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453 EphA2 transmembrane domain governs receptor membrane distribution and differentiation-associated signaling in keratinocytes
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Real time 3D in vivo pH imaging reveals tightly-regulated pH layers with distinct properties in mouse stratum corneum Y Furuichi1, T Matsui2 and M Amagai3 1 Dept.Dermatol., Keio Univ / RIKEN-IMS, Yokohama, Japan, 2 RIKEN-IMS, Yokohama, Japan and 3 Dept.Dermatol., Keio Univ / RIKENIMS, Tokyo, Japan The regulation of the pH is an important element in various biological functions. The pH distribution of the stratum corneum (SC) is considered as the gradient decreasing along surface in general while precise SC pH distribution and its dynamic changes under different environmental conditions remain to be clarified. The purpose of this study is to produce a transgenic mouse as a tool to visualize the intact SC pH in vivo. We generated a ratiometric pH-biosensor with pH-sensitive fluorescent protein, VenusH148G fused with pH-insensitive protein, mCherry (VenusH148G-mCherry), expressed specifically in the uppermost layer of stratum granulosum (SG1) using knock-in construct of SASPase gene by modified CRISPR/Cas9 system. Noninvasive confocal microscopic analysis of SC pH in living VenusH148G-mCherry mouse ear skin observed with dry objective demonstrated that upper SC is neutral compared to middle SC. Immersion of water onto SC surface enabled highresolution deeper range SC pH imaging and revealed that the SC and SG1 layer are divided into three zones with distinct pH properties rather than gradual changes across the layers. Namely, the SG1 was neutral, the middle SC was acidic, and neutral pH was again observed in the upper SC. Analysis of the 3D pH distribution of whole SC showed that the upper neutral SC zone distributed unevenly by differentiated units, and the acidic middle SC was wellmaintained. When ear skin was exposed to phosphate buffers with various pH (pH5.4, 6.6 and 7.4) by topical application, the pH of upper SC dynamically changed according to the pH applied, while the middle SC maintained its acidic pH. These findings indicated that the SC is a highly organized tissue with tightly-regulated pH layers having the distinct functions, and that the 3D in vivo pH imaging mice will provide a valuable tool to dissect the homeostatic mechanisms of SC by pH regulation.
Alterations to connexin 26 protein structure caused by the lethal keratitisichthyosis-deafness syndrome mutations A88V and G45E CG Bunick1, E Lilly2 and LM Milstone3 1 Yale University, New Haven, CT, 2 Massachusetts General Hospital, Boston, MA and 3 Yale School of Medicine, New Haven, CT Keratitis-ichthyosis-deafness (KID) syndrome is a rare autosomal dominant disorder caused by mutations in GJB2 [encodes connexin 26 (Cx26)]. Affected individuals are born with erythrokeratoderma and sensorineural deafness, and develop progressive vascularizing keratitis. Multiple mutations occur in GJB2 including A88V and G45E, which are termed “lethal variants” as most people affected do not live past infancy. Research by the Dale group has suggested a role for Cx26 in respiration and carbon dioxide sensing in humans. Our clinical experience with a child having Cx26A88V mutation who died after suffering hypoxia, respiratory failure, and central apnea encouraged us to further investigate the effects of Cx26 mutations on connexin/connexon structure. To correlate Cx26 protein structure with altered function and phenotype seen in patients with A88V mutation, we performed detailed computational structure analyses on the wildtype x-ray crystal structure of Cx26 (Protein Data Bank Code 2ZW3). We also generated two mutant models (Cx26A88V and Cx26G45E) of Cx26 to investigate whether these mutations affect CO2 sensing. Our analysis shows that a highly conserved proline residue (Pro87) across connexins exists adjacent to Ala88; it induces a proline kink in transmembrane helix 2, creating specific steric constraints on the Ala88 position. Substitution of any of the three possible rotamers of valine at position 88 results in steric clashes with adjacent residues. Rearrangement of the transmembrane helices in order to avoid this unfavorable stereochemistry plausibly disrupts the positioning of Arg104 and Lys125 necessary for Cx26 to bind CO2. We also predicted the change in folding free energy (DDG, kcal/mol) of Cx26 after A88V or G45E mutation. The DDG analysis showed that Cx26A88V is a destabilizing mutation, whereas Cx26G45E is stabilizing and would not appear to affect CO2 sensing.
Biological activity of phytol in skin C Hwang, J Idkowiak-Baldys and J Lyga Avon Products, Inc., Suffern, NY Phytol is a branched-chain fatty alcohol that was shown to have skin efficacy when applied topically in cosmetic formulations. When applied to skin, phytol is converted to phytanic acid (branched-chain fatty acid), which has biological activity through activating retinoic acid receptors and peroxisome proliferator activated receptors. The present study shows the effect of phytol on multiple biological pathways in skin both in vitro and in vivo. Phytol increased the production of pro-collagen-I and hyaluronic acid in cultured human dermal fibroblasts. Immunostaining of skin biopsy confirmed the increased levels of collagen and hyaluronic acid in the dermis of phytol-treated human skin. In addition, anti-inflammatory properties of phytol were evaluated in human epidermal keratinocytes. Phytol inhibited cytokine-mediated inflammatory response in keratinocytes. Skin biopsy study showed the effect of phytol on increasing epidermal proliferation as measured by Ki67 immunostaining. Finally, PCR array analysis indicated that phytol treatment led to change in expression of several skin-related genes when tested in 3D skin equivalents. In summary, this study showed biological basis for in vivo efficacy of phytol.
S78 Journal of Investigative Dermatology (2017), Volume 137
Human epidermal differentiation requires autophagy and lysosomal trafficking C Monteleon, T Agnihotri and T Ridky University of Pennsylvania, Philadelphia, PA Keratinocytes undergo significant structural remodeling during differentiation. This multistep program includes sweeping changes in the keratinocyte proteome coupled with a major reduction in cellular biomass. These changes suggest that lysosome-related activities are essential for normal epidermal homeostasis. Lysosomes are at the nexus of several vesicular trafficking pathways, including autophagy and lysosomal exocytosis. Using both genetic and pharmacologic approaches, we targeted these pathways individually in three-dimensional organotypic human skin. Inhibition of autophagosome assembly upstream of lysosomal fusion resulted in a complete block ofepidermal differentiation, suggesting that intracellular sequestration and degradation of cytoplasm components are required for epidermal homeostasis. Normal epidermal differentation also required proper fusion between secretory Lysosome Related Organelles (LROs) and the plasma membrane. These data suggest that both the autophagic and exocytic trafficking pathways are required for keratinocyte differentiation. As epidermal disorders including psoriasis and eczema are associated with dysregulation of lysosomal trafficking, a better understanding of these pathways may guide future therapeutic strategies.
EphA2 transmembrane domain governs receptor membrane distribution and differentiation-associated signaling in keratinocytes R Ventrella, N Kaplan, P Hoover, B Perez White, RM Lavker and S Getsios Northwestern University, Chicago, IL Abnormalities in lipid metabolism are present in atopic dermatitis in association with defective keratinocyte differentiation. In addition to impaired barrier function, these lipid defects may impact membrane-associated signaling events governing differentiation. To this end, we focused on the receptor tyrosine kinase EphA2, which elicits differentiationdependent signaling in keratinocytes upon binding to ephrin-A ligand on adjacent cells. Since ephrin-A ligands are GPI-linked, we hypothesized that lipid rafts would be important for EphA2 activation and its subsequent downregulation during keratinocyte differentiation. Sucrose density gradients and colocalization studies with lipid raft-associated proteins revealed that EphA2 was recruited to lipid raft fractions coincident with its activation upon calcium-induced differentiation of normal human epidermal keratinocytes (NHEKs). Lipid raft disruption by cholesterol depletion increased EphA2 levels in NHEKs and 3-D reconstituted human epidermis in association with impaired differentiation. To gain insight into the mechanisms governing EphA2 membrane localization and activation state in NHEKs, we genetically swapped the EphA2 extracellular, transmembrane (TM), and cytoplasmic domain with a related family member, EphA1, whose levels, activation state, and lipid raft association remain stable during keratinocyte differentiation. These chimera studies revealed a distinct role for the EphA2 TM domain in promoting receptor recruitment to cell-cell contacts. In addition to highlighting a previously unappreciated role for the EphA2 TM domain in organizing membrane-associated signaling events during keratinocyte differentiation, our work suggests that lipid alterations in diseases such as atopic dermatitis may negatively impact epidermal homeostasis via disruption of the EphA signaling axis.
Characterizing skin aging using two-photon fluorescence and second harmonic generation H Wang1, T Shyr1, G Stamatas2, M Fevola1 and G Cula1 1 Johnson & Johnson Consumer Inc., Skillman, NJ and 2 Johnson & Johnson Sante´ Beaute´, Issy Les Moulineaux, France Skin aging is a complicated process which involves remodeling of dermal fibers. Accurate characterization of skin aging enables researchers to better understand the underlying biology and provides technical insights to develop better anti-aging solutions. In vivo multiphoton microscopy (MPM) is a novel imaging technique which can provide unique contrast of elastic and collagen fibers through two-photon fluorescence (TPF) and second harmonic generation (SHG) processes,respectively.Therefore,non-invasive, direct visualization of dermal fibers can be achieved. Typically, analysis of MPM images is limited to averaging the TPF or SHG signal over the imaged area.The goal of the study is to explore new quantitative methods that account for the spatial distribution of the signal thereby establishing a better model to characterize skin aging. In this study,z-stacks of TPF and SHG images were taken at three different body sites including dorsal forearm, upper inner arm, and cheek.Two age groups (20-30 yr and above 50 yr) of healthy female volunteers were included. Image analysis characterizing dermal fiber alterations at different depths was performed. An algorithm based on a combination of k-means clustering and gradient was developed to generate orientation maps of the fibers and to derive the orientation distribution histogram. In addition,fast Fourier transform based fiber orientation analysis was performed to extract energy distribution in the frequency domain. We also demonstrated that consideration of the dermal depth information helps to better evaluate the aging state. In summary, with the capability of direct visualization of skin dermal fibers provided by MPM, novel algorithms have been developed to characterize skin aging.The derived image-based features can be combined to improve the accuracy of quantification, and can also be easily adapted to accommodate other imaging techniques.
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Real time 3D in vivo pH imaging reveals tightly-regulated pH layers with distinct properties in mouse stratum corneum Y Furuichi1, T Matsui2 and M Amagai3 1 Dept.Dermatol., Keio Univ / RIKEN-IMS, Yokohama, Japan, 2 RIKEN-IMS, Yokohama, Japan and 3 Dept.Dermatol., Keio Univ / RIKENIMS, Tokyo, Japan The regulation of the pH is an important element in various biological functions. The pH distribution of the stratum corneum (SC) is considered as the gradient decreasing along surface in general while precise SC pH distribution and its dynamic changes under different environmental conditions remain to be clarified. The purpose of this study is to produce a transgenic mouse as a tool to visualize the intact SC pH in vivo. We generated a ratiometric pH-biosensor with pH-sensitive fluorescent protein, VenusH148G fused with pH-insensitive protein, mCherry (VenusH148G-mCherry), expressed specifically in the uppermost layer of stratum granulosum (SG1) using knock-in construct of SASPase gene by modified CRISPR/Cas9 system. Noninvasive confocal microscopic analysis of SC pH in living VenusH148G-mCherry mouse ear skin observed with dry objective demonstrated that upper SC is neutral compared to middle SC. Immersion of water onto SC surface enabled highresolution deeper range SC pH imaging and revealed that the SC and SG1 layer are divided into three zones with distinct pH properties rather than gradual changes across the layers. Namely, the SG1 was neutral, the middle SC was acidic, and neutral pH was again observed in the upper SC. Analysis of the 3D pH distribution of whole SC showed that the upper neutral SC zone distributed unevenly by differentiated units, and the acidic middle SC was wellmaintained. When ear skin was exposed to phosphate buffers with various pH (pH5.4, 6.6 and 7.4) by topical application, the pH of upper SC dynamically changed according to the pH applied, while the middle SC maintained its acidic pH. These findings indicated that the SC is a highly organized tissue with tightly-regulated pH layers having the distinct functions, and that the 3D in vivo pH imaging mice will provide a valuable tool to dissect the homeostatic mechanisms of SC by pH regulation.
Alterations to connexin 26 protein structure caused by the lethal keratitisichthyosis-deafness syndrome mutations A88V and G45E CG Bunick1, E Lilly2 and LM Milstone3 1 Yale University, New Haven, CT, 2 Massachusetts General Hospital, Boston, MA and 3 Yale School of Medicine, New Haven, CT Keratitis-ichthyosis-deafness (KID) syndrome is a rare autosomal dominant disorder caused by mutations in GJB2 [encodes connexin 26 (Cx26)]. Affected individuals are born with erythrokeratoderma and sensorineural deafness, and develop progressive vascularizing keratitis. Multiple mutations occur in GJB2 including A88V and G45E, which are termed “lethal variants” as most people affected do not live past infancy. Research by the Dale group has suggested a role for Cx26 in respiration and carbon dioxide sensing in humans. Our clinical experience with a child having Cx26A88V mutation who died after suffering hypoxia, respiratory failure, and central apnea encouraged us to further investigate the effects of Cx26 mutations on connexin/connexon structure. To correlate Cx26 protein structure with altered function and phenotype seen in patients with A88V mutation, we performed detailed computational structure analyses on the wildtype x-ray crystal structure of Cx26 (Protein Data Bank Code 2ZW3). We also generated two mutant models (Cx26A88V and Cx26G45E) of Cx26 to investigate whether these mutations affect CO2 sensing. Our analysis shows that a highly conserved proline residue (Pro87) across connexins exists adjacent to Ala88; it induces a proline kink in transmembrane helix 2, creating specific steric constraints on the Ala88 position. Substitution of any of the three possible rotamers of valine at position 88 results in steric clashes with adjacent residues. Rearrangement of the transmembrane helices in order to avoid this unfavorable stereochemistry plausibly disrupts the positioning of Arg104 and Lys125 necessary for Cx26 to bind CO2. We also predicted the change in folding free energy (DDG, kcal/mol) of Cx26 after A88V or G45E mutation. The DDG analysis showed that Cx26A88V is a destabilizing mutation, whereas Cx26G45E is stabilizing and would not appear to affect CO2 sensing.
Biological activity of phytol in skin C Hwang, J Idkowiak-Baldys and J Lyga Avon Products, Inc., Suffern, NY Phytol is a branched-chain fatty alcohol that was shown to have skin efficacy when applied topically in cosmetic formulations. When applied to skin, phytol is converted to phytanic acid (branched-chain fatty acid), which has biological activity through activating retinoic acid receptors and peroxisome proliferator activated receptors. The present study shows the effect of phytol on multiple biological pathways in skin both in vitro and in vivo. Phytol increased the production of pro-collagen-I and hyaluronic acid in cultured human dermal fibroblasts. Immunostaining of skin biopsy confirmed the increased levels of collagen and hyaluronic acid in the dermis of phytol-treated human skin. In addition, anti-inflammatory properties of phytol were evaluated in human epidermal keratinocytes. Phytol inhibited cytokine-mediated inflammatory response in keratinocytes. Skin biopsy study showed the effect of phytol on increasing epidermal proliferation as measured by Ki67 immunostaining. Finally, PCR array analysis indicated that phytol treatment led to change in expression of several skin-related genes when tested in 3D skin equivalents. In summary, this study showed biological basis for in vivo efficacy of phytol.
S78 Journal of Investigative Dermatology (2017), Volume 137
Human epidermal differentiation requires autophagy and lysosomal trafficking C Monteleon, T Agnihotri and T Ridky University of Pennsylvania, Philadelphia, PA Keratinocytes undergo significant structural remodeling during differentiation. This multistep program includes sweeping changes in the keratinocyte proteome coupled with a major reduction in cellular biomass. These changes suggest that lysosome-related activities are essential for normal epidermal homeostasis. Lysosomes are at the nexus of several vesicular trafficking pathways, including autophagy and lysosomal exocytosis. Using both genetic and pharmacologic approaches, we targeted these pathways individually in three-dimensional organotypic human skin. Inhibition of autophagosome assembly upstream of lysosomal fusion resulted in a complete block ofepidermal differentiation, suggesting that intracellular sequestration and degradation of cytoplasm components are required for epidermal homeostasis. Normal epidermal differentation also required proper fusion between secretory Lysosome Related Organelles (LROs) and the plasma membrane. These data suggest that both the autophagic and exocytic trafficking pathways are required for keratinocyte differentiation. As epidermal disorders including psoriasis and eczema are associated with dysregulation of lysosomal trafficking, a better understanding of these pathways may guide future therapeutic strategies.
EphA2 transmembrane domain governs receptor membrane distribution and differentiation-associated signaling in keratinocytes R Ventrella, N Kaplan, P Hoover, B Perez White, RM Lavker and S Getsios Northwestern University, Chicago, IL Abnormalities in lipid metabolism are present in atopic dermatitis in association with defective keratinocyte differentiation. In addition to impaired barrier function, these lipid defects may impact membrane-associated signaling events governing differentiation. To this end, we focused on the receptor tyrosine kinase EphA2, which elicits differentiationdependent signaling in keratinocytes upon binding to ephrin-A ligand on adjacent cells. Since ephrin-A ligands are GPI-linked, we hypothesized that lipid rafts would be important for EphA2 activation and its subsequent downregulation during keratinocyte differentiation. Sucrose density gradients and colocalization studies with lipid raft-associated proteins revealed that EphA2 was recruited to lipid raft fractions coincident with its activation upon calcium-induced differentiation of normal human epidermal keratinocytes (NHEKs). Lipid raft disruption by cholesterol depletion increased EphA2 levels in NHEKs and 3-D reconstituted human epidermis in association with impaired differentiation. To gain insight into the mechanisms governing EphA2 membrane localization and activation state in NHEKs, we genetically swapped the EphA2 extracellular, transmembrane (TM), and cytoplasmic domain with a related family member, EphA1, whose levels, activation state, and lipid raft association remain stable during keratinocyte differentiation. These chimera studies revealed a distinct role for the EphA2 TM domain in promoting receptor recruitment to cell-cell contacts. In addition to highlighting a previously unappreciated role for the EphA2 TM domain in organizing membrane-associated signaling events during keratinocyte differentiation, our work suggests that lipid alterations in diseases such as atopic dermatitis may negatively impact epidermal homeostasis via disruption of the EphA signaling axis.
Characterizing skin aging using two-photon fluorescence and second harmonic generation H Wang1, T Shyr1, G Stamatas2, M Fevola1 and G Cula1 1 Johnson & Johnson Consumer Inc., Skillman, NJ and 2 Johnson & Johnson Sante´ Beaute´, Issy Les Moulineaux, France Skin aging is a complicated process which involves remodeling of dermal fibers. Accurate characterization of skin aging enables researchers to better understand the underlying biology and provides technical insights to develop better anti-aging solutions. In vivo multiphoton microscopy (MPM) is a novel imaging technique which can provide unique contrast of elastic and collagen fibers through two-photon fluorescence (TPF) and second harmonic generation (SHG) processes,respectively.Therefore,non-invasive, direct visualization of dermal fibers can be achieved. Typically, analysis of MPM images is limited to averaging the TPF or SHG signal over the imaged area.The goal of the study is to explore new quantitative methods that account for the spatial distribution of the signal thereby establishing a better model to characterize skin aging. In this study,z-stacks of TPF and SHG images were taken at three different body sites including dorsal forearm, upper inner arm, and cheek.Two age groups (20-30 yr and above 50 yr) of healthy female volunteers were included. Image analysis characterizing dermal fiber alterations at different depths was performed. An algorithm based on a combination of k-means clustering and gradient was developed to generate orientation maps of the fibers and to derive the orientation distribution histogram. In addition,fast Fourier transform based fiber orientation analysis was performed to extract energy distribution in the frequency domain. We also demonstrated that consideration of the dermal depth information helps to better evaluate the aging state. In summary, with the capability of direct visualization of skin dermal fibers provided by MPM, novel algorithms have been developed to characterize skin aging.The derived image-based features can be combined to improve the accuracy of quantification, and can also be easily adapted to accommodate other imaging techniques.