Theodora Mauro - Academia.edu (original) (raw)
Papers by Theodora Mauro
FEBS letters, Jan 9, 2015
The keratitis-ichthyosis-deafness (KID) syndrome is caused by mutations in the gap junctional cha... more The keratitis-ichthyosis-deafness (KID) syndrome is caused by mutations in the gap junctional channel protein connexin 26 (Cx26), among them the mutation Cx26S17F. Heterozygous Cx26S17F mice resemble the human KID syndrome, i.e. exhibiting epidermal hyperplasia and hearing impairments. Newborn Cx26S17F mice show a defective epidermal water barrier as well as altered epidermal lipid secretion and location. Linoleoyl ω-esterified ceramides are strongly decreased on the skin surface of Cx26S17F mice. Moreover, the epidermal calcium gradient is altered in the mutant mice. These alterations may be caused by an abnormal Cx26S17F channel function that leads to a defective epidermal water barrier, which in turn may trigger the hyperproliferation seen in the KID syndrome.
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Journal of dermatological science, Jan 19, 2015
The ceramide metabolite, sphingosine-1-phosphate (S1P), regulates multiple cellular functions in ... more The ceramide metabolite, sphingosine-1-phosphate (S1P), regulates multiple cellular functions in keratinocytes (KC). We recently discovered that production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), is stimulated via a NF-κB-dependent mechanism that is activated by S1P when S1P is generated by sphingosine kinase (SPHK) 1. We investigated whether pharmacological modulation of SPHK1 activity, using a novel synthetic SPHK1 activator, (S)-methyl 2-(hexanamide)-3-(4-hydroxyphenyl) propanoate (MHP), stimulates CAMP expression. MHP-mediated changes in both S1P and CAMP downstream mediators were analyzed in normal cultured human KC by qRT-PCR, Western immunoblot, ELISA, confocal microscopy for immunohistochemistry, HPLC and ESI-LC/MS/MS, and microbial pathogen invasion/colonization in a human epidermal organotypic model. Treatment with MHP directly activated SPHK1 and increased cellular S1P content in normal cultured human KC. Because MHP did not inhibit S1P ...
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Nano letters, Jan 27, 2015
Transdermal delivery of therapeutics is restricted by narrow limitations on size and hydrophobici... more Transdermal delivery of therapeutics is restricted by narrow limitations on size and hydrophobicity. Nanotopography has been shown to significantly enhance high molecular weight paracellular transport in vitro. Herein, we demonstrate for the first time that nanotopography applied to microneedles significantly enhances transdermal delivery of etanercept, a 150 kD therapeutic, in both rats and rabbits. We further show that this effect is mediated by remodeling of the tight junction proteins initiated via integrin binding to the nanotopography, followed by phosphorylation of myosin light chain (MLC) and activation of the actomyosin complex, which in turn increase paracellular permeability.
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Nature genetics, 2000
Hailey-Hailey disease (HHD, MIM 16960) is inherited in an autosomal dominant manner and character... more Hailey-Hailey disease (HHD, MIM 16960) is inherited in an autosomal dominant manner and characterized by persistent blisters and erosions of the skin. Impaired intercellular adhesion and epidermal blistering also occur in individuals with pemphigus (which is due to autoantibodies directed against desmosomal proteins) and in patients with Darier disease (DD, MIM 124200), which is caused by mutations in a gene encoding a sarco/endoplasmic reticulum (ER)-Golgi calcium pump. We report here the identification of mutations in ATP2C1, encoding the human homologue of an ATP-powered pump that sequesters calcium into the Golgi in yeast, in 21 HHD kindreds. Regulation of cytoplasmic calcium is impaired in cultured keratinocytes from HHD patients, and the normal epidermal calcium gradient is attenuated in vivo in HHD patients. Our findings not only provide an understanding of the molecular basis of HHD, but also underscore the importance of calcium control to the functioning of stratified squam...
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Molecular biology of the cell, 2004
Members of the serum- and glucocorticoid-regulated kinase (SGK) family are important mediators of... more Members of the serum- and glucocorticoid-regulated kinase (SGK) family are important mediators of growth factor and hormone signaling that, like their close relatives in the Akt family, are regulated by lipid products of phosphatidylinositol-3-kinase. SGK3 has been implicated in the control of cell survival and regulation of ion channel activity in cultured cells. To begin to dissect the in vivo functions of SGK3, we generated and characterized Sgk3 null mice. These mice are viable and fertile, and in contrast to mice lacking SGK1 or Akt2, respectively, display normal sodium handling and glucose tolerance. However, although normal at birth, by postpartum day 4 they have begun to display an unexpected defect in hair follicle morphogenesis. The abnormality in hair follicle development is preceded by a defect in proliferation and nuclear accumulation of beta-catenin in hair bulb keratinocytes. Furthermore, in cultured keratinocytes, heterologous expression of SGK3 potently modulates ac...
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The Journal of investigative dermatology, Jan 17, 2014
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The American Journal of Pathology, 2015
Although keratosis pilaris (KP) is common, its etiopathogenesis remains unknown. KP is associated... more Although keratosis pilaris (KP) is common, its etiopathogenesis remains unknown. KP is associated clinically with ichthyosis vulgaris and atopic dermatitis and molecular genetically with filaggrin-null mutations. In 20 KP patients and 20 matched controls, we assessed the filaggrin and claudin 1 genotypes, the phenotypes by dermatoscopy, and the morphology by light and transmission electron microscopy. Thirty-five percent of KP patients displayed filaggrin mutations, demonstrating that filaggrin mutations only partially account for the KP phenotype. Major histologic and dermatoscopic findings of KP were hyperkeratosis, hypergranulosis, mild T helper cell type 1-dominant lymphocytic inflammation, plugging of follicular orifices, striking absence of sebaceous glands, and hair shaft abnormalities in KP lesions but not in unaffected skin sites. Changes in barrier function and abnormal paracellular permeability were found in both interfollicular and follicular stratum corneum of lesional KP, which correlated ultrastructurally with impaired extracellular lamellar bilayer maturation and organization. All these features were independent of filaggrin genotype. Moreover, ultrastructure of corneodesmosomes and tight junctions appeared normal, immunohistochemistry for claudin 1 showed no reduction in protein amounts, and molecular analysis of claudin 1 was unremarkable. Our findings suggest that absence of sebaceous glands is an early step in KP pathogenesis, resulting in downstream hair shaft and epithelial barrier abnormalities.
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Journal of Investigative Dermatology Symposium Proceedings, 1998
The contents of epidermal lamellar bodies (LB) are delivered selectively to the intercellular spa... more The contents of epidermal lamellar bodies (LB) are delivered selectively to the intercellular spaces at the stratum granulosum (SG)-stratum corneum (SC) interface. We assessed the subcellular basis for LB secretion first by confocal microscopy, following labeling with Nile red or NBD-ceramide, which reveals a tubulo-reticular membrane system within the apical cytosol of the outermost SG cell layer under basal conditions, changing to a more peripheral staining pattern when secretion is stimulated. Ultrastructural study demonstrates that this network is composed of a widely disbursed trans-Golgi-like network (TGN), associated with arrays of contiguous LB, and deep invaginations of the SG-SC interface. Under basal conditions, limited fusion of apically directed LB leads to deep, interconnected invaginations of the apical plasma membrane, resulting in the formation of an extensive, honeycomb extension of the SG-SC interface. Still deeper invaginations and more extensive organelle fusion develop after the epidermis is acutely permeabilized by either acetone treatment, sonophoresis, or iontophoresis. Finally, nascent LB appear to bud off cisternae of the TGN, a process that appears to accelerate after barrier disruption. The deep invaginations of the SG-SC interface; the wide distribution of the TGN within the apical cytosol; the association of nascent LB with the TGN; and the rapid fusion of LB with these invaginations, deep within the cytosol, account for (i) the polarized secretion of LB from the apex of the outermost SG cell, and (ii) the rapid LB-secretory response to barrier perturbations. Finally, our results point to the outermost SG cell as a uniquely specialized secretory cell. We propose the term "secretory granulocyte" to encompass the specialized features of these cells.
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Stem cell reports, Jan 6, 2014
Cornification and epidermal barrier defects are associated with a number of clinically diverse sk... more Cornification and epidermal barrier defects are associated with a number of clinically diverse skin disorders. However, a suitable in vitro model for studying normal barrier function and barrier defects is still lacking. Here, we demonstrate the generation of human epidermal equivalents (HEEs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). HEEs are structurally similar to native epidermis, with a functional permeability barrier. We exposed a pure population of hESC/iPSC-derived keratinocytes, whose transcriptome corresponds to the gene signature of normal primary human keratinocytes (NHKs), to a sequential high-to-low humidity environment in an air/liquid interface culture. The resulting HEEs had all of the cellular strata of the human epidermis, with skin barrier properties similar to those of normal skin. Such HEEs generated from disease-specific iPSCs will be an invaluable tool not only for dissecting molecular mechanisms that lead to epiderma...
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Journal of Clinical Investigation, 2007
The skin is the first line of defense against microbial infection, and psychological stress (PS) ... more The skin is the first line of defense against microbial infection, and psychological stress (PS) has been shown to have adverse effects on cutaneous barrier function. Here we show that PS increased the severity of group A Streptococcus pyogenes (GAS) cutaneous skin infection in mice; this was accompanied by increased production of endogenous glucocorticoids (GCs), which inhibited epidermal lipid synthesis and decreased lamellar body (LB) secretion. LBs encapsulate antimicrobial peptides (AMPs), and PS or systemic or topical GC administration downregulated epidermal expression of murine AMPs cathelin-related AMP and beta-defensin 3. Pharmacological blockade of the stress hormone corticotrophin-releasing factor or of peripheral GC action, as well as topical administration of physiologic lipids, normalized epidermal AMP levels and delivery to LBs and decreased the severity of GAS infection during PS. Our results show that PS decreases the levels of 2 key AMPs in the epidermis and their delivery into LBs and that this is attributable to increased endogenous GC production. These data suggest that GC blockade and/or topical lipid administration could normalize cutaneous antimicrobial defense during PS or GC increase. We believe this to be the first mechanistic link between PS and increased susceptibility to infection by microbial pathogens.
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Femtosecond Laser Applications in Biology, 2004
Measurements of ionic concentrations in skin have traditionally been performed with an array of m... more Measurements of ionic concentrations in skin have traditionally been performed with an array of methods which either did not reveal detailed localization information, or only provided qualitative, not quantitative information. FLIM combines a number of advantages into a method ideally suited to visualize concentrations of ions such as H+ in intact, unperturbed epidermis and stratum corneum (SC). Fluorescence lifetime is dye concentration-independent, the method requires only low light intensities and is therefore not prone to photobleaching or phototoxic artifacts, and because multiphoton lasers of IR wavelength are used, light penetrates deep into intact tissue. The standard method to measure SC pH is the flat pH electrode, which provides reliable information only about surface pH changes, without further vertical or subcellular spatial resolution; i.e., specific microdomains such as the corneocyte interstices are not resolved, and the deeper SC is inaccessible without resorting to inherently disruptive stripping methods. Furthermore, the concept of a gradient of pH through the SC stems from such stripping experiments, but other confirmation for this concept is lacking. Our investigations into the SC pH distribution so far have revealed the crucial role of the Sodium/Hydrogen Antiporter NHE1 in generation of SC acidity, the colocalization of enzymatic lipid processing activity in the SC with acidic domains of the SC, and the timing and localization of emerging acidity in the SC of newborns. Together, these results have led to an improved understanding of the SC pH, its distribution, origin, and regulation. Future uses for this method include measurements of other ions important for epidermal processes, such as Ca2+, and a quantitative approach to topical drug penetration.
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The FASEB Journal, 2009
SGK3, which previously has been shown to play a key role in hair follicle development in mice, is... more SGK3, which previously has been shown to play a key role in hair follicle development in mice, is a member of the AGC family of serine-threonine kinases. Mice lacking SGK3 have abnormal follicle cycling, which begins shortly after birth and ameliorates substantially with age. However, this developmental abnormality is not recapitulated in mice lacking closely related kinases Akt1, Akt2, or Akt3. To examine whether Akt2 interacts with SGK3 in postnatal hair development, we have generated and characterized Akt2/SGK3 double knockouts (DKOs). We find that the DKO mice have a defect in hair growth that is markedly worse than that of SGK3(-/-) mice and does not ameliorate with age. Morphologically, this defect is characterized by accelerated entry into catagen and through anagen, irregular hair follicle orientation, and increased expression of sebaceous glands. The defect is preceded by a profound failure to increase follicle matrix cell nuclear beta-catenin accumulation and proliferation at the onset of morphogenesis. Furthermore, in cultured keratinocytes, transfected Akt2 and SGK3 both stimulate transcription of a beta-catenin-LEF1-dependent reporter gene. Thus, SGK3 and Akt2 both appear to play important roles in postnatal hair follicle morphogenesis, likely because of their redundant regulation of beta-catenin-dependent transcriptional processes, which control hair follicle cell proliferation.
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Oncogene, 2006
Focal adhesion kinase (FAK) is a critical component in transducing signals downstream of both int... more Focal adhesion kinase (FAK) is a critical component in transducing signals downstream of both integrins and growth factor receptors. To determine how the loss of FAK affects the epidermis in vivo, we have generated a mouse model with a keratinocyte-restricted deletion of fak (FAKK5 KO mice). FAK(K5 KO) mice displayed three major phenotypes--irregularities of hair cycle, sebaceous glands hypoplasia, and a thinner epidermis--pointing to defects in the proliferative capacity of multipotent stem cells found in the bulge. FAK-null keratinocytes in conventional primary culture undergo massive apoptosis hindering further analyses, whereas the defects observed in vivo do not shorten the mouse lifespan. These results suggest that the structure and the signaling environment of the native tissue may overcome the lack of signaling through FAK. Our findings point to the importance of in vivo and three-dimensional in vitro models in analyses of cell migration, proliferation, and survival. Surprisingly, the difference between FAKloxP/+ and FAKK5 KO mice in wound closure was not statistically significant, suggesting that in vivo loss of FAK does not affect migration/proliferation of basal keratinocytes in the same way as it affects multipotent stem cells of the skin.
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Journal of Investigative Dermatology, 2014
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Journal of Investigative Dermatology, 1997
K+ channel activation has been associated with growth or differentiation in many cells. We have p... more K+ channel activation has been associated with growth or differentiation in many cells. We have previously identified a 70-pS K+ channel that was found only in differentiated involucrin-positive cells. In this study we examined the role of K+ channels in Ca2+-induced keratinocyte differentiation. Consistent with our previous report, we found that a K+ conductance developed only in cells cultured in high extracellular Ca2+. Addition of charybdotoxin or verapamil blocked these K+ channels and inhibited Ca2+-induced differentiation, as assessed by cornified envelope formation or transglutaminase activity. These results suggest that K+ channel activation is necessary for Ca2+-induced differentiation. Finally, we used (125)I-labeled charybdotoxin to demonstrate the presence of K+ channels in intact human and mouse epidermis, hair follicles, and eccrine glands, indicating that these channels are found in keratinocytes both in vitro and in vivo. Thus K+ channels may moderate Ca2+ influx in more differentiated keratinocytes and may play a central role in keratinocyte differentiation.
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Journal of Investigative Dermatology, 2002
Ionic fluxes are important for critical aspects of keratinocyte differentiation, including synthe... more Ionic fluxes are important for critical aspects of keratinocyte differentiation, including synthesis of differentiation-specific proteins, enzymatic catalysis of protein cross-linking, post-transcriptional processing of profilaggrin, and lipid secretion. The epithelial sodium channel is expressed in epidermis and the expression of its alpha and beta subunits is enhanced as keratinocytes differentiate. In order to ascertain the role of the epithelial sodium channel in epidermal differentiation, we examined skin of mice in which the epithelial sodium channel alpha subunit had been deleted. Newborn -/- mice, in which the alpha subunit had been completely inactivated, demonstrated epithelial hyperplasia, abnormal nuclei, premature secretion of lipids, and abnormal keratohyaline granules. In addition, immunohistochemistry demonstrated that expression of the differentiation markers K1, K6, and involucrin were abnormal. These data suggest that the epithelial sodium channel modulates ionic signaling for specific aspects of epidermal differentiation, such as synthesis or processing of differentiation- specific proteins, and lipid secretion.
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Journal of Investigative Dermatology, 1998
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Journal of Investigative Dermatology, 2010
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Journal of Investigative Dermatology, 2003
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Journal of Investigative Dermatology, 2009
Neutralization of stratum corneum (SC) adversely impacts key epidermal functions, including perme... more Neutralization of stratum corneum (SC) adversely impacts key epidermal functions, including permeability barrier homeostasis and SC integrity. Conversely, acidification of SC improves these functions in developmentally impaired (neonatal or aged) skin, and enhances function in normal skin. Hence, we hypothesized that acidification could alter the course of inflammatory dermatoses, which invariably exhibit an increased SC pH. Maintenance of a low pH by topical applications of the polyhydroxyl acid, lactobionic acid, during the repeated-challenge phase inhibited the development of oxazolone-induced atopic dermatitis (AD). Neither gross/histological dermatitis nor altered barrier function developed, and emergence of epidermal hyperplasia was prevented; however, cytokine generation decreased. Acidification also largely normalized the development of hapten-induced changes in eosinophil/mast cell densities, density of chemoattractant receptor-homologous molecule expressed on TH2-positive lymphocytes, and serum IgE levels. The pH-induced improvement in barrier function most likely accounts for the anti-inflammatory activity, which could be further attributed to normalization of both lamellar body secretion and lamellar bilayer formation. Acidification of SC alone substantially prevents development of barrier abnormalities and downstream immune abnormalities during the elicitation phase of murine AD. These results provide direct evidence for the "outside-inside" pathogenesis of AD and further suggest that maintenance of an acidic SC pH could prevent the emergence of AD in humans.
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FEBS letters, Jan 9, 2015
The keratitis-ichthyosis-deafness (KID) syndrome is caused by mutations in the gap junctional cha... more The keratitis-ichthyosis-deafness (KID) syndrome is caused by mutations in the gap junctional channel protein connexin 26 (Cx26), among them the mutation Cx26S17F. Heterozygous Cx26S17F mice resemble the human KID syndrome, i.e. exhibiting epidermal hyperplasia and hearing impairments. Newborn Cx26S17F mice show a defective epidermal water barrier as well as altered epidermal lipid secretion and location. Linoleoyl ω-esterified ceramides are strongly decreased on the skin surface of Cx26S17F mice. Moreover, the epidermal calcium gradient is altered in the mutant mice. These alterations may be caused by an abnormal Cx26S17F channel function that leads to a defective epidermal water barrier, which in turn may trigger the hyperproliferation seen in the KID syndrome.
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Journal of dermatological science, Jan 19, 2015
The ceramide metabolite, sphingosine-1-phosphate (S1P), regulates multiple cellular functions in ... more The ceramide metabolite, sphingosine-1-phosphate (S1P), regulates multiple cellular functions in keratinocytes (KC). We recently discovered that production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), is stimulated via a NF-κB-dependent mechanism that is activated by S1P when S1P is generated by sphingosine kinase (SPHK) 1. We investigated whether pharmacological modulation of SPHK1 activity, using a novel synthetic SPHK1 activator, (S)-methyl 2-(hexanamide)-3-(4-hydroxyphenyl) propanoate (MHP), stimulates CAMP expression. MHP-mediated changes in both S1P and CAMP downstream mediators were analyzed in normal cultured human KC by qRT-PCR, Western immunoblot, ELISA, confocal microscopy for immunohistochemistry, HPLC and ESI-LC/MS/MS, and microbial pathogen invasion/colonization in a human epidermal organotypic model. Treatment with MHP directly activated SPHK1 and increased cellular S1P content in normal cultured human KC. Because MHP did not inhibit S1P ...
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Nano letters, Jan 27, 2015
Transdermal delivery of therapeutics is restricted by narrow limitations on size and hydrophobici... more Transdermal delivery of therapeutics is restricted by narrow limitations on size and hydrophobicity. Nanotopography has been shown to significantly enhance high molecular weight paracellular transport in vitro. Herein, we demonstrate for the first time that nanotopography applied to microneedles significantly enhances transdermal delivery of etanercept, a 150 kD therapeutic, in both rats and rabbits. We further show that this effect is mediated by remodeling of the tight junction proteins initiated via integrin binding to the nanotopography, followed by phosphorylation of myosin light chain (MLC) and activation of the actomyosin complex, which in turn increase paracellular permeability.
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Nature genetics, 2000
Hailey-Hailey disease (HHD, MIM 16960) is inherited in an autosomal dominant manner and character... more Hailey-Hailey disease (HHD, MIM 16960) is inherited in an autosomal dominant manner and characterized by persistent blisters and erosions of the skin. Impaired intercellular adhesion and epidermal blistering also occur in individuals with pemphigus (which is due to autoantibodies directed against desmosomal proteins) and in patients with Darier disease (DD, MIM 124200), which is caused by mutations in a gene encoding a sarco/endoplasmic reticulum (ER)-Golgi calcium pump. We report here the identification of mutations in ATP2C1, encoding the human homologue of an ATP-powered pump that sequesters calcium into the Golgi in yeast, in 21 HHD kindreds. Regulation of cytoplasmic calcium is impaired in cultured keratinocytes from HHD patients, and the normal epidermal calcium gradient is attenuated in vivo in HHD patients. Our findings not only provide an understanding of the molecular basis of HHD, but also underscore the importance of calcium control to the functioning of stratified squam...
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Molecular biology of the cell, 2004
Members of the serum- and glucocorticoid-regulated kinase (SGK) family are important mediators of... more Members of the serum- and glucocorticoid-regulated kinase (SGK) family are important mediators of growth factor and hormone signaling that, like their close relatives in the Akt family, are regulated by lipid products of phosphatidylinositol-3-kinase. SGK3 has been implicated in the control of cell survival and regulation of ion channel activity in cultured cells. To begin to dissect the in vivo functions of SGK3, we generated and characterized Sgk3 null mice. These mice are viable and fertile, and in contrast to mice lacking SGK1 or Akt2, respectively, display normal sodium handling and glucose tolerance. However, although normal at birth, by postpartum day 4 they have begun to display an unexpected defect in hair follicle morphogenesis. The abnormality in hair follicle development is preceded by a defect in proliferation and nuclear accumulation of beta-catenin in hair bulb keratinocytes. Furthermore, in cultured keratinocytes, heterologous expression of SGK3 potently modulates ac...
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The Journal of investigative dermatology, Jan 17, 2014
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The American Journal of Pathology, 2015
Although keratosis pilaris (KP) is common, its etiopathogenesis remains unknown. KP is associated... more Although keratosis pilaris (KP) is common, its etiopathogenesis remains unknown. KP is associated clinically with ichthyosis vulgaris and atopic dermatitis and molecular genetically with filaggrin-null mutations. In 20 KP patients and 20 matched controls, we assessed the filaggrin and claudin 1 genotypes, the phenotypes by dermatoscopy, and the morphology by light and transmission electron microscopy. Thirty-five percent of KP patients displayed filaggrin mutations, demonstrating that filaggrin mutations only partially account for the KP phenotype. Major histologic and dermatoscopic findings of KP were hyperkeratosis, hypergranulosis, mild T helper cell type 1-dominant lymphocytic inflammation, plugging of follicular orifices, striking absence of sebaceous glands, and hair shaft abnormalities in KP lesions but not in unaffected skin sites. Changes in barrier function and abnormal paracellular permeability were found in both interfollicular and follicular stratum corneum of lesional KP, which correlated ultrastructurally with impaired extracellular lamellar bilayer maturation and organization. All these features were independent of filaggrin genotype. Moreover, ultrastructure of corneodesmosomes and tight junctions appeared normal, immunohistochemistry for claudin 1 showed no reduction in protein amounts, and molecular analysis of claudin 1 was unremarkable. Our findings suggest that absence of sebaceous glands is an early step in KP pathogenesis, resulting in downstream hair shaft and epithelial barrier abnormalities.
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Journal of Investigative Dermatology Symposium Proceedings, 1998
The contents of epidermal lamellar bodies (LB) are delivered selectively to the intercellular spa... more The contents of epidermal lamellar bodies (LB) are delivered selectively to the intercellular spaces at the stratum granulosum (SG)-stratum corneum (SC) interface. We assessed the subcellular basis for LB secretion first by confocal microscopy, following labeling with Nile red or NBD-ceramide, which reveals a tubulo-reticular membrane system within the apical cytosol of the outermost SG cell layer under basal conditions, changing to a more peripheral staining pattern when secretion is stimulated. Ultrastructural study demonstrates that this network is composed of a widely disbursed trans-Golgi-like network (TGN), associated with arrays of contiguous LB, and deep invaginations of the SG-SC interface. Under basal conditions, limited fusion of apically directed LB leads to deep, interconnected invaginations of the apical plasma membrane, resulting in the formation of an extensive, honeycomb extension of the SG-SC interface. Still deeper invaginations and more extensive organelle fusion develop after the epidermis is acutely permeabilized by either acetone treatment, sonophoresis, or iontophoresis. Finally, nascent LB appear to bud off cisternae of the TGN, a process that appears to accelerate after barrier disruption. The deep invaginations of the SG-SC interface; the wide distribution of the TGN within the apical cytosol; the association of nascent LB with the TGN; and the rapid fusion of LB with these invaginations, deep within the cytosol, account for (i) the polarized secretion of LB from the apex of the outermost SG cell, and (ii) the rapid LB-secretory response to barrier perturbations. Finally, our results point to the outermost SG cell as a uniquely specialized secretory cell. We propose the term "secretory granulocyte" to encompass the specialized features of these cells.
Bookmarks Related papers MentionsView impact
Stem cell reports, Jan 6, 2014
Cornification and epidermal barrier defects are associated with a number of clinically diverse sk... more Cornification and epidermal barrier defects are associated with a number of clinically diverse skin disorders. However, a suitable in vitro model for studying normal barrier function and barrier defects is still lacking. Here, we demonstrate the generation of human epidermal equivalents (HEEs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). HEEs are structurally similar to native epidermis, with a functional permeability barrier. We exposed a pure population of hESC/iPSC-derived keratinocytes, whose transcriptome corresponds to the gene signature of normal primary human keratinocytes (NHKs), to a sequential high-to-low humidity environment in an air/liquid interface culture. The resulting HEEs had all of the cellular strata of the human epidermis, with skin barrier properties similar to those of normal skin. Such HEEs generated from disease-specific iPSCs will be an invaluable tool not only for dissecting molecular mechanisms that lead to epiderma...
Bookmarks Related papers MentionsView impact
Journal of Clinical Investigation, 2007
The skin is the first line of defense against microbial infection, and psychological stress (PS) ... more The skin is the first line of defense against microbial infection, and psychological stress (PS) has been shown to have adverse effects on cutaneous barrier function. Here we show that PS increased the severity of group A Streptococcus pyogenes (GAS) cutaneous skin infection in mice; this was accompanied by increased production of endogenous glucocorticoids (GCs), which inhibited epidermal lipid synthesis and decreased lamellar body (LB) secretion. LBs encapsulate antimicrobial peptides (AMPs), and PS or systemic or topical GC administration downregulated epidermal expression of murine AMPs cathelin-related AMP and beta-defensin 3. Pharmacological blockade of the stress hormone corticotrophin-releasing factor or of peripheral GC action, as well as topical administration of physiologic lipids, normalized epidermal AMP levels and delivery to LBs and decreased the severity of GAS infection during PS. Our results show that PS decreases the levels of 2 key AMPs in the epidermis and their delivery into LBs and that this is attributable to increased endogenous GC production. These data suggest that GC blockade and/or topical lipid administration could normalize cutaneous antimicrobial defense during PS or GC increase. We believe this to be the first mechanistic link between PS and increased susceptibility to infection by microbial pathogens.
Bookmarks Related papers MentionsView impact
Femtosecond Laser Applications in Biology, 2004
Measurements of ionic concentrations in skin have traditionally been performed with an array of m... more Measurements of ionic concentrations in skin have traditionally been performed with an array of methods which either did not reveal detailed localization information, or only provided qualitative, not quantitative information. FLIM combines a number of advantages into a method ideally suited to visualize concentrations of ions such as H+ in intact, unperturbed epidermis and stratum corneum (SC). Fluorescence lifetime is dye concentration-independent, the method requires only low light intensities and is therefore not prone to photobleaching or phototoxic artifacts, and because multiphoton lasers of IR wavelength are used, light penetrates deep into intact tissue. The standard method to measure SC pH is the flat pH electrode, which provides reliable information only about surface pH changes, without further vertical or subcellular spatial resolution; i.e., specific microdomains such as the corneocyte interstices are not resolved, and the deeper SC is inaccessible without resorting to inherently disruptive stripping methods. Furthermore, the concept of a gradient of pH through the SC stems from such stripping experiments, but other confirmation for this concept is lacking. Our investigations into the SC pH distribution so far have revealed the crucial role of the Sodium/Hydrogen Antiporter NHE1 in generation of SC acidity, the colocalization of enzymatic lipid processing activity in the SC with acidic domains of the SC, and the timing and localization of emerging acidity in the SC of newborns. Together, these results have led to an improved understanding of the SC pH, its distribution, origin, and regulation. Future uses for this method include measurements of other ions important for epidermal processes, such as Ca2+, and a quantitative approach to topical drug penetration.
Bookmarks Related papers MentionsView impact
The FASEB Journal, 2009
SGK3, which previously has been shown to play a key role in hair follicle development in mice, is... more SGK3, which previously has been shown to play a key role in hair follicle development in mice, is a member of the AGC family of serine-threonine kinases. Mice lacking SGK3 have abnormal follicle cycling, which begins shortly after birth and ameliorates substantially with age. However, this developmental abnormality is not recapitulated in mice lacking closely related kinases Akt1, Akt2, or Akt3. To examine whether Akt2 interacts with SGK3 in postnatal hair development, we have generated and characterized Akt2/SGK3 double knockouts (DKOs). We find that the DKO mice have a defect in hair growth that is markedly worse than that of SGK3(-/-) mice and does not ameliorate with age. Morphologically, this defect is characterized by accelerated entry into catagen and through anagen, irregular hair follicle orientation, and increased expression of sebaceous glands. The defect is preceded by a profound failure to increase follicle matrix cell nuclear beta-catenin accumulation and proliferation at the onset of morphogenesis. Furthermore, in cultured keratinocytes, transfected Akt2 and SGK3 both stimulate transcription of a beta-catenin-LEF1-dependent reporter gene. Thus, SGK3 and Akt2 both appear to play important roles in postnatal hair follicle morphogenesis, likely because of their redundant regulation of beta-catenin-dependent transcriptional processes, which control hair follicle cell proliferation.
Bookmarks Related papers MentionsView impact
Oncogene, 2006
Focal adhesion kinase (FAK) is a critical component in transducing signals downstream of both int... more Focal adhesion kinase (FAK) is a critical component in transducing signals downstream of both integrins and growth factor receptors. To determine how the loss of FAK affects the epidermis in vivo, we have generated a mouse model with a keratinocyte-restricted deletion of fak (FAKK5 KO mice). FAK(K5 KO) mice displayed three major phenotypes--irregularities of hair cycle, sebaceous glands hypoplasia, and a thinner epidermis--pointing to defects in the proliferative capacity of multipotent stem cells found in the bulge. FAK-null keratinocytes in conventional primary culture undergo massive apoptosis hindering further analyses, whereas the defects observed in vivo do not shorten the mouse lifespan. These results suggest that the structure and the signaling environment of the native tissue may overcome the lack of signaling through FAK. Our findings point to the importance of in vivo and three-dimensional in vitro models in analyses of cell migration, proliferation, and survival. Surprisingly, the difference between FAKloxP/+ and FAKK5 KO mice in wound closure was not statistically significant, suggesting that in vivo loss of FAK does not affect migration/proliferation of basal keratinocytes in the same way as it affects multipotent stem cells of the skin.
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Journal of Investigative Dermatology, 2014
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Journal of Investigative Dermatology, 1997
K+ channel activation has been associated with growth or differentiation in many cells. We have p... more K+ channel activation has been associated with growth or differentiation in many cells. We have previously identified a 70-pS K+ channel that was found only in differentiated involucrin-positive cells. In this study we examined the role of K+ channels in Ca2+-induced keratinocyte differentiation. Consistent with our previous report, we found that a K+ conductance developed only in cells cultured in high extracellular Ca2+. Addition of charybdotoxin or verapamil blocked these K+ channels and inhibited Ca2+-induced differentiation, as assessed by cornified envelope formation or transglutaminase activity. These results suggest that K+ channel activation is necessary for Ca2+-induced differentiation. Finally, we used (125)I-labeled charybdotoxin to demonstrate the presence of K+ channels in intact human and mouse epidermis, hair follicles, and eccrine glands, indicating that these channels are found in keratinocytes both in vitro and in vivo. Thus K+ channels may moderate Ca2+ influx in more differentiated keratinocytes and may play a central role in keratinocyte differentiation.
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Journal of Investigative Dermatology, 2002
Ionic fluxes are important for critical aspects of keratinocyte differentiation, including synthe... more Ionic fluxes are important for critical aspects of keratinocyte differentiation, including synthesis of differentiation-specific proteins, enzymatic catalysis of protein cross-linking, post-transcriptional processing of profilaggrin, and lipid secretion. The epithelial sodium channel is expressed in epidermis and the expression of its alpha and beta subunits is enhanced as keratinocytes differentiate. In order to ascertain the role of the epithelial sodium channel in epidermal differentiation, we examined skin of mice in which the epithelial sodium channel alpha subunit had been deleted. Newborn -/- mice, in which the alpha subunit had been completely inactivated, demonstrated epithelial hyperplasia, abnormal nuclei, premature secretion of lipids, and abnormal keratohyaline granules. In addition, immunohistochemistry demonstrated that expression of the differentiation markers K1, K6, and involucrin were abnormal. These data suggest that the epithelial sodium channel modulates ionic signaling for specific aspects of epidermal differentiation, such as synthesis or processing of differentiation- specific proteins, and lipid secretion.
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Journal of Investigative Dermatology, 1998
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Journal of Investigative Dermatology, 2010
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Journal of Investigative Dermatology, 2003
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Journal of Investigative Dermatology, 2009
Neutralization of stratum corneum (SC) adversely impacts key epidermal functions, including perme... more Neutralization of stratum corneum (SC) adversely impacts key epidermal functions, including permeability barrier homeostasis and SC integrity. Conversely, acidification of SC improves these functions in developmentally impaired (neonatal or aged) skin, and enhances function in normal skin. Hence, we hypothesized that acidification could alter the course of inflammatory dermatoses, which invariably exhibit an increased SC pH. Maintenance of a low pH by topical applications of the polyhydroxyl acid, lactobionic acid, during the repeated-challenge phase inhibited the development of oxazolone-induced atopic dermatitis (AD). Neither gross/histological dermatitis nor altered barrier function developed, and emergence of epidermal hyperplasia was prevented; however, cytokine generation decreased. Acidification also largely normalized the development of hapten-induced changes in eosinophil/mast cell densities, density of chemoattractant receptor-homologous molecule expressed on TH2-positive lymphocytes, and serum IgE levels. The pH-induced improvement in barrier function most likely accounts for the anti-inflammatory activity, which could be further attributed to normalization of both lamellar body secretion and lamellar bilayer formation. Acidification of SC alone substantially prevents development of barrier abnormalities and downstream immune abnormalities during the elicitation phase of murine AD. These results provide direct evidence for the "outside-inside" pathogenesis of AD and further suggest that maintenance of an acidic SC pH could prevent the emergence of AD in humans.
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