, only IL-1b enhances PPARD mRNA. Interestingly, abrogation of IL-1R signaling by anakinra abolishes SDS-mediated PPARG downregulation. Blocking of TNFa signaling by infliximab ameliorates SDS-induced PPARG downregulation in HEEs. Together, these findings show that epidermal barrier impairment alters PPAR expression in KCs. Furthermore, these data demonstrate that PPARG downregulation due to barrier impairment is likely mediated via IL-1b and partially via TNFa signaling.
www.jidonline.org S185
E-cadherin integrates EGFR signaling and mechanotransduction to control tissue polarization and barrier formation M Ru¨bsam1, A Mertz2, A Kubo3, ER Dufresne2, V Horsley2, W Ziegler5, SA Wickstro¨m4, M Amagai3 and CM Niessen1 1 Dermatology, University of Cologne, Cologne, Germany, 2 Yale University, New Haven, CT, 3 Dermatology, Keio University School of Medicine, Tokyo, Japan, 4 Max Planck Institute for Biology of Ageing, Cologne, Germany and 5 Hannover Medical School, Hannover, Germany Generation of a barrier in multi-layered epithelia like the epidermis requires restricted positioning of functional tight junctions (TJ) to the most suprabasal viable layer. This positioning necessitates tissue-level polarization of junctions and the cytoskeleton through unknown mechanisms. Previously, we showed that epidermal E-cadherin is essential for epidermal TJ barrier function. Using quantitative whole-mount imaging, genetic ablation, and traction force microscopy, we find that ubiquitously localized E-cadherin coordinates tissue polarization of vinculin positive, tension-bearing adherens junction (AJ) and F-actin organization and allows formation of an apical ZO1-EGFR-positive TJ network only in the granular layer 2 (SG2). The SG2 layer itself is highly polarized despite its flattened appearance with a lateral AJ network reaching up to a continuous ZO-1 positive apical tight junctional ring. We find that E-cadherin regulates spatiotemporal positioning of EGFR receptor during epidermal barrier development at embryonic day 16.5 and balances EGFR activity critical for in vitro TJ barrier function. In conclusion, our data link E-cadherin-dependent control of suprabasal EGFR activity and the polarized, tissue-level organization of junctions, tension, and the cytoskeleton to promote in vivo epidermal barrier formation. Our results further reveal a mechanistic role for EGFR at TJs and thereby uncover why EGFR inhibitors compromise skin barrier function in human cancer patients.
144
Deciphering the role of the hexosamine pathway in skin homeostasis K Allmeroth1, M Horn1, Y Hinze2 and MS Denzel1 1 Molecular Genetics of Ageing, Max Planck Institute for Biology of Ageing, Cologne, Germany and 2 Max Planck Insitute for Biology of Ageing, Cologne, Germany As a major structural and functional component of the skin, the extracellular matrix (ECM) plays a critical role in skin homeostasis. ECM components are not only a scaffold surrounding cells, but they exhibit pivotal functions in key cellular events such as migration, proliferation and survival. In addition to proteins like collagen and elastin, glycosaminoglycans (GAGs), such as hyaluronic acid (HA), are major constituents of the ECM. GAGs are long linear disaccharide chains that contain acetylated aminosugars. The hexosamine pathway (HP) generates these aminosugars, such as UDP-N-acetylglucosamine (UDP-GlcNAc), which is a substrate for N-glycosylation and mucin-type O-glycosylation in the endoplasmic reticulum and Golgi apparatus, as well as cytoplasmic O-GlcNAcylation. Interestingly, it was shown previously that HP activation leads to elevated aminosugar levels, improved protein homeostasis and extended lifespan in a nematode model. Thus, the HP might be linked to skin homeostasis in two ways: First, its effect on protein quality control might improve or maintain cellular functions in the skin and should thereby boost all-over robustness. In fact, HP activation significantly increases resistance to oxidative and UVB-induced stress in a murine neuroblastoma cell line. Second, activation of the HP modulates ECM composition and function. This is supported by our observation that HP activation by supplementation with a UDP-GlcNAc precursor, results in increased hyaluronic acid secretion. Therefore, I will further investigate the effect of HP activation on GAG production and matrix composition. Additionally, these matrix changes might affect the stem cell niche and thus stem cell function. Indeed, preliminary data suggest that HP activation promotes self-renewal of hair follicle stem cells in vitro. Furthermore, we have generated transgenic mice allowing for conditional HP activation. Therefore, we will be able to address molecular changes caused by HP activation in the skin, and test their relevance in vivo.
145
Skin barrier impairment downregulates PPARG via IL-1b S Blunder, V Moosbrugger-Martinz, R Gruber, M Schmuth and S Dubrac Dermatology, Innsbruck Medical University, Innsbruck, Austria Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear hormone receptors that comprises PPARa, PPARb/d and PPARg. In skin, PPARs modulate inflammation, lipid synthesis, keratinocyte differentiation and proliferation. Furthermore, activation of all three PPAR isoforms was found to improve cutaneous barrier recovery. In this study we aim (i) to establish easily reproducible models of epidermal barrier impairment using human epidermal equivalents (HEE), (ii) to assess PPAR and cytokine gene expression in these models and (iii) to evaluate potential modulatory effects of keratinocyte-derived cytokines on PPAR expression after skin barrier disruption. First, we show that SDS and acetone successfully impairs epidermal barrier function in HEEs as assessed by transepithelial electrical resistance (TEER). However, SDS but not acetone application renders HEEs permeable to Lucifer yellow fluorescent dye. Nile Red staining does not reveal major changes in the pattern and/or intensity of non-polar lipids. PPARG gene expression levels are decreased 24 hours after SDS- and acetone- mediated barrier impairment. In contrast, PPARD mRNA levels are increased 24 hours after SDS, but not after acetone application. PPARA mRNA levels are not altered with any of the treatments. TNFA mRNA levels are upregulated 6 hours and IL1B mRNA levels are increased 24 hours after both SDS- and acetone- mediated barrier impairment. In line with these results, both IL-1b and TNFa treatment lead to diminished PPARG gene expression levels in HEEs after 24 hours. Yet, only IL-1b enhances PPARD mRNA. Interestingly, abrogation of IL-1R signaling by anakinra abolishes SDS-mediated PPARG downregulation. Blocking of TNFa signaling by infliximab ameliorates SDS-induced PPARG downregulation in HEEs. Together, these findings show that epidermal barrier impairment alters PPAR expression in KCs. Furthermore, these data demonstrate that PPARG downregulation due to barrier impairment is likely mediated via IL-1b and partially via TNFa signaling.
www.jidonline.org S185