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305 Epidermal iron metabolism to prevent iron loss by desquamation
ABSTRACTS | Epidermal Structure & Barrier Function 303
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Protein kinase D1 regulates desmoglein-1 expression in mouse keratinocytes and skin WB Bollag1,2,3 and V Choudhary1,2,3 1 Charlie Norwood VAMC, Augusta, GA, 2 Physiology, Georgia Regents University, Augusta, GA and 3 Medicine (Dermatology), Georgia Regents University, Augusta, GA Proliferation, differentiation and adhesion are important in maintaining the healthy status of skin. These processes are regulated by many different molecules, such as protein kinase D (PKD), aquaporin 3 (AQP3) and desmogleins (Dsg). We have previously shown that PKD1 is pro-proliferative and anti-differentiative for epidermal keratinocytes, whereas AQP3 is a prodifferentiative factor in these cells. To determine if there is cross-talk between these signaling pathways, we investigated whether PKD1 affects keratinocyte expression of AQP3. Knocking down PKD1 by overexpressing adenoviral Cre-recombinase in floxed PKD1 keratinocytes increased mRNA and protein levels of AQP3. We further confirmed this regulation by overexpressing a dominant-negative PKD1 adenoviral construct in normal mouse keratinocytes and found that AQP3 mRNA was increased compared to the vector-infected group. Interestingly, the mRNA and protein levels of the cell adhesion protein, desmoglein 1 (Dsg1), were also increased in PKD1 knockdown keratinocytes, suggesting that PKD1 regulates Dsg1 expression. Importantly, skin from conditional PKD1 knockout mice also demonstrated increased Dsg1 protein levels. In contrast, other cell adhesion-related proteins, such as Dsg3, laminin 5 and Ajuba, were downregulated in keratinocytes lacking PKD1. In conclusion, we have identified regulation of Dsg1 by PKD1, with PKD1 normally repressing Dsg1 expression such that its loss results in increased Dsg1 levels. These data are consistent with the report of Ghazizadeh and colleagues demonstrating impaired wound healing in conditional epidermal-specific PKD1 knockout mice, characterized by reduced keratinocyte outgrowth from skin explants (i.e., decreased migration, as might be expected if the observed enhancement of Dsg1 levels promotes cell adhesion). Further studies are needed to determine the mechanism by which PKD1 regulates Dsg1 and AQP3 in the skin.
Mass spectrometry based proteomic investigation of epidermal and dermal skin aging V Newton1, I Riba-Garcia2, AV Rawlings3, R Voegeli4, C Griffiths1, R Unwin2, RE Watson1 and MJ Sherratt5 1 Centre for Dermatology Research, University of Manchester, Manchester, United Kingdom, 2 CADET, Manchester University Hospitals NHS Foundation Trust, Manchester, United Kingdom, 3 AVR Consulting, Northwich, United Kingdom, 4 DSM Nutritional Products Ltd, Kaiseraugst, Switzerland and 5 Tissue Injury & Repair, University of Manchester, Manchester, United Kingdom This study aimed to identify new biomarkers of skin ageing and to determine age-associated differences in the proteome of photoprotected versus photoexposed skin, specifically in the epidermis, dermal-epidermal junction (DEJ) and papillary dermis. Skin biopsies from the buttock (photoprotected) and extensor forearm (photoexposed) of volunteers (aged 18-30 or 65 yrs; n¼4-6/group) were transversely sectioned from the stratum corneum to a depth of 250 mm. Following SDS-PAGE, each sample lane was segmented into 11 prior to trypsin digestion and analyses by LC-MS/MS. Protein identification and relative quantification were determined using Mascot Scaffold and ProteinPilot software. Within the photoprotected skin, of the 366 proteins identified, 26 were 2-fold different in abundance in aged skin compared with young. Similarly, within photoexposed skin, of the 550 proteins identified 41 were 2fold different in abundance. Changes in both actin cytoskeleton and stress response pathways in aged photoprotected skin were identified using Ingenuity software. Similar pathway changes were also observed in photoexposed skin. To the best of our knowledge, this is the first study to directly compare the proteomes of the epidermis, DEJ and papillary dermis of skin biopsies from young and aged individuals. Other studies using whole-skin biopsies or cultured skin cells have detected greater numbers of proteins. However, our method excludes the majority of the reticular dermis and its high abundance of matrix molecules that otherwise could have masked less abundant proteins. This study therefore identifies changes in cutaneous protein abundance that occur with age and the effect of sunlight on the proteome of aged skin.
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Epidermal iron metabolism to prevent iron loss by desquamation M Asano, K Yamasaki, T Yamauchi and S Aiba Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan It is well known that the intracellular iron content in the epidermis is highest in the basal layer and declines towards the stratum corneum and that most of iron in epidermal keratinocytes is not lost by desquamation. These observations suggest that the epidermis is equipped with some mechanism by which intracellular iron in differentiated keratinocytes is excreted to extracellular space before leaching the stratum corneum. In this study, we first demonstrated a distinctive expression pattern of iron metabolism molecules in which molecules participating in iron import and storage are expressed in the lower epidermis while those used for iron release from heme or iron transport are expressed in the upper epidermis by immunohistochemistry and mRNA quantification of the lower and upper epidermis separately obtained by a laser capture microdissection. This expression pattern of iron metabolism molecules was confirmed by normal human epidermal keratinocytes (NHEKs) during in vitro differentiation. Consistent with their expression, keratinocyte differentiation induced by high calcium medium reduces the intracellular iron content. Next, to clarify whether these iron metabolism molecules are really functioning in the epidermis, we demonstrated reducing ferroportin expression in vitro by ferroportin-specific siRNAs and hepcidin significantly increased the intracellular iron content. Finally, we demonstrated that iron content of the epidermis and squames in the dorsal skin was significantly greater in FpnEpi-KO mice than that in control mice and that FpnEpi-KO decreased blood Hb concentration more rapidly than control mice on an iron low diet. These studies build upon the evidence that iron is absorbed in the lower epidermis and extruded in the upper epidermis by ferroportin before iron is lost as a part of shedding keratinocytes, consequently preventing iron being lost from the skin at the level of affecting the systemic iron homeostasis.
Form and function of keratins in the nucleus R Hobbs1, JT Jacob1, BG Poll1,2 and PA Coulombe1,2,3 1 Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, 2 Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD and 3 Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD Select keratin intermediate filament proteins, which are classically described as strictly cytoplasmic, have recently been shown to localize and function within the nucleus of tumorderived epithelial cells from skin and cervix. Of the 54 keratin proteins occurring in mammalian genomes, K7, K8, K17, and K18 have so far been shown to occur in the nucleus. Our efforts have focused on K17, which is robustly upregulated in inflammatory skin diseases and tumors originating in stratified and pseudostratified epithelia. Using qRT-PCR, ChIP, and EMSA analyses, we determined that K17 promotes pro-inflammatory cytokine expression in skin tumor keratinocytes, in part, by translocating to the nucleus and associating with autoimmune regulator (AIRE) at NF-kB consensus motifs within the promoter regions of several genes (e.g. CXCL10, CXCL11, CCL19, MMP9). Bioinformatic analyses show the presence of a hitherto unknown bipartite nuclear localization signal (NLS) at the boundary between the central rod and C-terminal tail domains. Site-directed mutagenesis of a di-lysine motif (p.K399K400) within the predicted NLS substantially abrogates the ability of K17 to localize to the nucleus and to induce expression of its target genes. Confocal immunofluorescence microscopy and other lines of evidence suggest that K17 does not adopt a conventional 10-nm filament form in the nucleus of A431 (skin derived), HeLa (cervix derived), or BT20 (breast derived) tumor epithelial cells, or in human BCC tissue in situ. Rather, nuclear K17 is diffuse and often occurs in discrete, rounded punctae adjacent PML bodies and in chromatinsparse regions. Nuclear-localized K17 has been evidenced by other researchers in cervical epithelial tumor cell lines and in cervical tumors in situ. The discovery of keratin presence and function in the nucleus adds a new dimension to cell biology, nuclear architecture and dynamics, and epithelial structure and function.
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UVB-induced HMGB1 and extracellular ATP increase Langerhans cell production of IL-23 implicated in ILC3 activation JL Turban1,2, J Lewis1 and M Girardi1 1 Dermatology, Yale School of Medicine, New Haven, CT and 2 Howard Hughes Medical Institute, Chevy Chase, MD Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) arise from epidermal keratinocytes (KC) following exposure to ultraviolet B (UVB) radiation. Situated adjacent to nearly all KC is a network of epidermal Langerhans cells (LC). Our group previously implicated LC in the development of UVB-induced KC cancers, with LC-deficient (huLangerinDTA) mice being relatively resistant to cancer formation induced by chronic UVB irradiation. Furthermore, LC-intact mice show augmented epidermal expression of IL-23 and IL-22 following nine weeks of UVB irradiation. Here we show that the UVB-induced damageassociated molecular patterns (DAMP) HMGB1 and extracellular ATP have the capacity to induce IL-23 production by the LC line XS106. HMGB1 treatment of XS106 increased IL-23 production, as measured by multiplex cytokine assay, 23.3x (p¼.0007). ATP exposure increased production 2.3x (p¼.0002). Freshly isolated murine LCs, purified with MHC-II magnetic beads from epidermal preparations, increased IL-23 production following HMGB1treatment >100x (p¼.0007). Notably, treatment with the UVB-induced DAMP beta-defensin 2 did not result in increased IL-23 production by XS106. We also show here the presence of an epidermal population of type three innate lymphoid cells (ILC3) by epidermal sheet immunofluorescence from nine-week UVB-irradiated T-cell deficient (b-/-d-/-) mice. ILC3 are known to produce the epidermal growth factor IL-22 in response to stimulation with IL-23. Together, this data suggests that activation of LC by UVB-induced DAMPs induces IL-23 production, which subsequently signals ILC3 to produce IL-22, delivering a proliferative signal to KC, including those harboring UVB-induced mutations. Targeting this DAMP-IL23IL22 pathway may ultimately prove a useful target in the prevention of skin cancer via the inhibition of mutant keratinocyte clonal expansion.
S54 Journal of Investigative Dermatology (2016), Volume 136
Heavy cigarette smokers in a Chinese population display a compromised permeability barrier S Xin2, L Ye2, G Man1, C Lv2, PM Elias1 and M Man1 1 Dermatology, VA Medical Ctr/UCSF, San Francisco, CA and 2 Skin disease, Dalian Hospital, Dalian, China Background/Objective: Cigarette smoking is associated with a higher prevalence of inflammatory disorders that display a defective epidermal permeability barrier, such as atopic dermatitis and psoriasis. Yet, whether cigarette smoking influences barrier function is largely unknown. The present study determined whether skin biophysical properties, including permeability barrier homeostasis, stratum corneum (SC) integrity, SC hydration, skin surface pH, and skin melanin/erythema index, are altered in cigarette smokers. Methods: A total of 99 male volunteers, including 63 smokers and 36 non-smokers, aged 41-65 years (mean 53.86 0.66), with no current skin disease, were enrolled in this study. Smokers were categorized as light-to-moderate (<20 cigarettes/day) or heavy smokers (20 cigarettes/day). A multifunctional skin physiology monitor (C&K MPA5) was used to measure SC hydration and skin melanin/erythema index on the dorsal hand, forehead and cheek. Basal transepidermal water loss (TEWL) and barrier recovery rates were assessed on the forearm. A Skin-pH-Meter pH900 was used to measure skin surface pH. Results: Despite showing no differences in basal TEWL, heavy cigarette smokers (20 cigarettes/day) exhibited delayed barrier recovery after acute abrogation (1.02% 13.06 vs. 16.48% 6.07 in non-smokers), and barrier recovery rates correlated negatively with the number of cigarettes smoked per day (r2¼0.1066, p¼0.0087). Differences in biophysical parameters between smokers and non-smokers, including surface pH, stratum corneum hydration and erythema index, varied with body sites. Conclusions: Heavy cigarette smokers display compromised permeability barrier homeostasis, which could contribute, in part, to the increased prevalence of certain cutaneous disorders characterized by defective epidermal permeability in smokers. Conversely, strategies that improve epidermal permeability barrier function should be considered for heavy cigarette smokers with a history of inflammatory dermatoses.
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Protein kinase D1 regulates desmoglein-1 expression in mouse keratinocytes and skin WB Bollag1,2,3 and V Choudhary1,2,3 1 Charlie Norwood VAMC, Augusta, GA, 2 Physiology, Georgia Regents University, Augusta, GA and 3 Medicine (Dermatology), Georgia Regents University, Augusta, GA Proliferation, differentiation and adhesion are important in maintaining the healthy status of skin. These processes are regulated by many different molecules, such as protein kinase D (PKD), aquaporin 3 (AQP3) and desmogleins (Dsg). We have previously shown that PKD1 is pro-proliferative and anti-differentiative for epidermal keratinocytes, whereas AQP3 is a prodifferentiative factor in these cells. To determine if there is cross-talk between these signaling pathways, we investigated whether PKD1 affects keratinocyte expression of AQP3. Knocking down PKD1 by overexpressing adenoviral Cre-recombinase in floxed PKD1 keratinocytes increased mRNA and protein levels of AQP3. We further confirmed this regulation by overexpressing a dominant-negative PKD1 adenoviral construct in normal mouse keratinocytes and found that AQP3 mRNA was increased compared to the vector-infected group. Interestingly, the mRNA and protein levels of the cell adhesion protein, desmoglein 1 (Dsg1), were also increased in PKD1 knockdown keratinocytes, suggesting that PKD1 regulates Dsg1 expression. Importantly, skin from conditional PKD1 knockout mice also demonstrated increased Dsg1 protein levels. In contrast, other cell adhesion-related proteins, such as Dsg3, laminin 5 and Ajuba, were downregulated in keratinocytes lacking PKD1. In conclusion, we have identified regulation of Dsg1 by PKD1, with PKD1 normally repressing Dsg1 expression such that its loss results in increased Dsg1 levels. These data are consistent with the report of Ghazizadeh and colleagues demonstrating impaired wound healing in conditional epidermal-specific PKD1 knockout mice, characterized by reduced keratinocyte outgrowth from skin explants (i.e., decreased migration, as might be expected if the observed enhancement of Dsg1 levels promotes cell adhesion). Further studies are needed to determine the mechanism by which PKD1 regulates Dsg1 and AQP3 in the skin.
Mass spectrometry based proteomic investigation of epidermal and dermal skin aging V Newton1, I Riba-Garcia2, AV Rawlings3, R Voegeli4, C Griffiths1, R Unwin2, RE Watson1 and MJ Sherratt5 1 Centre for Dermatology Research, University of Manchester, Manchester, United Kingdom, 2 CADET, Manchester University Hospitals NHS Foundation Trust, Manchester, United Kingdom, 3 AVR Consulting, Northwich, United Kingdom, 4 DSM Nutritional Products Ltd, Kaiseraugst, Switzerland and 5 Tissue Injury & Repair, University of Manchester, Manchester, United Kingdom This study aimed to identify new biomarkers of skin ageing and to determine age-associated differences in the proteome of photoprotected versus photoexposed skin, specifically in the epidermis, dermal-epidermal junction (DEJ) and papillary dermis. Skin biopsies from the buttock (photoprotected) and extensor forearm (photoexposed) of volunteers (aged 18-30 or 65 yrs; n¼4-6/group) were transversely sectioned from the stratum corneum to a depth of 250 mm. Following SDS-PAGE, each sample lane was segmented into 11 prior to trypsin digestion and analyses by LC-MS/MS. Protein identification and relative quantification were determined using Mascot Scaffold and ProteinPilot software. Within the photoprotected skin, of the 366 proteins identified, 26 were 2-fold different in abundance in aged skin compared with young. Similarly, within photoexposed skin, of the 550 proteins identified 41 were 2fold different in abundance. Changes in both actin cytoskeleton and stress response pathways in aged photoprotected skin were identified using Ingenuity software. Similar pathway changes were also observed in photoexposed skin. To the best of our knowledge, this is the first study to directly compare the proteomes of the epidermis, DEJ and papillary dermis of skin biopsies from young and aged individuals. Other studies using whole-skin biopsies or cultured skin cells have detected greater numbers of proteins. However, our method excludes the majority of the reticular dermis and its high abundance of matrix molecules that otherwise could have masked less abundant proteins. This study therefore identifies changes in cutaneous protein abundance that occur with age and the effect of sunlight on the proteome of aged skin.
305
306
Epidermal iron metabolism to prevent iron loss by desquamation M Asano, K Yamasaki, T Yamauchi and S Aiba Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan It is well known that the intracellular iron content in the epidermis is highest in the basal layer and declines towards the stratum corneum and that most of iron in epidermal keratinocytes is not lost by desquamation. These observations suggest that the epidermis is equipped with some mechanism by which intracellular iron in differentiated keratinocytes is excreted to extracellular space before leaching the stratum corneum. In this study, we first demonstrated a distinctive expression pattern of iron metabolism molecules in which molecules participating in iron import and storage are expressed in the lower epidermis while those used for iron release from heme or iron transport are expressed in the upper epidermis by immunohistochemistry and mRNA quantification of the lower and upper epidermis separately obtained by a laser capture microdissection. This expression pattern of iron metabolism molecules was confirmed by normal human epidermal keratinocytes (NHEKs) during in vitro differentiation. Consistent with their expression, keratinocyte differentiation induced by high calcium medium reduces the intracellular iron content. Next, to clarify whether these iron metabolism molecules are really functioning in the epidermis, we demonstrated reducing ferroportin expression in vitro by ferroportin-specific siRNAs and hepcidin significantly increased the intracellular iron content. Finally, we demonstrated that iron content of the epidermis and squames in the dorsal skin was significantly greater in FpnEpi-KO mice than that in control mice and that FpnEpi-KO decreased blood Hb concentration more rapidly than control mice on an iron low diet. These studies build upon the evidence that iron is absorbed in the lower epidermis and extruded in the upper epidermis by ferroportin before iron is lost as a part of shedding keratinocytes, consequently preventing iron being lost from the skin at the level of affecting the systemic iron homeostasis.
Form and function of keratins in the nucleus R Hobbs1, JT Jacob1, BG Poll1,2 and PA Coulombe1,2,3 1 Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, 2 Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD and 3 Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD Select keratin intermediate filament proteins, which are classically described as strictly cytoplasmic, have recently been shown to localize and function within the nucleus of tumorderived epithelial cells from skin and cervix. Of the 54 keratin proteins occurring in mammalian genomes, K7, K8, K17, and K18 have so far been shown to occur in the nucleus. Our efforts have focused on K17, which is robustly upregulated in inflammatory skin diseases and tumors originating in stratified and pseudostratified epithelia. Using qRT-PCR, ChIP, and EMSA analyses, we determined that K17 promotes pro-inflammatory cytokine expression in skin tumor keratinocytes, in part, by translocating to the nucleus and associating with autoimmune regulator (AIRE) at NF-kB consensus motifs within the promoter regions of several genes (e.g. CXCL10, CXCL11, CCL19, MMP9). Bioinformatic analyses show the presence of a hitherto unknown bipartite nuclear localization signal (NLS) at the boundary between the central rod and C-terminal tail domains. Site-directed mutagenesis of a di-lysine motif (p.K399K400) within the predicted NLS substantially abrogates the ability of K17 to localize to the nucleus and to induce expression of its target genes. Confocal immunofluorescence microscopy and other lines of evidence suggest that K17 does not adopt a conventional 10-nm filament form in the nucleus of A431 (skin derived), HeLa (cervix derived), or BT20 (breast derived) tumor epithelial cells, or in human BCC tissue in situ. Rather, nuclear K17 is diffuse and often occurs in discrete, rounded punctae adjacent PML bodies and in chromatinsparse regions. Nuclear-localized K17 has been evidenced by other researchers in cervical epithelial tumor cell lines and in cervical tumors in situ. The discovery of keratin presence and function in the nucleus adds a new dimension to cell biology, nuclear architecture and dynamics, and epithelial structure and function.
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UVB-induced HMGB1 and extracellular ATP increase Langerhans cell production of IL-23 implicated in ILC3 activation JL Turban1,2, J Lewis1 and M Girardi1 1 Dermatology, Yale School of Medicine, New Haven, CT and 2 Howard Hughes Medical Institute, Chevy Chase, MD Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) arise from epidermal keratinocytes (KC) following exposure to ultraviolet B (UVB) radiation. Situated adjacent to nearly all KC is a network of epidermal Langerhans cells (LC). Our group previously implicated LC in the development of UVB-induced KC cancers, with LC-deficient (huLangerinDTA) mice being relatively resistant to cancer formation induced by chronic UVB irradiation. Furthermore, LC-intact mice show augmented epidermal expression of IL-23 and IL-22 following nine weeks of UVB irradiation. Here we show that the UVB-induced damageassociated molecular patterns (DAMP) HMGB1 and extracellular ATP have the capacity to induce IL-23 production by the LC line XS106. HMGB1 treatment of XS106 increased IL-23 production, as measured by multiplex cytokine assay, 23.3x (p¼.0007). ATP exposure increased production 2.3x (p¼.0002). Freshly isolated murine LCs, purified with MHC-II magnetic beads from epidermal preparations, increased IL-23 production following HMGB1treatment >100x (p¼.0007). Notably, treatment with the UVB-induced DAMP beta-defensin 2 did not result in increased IL-23 production by XS106. We also show here the presence of an epidermal population of type three innate lymphoid cells (ILC3) by epidermal sheet immunofluorescence from nine-week UVB-irradiated T-cell deficient (b-/-d-/-) mice. ILC3 are known to produce the epidermal growth factor IL-22 in response to stimulation with IL-23. Together, this data suggests that activation of LC by UVB-induced DAMPs induces IL-23 production, which subsequently signals ILC3 to produce IL-22, delivering a proliferative signal to KC, including those harboring UVB-induced mutations. Targeting this DAMP-IL23IL22 pathway may ultimately prove a useful target in the prevention of skin cancer via the inhibition of mutant keratinocyte clonal expansion.
S54 Journal of Investigative Dermatology (2016), Volume 136
Heavy cigarette smokers in a Chinese population display a compromised permeability barrier S Xin2, L Ye2, G Man1, C Lv2, PM Elias1 and M Man1 1 Dermatology, VA Medical Ctr/UCSF, San Francisco, CA and 2 Skin disease, Dalian Hospital, Dalian, China Background/Objective: Cigarette smoking is associated with a higher prevalence of inflammatory disorders that display a defective epidermal permeability barrier, such as atopic dermatitis and psoriasis. Yet, whether cigarette smoking influences barrier function is largely unknown. The present study determined whether skin biophysical properties, including permeability barrier homeostasis, stratum corneum (SC) integrity, SC hydration, skin surface pH, and skin melanin/erythema index, are altered in cigarette smokers. Methods: A total of 99 male volunteers, including 63 smokers and 36 non-smokers, aged 41-65 years (mean 53.86 0.66), with no current skin disease, were enrolled in this study. Smokers were categorized as light-to-moderate (<20 cigarettes/day) or heavy smokers (20 cigarettes/day). A multifunctional skin physiology monitor (C&K MPA5) was used to measure SC hydration and skin melanin/erythema index on the dorsal hand, forehead and cheek. Basal transepidermal water loss (TEWL) and barrier recovery rates were assessed on the forearm. A Skin-pH-Meter pH900 was used to measure skin surface pH. Results: Despite showing no differences in basal TEWL, heavy cigarette smokers (20 cigarettes/day) exhibited delayed barrier recovery after acute abrogation (1.02% 13.06 vs. 16.48% 6.07 in non-smokers), and barrier recovery rates correlated negatively with the number of cigarettes smoked per day (r2¼0.1066, p¼0.0087). Differences in biophysical parameters between smokers and non-smokers, including surface pH, stratum corneum hydration and erythema index, varied with body sites. Conclusions: Heavy cigarette smokers display compromised permeability barrier homeostasis, which could contribute, in part, to the increased prevalence of certain cutaneous disorders characterized by defective epidermal permeability in smokers. Conversely, strategies that improve epidermal permeability barrier function should be considered for heavy cigarette smokers with a history of inflammatory dermatoses.