Polymeric membranes modulate human keratinocyte differentiation in specific epidermal layers.

PubMed

Salerno, Simona; Morelli, Sabrina; Giordano, Francesca; Gordano, Amalia; Bartolo, Loredana De

2016-10-01

In vitro models of human bioengineered skin substitutes are an alternative to animal experimentation for testing the effects and toxicity of drugs, cosmetics and pollutants. For the first time specific and distinct human epidermal strata were engineered by using membranes and keratinocytes. To this purpose, biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT-PCL were prepared by phase-inversion technique and characterized in order to evaluate their morphological, physico-chemical and mechanical properties. The capability of membranes to modulate keratinocyte differentiation inducing specific interactions in epidermal membrane systems was investigated. The overall results demonstrated that the membrane properties strongly influence the cell morpho-functional behaviour of human keratinocytes, modulating their terminal differentiation, with the creation of specific epidermal strata or a fully proliferative epidermal multilayer system. In particular, human keratinocytes adhered on CHT and CHT-PCL membranes, forming the structure of the epidermal top layers, such as the corneum and granulosum strata, characterized by withdrawal or reduction from the cell cycle and cell proliferation. On the PCL membrane, keratinocytes developed an epidermal basal lamina, with high proliferating cells that stratified and migrated over time to form a complete differentiating epidermal multilayer system. Copyright © 2016 Elsevier B.V. All rights reserved.

Esterase Activity and Intracellular Localization in Reconstructed Human Epidermal Cultured Skin Models.

PubMed

Tokudome, Yoshihiro; Katayanagi, Mishina; Hashimoto, Fumie

2015-06-01

Reconstructed human epidermal culture skin models have been developed for cosmetic and pharmaceutical research. This study evaluated the total and carboxyl esterase activities (i.e., Km and Vmax , respectively) and localization in two reconstructed human epidermal culture skin models (LabCyte EPI-MODEL [Japan Tissue Engineering] and EpiDerm [MatTek/Kurabo]). The usefulness of the reconstruction cultured epidermis was also verified by comparison with human and rat epidermis. Homogenized epidermal samples were fractioned by centrifugation. p-nitrophenyl acetate and 4-methylumbelliferyl acetate were used as substrates of total esterase and carboxyl esterase, respectively. Total and carboxyl esterase activities were present in the reconstructed human epidermal culture skin models and were localized in the cytosol. Moreover, the activities and localization were the same as those in human and rat epidermis. LabCyte EPI-MODEL and EpiDerm are potentially useful for esterase activity prediction in human epidermis.

Esterase Activity and Intracellular Localization in Reconstructed Human Epidermal Cultured Skin Models

PubMed Central

Katayanagi, Mishina; Hashimoto, Fumie

2015-01-01

Background Reconstructed human epidermal culture skin models have been developed for cosmetic and pharmaceutical research. Objective This study evaluated the total and carboxyl esterase activities (i.e., Km and Vmax, respectively) and localization in two reconstructed human epidermal culture skin models (LabCyte EPI-MODEL [Japan Tissue Engineering] and EpiDerm [MatTek/Kurabo]). The usefulness of the reconstruction cultured epidermis was also verified by comparison with human and rat epidermis. Methods Homogenized epidermal samples were fractioned by centrifugation. p-nitrophenyl acetate and 4-methylumbelliferyl acetate were used as substrates of total esterase and carboxyl esterase, respectively. Results Total and carboxyl esterase activities were present in the reconstructed human epidermal culture skin models and were localized in the cytosol. Moreover, the activities and localization were the same as those in human and rat epidermis. Conclusion LabCyte EPI-MODEL and EpiDerm are potentially useful for esterase activity prediction in human epidermis. PMID:26082583

Angiopoietin-Like 4 Regulates Epidermal Differentiation

PubMed Central

Huang, Royston-Luke; Goh, Yan Yih; Wang, Xiao Ling; Tang, Mark Boon Yang; Tan, Nguan Soon

2011-01-01

The nuclear hormone receptor PPARβ/δ is integral to efficient wound re-epithelialization and implicated in epidermal maturation. However, the mechanism underlying the latter process of epidermal differentiation remains unclear. We showed that ligand-activated PPARβ/δ indirectly stimulated keratinocyte differentiation, requiring de novo gene transcription and protein translation. Using organotypic skin cultures constructed from PPARβ/δ- and angiopoietin-like 4 (ANGPTL4)-knockdown human keratinocytes, we showed that the expression of ANGPTL4, a PPARβ/δ target gene, is essential for the receptor mediated epidermal differentiation. The pro-differentiation effect of PPARβ/δ agonist GW501516 was also abolished when keratinocytes were co-treated with PPARβ/δ antagonist GSK0660 and similarly in organotypic skin culture incubated with blocking ANGPTL4 monoclonal antibody targeted against the C-terminal fibrinogen-like domain. Our focused real-time PCR gene expression analysis comparing the skin biopsies from wildtype and ANGPTL4-knockout mice confirmed a consistent down-regulation of numerous genes involved in epidermal differentiation and proliferation in the ANGPTL4-knockout skin. We further showed that the deficiency of ANGPTL4 in human keratinocytes and mice skin have diminished expression of various protein kinase C isotypes and phosphorylated transcriptional factor activator protein-1, which are well-established for their roles in keratinocyte differentiation. Chromatin immunoprecipitation confirmed that ANGPTL4 stimulated the activation and binding of JUNB and c-JUN to the promoter region of human involucrin and transglutaminase type 1 genes, respectively. Taken together, we showed that PPARβ/δ regulates epidermal maturation via ANGPTL4-mediated signalling pathway. PMID:21966511

The temporal and spatial expression of Claudins in epidermal development and the accelerated program of epidermal differentiation in K14-CaSR transgenic mice.

PubMed

Troy, Tammy-Claire; Li, Yuhua; O'Malley, Lauren; Turksen, Kursad

2007-02-01

The importance of the epidermal permeability barrier (EPB) in protecting the mammalian species against harmful UV irradiation, microorganism invasion and water loss is well recognized, as is the role of calcium (Ca(2+)) in keratinocyte differentiation, cell-cell contact and the EPB. In a previous study, we reported that the overexpression of the Ca(2+)-sensing receptor (CaSR) in the undifferentiated basal cells of the epidermis induced a modified epidermal differentiation program including an accelerated EPB formation in transgenic mice, suggesting a role for CaSR signaling in the differentiation of embryonic epidermal cells during development. We now describe the expression profile of claudins (Cldns) and keratin markers in the accelerated EPB formation of K14-CaSR transgenic mice during development as compared to the wild type from E12.5 to newborn stages. Our data show that the transgenic epidermis undergoes an advanced epidermal differentiation program as compared to the wild type as evidenced morphologically as well as by the expression of K14, K1, loricrin, Cldn6, Cldn18 and Cldn11. In addition, we report for the first time the sequential expression of Cldns in epidermal development and describe that the localization of some Cldns change within the epidermis as it matures. Furthermore, we demonstrate that Cldn6 is expressed very early in epidermal morphogenesis, followed by Cldn18, Cldn11 and Cldn1.

Epidermal cornification is preceded by the expression of a keratinocyte-specific set of pyroptosis-related genes.

PubMed

Lachner, Julia; Mlitz, Veronika; Tschachler, Erwin; Eckhart, Leopold

2017-12-12

The homeostasis of the epidermis depends on keratinocyte differentiation and cornification, a mode of programmed cell death that does not elicit inflammation. Here, we report that cornification is associated with the expression of specific genes that control multiple steps of pyroptosis, another form of cell death that involves the processing and release of interleukin-1 family (IL1F) cytokines. Expression levels of pro-inflammatory IL1A and IL1B and of the pyroptotic pore-forming gasdermin (GSDM) D were downregulated during terminal differentiation of human keratinocytes in vitro. By contrast, negative regulators of IL-1 processing, including NLR family pyrin domain containing 10 (NLRP10) and pyrin domain-containing 1 (PYDC1), the anti-inflammatory IL1F members IL-37 (IL1F7) and IL-38 (IL1F10), and GSDMA, were strongly induced in differentiated keratinocytes. In human tissues, these keratinocyte differentiation-associated genes are expressed in the skin at higher levels than in any other organ, and mammalian species, that have lost the epidermal cornification program during evolution, i.e. whales and dolphins, lack homologs of these genes. Together, our results suggest that human epidermal cornification is accompanied by a tight control of pyroptosis and warrant further studies of potential defects in the balance between cornification and pyroptosis in skin pathologies.

Epidermal Expression and Regulation of Interleukin-33 during Homeostasis and Inflammation: Strong Species Differences.

PubMed

Sundnes, Olav; Pietka, Wojciech; Loos, Tamara; Sponheim, Jon; Rankin, Andrew L; Pflanz, Stefan; Bertelsen, Vibeke; Sitek, Jan C; Hol, Johanna; Haraldsen, Guttorm; Khnykin, Denis

2015-07-01

IL-33 is a novel IL-1 family member with a putative role in inflammatory skin disorders and a complex biology. Therefore, recent conflicting data regarding its function in experimental models justify a close assessment of its tissue expression and regulation. Indeed, we report here that there are strong species differences in the expression and regulation of epidermal IL-33. In murine epidermis, IL-33 behaved similar to an alarmin, being constitutively expressed in keratinocyte nuclei and rapidly lost during acute inflammation. By contrast, human and porcine IL-33 were weakly expressed or absent in keratinocytes of noninflamed skin but induced during acute inflammation. To this end, we observed that expression of IL-33 in human keratinocytes but not murine keratinocytes was strongly induced by IFN-γ, and this upregulation completely depended on the presence of EGFR ligands. Accordingly, IFN-γ increased the expression of IL-33 in the basal layers of the epidermis in human ex vivo skin cultures only, despite good evidence of IFN-γ activity in cultures from both species. Together these findings demonstrate that a full understanding of IL-33 function in clinical settings must take species-specific differences into account.

Psoriatic T cells reduce epidermal turnover time and affect cell proliferation contributed from differential gene expression.

PubMed

Li, Junqin; Li, Xinhua; Hou, Ruixia; Liu, Ruifeng; Zhao, Xincheng; Dong, Feng; Wang, Chunfang; Yin, Guohua; Zhang, Kaiming

2015-09-01

Psoriasis is mediated primarily by T cells, which reduce epidermal turnover time and affect keratinocyte proliferation. We aimed to identify differentially expressed genes (DEG) in T cells from normal, five pairs of monozygotic twins concordant or discordant for psoriasis, to determine whether these DEG may account for the influence to epidermal turnover time and keratinocyte proliferation. The impact of T cells on keratinocyte proliferation and epidermal turnover time were investigated separately by immunohistochemistry and cultured with (3) H-TdR. mRNA expression patterns were investigated by RNA sequencing and verified by real-time reverse transcription polymerase chain reaction. After co-culture with psoriatic T cells, the expression of Ki-67, c-Myc and p53 increased, while expression of Bcl-2 and epidermal turnover time decreased. There were 14 DEG which were found to participate in the regulation of cell proliferation or differentiation. Psoriatic T cells exhibited the ability to decrease epidermal turnover time and affect keratinocyte proliferation because of the differential expression of PPIL1, HSPH1, SENP3, NUP54, FABP5, PLEKHG3, SLC9A9 and CHCHD4. © 2015 Japanese Dermatological Association.

Human Epidermal Growth Factor Receptor 2 Expression in Unresectable Gastric Cancers: Relationship with CT Characteristics.

PubMed

Lee, Jeong Sub; Kim, Se Hyung; Im, Seock-Ah; Kim, Min A; Han, Joon Koo

2017-01-01

To retrospectively analyze the qualitative CT features that correlate with human epidermal growth factor receptor 2 (HER2)-expression in pathologically-proven gastric cancers. A total of 181 patients with pathologically-proven unresectable gastric cancers with HER2-expression (HER2-positive [n = 32] and negative [n = 149]) were included. CT features of primary gastric and metastatic tumors were reviewed. The prevalence of each CT finding was compared in both groups. Thereafter, binary logistic regression determined the most significant differential CT features. Clinical outcomes were compared using Kaplan-Meier method. HER2-postive cancers showed lower clinical T stage (21.9% vs. 8.1%; p = 0.015), hyperattenuation on portal phase (62.5% vs. 30.9%; p = 0.003), and was more frequently metastasized to the liver (62.5% vs. 32.2%; p = 0.001), than HER2-negative cancers. On binary regression analysis, hyperattenuation of the tumor (odds ratio [OR], 4.68; p < 0.001) and hepatic metastasis (OR, 4.43; p = 0.001) were significant independent factors that predict HER2-positive cancers. Median survival of HER2-positive cancers (13.7 months) was significantly longer than HER2-negative cancers (9.6 months) ( p = 0.035). HER2-positive gastric cancers show less-advanced T stage, hyperattenuation on the portal phase, and frequently metastasize to the liver, as compared to HER2-negative cancers.

Epidermal stem cells: location, potential and contribution to cancer.

PubMed

Ambler, C A; Määttä, A

2009-01-01

Epidermal stem cells have been classically characterized as slow-cycling, long-lived cells that reside in discrete niches in the skin. Gene expression studies of niche-resident cells have revealed a number of stem cell markers and regulators, including the Wnt/beta-catenin, Notch, p63, c-Myc and Hedgehog pathways. A new study challenges the traditional developmental paradigm of slow-cycling stem cells and rapid-cycling transit amplifying cells in some epidermal regions, and there is mounting evidence to suggest that multi-lineage epidermal progenitors can be isolated from highly proliferative, non-niche regions. Whether there is a unique microenvironment surrounding these progenitors remains to be determined. Interestingly, cancer stem cells derived from epidermal tumours exist independent of the classic skin stem cell niche, yet also have stem cell properties, including multi-lineage differentiation. This review summarizes recent studies identifying the location and regulators of mouse and human epidermal stem cells and highlights the strategies used to identify cancer stem cells, including expression of normal epidermal stem cell markers, expression of cancer stem cell markers identified in other epidermal tumours and characterization of side-population tumour cells.

UV radiation induces CXCL5 expression in human skin.

PubMed

Reichert, Olga; Kolbe, Ludger; Terstegen, Lara; Staeb, Franz; Wenck, Horst; Schmelz, Martin; Genth, Harald; Kaever, Volkhard; Roggenkamp, Dennis; Neufang, Gitta

2015-04-01

CXCL5 has recently been identified as a mediator of UVB-induced pain in rodents. To compare and to extend previous knowledge of cutaneous CXCL5 regulation, we performed a comprehensive study on the effects of UV radiation on CXCL5 regulation in human skin. Our results show a dose-dependent increase in CXCL5 protein in human skin after UV radiation. CXCL5 can be released by different cell types in the skin. We presumed that, in addition to immune cells, non-immune skin cells also contribute to UV-induced increase in CXCL5 protein. Analysis of monocultured dermal fibroblasts and keratinocytes revealed that only fibroblasts but not keratinocytes displayed up regulated CXCL5 levels after UV stimulation. Whereas UV treatment of human skin equivalents, induced epidermal CXCL5 mRNA and protein expression. Up regulation of epidermal CXCL5 was independent of keratinocyte differentiation and keratinocyte-keratinocyte interactions in epidermal layers. Our findings provide first evidence on the release of CXCL5 in UV-radiated human skin and the essential role of fibroblast-keratinocyte interaction in the regulation of epidermal CXCL5. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Oral mucosa: an alternative epidermic cell source to develop autologous dermal-epidermal substitutes from diabetic subjects.

PubMed

Guzmán-Uribe, Daniela; Alvarado-Estrada, Keila Neri; Pierdant-Pérez, Mauricio; Torres-Álvarez, Bertha; Sánchez-Aguilar, Jesus Martin; Rosales-Ibáñez, Raúl

2017-01-01

The aim of this study was to obtain autologous dermal-epidermal skin substitutes from oral mucosa from diabetic subjects as a first step towards a possible clinical application for cases of diabetic foot. Oral mucosa was obtained from diabetic and healthy subjects (n=20 per group). Epidermal cells were isolated and cultured using autologous fibrin to develop dermal-epidermal in vitro substitutes by the air-liquid technique with autologous human serum as a supplement media. Substitutes were immunocharacterized with collagen IV and cytokeratin 5-14 as specific markers. A Student´s t- test was performed to assess the differences between both groups. It was possible to isolate epidermal cells from the oral mucosa of diabetic and healthy subjects and develop autologous dermal-epidermal skin substitutes using autologous serum as a supplement. Differences in the expression of specific markers were observed and the cytokeratin 5-14 expression was lower in the diabetic substitutes, and the collagen IV expression was higher in the diabetic substitutes when compared with the healthy group, showing a significant difference. Cells from oral mucosa could be an alternative and less invasive source for skin substitutes and wound healing. A difference in collagen production of diabetic cells suggests diabetic substitutes could improve diabetic wound healing. More research is needed to determine the crosstalk between components of these skin substitutes and damaged tissues.

The organization of human epidermis: functional epidermal units and phi proportionality.

PubMed

Hoath, Steven B; Leahy, D G

2003-12-01

The concept that mammalian epidermis is structurally organized into functional epidermal units has been proposed on the basis of stratum corneum (SC) architecture, proliferation kinetics, melanocyte:keratinocyte ratios (1:36), and, more recently, Langerhans cell: epidermal cell ratios (1:53). This article examines the concept of functional epidermal units in human skin in which the maintenance of phi (1.618034) proportionality provides a central organizing principle. The following empirical measurements were used: 75,346 nucleated epidermal cells per mm2, 1394 Langerhans cells per mm2, 1999 melanocytes per mm2, 16 (SC) layers, 900-microm2 corneocyte surface area, 17,778 corneocytes per mm2, 14-d (SC) turnover time, and 93,124 per mm2 total epidermal cells. Given these empirical data: (1) the number of corneocytes is a mean proportional between the sum of the Langerhans cell + melanocyte populations and the number of epidermal cells, 3393/17,778-17,778/93,124; (2) the ratio of nucleated epidermal cells over corneocytes is phi proportional, 75,346/17,778 approximately phi3; (3) assuming similar 14-d turnover times for the (SC) and Malpighian epidermis, the number of corneocytes results from subtraction of a cellular fraction equal to approximately 2/phi2 x the number of living cells, 75,436 - (2/phi2 x 75,346) approximately 17,778; and (4) if total epidermal turnover time equals (SC) turnover time x the ratio of living/dead cells, then compartmental turnover times are unequal (14 d for (SC) to 45.3 d for nucleated epidermis approximately 1/2phi) and cellular replacement rates are 52.9 corneocytes/69.3 keratinocytes per mm2 per h approximately 2/phi2. These empirically derived equivalences provide logicomathematical support for the presence of functional epidermal units in human skin. Validation of a phi proportional unit architecture in human epidermis will be important for tissue engineering of skin and the design of instruments for skin measurement.

Hyaluronan Does Not Regulate Human Epidermal Keratinocyte Proliferation and Differentiation*

PubMed Central

Malaisse, Jérémy; Pendaries, Valérie; Hontoir, Fanny; De Glas, Valérie; Van Vlaender, Daniel; Simon, Michel; Lambert de Rouvroit, Catherine; Poumay, Yves; Flamion, Bruno

2016-01-01

Hyaluronan (HA) is synthesized by three HA synthases (HAS1, HAS2, and HAS3) and secreted in the extracellular matrix. In human skin, large amounts of HA are found in the dermis. HA is also synthesized by keratinocytes in the epidermis, although its epidermal functions are not clearly identified yet. To investigate HA functions, we studied the effects of HA depletion on human keratinocyte physiology within in vitro reconstructed human epidermis. Inhibition of HA synthesis with 4-methylumbelliferone (4MU) did not modify the expression profile of the epidermal differentiation markers involucrin, keratin 10, and filaggrin during tissue reconstruction. In contrast, when keratinocytes were incubated with 4MU, cell proliferation was decreased. In an attempt to rescue the proliferation function, HA samples of various mean molecular masses were added to keratinocyte cultures treated with 4MU. These samples were unable to rescue the initial proliferation rate. Furthermore, treatments with HA-specific hyaluronidase, although removing almost all HA from keratinocyte cultures, did not alter the differentiation or proliferation processes. The differences between 4MU and hyaluronidase effects did not result from differences in intracellular HA, sulfated glycosaminoglycan concentration, apoptosis, or levels of HA receptors, all of which remained unchanged. Similarly, knockdown of UDP-glucose 6-dehydrogenase (UGDH) using lentiviral shRNA effectively decreased HA production but did not affect proliferation rate. Overall, these data suggest that HA levels in the human epidermis are not directly correlated with keratinocyte proliferation and differentiation and that incubation of cells with 4MU cannot equate with HA removal. PMID:26627828

Imaging sulfur mustard lesions in human epidermal tissues and keratinocytes by confocal and multiphoton microscopy

NASA Astrophysics Data System (ADS)

Werrlein, Robert; Madren-Whalley, Janna S.

2002-06-01

Topical exposure to sulfur mustard (HD), a known theat agent, produces persistent and debilitating cutaneous blisters. The blisters occur at the dermal-epidermal junction following a dose-dependent latent period of 8-24 h, however, the primary lesions causing vesication remain uncertain. Immunofluorescent images reveal that a 5-min exposure to 400 (mu) M HD disrupts molecules that are also disrupted by epidermolysis bullosa-type blistering diseases of the skin. Using keratinocyte cultures and fluorochomes conjugated to two different keratin-14 (K14) antibodies (clones CKB1 and LL002), results have shown a statistically significant (p<0.1) 1-h decrease of 29.2% in expression of the CKB1 epitope, a nearly complete loss of CKB1 expression within 2 h, and progressive cytoskeletal (K14) collapse without loss in expression of the LL002 epitope. With human epidermal tissues, multi-photon images of (alpha) 6 integrin and laminin 5 showed disruptive changes in the cell-surface organization and integrity of these adhesion molecules. At 1 H postexposure, analyses showed a statistically significant (p<0.1) decrease of 27.3% in (alpha) 6 integrin emissions, and a 32% decrease in laminin 5 volume. Multi-photon imaging indicates that molecules essential for epidermal-dermal attachment are early targets in the alkylating events leading to HD-induced vesication.

Oral mucosa: an alternative epidermic cell source to develop autologous dermal-epidermal substitutes from diabetic subjects

PubMed Central

GUZMÁN-URIBE, Daniela; ALVARADO-ESTRADA, Keila Neri; PIERDANT-PÉREZ, Mauricio; TORRES-ÁLVAREZ, Bertha; SÁNCHEZ-AGUILAR, Jesus Martin; ROSALES-IBÁÑEZ, Raúl

2017-01-01

Abstract Oral mucosa has been highlighted as a suitable source of epidermal cells due to its intrinsic characteristics such as its higher proliferation rate and its obtainability. Diabetic ulcers have a worldwide prevalence that is variable (1%-11%), meanwhile treatment of this has been proven ineffective. Tissue-engineered skin plays an important role in wound care focusing on strategies such autologous dermal-epidermal substitutes. Objective The aim of this study was to obtain autologous dermal-epidermal skin substitutes from oral mucosa from diabetic subjects as a first step towards a possible clinical application for cases of diabetic foot. Material and Methods Oral mucosa was obtained from diabetic and healthy subjects (n=20 per group). Epidermal cells were isolated and cultured using autologous fibrin to develop dermal-epidermal in vitro substitutes by the air-liquid technique with autologous human serum as a supplement media. Substitutes were immunocharacterized with collagen IV and cytokeratin 5-14 as specific markers. A Student´s t- test was performed to assess the differences between both groups. Results It was possible to isolate epidermal cells from the oral mucosa of diabetic and healthy subjects and develop autologous dermal-epidermal skin substitutes using autologous serum as a supplement. Differences in the expression of specific markers were observed and the cytokeratin 5-14 expression was lower in the diabetic substitutes, and the collagen IV expression was higher in the diabetic substitutes when compared with the healthy group, showing a significant difference. Conclusion Cells from oral mucosa could be an alternative and less invasive source for skin substitutes and wound healing. A difference in collagen production of diabetic cells suggests diabetic substitutes could improve diabetic wound healing. More research is needed to determine the crosstalk between components of these skin substitutes and damaged tissues. PMID:28403359

Zinc oxide nanoparticles-induced epigenetic change and G2/M arrest are associated with apoptosis in human epidermal keratinocytes

PubMed Central

Gao, Fei; Ma, Ningjie; Zhou, Hong; Wang, Qing; Zhang, Hao; Wang, Pu; Hou, Haoli; Wen, Huan; Li, Lijia

2016-01-01

As an engineered nanomaterial, zinc oxide nanoparticles (ZnO NPs) are used frequently in biological applications and can make contact with human skin. Here, we systematically investigated the effects of ZnO NPs on non-tumorigenic human epidermal keratinocytes, which were used as a test model for this in vitro study, at the epigenetic and molecular levels. Our results showed that ZnO NPs induced cell cycle arrest at the G2/M checkpoint before the viability of human epidermal keratinocytes was reduced, which was associated with the chromatin changes at the epigenetic level, including increased methylation of histone H3K9 and decreased acetylation of histone H4K5 accompanied by chromatin condensation at 24 hours. The mRNA expression of the methyltransferase genes G9a and GLP was also increased upon treatment with ZnO NPs, and the acetyltransferase genes GCN5, P300, and CBP were downregulated. Reactive oxygen species were found to be more abundant after treatment with ZnO NPs for 6 hours, and DNA damage was observed at 24 hours. Transmission electron microscopy and flow cytometry confirmed that ZnO NPs were absorbed into the cell when they were added to the medium. Apoptotic human epidermal keratinocytes were detected, and the expression of the proapoptotic genes Bax, Noxa, and Puma increased significantly, while the expression of the antiapoptotic gene Bcl-xl decreased 24 hours after exposure to ZnO NPs. These findings suggest that the ZnO NPs induced cell cycle arrest at G2/M, which was associated with epigenetic changes and accompanied by p53-Bax mitochondrial pathway-mediated apoptosis. PMID:27570453

Zinc oxide nanoparticles-induced epigenetic change and G2/M arrest are associated with apoptosis in human epidermal keratinocytes.

PubMed

Gao, Fei; Ma, Ningjie; Zhou, Hong; Wang, Qing; Zhang, Hao; Wang, Pu; Hou, Haoli; Wen, Huan; Li, Lijia

2016-01-01

As an engineered nanomaterial, zinc oxide nanoparticles (ZnO NPs) are used frequently in biological applications and can make contact with human skin. Here, we systematically investigated the effects of ZnO NPs on non-tumorigenic human epidermal keratinocytes, which were used as a test model for this in vitro study, at the epigenetic and molecular levels. Our results showed that ZnO NPs induced cell cycle arrest at the G2/M checkpoint before the viability of human epidermal keratinocytes was reduced, which was associated with the chromatin changes at the epigenetic level, including increased methylation of histone H3K9 and decreased acetylation of histone H4K5 accompanied by chromatin condensation at 24 hours. The mRNA expression of the methyltransferase genes G9a and GLP was also increased upon treatment with ZnO NPs, and the acetyltransferase genes GCN5, P300, and CBP were downregulated. Reactive oxygen species were found to be more abundant after treatment with ZnO NPs for 6 hours, and DNA damage was observed at 24 hours. Transmission electron microscopy and flow cytometry confirmed that ZnO NPs were absorbed into the cell when they were added to the medium. Apoptotic human epidermal keratinocytes were detected, and the expression of the proapoptotic genes Bax, Noxa, and Puma increased significantly, while the expression of the antiapoptotic gene Bcl-xl decreased 24 hours after exposure to ZnO NPs. These findings suggest that the ZnO NPs induced cell cycle arrest at G2/M, which was associated with epigenetic changes and accompanied by p53-Bax mitochondrial pathway-mediated apoptosis.

Mitochondrial gene expression changes in cultured human skin cells following simulated sunlight irradiation.

PubMed

Kelly, J; Murphy, J E

2018-02-01

Exposure of skin to simulated sunlight irradiation (SSI) has being extensively researched and shown to be the main cause for changes in the skin including changes in cellular function and generation of reactive oxygen species (ROS). This oxidative stress can subsequently exert downstream effects and the subcellular compartments most affected by this oxidative stress are mitochondria. The importance of functional mitochondrial morphology is apparent as morphological defects are related to many human diseases including diabetes mellitus, liver disease, neurodegenerative diseases, aging and cancer. The main objective of this study was to evaluate solar radiation-induced changes in mitochondrial gene expression in human skin cells using a Q-Sun solar simulator to deliver a close match to the intensity of summer sunlight. Spontaneously immortalised human skin epidermal keratinocytes (HaCaT) and Human Dermal Fibroblasts (HDFn) were divided into two groups. Group A were irradiated once and Group B twice 7days apart; following irradiation, mitochondrial gene expression was evaluated 1, 4 and 7days post primary exposure for group A and 1, 4, 7 and 14days post-secondary exposure for group B. Both the epidermal and dermal cells displayed significant reduced expression of the genes analysed for mitochondrial morphology and function; however, epidermal cells displayed this reduction post SSI earlier then dermal cells at multiple time points. The data presented here reinforces the fact that epidermal cells, while displaying a heightened sensitivity to sunlight, are less prone to changes in gene expression, while dermal cells, which appear to be more resilient are possibly more prone to genomic instability and mitochondrial damage. Copyright © 2017 Elsevier B.V. All rights reserved.

High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli.

PubMed

Ma, Yi; Yu, Jieying; Lin, Jinglian; Wu, Shaomin; Li, Shan; Wang, Jufang

2016-01-01

Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H 10 . The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

Temporal variations in sirtuin expression under normal and ultraviolet B-induced conditions and their correlation to energy levels in normal human epidermal keratinocytes.

PubMed

Pelle, Edward; Dong, Kelly; Pernodet, Nadine

2015-01-01

Sirtuins are post-translational modifiers that affect transcriptional signaling, metabolism, and DNA repair. Although originally identified as gene silencers capable of extending cell lifespan, the involvement of sirtuins in many different areas of cell biology has now become widespread. Our approach has been to study the temporal variation and also the effect of environmental stressors, such as ultraviolet B (UVB) and ozone, on sirtuin expression in human epidermal keratinocytes. In this report, we measured the variation in expression of several sirtuins over time and also show how a low dose of UVB can affect this pattern of expression. Moreover, we correlated these changes to variations in hydrogen peroxide (H2O2) and ATP levels. Our data show significant variations in normal sirtuin expression, which may indicate a generalized response by sirtuins to cell cycle kinetics. These results also demonstrate that sirtuins as a family of molecules are sensitive to UVB-induced disruption and may suggest a new paradigm for determining environmental stress on aging and provide direction for the development of new cosmetic products.

microRNA-184 Induces a Commitment Switch to Epidermal Differentiation.

PubMed

Nagosa, Sara; Leesch, Friederike; Putin, Daria; Bhattacharya, Swarnabh; Altshuler, Anna; Serror, Laura; Amitai-Lange, Aya; Nasser, Waseem; Aberdam, Edith; Rouleau, Matthieu; Tattikota, Sudhir G; Poy, Matthew N; Aberdam, Daniel; Shalom-Feuerstein, Ruby

2017-12-12

miR-184 is a highly evolutionary conserved microRNA (miRNA) from fly to human. The importance of miR-184 was underscored by the discovery that point mutations in miR-184 gene led to corneal/lens blinding disease. However, miR-184-related function in vivo remained unclear. Here, we report that the miR-184 knockout mouse model displayed increased p63 expression in line with epidermal hyperplasia, while forced expression of miR-184 by stem/progenitor cells enhanced the Notch pathway and induced epidermal hypoplasia. In line, miR-184 reduced clonogenicity and accelerated differentiation of human epidermal cells. We showed that by directly repressing cytokeratin 15 (K15) and FIH1, miR-184 induces Notch activation and epidermal differentiation. The disease-causing miR-184 C57U mutant failed to repress K15 and FIH1 and to induce Notch activation, suggesting a loss-of-function mechanism. Altogether, we propose that, by targeting K15 and FIH1, miR-184 regulates the transition from proliferation to early differentiation, while mis-expression or mutation in miR-184 results in impaired homeostasis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Nuclear expression of IL-33 in epidermal keratinocytes promotes wound healing in mice.

PubMed

Oshio, Tomoyuki; Komine, Mayumi; Tsuda, Hidetoshi; Tominaga, Shin-Ichi; Saito, Hirohisa; Nakae, Susumu; Ohtsuki, Mamitaro

2017-02-01

Skin is the outermost tissue of the human body, and works as a mechanical, chemical, and biological barrier. The epidermis is the uppermost layer of the skin, and keratinocytes constitute the majority of epidermal cells. Wounds are disruptions of skin integrity, and cause tremendous disadvantages to humans; accordingly, rapid wound healing is very important. Interleukin (IL)-33 is expressed in barrier tissue cells, such as epithelial and endothelial cells. Upon injury, IL-33 is released to stimulate immune cells, functioning as an "alarmin." ST2 is a receptor for IL-33; its soluble form (s)ST2 acts as a decoy receptor and competes for IL-33 binding. We aimed to clarify the role of IL-33 in wound healing. Wild-type (WT), IL-33 knockout (IL33 KO) mice, and sST2 transgenic (Tg) mice were wounded with a 4-mm punch, and the wound healing process was compared. Immunohistochemical analyses were performed to detect macrophages, neutrophils, and mast cells. Total RNA was extracted from the skin samples and real-time PCR was performed. An in vitro scratch wound assay was performed. Wound healing was delayed in IL33 KO mice compared to WT mice, while wound healing in sST2 Tg mice was comparable to that of WT mice. A histological examination showed delayed elongation of the epidermal tongue in IL-33 KO mice. An immunohistochemical study revealed prolonged neutrophilic infiltration at a later stage in IL-33 KO mice. IL-6, IL-1β, and CXCL1 transcripts were more abundant in the wounds of IL-33 KO mice than WT mice. Intraperitoneal administration of an NFκB inhibitor to IL-33 KO mice normalized the delayed wound healing and the enhanced expression of IL-6 in IL-33 KO mice. Epidermal keratinocytes from IL-33 KO mice showed delayed wound closure compared to those from WT mice. Our results indicate that nuclear IL-33, but not IL-33 as a cytokine, has beneficial effects on wound healing in mice, probably by suppressing NFκB to inhibit excessive inflammation and by maintaining

Identification of Rice Koji Extract Components that Increase β-Glucocerebrosidase Levels in Human Epidermal Keratinocytes.

PubMed

Maeda, Kazuhisa; Ogino, Yuuka; Nakamura, Ayano; Nakata, Keiji; Kitagawa, Manabu; Ito, Seiki

2018-06-18

Rice miso contains many ingredients derived from rice koji and has been a valuable source of nutrition since ancient times. We found that the consumption of rice miso led to improvements in the moisture content of cheek stratum corneum, skin viscoelasticity, and skin texture. Further, rice miso extract was found to increase the mRNA expression and activity of β-glucocerebrosidase (β-GCase), an enzyme involved in ceramide synthesis in the stratum corneum, in cultures. In this study, we identified the lipid-derived components of rice koji that increase the β-GCase activity in cultured human epidermal keratinocytes. The methanol fraction of rice koji extract induced an increase in the mRNA expression and activity of β-GCase in keratinocytes. The active fraction of rice koji was found to contain phosphatidic acid (PA) and lysophosphatidic acid (LPA). The total PA concentration in rice koji was 973.9 ng/mg dry weight, which was 17.5 times higher than that in steamed rice. Among the molecular species, PA_18:2/18:2 was the most frequently found. The total LPA concentration in rice koji was 29.6 ng/mg dry weight, and 2-LPA_18:2 was the most frequently found LPA. Since PA and LPA increase the mRNA expression and activity of β-GCase in keratinocytes, they are thought to be the active ingredients in rice koji that increase the β-GCase levels in human epidermal keratinocytes.

Genetic and pharmacological analysis identifies a physiological role for the AHR in epidermal differentiation

PubMed Central

van den Bogaard, Ellen; Podolsky, Michael; Smits, Jos; Cui, Xiao; John, Christian; Gowda, Krishne; Desai, Dhimant; Amin, Shantu; Schalkwijk, Joost; Perdew, Gary H.

2015-01-01

Stimulation of the aryl hydrocarbon receptor (AHR) by xenobiotics is known to affect epidermal differentiation and skin barrier formation. The physiological role of endogenous AHR signaling in keratinocyte differentiation is not known. We used murine and human skin models to address the hypothesis that AHR activation is required for normal keratinocyte differentiation. Using transcriptome analysis of Ahr-/- and Ahr+/+ murine keratinocytes, we found significant enrichment of differentially expressed genes linked to epidermal differentiation. Primary Ahr-/- keratinocytes showed a significant reduction in terminal differentiation gene and protein expression, similar to Ahr+/+ keratinocytes treated with AHR antagonists GNF351 and CH223191, or the selective AHR modulator (SAhRM), SGA360. In vitro keratinocyte differentiation led to increased AHR levels and subsequent nuclear translocation, followed by induced CYP1A1 gene expression. Monolayer cultured primary human keratinocytes treated with AHR antagonists also showed an impaired terminal differentiation program. Inactivation of AHR activity during human skin equivalent development severely impaired epidermal stratification, terminal differentiation protein expression and stratum corneum formation. As disturbed epidermal differentiation is a main feature of many skin diseases, pharmacological agents targeting AHR signaling or future identification of endogenous keratinocyte-derived AHR ligands should be considered as potential new drugs in dermatology. PMID:25602157

Niclosamide inhibits epithelial-mesenchymal transition and tumor growth in lapatinib-resistant human epidermal growth factor receptor 2-positive breast cancer.

PubMed

Liu, Junjun; Chen, Xiaosong; Ward, Toby; Mao, Yan; Bockhorn, Jessica; Liu, Xiaofei; Wang, Gen; Pegram, Mark; Shen, Kunwei

2016-02-01

Acquired resistance to lapatinib, a human epidermal growth factor receptor 2 kinase inhibitor, remains a clinical problem for women with human epidermal growth factor receptor 2-positive advanced breast cancer, as metastasis is commonly observed in these patients. Niclosamide, an anti-helminthic agent, has recently been shown to exhibit cytotoxicity to tumor cells with stem-like characteristics. This study was designed to identify the mechanisms underlying lapatinib resistance and to determine whether niclosamide inhibits lapatinib resistance by reversing epithelial-mesenchymal transition. Here, two human epidermal growth factor receptor 2-positive breast cancer cell lines, SKBR3 and BT474, were exposed to increasing concentrations of lapatinib to establish lapatinib-resistant cultures. Lapatinib-resistant SKBR3 and BT474 cells exhibited up-regulation of the phenotypic epithelial-mesenchymal transition markers Snail, vimentin and α-smooth muscle actin, accompanied by activation of nuclear factor-кB and Src and a concomitant increase in stem cell marker expression (CD44(high)/CD24(low)), compared to naive lapatinib-sensitive SKBR3 and BT474 cells, respectively. Interestingly, niclosamide reversed epithelial-mesenchymal transition, induced apoptosis and inhibited cell growth by perturbing aberrant signaling pathway activation in lapatinib-resistant human epidermal growth factor receptor 2-positive cells. The ability of niclosamide to alleviate stem-like phenotype development and invasion was confirmed. Collectively, our results demonstrate that lapatinib resistance correlates with epithelial-mesenchymal transition and that niclosamide inhibits lapatinib-resistant cell viability and epithelial-mesenchymal transition. These findings suggest a role of niclosamide or derivatives optimized for more favorable bioavailability not only in reversing lapatinib resistance but also in reducing metastatic potential during the treatment of human epidermal growth factor receptor

Considerations in the improvement of human epidermal keratinocyte culture in vitro.

PubMed

Kaviani, Maryam; Geramizadeh, Bita; Rahsaz, Marjan; Marzban, Saeed

2015-04-01

Large-scale expansion of epidermal keratinocytes is essential in the application of these cells for severe burn treatment in patients. Therefore, this study was designed to evaluate various conditions in the expansion of human epidermal keratinocytes. The epidermis was separated from the dermis of skin samples using dispase. The epidermis was trypsinized for keratinocyte isolation. Keratinocytes were cultured in various conditions, with or without a human dermal fibroblast feeder layer, mitomycin C treatment, and different culture media. Our results suggest that keratinocytes cultured on a human dermal fibroblast feeder layer were grown for several passages. Extensive deformation and rapid deterioration were observed in the cultured cells without a feeder layer and in serum-free medium. Human dermal fibroblasts treated with mitomycin C can provide optimal conditions for proliferation of keratinocytes.

Combined caveolin-1 and epidermal growth factor receptor expression as a prognostic marker for breast cancer.

PubMed

Liang, Ya-Nan; Liu, Yu; Wang, Letian; Yao, Guodong; Li, Xiaobo; Meng, Xiangning; Wang, Fan; Li, Ming; Tong, Dandan; Geng, Jingshu

2018-06-01

Previous studies have indicated that caveolin-1 (Cav-1) is able to bind the signal transduction factor epidermal growth factor receptor (EGFR) to regulate its tyrosine kinase activity. The aim of the present study was to evaluate the clinical significance of Cav-1 gene expression in association with the expression of EGFR in patients with breast cancer. Primary breast cancer samples from 306 patients were analyzed for Cav-1 and EGFR expression using immunohistochemistry, and clinical significance was assessed using multivariate Cox regression analysis, Kaplan-Meier estimator curves and the log-rank test. Stromal Cav-1 was downregulated in 38.56% (118/306) of tumor tissues, whereas cytoplasmic EGFR and Cav-1 were overexpressed in 53.92% (165/306) and 44.12% (135/306) of breast cancer tissues, respectively. EGFR expression was positively associated with cytoplasmic Cav-1 and not associated with stromal Cav-1 expression in breast cancer samples; however, low expression of stromal Cav-1 was negatively associated with cytoplasmic Cav-1 expression in total tumor tissues, and analogous results were identified in the chemotherapy group. Multivariate Cox's proportional hazards model analysis revealed that, for patients in the estrogen receptor (ER)(+) group, the expression of stromal Cav-1 alone was a significant prognostic marker of breast cancer. However, in the chemotherapy, human epidermal growth factor receptor 2 (HER-2)(-), HER-2(+) and ER(-) groups, the use of combined markers was more effective prognostic marker. Stromal Cav-1 has a tumor suppressor function, and the combined marker stromal Cav-1/EGFR expression was identified as an improved prognostic marker in the diagnosis of breast cancer. Parenchymal expression of Cav-1 is able to promote EGFR signaling in breast cancer, potentially being required for EGFR-mediated initiation of mitosis.

Improvement of hydration and epidermal barrier function in human skin by a novel compound isosorbide dicaprylate.

PubMed

Chaudhuri, R K; Bojanowski, K

2017-10-01

The study involved the synthesis of a novel derivative of caprylic acid - isosorbide dicaprylate (IDC) - and the evaluation of its potential in improving water homoeostasis and epidermal barrier function in human skin. The effect of IDC on gene expression was assayed in skin organotypic cultures by DNA microarrays. The results were then confirmed for a few key genes by quantitative PCR, immuno- and cytochemistry. Final validation of skin hydration properties was obtained by four separate clinical studies. Level of hydration was measured by corneometer either by using 2% IDC lotion alone vs placebo or in combination with 2% glycerol lotion vs 2% glycerol only. A direct comparison in skin hydration between 2% IDC and 2% glycerol lotions was also carried out. The epidermal barrier function improvement was assessed by determining changes in transepidermal water loss (TEWL) on the arms before and after treatment with 2% IDC lotion versus placebo. IDC was found to upregulate the expression of AQP3, CD44 and proteins involved in keratinocyte differentiation as well as the formation and function of stratum corneum. A direct comparison between 2% IDC versus 2% glycerol lotions revealed a three-fold advantage of IDC in providing skin hydration. Severely dry skin treated with 2% IDC in combination with 2% glycerol showed 133% improvement, whereas 35% improvement was observed with moderately dry human skin. Topical isosorbide dicaprylate favourably modulates genes involved in the maintenance of skin structure and function, resulting in superior clinical outcomes. By improving skin hydration and epidermal permeability barrier, it offers therapeutic applications in skin ageing. © 2017 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

GATA3 Expression Is Decreased in Psoriasis and during Epidermal Regeneration; Induction by Narrow-Band UVB and IL-4

PubMed Central

Kant, Marius; Baerveldt, Ewout M.; Florencia, Edwin; Mourits, Sabine; de Ridder, Dick; Laman, Jon D.; van der Fits, Leslie; Prens, Errol P.

2011-01-01

Psoriasis is characterized by hyperproliferation of keratinocytes and by infiltration of activated Th1 and Th17 cells in the (epi)dermis. By expression microarray, we previously found the GATA3 transcription factor significantly downregulated in lesional psoriatic skin. Since GATA3 serves as a key switch in both epidermal and T helper cell differentiation, we investigated its function in psoriasis. Because psoriatic skin inflammation shares many characteristics of epidermal regeneration during wound healing, we also studied GATA3 expression under such conditions. Psoriatic lesional skin showed decreased GATA3 mRNA and protein expression compared to non-lesional skin. GATA3 expression was also markedly decreased in inflamed skin of mice with a psoriasiform dermatitis induced with imiquimod. Tape-stripping of non-lesional skin of patients with psoriasis, a standardized psoriasis-triggering and skin regeneration-inducing technique, reduced the expression of GATA3. In wounded skin of mice, low GATA3 mRNA and protein expression was detected. Taken together, GATA3 expression is downregulated under regenerative and inflammatory hyperproliferative skin conditions. GATA3 expression could be re-induced by successful narrow-band UVB treatment of both human psoriasis and imiquimod-induced psoriasiform dermatitis in mice. The prototypic Th2 cytokine IL-4 was the only cytokine capable of inducing GATA3 in skin explants from healthy donors. Based on these findings we argue that GATA3 serves as a key regulator in psoriatic inflammation, keratinocyte hyperproliferation and skin barrier dysfunction. PMID:21611195

Mechanisms of resistance to anti-human epidermal growth factor receptor 2 agents in breast cancer.

PubMed

Mukohara, Toru

2011-01-01

Approximately 20% of breast cancers are characterized by overexpression of human epidermal growth factor receptor 2 (HER2) protein and associated gene amplification, and the receptor tyrosine kinase is believed to play a critical role in the pathogenesis of these tumors. The development and implementation of trastuzumab, a humanized monoclonal antibody against the extracellular domain of HER2 protein, has significantly improved treatment outcomes in patients with HER2-overexpressing breast cancer. However, despite this clinical usefulness, unmet needs for better prediction of trastuzumab's response and overcoming primary and acquired resistance remain. In this review, we discuss several potential mechanisms of resistance to trastuzumab that have been closely studied over the last decade. Briefly, these mechanisms include: impaired access of trastuzumab to HER2 by expression of extracellular domain-truncated HER2 (p95 HER2) or overexpression of MUC4; alternative signaling from insulin-like growth factor-1 receptor, other epidermal growth factor receptor family members, or MET; aberrant downstream signaling caused by loss of phosphatase and tensin homologs deleted from chromosome 10 (PTEN), PIK3CA mutation, or downregulation of p27; or FCGR3A polymorphisms. In addition, we discuss potential strategies for overcoming resistance to trastuzumab. Specifically, the epidermal growth factor receptor/HER2 tyrosine kinase inhibitor lapatinib partially overcame trastuzumab resistance in a clinical setting, so its efficacy results and limited data regarding potential mechanisms of resistance to the drug are also discussed. © 2010 Japanese Cancer Association.

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

PubMed

Gaviglio, Angela L; Knelson, Erik H; Blobe, Gerard C

2017-05-01

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation. © FASEB.

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

PubMed Central

Gaviglio, Angela L.; Knelson, Erik H.; Blobe, Gerard C.

2017-01-01

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor–like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.—Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation. PMID:28174207

Indirubin, an acting component of indigo naturalis, inhibits EGFR activation and EGF-induced CDC25B gene expression in epidermal keratinocytes.

PubMed

Hsieh, Wan-Ling; Lin, Yin-Ku; Tsai, Chi-Neu; Wang, Ta-Min; Chen, Tzu-Ya; Pang, Jong-Hwei S

2012-08-01

Topical indigo naturalis ointment is clinically proved to be an effective therapy for plaque-type psoriasis. Indirubin, as the active component of indigo naturalis, inhibits cell proliferation of epidermal keratinocytes. However, the detailed underlying mechanism is not fully understood. To further investigate the anti-proliferating effects of indigo naturalis and indirubin on epidermal keratinocytes. The decreased expression of CDC25B in indigo naturalis- or indirubin-treated epidermal keratinocytes, as revealed by cDNA microarray analysis, was studied. The CDC25B expression was examined under different serum concentrations and compared between primary and immortalized keratinocytes. The activation of EGFR and the effect of EGF on the cell proliferation and CDC25B expression were also investigated in epidermal keratinocytes. RT/real-time PCR and western blot method were used to analyze the CDC25B expression at the mRNA and protein levels, respectively. Indigo naturalis and indirubin were confirmed to down-regulate CDC25B expression significantly at both the mRNA and protein levels. The growth-dependent expression of CDC25B was demonstrated by the increased expression in serum-stimulated and immortalized keratinocytes. The activation of EGF receptor, known to be highly expressed in psoriatic lesions, was inhibited by indigo naturalis or indirubin. The cell proliferation and CDC25B expression of epidermal keratinocytes were induced by EGF alone and confirmed to be inhibited by indigo naturalis or indirubin. Except being a common therapeutic target in various cancers, CDC25B also plays an important role in the hyper-proliferation of epidermal keratinocytes which can be suppressed by anti-psoriatic drug indigo naturalis and its component, indirubin. Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Effects of ozone in normal human epidermal keratinocytes.

PubMed

McCarthy, James T; Pelle, Edward; Dong, Kelly; Brahmbhatt, Krupa; Yarosh, Dan; Pernodet, Nadine

2013-05-01

Ozone is a tropospheric pollutant that can form at ground level as a result of an interaction between sunlight and hydrocarbon engine emissions. As ozone is an extremely oxidative reaction product, epidermal cells are in the outer layer of defense against ozone. We exposed normal human epidermal keratinocytes (NHEK) to concentrations of ozone that have been measured in cities and assayed for its effects. Hydrogen peroxide and IL-1α levels both increased while ATP levels decreased. We found a decrease in the NAD-dependent histone deacetylase, sirtuin 3. Lastly, we found that ozone increased DNA damage as evaluated by Comet assay. Taken together, our results show increased damage to NHEK that will ultimately impair normal cellular function as a result of an environmentally relevant ozone exposure. © 2013 John Wiley & Sons A/S.

IFI27, a novel epidermal growth factor-stabilized protein, is functionally involved in proliferation and cell cycling of human epidermal keratinocytes.

PubMed

Hsieh, W-L; Huang, Y-H; Wang, T-M; Ming, Y-C; Tsai, C-N; Pang, J-H S

2015-04-01

IFI27 is highly expressed in psoriatic lesions but its function has not been known. The present study aimed to explore its role in proliferation of epidermal keratinocytes. IFI27 knockdown and over-expression in keratinocytes were used to compare their proliferation, by MTT assay, apoptosis (by annexin V binding) and cell cycle progression by flow cytometry. Formation of cyclin A/CDK1 complex was examined by a co-immunoprecipitaion method. Anti-proliferation effects of IFI27 were also examined in vivo by topical application of IFI27 siRNA on imiquimod-induced psoriatic lesions, in a mouse model. Epidermal growth factor was demonstrated to increase IFI27 expression by prolonging half-life of IFI27 protein. The IFI27 knockdown in keratinocytes reduced the proliferation rate, but had no effect on apoptosis nor on apoptosis-related genes. Interestingly, IFI27 knockdown resulted in S-phase arrest that was found to be associated with increased Tyr15 phosphorylation of CDK1, reduced CDC25B and reduced formation of cyclin A/CDK1 complex. In addition, IFI27 knockdown was also shown to activate p53 by Ser15 phosphorylation and increase p21 expression. Topical application of IFI27 siRNA on imiquimod-induced psoriatic lesion in a mouse model reduced epidermal thickness, formation of rete ridges and PCNA expression. Our study demonstrates for the first time, that cell function of IFI27 is involved in proliferation of skin keratinocytes both in vitro and in vivo. It suggests that IFI27 might be a suitable target for development of a novel anti-psoriasis therapy. © 2015 John Wiley & Sons Ltd.

E5 can be expressed in anal cancer and leads to epidermal growth factor receptor-induced invasion in a human papillomavirus 16-transformed anal epithelial cell line.

PubMed

Wechsler, Erin Isaacson; Tugizov, Sharof; Herrera, Rossana; Da Costa, Maria; Palefsky, Joel M

2018-05-01

We detected the first human papillomavirus (HPV)-16-immortalized anal epithelial cell line, known as AKC2 cells to establish an in vitro model of HPV-16-induced anal carcinogenesis. Consistent with detection of E6, E7 and E5 expression in anal cancer biopsies, AKC2 cells expressed high levels of all three HPV oncogenes. Also, similar to findings in anal cancer biopsies, epidermal growth factor receptor (EGFR) was overexpressed in AKC2 cells. AKC2 cells exhibited a poorly differentiated and invasive phenotype in three-dimensional raft culture and inhibition of EGFR function abrogated AKC2 invasion. Reducing E5 expression using E5-targeted siRNAs in AKC2 cells led to knockdown of E5 expression, but also HPV-16 E2, E6 and E7 expression. AKC2 cells treated with E5-targeted siRNA had reduced levels of total and phosphorylated EGFR, and reduced invasion. Rescue of E6/E7 expression with simultaneous E5 knockdown confirmed that E5 plays a key role in EGFR overexpression and EGFR-induced invasion.

Repeated short climatic change affects the epidermal differentiation program and leads to matrix remodeling in a human organotypic skin model.

PubMed

Boutrand, Laetitia-Barbollat; Thépot, Amélie; Muther, Charlotte; Boher, Aurélie; Robic, Julie; Guéré, Christelle; Vié, Katell; Damour, Odile; Lamartine, Jérôme

2017-01-01

Human skin is subject to frequent changes in ambient temperature and humidity and needs to cope with these environmental modifications. To decipher the molecular response of human skin to repeated climatic change, a versatile model of skin equivalent subject to "hot-wet" (40°C, 80% relative humidity [RH]) or "cold-dry" (10°C, 40% RH) climatic stress repeated daily was used. To obtain an exhaustive view of the molecular mechanisms elicited by climatic change, large-scale gene expression DNA microarray analysis was performed and modulated function was determined by bioinformatic annotation. This analysis revealed several functions, including epidermal differentiation and extracellular matrix, impacted by repeated variations in climatic conditions. Some of these molecular changes were confirmed by histological examination and protein expression. Both treatments (hot-wet and cold-dry) reduced the expression of genes encoding collagens, laminin, and proteoglycans, suggesting a profound remodeling of the extracellular matrix. Strong induction of the entire family of late cornified envelope genes after cold-dry exposure, confirmed at protein level, was also observed. These changes correlated with an increase in epidermal differentiation markers such as corneodesmosin and a thickening of the stratum corneum, indicating possible implementation of defense mechanisms against dehydration. This study for the first time reveals the complex pattern of molecular response allowing adaption of human skin to repeated change in its climatic environment.

Dermal-epidermal membrane systems by using human keratinocytes and mesenchymal stem cells isolated from dermis.

PubMed

Salerno, Simona; Messina, Antonietta; Giordano, Francesca; Bader, Augustinus; Drioli, Enrico; De Bartolo, Loredana

2017-02-01

Dermal-epidermal membrane systems were developed by co-culturing human keratinocytes with Skin derived Stem Cells (SSCs), which are Mesenchymal Stem Cells (MSCs) isolated from dermis, on biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT and PCL. The membranes display physico-chemical, morphological, mechanical and biodegradation properties that could satisfy and fulfil specific requirements in skin tissue engineering. CHT membrane exhibits an optimal biodegradation rate for acute wounds; CHT-PCL for the chronic ones. On the other hand, PCL membrane in spite of its very slow biodegradation rate exhibits mechanical properties similar to in vivo dermis, a lower hydrophilic character, and a surface roughness, all properties that make it able to sustain cell adhesion and proliferation for in vitro skin models. Both CHT-PCL and PCL membranes guided epidermal and dermal differentiation of SSCs as pointed out by the expression of cytokeratins and the deposition of the ECM protein fibronectin, respectively. In the dermal-epidermal membrane systems, a more suitable microenvironment for the SSCs differentiation was promoted by the interactions and the mutual interplay with keratinocytes. Being skin tissue-biased stem cells committed to their specific final dermal and/or epidermal cell differentiation, SSCs are more suitable for skin tissue engineering than other adult MSCs with different origin. For this reason, they represent a useful autologous cell source for engineering skin substitutes for both in vivo and in vitro applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression of transforming growth factor alpha and epidermal growth factor receptor messenger RNA in neoplastic and nonneoplastic human kidney tissue.

PubMed

Mydlo, J H; Michaeli, J; Cordon-Cardo, C; Goldenberg, A S; Heston, W D; Fair, W R

1989-06-15

Using Northern blot analysis, we have demonstrated that mRNA for transforming growth factor alpha (TGF-alpha) was expressed in five malignant kidney tissue specimens but was not detected in their autologous nonneoplastic homologues. In addition, the expression of epidermal growth factor (EGF) receptor mRNA in these malignant tissues was 2- to 3-fold greater than in nontransformed tissues. In two cases examined using immunohistochemistry, we were able to correlate the increased expression of the mRNA with an increase in protein expression. Since TGF-alpha is known to bind to the EGF receptor, the finding of an increased expression of both TGF-alpha and EGF receptor mRNA in kidney tumor tissue suggests that interaction between TGF-alpha and the EGF receptor may play a role in promoting transformation and/or proliferation of kidney neoplasms, perhaps by an autocrine mechanism.

Intralaboratory and interlaboratory evaluation of the EpiDerm 3D human reconstructed skin micronucleus (RSMN) assay.

PubMed

Hu, Ting; Kaluzhny, Yulia; Mun, Greg C; Barnett, Brenda; Karetsky, Viktor; Wilt, Nathan; Klausner, Mitchell; Curren, Rodger D; Aardema, Marilyn J

2009-03-17

A novel in vitro human reconstructed skin micronucleus (RSMN) assay has been developed using the EpiDerm 3D human skin model [R. D. Curren, G. C. Mun, D. P. Gibson, and M. J. Aardema, Development of a method for assessing micronucleus induction in a 3D human skin model EpiDerm, Mutat. Res. 607 (2006) 192-204]. The RSMN assay has potential use in genotoxicity assessments as a replacement for in vivo genotoxicity assays that will be banned starting in 2009 according to the EU 7th Amendment to the Cosmetics Directive. Utilizing EpiDerm tissues reconstructed with cells from four different donors, intralaboratory and interlaboratory reproducibility of the RSMN assay were examined. Seven chemicals were evaluated in three laboratories using a standard protocol. Each chemical was evaluated in at least two laboratories and in EpiDerm tissues from at least two different donors. Three model genotoxins, mitomycin C (MMC), vinblastine sulfate (VB) and methyl methanesulfonate (MMS) induced significant, dose-related increases in cytotoxicity and MN induction in EpiDerm tissues. Conversely, four dermal non-carcinogens, 4-nitrophenol (4-NP), trichloroethylene (TCE), 2-ethyl-1,3-hexanediol (EHD), and 1,2-epoxydodecane (EDD) were negative in the RSMN assay. Results between tissues reconstructed from different donors were comparable. These results indicate the RSMN assay using the EpiDerm 3D human skin model is a promising new in vitro genotoxicity assay that allows evaluation of chromosome damage following "in vivo-like" dermal exposures.

Epidermal growth factor receptor expression in radiation-induced dog lung tumors by immunocytochemical localization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leung, F.L.; Park, J.F.; Dagle, G.E.

1993-06-01

In studies to determine the role of growth factors in radiation-induced lung cancer, epidermal growth factor (EGFR) expression was examined by immunocytochemistry in 51 lung tumors from beagle dogs exposed to inhaled plutonium; 21 of 51 (41%) tumors were positive for EGFR. The traction of tumors positive for EGFR and the histological type of EGFR-positive tumors in the plutonium-exposed dogs were not different from spontaneous dog lung tumors, In which 36% were positive for EGFR. EGFR involvement in Pu-induced lung tumors appeared to be similar to that in spontaneous lung tumors. However, EGFR-positive staining was observed in only 1 ofmore » 16 tumors at the three lowest Pu exposure levels, compared to 20 of 35 tumors staining positive at the two highest Pu exposure levels. The results in dogs were in good agreement with the expression of EGFR reported in human non-small cell carcinoma of the lung, suggesting that Pu-induced lung tumors in the dog may be a suitable animal model to investigate the role of EGFR expression in lung carcinogenesis. In humans, EGFR expression in lung tumors has been primarily related to histological tumor types. In individual dogs with multiple primary lung tumors, the tumors were either all EGFR positive or EGFR negative, suggesting that EGFR expression may be related to the response of the individual dog as well as to the histological type of tumor.« less

Human Papilloma Viral DNA Replicates as a Stable Episome in Cultured Epidermal Keratinocytes

NASA Astrophysics Data System (ADS)

Laporta, Robert F.; Taichman, Lorne B.

1982-06-01

Human papilloma virus (HPV) is poorly understood because systems for its growth in tissue culture have not been developed. We report here that cultured human epidermal keratinocytes could be infected with HPV from plantar warts and that the viral DNA persisted and replicated as a stable episome. There were 50-200 copies of viral DNA per cell and there was no evidence to indicate integration of viral DNA into the cellular genome. There was also no evidence to suggest that viral DNA underwent productive replication. We conclude that cultured human epidermal keratinocytes may be a model for the study of certain aspects of HPV biology.

Upregulated expression of La ribonucleoprotein domain family member 6 and collagen type I gene following water-filtered broad-spectrum near-infrared irradiation in a 3-dimensional human epidermal tissue culture model as revealed by microarray analysis.

PubMed

Tanaka, Yohei; Nakayama, Jun

2018-05-01

Water-filtered broad-spectrum near-infrared irradiation can induce various biological effects, as our previous clinical, histological, and biochemical investigations have shown. However, few studies that examined the changes thus induced in gene expression. The aim was to investigate the changes in gene expression in a 3-dimensional reconstructed epidermal tissue culture exposed to water-filtered broad-spectrum near-infrared irradiation. DNA microarray and quantitative real-time polymerase chain reaction (PCR) analysis was used to assess gene expression levels in a 3-dimensional reconstructed epidermal model composed of normal human epidermal cells exposed to water-filtered broad-spectrum near-infrared irradiation. The water filter allowed 1000-1800 nm wavelengths and excluded 1400-1500 nm wavelengths, and cells were exposed to 5 or 10 rounds of near-infrared irradiation at 10 J/cm 2 . A DNA microarray with over 50 000 different probes showed 18 genes that were upregulated or downregulated by at least twofold after irradiation. Quantitative real-time PCR revealed that, relative to control cells, the gene encoding La ribonucleoprotein domain family member 6 (LARP6), which regulates collagen expression, was significantly and dose-dependently upregulated (P < 0.05) by water-filtered broad-spectrum near-infrared exposure. Gene encoding transcripts of collagen type I were significantly upregulated compared with controls (P < 0.05). This study demonstrates the ability of water-filtered broad-spectrum near-infrared irradiation to stimulate the production of type I collagen. © 2017 The Australasian College of Dermatologists.

Prevention of UVB Radiation-induced Epidermal Damage by Expression of Heat Shock Protein 70*

PubMed Central

Matsuda, Minoru; Hoshino, Tatsuya; Yamashita, Yasuhiro; Tanaka, Ken-ichiro; Maji, Daisuke; Sato, Keizo; Adachi, Hiroaki; Sobue, Gen; Ihn, Hironobu; Funasaka, Yoko; Mizushima, Tohru

2010-01-01

Irradiation with UV light, especially UVB, causes epidermal damage via the induction of apoptosis, inflammatory responses, and DNA damage. Various stressors, including UV light, induce heat shock proteins (HSPs) and the induction, particularly that of HSP70, provides cellular resistance to such stressors. The anti-inflammatory activity of HSP70, such as its inhibition of nuclear factor kappa B (NF-κB), was recently revealed. These in vitro results suggest that HSP70 protects against UVB-induced epidermal damage. Here we tested this idea by using transgenic mice expressing HSP70 and cultured keratinocytes. Irradiation of wild-type mice with UVB caused epidermal damage such as induction of apoptosis, which was suppressed in transgenic mice expressing HSP70. UVB-induced apoptosis in cultured keratinocytes was suppressed by overexpression of HSP70. Irradiation of wild-type mice with UVB decreased the cutaneous level of IκB-α (an inhibitor of NF-κB) and increased the infiltration of leukocytes and levels of pro-inflammatory cytokines and chemokines in the epidermis. These inflammatory responses were suppressed in transgenic mice expressing HSP70. In vitro, the overexpression of HSP70 suppressed the expression of pro-inflammatory cytokines and chemokines and increased the level of IκB-α in keratinocytes irradiated with UVB. UVB induced an increase in cutaneous levels of cyclobutane pyrimidine dimers and 8-hydroxy-2′-deoxyguanosine, both of which were suppressed in transgenic mice expressing HSP70. This study provides genetic evidence that HSP70 protects the epidermis from UVB-induced radiation damage. The findings here also suggest that the protective action of HSP70 is mediated by anti-apoptotic, anti-inflammatory, and anti-DNA damage effects. PMID:20018843

Use of a collagen-elastin matrix as transport carrier system to transfer proliferating epidermal cells to human dermis in vitro.

PubMed

Waaijman, Taco; Breetveld, Melanie; Ulrich, Magda; Middelkoop, Esther; Scheper, Rik J; Gibbs, Susan

2010-01-01

This in vitro study describes a novel cell culture, transport, and transfer protocol that may be highly suitable for delivering cultured proliferating keratinocytes and melanocytes to large open skin wounds (e.g., burns). We have taken into account previous limitations identified using other keratinocyte transfer techniques, such as regulatory issues, stability of keratinocytes during transport (single cell suspensions undergo terminal differentiation), ease of handling during application, and the degree of epidermal blistering resulting after transplantation (both related to transplanting keratinocyte sheets). Large numbers of proliferating epidermal cells (EC) (keratinocytes and melanocytes) were generated within 10-14 days and seeded onto a three-dimensional matrix composed of elastin and collagen types I, III, and V (Matriderm®), which enabled easy and stable transport of the EC for up to 24 h under ambient conditions. All culture conditions were in accordance with the regulations set by the Dutch Central Committee on Research Involving Human Subjects (CCMO). As an in vitro model system for clinical in vivo transfer, the EC were then transferred from Matriderm onto human acellular dermis during a period of 3 days. After transfer the EC maintained the ability to regenerate into a fully differentiated epidermis containing melanocytes on the human dermis. Proliferating keratinocytes were located in the basal layer and keratin-10 expression was located in differentiating suprabasal layers similar to that found in human epidermis. No blistering was observed (separation of the epidermis from the basement membrane). Keratin-6 expression was strongly upregulated in the regenerating epidermis similar to normal wound healing. In summary, we show that EC-Matriderm contains viable, metabolically active keratinocytes and melanocytes cultured in a manner that permits easy transportation and contains epidermal cells with the potential to form a pigmented reconstructed

Cell motion predicts human epidermal stemness

PubMed Central

Toki, Fujio; Tate, Sota; Imai, Matome; Matsushita, Natsuki; Shiraishi, Ken; Sayama, Koji; Toki, Hiroshi; Higashiyama, Shigeki

2015-01-01

Image-based identification of cultured stem cells and noninvasive evaluation of their proliferative capacity advance cell therapy and stem cell research. Here we demonstrate that human keratinocyte stem cells can be identified in situ by analyzing cell motion during their cultivation. Modeling experiments suggested that the clonal type of cultured human clonogenic keratinocytes can be efficiently determined by analysis of early cell movement. Image analysis experiments demonstrated that keratinocyte stem cells indeed display a unique rotational movement that can be identified as early as the two-cell stage colony. We also demonstrate that α6 integrin is required for both rotational and collective cell motion. Our experiments provide, for the first time, strong evidence that cell motion and epidermal stemness are linked. We conclude that early identification of human keratinocyte stem cells by image analysis of cell movement is a valid parameter for quality control of cultured keratinocytes for transplantation. PMID:25897083

Epidermal growth factor- and hepatocyte growth factor-receptor activity in serum-free cultures of human hepatocytes.

PubMed

Runge, D M; Runge, D; Dorko, K; Pisarov, L A; Leckel, K; Kostrubsky, V E; Thomas, D; Strom, S C; Michalopoulos, G K

1999-02-01

Serum-free primary cultures of hepatocytes are a useful tool to study factors triggering hepatocyte proliferation and regeneration. We have developed a chemically defined serum-free system that allows human hepatocyte proliferation in the presence of epidermal growth factor and hepatocyte growth factor. DNA synthesis and accumulation were determined by [3H]thymidine incorporation and fluorometry, respectively. Western blot analyses and co-immunoprecipitations were used to investigate the association of proteins involved in epidermal growth factor and hepatocyte growth factor activation and signaling: epidermal growth factor receptor, hepatocyte growth factor receptor (MET), urokinase-type plasminogen activator and its receptor, and a member of the signal transducer and activator of transcription family, STAT-3. Primary human hepatocytes proliferated under serum-free conditions in a chemically defined medium for up to 12 days. Epidermal growth factor-receptor and MET were present and functional, decreasing over time. MET, urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor co-precipitated to varying degrees during the culture period. STAT-3 co-precipitated with epidermal growth factor-receptor and MET to varying degrees. Proliferation of human hepatocytes can improve by modification of a chemically defined medium originally used for rat hepatocyte cultures. In these long-term cultures of human hepatocytes, hepatocyte growth factor and epidermal growth factor can stimulate growth and differentiation by interacting with their receptors and initiating downstream signaling. This involves complex formation of the receptors with other plasma membrane components for MET (urokinase-type plasminogen activator in context of its receptor) and activation of STAT-3 for both receptors.

Prognostic significance of epidermal growth factor receptor (EGFR) over expression in urothelial carcinoma of urinary bladder.

PubMed

Hashmi, Atif Ali; Hussain, Zubaida Fida; Irfan, Muhammad; Khan, Erum Yousuf; Faridi, Naveen; Naqvi, Hanna; Khan, Amir; Edhi, Muhammad Muzzammil

2018-06-07

Epidermal growth factor receptor (EGFR) has been shown to have abnormal expression in many human cancers and is considered as a marker of poor prognosis. Frequency of over expression in bladder cancer has not been studied in our population; therefore we aimed to evaluate the frequency and prognostic significance of EGFR immunohistochemical expression in locoregional population. We performed EGFR immunohistochemistry on 126 cases of bladder cancer and association of EGFR expression with tumor grade, lamina propria invasion, deep muscle invasion and recurrence of disease was evaluated. High EGFR expression was noted in 26.2% (33 cases), 15.1% (19 cases) and 58.7% (74 cases) revealed low and no EGFR expression respectively. Significant association of EGFR expression was noted with tumor grade, lamina propria invasion, deep muscle invasion and recurrence status while no significant association was seen with age, gender and overall survival. Kaplan- Meier curves revealed significant association of EGFR expression with recurrence while no significant association was seen with overall survival. Significant association of EGFR overexpression with tumor grade, muscularis propria invasion and recurrence signifies its prognostic value; therefore EGFR can be used as a prognostic biomarker in Urothelial bladder carcinoma.

Upregulated epidermal growth factor receptor expression following near-infrared irradiation simulating solar radiation in a three-dimensional reconstructed human corneal epithelial tissue culture model

PubMed Central

Tanaka, Yohei; Nakayama, Jun

2016-01-01

Background and objective Humans are increasingly exposed to near-infrared (NIR) radiation from both natural (eg, solar) and artificial (eg, electrical appliances) sources. Although the biological effects of sun and ultraviolet (UV) exposure have been extensively investigated, the biological effect of NIR radiation is still unclear. We previously reported that NIR as well as UV induces photoaging and standard UV-blocking materials, such as sunglasses, do not sufficiently block NIR. The objective of this study was to investigate changes in gene expression in three-dimensional reconstructed corneal epithelial tissue culture exposed to broad-spectrum NIR irradiation to simulate solar NIR radiation that reaches human tissues. Materials and methods DNA microarray and quantitative real-time polymerase chain reaction analysis were used to assess gene expression levels in a three-dimensional reconstructed corneal epithelial model composed of normal human corneal epithelial cells exposed to water-filtered broad-spectrum NIR irradiation with a contact cooling (20°C). The water-filter allowed 1,000–1,800 nm wavelengths and excluded 1,400–1,500 nm wavelengths. Results A DNA microarray with >62,000 different probes showed 25 and 150 genes that were up- or downregulated by at least fourfold and twofold, respectively, after NIR irradiation. In particular, epidermal growth factor receptor (EGFR) was upregulated by 19.4-fold relative to control cells. Quantitative real-time polymerase chain reaction analysis revealed that two variants of EGFR in human corneal epithelial tissue were also significantly upregulated after five rounds of 10 J/cm2 irradiation (P<0.05). Conclusion We found that NIR irradiation induced the upregulated expression of EGFR in human corneal cells. Since over half of the solar energy reaching the Earth is in the NIR region, which cannot be adequately blocked by eyewear and thus can induce eye damage with intensive or long-term exposure, protection from both

Upregulated epidermal growth factor receptor expression following near-infrared irradiation simulating solar radiation in a three-dimensional reconstructed human corneal epithelial tissue culture model.

PubMed

Tanaka, Yohei; Nakayama, Jun

2016-01-01

Humans are increasingly exposed to near-infrared (NIR) radiation from both natural (eg, solar) and artificial (eg, electrical appliances) sources. Although the biological effects of sun and ultraviolet (UV) exposure have been extensively investigated, the biological effect of NIR radiation is still unclear. We previously reported that NIR as well as UV induces photoaging and standard UV-blocking materials, such as sunglasses, do not sufficiently block NIR. The objective of this study was to investigate changes in gene expression in three-dimensional reconstructed corneal epithelial tissue culture exposed to broad-spectrum NIR irradiation to simulate solar NIR radiation that reaches human tissues. DNA microarray and quantitative real-time polymerase chain reaction analysis were used to assess gene expression levels in a three-dimensional reconstructed corneal epithelial model composed of normal human corneal epithelial cells exposed to water-filtered broad-spectrum NIR irradiation with a contact cooling (20°C). The water-filter allowed 1,000-1,800 nm wavelengths and excluded 1,400-1,500 nm wavelengths. A DNA microarray with >62,000 different probes showed 25 and 150 genes that were up- or downregulated by at least fourfold and twofold, respectively, after NIR irradiation. In particular, epidermal growth factor receptor (EGFR) was upregulated by 19.4-fold relative to control cells. Quantitative real-time polymerase chain reaction analysis revealed that two variants of EGFR in human corneal epithelial tissue were also significantly upregulated after five rounds of 10 J/cm(2) irradiation (P<0.05). We found that NIR irradiation induced the upregulated expression of EGFR in human corneal cells. Since over half of the solar energy reaching the Earth is in the NIR region, which cannot be adequately blocked by eyewear and thus can induce eye damage with intensive or long-term exposure, protection from both UV and NIR radiation may prevent changes in gene expression and in

Retinoic acid influences anteroposterior positioning of epidermal sensory neurons and their gene expression in a developing chordate (amphioxus)

PubMed Central

Schubert, Michael; Holland, Nicholas D.; Escriva, Hector; Holland, Linda Z.; Laudet, Vincent

2004-01-01

In developing chordates, retinoic acid (RA) signaling patterns the rostrocaudal body axis globally and affects gene expression locally in some differentiating cell populations. Here we focus on development of epidermal sensory neurons in an invertebrate chordate (amphioxus) to determine how RA signaling influences their rostrocaudal distribution and gene expression (for AmphiCoe, a neural precursor gene; for amphioxus islet and AmphiERR, two neural differentiation genes; and for AmphiHox1, -3, -4, and -6). Treatments with RA or an RA antagonist (BMS009) shift the distribution of developing epidermal neurons anteriorly or posteriorly, respectively. These treatments also affect gene expression patterns in the epidermal neurons, suggesting that RA levels may influence specification of neuronal subtypes. Although colinear expression of Hox genes is well known for the amphioxus central nervous system, we find an unexpected comparable colinearity for AmphiHox1, -3, -4, and -6 in the developing epidermis; moreover, RA levels affect the anteroposterior extent of these Hox expression domains, suggesting that RA signaling controls a colinear Hox code for anteroposterior patterning of the amphioxus epidermis. Thus, in amphioxus, the developing peripheral nervous system appears to be structured by mechanisms parallel to those that structure the central nervous system. One can speculate that, during evolution, an ancestral deuterostome that structured its panepidermal nervous system with an RA-influenced Hox code gave rise to chordates in which this patterning mechanism persisted within the epidermal elements of the peripheral nervous system and was transferred to the neuroectoderm as the central nervous system condensed dorsally. PMID:15226493

Epidermal growth factor expression in esophageal adenocarcinoma: a clinically relevant target?

PubMed

Harper, Nicholas; Li, Yan; Farmer, Russell; Martin, Robert C G

2012-05-01

There has been recent widespread enthusiasm in epidermal growth factor (EGFR) as a molecularly active target in esophageal adenocarcinoma (EAC). However, there is limited data on the extent of EGFR expression in EAC. Thus, the aim of this study was to evaluated EGFR, pErk1/2, and total Erk1/2 expression in malignant and benign specimens. Baseline expression of EGFR in the human normal squamous, Barrett's, and EAC cell lines were determined as well as after bile acid treatment and curcumin pretreatment. In addition, EGFR expression was also evaluated in 60 matched normal and malignant EAC resected specimens. The in vitro studies in the Het-1a, BarT, and OE19 cell lines failed to show any measurable expression of EGFR via Western blot technique. The marker serving as the positive control for the study, MnSOD, showed expression in each cell line for all three treatment regimens at approximately 24 kDa EGFR, showing moderate staining in the malignant tumor specimens and low staining in the benign tissue specimens. pErk1/2 showed low staining in the malignant tumor specimens and no staining in the benign tissue specimens. Total Erk1/2 showed high staining in both the malignant tumor specimens and benign tissue specimens. The differences in the mean staining scores for the malignant versus benign tissue specimens for pErk1/2 and total Erk1/2 are not statistically significant (p = 0.0726 and p = 0.7054, respectively). Thus, in conclusion, EGFR expression has been confirmed to be limited to non-existent in EAC and thus its use as a clinically active target is limited at best. Prior to the use of these expensive anti-EGFR therapies, confirmation of overexpression should be verified.

Human eccrine sweat gland cells turn into melanin-uptaking keratinocytes in dermo-epidermal skin substitutes.

PubMed

Böttcher-Haberzeth, Sophie; Biedermann, Thomas; Pontiggia, Luca; Braziulis, Erik; Schiestl, Clemens; Hendriks, Bart; Eichhoff, Ossia M; Widmer, Daniel S; Meuli-Simmen, Claudia; Meuli, Martin; Reichmann, Ernst

2013-02-01

Recently, Biedermann et al. (2010) have demonstrated that human eccrine sweat gland cells can develop a multilayered epidermis. The question still remains whether these cells can fulfill exclusive and very specific functional properties of epidermal keratinocytes, such as the incorporation of melanin, a feature absent in sweat gland cells. We added human melanocytes to eccrine sweat gland cells to let them develop into an epidermal analog in vivo. The interaction between melanocytes and sweat gland-derived keratinocytes was investigated. The following results were gained: (1) macroscopically, a pigmentation of the substitutes was seen 2-3 weeks after transplantation; (2) we confirmed the development of a multilayered, stratified epidermis with melanocytes distributed evenly throughout the basal layer; (3) melanocytic dendrites projected to suprabasal layers; and (4) melanin was observed to be integrated into former eccrine sweat gland cells. These skin substitutes were similar or equal to skin substitutes cultured from human epidermal keratinocytes. The only differences observed were a delay in pigmentation and less melanin uptake. These data suggest that eccrine sweat gland cells can form a functional epidermal melanin unit, thereby providing striking evidence that they can assume one of the most characteristic keratinocyte properties.

Induction of cyclooxygenase-2 expression by prostaglandin E2 stimulation of the prostanoid EP4 receptor via coupling to Gαi and transactivation of the epidermal growth factor receptor in HCA-7 human colon cancer cells.

PubMed

Yoshida, Kenji; Fujino, Hiromichi; Otake, Sho; Seira, Naofumi; Regan, John W; Murayama, Toshihiko

2013-10-15

Increased expressions of cyclooxygenase-2 (COX-2) and its downstream metabolite, prostaglandin E2 (PGE2), are well documented events in the development of colorectal cancer. Interestingly, PGE2 itself can induce the expression of COX-2 thereby creating the potential for positive feedback. Although evidence for such a positive feedback has been previously described, the specific E-type prostanoid (EP) receptor subtype that mediates this response, as well as the relevant signaling pathways, remain unclear. We now report that the PGE2 stimulated induction of COX-2 expression in human colon cancer HCA-7 cells is mediated by activation of the prostanoid EP4 receptor subtype and is followed by coupling of the receptor to Gαi and the activation of phosphatidylinositol 3-kinase. Subsequent activation of metalloproteinases releases membrane bound heparin-binding epidermal growth factor-like growth factor resulting in the transactivation of epidermal growth factor receptors and the activation of the extracellular signal-regulated kinases and induction of COX-2 expression. This induction of COX-2 expression by PGE2 stimulation of the prostanoid EP4 receptor may underlie the upregulation of COX-2 during colorectal cancer and appears to be an early event in the process of tumorigenesis. © 2013 Elsevier B.V. All rights reserved.

Human Langerhans cells express E-cadherin.

PubMed

Blauvelt, A; Katz, S I; Udey, M C

1995-02-01

Murine Langerhans cells (LC) synthesize and express E-cadherin, a Ca(++)-dependent homophilic cell adhesion molecule that mediates LC-keratinocyte (KC) binding in vitro. In vivo, E-cadherin expression by LC may promote localization and persistence of LC within the epidermis through LC-KC adhesion. In addition, changes in LC E-cadherin expression or affinity may be an important factor in the egress of LC from the epidermis after exposure to antigen. The aim of the present study was to determine if human LC also express E-cadherin. Suction blister roofs were obtained from normal volunteers and epidermal cell (EC) suspensions were prepared by limited trypsinization in the presence of 1 mM Ca++. EC were then incubated with antibodies to E-cadherin and CD1a or HLA-DR, and examined by two-color analytical flow cytometry or immunofluorescence microscopy. Most (82.9% +/- 7.4% [mean +/- SD], range 67-89%, n = 7) freshly prepared human LC expressed E-cadherin, as did the majority of KC. The amount of E-cadherin (as determined by mean fluorescence intensity) expressed by LC and KC was similar. Trypsin/EDTA treatment of freshly prepared EC abrogated expression of E-cadherin by LC and KC, whereas E-cadherin was not degraded by trypsin in the presence of Ca++. LC expressed lower levels of E-cadherin after 3 d in culture. Thus, human LC, like murine LC, express the homophilic adhesion molecule E-cadherin, which may be important in establishing and maintaining interactions between LC and KC in mammalian epidermis.

Intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts.

PubMed

Pomari, Elena; Dalla Valle, Luisa; Pertile, Paolo; Colombo, Lorenzo; Thornton, M Julie

2015-02-01

Peripheral intracrine sex steroid synthesis from adrenal precursors dehydroepiandrosterone (DHEA) and DHEA-sulfate has evolved in humans. We sought to establish if there are differences in intracrine, paracrine, and endocrine regulation of sex steroids by primary cultures of human skin epidermal keratinocytes and dermal fibroblasts. Microarray analysis identified multifunctional genes modulated by steroids, quantitative RT-PCR (qRT-PCR) mRNA expression, enzymatic assay aromatase activity, scratch assay cell migration, immunocytochemistry α-smooth muscle actin (α-SMA), and collagen gel fibroblast contraction. All steroidogenic components were present, although only keratinocytes expressed the organic anion organic anion transporter protein (OATP) 2B1 transporter. Both expressed the G-protein-coupled estrogen receptor (GPER1). Steroids modulated multifunctional genes, up-regulating genes important in repair and aging [angiopoietin-like 4 (ANGPTL4), chemokine (C-X-C motif) ligand 1 (CXCL1), lamin B1 (LMNB1), and thioredoxin interacting protein (TXNIP)]. DHEA-sulfate (DHEA-S), DHEA, and 17β-estradiol stimulated keratinocyte and fibroblast migration at early (4 h) and late (24-48 h) time points, suggesting involvement of genomic and nongenomic signaling. Migration was blocked by aromatase and steroid sulfatase (STS) inhibitors confirming intracrine synthesis to estrogen. Testosterone had little effect, implying it is not an intermediate. Steroids stimulated fibroblast contraction but not α-SMA expression. Mechanical wounding reduced fibroblast aromatase activity but increased keratinocyte activity, amplifying the bioavailability of intracellular estrogen. Cultured fibroblasts and keratinocytes provide a biologically relevant model system to investigate the complex pathways of sex steroid intracrinology in human skin. © FASEB.

Carbon Dioxide Metabolism in Leaf Epidermal Tissue 1

PubMed Central

Willmer, C. M.; Pallas, J. E.; Black, C. C.

1973-01-01

A number of plant species were surveyed to obtain pure leaf epidermal tissue in quantity. Commelina communis L. and Tulipa gesnariana L. (tulip) were chosen for further work. Chlorophyll a/b ratios of epidermal tissues were 2.41 and 2.45 for C. communis and tulip, respectively. Phosphoenolpyruvate carboxylase, ribulose-1,5-diphosphate carboxylase, malic enzyme, and NAD+ and NADP+ malate dehydrogenases were assayed with epidermal tissue and leaf tissue minus epidermal tissue. In both species, there was less ribulose 1,5-diphosphate than phosphoenolpyruvate carboxylase activity in epidermal tissue whether expressed on a protein or chlorophyll basis whereas the reverse was true for leaf tissue minus epidermal tissue. In both species, malic enzyme activities were higher in epidermal tissue than in the remaining leaf tissue when expressed on a protein or chlorophyll basis. In both species, NAD+ and NADP+ malate dehydrogenase activities were higher in the epidermal tissue when expressed on a chlorophyll basis; however, on a protein basis, the converse was true. Microautoradiography of C. communis epidermis and histochemical tests for keto acids suggested that CO2 fixation occurred predominantly in the guard cells. The significance and possible location of the enzymes are discussed in relation to guard cell metabolism. Images PMID:16658581

Oncogenic Ras: A double-edged sword for human epidermal stem and transient amplifying cells

PubMed Central

Dellambra, Elena

2016-01-01

ABSTRACT The human epidermal clonal evolution, i.e. the transition from stem cells (SCs) to transient amplifying (TA)-cells and post-mitotic cells, is a continuous and tightly regulated process that ensures physiologic tissue homeostasis. The Ras family of small GTPases has a key role in skin homeostasis and tumorigenesis. Indeed, activating mutations in Ras genes have been found in human cutaneous squamous cell carcinomas (cSCCs) and in experimentally-induced murine cSCCs. In mouse models, the Ras signaling might lead to hyperproliferative phenotypes, including the development of cSCCs, depending on the nature of the founding cells. Tumor-initiating cells or Cancer Stem Cells (CSCs) have been demonstrated in murine and human cSCCs even if the mechanism of their development from normal SCs or TA-cells is not completely elucidated. Here, the relation between the Ras expression outcome and the clonogenic potential of the target keratinocyte is discussed. PMID:27111451

Heavy metal-induced cytotoxicity to cultured human epidermal keratinocytes and effects of antioxidants.

PubMed

Kappus, H; Reinhold, C

1994-04-01

Human epidermal keratinocytes which have been cultured were treated with the heavy metal ions of cadmium, mercury, copper and zinc. Cytotoxicity was measured either by protein estimation or by using the neutral red assay. Antioxidants were added in order to find out whether heavy metal-induced cytotoxicity is related to oxidative stress. All metals used showed considerable cytotoxic effects within 24 h in moderate concentrations. None of the antioxidants vitamin E (alpha-tocopherol), pyrogallol, propyl gallate, BHT or ebselen showed any protective or preventive effect. This indicates that oxidative stress may not be involved in the cytotoxicity induced by heavy metals in human epidermal keratinocytes. The cells used are, however, a valuable tool to study mechanisms of cytotoxicity.

Toxicity Assessment of Six Titanium Dioxide Nanoparticles in Human Epidermal Keratinocytes

EPA Science Inventory

Toxicity Assessment of Six Titanium Dioxide Nanoparticles in Human Epidermal Keratinocytes Nanoparticle uptake in cells may be an important determinant of their potential cytotoxic and inflammatory effects. Six commercial TiO2 NP (A=Alfa Aesar,10nm, A*=Alfa Aesar 32nm, B=P25 27...

Beneficial Effects of the Genus Aloe on Wound Healing, Cell Proliferation, and Differentiation of Epidermal Keratinocytes

PubMed Central

Uda, Junki; Kubo, Hirokazu; Nakajima, Yuka; Goto, Arisa; Akaki, Junji; Yoshida, Ikuyo; Matsuoka, Nobuya; Hayakawa, Takao

2016-01-01

Aloe has been used as a folk medicine because it has several important therapeutic properties. These include wound and burn healing, and Aloe is now used in a variety of commercially available topical medications for wound healing and skin care. However, its effects on epidermal keratinocytes remain largely unclear. Our data indicated that both Aloe vera gel (AVG) and Cape aloe extract (CAE) significantly improved wound healing in human primary epidermal keratinocytes (HPEKs) and a human skin equivalent model. In addition, flow cytometry analysis revealed that cell surface expressions of β1-, α6-, β4-integrin, and E-cadherin increased in HPEKs treated with AVG and CAE. These increases may contribute to cell migration and wound healing. Treatment with Aloe also resulted in significant changes in cell-cycle progression and in increases in cell number. Aloe increased gene expression of differentiation markers in HPEKs, suggesting roles for AVG and CAE in the improvement of keratinocyte function. Furthermore, human skin epidermal equivalents developed from HPEKs with medium containing Aloe were thicker than control equivalents, indicating the effectiveness of Aloe on enhancing epidermal development. Based on these results, both AVG and CAE have benefits in wound healing and in treatment of rough skin. PMID:27736988

Induction of keratinocyte IL-8 expression and secretion by IgG autoantibodies as a novel mechanism of epidermal neutrophil recruitment in a pemphigus variant

PubMed Central

O'toole, E A; Mak, L L; Guitart, J; Woodley, D T; Hashimoto, T; Amagai, M; Chan, L S

2000-01-01

A subset of pemphigus herpetiformis, a rare pemphigus variant, is characterized histopathologically by subcorneal acantholysis and neutrophilic infiltration. The mechanism of neutrophil infiltration is unknown, but chemokines such as IL-8 may play a role. We investigated the possible role of IL-8 in two such cases. Direct and indirect immunofluorescence studies demonstrated in vivo-bound and circulating IgG epithelial cell surface-binding autoantibodies, both predominated by IgG4 subclass. ELISA and immunoblotting studies revealed that the patients' IgG autoantibodies recognized recombinant desmoglein 1 but not desmoglein 3. Preadsorption of the patients' sera with recombinant desmoglein 1 completely removed the epidermal cell surface immunostaining. Significantly, immunohistochemistry demonstrated intense expression of IL-8, co-localized with in vivo-bound IgG, in the upper epidermis, where the acantholysis took place. Affinity-purified sera IgG from these two patients, a normal individual, and a pemphigus vulgaris patient containing desmoglein 1 autoantibodies, were incubated with normal human keratinocytes in vitro. Cells treated with these patients' IgG secreted a seven-to-nine-fold increase of IL-8 (30–37 pg/ml) compared with the controls (2–4 pg/ml) and expressed a higher intensity of cytoplasmic IL-8 staining. These data demonstrate a novel functional role for IL-8 in the pathogenesis of the neutrophil-dominant subset of pemphigus herpetiformis. The autoantibody-induced epidermal cell IL-8 expression may represent a novel mechanism of epidermal neutrophil recruitment. PMID:10606986

Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells.

PubMed

Alper, O; De Santis, M L; Stromberg, K; Hacker, N F; Cho-Chung, Y S; Salomon, D S

2000-11-15

Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness. Copyright 2000 Wiley-Liss, Inc.

Lowering relative humidity level increases epidermal protein deimination and drives human filaggrin breakdown.

PubMed

Cau, Laura; Pendaries, Valérie; Lhuillier, Emeline; Thompson, Paul R; Serre, Guy; Takahara, Hidenari; Méchin, Marie-Claire; Simon, Michel

2017-05-01

Deimination (also known as citrullination), the conversion of arginine in a protein to citrulline, is catalyzed by a family of enzymes called peptidylarginine deiminases (PADs). Three PADs are expressed in the epidermis, one of their targets being filaggrin. Filaggrin plays a central role in atopic dermatitis and is a key protein for the epidermal barrier. It aggregates keratins and is cross-linked to cornified envelopes. Following its deimination, it is totally degraded to release free amino acids, contributing to the natural moisturizing factor (NMF). The mechanisms controlling this multistep catabolism in human are unknown. To test whether external humidity plays a role, and investigate the molecular mechanisms involved. Specimens of reconstructed human epidermis (RHEs) produced in humid or dry conditions (>95% or 30-50% relative humidity) were compared. RHEs produced in the dry condition presented structural changes, including a thicker stratum corneum and a larger amount of keratohyalin granules. The transepidermal water loss and the stratum corneum pH were decreased whereas the quantity of NMF was greater. This highly suggested that filaggrin proteolysis was up-regulated. The expression/activity of the proteases involved in filaggrin breakdown did not increase while PAD1 expression and the deimination rate of proteins, including filaggrin, were drastically enhanced. Partial inhibition of PADs with Cl-amidine reversed the effect of dryness on filaggrin breakdown. These results demonstrate the importance of external humidity in the control of human filaggrin metabolism, and suggest that deimination plays a major role in this regulation. Copyright © 2017 Japanese Society for Investigative Dermatology. All rights reserved.

Barrier Requirements as the Evolutionary “Driver” of Epidermal Pigmentation in Humans

PubMed Central

ELIAS, PETER M.; MENON, GOPINATHAN; WETZEL, BRUCE K.; WILLIAMS, JOHN (JACK) W.

2011-01-01

Current explanations for the development of epidermal pigmentation during human evolution are not tenable as stand-alone hypotheses. Accordingly, we assessed instead whether xeric- and UV-B-induced stress to the epidermal permeability barrier, critical to survival in a terrestrial environment, could have “driven” the development of epidermal pigmentation. (1) Megadroughts prevailed in central Africa when hominids expanded into open savannahs [≈1.5–0.8 million years ago], resulting in sustained exposure to both extreme aridity and erythemogenic UV-B, correlating with genetic evidence that pigment developed ≈1.2 million years ago. (2) Pigmented skin is endowed with enhanced permeability barrier function, stratum corneum integrity/cohesion, and a reduced susceptibility to infections. The enhanced function of pigmented skin can be attributed to the lower pH of the outer epidermis, likely due to the persistence of (more-acidic) melanosomes into the outer epidermis, as well as the conservation of genes associated with eumelanin synthesis and melanosome acidification (e.g., TYR, OCA2 [p protein], SLC24A5, SLC45A2, MATP) in pigmented populations. Five keratinocyte-derived signals (stem cell factor⇒KIT; FOXn1⇒FGF2; IL-1α, NGF, and p53) are potential candidates to have stimulated the sequential development of epidermal pigmentation in response to stress to the barrier. We summarize evidence here that epidermal interfollicular pigmentation in early hominids likely evolved in response to stress to the permeability barrier. PMID:20209486

Granulocyte colony-stimulating-factor-induced psoriasiform dermatitis resembles psoriasis with regard to abnormal cytokine expression and epidermal activation.

PubMed

Mössner, R; Beckmann, I; Hallermann, C; Neumann, C; Reich, K

2004-06-01

Psoriasis is a chronic inflammatory skin disorder characterized by accumulation of Th1-type T cells and neutrophils, regenerative keratinocyte proliferation and differentiation, and enhanced epidermal production of antimicrobial peptides. The underlying cause is unknown, but there are some similarities with the immunologic defense program against bacteria. Development of psoriasiform skin lesions has been reported after administration of granulocyte colony-stimulating factor (G-CSF), a cytokine induced in monocytes by bacterial antigens. To further investigate the relation between this type of cytokine-induced dermatitis and psoriasis, we analyzed the cutaneous cytokine profile [tumor necrosis factor-alpha (TNF-alpha), interferon-gamma, transforming growth factor-beta1 (TGF-beta1), interleukin-10 (IL-10), IL-12p35 and p40, and IL-8] and expression of markers of epidermal activation [Ki-67, cytokeratin-16, major histocompatibility complex (MHC) class II, intercellular adhesion molecule-1 (ICAM-1)] in a patient who developed G-CSF-induced psoriasiform dermatitis by using quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistology. The histologic picture resembled psoriasis with regard to epidermal hyperparakeratosis and the accumulation of lymphocytes in the upper corium. CD8(+) T cells were found to infiltrate the epidermis which was associated with an aberrant expression of Ki-67, cytokeratin-16, MHC class II, and ICAM-1 on adjacent keratinocytes. As compared to normal skin (n = 7), there was an increased expression of TNF-alpha, IL-12p40, and IL-8, a decreased expression of TGF-beta1, and a lack of IL-10, similar to the findings in active psoriasis (n = 8). Therefore, G-CSF may cause a lymphocytic dermatitis that, similar to psoriasis, is characterized by a pro-inflammatory Th1-type cytokine milieu and an epidermal phenotype indicative of aberrant maturation and acquisition of non-professional immune functions.

Effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro.

PubMed

Lee, J S; Kim, J M; Hong, E K; Kim, S-O; Yoo, Y-J; Cha, J-H

2009-02-01

A growing amount of attention has been placed on periodontal regeneration and wound healing for periodontal therapy. This study was conducted in an effort to determine the effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro. Human periodontal ligament cells were acquired from explant tissue of human healthy periodontal ligament. After the wounding of periodontal ligament cells, the change in expression of heparin-binding epidermal growth factor-like growth factor and epidermal growth factor receptors 1-4 mRNA was assessed. The effects of heparin-binding epidermal growth factor-like growth factor on periodontal ligament cell proliferation and repopulation were assessed in vitro via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and by photographing the injuries, respectively. Extracellular signal-regulated kinase (Erk)1/2, p38 and Akt phosphorylation was characterized via western blotting. Scratch wounding resulted in a significant up-regulation of heparin-binding epidermal growth factor-like growth factor mRNA expression, whereas wounding had no effect on the expression levels of epidermal growth factor receptors 1-4. Interestingly, no expression of epidermal growth factor receptors 2 and 4 was detectable prior to or after wounding. Heparin-binding epidermal growth factor-like growth factor treatment promoted the proliferation and repopulation of periodontal ligament cells. The scratch wounding also stimulated the phosphorylation of Erk1/2 and p38, but not of Akt, in periodontal ligament cells, and heparin-binding epidermal growth factor-like growth factor treatment applied after wounding amplified and extended the activations of Erk1/2 and p38, but not of Akt. Furthermore, Erk1/2 inhibition blocked the process of cell repopulation induced by heparin-binding epidermal growth factor-like growth factor, whereas the

Isolation, Culture, and Motility Measurements of Epidermal Melanocytes from GFP-Expressing Reporter Mice.

PubMed

Dagnino, Lina; Crawford, Melissa

2018-03-27

In this article, we provide a method to isolate primary epidermal melanocytes from reporter mice, which also allow targeted gene inactivation. The mice from which these cells are isolated are bred into a Rosa26 mT/mG reporter background, which results in GFP expression in the targeted melanocytic cell population. These cells are isolated and cultured to >95% purity. The cells can be used for gene expression studies, clonogenic experiments, and biological assays, such as capacity for migration. Melanocytes are slow moving cells, and we also provide a method to measure motility using individual cell tracking and data analysis.

Epidermal growth factor-containing fibulin-like extracellular matrix protein 1 expression and regulation in uterine leiomyoma.

PubMed

Marsh, Erica E; Chibber, Shani; Wu, Ju; Siegersma, Kendra; Kim, Julie; Bulun, Serdar

2016-04-01

To determine the presence, differential expression, and regulation of epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) in uterine leiomyomas. Laboratory in vivo and in vitro study with the use of human leiomyoma and myometrial tissue and primary cells. Academic medical center. Leiomyoma and myometrial tissue samples and cultured cells. 5-Aza-2'-deoxycytidine (5-aza-dC) treatment. Fold-change difference between EFEMP1 and fibulin-3 expression in leiomyoma tissue and cells compared with matched myometrial samples, and fold-change difference in EFEMP1 expression with 5-Aza-dC treatment. In vivo, EFEMP1 expression was 3.19-fold higher in myometrial tissue than in leiomyoma tissue. EFEMP1 expression in vitro was 5.03-fold higher in myometrial cells than in leiomyoma cells. Western blot and immunohistochemistry staining of tissue and cells confirmed similar findings in protein expression. Treatment of leiomyoma cells with 5-Aza-dC resulted in increased expression of EFEMP1 in vitro. The EFEMP1 gene and its protein product, fibulin-3, are both significantly down-regulated in leiomyoma compared with myometrium when studied both in vivo and in vitro. The increase in EFEMP1 expression in leiomyoma cells with 5-Aza-dC treatment suggest that differential methylation is responsible, in part, for the differences seen in gene expression. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation

NASA Technical Reports Server (NTRS)

Leong, J.; Hughes-Fulford, M.; Rakhlin, N.; Habib, A.; Maclouf, J.; Goldyne, M. E.

1996-01-01

Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

Expression, purification, and characterization of recombinant human and murine milk fat globule-epidermal growth factor-factor 8.

PubMed

Castellanos, Erick R; Ciferri, Claudio; Phung, Wilson; Sandoval, Wendy; Matsumoto, Marissa L

2016-08-01

Milk fat globule-epidermal growth factor-factor 8 (MFG-E8), as its name suggests, is a major glycoprotein component of milk fat globules secreted by the mammary epithelium. Although its role in milk fat production is unclear, MFG-E8 has been shown to act as a bridge linking apoptotic cells to phagocytes for removal of these dying cells. MFG-E8 is capable of bridging these two very different cell types via interactions through both its epidermal growth factor (EGF)-like domain(s) and its lectin-type C domains. The EGF-like domain interacts with αVβ3 and αVβ5 integrins on the surface of phagocytes, whereas the C domains bind phosphatidylserine found on the surface of apoptotic cells. In an attempt to purify full-length, recombinant MFG-E8 expressed in either insect cells or CHO cells, we find that it is highly aggregated. Systematic truncation of the domain architecture of MFG-E8 indicates that the C domains are mainly responsible for the aggregation propensity. Addition of Triton X-100 to the conditioned cell culture media allowed partial recovery of non-aggregated, full-length MFG-E8. A more comprehensive detergent screen identified CHAPS as a stabilizer of MFG-E8 and allowed purification of a significant portion of non-aggregated, full-length protein. The CHAPS-stabilized recombinant MFG-E8 retained its natural ability to bind both αVβ3 and αVβ5 integrins and phosphatidylserine suggesting that it is properly folded and active. Herein we describe an efficient purification method for production of non-aggregated, full-length MFG-E8. Copyright © 2016 Elsevier Inc. All rights reserved.

Ligand-Independent Epidermal Growth Factor Receptor Overexpression Correlates with Poor Prognosis in Colorectal Cancer.

PubMed

Yun, Sumi; Kwak, Yoonjin; Nam, Soo Kyung; Seo, An Na; Oh, Heung-Kwon; Kim, Duck-Woo; Kang, Sung-Bum; Lee, Hye Seung

2018-01-17

Molecular treatments targeting epidermal growth factor receptors (EGFRs) are important strategies for advanced colorectal cancer (CRC). However, clinicopathologic implications of EGFRs and EGFR ligand signaling have not been fully evaluated. We evaluated the expression of EGFR ligands and correlation with their receptors, clinicopathologic factors, and patients' survival with CRC. The expression of EGFR ligands, including heparin binding epidermal growth factor like growth factor (HBEGF), transforming growth factor (TGF), betacellulin, and epidermal growth factor (EGF), were evaluated in 331 consecutive CRC samples using mRNA in situ hybridization (ISH). We also evaluated the expression status of EGFR, human epidermal growth factor receptor 2 (HER2), HER3, and HER4 using immunohistochemistry and/or silver ISH. Unlike low incidences of TGF (38.1%), betacellulin (7.9%), and EGF (2.1%), HBEGF expression was noted in 62.2% of CRC samples. However, the expression of each EGFR ligand did not reveal significant correlations with survival. The combined analyses of EGFR ligands and EGFR expression indicated that the ligands‒/EGFR+ group showed a significant association with the worst disease-free survival (DFS, p=0.018) and overall survival (OS, p=0.005). It was also an independent, unfavorable prognostic factor for DFS (p=0.026) and OS (p=0.007). Additionally, HER4 nuclear expression, regardless of ligand expression, was an independent, favorable prognostic factor for DFS (p=0.034) and OS (p=0.049), by multivariate analysis. Ligand-independent EGFR overexpression was suggested to have a significant prognostic impact; thus, the expression status of EGFR ligands, in addition to EGFR, might be necessary for predicting patients' outcome in CRC.

Human papillomavirus E6/E7 oncogenes promote mouse ear regeneration by increasing the rate of wound re-epithelization and epidermal growth.

PubMed

Valencia, Concepción; Bonilla-Delgado, José; Oktaba, Katarzyna; Ocádiz-Delgado, Rodolfo; Gariglio, Patricio; Covarrubias, Luis

2008-12-01

Mammals have limited regeneration capacity. We report here that, in transgenic mice (Tg(bK6-E6/E7)), the expression of the E6/E7 oncogenes of human papilloma virus type 16 (HPV16) under the control of the bovine keratin 6 promoter markedly improves the mouse's capacity to repair portions of the ear after being wounded. Increased repair capacity correlates with an increased number of epidermal proliferating cells. In concordance with the expected effects of the E6 and E7 oncogenes, levels of p53 decreased and those of p16 in epidermal cells increased. In addition, we observed that wound re-epithelization proceeded faster in transgenic than in wild-type animals. After the initial re-epithelization, epidermal cell migration from the intact surrounding tissue appears to be a major contributor to the growing epidermis, especially in the repairing tissue of transgenic mice. We also found that there is a significantly higher number of putative epidermal stem cells in Tg(bK6-E6/E7) than in wild-type mice. Remarkably, hair follicles and cartilage regenerated within the repaired ear tissue, without evidence of tumor formation. We propose that the ability to regenerate ear portions is limited by the capacity of the epidermis to repair itself and grow.

Terminal epidermal differentiation is regulated by the interaction of Fra-2/AP-1 with Ezh2 and ERK1/2

PubMed Central

Wurm, Stefanie; Zhang, Jisheng; Guinea-Viniegra, Juan; García, Fernando; Muñoz, Javier; Bakiri, Latifa; Ezhkova, Elena

2015-01-01

Altered epidermal differentiation characterizes numerous skin diseases affecting >25% of the human population. Here we identified Fra-2/AP-1 as a key regulator of terminal epidermal differentiation. Epithelial-restricted, ectopic expression of Fra-2 induced expression of epidermal differentiation genes located within the epidermal differentiation complex (EDC). Moreover, in a papilloma-prone background, a reduced tumor burden was observed due to precocious keratinocyte differentiation by Fra-2 expression. Importantly, loss of Fra-2 in suprabasal keratinocytes is sufficient to cause skin barrier defects due to reduced expression of differentiation genes. Mechanistically, Fra-2 binds and transcriptionally regulates EDC gene promoters, which are co-occupied by the transcriptional repressor Ezh2. Fra-2 remains transcriptionally inactive in nondifferentiated keratinocytes, where it was found monomethylated and dimethylated on Lys104 and interacted with Ezh2. Upon keratinocyte differentiation, Fra-2 is C-terminally phosphorylated on Ser320 and Thr322 by ERK1/2, leading to transcriptional activation. Thus, the induction of epidermal differentiation by Fra-2 is controlled by a dual mechanism involving Ezh2-dependent methylation and activation by ERK1/2-dependent phosphorylation. PMID:25547114

Human epidermis is a novel site of phospholipase B expression.

PubMed

Maury, Eric; Prévost, Marie Claude; Nauze, Michel; Redoulès, Daniel; Tarroux, Roger; Charvéron, Marie; Salles, Jean Pierre; Perret, Bertrand; Chap, Hugues; Gassama-Diagne, Ama

2002-07-12

Phospholipase B (PLB) is an enzyme that displays both phospholipase A(2) and lysophospholipase activities. Analysis of human epidermis homogenates indicated the presence of a 97 kDa PLB protein, as well as a phospholipase A(2) activity, both being enriched in the soluble fraction. Immunolabelling and in situ hybridization experiments showed that this enzyme is expressed in the different layers of epidermis with an accumulation at the dermo-epidermis junction. RT-PCR data indicated that PLB is specifically expressed in natural and reconstructed epidermis. By 3'-RACE-PCR and screening of human genome databases, we obtained a 3600 bp cDNA coding for human PLB highly homologous to already described intestinal brush border PLBs. These data led us to conclude that the soluble PLB corresponds to a proteolytic cleavage of the membrane anchored protein. Altogether, our results provide the first characterization of human PLB which should play an important role in epidermal barrier function.

The expression of proinflammatory genes in epidermal keratinocytes is regulated by hydration status.

PubMed

Xu, Wei; Jia, Shengxian; Xie, Ping; Zhong, Aimei; Galiano, Robert D; Mustoe, Thomas A; Hong, Seok J

2014-04-01

Mucosal wounds heal more rapidly, exhibit less inflammation, and are associated with minimal scarring when compared with equivalent cutaneous wounds. We previously demonstrated that cutaneous epithelium exhibits an exaggerated response to injury compared with mucosal epithelium. We hypothesized that treatment of injured skin with a semiocclusive dressing preserves the hydration of the skin and results in a wound healing phenotype that more closely resembles that of mucosa. Here we explored whether changes in hydration status alter epidermal gene expression patterns in rabbit partial-thickness incisional wounds. Using microarray studies on injured epidermis, we showed that global gene expression patterns in highly occluded versus non-occluded wounds are distinct. Many genes including IL-1β, IL-8, TNF-α (tumor necrosis factor-α), and COX-2 (cyclooxygenase 2) are upregulated in non-occluded wounds compared with highly occluded wounds. In addition, decreased levels of hydration resulted in an increased expression of proinflammatory genes in human ex vivo skin culture (HESC) and stratified keratinocytes. Hierarchical analysis of genes using RNA interference showed that both TNF-α and IL-1β regulate the expression of IL-8 through independent pathways in response to reduced hydration. Furthermore, both gene knockdown and pharmacological inhibition studies showed that COX-2 mediates the TNF-α/IL-8 pathway by increasing the production of prostaglandin E2 (PGE2). IL-8 in turn controls the production of matrix metalloproteinase-9 in keratinocytes. Our data show that hydration status directly affects the expression of inflammatory signaling in the epidermis. The identification of genes involved in the epithelial hydration pathway provides an opportunity to develop strategies to reduce scarring and optimize wound healing.

Protection on Skin Aging Mediated by Antiapoptosis Effects of the Water Lily (Nymphaea Tetragona Georgi) via Reactive Oxygen Species Scavenging in Human Epidermal Keratinocytes.

PubMed

Park, Gunhyuk; Sim, Yeomoon; Lee, Wonil; Sung, Sang Hyun; Oh, Myung Sook

2016-01-01

The water lily (WL) is found in Europe, Asia, and North America. WL reportedly has various pharmacological activities that improve the activities of daily life in humans. To our knowledge, no previous study has investigated about the aspect of protection on skin aging due to the mitochondria-mediated antiapoptosis effects of WL rhizome extract (WLRE) on human epidermal keratinocytes. Human epidermal keratinocytes cells were treated with WLRE (100, 200, and 400 μg/ml) for 1 h and then with ultraviolet radiation B (UVB) (50 mJ/cm2) for another 23 h. The levels of lactate dehydrogenase, reactive oxygen species (ROS), MitoTracker, caspase-3, and glutathione were analyzed spectrophotometrically. Also, the levels of B-cell lymphoma 2 (Bcl-2) family proteins were determined with immunohistochemistry or western blotting. We investigated the protective effects of WLRE against UVB-induced mitochondria-mediated apoptosis. WLRE significantly and concentrations-dependently reduced UVB-induced apoptotic cytotoxicity. Furthermore, WLRE decreased ROS generation, mitochondrial dysfunction, Bcl-2-associated X protein levels, and cytochrome c release from mitochondria while increasing Bcl-2 protein levels as assessed. Moreover, WLRE inhibited caspase-3 activity and expression, indicating the inhibition of the apoptotic cascade, and induced increased levels of total glutathione, heme oxygenase 1, and radical-scavenging activity. Together, these results demonstrate that WLRE can protect human epidermal keratinocytes against UVB-induced mitochondria-mediated apoptosis by regulating ROS-eliminating pathways. © 2016 S. Karger AG, Basel.

Photoacoustic measurement of epidermal melanin

NASA Astrophysics Data System (ADS)

Viator, John A.; Svaasand, Lars O.; Aguilar, Guillermo; Choi, Bernard; Nelson, J. Stuart

2003-06-01

Most dermatologic laser procedures must consider epidermal melanin, as it is a broadband optical absorber which affects subsurface fluence, effectively limiting the amount of light reaching the dermis and targeted chromophores. An accurate method for quantifying epidermal melanin content would aid clinicians in determining proper light dosage for therapeutic laser procedures. While epidermal melanin content has been quantified non-invasively using optical methods, there is currently no way to determine the melanin distribution in the epidermis. We have developed a photoacoustic probe that uses a Q-switched, frequency doubled Nd:YAG laser operating at 532nm to generate acoustic pulses in skin in vivo. The probe contained a piezoelectric element that detected photoacoustic waves which were then analyzed for epidermal melanin content, using a photoacoustic melanin index (PAMI). We tested 15 human subjects with skin types I--VI using the photoacoustic probe. We also present photoacoustic data for a human subject with vitiligo. Photoacoustic measurement showed melanin in the vitiligo subject was almost completely absent.

Epidermal growth factor increases cortisol production and type II 3 beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase expression in human adrenocortical carcinoma cells: evidence for a Stat5-dependent mechanism.

PubMed

Feltus, F Alex; Kovacs, William J; Nicholson, Wendell; Silva, Corrine M; Nagdas, Subir K; Ducharme, Nicole A; Melner, Michael H

2003-05-01

We tested the ability of epidermal growth factor (EGF) to regulate a key enzyme in the adrenal synthesis of glucocorticoids: human type II 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3 beta HSD). EGF treatment (25 ng/ml) of human adrenocortical carcinoma cells (H295R) resulted in a 5-fold increase in cortisol production and a corresponding 2-fold increase in 3 beta HSD mRNA. Experiments were performed to determine whether EGF is acting through a previously identified signal transducer and activator of transcription 5 (Stat5)-responsive element located from -110 to -118 in the human type II 3 beta HSD promoter. A Stat5 expression construct was cotransfected with a 3 beta HSD-chloramphenol acetyltransferase (CAT) reporter construct comprised of nucleotides -301-->+45 of the human type II 3 beta HSD promoter linked to the CAT reporter gene sequence. The addition of EGF at doses as low as 10 ng/ml resulted in an 11- to 15-fold increase in CAT activity. The introduction of 3-bp point mutations into critical nucleotides in the Stat5 response element obviated the EGF response. Either Stat5a or Stat5b isoforms induced CAT reporter expression upon treatment with EGF. These results demonstrate the ability of EGF to regulate the expression of a critical enzyme (3 beta HSD) in the production of cortisol and suggest a molecular mechanism by which this regulation occurs.

Epidermal elafin expression is an indicator of poor prognosis in cutaneous graft-versus-host disease.

PubMed

Brüggen, Marie-Charlotte; Petzelbauer, Peter; Greinix, Hildegard; Contassot, Emmanuel; Jankovic, Dragana; French, Lars; Socié, Gérard; Rabitsch, Werner; Kuzmina, Zoya; Kalhs, Peter; Knobler, Robert; Stingl, Georg; Stary, Georg

2015-04-01

Graft-versus-host disease (GVHD) remains a common and potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation. In the skin, GVHD can present in an acute (aGVHD), chronic lichenoid (clGVHD), or chronic sclerotic form (csGVHD). Measuring peripheral blood levels of the keratinocyte-derived protease inhibitor elafin has recently emerged as a promising tool for the diagnosis of cutaneous aGVHD. We evaluated whether the analysis of elafin expression in skin would allow distinguishing aGVHD from drug hypersensitivity rashes (DHR) and whether cutaneous elafin expression would correlate with disease severity or altered prognosis of aGVHD and clGVHD/csGVHD. Skin biopsies from aGVHD (n=22), clGVHD (n=15), csGVHD (n=7), and DHR (n=10) patients were collected and epidermal elafin expression and its association with diverse clinical/histological parameters were analyzed. Acute GVHD and DHR displayed varying degrees of elafin expression. No elafin was detectable in csGVHD, whereas the molecule was increased in clGVHD as compared with aGVHD. Elafin-high aGVHD/clGVHD lesions presented with epidermal thickening and were associated with poor prognosis-i.e., decreased overall survival in aGVHD and corticosteroid resistance in clGVHD. Although cutaneous elafin does not seem to discriminate aGVHD from DHR lesions, our study strongly suggests an association between cutaneous elafin expression and poor prognosis for patients with cutaneous GVHD.

Human Adipose Mesenchymal Cells Inhibit Melanocyte Differentiation and the Pigmentation of Human Skin via Increased Expression of TGF-β1.

PubMed

Klar, Agnes S; Biedermann, Thomas; Michalak, Katarzyna; Michalczyk, Teresa; Meuli-Simmen, Claudia; Scherberich, Arnaud; Meuli, Martin; Reichmann, Ernst

2017-12-01

There is accumulating evidence that interactions between epidermal melanocytes and stromal cells play an important role in the regulation of skin pigmentation. In this study we established a pigmented dermo-epidermal skin model, melDESS, of human origin to investigate the effects of distinct stromal cells on melanogenesis. melDESS is a complex, clinically relevant skin equivalent composed of an epidermis containing both melanocytes and keratinocytes. Its dermal compartment consists either of adipose tissue-derived stromal cells, dermal fibroblasts (Fbs), or a mixture of both cell types. These skin substitutes were transplanted for 5 weeks on the backs of immuno-incompetent rats and analyzed. Gene expression and Western blot analyses showed a significantly higher expression of transforming growth factor-β1 by adipose tissue-derived stromal cells compared with dermal Fbs. In addition, we showed that melanocytes responded to the increased levels of transforming growth factor-β1 by down-regulating the expression of key melanogenic enzymes such as tyrosinase. This caused decreased melanin synthesis and, consequently, greatly reduced pigmentation of melDESS. The conclusions are of utmost clinical relevance, namely that adipose tissue-derived stromal cells derived from the hypodermis fail to appropriately interact with epidermal melanocytes, thus preventing the sustainable restoration of the patient's native skin color in bioengineered skin grafts. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Recombinant expression of extracellular domain of mutant Epidermal Growth Factor Receptor in prokaryotic and baculovirus expression systems.

PubMed

Vettath, Sunitha Kodengil; Shivashankar, Gaganashree; Menon, Krishnakumar N; Vijayachandran, Lakshmi S

2018-04-15

Epidermal Growth Factor Receptor variant III (EGFRvIII) is a tumor specific antigen detected in various tumors including gliomas, breast cancer, lung cancer, head and neck squamous cell carcinoma (HNSCC). Screening of EGFRvIII targeting drug molecules can be accelerated by developing drug screening platforms using recombinantly expressed protein. Choice of expression system is one of the major factors deciding the success of recombinant expression of a protein. In our study, we have tried to express and purify the extracellular domain (ECD) of this highly unstable protein using bacterial and baculovirus expression systems to select the expression system suited for our purpose. Even though the protein was successfully expressed in prokaryotic system, purification could be done only under denaturing conditions. But in the baculovirus expression system, the protein was expressed in soluble form and could be purified under native conditions, with single step of purification. Based on our results, we conclude that insect cells are better choice over E. coli cells for expressing EGFRvIII ECD in soluble form. This study provides insights for other researchers involved in expression of similar unstable membrane proteins, on selecting the best expression system and challenges involved. Copyright © 2018 Elsevier B.V. All rights reserved.

Induction of IL-10 gene expression in human keratinocytes by UVB exposure in vivo and in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Enk, C.D.; Blauvelt, A.; Katz, S.I.

Numerous studies have demonstrated that ultraviolet B (UVB) irradiation has profound effects on the skin and systemic immune systems. Because many of the effects of UVB result in suppression of contact sensitivity responses and because IL-10 induces a Th2 rather than a Th1 response, we sought to determine whether UVB irradiation induces IL-10 transcription and subsequent protein secretion by human epidermal cells. Skin of nine volunteers was exposed to UVB or sham irradiation, and epidermal cell suspensions were prepared from suction blister roofs 24 h thereafter. mRNA was extracted using oligo dT-coated magnetic beads, and IL-10 cDNA was amplified withmore » a sensitive RT-PCR technique. We found that IL-10 was constitutively expressed by epidermal cells in 5 of 9 volunteers and that IL-10 message was up-regulated by UVB exposure in all experiments. Since epidermis consists of a heterogeneous cell population with distinct cytokine profiles, we determined whether UVB caused enhanced IL-10 transcription and protein secretion in human keratinocyte cultures. In these experiments, IL-10 was constitutively expressed by keratinocytes and UVB up-regulated IL-10 gene expression in a dose-dependent manner 24 h after in vitro irradiation, coinciding with IL-10 protein secretion into the culture supernatants. Taken together, the findings indicate that UVB irradiation induces IL-10 in human keratinocytes and suggest that keratinocyte-derived IL-10 may be an important component of the immunosuppression that results from UVB irradiation. 55 refs., 5 figs.« less

Evaluation of cultured human dermal- and dermo-epidermal substitutes focusing on extracellular matrix components: Comparison of protein and RNA analysis.

PubMed

Oostendorp, Corien; Meyer, Sarah; Sobrio, Monia; van Arendonk, Joyce; Reichmann, Ernst; Daamen, Willeke F; van Kuppevelt, Toin H

2017-05-01

Treatment of full-thickness skin defects with split-thickness skin grafts is generally associated with contraction and scar formation and cellular skin substitutes have been developed to improve skin regeneration. The evaluation of cultured skin substitutes is generally based on qualitative parameters focusing on histology. In this study we focused on quantitative evaluation to provide a template for comparison of human bio-engineered skin substitutes between clinical and/or research centers, and to supplement histological data. We focused on extracellular matrix proteins since these components play an important role in skin regeneration. As a model we analyzed the human dermal substitute denovoDerm and the dermo-epidermal skin substitute denovoSkin. The quantification of the extracellular matrix proteins type III collagen and laminin 5 in tissue homogenates using western blotting analysis and ELISA was not successful. The same was true for assaying lysyl oxidase, an enzyme involved in crosslinking of matrix molecules. As an alternative, gene expression levels were measured using qPCR. Various RNA isolation procedures were probed. The gene expression profile for specific dermal and epidermal genes could be measured reliably and reproducibly. Differences caused by changes in the cell culture conditions could easily be detected. The number of cells in the skin substitutes was measured using the PicoGreen dsDNA assay, which was found highly quantitative and reproducible. The (dis) advantages of assays used for quantitative evaluation of skin substitutes are discussed. Copyright © 2016 Elsevier Ltd and ISBI. All rights reserved.

Age-associated decrease in GDNF and its cognate receptor GFRα-1 protein expression in human skin.

PubMed

Adly, Mohamed A; Assaf, Hanan A; Hussein, Mahmoud Rezk Abdelwahed

2016-06-01

Glial cell line-derived neurotrophic factor (GDNF) and its cognate receptor (GFRα-1) are expressed in normal human skin. They are involved in murine hair follicle morphogenesis and cycling control. We hypothesize that 'GDNF and GFRα-1 protein expression in human skin undergoes age-associated alterations. To test our hypothesis, the expression of these proteins was examined in human skin specimens obtained from 30 healthy individuals representing three age groups: children (5-18 years), adults (19-60 years) and the elderly (61-81 years). Immunofluorescent and light microscopic immunohistologic analyses were performed using tyramide signal amplification and avidin-biotin complex staining methods respectively. GDNF mRNA expression was examined by RT-PCR analysis. GDNF mRNA and protein as well as GFRα-1 protein expressions were detected in normal human skin. We found significantly reduced epidermal expression of these proteins with ageing. In the epidermis, the expression was strong in the skin of children and declined gradually with ageing, being moderate in adults and weak in the elderly. In children and adults, the expression of both GDNF and GFRα-1 proteins was strongest in the stratum basale and decreased gradually towards the surface layers where it was completely absent in the stratum corneum. In the elderly, GDNF and GFRα-1 protein expression was confined to the stratum basale. In the dermis, both GDNF and GFRα-1 proteins had strong expressions in the fibroblasts, sweat glands, sebaceous glands, hair follicles and blood vessels regardless of the age. Thus there is a decrease in epidermal GDNF and GFRα-1 protein expression in normal human skin with ageing. Our findings suggest that the consequences of this is that GFRα-1-mediated signalling is altered during the ageing process. The clinical and therapeutic ramifications of these observations mandate further investigations. © 2016 The Authors. International Journal of Experimental Pathology © 2016

The mysterious human epidermal cell cycle, or an oncogene-induced differentiation checkpoint

PubMed Central

Gandarillas, Alberto

2012-01-01

Fifteen years ago, we reported that proto-oncogene MYC promoted differentiation of human epidermal stem cells, a finding that was surprising to the MYC and the skin research communities. MYC was one of the first human oncogenes identified, and it had been strongly associated with proliferation. However, it was later shown that MYC could induce apoptosis under low survival conditions. Currently, the notion that MYC promotes epidermal differentiation is widely accepted, but the cell cycle mechanisms that elicit this function remain unresolved. We have recently reported that keratinocytes respond to cell cycle deregulation and DNA damage by triggering terminal differentiation. This mechanism might constitute a homeostatic protection face to cell cycle insults. Here, I discuss recent and not-so-recent evidence suggesting the existence of a largely unexplored oncogene-induced differentiation response (OID) analogous to oncogene-induced apoptosis (OIA) or senescence (OIS). In addition, I propose a model for the role of the cell cycle in skin homeostasis maintenance and for the dual role of MYC in differentiation. PMID:23114621

Regenerative and reparative effects of human chorion-derived stem cell conditioned medium on photo-aged epidermal cells

PubMed Central

Li, Qiankun; Chen, Yan; Ma, Kui; Zhao, Along; Zhang, Cuiping; Fu, Xiaobing

2016-01-01

ABSTRACT Epidermal cells are an important regenerative source for skin wound healing. Aged epidermal cells have a low ability to renew themselves and repair skin injury. Ultraviolet (UV) radiation, particularly UVB, can cause photo-aging of the skin by suppressing the viability of human epidermal cells. A chorion-derived stem cell conditioned medium (CDSC-CNM) is thought to have regenerative properties. This study aimed to determine the regenerative effects of CDSC-CNM on UVB-induced photo-aged epidermal cells. Epidermal cells were passaged four times and irradiated with quantitative UVB, and non-irradiated cells served as a control group. Cells were then treated with different concentrations of CDSC-CNM. Compared to the non-irradiated group, the proliferation rates and migration rates of UVB-induced photo-aged epidermal cells significantly decreased (p < 0.05) with increasing intracellular radical oxygen species (ROS) generation and DNA damage. After treatment with CDSC-CNM, photo-aged epidermal cells significantly improved their viability, and their ROS generation and DNA damage decreased. The secretory factors in CDSC-CNM, including epidermal growth factor (EGF), transforming growth factor-β (TGF-β), interleukin (IL)-6, and IL-8 and the related signaling pathway protein levels, increased compared to the control medium (CM). The potential regenerative and reparative effects of CDSC-CNM indicate that it may be a candidate material for the treatment of prematurely aged skin. The functions of the secretory factors and the mechanisms of CDSC-CNM therapy deserve further attention. PMID:27097375

Local Adaptation of Sun-Exposure-Dependent Gene Expression Regulation in Human Skin.

PubMed

Kita, Ryosuke; Fraser, Hunter B

2016-10-01

Sun-exposure is a key environmental variable in the study of human evolution. Several skin-pigmentation genes serve as classical examples of positive selection, suggesting that sun-exposure has significantly shaped worldwide genomic variation. Here we investigate the interaction between genetic variation and sun-exposure, and how this impacts gene expression regulation. Using RNA-Seq data from 607 human skin samples, we identified thousands of transcripts that are differentially expressed between sun-exposed skin and non-sun-exposed skin. We then tested whether genetic variants may influence each individual's gene expression response to sun-exposure. Our analysis revealed 10 sun-exposure-dependent gene expression quantitative trait loci (se-eQTLs), including genes involved in skin pigmentation (SLC45A2) and epidermal differentiation (RASSF9). The allele frequencies of the RASSF9 se-eQTL across diverse populations correlate with the magnitude of solar radiation experienced by these populations, suggesting local adaptation to varying levels of sunlight. These results provide the first examples of sun-exposure-dependent regulatory variation and suggest that this variation has contributed to recent human adaptation.

Heparin inhibits melanosome uptake and inflammatory response coupled with phagocytosis through blocking PI3k/Akt and MEK/ERK signaling pathways in human epidermal keratinocytes.

PubMed

Makino-Okamura, Chieko; Niki, Yoko; Takeuchi, Seiji; Nishigori, Chikako; Declercq, Lieve; Yaroch, Daniel B; Saito, Naoaki

2014-11-01

To gain insight for the role of mast cell-produced heparin in the regulation of epidermal homeostasis and skin pigmentation, we have investigated the effect of heparin on melanosome uptake and proinflammatory responses in normal human epidermal keratinocytes (NHEKs). We quantified phagocytic activity of NHEKs with uptake of melanosomes or fluorescent microspheres. Heparin exhibited the inhibitory effect on keratinocyte phagocytosis through blocking PI3k/Akt and MEK/ERK signaling pathways. In fact, the heparin-treated NHEKs showed impaired activation of Akt and ERK during phagocytosis, whereas PI3k and MEK inhibitors significantly suppressed melanosome uptake by NHEKs. In addition, the inflammation marker cycloxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2 ) production were induced during phagocytosis, while these effects were downregulated in the presence of heparin. Our observations suggest that heparin may play an antiphagocytic and anti-inflammation role in epidermis of human skin. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions.

PubMed

Perez White, Bethany E; Ventrella, Rosa; Kaplan, Nihal; Cable, Calvin J; Thomas, Paul M; Getsios, Spiro

2017-01-01

EphA2 is a receptor tyrosine kinase that helps to maintain epidermal tissue homeostasis. A proximity-dependent biotin identification (BioID) approach was used to identify proteins in close proximity to EphA2 within primary human keratinocytes and three-dimensional (3D) reconstituted human epidermis (RHE) cultures to map a putative protein interaction network for this membrane receptor that exhibits a polarized distribution in stratified epithelia. Although a subset of known EphA2 interactors were identified in the BioID screen, >97% were uniquely detected in keratinocytes with over 50% of these vicinal proteins only present in 3D human epidermal culture. Afadin (AFDN), a cytoskeletal and junction-associated protein, was present in 2D and 3D keratinocyte cultures, and validated as a so-far-unknown EphA2-interacting protein. Loss of EphA2 protein disrupted the subcellular distribution of afadin and occludin in differentiated keratinocytes, leading to impairment of tight junctions. Collectively, these studies illustrate the use of the BioID approach in order to map receptor interaction networks in 3D human epithelial cultures, and reveal a positive regulatory role for EphA2 in the organization of afadin and epidermal tight junctions. © 2017. Published by The Company of Biologists Ltd.

EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions

PubMed Central

Perez White, Bethany E.; Ventrella, Rosa; Kaplan, Nihal; Cable, Calvin J.; Thomas, Paul M.

2017-01-01

ABSTRACT EphA2 is a receptor tyrosine kinase that helps to maintain epidermal tissue homeostasis. A proximity-dependent biotin identification (BioID) approach was used to identify proteins in close proximity to EphA2 within primary human keratinocytes and three-dimensional (3D) reconstituted human epidermis (RHE) cultures to map a putative protein interaction network for this membrane receptor that exhibits a polarized distribution in stratified epithelia. Although a subset of known EphA2 interactors were identified in the BioID screen, >97% were uniquely detected in keratinocytes with over 50% of these vicinal proteins only present in 3D human epidermal culture. Afadin (AFDN), a cytoskeletal and junction-associated protein, was present in 2D and 3D keratinocyte cultures, and validated as a so-far-unknown EphA2-interacting protein. Loss of EphA2 protein disrupted the subcellular distribution of afadin and occludin in differentiated keratinocytes, leading to impairment of tight junctions. Collectively, these studies illustrate the use of the BioID approach in order to map receptor interaction networks in 3D human epithelial cultures, and reveal a positive regulatory role for EphA2 in the organization of afadin and epidermal tight junctions. PMID:27815408

Epidermal Growth Factor and Interleukin-1β Utilize Divergent Signaling Pathways to Synergistically Upregulate Cyclooxygenase-2 Gene Expression in Human Amnion-Derived WISH Cells1

PubMed Central

Ackerman, William E.; Rovin, Brad H.; Kniss, Douglas A.

2006-01-01

In human parturition, uterotonic prostaglandins (PGs) arise predominantly via increased expression of cyclooxygenase-2 (COX-2 [also known as prostaglandin synthase 2]) within intra-uterine tissues. Interleukin-1 (IL-1) and epidermal growth factor (EGF), both inducers of COX-2 transcription, are among numerous factors that accumulate within amniotic fluid with advancing gestation. It was previously demonstrated that EGF could potentiate IL-1β-driven PGE2 production in amnion and amnion-derived (WISH) cells. To define the mechanism for this observation, we hypothesized that EGF and IL-1β might exhibit synergism in regulating COX-2 gene expression. In WISH cells, combined treatment with EGF and IL-1β resulted in a greater-than-additive increase in COX-2 mRNA relative to challenge with either agent independently. Augmentation of IL-1β-induced transactivation by EGF was not observed in cells harboring reporter plasmids bearing nuclear factor-kappa B (NFκB) regulatory elements alone, but was evident when a fragment (−891/+9) of the COX-2 gene 5′-promoter was present. Both agents transiently activated intermediates of multiple signaling pathways potentially involved in the regulation of COX-2 gene expression. The 26 S proteasome inhibitor, MG-132, selectively abrogated IL-1β-driven NFκB activation and COX-2 mRNA expression. Only pharmacologic blockade of the p38 mitogen-activated protein kinase eliminated COX-2 expression following EGF stimulation. We conclude that EGF and IL-1β appear to signal through different signaling cascades leading to COX-2 gene expression. IL-1β employs the NFκB pathway predominantly, while the spectrum of EGF signaling is broader and includes p38 kinase. The synergism observed between IL-1β and EGF does not rely on augmented NFκB function, but rather, occurs through differential use of independent response elements within the COX-2 promoter. PMID:15329330

Cultivation of human dermal fibroblasts and epidermal keratinocytes on keratin-coated silica bead substrates.

PubMed

Tan, Bee Yi; Nguyen, Luong T H; Kim, Hyo-Sop; Kim, Jae-Ho; Ng, Kee Woei

2017-10-01

Human hair keratin is promising as a bioactive material platform for various biomedical applications. To explore its versatility further, human hair keratin was coated onto monolayers of silica beads to produce film-like substrates. This combination was hypothesized to provide a synergistic effect in improving the biochemical properties of the resultant composite. Atomic force microscopy analysis showed uniform coatings of keratin on the silica beads with a slight increase in the resulting surface roughness. Keratin-coated silica beads had higher surface energy and relatively lower negative charge than those of bare silica beads. To investigate cell response, human dermal fibroblasts (HDFs), and human epidermal keratinocytes (HEKs) were cultured on the substrates over 4 days. Results showed that keratin coatings significantly enhanced the metabolic activity of HDFs and encouraged cell spreading but did not exert any significant effects on HEKs. HDF expression of collagen I was significantly more intense on the keratin-coated compared to the bare silica substrates. Furthermore, HDF secretion of various cytokines suggested that keratin coatings triggered active cell responses related to wound healing. Collectively, our study demonstrated that human hair keratin-coated silica bead monolayers have the potential to modulate HDF behavior in culture and may be exploited further. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2789-2798, 2017. © 2017 Wiley Periodicals, Inc.

Filamin A Modulates Kinase Activation and Intracellular Trafficking of Epidermal Growth Factor Receptors in Human Melanoma Cells

PubMed Central

Fiori, Jennifer L.; Zhu, Tie-Nian; O'Connell, Michael P.; Hoek, Keith S.; Indig, Fred E.; Frank, Brittany P.; Morris, Christa; Kole, Sutapa; Hasskamp, Joanne; Elias, George; Weeraratna, Ashani T.; Bernier, Michel

2009-01-01

The actin-binding protein filamin A (FLNa) affects the intracellular trafficking of various classes of receptors and has a potential role in oncogenesis. However, it is unclear whether FLNa regulates the signaling capacity and/or down-regulation of the activated epidermal growth factor receptor (EGFR). Here it is shown that partial knockdown of FLNa gene expression blocked ligand-induced EGFR responses in metastatic human melanomas. To gain greater insights into the role of FLNa in EGFR activation and intracellular sorting, we used M2 melanoma cells that lack endogenous FLNa and a subclone in which human FLNa cDNA has been stably reintroduced (M2A7 cells). Both tyrosine phosphorylation and ubiquitination of EGFR were significantly lower in epidermal growth factor (EGF)-stimulated M2 cells when compared with M2A7 cells. Moreover, the lack of FLNa interfered with EGFR interaction with the ubiquitin ligase c-Cbl. M2 cells exhibited marked resistance to EGF-induced receptor degradation, which was very active in M2A7 cells. Despite comparable rates of EGF-mediated receptor endocytosis, internalized EGFR colocalized with the lysosomal marker lysosome-associated membrane protein-1 in M2A7 cells but not M2 cells, in which EGFR was found to be sequestered in large vesicles and subsequently accumulated in punctated perinuclear structures after EGF stimulation. These results suggest the requirement of FLNa for efficient EGFR kinase activation and the sorting of endocytosed receptors into the degradation pathway. PMID:19213840

HER2 induces expression of leptin in human breast epithelial cells.

PubMed

Cha, Yujin; Kang, Youjin; Moon, Aree

2012-12-01

A close association between the obesity hormone leptin and breast cancer progression has been suggested. The present study investigated the molecular mechanism for enhanced leptin expression in breast cancer cells and its functional significance in breast cancer aggressiveness. We examined whether leptin expression level is affected by the oncoprotein human epidermal growth factor receptor2 (HER2), which is overexpressed in ∼30% of breast tumors. Here, we report, for the first time, that HER2 induces transcriptional activation of leptin in MCF10A human breast epithelial cells. We also showed that p38 mitogen-activated protein kinase signaling was involved in leptin expression induced by HER2. We showed a crucial role of leptin in the invasiveness of HER2-MCF10A cells using an siRNA molecule targeting leptin. Taken together, the results indicate a molecular link between HER2 and leptin, providing supporting evidence that leptin represents a target for breast cancer therapy. [BMB Reports 2012; 45(12): 719-723].

HER2 induces expression of leptin in human breast epithelial cells

PubMed Central

Cha, Yujin; Kang, Youjin; Moon, Aree

2012-01-01

A close association between the obesity hormone leptin and breast cancer progression has been suggested. The present study investigated the molecular mechanism for enhanced leptin expression in breast cancer cells and its functional significance in breast cancer aggressiveness. We examined whether leptin expression level is affected by the oncoprotein human epidermal growth factor receptor2 (HER2), which is overexpressed in ∼30% of breast tumors. Here, we report, for the first time, that HER2 induces transcriptional activation of leptin in MCF10A human breast epithelial cells. We also showed that p38 mitogen-activated protein kinase signaling was involved in leptin expression induced by HER2. We showed a crucial role of leptin in the invasiveness of HER2-MCF10A cells using an siRNA molecule targeting leptin. Taken together, the results indicate a molecular link between HER2 and leptin, providing supporting evidence that leptin represents a target for breast cancer therapy. [BMB Reports 2012; 45(12): 719-723] PMID:23261058

Expression of a functional epidermal growth factor receptor on human adipose-derived mesenchymal stem cells and its signaling mechanism.

PubMed

Baer, Patrick C; Schubert, Ralf; Bereiter-Hahn, Jürgen; Plösser, Michaela; Geiger, Helmut

2009-05-01

Adult stem cells act as a pluripotent source of regenerative cells during tissue injury. Despite expanded research in stem cell biology, understanding how growth and migration of adipose-derived adult mesenchymal stem cells (ASC) are governed by interactions with growth factors is very limited. One important property of ASC is the presence of the epidermal growth factor (EGF) receptor and the cellular response to soluble EGF. Expression of the EGF receptor was proven by PCR and Western blotting. Signal transduction was analyzed by Western blotting and PhosFlow assay. EGF caused robust phosphorylation of SHC and ERK1/2, which could be inhibited by EGF receptor antagonist AG1478 and MEK inhibitor PD98059. ASC proliferation was determined by MTT assay. Stem cell migration was analyzed in a modified Boyden chamber. Incubation with EGF led to cell proliferation and induced cell migration, but did not change the undifferentiated state of the cells. In the kidney, injured renal tubular cells express high amounts of EGF. Therefore, our results may highlight a mechanism underlying renal regeneration. Thus, future in vivo studies that focus on the effects of EGF on recruitment of ASC to sites of injury are necessary.

Development of 3D imaging technique of reconstructed human epidermis with immortalized human epidermal cell line.

PubMed

Inoue, Yu; Hasegawa, Seiji; Miyachi, Katsuma; Yamada, Takaaki; Nakata, Satoru; Ipponjima, Sari; Hibi, Terumasa; Nemoto, Tomomi; Tanaka, Masahiko; Suzuki, Ryo; Hirashima, Naohide

2018-05-01

The epidermis, the outermost layer of the skin, retains moisture and functions as a physical barrier against the external environment. Epidermal cells are continuously replaced by turnover, and thus to understand in detail the dynamic cellular events in the epidermis, techniques to observe live tissues in 3D are required. Here, we established a live 3D imaging technique for epidermis models. We first obtained immortalized human epidermal cell lines which have a normal differentiation capacity and fluorescence-labelled cytoplasm or nuclei. The reconstituted 3D epidermis was prepared with these lines. Using this culture system, we were able to observe the structure of the reconstituted epidermis live in 3D, which was similar to an in vivo epidermis, and evaluate the effect of a skin irritant. This technique may be useful for dermatological science and drug development. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Gene expression profiling of histologically normal breast tissue in females with human epidermal growth factor receptor 2‑positive breast cancer.

PubMed

Zubor, Pavol; Hatok, Jozef; Moricova, Petra; Kapustova, Ivana; Kajo, Karol; Mendelova, Andrea; Sivonova, Monika Kmetova; Danko, Jan

2015-02-01

Gene expression profile‑based taxonomy of breast cancer (BC) has been described as a significant breakthrough in comprehending the differences in the origin and behavior of cancer to allow individually tailored therapeutic approaches. In line with this, we hypothesized that the gene expression profile of histologically normal epithelium (HNEpi) could harbor certain genetic abnormalities predisposing breast tissue cells to develop human epidermal growth factor receptor 2 (HER2)‑positive BC. Thus, the aim of the present study was to assess gene expression in normal and BC tissue (BCTis) from patients with BC in order to establish its value as a potential diagnostic marker for cancer development. An array study evaluating a panel of 84 pathway‑ and disease‑specific genes in HER2‑positive BC and tumor‑adjacent HNEpi was performed using quantitative polymerase chain reaction in 12 patients using microdissected samples from frozen tissue. Common prognostic and predictive parameters of BC were assessed by immunohistochemistry and in situ hybridization. In the BCTis and HNEpi samples of 12 HER2‑positive subjects with BC, the expression of 2,016 genes was assessed. A total of 39.3% of genes were deregulated at a minimal two‑fold deregulation rate and 10.7% at a five‑fold deregulation rate in samples of HNEpi or BCTis. Significant differences in gene expression between BCTis and HNEpi samples were revealed for BCL2L2, CD44, CTSD, EGFR, ERBB2, ITGA6, NGFB, RPL27, SCBG2A1 and SCGB1D2 genes (P<0.05), as well as GSN, KIT, KLK5, SERPINB5 and STC2 genes (P<0.01). Insignificant differences (P<0.07) were observed for CCNA1, CLU, DLC1, GABRP and IL6 genes. The ontological gene analyses revealed that the majority of the deregulated genes in the HNEpi samples were part of the functional gene group directly associated with BC origin and prognosis. Functional analysis showed that the most frequent gene deregulations occurred in genes associated with apoptosis and cell

Epidermal Growth Factor-Dependent Transformation by a Human EGF Receptor Proto-Oncogene

NASA Astrophysics Data System (ADS)

Velu, Thierry J.; Beguinot, Laura; Vass, William C.; Willingham, Mark C.; Merlino, Glenn T.; Pastan, Ira; Lowy, Douglas R.

1987-12-01

The epidermal growth factor (EGF) receptor gene EGFR has been placed in a retrovirus vector to examine the growth properties of cells that experimentally overproduce a full-length EGF receptor. NIH 3T3 cells transfected with the viral DNA or infected with the corresponding rescued retrovirus developed a fully transformed phenotype in vitro that required both functional EGFR expression and the presence of EGF in the growth medium. Cells expressing 4 × 105 EGF receptors formed tumors in nude mice, while control cells did not. Therefore, the EGFR retrovirus, which had a titer on NIH 3T3 cells that was greater than 107 focus-forming units per milliliter, can efficiently transfer and express this gene, and increased numbers of EGF receptors can contribute to the transformed phenotype.

Immunohistochemical demonstration of epidermal growth factor in human gastric cancer xenografts of nude mice.

PubMed

Yoshiyuki, T; Shimizu, Y; Onda, M; Tokunaga, A; Kiyama, T; Nishi, K; Mizutani, T; Matsukura, N; Tanaka, N; Akimoto, M

1990-02-15

Thirty-two surgical specimens and three cell lines of human gastric cancers were used for subcutaneous transplantation into nude mice, resulting in the establishment of eight (25%) xenografts from the surgical specimens and two (67%) from the cell lines. The localization of epidermal growth factor (EGF) in the surgical specimens and cell lines of the gastric cancers and their xenografts in nude mice was then investigated immunohistochemically. Epidermal growth factor was stained in the cytoplasm of the cancer cells, being detected in 16 (50%) of the 32 surgical specimens and in all of the cell lines. Seven (44%) of the sixteen EGF-positive surgical specimens and one (6%) of the 16 EGF-negative ones were tumorigenic in nude mice. All of the xenografts in nude mice were positive for EGF. The tumorigenicity of human gastric cancer xenografts in nude mice may, therefore, be correlated with the presence of EGF in cancer cells.

Epidermal Overexpression of Xenobiotic Receptor PXR Impairs the Epidermal Barrier and Triggers Th2 Immune Response.

PubMed

Elentner, Andreas; Schmuth, Matthias; Yannoutsos, Nikolaos; Eichmann, Thomas O; Gruber, Robert; Radner, Franz P W; Hermann, Martin; Del Frari, Barbara; Dubrac, Sandrine

2018-01-01

The skin is in daily contact with environmental pollutants, but the long-term effects of such exposure remain underinvestigated. Many of these toxins bind and activate the pregnane X receptor (PXR), a ligand-activated transcription factor that regulates genes central to xenobiotic metabolism. The objective of this work was to investigate the effect of constitutive activation of PXR in the basal layer of the skin to mimic repeated skin exposure to noxious molecules. We designed a transgenic mouse model that overexpresses the human PXR gene linked to the herpes simplex VP16 domain under the control of the keratin 14 promoter. We show that transgenic mice display increased transepidermal water loss and elevated skin pH, abnormal stratum corneum lipids, focal epidermal hyperplasia, activated keratinocytes expressing more thymic stromal lymphopoietin, a T helper type 2/T helper type 17 skin immune response, and increased serum IgE. Furthermore, the cutaneous barrier dysfunction precedes development of the T helper type 2/T helper type 17 inflammation in transgenic mice, thereby mirroring the time course of atopic dermatitis development in humans. Moreover, further experiments suggest increased PXR signaling in the skin of patients with atopic dermatitis when compared with healthy skin. Thus, PXR activation by environmental pollutants may compromise epidermal barrier function and favor an immune response resembling atopic dermatitis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Arctigenin induced gallbladder cancer senescence through modulating epidermal growth factor receptor pathway.

PubMed

Zhang, Mingdi; Cai, Shizhong; Zuo, Bin; Gong, Wei; Tang, Zhaohui; Zhou, Di; Weng, Mingzhe; Qin, Yiyu; Wang, Shouhua; Liu, Jun; Ma, Fei; Quan, Zhiwei

2017-05-01

Gallbladder cancer has poor prognosis and limited therapeutic options. Arctigenin, a representative dibenzylbutyrolactone lignan, occurs in a variety of plants. However, the molecular mechanisms involved in the antitumor effect of arctigenin on gallbladder cancer have not been fully elucidated. The expression levels of epidermal growth factor receptor were examined in 100 matched pairs of gallbladder cancer tissues. A positive correlation between high epidermal growth factor receptor expression levels and poor prognosis was observed in gallbladder cancer tissues. Pharmacological inhibition or inhibition via RNA interference of epidermal growth factor receptor induced cellular senescence in gallbladder cancer cells. The antitumor effect of arctigenin on gallbladder cancer cells was primarily achieved by inducing cellular senescence. In gallbladder cancer cells treated with arctigenin, the expression level of epidermal growth factor receptor significantly decreased. The analysis of the activity of the kinases downstream of epidermal growth factor receptor revealed that the RAF-MEK-ERK signaling pathway was significantly inhibited. Furthermore, the cellular senescence induced by arctigenin could be reverted by pcDNA-epidermal growth factor receptor. Arctigenin also potently inhibited the growth of tumor xenografts, which was accompanied by the downregulation of epidermal growth factor receptor and induction of senescence. This study demonstrates arctigenin could induce cellular senescence in gallbladder cancer through the modulation of epidermal growth factor receptor pathway. These data identify epidermal growth factor receptor as a key regulator in arctigenin-induced gallbladder cancer senescence.

Epidermal electronic systems for sensing and therapy

NASA Astrophysics Data System (ADS)

Lu, Nanshu; Ameri, Shideh K.; Ha, Taewoo; Nicolini, Luke; Stier, Andrew; Wang, Pulin

2017-04-01

Epidermal electronic system is a class of hair thin, skin soft, stretchable sensors and electronics capable of continuous and long-term physiological sensing and clinical therapy when applied on human skin. The high cost of manpower, materials, and photolithographic facilities associated with its manufacture limit the availability of disposable epidermal electronics. We have invented a cost and time effective, completely dry, benchtop "cut-and-paste" method for the green, freeform and portable manufacture of epidermal electronics within minutes. We have applied the "cut-and-paste" method to manufacture epidermal electrodes, hydration and temperature sensors, conformable power-efficient heaters, as well as cuffless continuous blood pressure monitors out of metal thin films, two-dimensional (2D) materials, and piezoelectric polymer sheets. For demonstration purpose, we will discuss three examples of "cut-and-pasted" epidermal electronic systems in this paper. The first will be submicron thick, transparent epidermal graphene electrodes that can be directly transferred to human skin like a temporary transfer tattoo and can measure electrocardiogram (ECG) with signal-to-noise ratio and motion artifacts on par with conventional gel electrodes. The second will be a chest patch which houses both electrodes and pressure sensors for the synchronous measurements of ECG and seismocardiogram (SCG) such that beat-to-beat blood pressure can be inferred from the time interval between the R peak of the ECG and the AC peak of the SCG. The last example will be a highly conformable, low power consumption epidermal heater for thermal therapy.

Inflammation-mediated skin tumorigenesis induced by epidermal c-Fos

PubMed Central

Briso, Eva M.; Guinea-Viniegra, Juan; Bakiri, Latifa; Rogon, Zbigniew; Petzelbauer, Peter; Eils, Roland; Wolf, Ronald; Rincón, Mercedes; Angel, Peter; Wagner, Erwin F.

2013-01-01

Skin squamous cell carcinomas (SCCs) are the second most prevalent skin cancers. Chronic skin inflammation has been associated with the development of SCCs, but the contribution of skin inflammation to SCC development remains largely unknown. In this study, we demonstrate that inducible expression of c-fos in the epidermis of adult mice is sufficient to promote inflammation-mediated epidermal hyperplasia, leading to the development of preneoplastic lesions. Interestingly, c-Fos transcriptionally controls mmp10 and s100a7a15 expression in keratinocytes, subsequently leading to CD4 T-cell recruitment to the skin, thereby promoting epidermal hyperplasia that is likely induced by CD4 T-cell-derived IL-22. Combining inducible c-fos expression in the epidermis with a single dose of the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) leads to the development of highly invasive SCCs, which are prevented by using the anti-inflammatory drug sulindac. Moreover, human SCCs display a correlation between c-FOS expression and elevated levels of MMP10 and S100A15 proteins as well as CD4 T-cell infiltration. Our studies demonstrate a bidirectional cross-talk between premalignant keratinocytes and infiltrating CD4 T cells in SCC development. Therefore, targeting inflammation along with the newly identified targets, such as MMP10 and S100A15, represents promising therapeutic strategies to treat SCCs. PMID:24029918

Sulindac metabolites inhibit epidermal growth factor receptor activation and expression.

PubMed

Pangburn, Heather A; Kraus, Hanna; Ahnen, Dennis J; Rice, Pamela L

2005-09-02

Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a decreased mortality from colorectal cancer (CRC). NSAIDs induce apoptotic cell death in colon cancer cells in vitro and inhibit growth of neoplastic colonic mucosa in vivo however, the biochemical mechanisms required for these growth inhibitory effects are not well defined. We previously reported that metabolites of the NSAID sulindac downregulate extracellular-signal regulated kinase 1/2 (ERK1/2) signaling and that this effect is both necessary and sufficient for the apoptotic effects of these drugs. The goal of this project was to specifically test the hypothesis that sulindac metabolites block activation and/or expression of the epidermal growth factor (EGF) receptor (EGFR). HT29 human colon cancer cells were treated with EGF, alone, or in the presence of sulindac sulfide or sulindac sulfone. Cells lysates were assayed by immunoblotting for phosphorylated EGFR (pEGFR, pY1068), total EGFR, phosphorylated ERK1/2 (pERK1/2), total ERK1/2, activated caspase-3, and alpha-tubulin. EGF treatment rapidly induced phosphorylation of both EGFR and ERK1/2 in HT29 colon cancer cells. Pretreatment with sulindac metabolites for 24 h blocked EGF-induced phosphorylation of both EGFR and ERK1/2 and decreased total EGFR protein expression. Under basal conditions, downregulation of pEGFR and total EGFR was detected as early as 12 h following sulindac sulfide treatment and persisted through at least 48 h. Sulindac sulfone induced downregulation of pEGFR and total EGFR was detected as early as 1 h and 24 h, respectively, following drug treatment, and persisted through at least 72 h. EGFR downregulation by sulindac metabolites was observed in three different CRC cell lines, occurred prior to the observed downregulation of pERK1/2 and induction of apoptosis by these drugs, and was not dependent of caspase activation. These results suggest that downregulation of EGFR signaling by sulindac metabolites may

Role of Pin1 in UVA-induced cell proliferation and malignant transformation in epidermal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Chang Yeob; Hien, Tran Thi; Lim, Sung Chul

2011-06-24

Highlights: {yields} Pin1 expression is enhanced by low energy UVA irradiation in both skin tissues of hairless mice and JB6 C141 epidermal cells. {yields} UVA irradiation increases activator protein-1 activity and cyclin D1 in a Pin1-dependent manner. {yields} UVA potentiates EGF-inducible, anchorage-independent growth of epidermal cells, and this is suppressed by Pin1 inhibition or by anti-oxidant. -- Abstract: Ultraviolet A (UVA) radiation ({lambda} = 320-400 nm) is considered a major cause of human skin cancer. Pin1, a peptidyl prolyl isomerase, is overexpressed in most types of cancer tissues and plays an important role in cell proliferation and transformation. Here, wemore » demonstrated that Pin1 expression was enhanced by low energy UVA (300-900 mJ/cm{sup 2}) irradiation in both skin tissues of hairless mice and JB6 C141 epidermal cells. Exposure of epidermal cells to UVA radiation increased cell proliferation and cyclin D1 expression, and these changes were blocked by Pin1 inhibition. UVA irradiation also increased activator protein-1 (AP-1) minimal reporter activity and nuclear levels of c-Jun, but not c-Fos, in a Pin1-dependent manner. The increases in Pin1 expression and in AP-1 reporter activity in response to UVA were abolished by N-acetylcysteine (NAC) treatment. Finally, we found that pre-exposure of JB6 C141 cells to UVA potentiated EGF-inducible, anchorage-independent growth, and this effect was significantly suppressed by Pin1inhibition or by NAC.« less

Epidermal growth factor receptor expression in different subtypes of oral lichenoid disease.

PubMed

Cortés-Ramírez, Dionisio-Alejandro; Rodríguez-Tojo, María-Jose; Coca-Meneses, Juan-Carlos; Marichalar-Mendia, Xabier; Aguirre-Urizar, José-Manuel

2014-09-01

The oral lichenoid disease (OLD) includes different chronic inflammatory processes such as oral lichen planus (OLP) and oral lichenoid lesions (OLL), both entities with controversial diagnosis and malignant potential. Epidermal growth factor receptor (EFGR) is an important oral carcinogenesis biomarker and overexpressed in several oral potentially malignant disorders. To analyze the EGFR expression in the OLD to find differences between OLP and OLL, and to correlate it with the main clinical and pathological features. Forty-four OLD cases were studied and classified according to their clinical (Group C1: only papular lesions / Group C2: papular and other lesions) and histopathological features (Group HT: OLP-typical / Group HC: OLP-compatible) based in previous published criteria. Standard immunohistochemical identification of EGFR protein was performed. Comparative and descriptive statistical analyses were performed. Thirty-five cases (79.5%) showed EGFR overexpression without significant differences between clinical and histopathological groups (p<0.05). Histological groups showed significant differences in the EGFR expression pattern (p=0.016). Conlusions: All OLD samples showed high EGFR expression. The type of clinical lesion was not related with EGFR expression; however, there are differences in the EGFR expression pattern between histological groups that may be related with a different biological profile and malignant risk.

Oral pathogens change proliferation properties of oral tumor cells by affecting gene expression of human defensins.

PubMed

Hoppe, T; Kraus, D; Novak, N; Probstmeier, R; Frentzen, M; Wenghoefer, M; Jepsen, S; Winter, J

2016-10-01

The impact of oral pathogens onto the generation and variability of oral tumors has only recently been investigated. To get further insights, oral cancer cells were treated with pathogens and additionally, as a result of this bacterial cellular infection, with human defensins, which are as anti-microbial peptide members of the innate immune system. After cell stimulation, proliferation behavior, expression analysis of oncogenic relevant defensin genes, and effects on EGFR signaling were investigated. The expression of oncogenic relevant anti-microbial peptides was analyzed with real-time PCR and immunohistochemistry. Cell culture experiments were performed to examine cellular impacts caused by stimulation, i.e., altered gene expression, proliferation rate, and EGF receptor-dependent signaling. Incubation of oral tumor cells with an oral pathogen (Porphyromonas gingivalis) and human α-defensins led to an increase in cell proliferation. In contrast, another oral bacterium used, Aggregatibacter actinomycetemcomitans, enhanced cell death. The bacteria and anti-microbial peptides exhibited diverse effects on the transcript levels of oncogenic relevant defensin genes and epidermal growth factor receptor signaling. These two oral pathogens exhibited opposite primary effects on the proliferation behavior of oral tumor cells. Nevertheless, both microbe species led to similar secondary impacts on the proliferation rate by modifying expression levels of oncogenic relevant α-defensin genes. In this respect, oral pathogens exerted multiplying effects on tumor cell proliferation. Additionally, human defensins were shown to differently influence epidermal growth factor receptor signaling, supporting the hypothesis that these anti-microbial peptides serve as ligands of EGFR, thus modifying the proliferation behavior of oral tumor cells.

Cellular and Tumor Radiosensitivity is Correlated to Epidermal Growth Factor Receptor Protein Expression Level in Tumors Without EGFR Amplification;Epidermal growth factor receptor; Radiotherapy; Squamous cell carcinoma; Biomarker; Local tumor control

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kasten-Pisula, Ulla; Saker, Jarob; Eicheler, Wolfgang

2011-07-15

Purpose: There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. Methods and Materials: The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blotmore » and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. Results: EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. Conclusions: Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.« less

TERATOGENIC RESPONSES ARE MODULATED IN MICE LACKING EXPRESSION OF EPIDERMAL GROWTH FACTOR (EGF) AND TRANSFORMING GROWTH FACTOR-ALPHA (TGF)

EPA Science Inventory

TITLE:
TERATOGENIC RESPONSES ARE MODULATED IN MICE LACKING EXPRESSION OF EPIDERMAL GROWTH FACTOR (EGF) AND TRANSFORMING GROWTH FACTOR-ALPHA (TGF). AUTHORS (ALL): Abbott, Barbara D.1; Best, Deborah S.1; Narotsky, Michael G.1. SPONSOR NAME: None INSTITUTIONS (ALL): 1. Repro Tox ...

Expression of epidermal growth factor receptor and vascular endothelial growth factor in malignant canine epithelial nasal tumours.

PubMed

Shiomitsu, K; Johnson, C L; Malarkey, D E; Pruitt, A F; Thrall, D E

2009-06-01

Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) signalling pathways play a role in carcinogenesis. Inhibition of EGF receptor (EGFR) and of VEGF is effective in increasing the radiation responsiveness of neoplastic cells both in vitro and in human trials. In this study, immunohistochemical evaluation was employed to determine and characterize the potential protein expression levels and patterns of EGFR and VEGF in a variety of canine malignant epithelial nasal tumours. Of 24 malignant canine nasal tumours, 13 (54.2%) were positive for EGFR staining and 22 (91.7%) were positive for VEGF staining. The intensity and percentage of immunohistochemically positive neoplastic cells for EGFR varied. These findings indicate that EGFR and VEGF proteins were present in some malignant epithelial nasal tumours in the dogs, and therefore, it may be beneficial to treat canine patients with tumours that overexpress EGFR and VEGF with specific inhibitors in conjunction with radiation.

UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling

PubMed Central

Moore, Carlene; Cevikbas, Ferda; Pasolli, H. Amalia; Chen, Yong; Kong, Wei; Kempkes, Cordula; Parekh, Puja; Lee, Suk Hee; Kontchou, Nelly-Ange; Yeh, Iwei; Jokerst, Nan Marie; Fuchs, Elaine; Steinhoff, Martin; Liedtke, Wolfgang B.

2013-01-01

At our body surface, the epidermis absorbs UV radiation. UV overexposure leads to sunburn with tissue injury and pain. To understand how, we focus on TRPV4, a nonselective cation channel highly expressed in epithelial skin cells and known to function in sensory transduction, a property shared with other transient receptor potential channels. We show that following UVB exposure mice with induced Trpv4 deletions, specifically in keratinocytes, are less sensitive to noxious thermal and mechanical stimuli than control animals. Exploring the mechanism, we find that epidermal TRPV4 orchestrates UVB-evoked skin tissue damage and increased expression of the proalgesic/algogenic mediator endothelin-1. In culture, UVB causes a direct, TRPV4-dependent Ca2+ response in keratinocytes. In mice, topical treatment with a TRPV4-selective inhibitor decreases UVB-evoked pain behavior, epidermal tissue damage, and endothelin-1 expression. In humans, sunburn enhances epidermal expression of TRPV4 and endothelin-1, underscoring the potential of keratinocyte-derived TRPV4 as a therapeutic target for UVB-induced sunburn, in particular pain. PMID:23929777

UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling.

PubMed

Moore, Carlene; Cevikbas, Ferda; Pasolli, H Amalia; Chen, Yong; Kong, Wei; Kempkes, Cordula; Parekh, Puja; Lee, Suk Hee; Kontchou, Nelly-Ange; Yeh, Iwei; Ye, Iwei; Jokerst, Nan Marie; Fuchs, Elaine; Steinhoff, Martin; Liedtke, Wolfgang B

2013-08-20

At our body surface, the epidermis absorbs UV radiation. UV overexposure leads to sunburn with tissue injury and pain. To understand how, we focus on TRPV4, a nonselective cation channel highly expressed in epithelial skin cells and known to function in sensory transduction, a property shared with other transient receptor potential channels. We show that following UVB exposure mice with induced Trpv4 deletions, specifically in keratinocytes, are less sensitive to noxious thermal and mechanical stimuli than control animals. Exploring the mechanism, we find that epidermal TRPV4 orchestrates UVB-evoked skin tissue damage and increased expression of the proalgesic/algogenic mediator endothelin-1. In culture, UVB causes a direct, TRPV4-dependent Ca(2+) response in keratinocytes. In mice, topical treatment with a TRPV4-selective inhibitor decreases UVB-evoked pain behavior, epidermal tissue damage, and endothelin-1 expression. In humans, sunburn enhances epidermal expression of TRPV4 and endothelin-1, underscoring the potential of keratinocyte-derived TRPV4 as a therapeutic target for UVB-induced sunburn, in particular pain.

Novel targeted approaches to treating biliary tract cancer: the dual epidermal growth factor receptor and ErbB-2 tyrosine kinase inhibitor NVP-AEE788 is more efficient than the epidermal growth factor receptor inhibitors gefitinib and erlotinib.

PubMed

Wiedmann, Marcus; Feisthammel, Jürgen; Blüthner, Thilo; Tannapfel, Andrea; Kamenz, Thomas; Kluge, Annett; Mössner, Joachim; Caca, Karel

2006-08-01

Aberrant activation of the epidermal growth factor receptor is frequently observed in neoplasia, notably in tumors of epithelial origin. Attempts to treat such tumors with epidermal growth factor receptor antagonists resulted in remarkable success in recent studies. Little is known, however, about the efficacy of this therapy in biliary tract cancer. Protein expression of epidermal growth factor receptor, ErbB-2, and vascular endothelial growth factor receptor-2 was assessed in seven human biliary tract cancer cell lines by immunoblotting. In addition, histological sections from 19 patients with extrahepatic cholangiocarcinoma were analyzed for epidermal growth factor receptor, ErbB-2 and vascular endothelial growth factor receptor-2 expression by immunohistochemistry. Moreover, we sequenced the cDNA products representing the entire epidermal growth factor receptor coding region of the seven cell lines, and searched for genomic epidermal growth factor receptor amplifications and polysomy by fluorescence in-situ hybridization. Cell growth inhibition by gefitinib erlotinib and NVP-AEE788 was studied in vitro by automated cell counting. In addition, the anti-tumoral effect of erlotinib and NVP-AEE788 was studied in a chimeric mouse model. The anti-tumoral drug mechanism in this model was assessed by MIB-1 antibody staining, terminal deoxynucleotidyl transfer-mediated dUTP nick end-labelling assay, von Willebrand factor staining, and immunoblotting for p-p42/44 (p-Erk1/2, p-MAPK) and p-AKT. Immunoblotting revealed expression of epidermal growth factor receptor, ErbB-2, and vascular endothelial growth factor receptor-2 in all biliary tract cancer cell lines. EGFR was detectable in six of 19 (32%) extrahepatic human cholangiocarcinoma tissue samples, ErbB-2 in 16 of 19 (84%), and vascular endothelial growth factor receptor-2 in nine of 19 (47%). Neither epidermal growth factor receptor mutations nor amplifications or polysomy were found in the seven biliary tract cancer

Epidermal Notch signalling: differentiation, cancer and adhesion.

PubMed

Watt, Fiona M; Estrach, Soline; Ambler, Carrie A

2008-04-01

The Notch pathway plays an important role in regulating epidermal differentiation. Notch ligands, receptors and effectors are expressed in a complex and dynamic pattern in embryonic and adult skin. Genetic ablation or activation of the pathway reveals that Notch signalling promotes differentiation of the hair follicle, sebaceous gland and interfollicular epidermal lineages and that Notch acts as an epidermal tumour suppressor. Notch signalling interacts with a range of other pathways to fulfil these functions and acts via RBP-Jkappa dependent and independent mechanisms. The effects on differentiation can be cell autonomous and non-autonomous, and Notch contributes to stem cell clustering via modulation of cell adhesion.

Inhibition of Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Phosphorylation on Tumor-Associated Endothelial Cells Leads to Treatment of Orthotopic Human Colon Cancer in Nude Mice1

PubMed Central

Sasaki, Takamitsu; Kitadai, Yasuhiko; Nakamura, Toru; Kim, Jang-Seong; Tsan, Rachel Z; Kuwai, Toshio; Langley, Robert R; Fan, Dominic; Kim, Sun-Jin; Fidler, Isaiah J

2007-01-01

The purpose of our study was to determine whether the dual inhibition of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) signaling pathways in tumor-associated endothelial cells can inhibit the progressive growth of human colon carcinoma in the cecum of nude mice. SW620CE2 human colon cancer cells growing in culture and orthotopically in the cecum of nude mice expressed a high level of transforming growth factor alpha (TGF-α) and vascular endothelial growth factor (VEGF) but were negative for EGFR, human epidermal growth factor receptor 2 (HER2), and VEGFR. Double immunofluorescence staining revealed that tumor-associated endothelial cells expressed EGFR, VEGFR2, phosphorylated EGFR (pEGFR), and phosphorylated VEGFR (pVEGFR). Treatment of mice with either 7H-pyrrolo [2,3-d]-pyrimidine lead scaffold (AEE788; an inhibitor of EGFR and VEGFR tyrosine kinase) or CPT-11 as single agents significantly inhibited the growth of cecal tumors (P < .01); this decrease was even more pronounced with AEE788 combined with CPT-11 (P < .001). AEE788 alone or combined with CPT-11 also inhibited the expression of pEGFR and pVEGFR on tumor-associated endothelial cells, significantly decreased vascularization and tumor cell proliferation, and increased the level of apoptosis in both tumor-associated endothelial cells and tumor cells. These data demonstrate that targeting EGFR and VEGFR signaling on tumor-associated endothelial cells provides a viable approach for the treatment of colon cancer. PMID:18084614

Epidermal growth factor receptor expression in different subtypes of oral lichenoid disease

PubMed Central

Cortés-Ramírez, Dionisio A.; Rodríguez-Tojo, María J.; Coca-Meneses, Juan C.; Marichalar-Mendia, Xabier

2014-01-01

The oral lichenoid disease (OLD) includes different chronic inflammatory processes such as oral lichen planus (OLP) and oral lichenoid lesions (OLL), both entities with controversial diagnosis and malignant potential. Epidermal growth factor receptor (EFGR) is an important oral carcinogenesis biomarker and overexpressed in several oral potentially malignant disorders. Objectives: To analyze the EGFR expression in the OLD to find differences between OLP and OLL, and to correlate it with the main clinical and pathological features. Material and Methods: Forty-four OLD cases were studied and classified according to their clinical (Group C1: only papular lesions / Group C2: papular and other lesions) and histopathological features (Group HT: OLP-typical / Group HC: OLP-compatible) based in previous published criteria. Standard immunohistochemical identification of EGFR protein was performed. Comparative and descriptive statistical analyses were performed. Results: Thirty-five cases (79.5%) showed EGFR overexpression without significant differences between clinical and histopathological groups (p<0.05). Histological groups showed significant differences in the EGFR expression pattern (p=0.016). Conlusions: All OLD samples showed high EGFR expression. The type of clinical lesion was not related with EGFR expression; however, there are differences in the EGFR expression pattern between histological groups that may be related with a different biological profile and malignant risk. Key words:Oral lichenoid disease, oral lichen planus, oral lichenoid lesion, oral carcinogenesis, EGFR. PMID:24880441

Overexpression of Transcription Factor Sp2 Inhibits Epidermal Differentiation and Increases Susceptibility to Wound and Carcinogen-Induced Tumorigenesis

PubMed Central

Kim, Tae-Hyung; Chiera, Shannon L.; Linder, Keith E.; Trempus, Carol S.; Smart, Robert C.; Horowitz, Jonathan M.

2010-01-01

Sp proteins are evolutionarily-conserved transcription factors required for the expression of a wide variety of genes that are critical for development and cell-cycle progression. De-regulated expression of certain Sp proteins is associated with the formation of a variety of human tumors, however direct evidence that any given Sp protein is oncogenic has been lacking. Here we report that Sp2 protein abundance in mice increases in concert with the progression of carcinogen-induced murine squamous cell carcinomas. Transgenic mice specifically overexpressing murine Sp2 in epidermal basal keratinocytes were highly susceptible to wound- and carcinogen-induced papillomagenesis. Transgenic animals that were homozygous rather than hemizygous for the Sp2 transgene exhibited a striking arrest in the epidermal differentiation program, perishing within two weeks of birth. Our results directly support the likelihood that Sp2 overexpression occurring in various human cancers has significant functional impact. PMID:20959487

The DP-1 transcription factor is required for keratinocyte growth and epidermal stratification.

PubMed

Chang, Wing Y; Bryce, Dawn M; D'Souza, Sudhir J A; Dagnino, Lina

2004-12-03

The epidermis is a stratified epithelium constantly replenished through the ability of keratinocytes in its basal layer to proliferate and self-renew. The epidermis arises from a single-cell layer ectoderm during embryogenesis. Large proliferative capacity is central to ectodermal cell and basal keratinocyte function. DP-1, a heterodimeric partner of E2F transcription factors, is highly expressed in the ectoderm and all epidermal layers during embryogenesis. To investigate the role of DP-1 in epidermal morphogenesis, we inhibited DP-1 activity through exogenous expression of a dominant-negative mutant (dnDP-1). Expression of the dnDP-1 mutant interferes with binding of E2F/DP-1 heterodimers to DNA and inhibits DNA replication, as well as cyclin A mRNA and protein expression. Chromatin immunoprecipitation analysis demonstrated that the cyclin A promoter is predominantly bound in proliferating keratinocytes by complexes containing E2F-3 and E2F-4. Thus, the mechanisms of decreased expression of cyclin A in the presence of dnDP-1 seem to involve inactivation of DP-1 complexes containing E2F-3 and E2F-4. To assess the consequences on epidermal morphogenesis of inhibiting DP-1 activity, we expressed dnDP-1 in rat epithelial keratinocytes in organotypic culture and observed that DP-1 inhibition negatively affected stratification of these cells. Likewise, expression of dnDP-1 in embryonic ectoderm explants produced extensive disorganization of subsequently formed epidermal basal and suprabasal layers, interfering with normal epidermal formation. We conclude that DP-1 activity is required for normal epidermal morphogenesis and ectoderm-to-epidermis transition.

Antimicrobial peptides and pro-inflammatory cytokines are differentially regulated across epidermal layers following bacterial stimuli.

PubMed

Percoco, Giuseppe; Merle, Chloé; Jaouen, Thomas; Ramdani, Yasmina; Bénard, Magalie; Hillion, Mélanie; Mijouin, Lily; Lati, Elian; Feuilloley, Marc; Lefeuvre, Luc; Driouich, Azeddine; Follet-Gueye, Marie-Laure

2013-12-01

The skin is a natural barrier between the body and the environment and is colonised by a large number of microorganisms. Here, we report a complete analysis of the response of human skin explants to microbial stimuli. Using this ex vivo model, we analysed at both the gene and protein level the response of epidermal cells to Staphylococcus epidermidis (S. epidermidis) and Pseudomonas fluorescens (P. fluorescens), which are present in the cutaneous microbiota. We showed that both bacterial species affect the structure of skin explants without penetrating the living epidermis. We showed by real-time quantitative polymerase chain reaction (qPCR) that S. epidermidis and P. fluorescens increased the levels of transcripts that encode antimicrobial peptides (AMPs), including human β defensin (hBD)2 and hBD3, and the pro-inflammatory cytokines interleukin (IL)-1α and (IL)-1-β, as well as IL-6. In addition, we analysed the effects of bacterial stimuli on the expression profiles of genes related to innate immunity and the inflammatory response across the epidermal layers, using laser capture microdissection (LCM) coupled to qPCR. We showed that AMP transcripts were principally upregulated in suprabasal keratinocytes. Conversely, the expression of pro-inflammatory cytokines was upregulated in the lower epidermis. These findings were confirmed by protein localisation using specific antibodies coupled to optical or electron microscopy. This work underscores the potential value of further studies that use LCM on human skin explants model to study the roles and effects of the epidermal microbiota on human skin physiology. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Temporal Gene Expression Kinetics for Human Keratinocytes Exposed to Hyperthermic Stress

PubMed Central

Echchgadda, Ibtissam; Roth, Caleb C.; Cerna, Cesario Z.; Wilmink, Gerald J.

2013-01-01

The gene expression kinetics for human cells exposed to hyperthermic stress are not well characterized. In this study, we identified and characterized the genes that are differentially expressed in human epidermal keratinocyte (HEK) cells exposed to hyperthermic stress. In order to obtain temporal gene expression kinetics, we exposed HEK cells to a heat stress protocol (44 °C for 40 min) and used messenger RNA (mRNA) microarrays at 0 h, 4 h and 24 h post-exposure. Bioinformatics software was employed to characterize the chief biological processes and canonical pathways associated with these heat stress genes. The data shows that the genes encoding for heat shock proteins (HSPs) that function to prevent further protein denaturation and aggregation, such as HSP40, HSP70 and HSP105, exhibit maximal expression immediately after exposure to hyperthermic stress. In contrast, the smaller HSPs, such as HSP10 and HSP27, which function in mitochondrial protein biogenesis and cellular adaptation, exhibit maximal expression during the “recovery phase”, roughly 24 h post-exposure. These data suggest that the temporal expression kinetics for each particular HSP appears to correlate with the cellular function that is required at each time point. In summary, these data provide additional insight regarding the expression kinetics of genes that are triggered in HEK cells exposed to hyperthermic stress. PMID:24709698

The impact of epidermal melanin on objective measurements of human skin colour.

PubMed

Alaluf, Simon; Atkins, Derek; Barrett, Karen; Blount, Margaret; Carter, Nik; Heath, Alan

2002-04-01

Objective measurements of human skin colour were made with a tristimulus (L*a*b*) chromameter in a range of different ethnic skin types. These were compared with biochemical measurements of melanin content, melanin composition and melanosome size in skin biopsies obtained from the same sites. L*, a* and b* values were found to vary significantly with ethnicity. In general, constitutively dark skin types have lower L* values, higher a* values and higher b* values than constitutively light skin types. Total epidermal melanin content appears to be the primary determinant of L* values in human skin (r = -0.88; P < 0.00001), whilst melanosome size also has a significant but more subtle influence on L* values (r = -0.73; P < 0.00001). There is also a strong positive contribution to a* values from epidermal melanin (r = 0.66, P < 0.00001), which accounts for the ethnic variation in a* values observed in this study. Melanin is also a major contributor to b* values in lighter skin types (r = 0.71, P < 0.00001). However, this relationship breaks down in darker skin types where b* values actually reach a maximum and then decrease as the concentration of melanin in the skin increases. This appears to be because of optical masking of yellow light by high concentrations of melanin in the epidermis. Analysis of the relationships between L*, a* and b* values in human skin indicate that they are very closely interrelated, and suggest that the optical properties of melanin in the epidermis are very similar to those of a dye on a fabric substrate.

Bioengineering a Human Plasma-Based Epidermal Substitute With Efficient Grafting Capacity and High Content in Clonogenic Cells

PubMed Central

Alexaline, Maia M.; Trouillas, Marina; Nivet, Muriel; Bourreau, Emilie; Leclerc, Thomas; Duhamel, Patrick; Martin, Michele T.; Doucet, Christelle; Fortunel, Nicolas O.

2015-01-01

Cultured epithelial autografts (CEAs) produced from a small, healthy skin biopsy represent a lifesaving surgical technique in cases of full-thickness skin burn covering >50% of total body surface area. CEAs also present numerous drawbacks, among them the use of animal proteins and cells, the high fragility of keratinocyte sheets, and the immaturity of the dermal-epidermal junction, leading to heavy cosmetic and functional sequelae. To overcome these weaknesses, we developed a human plasma-based epidermal substitute (hPBES) for epidermal coverage in cases of massive burn, as an alternative to traditional CEA, and set up critical quality controls for preclinical and clinical studies. In this study, phenotypical analyses in conjunction with functional assays (clonal analysis, long-term culture, or in vivo graft) showed that our new substitute fulfills the biological requirements for epidermal regeneration. hPBES keratinocytes showed high potential for cell proliferation and subsequent differentiation similar to healthy skin compared with a well-known reference material, as ascertained by a combination of quality controls. This work highlights the importance of integrating relevant multiparameter quality controls into the bioengineering of new skin substitutes before they reach clinical development. Significance This work involves the development of a new bioengineered epidermal substitute with pertinent functional quality controls. The novelty of this work is based on this quality approach. PMID:25848122

Bioengineering a human plasma-based epidermal substitute with efficient grafting capacity and high content in clonogenic cells.

PubMed

Alexaline, Maia M; Trouillas, Marina; Nivet, Muriel; Bourreau, Emilie; Leclerc, Thomas; Duhamel, Patrick; Martin, Michele T; Doucet, Christelle; Fortunel, Nicolas O; Lataillade, Jean-Jacques

2015-06-01

Cultured epithelial autografts (CEAs) produced from a small, healthy skin biopsy represent a lifesaving surgical technique in cases of full-thickness skin burn covering >50% of total body surface area. CEAs also present numerous drawbacks, among them the use of animal proteins and cells, the high fragility of keratinocyte sheets, and the immaturity of the dermal-epidermal junction, leading to heavy cosmetic and functional sequelae. To overcome these weaknesses, we developed a human plasma-based epidermal substitute (hPBES) for epidermal coverage in cases of massive burn, as an alternative to traditional CEA, and set up critical quality controls for preclinical and clinical studies. In this study, phenotypical analyses in conjunction with functional assays (clonal analysis, long-term culture, or in vivo graft) showed that our new substitute fulfills the biological requirements for epidermal regeneration. hPBES keratinocytes showed high potential for cell proliferation and subsequent differentiation similar to healthy skin compared with a well-known reference material, as ascertained by a combination of quality controls. This work highlights the importance of integrating relevant multiparameter quality controls into the bioengineering of new skin substitutes before they reach clinical development. This work involves the development of a new bioengineered epidermal substitute with pertinent functional quality controls. The novelty of this work is based on this quality approach. ©AlphaMed Press.

Characterization of xenobiotic metabolizing enzymes of a reconstructed human epidermal model from adult hair follicles.

PubMed

Bacqueville, Daniel; Jacques, Carine; Duprat, Laure; Jamin, Emilien L; Guiraud, Beatrice; Perdu, Elisabeth; Bessou-Touya, Sandrine; Zalko, Daniel; Duplan, Hélène

2017-08-15

In this study, a comprehensive characterization of xenobiotic metabolizing enzymes (XMEs) based on gene expression and enzyme functionality was made in a reconstructed skin epidermal model derived from the outer root sheath (ORS) of hair follicles (ORS-RHE). The ORS-RHE model XME gene profile was consistent with native human skin. Cytochromes P450 (CYPs) consistently reported to be detected in native human skin were also present at the gene level in the ORS-RHE model. The highest Phase I XME gene expression levels were observed for alcohol/aldehyde dehydrogenases and (carboxyl) esterases. The model was responsive to the CYP inducers, 3-methylcholanthrene (3-MC) and β-naphthoflavone (βNF) after topical and systemic applications, evident at the gene and enzyme activity level. Phase II XME levels were generally higher than those of Phase I XMEs, the highest levels were GSTs and transferases, including NAT1. The presence of functional CYPs, UGTs and SULTs was confirmed by incubating the models with 7-ethoxycoumarin, testosterone, benzo(a)pyrene and 3-MC, all of which were rapidly metabolized within 24h after topical application. The extent of metabolism was dependent on saturable and non-saturable metabolism by the XMEs and on the residence time within the model. In conclusion, the ORS-RHE model expresses a number of Phase I and II XMEs, some of which may be induced by AhR ligands. Functional XME activities were also demonstrated using systemic or topical application routes, supporting their use in cutaneous metabolism studies. Such a reproducible model will be of interest when evaluating the cutaneous metabolism and potential toxicity of innovative dermo-cosmetic ingredients. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparative Genomics Identifies Epidermal Proteins Associated with the Evolution of the Turtle Shell

PubMed Central

Holthaus, Karin Brigit; Strasser, Bettina; Sipos, Wolfgang; Schmidt, Heiko A.; Mlitz, Veronika; Sukseree, Supawadee; Weissenbacher, Anton; Tschachler, Erwin; Alibardi, Lorenzo; Eckhart, Leopold

2016-01-01

The evolution of reptiles, birds, and mammals was associated with the origin of unique integumentary structures. Studies on lizards, chicken, and humans have suggested that the evolution of major structural proteins of the outermost, cornified layers of the epidermis was driven by the diversification of a gene cluster called Epidermal Differentiation Complex (EDC). Turtles have evolved unique defense mechanisms that depend on mechanically resilient modifications of the epidermis. To investigate whether the evolution of the integument in these reptiles was associated with specific adaptations of the sequences and expression patterns of EDC-related genes, we utilized newly available genome sequences to determine the epidermal differentiation gene complement of turtles. The EDC of the western painted turtle (Chrysemys picta bellii) comprises more than 100 genes, including at least 48 genes that encode proteins referred to as beta-keratins or corneous beta-proteins. Several EDC proteins have evolved cysteine/proline contents beyond 50% of total amino acid residues. Comparative genomics suggests that distinct subfamilies of EDC genes have been expanded and partly translocated to loci outside of the EDC in turtles. Gene expression analysis in the European pond turtle (Emys orbicularis) showed that EDC genes are differentially expressed in the skin of the various body sites and that a subset of beta-keratin genes within the EDC as well as those located outside of the EDC are expressed predominantly in the shell. Our findings give strong support to the hypothesis that the evolutionary innovation of the turtle shell involved specific molecular adaptations of epidermal differentiation. PMID:26601937

Transient gene expression in epidermal cells of plant leaves by biolistic DNA delivery.

PubMed

Ueki, Shoko; Magori, Shimpei; Lacroix, Benoît; Citovsky, Vitaly

2013-01-01

Transient gene expression is a useful approach for studying the functions of gene products. In the case of plants, Agrobacterium infiltration is a method of choice for transient introduction of genes for many species. However, this technique does not work efficiently in some species, such as Arabidopsis thaliana. Moreover, the infection of Agrobacterium is known to induce dynamic changes in gene expression patterns in the host plants, possibly affecting the function and localization of the proteins to be tested. These problems can be circumvented by biolistic delivery of the genes of interest. Here, we present an optimized protocol for biolistic delivery of plasmid DNA into epidermal cells of plant leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol allows efficient and reproducible transient expression of diverse genes in Arabidopsis, Nicotiana benthamiana and N. tabacum, and is suitable for studies of the biological function and subcellular localization of the gene products directly in planta. The protocol also can be easily adapted to other species by optimizing the delivery gas pressure.

Pharmacologic induction of epidermal melanin and protection against sunburn in a humanized mouse model.

PubMed

Amaro-Ortiz, Alexandra; Vanover, Jillian C; Scott, Timothy L; D'Orazio, John A

2013-09-07

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection (1). Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Identification of a Population of Epidermal Squamous Cell Carcinoma Cells with Enhanced Potential for Tumor Formation

PubMed Central

Adhikary, Gautam; Grun, Dan; Kerr, Candace; Balasubramanian, Sivaprakasam; Rorke, Ellen A.; Vemuri, Mohan; Boucher, Shayne; Bickenbach, Jackie R.; Hornyak, Thomas; Xu, Wen; Fisher, Matthew L.; Eckert, Richard L.

2013-01-01

Epidermal squamous cell carcinoma is among the most common cancers in humans. These tumors are comprised of phenotypically diverse populations of cells that display varying potential for proliferation and differentiation. An important goal is identifying cells from this population that drive tumor formation. To enrich for tumor-forming cells, cancer cells were grown as spheroids in non-attached conditions. We show that spheroid-selected cells form faster growing and larger tumors in immune-compromised mice as compared to non-selected cells. Moreover, spheroid-selected cells gave rise to tumors following injection of as few as one hundred cells, suggesting these cells have enhanced tumor-forming potential. Cells isolated from spheroid-selected tumors retain an enhanced ability to grow as spheroids when grown in non-attached culture conditions. Thus, these tumor-forming cells retain their phenotype following in vivo passage as tumors. Detailed analysis reveals that spheroid-selected cultures are highly enriched for expression of epidermal stem cell and embryonic stem cell markers, including aldehyde dehydrogenase 1, keratin 15, CD200, keratin 19, Oct4, Bmi-1, Ezh2 and trimethylated histone H3. These studies indicate that a subpopulation of cells that possess stem cell-like properties and express stem cell markers can be derived from human epidermal cancer cells and that these cells display enhanced ability to drive tumor formation. PMID:24376802

Autocrine expression of the epidermal growth factor receptor ligand heparin-binding EGF-like growth factor in cervical cancer.

PubMed

Schrevel, Marlies; Osse, E Michelle; Prins, Frans A; Trimbos, J Baptist M Z; Fleuren, Gert Jan; Gorter, Arko; Jordanova, Ekaterina S

2017-06-01

In cervical cancer, the epidermal growth factor receptor (EGFR) is overexpressed in 70-90% of the cases and has been associated with poor prognosis. EGFR-based therapy is currently being explored in cervical cancer. We investigated which EGFR ligand is primarily expressed in cervical cancer and which cell type functions as the major source of this ligand. We hypothesized that macrophages are the main source of EGFR ligands and that a paracrine loop between tumor cells and macrophages is responsible for ligand expression. mRNA expression analysis was performed on 32 cervical cancer cases to determine the expression of the EGFR ligands amphiregulin, β-cellulin, epidermal growth factor (EGF), epiregulin, heparin-binding EGF-like growth factor (HB‑EGF) and transforming growth factor α (TGFα). Subsequently, protein expression was determined immunohistochemically on 36 additional cases. To assess whether macrophages are the major source of EGFR ligands, immunohistochemical double staining was performed on four representative tissue slides. Expression of the chemokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and C-C motif ligand 2 (CCL2) was determined by mRNA in situ hybridization. Of the known EGFR ligands, HB‑EGF had the highest mRNA expression and HB‑EGF and EGFR protein expression were highly correlated. Tumor specimens with high EGFR expression showed higher numbers of macrophages, and higher expression of GM-CSF and CCL2, but only a small subset (9%) of macrophages was found to be HB‑EGF-positive. Strikingly, 78% of cervical cancer specimens were found to express HB‑EGF. Standardized assessment of staining intensity, using spectral imaging analysis, showed that HB‑EGF expression was higher in the tumor compartment than in the stromal compartment. These results suggest that HB‑EGF is an important EGFR ligand in cervical cancer and that cervical cancer cells are the predominant source of HB‑EGF. Therefore, we propose an autocrine

Amphiregulin enhances intercellular adhesion molecule-1 expression and promotes tumor metastasis in human osteosarcoma

PubMed Central

Liu, Ju-Fang; Tsao, Ya-Ting; Hou, Chun-Han

2015-01-01

Osteosarcoma is a common, high malignant, and metastatic bone cancer. Amphiregulin (AREG) has been associated with cancer cellular activities. However, the effect of AREG on metastasis activity in human osteosarcoma cells has yet to be determined. We determined that AREG increases the expression of intercellular adhesion molecule-1 (ICAM-1) through PI3K/Akt signaling pathway via its interaction with the epidermal growth factor receptor, thus resulting in the enhanced cell migration of osteosarcoma. Furthermore, AREG stimulation increased the association of NF-κB to ICAM-1 promoter which then up-regulated ICAM-1 expression. Finally, we observed that shRNA silencing of AREG decreased osteosarcoma metastasis in vivo. Our findings revealed a relationship between osteosarcoma metastatic potential and AREG expression and the modulating effect of AREG on ICAM-1 expression. PMID:26503469

Diminished survival of human cytotrophoblast cells exposed to hypoxia/reoxygenation injury and associated reduction of heparin-binding epidermal growth factor-like growth factor.

PubMed

Leach, Richard E; Kilburn, Brian A; Petkova, Anelia; Romero, Roberto; Armant, D Randall

2008-04-01

The antiapoptotic action of heparin-binding epidermal growth factor (HBEGF)-like growth factor and its regulation by O(2) constitutes a key factor for trophoblast survival. The hypothesis that cytotrophoblast survival is compromised by exposure to hypoxia-reoxygenation (H/R) injury, which may contribute to preeclampsia and some missed abortions, prompted us to investigate HBEGF regulation and its role as a survival factor during H/R in cytotrophoblast cells. A transformed human first-trimester cytotrophoblast cell line HTR-8/SVneo was exposed to H/R (2% O(2) followed by 20% O(2)) and assessed for HBEGF expression and cell death. Cellular HBEGF declined significantly within 30 minutes of reoxygenation after culture at 2% O(2). H/R significantly reduced proliferation and increased cell death when compared with trophoblast cells cultured continuously at 2% or 20% O(2). Restoration of cell survival also was achieved by adding recombinant HBEGF during reoxygenation. HBEGF inhibited apoptosis through its binding to either human epidermal receptor (HER)-1 or HER4, its cognate receptors. These results provide evidence that cytotrophoblast exposure to H/R induces apoptosis and decreased cell proliferation. HBEGF accumulation is diminished under these conditions, whereas restoration of HBEGF signaling improves trophoblast survival.

Toxic epidermal necrolysis successfully treated with etanercept.

PubMed

Gubinelli, Emanuela; Canzona, Flora; Tonanzi, Tiziano; Raskovic, Desanka; Didona, Biagio

2009-03-01

Toxic epidermal necrolysis (TEN) is a rare and acute severe adverse reaction to drugs, characterised by massive apoptosis and widespread epidermal and mucosal detachment. Although no gold standard therapy exists, human i.v. immunoglobulins have recently been described as an effective treatment for this disease. We report a case of phenobarbital-induced TEN in a 59-year-old white woman where the epidermal detachment stopped 48 h after beginning the etanercept treatment with complete healing after 20 days. To the best of our knowledge, this is only the second reported case of TEN successfully treated with etanercept.

Alterations of epidermal proliferation and cytokeratin expression in skin biopsies from heavy draught horses with chronic pastern dermatitis.

PubMed

Geburek, Florian; Ohnesorge, Bernhard; Deegen, Eckehard; Doeleke, Renate; Hewicker-Trautwein, Marion

2005-12-01

We report the historical, clinical and histopathological characteristics of skin lesions in biopsies from 37 heavy draught horses with chronic pastern dermatitis. The skin lesions were divided into four macroscopic groups: scaling (group I, n=5), hyperkeratotic and hyperplastic plaque-like lesions (group II, n=14), nodular skin masses (group III, n=16) and verrucous skin lesions (group IV, n=2). The principal histological findings were hyperkeratosis and epidermal hyperplasia. There was a gradual increase in epidermal hyperplasia from groups I to IV, suggesting that the lesions represent different stages of disease. In all cases, there was perivascular dermatitis dominated by T lymphocytes with an increase in MHC class II-positive dendritic-like cells. Immunohistochemical labelling for cytokeratins CK5/6(4), CK10 and CK14 indicated a change in their expression pattern. This correlated with the degree of epidermal hyperplasia, indicating abnormal differentiation of keratinocytes. There was a statistically significant correlation between the severity of skin lesions and several other factors including increasing age, increasing cannon circumference, prominence of anatomical structures such as fetlock tufts of hairs, ergots and chestnuts, and bulges in the fetlock region.

Development of Transgenic Minipigs with Expression of Antimorphic Human Cryptochrome 1

PubMed Central

Liu, Chunxin; Bolund, Lars; Vajta, Gábor; Dou, Hongwei; Yang, Wenxian; Xu, Ying; Luan, Jing; Wang, Jun; Yang, Huanming; Staunstrup, Nicklas Heine; Du, Yutao

2013-01-01

Minipigs have become important biomedical models for human ailments due to similarities in organ anatomy, physiology, and circadian rhythms relative to humans. The homeostasis of circadian rhythms in both central and peripheral tissues is pivotal for numerous biological processes. Hence, biological rhythm disorders may contribute to the onset of cancers and metabolic disorders including obesity and type II diabetes, amongst others. A tight regulation of circadian clock effectors ensures a rhythmic expression profile of output genes which, depending on cell type, constitute about 3–20% of the transcribed mammalian genome. Central to this system is the negative regulator protein Cryptochrome 1 (CRY1) of which the dysfunction or absence has been linked to the pathogenesis of rhythm disorders. In this study, we generated transgenic Bama-minipigs featuring expression of the Cys414-Ala antimorphic human Cryptochrome 1 mutant (hCRY1AP). Using transgenic donor fibroblasts as nuclear donors, the method of handmade cloning (HMC) was used to produce reconstructed embryos, subsequently transferred to surrogate sows. A total of 23 viable piglets were delivered. All were transgenic and seemingly healthy. However, two pigs with high transgene expression succumbed during the first two months. Molecular analyzes in epidermal fibroblasts demonstrated disturbances to the expression profile of core circadian clock genes and elevated expression of the proinflammatory cytokines IL-6 and TNF-α, known to be risk factors in cancer and metabolic disorders. PMID:24146819

Resveratrol modulates MED28 (Magicin/EG-1) expression and inhibits epidermal growth factor (EGF)-induced migration in MDA-MB-231 human breast cancer cells.

PubMed

Lee, Ming-Fen; Pan, Min-Hsiung; Chiou, Yi-Siou; Cheng, An-Chin; Huang, Han

2011-11-09

Resveratrol and pterostilbene exhibit diverse biological activities. MED28, a subunit of the mammalian Mediator complex for transcription, was also identified as magicin, an actin cytoskeleton Grb2-associated protein, and as endothelial-derived gene (EG-1). Several tumors exhibit aberrant MED28 expression, whereas the underlying mechanism is unclear. Triple-negative breast cancers, often expressing epidermal growth factor (EGF) receptor (EGFR), are associated with metastasis and poor survival. The objective of this study is to compare the effect of resveratrol and pterostilbene and to investigate the role of MED28 in EGFR-overexpressing MDA-MB-231 breast cancer cells. Pretreatment of resveratrol, but not pterostlbene, suppressed EGF-mediated migration and expression of MED28 and matrix metalloproteinase (MMP)-9 in MDA-MB-231 cells. Moreover, overexpression of MED28 increased migration, and the addition of EGF further enhanced migration. Our data indicate that resveratrol modulates the effect of MED28 on cellular migration, presumably through the EGFR/phosphatidylinositol 3-kinase (PI3K) signaling pathway, in breast cancer cells.

Basic Fibroblast Growth Factor Influences Epidermal Homeostasis of Living Skin Equivalents through Affecting Fibroblast Phenotypes and Functions.

PubMed

Yang, Lujun; Zhang, Dangui; Wu, Hongjuan; Xie, Sitian; Zhang, Mingjun; Zhang, Bingna; Tang, Shijie

2018-05-30

To elucidate the possible mechanisms of how basic fibroblast growth factor (bFGF) influences epidermal homeostasis in a living skin equivalent (LSE) model. Several wound healing-related growth factors were analyzed at protein and mRNA levels for dermal fibroblasts of induced alpha-smooth muscle actin (α-SMA)-positive or α-SMA-negative phenotypes. During culturing an LSE model by seeding normal human keratinocytes on a fibroblast-populated type I collagen gel, bFGF or neutralizing antibody for keratinocyte growth factor (KGF) was added to investigate its effects on fibroblast phenotypes and, subsequently, epidermal homeostasis by histology and immunohistochemistry. The α-SMA-positive phenotype of fibroblasts induced by transforming growth factor beta-1 (TGF-β1) markedly suppressed the expression of KGF and hepatocyte growth factor (HGF), and slightly upregulated vascular endothelial growth factor (VEGF) and TGF-β1 at mRNA and protein levels, compared with α-SMA-negative fibroblasts treated with bFGF. α-SMA expression of fibroblasts at the epidermal-mesenchymal junction of the LSEs was suppressed by the addition of bFGF, and a better-differentiated epidermis was presented. The abrogation of KGF from fibroblasts by the addition of the KGF neutralizing antibody disenabled the LSE culturing system to develop an epidermis. bFGF, through affecting the phenotypes and functions of fibroblasts, especially KGF expression, influenced epidermal homeostasis in an LSE model. © 2018 S. Karger AG, Basel.

Epidermal growth factor receptor expression is related to post-mitotic events in cerebellar development: regulation by thyroid hormone.

PubMed

Carrasco, Emilce; Blum, Mariann; Weickert, Cynthia Shannon; Casper, Diana

2003-01-10

It has been established that thyroid hormone and neurotrophic factors both orchestrate developmental events in the brain. However, it is not clear how these two influences are related. In this study, we investigated the effects of thyroid hormone on cerebellar development and the coincident expression of transforming growth factor-alpha (TGF-alpha), a ligand in the epidermal growth factor (EGF) family, and the epidermal growth factor receptor (EGFR). Profiles of thyroid hormone expression were measured in postnatal animals and were found to peak at postnatal day 15 (P15). These levels dropped below detectable levels when mice were made hypothyroid with propylthiouracil (PTU). TGF-alpha and EGFR expression, as determined by RNAse protection assay, was maximal at P6 in normal animals, but remained low in hypothyroid animals, suggesting that thyroid hormone was responsible for their induction. In situ hybridization and immunohistochemical analysis of EGFR expression revealed that this receptor was present on granule cells within the inner zone of the external granule cell layer (EGL), suggesting that EGFR-ligands were not inducing granule cell proliferation. The persistence of EGFR expression on migrating granule cells and subsequent down-regulation of expression in the internal granule cell layer (IGL) implicates a role for EGFR-ligands in differentiation and/or migration. In hypothyroid animals, we observed a delayed progression of granule cell migration, consistent with the persistence of EGFR labeling in the EGL, and in the 'pile-up' of labeled cells at the interface between the molecular layer and the Purkinje cell layer. Taken together, these results implicate thyroid hormone in the coordinated expression of TGF-alpha and EGFR, which are positioned to play a role in post-mitotic developmental events in the cerebellum.

Discovery of Novel Human Epidermal Growth Factor Receptor-2 Inhibitors by Structure-based Virtual Screening.

PubMed

Shi, Zheng; Yu, Tian; Sun, Rong; Wang, Shan; Chen, Xiao-Qian; Cheng, Li-Jia; Liu, Rong

2016-01-01

Human epidermal growth factor receptor-2 (HER2) is a trans-membrane receptor like protein, and aberrant signaling of HER2 is implicated in many human cancers, such as ovarian cancer, gastric cancer, and prostate cancer, most notably breast cancer. Moreover, it has been in the spotlight in the recent years as a promising new target for therapy of breast cancer. Since virtual screening has become an integral part of the drug discovery process, it is of great significant to identify novel HER2 inhibitors by structure-based virtual screening. In this study, we carried out a series of elegant bioinformatics approaches, such as virtual screening and molecular dynamics (MD) simulations to identify HER2 inhibitors from Food and Drug Administration-approved small molecule drug as potential "new use" drugs. Molecular docking identified top 10 potential drugs which showed spectrum affinity to HER2. Moreover, MD simulations suggested that ZINC08214629 (Nonoxynol-9) and ZINC03830276 (Benzonatate) might exert potential inhibitory effects against HER2-targeted anti-breast cancer therapeutics. Together, our findings may provide successful application of virtual screening studies in the lead discovery process, and suggest that our discovered small molecules could be effective HER2 inhibitor candidates for further study. A series of elegant bioinformatics approaches, including virtual screening and molecular dynamics (MD) simulations were took advantage to identify human epidermal growth factor receptor-2 (HER2) inhibitors. Molecular docking recognized top 10 candidate compounds, which showed spectrum affinity to HER2. Further, MD simulations suggested that ZINC08214629 (Nonoxynol-9) and ZINC03830276 (Benzonatate) in candidate compounds were identified as potential "new use" drugs against HER2-targeted anti-breast cancer therapeutics. Abbreviations used: HER2: Human epidermal growth factor receptor-2, FDA: Food and Drug Administration, PDB: Protein Database Bank, RMSDs: Root mean

C/EBPα expression is downregulated in human nonmelanoma skin cancers and inactivation of C/EBPα confers susceptibility to UVB-induced skin squamous cell carcinomas.

PubMed

Thompson, Elizabeth A; Zhu, Songyun; Hall, Jonathan R; House, John S; Ranjan, Rakesh; Burr, Jeanne A; He, Yu-Ying; Owens, David M; Smart, Robert C

2011-06-01

Human epidermis is routinely subjected to DNA damage induced by UVB solar radiation. Cell culture studies have revealed an unexpected role for C/EBPα (CCAAT/enhancer-binding protein-α) in the DNA damage response network, where C/EBPα is induced following UVB DNA damage, regulates the G(1) checkpoint, and diminished or ablated expression of C/EBPα results in G(1) checkpoint failure. In the current study we observed that C/EBPα is induced in normal human epidermal keratinocytes and in the epidermis of human subjects exposed to UVB radiation. The analysis of human skin precancerous and cancerous lesions (47 cases) for C/EBPα expression was conducted. Actinic keratoses, a precancerous benign skin growth and precursor to squamous cell carcinoma (SCC), expressed levels of C/EBPα similar to normal epidermis. Strikingly, all invasive SCCs no longer expressed detectable levels of C/EBPα. To determine the significance of C/EBPα in UVB-induced skin cancer, SKH-1 mice lacking epidermal C/EBPα (CKOα) were exposed to UVB. CKOα mice were highly susceptible to UVB-induced SCCs and exhibited accelerated tumor progression. CKOα mice displayed keratinocyte cell cycle checkpoint failure in vivo in response to UVB that was characterized by abnormal entry of keratinocytes into S phase. Our results demonstrate that C/EBPα is silenced in human SCC and loss of C/EBPα confers susceptibility to UVB-induced skin SCCs involving defective cell cycle arrest in response to UVB.

C/EBPα Expression Is Downregulated in Human Nonmelanoma Skin Cancers and Inactivation of C/EBPα Confers Susceptibility to UVB-Induced Skin Squamous Cell Carcinomas

PubMed Central

Thompson, Elizabeth A.; Zhu, Songyun; Hall, Jonathan R.; House, John S.; Ranjan, Rakesh; Burr, Jeanne A.; He, Yu-Ying; Owens, David M.; Smart, Robert C.

2012-01-01

Human epidermis is routinely subjected to DNA damage induced by UVB solar radiation. Cell culture studies have revealed an unexpected role for C/EBPα (CCAAT/enhancer-binding protein-α) in the DNA damage response network, where C/EBPα is induced following UVB DNA damage, regulates the G1 checkpoint, and diminished or ablated expression of C/EBPα results in G1 checkpoint failure. In the current study we observed that C/EBPα is induced in normal human epidermal keratinocytes and in the epidermis of human subjects exposed to UVB radiation. The analysis of human skin precancerous and cancerous lesions (47 cases) for C/EBPα expression was conducted. Actinic keratoses, a precancerous benign skin growth and precursor to squamous cell carcinoma (SCC), expressed levels of C/EBPα similar to normal epidermis. Strikingly, all invasive SCCs no longer expressed detectable levels of C/EBPα. To determine the significance of C/EBPα in UVB-induced skin cancer, SKH-1 mice lacking epidermal C/EBPα (CKOα) were exposed to UVB. CKOα mice were highly susceptible to UVB-induced SCCs and exhibited accelerated tumor progression. CKOα mice displayed keratinocyte cell cycle checkpoint failure in vivo in response to UVB that was characterized by abnormal entry of keratinocytes into S phase. Our results demonstrate that C/EBPα is silenced in human SCC and loss of C/EBPα confers susceptibility to UVB-induced skin SCCs involving defective cell cycle arrest in response to UVB. PMID:21346772

New Whitening Constituents from Taiwan-Native Pyracantha koidzumii: Structures and Tyrosinase Inhibitory Analysis in Human Epidermal Melanocytes.

PubMed

Lin, Rong-Dih; Chen, Mei-Chuan; Liu, Yan-Ling; Lin, Yi-Tzu; Lu, Mei-Kuang; Hsu, Feng-Lin; Lee, Mei-Hsien

2015-12-02

Nontoxic natural products useful in skin care cosmetics are of considerable interest. Tyrosinase is a rate-limiting enzyme for which its inhibitor is useful in developing whitening cosmetics. Pyracantha koidzumii (Hayata) Rehder is an endemic species in Taiwan that exhibits tyrosinase-inhibitory activity. To find new active natural compounds from P. koidzumii, we performed bioguided isolation and studied the related activity in human epidermal melanocytes. In total, 13 compounds were identified from P. koidzumii in the present study, including two new compounds, 3,6-dihydroxy-2,4-dimethoxy-dibenzofuran (9) and 3,4-dihydroxy-5-methoxybiphenyl-2'-O-β-d-glucopyranoside (13), as well as 11 known compounds. The new compound 13 exhibited maximum potency in inhibiting cellular tyrosinase activity, the protein expression of cellular tyrosinase and tyrosinase-related protein-2, as well as the mRNA expression of Paired box 3 and microphthalmia-associated transcription factor in a concentration-dependent manner. In the enzyme kinetic assay, the new compound 13 acted as an uncompetitive mixed-type inhibitor against the substrate l-3,4-dihydroxyphenylalanine and had a Km value against this substrate of 0.262 mM, as calculated using the Lineweaver-Burk plots. Taken together, our findings show compound 13 exhibits tyrosinase inhibition in human melanocytes and compound 13 may be a potential candidate for use in cosmetics.

New Whitening Constituents from Taiwan-Native Pyracantha koidzumii: Structures and Tyrosinase Inhibitory Analysis in Human Epidermal Melanocytes

PubMed Central

Lin, Rong-Dih; Chen, Mei-Chuan; Liu, Yan-Ling; Lin, Yi-Tzu; Lu, Mei-Kuang; Hsu, Feng-Lin; Lee, Mei-Hsien

2015-01-01

Nontoxic natural products useful in skin care cosmetics are of considerable interest. Tyrosinase is a rate-limiting enzyme for which its inhibitor is useful in developing whitening cosmetics. Pyracantha koidzumii (Hayata) Rehder is an endemic species in Taiwan that exhibits tyrosinase-inhibitory activity. To find new active natural compounds from P. koidzumii, we performed bioguided isolation and studied the related activity in human epidermal melanocytes. In total, 13 compounds were identified from P. koidzumii in the present study, including two new compounds, 3,6-dihydroxy-2,4-dimethoxy-dibenzofuran (9) and 3,4-dihydroxy-5-methoxybiphenyl-2ʹ-O-β-d-glucopyranoside (13), as well as 11 known compounds. The new compound 13 exhibited maximum potency in inhibiting cellular tyrosinase activity, the protein expression of cellular tyrosinase and tyrosinase-related protein-2, as well as the mRNA expression of Paired box 3 and microphthalmia-associated transcription factor in a concentration-dependent manner. In the enzyme kinetic assay, the new compound 13 acted as an uncompetitive mixed-type inhibitor against the substrate l-3,4-dihydroxyphenylalanine and had a Km value against this substrate of 0.262 mM, as calculated using the Lineweaver–Burk plots. Taken together, our findings show compound 13 exhibits tyrosinase inhibition in human melanocytes and compound 13 may be a potential candidate for use in cosmetics. PMID:26633381

Comparison of in vivo immune responses following transplantation of vascularized and non-vascularized human dermo-epidermal skin substitutes.

PubMed

Klar, Agnes S; Biedermann, Thomas; Simmen-Meuli, Claudia; Reichmann, Ernst; Meuli, Martin

2017-03-01

Autologous bio-engineered dermo-epidermal skin substitutes (DESS) represent an alternative therapeutic option for a definitive treatment of skin defects in human patients. Largely, the interaction of host immune cells with transplanted DESS is considered to be essential for the granulation tissue formation, graft take, and its functionality. The aim of this study was to compare the spatiotemporal distribution and density of host-derived monocytes/macrophages and granulocytes in vascularized (vascDESS) versus non-vascularized DESS (non-vascDESS) in a rat model. Keratinocytes and the stromal vascular fraction (SVF) were derived from human skin or human adipose tissue, respectively. Human SVF containing both endothelial and mesenchymal/stromal progenitors was used to develop a vascularized collagen type I-based dermal component in vitro. The donor-matched, monolayer-expanded adipose stromal cells lacking endothelial cells were used as a negative control. Subsequently, human keratinocytes were seeded on top of hydrogels to build dermo-epidermal skin grafts. After transplantation onto full-thickness skin wounds on the back of immuno-incompetent rats, grafts were excised and analyzed after 1 and 3 weeks. The expression of distinct inflammatory cell markers specific for host-derived monocytes/macrophages (CD11b, CD68) or granulocytes (HIS48) was analyzed by immunofluorescence microscopy. All skin grafts were infiltrated by host-derived monocytes/macrophages (CD11b + , CD68 + ) and granulocytes (HIS48 + ) between 1-3 week post-transplantation. When compared to non-vascDESS, the vascDESS showed an increased granulocyte infiltration at all time points analyzed with the majority of cells scattered throughout the whole dermal part. Whereas a moderate number of rat monocytes/macrophages (CD11b + , CD68 + ) were found in vascDESS at 1 week, only a few cells were detected in non-vascDESS. We observed a time-dependent decrease of monocytes/macrophages in all transplants at 3�

Adult epidermal Notch activity induces dermal accumulation of T cells and neural crest derivatives through upregulation of jagged 1

PubMed Central

Ambler, Carrie A.; Watt, Fiona M.

2010-01-01

Notch signalling regulates epidermal differentiation and tumour formation via non-cell autonomous mechanisms that are incompletely understood. This study shows that epidermal Notch activation via a 4-hydroxy-tamoxifen-inducible transgene caused epidermal thickening, focal detachment from the underlying dermis and hair clumping. In addition, there was dermal accumulation of T lymphocytes and stromal cells, some of which localised to the blisters at the epidermal-dermal boundary. The T cell infiltrate was responsible for hair clumping but not for other Notch phenotypes. Notch-induced stromal cells were heterogeneous, expressing markers of neural crest, melanocytes, smooth muscle and peripheral nerve. Although Slug1 expression was expanded in the epidermis, the stromal cells did not arise through epithelial-mesenchymal transition. Epidermal Notch activation resulted in upregulation of jagged 1 in both epidermis and dermis. When Notch was activated in the absence of epidermal jagged 1, jagged 1 was not upregulated in the dermis, and epidermal thickening, blister formation, accumulation of T cells and stromal cells were inhibited. Gene expression profiling revealed that epidermal Notch activation resulted in upregulation of several growth factors and cytokines, including TNFα, the expression of which was dependent on epidermal jagged 1. We conclude that jagged 1 is a key mediator of non-cell autonomous Notch signalling in skin. PMID:20940224

Adult epidermal Notch activity induces dermal accumulation of T cells and neural crest derivatives through upregulation of jagged 1.

PubMed

Ambler, Carrie A; Watt, Fiona M

2010-11-01

Notch signalling regulates epidermal differentiation and tumour formation via non-cell autonomous mechanisms that are incompletely understood. This study shows that epidermal Notch activation via a 4-hydroxy-tamoxifen-inducible transgene caused epidermal thickening, focal detachment from the underlying dermis and hair clumping. In addition, there was dermal accumulation of T lymphocytes and stromal cells, some of which localised to the blisters at the epidermal-dermal boundary. The T cell infiltrate was responsible for hair clumping but not for other Notch phenotypes. Notch-induced stromal cells were heterogeneous, expressing markers of neural crest, melanocytes, smooth muscle and peripheral nerve. Although Slug1 expression was expanded in the epidermis, the stromal cells did not arise through epithelial-mesenchymal transition. Epidermal Notch activation resulted in upregulation of jagged 1 in both epidermis and dermis. When Notch was activated in the absence of epidermal jagged 1, jagged 1 was not upregulated in the dermis, and epidermal thickening, blister formation, accumulation of T cells and stromal cells were inhibited. Gene expression profiling revealed that epidermal Notch activation resulted in upregulation of several growth factors and cytokines, including TNFα, the expression of which was dependent on epidermal jagged 1. We conclude that jagged 1 is a key mediator of non-cell autonomous Notch signalling in skin.

A genome-wide screen identifies YAP/WBP2 interplay conferring growth advantage on human epidermal stem cells

PubMed Central

Walko, Gernot; Woodhouse, Samuel; Pisco, Angela Oliveira; Rognoni, Emanuel; Liakath-Ali, Kifayathullah; Lichtenberger, Beate M.; Mishra, Ajay; Telerman, Stephanie B.; Viswanathan, Priyalakshmi; Logtenberg, Meike; Renz, Lisa M.; Donati, Giacomo; Quist, Sven R.; Watt, Fiona M.

2017-01-01

Individual human epidermal cells differ in their self-renewal ability. To uncover the molecular basis for this heterogeneity, we performed genome-wide pooled RNA interference screens and identified genes conferring a clonal growth advantage on normal and neoplastic (cutaneous squamous cell carcinoma, cSCC) human epidermal cells. The Hippo effector YAP was amongst the top positive growth regulators in both screens. By integrating the Hippo network interactome with our data sets, we identify WW-binding protein 2 (WBP2) as an important co-factor of YAP that enhances YAP/TEAD-mediated gene transcription. YAP and WPB2 are upregulated in actively proliferating cells of mouse and human epidermis and cSCC, and downregulated during terminal differentiation. WBP2 deletion in mouse skin results in reduced proliferation in neonatal and wounded adult epidermis. In reconstituted epidermis YAP/WBP2 activity is controlled by intercellular adhesion rather than canonical Hippo signalling. We propose that defective intercellular adhesion contributes to uncontrolled cSCC growth by preventing inhibition of YAP/WBP2. PMID:28332498

Comparative Genomics Identifies Epidermal Proteins Associated with the Evolution of the Turtle Shell.

PubMed

Holthaus, Karin Brigit; Strasser, Bettina; Sipos, Wolfgang; Schmidt, Heiko A; Mlitz, Veronika; Sukseree, Supawadee; Weissenbacher, Anton; Tschachler, Erwin; Alibardi, Lorenzo; Eckhart, Leopold

2016-03-01

The evolution of reptiles, birds, and mammals was associated with the origin of unique integumentary structures. Studies on lizards, chicken, and humans have suggested that the evolution of major structural proteins of the outermost, cornified layers of the epidermis was driven by the diversification of a gene cluster called Epidermal Differentiation Complex (EDC). Turtles have evolved unique defense mechanisms that depend on mechanically resilient modifications of the epidermis. To investigate whether the evolution of the integument in these reptiles was associated with specific adaptations of the sequences and expression patterns of EDC-related genes, we utilized newly available genome sequences to determine the epidermal differentiation gene complement of turtles. The EDC of the western painted turtle (Chrysemys picta bellii) comprises more than 100 genes, including at least 48 genes that encode proteins referred to as beta-keratins or corneous beta-proteins. Several EDC proteins have evolved cysteine/proline contents beyond 50% of total amino acid residues. Comparative genomics suggests that distinct subfamilies of EDC genes have been expanded and partly translocated to loci outside of the EDC in turtles. Gene expression analysis in the European pond turtle (Emys orbicularis) showed that EDC genes are differentially expressed in the skin of the various body sites and that a subset of beta-keratin genes within the EDC as well as those located outside of the EDC are expressed predominantly in the shell. Our findings give strong support to the hypothesis that the evolutionary innovation of the turtle shell involved specific molecular adaptations of epidermal differentiation. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Genome-wide expression profiling of five mouse models identifies similarities and differences with human psoriasis.

PubMed

Swindell, William R; Johnston, Andrew; Carbajal, Steve; Han, Gangwen; Wohn, Christian; Lu, Jun; Xing, Xianying; Nair, Rajan P; Voorhees, John J; Elder, James T; Wang, Xiao-Jing; Sano, Shigetoshi; Prens, Errol P; DiGiovanni, John; Pittelkow, Mark R; Ward, Nicole L; Gudjonsson, Johann E

2011-04-04

Development of a suitable mouse model would facilitate the investigation of pathomechanisms underlying human psoriasis and would also assist in development of therapeutic treatments. However, while many psoriasis mouse models have been proposed, no single model recapitulates all features of the human disease, and standardized validation criteria for psoriasis mouse models have not been widely applied. In this study, whole-genome transcriptional profiling is used to compare gene expression patterns manifested by human psoriatic skin lesions with those that occur in five psoriasis mouse models (K5-Tie2, imiquimod, K14-AREG, K5-Stat3C and K5-TGFbeta1). While the cutaneous gene expression profiles associated with each mouse phenotype exhibited statistically significant similarity to the expression profile of psoriasis in humans, each model displayed distinctive sets of similarities and differences in comparison to human psoriasis. For all five models, correspondence to the human disease was strong with respect to genes involved in epidermal development and keratinization. Immune and inflammation-associated gene expression, in contrast, was more variable between models as compared to the human disease. These findings support the value of all five models as research tools, each with identifiable areas of convergence to and divergence from the human disease. Additionally, the approach used in this paper provides an objective and quantitative method for evaluation of proposed mouse models of psoriasis, which can be strategically applied in future studies to score strengths of mouse phenotypes relative to specific aspects of human psoriasis.

Epidermal Growth Factor Receptor Mutation Enhances Expression of Cadherin-5 in Lung Cancer Cells.

PubMed

Hung, Ming-Szu; Chen, I-Chuan; Lung, Jr-Hau; Lin, Paul-Yann; Li, Ya-Chin; Tsai, Ying-Huang

2016-01-01

Epidermal growth factor receptor (EGFR) activation has been shown to play a critical role in tumor angiogenesis. In this study, we investigate the correlation between EGFR mutations and cadherin-5 (CDH5), which is an angiogenic factor, in lung cancer cells. Increased expression CDH5 is observed in lung cancer cells with EGFR mutations. Stable lung cancer cell lines expressing mutant (exon 19 deletion E746-A750, and exon 21 missense mutation L858R) and wild type EGFR genes are established. A significantly higher expression of CDH5 is observed in exon 19 deletion stable lung cancer cells and mouse xenografts. Further studies show that expression of CDH5 is decreased after the inhibition of EGFR and downstream Akt pathways in lung cancer cells with EGFR mutation. In addition, mutant EGFR genes potentiates angiogenesis in lung cancer cells, which is inhibited by CDH5 siRNA, and potentiates migration and invasion in lung cancer cells. Our study shows that mutant EGFR genes are associated with overexpression of CDH5 through increased phosphorylation of EGFR and downstream Akt pathways. Our result may provide an insight into the association of mutant EGFR and CDH5 expression in lung cancer and aid further development of target therapy for NSCLC in the future.

Epidermal Growth Factor Receptor Mutation Enhances Expression of Cadherin-5 in Lung Cancer Cells

PubMed Central

Hung, Ming-Szu; Chen, I-Chuan; Lung, Jr-Hau; Lin, Paul-Yann; Li, Ya-Chin; Tsai, Ying-Huang

2016-01-01

Epidermal growth factor receptor (EGFR) activation has been shown to play a critical role in tumor angiogenesis. In this study, we investigate the correlation between EGFR mutations and cadherin-5 (CDH5), which is an angiogenic factor, in lung cancer cells. Increased expression CDH5 is observed in lung cancer cells with EGFR mutations. Stable lung cancer cell lines expressing mutant (exon 19 deletion E746-A750, and exon 21 missense mutation L858R) and wild type EGFR genes are established. A significantly higher expression of CDH5 is observed in exon 19 deletion stable lung cancer cells and mouse xenografts. Further studies show that expression of CDH5 is decreased after the inhibition of EGFR and downstream Akt pathways in lung cancer cells with EGFR mutation. In addition, mutant EGFR genes potentiates angiogenesis in lung cancer cells, which is inhibited by CDH5 siRNA, and potentiates migration and invasion in lung cancer cells. Our study shows that mutant EGFR genes are associated with overexpression of CDH5 through increased phosphorylation of EGFR and downstream Akt pathways. Our result may provide an insight into the association of mutant EGFR and CDH5 expression in lung cancer and aid further development of target therapy for NSCLC in the future. PMID:27362942

Mu-opiate receptor and Beta-endorphin expression in nerve endings and keratinocytes in human skin.

PubMed

Bigliardi-Qi, M; Sumanovski, L T; Büchner, S; Rufli, T; Bigliardi, P L

2004-01-01

We have previously shown that human epidermal keratinocytes express a functionally active micro-opiate receptor, which adds a new dimension to the recently developed research in neuroimmunodermatology and neurogenic inflammation in skin diseases. Human keratinocytes specifically bind and also produce beta-endorphin, the endogenous micro-opiate receptor ligand. Using confocal imaging microscopy, we could now demonstrate that micro-opiate receptors are not only expressed in keratinocytes, but also on unmyelinated peripheral nerve fibers in the dermis and epidermis. Some of the peripheral nerve fibers also express the ligand beta-endorphin. The keratinocytes positive for beta-endorphin staining are clustered around the terminal ends of the unmyelinated nerve fibers. Therefore the opiate receptor system seems to be crucial in the direct communication between nerves and skin. The keratinocytes can influence the unmyelinated nerve fibers in the epidermis directly via secreting beta-endorphin. On the other hand, nerve fibers can also secrete beta-endorphin and influence the migration, differentiation and probably also the cytokine production pattern of keratinocytes.

An Automated and Minimally Invasive Tool for Generating Autologous Viable Epidermal Micrografts.

PubMed

Osborne, Sandra N; Schmidt, Marisa A; Harper, John R

2016-02-01

A new epidermal harvesting tool (CelluTome; Kinetic Concepts, Inc, San Antonio, Texas) created epidermal micrografts with minimal donor site damage, increased expansion ratios, and did not require the use of an operating room. The tool, which applies both heat and suction concurrently to normal skin, was used to produce epidermal micrografts that were assessed for uniform viability, donor-site healing, and discomfort during and after the epidermal harvesting procedure. This study was a prospective, noncomparative institutional review board-approved healthy human study to assess epidermal graft viability, donor-site morbidity, and patient experience. These studies were conducted at the multispecialty research facility, Clinical Trials of Texas, Inc, San Antonio. The participants were 15 healthy human volunteers. The average viability of epidermal micrografts was 99.5%. Skin assessment determined that 76% to 100% of the area of all donor sites was the same in appearance as the surrounding skin within 14 days after epidermal harvest. A mean pain of 1.3 (on a scale of 1 to 5) was reported throughout the harvesting process. Use of this automated, minimally invasive harvesting system provided a simple, low-cost method of producing uniformly viable autologous epidermal micrografts with minimal patient discomfort and superficial donor-site wound healing within 2 weeks.

ZNF750 is a p63 Target Gene that Induces KLF4 to Drive Terminal Epidermal Differentiation

PubMed Central

Sen, George L.; Boxer, Lisa D.; Webster, Dan E.; Bussat, Rose T.; Qu, Kun; Zarnegar, Brian J.; Johnston, Danielle; Siprashvili, Zurab; Khavari, Paul A.

2012-01-01

SUMMARY Disrupted epidermal differentiation characterizes numerous diseases that impact >25% of the population. In a search for dominant mediators of differentiation, we defined a requirement for ZNF750 in terminal epidermal differentiation. ZNF750 controlled genes mutated in numerous human skin diseases, including FLG, LOR, LCE3B, ALOXE3, and SPINK5. ZNF750 induced progenitor differentiation via an evolutionarily conserved C2H2 zinc finger motif. The epidermal master regulator, p63, bound the ZNF750 promoter and was necessary for its induction. ZNF750 restored differentiation to p63-deficient tissue, suggesting it acts downstream of p63. A search for functionally important ZNF750 targets via analysis of ZNF750-regulated genes identified KLF4, a transcription factor that activates late epidermal differentiation. ZNF750 binds to KLF4 at multiple sites flanking the transcriptional start site and controls its expression. ZNF750 thus directly links a tissue-specifying factor, p63, to an effector of terminal differentiation, KLF4, and represents a potential future target for disorders of this process. PMID:22364861

A mitosis block links active cell cycle with human epidermal differentiation and results in endoreplication.

PubMed

Zanet, Jennifer; Freije, Ana; Ruiz, María; Coulon, Vincent; Sanz, J Ramón; Chiesa, Jean; Gandarillas, Alberto

2010-12-20

How human self-renewal tissues co-ordinate proliferation with differentiation is unclear. Human epidermis undergoes continuous cell growth and differentiation and is permanently exposed to mutagenic hazard. Keratinocytes are thought to arrest cell growth and cell cycle prior to terminal differentiation. However, a growing body of evidence does not satisfy this model. For instance, it does not explain how skin maintains tissue structure in hyperproliferative benign lesions. We have developed and applied novel cell cycle techniques to human skin in situ and determined the dynamics of key cell cycle regulators of DNA replication or mitosis, such as cyclins E, A and B, or members of the anaphase promoting complex pathway: cdc14A, Ndc80/Hec1 and Aurora kinase B. The results show that actively cycling keratinocytes initiate terminal differentiation, arrest in mitosis, continue DNA replication in a special G2/M state, and become polyploid by mitotic slippage. They unambiguously demonstrate that cell cycle progression coexists with terminal differentiation, thus explaining how differentiating cells increase in size. Epidermal differentiating cells arrest in mitosis and a genotoxic-induced mitosis block rapidly pushes epidermal basal cells into differentiation and polyploidy. These observations unravel a novel mitosis-differentiation link that provides new insight into skin homeostasis and cancer. It might constitute a self-defence mechanism against oncogenic alterations such as Myc deregulation.

A Mitosis Block Links Active Cell Cycle with Human Epidermal Differentiation and Results in Endoreplication

PubMed Central

Zanet, Jennifer; Freije, Ana; Ruiz, María; Coulon, Vincent; Sanz, J. Ramón; Chiesa, Jean; Gandarillas, Alberto

2010-01-01

How human self-renewal tissues co-ordinate proliferation with differentiation is unclear. Human epidermis undergoes continuous cell growth and differentiation and is permanently exposed to mutagenic hazard. Keratinocytes are thought to arrest cell growth and cell cycle prior to terminal differentiation. However, a growing body of evidence does not satisfy this model. For instance, it does not explain how skin maintains tissue structure in hyperproliferative benign lesions. We have developed and applied novel cell cycle techniques to human skin in situ and determined the dynamics of key cell cycle regulators of DNA replication or mitosis, such as cyclins E, A and B, or members of the anaphase promoting complex pathway: cdc14A, Ndc80/Hec1 and Aurora kinase B. The results show that actively cycling keratinocytes initiate terminal differentiation, arrest in mitosis, continue DNA replication in a special G2/M state, and become polyploid by mitotic slippage. They unambiguously demonstrate that cell cycle progression coexists with terminal differentiation, thus explaining how differentiating cells increase in size. Epidermal differentiating cells arrest in mitosis and a genotoxic-induced mitosis block rapidly pushes epidermal basal cells into differentiation and polyploidy. These observations unravel a novel mitosis-differentiation link that provides new insight into skin homeostasis and cancer. It might constitute a self-defence mechanism against oncogenic alterations such as Myc deregulation. PMID:21187932

[Verrucose epidermal nevus with belated grow and pregnancy. Case report].

PubMed

Aguilera Martínez, Verónica; Cervantes Villarreal, Gustavo Enrique; Ramos Garibay, Alberto; Ruiz Mondragón, María Eugenia

2007-10-01

Verrucose epidermal nevus is a benign and congenital hyperplasia of the superficial epidermis and annexes. It expresses itself during the firs year of life, grows during childhood and in adolescence reaches its largest size. It can appear everywhere in skin surface. We present a case of late verrucose epidermal nevus with genital onset. Differential diagnosis was done with acuminated condylomas.

Different response of human glioma tumor-initiating cells to epidermal growth factor receptor kinase inhibitors.

PubMed

Griffero, Fabrizio; Daga, Antonio; Marubbi, Daniela; Capra, Maria Cristina; Melotti, Alice; Pattarozzi, Alessandra; Gatti, Monica; Bajetto, Adriana; Porcile, Carola; Barbieri, Federica; Favoni, Roberto E; Lo Casto, Michele; Zona, Gianluigi; Spaziante, Renato; Florio, Tullio; Corte, Giorgio

2009-03-13

Because a subpopulation of cancer stem cells (tumor-initiating cells, TICs) is believed to be responsible for the development, progression, and recurrence of many tumors, we evaluated the in vitro sensitivity of human glioma TICs to epidermal growth factor receptor (EGFR) kinase inhibitors (erlotinib and gefitinib) and possible molecular determinants for their effects. Cells isolated from seven glioblastomas (GBM 1-7) and grown using neural stem cell permissive conditions were characterized for in vivo tumorigenicity, expression of tumor stem cell markers (CD133, nestin), and multilineage differentiation properties, confirming that these cultures are enriched in TICs. TIC cultures were challenged with increasing concentrations of erlotinib and gefitinib, and their survival was evaluated after 1-4 days. In most cases, a time- and concentration-dependent cell death was observed, although GBM 2 was completely insensitive to both drugs, and GBM 7 was responsive only to the highest concentrations tested. Using a radioligand binding assay, we show that all GBM TICs express EGFR. Erlotinib and gefitinib inhibited EGFR and ERK1/2 phosphorylation/activation in all GBMs, irrespective of the antiproliferative response observed. However, under basal conditions GBM 2 showed a high Akt phosphorylation that was completely insensitive to both drugs, whereas GBM 7 was completely insensitive to gefitinib, and Akt inactivation occurred only for the highest erlotinib concentration tested, showing a precise relationship with the antiproliferative effects of the drug. Interestingly, in GBM 2, phosphatase and tensin homolog expression was significantly down-regulated, possibly accounting for the insensitivity to the drugs. In conclusion, glioma TICs are responsive to anti-EGFR drugs, but phosphatase and tensin homolog expression and Akt inhibition seem to be necessary for such effect.

Inactivated Wnt signaling in resveratrol-treated epidermal squamous cancer cells and its biological implication

PubMed Central

Liu, Zhi-Li; Li, Hong; Liu, Jia; Wu, Mo-Li; Chen, Xiao-Yan; Liu, Li-Hong; Wang, Qian

2017-01-01

Squamous cell carcinoma (SCC) is the most common epidermal malignancy, and Wnt/β-catenin signaling is frequently activated in SCC. Resveratrol prevents rodent epidermal carcinogenesis, while its effect on human epidermal cancer remains unknown. To address this issue, the impact of resveratrol on the growth and Wnt signaling of skin SCC Colo16 cells were investigated at the cellular and molecular biology levels by flow cytometry, immunocytochemistry, reverse transcription-polymerase chain reaction, western blotting and β-catenin-specific small interfering RNA (siRNA) transfection. Resveratrol (100 µM) suppressed cell growth and induced apoptosis in Colo16 cells. Wnt2 and its downstream genes were downregulated, which was accompanied by increased expression of the Wnt signaling inhibitor Axin2. Transfection with a β-catenin-specific siRNA did not affect cell growth but enhanced the resveratrol susceptibility of Colo16 transfectants. The present results suggest the inhibitory effects of resveratrol on epidermal SCCs and inactivation of Wnt signaling as one of the resveratrol-caused molecular events in Colo16 cells. β-catenin oriented siRNA is insufficient to induce cell crisis, implicating the presence of more critical cancer-associated element(s) as the target in Colo16 cells. PMID:28781663

Heterotrimeric G Protein Signaling Is Required for Epidermal Cell Death in Rice[W][OA

PubMed Central

Steffens, Bianka; Sauter, Margret

2009-01-01

In rice (Oryza sativa) adventitious root primordia are formed at the nodes as part of normal development. Upon submergence of rice plants, adventitious roots emerge from the nodes preceded by death of epidermal cells above the root primordia. Cell death is induced by ethylene and mediated by hydrogen peroxide (H2O2). Pharmacological experiments indicated that epidermal cell death was dependent on signaling through G proteins. Treatment with GTP-γ-S induced epidermal cell death, whereas GDP-β-S partially inhibited ethylene-induced cell death. The dwarf1 (d1) mutant of rice has repressed expression of the Gα subunit RGA1 of heterotrimeric G protein. In d1 plants, cell death in response to ethylene and H2O2 was nearly completely abolished, indicating that signaling through Gα is essential. Ethylene and H2O2 were previously shown to alter gene expression in epidermal cells that undergo cell death. Transcriptional regulation was not generally affected in the d1 mutant, indicating that altered gene expression is not sufficient to trigger cell death in the absence of Gα. Analysis of genes encoding proteins related to G protein signaling revealed that four small GTPase genes, two GTPase-activating protein genes, and one GDP dissociation inhibitor gene but not RGA1 were differentially expressed in epidermal cells above adventitious roots, indicating that Gα activity is regulated posttranscriptionally. PMID:19656904

Increased hydrophobicity in Malassezia species correlates with increased proinflammatory cytokine expression in human keratinocytes.

PubMed

Akaza, Narifumi; Akamatsu, Hirohiko; Takeoka, Shiori; Mizutani, Hiroshi; Nakata, Satoru; Matsunaga, Kayoko

2012-11-01

Malassezia cells stimulate cytokine production by keratinocytes, although this ability differs among Malassezia species for unknown reasons. The aim of this study was to clarify the factors determining the ability to induce cytokine production by human keratinocytes in response to Malassezia species. M. furfur NBRC 0656, M. sympodialis CBS 7222, M. dermatis JCM 11348, M. globosa CBS 7966, M. restricta CBS 7877, and three strains each of M. globosa, M. restricta, M. dermatis, M. sympodialis, and M. furfur maintained under various culture conditions were used. Normal human epidermal keratinocytes (NHEKs) (1 × 10(5) cells) and the Malassezia species (1 × 10(6) cells) were co-cultured, and IL-1α, IL-6, and IL-8 mRNA levels were determined. Moreover, the hydrophobicity and β-1,3-glucan expression at the surface of Malassezia cells were analyzed. The ability of Malassezia cells to trigger the mRNA expression of proinflammatory cytokines in NHEKs differed with the species and conditions and was dependent upon the hydrophobicity of Malassezia cells not β-1,3-glucan expression.

An Automated and Minimally Invasive Tool for Generating Autologous Viable Epidermal Micrografts

PubMed Central

Osborne, Sandra N.; Schmidt, Marisa A.; Harper, John R.

2016-01-01

ABSTRACT OBJECTIVE: A new epidermal harvesting tool (CelluTome; Kinetic Concepts, Inc, San Antonio, Texas) created epidermal micrografts with minimal donor site damage, increased expansion ratios, and did not require the use of an operating room. The tool, which applies both heat and suction concurrently to normal skin, was used to produce epidermal micrografts that were assessed for uniform viability, donor-site healing, and discomfort during and after the epidermal harvesting procedure. DESIGN: This study was a prospective, noncomparative institutional review board–approved healthy human study to assess epidermal graft viability, donor-site morbidity, and patient experience. SETTING: These studies were conducted at the multispecialty research facility, Clinical Trials of Texas, Inc, San Antonio. PATIENTS: The participants were 15 healthy human volunteers. RESULTS: The average viability of epidermal micrografts was 99.5%. Skin assessment determined that 76% to 100% of the area of all donor sites was the same in appearance as the surrounding skin within 14 days after epidermal harvest. A mean pain of 1.3 (on a scale of 1 to 5) was reported throughout the harvesting process. CONCLUSIONS: Use of this automated, minimally invasive harvesting system provided a simple, low-cost method of producing uniformly viable autologous epidermal micrografts with minimal patient discomfort and superficial donor-site wound healing within 2 weeks. PMID:26765157

Local Burn Injury Promotes Defects in the Epidermal Lipid and Antimicrobial Peptide Barriers in Human Autograft Skin and Burn Margin: Implications for Burn Wound Healing and Graft Survival

PubMed Central

Plichta, Jennifer K.; Holmes, Casey J.; Gamelli, Richard L.; Radek, Katherine A.

2016-01-01

Burn injury increases the risk of morbidity and mortality by promoting severe hemodynamic shock and risk for local or systemic infection. Graft failure due to poor wound healing or infection remains a significant problem for burn subjects. The mechanisms by which local burn injury compromises the epithelial antimicrobial barrier function in the burn margin, containing the elements necessary for healing of the burn site, and in distal unburned skin, which serves as potential donor tissue, are largely unknown. The objective of this study was to establish defects in epidermal barrier function in human donor skin and burn margin, in order to identify potential mechanisms that may lead to graft failure and/or impaired burn wound healing. In the present study, we established that epidermal lipids and respective lipid synthesis enzymes were significantly reduced in both donor skin and burn margin. We further identified diverse changes in the gene expression and protein production of several candidate skin antimicrobial peptides (AMPs) in both donor skin and burn margin. These results also parallel changes in cutaneous AMP activity against common burn wound pathogens, aberrant production of epidermal proteases known to regulate barrier permeability and AMP activity, and greater production of pro-inflammatory cytokines known to be induced by AMPs. These findings suggest that impaired epidermal lipid and AMP regulation could contribute to graft failure and infectious complications in subjects with burn or other traumatic injury. PMID:27183442

Harnessing Integrative Omics to Facilitate Molecular Imaging of the Human Epidermal Growth Factor Receptor Family for Precision Medicine.

PubMed

Pool, Martin; de Boer, H Rudolf; Hooge, Marjolijn N Lub-de; van Vugt, Marcel A T M; de Vries, Elisabeth G E

2017-01-01

Cancer is a growing problem worldwide. The cause of death in cancer patients is often due to treatment-resistant metastatic disease. Many molecularly targeted anticancer drugs have been developed against 'oncogenic driver' pathways. However, these treatments are usually only effective in properly selected patients. Resistance to molecularly targeted drugs through selective pressure on acquired mutations or molecular rewiring can hinder their effectiveness. This review summarizes how molecular imaging techniques can potentially facilitate the optimal implementation of targeted agents. Using the human epidermal growth factor receptor (HER) family as a model in (pre)clinical studies, we illustrate how molecular imaging may be employed to characterize whole body target expression as well as monitor drug effectiveness and the emergence of tumor resistance. We further discuss how an integrative omics discovery platform could guide the selection of 'effect sensors' - new molecular imaging targets - which are dynamic markers that indicate treatment effectiveness or resistance.

Apoptosis as a Mechanism for Keratinocyte Death in Canine Toxic Epidermal Necrolysis.

PubMed

Banovic, F; Dunston, S; Linder, K E; Rakich, P; Olivry, T

2017-03-01

In humans and dogs, toxic epidermal necrolysis (TEN) is a life-threatening dermatosis characterized by sudden epidermal death resulting in extensive skin detachment. There is little information on the pathogenesis of keratinocyte cell death in canine TEN. We studied the occurrence of apoptosis in skin lesions of dogs with TEN to determine if apoptosis contributes to the pathogenesis of this disease. Immunostaining with antibodies to activated caspase-3 and the terminal deoxynucleotidyl-transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling technique revealed positive apoptotic keratinocytes in basal and suprabasal epidermal compartments in 17 biopsy specimens collected from 3 dogs with TEN and 16 from 3 dogs with erythema multiforme (EM). There was no significant difference in the number of positively stained epidermal cells between TEN and EM. These results suggest that apoptosis of epidermal keratinocytes and lymphocytic satellitosis represent one of the early steps in the pathogenesis of canine TEN, as in the human disease counterpart.

Genome-Wide Expression Profiling of Five Mouse Models Identifies Similarities and Differences with Human Psoriasis

PubMed Central

Swindell, William R.; Johnston, Andrew; Carbajal, Steve; Han, Gangwen; Wohn, Christian; Lu, Jun; Xing, Xianying; Nair, Rajan P.; Voorhees, John J.; Elder, James T.; Wang, Xiao-Jing; Sano, Shigetoshi; Prens, Errol P.; DiGiovanni, John; Pittelkow, Mark R.; Ward, Nicole L.; Gudjonsson, Johann E.

2011-01-01

Development of a suitable mouse model would facilitate the investigation of pathomechanisms underlying human psoriasis and would also assist in development of therapeutic treatments. However, while many psoriasis mouse models have been proposed, no single model recapitulates all features of the human disease, and standardized validation criteria for psoriasis mouse models have not been widely applied. In this study, whole-genome transcriptional profiling is used to compare gene expression patterns manifested by human psoriatic skin lesions with those that occur in five psoriasis mouse models (K5-Tie2, imiquimod, K14-AREG, K5-Stat3C and K5-TGFbeta1). While the cutaneous gene expression profiles associated with each mouse phenotype exhibited statistically significant similarity to the expression profile of psoriasis in humans, each model displayed distinctive sets of similarities and differences in comparison to human psoriasis. For all five models, correspondence to the human disease was strong with respect to genes involved in epidermal development and keratinization. Immune and inflammation-associated gene expression, in contrast, was more variable between models as compared to the human disease. These findings support the value of all five models as research tools, each with identifiable areas of convergence to and divergence from the human disease. Additionally, the approach used in this paper provides an objective and quantitative method for evaluation of proposed mouse models of psoriasis, which can be strategically applied in future studies to score strengths of mouse phenotypes relative to specific aspects of human psoriasis. PMID:21483750

Lysophosphatidic acid stimulates epidermal growth factor-family ectodomain shedding and paracrine signaling from human lung fibroblasts.

PubMed

Shiomi, Tetsuya; Boudreault, Francis; Padem, Nurcicek; Higashiyama, Shigeki; Drazen, Jeffrey M; Tschumperlin, Daniel J

2011-01-01

Lysophospatidic acid (LPA) is a bioactive lipid mediator implicated in tissue repair and wound healing. It mediates diverse functional effects in fibroblasts, including proliferation, migration and contraction, but less is known about its ability to evoke paracrine signaling to other cell types involved in wound healing. We hypothesized that human pulmonary fibroblasts stimulated by LPA would exhibit ectodomain shedding of epidermal growth factor receptor (EGFR) ligands that signal to lung epithelial cells. To test this hypothesis, we used alkaline phosphatase-tagged EGFR ligand plasmids transfected into lung fibroblasts, and enzyme-linked immunosorbent assays to detect shedding of native ligands. LPA induced shedding of alkaline phosphatase-tagged heparin-binding epidermal growth factor (HB-EGF), amphiregulin, and transforming growth factor-a; non-transfected fibroblasts shed amphiregulin and HBEGF under baseline conditions, and increased shedding of HB-EGF in response to LPA. Treatment of fibroblasts with LPA resulted in elevated phosphorylation of extracellular signal-regulated kinase 1/2, enhanced expression of mRNA for c-fos, HB-EGF and amphiregulin, and enhanced proliferation at 96 hours. However, none of these fibroblast responses to LPA required ectodomain shedding or EGFR activity. To test the ability of LPA to stimulate paracrine signaling from fibroblasts, we transferred conditioned medium from LPA-stimulated cells, and found enhanced EGFR and extracellular signal-regulated kinase 1/2 phosphorylation in reporter A549 cells in excess of what could be accounted for by transferred LPA alone. These data show that LPA mediates EGF-family ectodomain shedding, resulting in enhanced paracrine signaling from lung fibroblasts to epithelial cells. © 2011 by the Wound Healing Society.

Periostin contributes to epidermal hyperplasia in psoriasis common to atopic dermatitis

PubMed Central

Arima, Kazuhiko; Ohta, Shoichiro; Takagi, Atsushi; Shiraishi, Hiroshi; Masuoka, Miho; Ontsuka, Kanako; Suto, Hajime; Suzuki, Shoichi; Yamamoto, Ken-ichi; Ogawa, Masahiro; Simmons, Olga; Yamaguchi, Yukie; Toda, Shuji; Aihara, Michiko; Conway, Simon J.; Ikeda, Shigaku; Izuhara, Kenji

2016-01-01

Background Epidermal hyperplasia is a histological hallmark observed in both atopic dermatitis (AD) and psoriasis, although the clinical features and the underlying immunological disorders of these diseases are different. We previously showed that periostin, a matricellular protein, plays a critical role in epidermal hyperplasia in AD, using a mouse model and a 3-dimensional organotypic coculture system. In this study, we explore the hypothesis that periostin is involved in epidermal hyperplasia in psoriasis. Methods To examine expression of periostin in psoriasis patients, we performed immunohistochemical analysis on skin biopsies from six such patients. To investigate periostin’s role in the pathogenesis of psoriasis, we evaluated periostin-deficient mice in a psoriasis mouse model induced by topical treatment with imiquimod (IMQ). Results Periostin was substantially expressed in the dermis of all investigated psoriasis patients. Epidermal hyperplasia induced by IMQ treatment was impaired in periostin-deficient mice, along with decreased skin swelling. However, upon treatment with IMQ, periostin deficiency did not alter infiltration of inflammatory cells such as neutrophils; production of IL-17, –22, or –23; or induction/expansion of IL-17– and IL-22–producing group 3 innate lymphoid cells. Conclusions Periostin plays an important role during epidermal hyperplasia in IMQ-induced skin inflammation, independently of the IL-23–IL-17/IL-22 axis. Periostin appears to be a mediator for epidermal hyperplasia that is common to AD and psoriasis. PMID:25572557

Epidermal Growth Factor Receptor (EGFR) mutation analysis, gene expression profiling and EGFR protein expression in primary prostate cancer

PubMed Central

2011-01-01

Background Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease. Methods Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples. Results EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression. Conclusions Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series. PMID:21266046

Epidermal Growth Factor Receptor (EGFR) mutation analysis, gene expression profiling and EGFR protein expression in primary prostate cancer.

PubMed

Peraldo-Neia, Caterina; Migliardi, Giorgia; Mello-Grand, Maurizia; Montemurro, Filippo; Segir, Raffaella; Pignochino, Ymera; Cavalloni, Giuliana; Torchio, Bruno; Mosso, Luciano; Chiorino, Giovanna; Aglietta, Massimo

2011-01-25

Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease. Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples. EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression. Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series.

Aberrant Wound Healing in an Epidermal Interleukin-4 Transgenic Mouse Model of Atopic Dermatitis

PubMed Central

Zhao, Yan; Bao, Lei; Chan, Lawrence S.; DiPietro, Luisa A.; Chen, Lin

2016-01-01

Wound healing in a pre-existing Th2-dominated skin milieu was assessed by using an epidermal specific interleukin-4 (IL-4) transgenic (Tg) mouse model, which develops a pruritic inflammatory skin condition resembling human atopic dermatitis. Our results demonstrated that IL-4 Tg mice had delayed wound closure and re-epithelialization even though these mice exhibited higher degrees of epithelial cell proliferation. Wounds in IL-4 Tg mice also showed a marked enhancement in expression of inflammatory cytokines/chemokines, elevated infiltration of inflammatory cells including neutrophils, macrophages, CD3+ lymphocytes, and epidermal dendritic T lymphocytes. In addition, these mice exhibited a significantly higher level of angiogenesis as compared to wild type mice. Furthermore, wounds in IL-4 Tg mice presented with larger amounts of granulation tissue, but had less expression and deposition of collagen. Taken together, an inflamed skin condition induced by IL-4 has a pronounced negative influence on the healing process. Understanding more about the pathogenesis of wound healing in a Th2- dominated environment may help investigators explore new potential therapeutic strategies. PMID:26752054

Selective Ablation of Ctip2/Bcl11b in Epidermal Keratinocytes Triggers Atopic Dermatitis-Like Skin Inflammatory Responses in Adult Mice

PubMed Central

Guha, Gunjan; Li, Shan; Kyrylkova, Kateryna; Kioussi, Chrissa; Leid, Mark; Ganguli-Indra, Gitali; Indra, Arup K.

2012-01-01

Background Ctip2 is crucial for epidermal homeostasis and protective barrier formation in developing mouse embryos. Selective ablation of Ctip2 in epidermis leads to increased transepidermal water loss (TEWL), impaired epidermal proliferation, terminal differentiation, as well as altered lipid composition during development. However, little is known about the role of Ctip2 in skin homeostasis in adult mice. Methodology/Principal Findings To study the role of Ctip2 in adult skin homeostasis, we utilized Ctip2ep−/− mouse model in which Ctip2 is selectively deleted in epidermal keratinocytes. Measurement of TEWL, followed by histological, immunohistochemical, and RT-qPCR analyses revealed an important role of Ctip2 in barrier maintenance and in regulating adult skin homeostasis. We demonstrated that keratinocytic ablation of Ctip2 leads to atopic dermatitis (AD)-like skin inflammation, characterized by alopecia, pruritus and scaling, as well as extensive infiltration of immune cells including T lymphocytes, mast cells, and eosinophils. We observed increased expression of T-helper 2 (Th2)-type cytokines and chemokines in the mutant skin, as well as systemic immune responses that share similarity with human AD patients. Furthermore, we discovered that thymic stromal lymphopoietin (TSLP) expression was significantly upregulated in the mutant epidermis as early as postnatal day 1 and ChIP assay revealed that TSLP is likely a direct transcriptional target of Ctip2 in epidermal keratinocytes. Conclusions/Significance Our data demonstrated a cell-autonomous role of Ctip2 in barrier maintenance and epidermal homeostasis in adult mice skin. We discovered a crucial non-cell autonomous role of keratinocytic Ctip2 in suppressing skin inflammatory responses by regulating the expression of Th2-type cytokines. It is likely that the epidermal hyperproliferation in the Ctip2-lacking epidermis may be secondary to the compensatory response of the adult epidermis that is defective in

Transcriptional Profiling of Ectoderm Specification to Keratinocyte Fate in Human Embryonic Stem Cells

PubMed Central

Tadeu, Ana Mafalda Baptista; Lin, Samantha; Hou, Lin; Chung, Lisa; Zhong, Mei; Zhao, Hongyu; Horsley, Valerie

2015-01-01

In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ–secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions. PMID:25849374

miR-203 modulates epithelial differentiation of human embryonic stem cells towards epidermal stratification.

PubMed

Nissan, Xavier; Denis, Jérôme Alexandre; Saidani, Manoubia; Lemaitre, Gilles; Peschanski, Marc; Baldeschi, Christine

2011-08-15

The molecular mechanisms controlling the differentiation of human basal keratinocyte stem cells towards the epidermis are well characterized, whereas the earliest process leading to the specification of embryonic stem cells into keratinocytes is still not well understood. MicroRNAs are regulators of many cellular events, but evidence for microRNA acting on the differentiation of human embryonic stem cells into a specific lineage has been elusive. By using our recent protocol for obtaining functional keratinocytes from hESC, we attempted to analyze the role of microRNAs in the early stages of epidermal differentiation. Thus, we identified a set of 5 microRNAs, namely miR-200a, miR-200b, miR-203, miR-205 and miR-429, that are specifically overexpressed during the early stages of the differentiation process. Interestingly, our functional analyses revealed an instrumental role of miR-203, which had been previously shown to play a key role during the formation of the pluristratified epidermis by basal keratinocyte stem cells, in the early keratinocyte commitment. These results highlight the determinant and unique role of miR-203 during the entire process of epidermal development by extending its spectrum of action from the early commitment of embryonic stem cells to ultimate differentiation of the organ. Copyright © 2011 Elsevier Inc. All rights reserved.

Assessment of the human epidermal model LabCyte EPI-MODEL for In vitro skin corrosion testing according to the OECD test guideline 431.

PubMed

Katoh, Masakazu; Hamajima, Fumiyasu; Ogasawara, Takahiro; Hata, Ken-Ichiro

2010-06-01

A new OECD test guideline 431 (TG431) for in vitro skin corrosion tests using human reconstructed skin models was adopted by OECD in 2004. TG431 defines the criteria for the general function and performance of applicable skin models. In order to confirm that the new reconstructed human epidermal model, LabCyte EPI-MODEL is applicable for the skin corrosion test according to TG431, the predictability and repeatability of the model for the skin corrosion test was evaluated. The test was performed according to the test protocol described in TG431. Based on the knowledge that LabCyte EPI-MODEL is an epidermal model as well as EpiDerm, we decided to adopt the the Epiderm prediction model of skin corrosion for the LabCyte EPI-MODEL, using twenty test chemicals (10 corrosive chemicals and 10 non-corrosive chemicals) in the 1(st) stage. The prediction model results showed that the distinction of non-corrosion to corrosion corresponded perfectly. Therefore, it was judged that the prediction model of EpiDerm could be applied to the LabCyte EPI-MODEL. In the 2(nd) stage, the repeatability of this test protocol with the LabCyte EPI-MODEL was examined using twelve chemicals (6 corrosive chemicals and 6 non-corrosive chemicals) that are described in TG431, and these results recognized a high repeatability and accurate predictability. It was concluded that LabCyte EPI-MODEL is applicable for the skin corrosive test protocol according to TG431.

Hybrid Enhanced Epidermal SpaceSuit Design Approaches

NASA Astrophysics Data System (ADS)

Jessup, Joseph M.

A Space suit that does not rely on gas pressurization is a multi-faceted problem that requires major stability controls to be incorporated during design and construction. The concept of Hybrid Epidermal Enhancement space suit integrates evolved human anthropomorphic and physiological adaptations into its functionality, using commercially available bio-medical technologies to address shortcomings of conventional gas pressure suits, and the impracticalities of MCP suits. The prototype HEE Space Suit explored integumentary homeostasis, thermal control and mobility using advanced bio-medical materials technology and construction concepts. The goal was a space suit that functions as an enhanced, multi-functional bio-mimic of the human epidermal layer that works in attunement with the wearer rather than as a separate system. In addressing human physiological requirements for design and construction of the HEE suit, testing regimes were devised and integrated into the prototype which was then subject to a series of detailed tests using both anatomical reproduction methods and human subject.

NASA-approved rotary bioreactor enhances proliferation of human epidermal stem cells and supports formation of 3D epidermis-like structure.

PubMed

Lei, Xiao-hua; Ning, Li-na; Cao, Yu-jing; Liu, Shuang; Zhang, Shou-bing; Qiu, Zhi-fang; Hu, Hui-min; Zhang, Hui-shan; Liu, Shu; Duan, En-kui

2011-01-01

The skin is susceptible to different injuries and diseases. One major obstacle in skin tissue engineering is how to develop functional three-dimensional (3D) substitute for damaged skin. Previous studies have proved a 3D dynamic simulated microgravity (SMG) culture system as a "stimulatory" environment for the proliferation and differentiation of stem cells. Here, we employed the NASA-approved rotary bioreactor to investigate the proliferation and differentiation of human epidermal stem cells (hEpSCs). hEpSCs were isolated from children foreskins and enriched by collecting epidermal stem cell colonies. Cytodex-3 micro-carriers and hEpSCs were co-cultured in the rotary bioreactor and 6-well dish for 15 days. The result showed that hEpSCs cultured in rotary bioreactor exhibited enhanced proliferation and viability surpassing those cultured in static conditions. Additionally, immunostaining analysis confirmed higher percentage of ki67 positive cells in rotary bioreactor compared with the static culture. In contrast, comparing with static culture, cells in the rotary bioreactor displayed a low expression of involucrin at day 10. Histological analysis revealed that cells cultured in rotary bioreactor aggregated on the micro-carriers and formed multilayer 3D epidermis structures. In conclusion, our research suggests that NASA-approved rotary bioreactor can support the proliferation of hEpSCs and provide a strategy to form multilayer epidermis structure.

Embryonic control of epidermal cell patterning in the root and hypocotyl of Arabidopsis.

PubMed

Lin, Y; Schiefelbein, J

2001-10-01

A position-dependent pattern of epidermal cell types is produced during the development of the Arabidopsis seedling root and hypocotyl. To understand the origin and regulation of this patterning mechanism, we have examined the embryonic expression of the GLABRA2 (GL2) gene, which encodes a cell-type-specific transcription factor. Using in situ RNA hybridization and a sensitive GL2::GFP reporter, we discovered that a position-dependent pattern of GL2 expression is established within protodermal cells at the heart stage and is maintained throughout the remainder of embryogenesis. In addition, we show that an exceptional GL2 expression character and epidermal cell pattern arises during development of the root-hypocotyl junction, which represents an anatomical transition zone. Furthermore, we find that two of the genes regulating seedling epidermal patterning, TRANSPARENT TESTA GLABRA (TTG) and WEREWOLF (WER), also control the embryonic GL2 pattern, whereas the CAPRICE (CPC) and GL2 genes are not required to establish this pattern. These results indicate that position-dependent patterning of epidermal cell types begins at an early stage of embryogenesis, before formation of the apical meristems and shortly after the cellular anatomy of the protoderm and outer ground tissue layer is established. Thus, epidermal cell specification in the Arabidopsis seedling relies on the embryonic establishment of a patterning mechanism that is perpetuated postembryonically.

De novo epidermal regeneration using human eccrine sweat gland cells: higher competence of secretory over absorptive cells.

PubMed

Pontiggia, Luca; Biedermann, Thomas; Böttcher-Haberzeth, Sophie; Oliveira, Carol; Braziulis, Erik; Klar, Agnieszka S; Meuli-Simmen, Claudia; Meuli, Martin; Reichmann, Ernst

2014-06-01

In our previous work, we showed that human sweat gland-derived epithelial cells represent an alternative source of keratinocytes to grow a near normal autologous epidermis. The role of subtypes of sweat gland cells in epidermal regeneration and maintenance remained unclear. In this study, we compare the regenerative potential of both secretory and absorptive sweat gland cell subpopulations. We demonstrate the superiority of secretory over absorptive cells in forming a new epidermis on two levels: first, the proliferative and colony-forming efficiencies in vitro are significantly higher for secretory cells (SCs), and second, SCs show a higher frequency of successful epidermis formation as well as an increase in the thickness of the formed epidermis in the in vitro and in vivo functional analyses using a 3D dermo-epidermal skin model. However, the ability of forming functional skin substitutes is not limited to SCs, which supports the hypothesis that multiple subtypes of sweat gland epithelial cells hold regenerative properties, while the existence and exact localization of a keratinocyte stem cell population in the human eccrine sweat gland remain elusive.

Identification of Candidate Transcriptional Regulators of Epidermal Transfer Cell Development in Vicia faba Cotyledons

PubMed Central

Arun-Chinnappa, Kiruba S.; McCurdy, David W.

2016-01-01

Transfer cells (TCs) are anatomically-specialized cells formed at apoplasmic-symplasmic bottlenecks in nutrient transport pathways in plants. TCs form invaginated wall ingrowths which provide a scaffold to amplify plasma membrane surface area and thus increase the density of nutrient transporters required to achieve enhanced nutrient flow across these bottlenecks. Despite their importance to nutrient transport in plants, little is known of the transcriptional regulation of wall ingrowth formation. Here, we used RNA-Seq to identify transcription factors putatively involved in regulating epidermal TC development in cotyledons of Vicia faba. Comparing cotyledons cultured for 0, 3, 9, and 24 h to induce trans-differentiation of epidermal TCs identified 43 transcription factors that showed either epidermal-specific or epidermal–enhanced expression, and 10 that showed epidermal-specific down regulation. Members of the WRKY and ethylene-responsive families were prominent in the cohort of transcription factors showing epidermal-specific or epidermal–enhanced expression, consistent with the initiation of TC development often representing a response to stress. Members of the MYB family were also prominent in these categories, including orthologs of MYB genes involved in localized secondary wall deposition in Arabidopsis thaliana. Among the group of transcription factors showing down regulation were various homeobox genes and members of the MADs-box and zinc-finger families of poorly defined functions. Collectively, this study identified several transcription factors showing expression characteristics and orthologous functions that indicate likely participation in transcriptional regulation of epidermal TC development in V. faba cotyledons. PMID:27252730

Heparin binding epidermal growth factor in renal ischaemia/reperfusion injury.

PubMed

Mulder, Gemma M; Nijboer, Willemijn N; Seelen, Marc A; Sandovici, Maria; Bos, Eelke M; Melenhorst, Wynand B W H; Trzpis, Monika; Kloosterhuis, Niels J; Visser, Lydia; Henning, Rob H; Leuvenink, Henri G D; Ploeg, Rutger J; Sunnarborg, Susan W; van Goor, Harry

2010-06-01

The epidermal growth factor (EGF) receptor and its ligands are crucially involved in the renal response to ischaemia. We studied the heparin binding-epidermal growth factor (HB-EGF), a major ligand for the EGF receptor, in experimental and human ischaemia/reperfusion injury (IRI). HB-EGF mRNA and protein expression was studied in rat kidneys and cultured human tubular (HK-2) cells that were subjected to IRI and in human donor kidneys during transplantation. The effect of EGF receptor inhibition was investigated in vivo and in vitro. Furthermore, urinary HB-EGF protein excretion was studied after renal transplantation. Finally, HB-EGF KO and WT mice were subjected to IRI to study the role of HB-EGF in renal injury. HB-EGF mRNA was significantly up-regulated in the early phase of IRI in rats, cells, and human donor biopsies. Treatment with PKI-166 reduces macrophage accumulation and interstitial alpha-SMA in the early phase of IRI in rats. In vitro, PKI-166 causes a marked reduction in HB-EGF-induced cellular proliferation. Urinary HB-EGF is increased after transplantation compared with control urines from healthy subjects. HB-EGF KO mice subjected to IRI revealed significantly less morphological damage after IRI, compared with WT mice. We conclude that IRI results in early induction of HB-EGF mRNA and protein in vivo and in vitro. Absence of HB-EGF and inhibition of the EGF receptor in the early phase of IRI has protective effects, suggesting a modulating role for HB-EGF.

Expression and analysis of exogenous proteins in epidermal cells.

PubMed

Dagnino, Lina; Ho, Ernest; Chang, Wing Y

2010-01-01

In this chapter we review protocols for transient transfection of primary keratinocytes. The ability to transfect primary epidermal cells regardless of their differentiation status allows the biochemical and molecular characterization of multiple proteins. We review methods to analyze exogenous protein abundance in transfected keratinocytes by immunoblot and immunoprecipitation. We also present protocols to determine the subcellular distribution of these proteins by indirect immunofluorescence microscopy approaches.

In Vitro Expression of Cytokeratin 19 in Adipose-Derived Stem Cells Is Induced by Epidermal Growth Factor.

PubMed

Chen, Shangliang; Wang, Mingzhu; Chen, Xinglu; Chen, Shaolian; Liu, Li; Zhu, Jianbin; Wang, Jinhui; Yang, Xiaorong; Cai, Xiangsheng

2018-06-21

BACKGROUND Cytokeratin 19 (CK19) is a typical epithelial marker. In this study, we determined whether epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) could enhance CK19 expression in adipose-derived stem cells (ADSCs), thereby inducing the differentiation of ADSCs into epithelial-like cells. MATERIAL AND METHODS ADSCs were isolated from perinephric fat, and the expression of CD29, CD90, and CD105 was confirmed. Following isolation, ADSCs were cultured in static medium or medium containing EGF or bFGF. RESULTS Flow cytometry revealed that EGF and bFGF could alter mesenchymal stem cell markers as well as the cell cycle of ADSCs. Western blotting and immunofluorescence revealed that after 14 days, EGF treatment enhanced the expression of CK19 in ADSCs. CONCLUSIONS Our findings offer important insight for the clinical use of ADSCs in the generation of epithelial-like cells in the future.

In vitro study of the effects of ELF electric fields on gene expression in human epidermal cells.

PubMed

Collard, Jean-Francois; Mertens, Benjamin; Hinsenkamp, Maurice

2011-01-01

An acceleration of differentiation, at the expense of proliferation, is observed after exposure of various biological models to low frequency and low amplitude electric and electromagnetic fields. Following these results showing significant modifications, we try to identify the biological mechanism involved at the cell level through microarray screening. For this study, we use epidermis cultures harvested from human abdominoplasty. Two platinum electrodes are used to apply the electric signal. The gene expressions of 38,500 well-characterized human genes are analyzed using Affymetrix(®) microarray U133 Plus 2.0 chips. The protocol is repeated on three different patients. After three periods of exposure, a total of 24 chips have been processed. After the application of ELF electric fields, the microarray analysis confirms a modification of the gene expression of epidermis cells. Particularly, four up-regulated genes (DKK1, TXNRD1, ATF3, and MME) and one down-regulated gene (MACF1) are involved in the regulation of proliferation and differentiation. Expression of these five genes was also confirmed by real-time rtPCR in all samples used for microarray analysis. These results corroborate an acceleration of cell differentiation at the expense of cell proliferation. © 2010 Wiley-Liss, Inc.

Inhibition of epidermal growth factor receptor by ferulic acid and 4-vinylguaiacol in human breast cancer cells.

PubMed

Sudhagar, S; Sathya, S; Anuradha, R; Gokulapriya, G; Geetharani, Y; Lakshmi, B S

2018-02-01

To examine the potential of ferulic acid and 4-vinylguaiacol for inhibiting epidermal growth factor receptor (EGFR) in human breast cancer cells in vitro. Ferulic acid and 4-vinylguaiacol limit the EGF (epidermal growth factor)-induced breast cancer proliferation and new DNA synthesis. Western blot analysis revealed both ferulic acid and 4-vinylguaiacol exhibit sustained inhibition of EGFR activation through down-regulation of Tyr 1068 autophosphorylation. Molecular docking analysis shows ferulic acid forming hydrogen bond interaction with Lys 745 and Met 793 whereas, 4-vinylguaiacol forms two hydrogen bonds with Phe 856 and exhibits stronger hydrophobic interactions with multiple amino acid residues at the EGFR kinase domain. Ferulic acid and 4-vinylguaiacol could serve as a potential structure for the development of new small molecule therapeutics against EGFR.

The peripheral clock regulates human pigmentation.

PubMed

Hardman, Jonathan A; Tobin, Desmond J; Haslam, Iain S; Farjo, Nilofer; Farjo, Bessam; Al-Nuaimi, Yusur; Grimaldi, Benedetto; Paus, Ralf

2015-04-01

Although the regulation of pigmentation is well characterized, it remains unclear whether cell-autonomous controls regulate the cyclic on-off switching of pigmentation in the hair follicle (HF). As human HFs and epidermal melanocytes express clock genes and proteins, and given that core clock genes (PER1, BMAL1) modulate human HF cycling, we investigated whether peripheral clock activity influences human HF pigmentation. We found that silencing BMAL1 or PER1 in human HFs increased HF melanin content. Furthermore, tyrosinase expression and activity, as well as TYRP1 and TYRP2 mRNA levels, gp100 protein expression, melanocyte dendricity, and the number gp100+ HF melanocytes, were all significantly increased in BMAL1 and/or PER1-silenced HFs. BMAL1 or PER1 silencing also increased epidermal melanin content, gp100 protein expression, and tyrosinase activity in human skin. These effects reflect direct modulation of melanocytes, as BMAL1 and/or PER1 silencing in isolated melanocytes increased tyrosinase activity and TYRP1/2 expression. Mechanistically, BMAL1 knockdown reduces PER1 transcription, and PER1 silencing induces phosphorylation of the master regulator of melanogenesis, microphthalmia-associated transcription factor, thus stimulating human melanogenesis and melanocyte activity in situ and in vitro. Therefore, the molecular clock operates as a cell-autonomous modulator of human pigmentation and may be targeted for future therapeutic strategies.

Antisense imaging of epidermal growth factor-induced p21(WAF-1/CIP-1) gene expression in MDA-MB-468 human breast cancer xenografts.

PubMed

Wang, Judy; Chen, Paul; Mrkobrada, Marko; Hu, Meiduo; Vallis, Katherine A; Reilly, Raymond M

2003-09-01

Molecular imaging of the expression of key genes which determine the response to DNA damage following cancer treatment may predict the effectiveness of a particular treatment strategy. A prominent early response gene for DNA damage is the gene encoding p21(WAF-1/CIP-1), a cyclin-dependent kinase inhibitor that regulates progression through the cell cycle. In this study, we explored the feasibility of imaging p21(WAF-1/CIP-1) gene expression at the mRNA level using an 18-mer phosphorothioated antisense oligodeoxynucleotide (ODN) labeled with (111)In. The known induction of the p21(WAF-1/CIP-1) gene in MDA-MB-468 human breast cancer cells following exposure to epidermal growth factor (EGF) was used as an experimental tool. Treatment of MDA-MB-468 cells in vitro with EGF (20 n M) increased the ratio of p21(WAF-1/CIP-1) mRNA/beta-actin mRNA threefold within 2 h as measured by the reverse transcription polymerase chain reaction (RT-PCR). A concentration-dependent inhibition of EGF-induced p21(WAF-1/CIP-1) protein expression was achieved in MDA-MB-468 cells by treatment with antisense ODNs with up to a tenfold decrease observed at 1 microM. There was a fourfold lower inhibition of p21(WAF-1/CIP-1) protein expression by control sense or random sequence ODNs. Intratumoral injections of EGF (15 microg/dayx3 days) were employed to induce p21(WAF-1/CIP-1) gene expression in MDA-MB-468 xenografts implanted subcutaneously into athymic mice. RT-PCR of explanted tumors showed a threefold increased level of p21(WAF-1/CIP-1) mRNA compared with normal saline-treated tumors. Successful imaging of EGF-induced p21(WAF-1/CIP-1) gene expression in MDA-MB-468 xenografts was achieved at 48 h post injection of (111)In-labeled antisense ODNs (3.7 MBq; 2 microg). Tumors displaying basal levels of p21(WAF-1/CIP-1) gene expression in the absence of EGF treatment could not be visualized. Biodistribution studies showed a significantly higher tumor accumulation of (111)In-labeled antisense ODNs in

Dermal penetration and metabolism of p-aminophenol and p-phenylenediamine: application of the EpiDerm human reconstructed epidermis model.

PubMed

Hu, Ting; Bailey, Ruth E; Morrall, Stephen W; Aardema, Marilyn J; Stanley, Lesley A; Skare, Julie A

2009-07-24

To address the provision of the 7th Amendment to the EU Cosmetics Directive banning the use of in vivo genotoxicity assays for testing cosmetic ingredients in 2009, the 3D EpiDerm reconstructed human skin micronucleus assay has been developed. To further characterise the EpiDerm tissue for potential use in genotoxicity testing, we have evaluated the dermal penetration and metabolism of two hair dye ingredients, p-aminophenol (PAP) and p-phenylenediamine (PPD) in this reconstructed epidermis model. When EpiDerm tissue was topically exposed to PAP or PPD for 30 min (typical for a hair dye exposure), the majority (80->90%) of PAP or PPD was excluded from skin tissue and removed by rinsing. After a 23.5h recovery period, the PAP fraction that did penetrate was completely N-acetylated to acetaminophen (APAP). Similarly, 30 min topical application of PPD resulted in the formation of the N-mono- and N,N'-diacetylated metabolites of PPD. These results are consistent with published data on the dermal metabolism of these compounds from other in vitro systems as well as from in vivo studies. When tissue was exposed topically (PAP) or via the culture media (PPD) for 24h, there was good batch-to-batch and donor-to-donor reproducibility in the penetration and metabolism of PAP and PPD. Overall, the results demonstrate that these two aromatic amines are biotransformed in 3D EpiDerm tissue via N-acetylation. Characterising the metabolic capability of EpiDerm tissue is important for the evaluation of this model for use in genotoxicity testing.

Tumor formation initiated by nondividing epidermal cells via an inflammatory infiltrate.

PubMed

Arwert, Esther N; Lal, Rohit; Quist, Sven; Rosewell, Ian; van Rooijen, Nico; Watt, Fiona M

2010-11-16

In mammalian epidermis, integrin expression is normally confined to the basal proliferative layer that contains stem cells. However, in epidermal hyperproliferative disorders and tumors, integrins are also expressed by suprabasal cells, with concomitant up-regulation of Erk mitogen-activated protein kinase (MAPK) signaling. In transgenic mice, expression of activated MAPK kinase 1 (MEK1) in the suprabasal, nondividing, differentiated cell layers (InvEE transgenics) results in epidermal hyperproliferation and skin inflammation. We now demonstrate that wounding induces benign tumors (papillomas and keratoacanthomas) in InvEE mice. By generating chimeras between InvEE mice and mice that lack the MEK1 transgene, we demonstrate that differentiating, nondividing cells that express MEK1 stimulate adjacent transgene-negative cells to divide and become incorporated into the tumor mass. Dexamethasone treatment inhibits tumor formation, suggesting that inflammation is involved. InvEE skin and tumors express high levels of IL1α; treatment with an IL1 receptor antagonist delays tumor onset and reduces incidence. Depletion of γδ T cells and macrophages also reduces tumor incidence. Because a hallmark of cancer is uncontrolled proliferation, it is widely assumed that tumors arise only from dividing cells. In contrast, our studies show that differentiated epidermal cells can initiate tumor formation without reacquiring the ability to divide and that they do so by triggering an inflammatory infiltrate.

A role for the epidermal growth factor receptor signaling in development of intestinal serrated polyps in mice and humans.

PubMed

Bongers, Gerold; Muniz, Luciana R; Pacer, Michelle E; Iuga, Alina C; Thirunarayanan, Nanthakumar; Slinger, Erik; Smit, Martine J; Reddy, E Premkumar; Mayer, Lloyd; Furtado, Glaucia C; Harpaz, Noam; Lira, Sergio A

2012-09-01

Epithelial cancers can be initiated by activating mutations in components of the mitogen-activated protein kinase signaling pathway such as v-raf murine sarcoma viral oncogene homolog B1 (BRAF), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), or epidermal growth factor receptor (EGFR). Human intestinal serrated polyps are a heterogeneous group of benign lesions, but some progress to colorectal cancer. Tumors that arise from these polyps frequently contain activating mutations in BRAF or KRAS, but little is known about the role of EGFR activation in their development. Polyp samples were obtained from adults during screening colonoscopies at Mount Sinai Hospital in New York. We measured levels of EGFR protein and phosphorylation in human serrated polyps by immunohistochemical and immunoblot analyses. We generated transgenic mice that express the ligand for EGFR, Heparin-binding EGF-like growth factor (HB-EGF), in the intestine. EGFR and the extracellular-regulated kinases (ERK)1/2 were phosphorylated in serrated areas of human hyperplastic polyps (HPPs), sessile serrated adenomas, and traditional serrated adenomas. EGFR and ERK1/2 were phosphorylated in the absence of KRAS or BRAF activating mutations in a subset of HPP. Transgenic expression of the EGFR ligand HB-EGF in the intestines of mice promoted development of small cecal serrated polyps. Mice that expressed a combination of HB-EGF and US28 (a constitutively active, G-protein-coupled receptor that increases processing of HB-EGF from the membrane) rapidly developed large cecal serrated polyps. These polyps were similar to HPPs and had increased phosphorylation of EGFR and ERK1/2 within the serrated epithelium. Administration of pharmacologic inhibitors of EGFR or MAPK to these transgenic mice significantly reduced polyp development. Activation of EGFR signaling in the intestine of mice promotes development of serrated polyps. EGFR signaling also is activated in human HPPs, sessile serrated adenomas

"Cut-and-paste" manufacture of multiparametric epidermal electronic systems

NASA Astrophysics Data System (ADS)

Lu, Nanshu; Yang, Shixuan; Wang, Pulin

2016-05-01

Epidermal electronics is a class of noninvasive and unobstructive skin-mounted, tattoo-like sensors and electronics capable of vital sign monitoring and establishing human-machine interface. The high cost of manpower, materials, vacuum equipment, and photolithographic facilities associated with its manufacture greatly hinders the widespread use of disposable epidermal electronics. Here we report a cost and time effective, completely dry, benchtop "cut-and-paste" method for the freeform and portable manufacture of multiparametric epidermal sensor systems (ESS) within minutes. This versatile method works for all types of thin metal and polymeric sheets and is compatible with any tattoo adhesives or medical tapes. The resulting ESS are multimaterial and multifunctional and have been demonstrated to noninvasively but accurately measure electrophysiological signals, skin temperature, skin hydration, as well as respiratory rate. In addition, planar stretchable coils exploiting double-stranded serpentine design have been successfully applied as wireless, passive epidermal strain sensors.

Langerhans' cell expression of the selectin ligand, sialyl Lewis x.

PubMed Central

Ross, E L; Barker, J N; Allen, M H; Chu, A C; Groves, R W; MacDonald, D M

1994-01-01

Cellular adhesion molecules play a central role in leucocyte migration through peripheral blood and tissues. A crucial stage in these events in selectin-mediated adhesion involving E-selectin expressed on activated endothelium interacting with a range of carbohydrate ligands expressed by specific subpopulations of leucocytes. As such mechanisms may be relevant to bone marrow-derived dendritic epidermal Langerhans' cell (LC) migration, expression of these carbohydrate ligands was assessed immunocytochemically in whole skin biopsies and in epidermal cell suspensions obtained from adult humans. Double-labelling experiments revealed that sialyl Lewis x, recognized by the monoclonal antibody CSLEX1, was expressed on epidermal LC (n = 9). Furthermore, expression was enhanced at 24 hr following epicutaneous application of antigen and in the inflammatory disorder psoriasis (n = 10). E-selectin was concomitantly strongly expressed on dermal endothelium in psoriasis and allergic contact dermatitis. Intradermal injection of the T-cell-derived cytokine interferon-gamma (IFN-gamma) led to increased LC expression of sialyl Lewis x. In epidermal cell suspensions, in contrast to keratinocytes, CD1a+ cells expressed sialyl Lewis x, intensity of which was enhanced after 4 days in culture. CSLEX1 staining could be abolished and CD15 (non-sialated Lewis x) expression induced by saponification and treatment with neuraminidase. Expression of other selectin ligands was also examined. While the cutaneous lymphocyte antigen defined by the monoclonal antibody HECA-452 reacted with a small minority of LC, sialyl Lewis a and sulphatide were not expressed under any experimental conditions. These studies indicate that E-selectin-sialyl Lewis x interactions are potentially important in LC migration, both into and out of skin. Images Figure 2 Figure 3 Figure 5 Figure 6 PMID:7512530

Effects of soap-water wash on human epidermal penetration.

PubMed

Zhu, Hanjiang; Jung, Eui-Chang; Phuong, Christina; Hui, Xiaoying; Maibach, Howard

2016-08-01

Skin decontamination is a primary interventional method used to decrease dermal absorption of hazardous contaminants, including chemical warfare agents, pesticides and industrial pollutants. Soap and water wash, the most common and readily available decontamination system, may enhance percutaneous absorption through the "wash-in effect." To understand better the effect of soap-water wash on percutaneous penetration, and provide insight to improving skin decontamination methods, in vitro human epidermal penetration rates of four C(14) -labeled model chemicals (hydroquinone, clonidine, benzoic acid and paraoxon) were assayed using flow-through diffusion cells. Stratum corneum (SC) absorption rates of these chemicals at various hydration levels (0-295% of the dry SC weights) were determined and compared with the results of the epidermal penetration study to clarify the effect of SC hydration on skin permeability. Results showed accelerated penetration curves of benzoic acid and paraoxon after surface wash at 30 min postdosing. Thirty minutes after washing (60 min postdosing), penetration rates of hydroquinone and benzoic acid decreased due to reduced amounts of chemical on the skin surface and in the SC. At the end of the experiment (90 min postdosing), a soap-water wash resulted in lower hydroquinone penetration, greater paraoxon penetration and similar levels of benzoic acid and clonidine penetration compared to penetration levels in the non-wash groups. The observed wash-in effect agrees with the enhancement effect of SC hydration on the SC chemical absorption rate. These results suggest SC hydration derived from surface wash to be one cause of the wash-in effect. Further, the occurrence of a wash-in effect is dependent on chemical identity and elapsed time between exposure and onset of decontamination. By reducing chemical residue quantity on skin surface and in the SC reservoir, the soap-water wash may decrease the total quantity of chemical absorbed in the

Antioxidant capacity of 3D human skin EpiDerm model: effects of skin moisturizers.

PubMed

Grazul-Bilska, A T; Bilski, J J; Redmer, D A; Reynolds, L P; Abdullah, K M; Abdullah, A

2009-06-01

The objective of this study was to determine the effects of skin moisturizers on total antioxidant capacity (TAC) of human skin using EpiDerm model. Three different skin moisturizers containing antioxidant ingredients (samples 1-3) or aloe vera extract were topically applied to EpiDerm units and incubated for 2 and 24 h to determine acute and longer-term effects of applied samples on TAC and glutathione peroxidase activity in medium and/or homogenized skin tissues. Total antioxidant capacity in medium and skin homogenates was enhanced (P < 0.0001) by gel containing antioxidant ingredients (sample 2) after 2 and 24 h of incubation. Total antioxidant capacity in medium was also enhanced (P < 0.001) by cream containing antioxidant ingredients (sample 3) after 24 h of incubation. Overall, TAC in medium was greater (P < 0.02) after 24 h than 2 h of incubation. Skin moisturizer cream with high antioxidant levels determined by using oxygen radical absorbance capacity testing (sample 1) and aloe vera extract did not affect TAC. Glutathione peroxidase activity was enhanced (P < 0.0001) in medium and skin homogenates by sample 2 but not by any other sample. These data demonstrate high potential of gel and cream (samples 2 and 3) containing antioxidant ingredients in enhancing antioxidant capacity of EpiDerm which will likely contribute to overall skin health. Results of this experiment will help to better understand mechanisms of effects of skin moisturizers containing antioxidant ingredients on skin function at the tissue level and to establish effective strategies for skin protection and clinical treatments of skin disorders and possibly healing wounds.

Steroid hormone and epidermal growth factor receptors in meningiomas.

PubMed

Horsfall, D J; Goldsmith, K G; Ricciardelli, C; Skinner, J M; Tilley, W D; Marshall, V R

1989-11-01

A prospective study of steroid hormone and epidermal growth factor receptor expression in 57 meningiomas is presented. Scatchard analysis of radioligand binding identified 20% of meningiomas as expressing classical oestrogen receptors (ER) at levels below that normally accepted for positivity, the remainder being negative. ER could not be visualized in any meningioma using immunocytochemistry. Alternatively, 74% of meningiomas demonstrated the presence of progesterone receptors (PR) by Scatchard analysis, the specificity of which could not be attributed to glucocorticoid or androgen receptors. Confirmation of classical PR presence was determined by immunocytochemical staining. The presence of epidermal growth factor receptor (EGFR) was demonstrated in 100% of meningiomas using immunocytochemical staining. These data are reviewed in the context of previously reported results and are discussed in relation to the potential for medical therapy as an adjunct to surgery.

Protective effects of amifostine and cyclooxygenase-1 inhibitor against normal human epidermal keratinocyte toxicity induced by methotrexate and 5-fluorouracil.

PubMed

Maiguma, Takayoshi; Kaji, Hiroaki; Makino, Kazutaka; Teshima, Daisuke

2009-07-01

Our study aimed to find more effective protective agents against mucosa toxicity induced by methotrexate and 5-fluorouracil. We focused on the relationship between oral mucositis and keratinocyte injury and examined methotrexate and 5-fluorouracil-induced cytotoxicity in normal human epidermal keratinocyte cell lines. Cell viability and superoxide radical activity were measured based on converting WST-1 (4-[3-(4-indophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzen disulfonate) to a water-soluble formazan dye. DNA synthesis by 5-bromo-2'-deoxyuridine incorporation was measured as an indirect parameter of cell proliferation. Allopurinol and amifostine were used as the radical scavengers. l-glutamine was used as a mucosa-protective agent. A cyclooxygenase inhibitor interrupting the production of hydroxyl radicals in the arachidonic acid cascade was also examined. 5-fluorouracil and methotrexate caused cytotoxicity due to the activation of intracellular superoxide radicals specifically on normal human epidermal keratinocytes. From the electron spin resonance study, it was found that allopurinol was a superoxide radical scavenger, while amifostine was hydroxyl radical scavenger. Allopurinol showed no effect on the cytotoxicity due to 5-fluorouracil and methotrexate. The cell injury induced by methotrexate was restored by amifostine. However, the cell injury induced by 5-fluorouracil was markedly recovered by a selective cyclooxygenase-1 inhibitor compared to amifostine. It was suggested that amifostine and cyclooxygenase-1 inhibitor could be useful protective agents against methotrexate and 5-fluorouracil chemotherapeutic toxicity. Additionally, this in vitro cell injury model using normal human epidermal keratinocytes may be useful for understanding the pathophysiology of oral mucositis induced by chemotherapeutic agents.

Toll like receptors gene expression of human keratinocytes cultured of severe burn injury.

PubMed

Cornick, Sarita Mac; Noronha, Silvana Aparecida Alves Corrêa de; Noronha, Samuel Marcos Ribeiro de; Cezillo, Marcus V B; Ferreira, Lydia Masako; Gragnani, Alfredo

2014-01-01

To evaluate the expression profile of genes related to Toll Like Receptors (TLR) pathways of human Primary Epidermal keratinocytes of patients with severe burns. After obtaining viable fragments of skin with and without burning, culture hKEP was initiated by the enzymatic method using Dispase (Sigma-Aldrich). These cells were treated with Trizol(r) (Life Technologies) for extraction of total RNA. This was quantified and analyzed for purity for obtaining cDNA for the analysis of gene expression using specific TLR pathways PCR Arrays plates (SA Biosciences). After the analysis of gene expression we found that 21% of these genes were differentially expressed, of which 100% were repressed or hyporegulated. Among these, the following genes (fold decrease): HSPA1A (-58), HRAS (-36), MAP2K3 (-23), TOLLIP (-23), RELA (-18), FOS (-16), and TLR1 (-6.0). This study contributes to the understanding of the molecular mechanisms related to TLR pathways and underlying wound infection caused by the burn. Furthermore, it may provide new strategies to restore normal expression of these genes and thereby change the healing process and improve clinical outcome.

Epidermal Phytochrome B Inhibits Hypocotyl Negative Gravitropism Non-Cell-Autonomously

PubMed Central

Kim, Jaewook; Song, Kijong; Park, Eunae; Kim, Keunhwa; Choi, Giltsu

2016-01-01

Seedling hypocotyls display negative gravitropism in the dark but agravitropism in the light. The Arabidopsis thaliana pif quadruple mutant (pifQ), which lacks four PHYTOCHROME-INTERACTING FACTORS (PIFs), is agravitropic in the dark. Endodermis-specific expression of PIF1 rescues gravitropism in pifQ mutant seedlings. Since phytochromes induce light responses by inhibiting PIFs and the COP1-SPA ubiquitin E3 ligase complex in the nucleus, we asked whether phyB can cell autonomously inhibit hypocotyl negative gravitropism in the endodermis. We found that while epidermis-specific expression of PHYB rescues hypocotyl negative gravitropism and all other phyB mutant phenotypes, endodermis-specific expression of PHYB does not. Epidermal phyB induces the phosphorylation and degradation of endodermal PIFs in response to red light. This induces a global gene expression pattern similar to that induced by red light treatment of seedlings expressing PHYB under the control of its own endogenous promoter. Our results imply that epidermal phyB generates an unidentified mobile signal that travels to the endodermis where it promotes PIF degradation and inhibits hypocotyl negative gravitropism. PMID:27758895

Effects of Asterias amurensis-derived Sphingoid Bases on the de novo Ceramide Synthesis in Cultured Normal Human Epidermal Keratinocytes.

PubMed

Mikami, Daisuke; Sakai, Shota; Sasaki, Shigefumi; Igarashi, Yasuyuki

2016-08-01

Asterias amurensis starfish provide several bioactive species in addition to being fishery waste. Glucosyl ceramides (GlcCers) were extracted from the viscera of these starfish and were isolated by silica gel column chromatography. Degraded GlcCers generated A. amurensis sphingoid bases (ASBs) that mainly consisted of the triene-type bases d18:3 and 9-methyl-d18:3. The effect of these bases on ceramide synthesis and content were analyzed using normal human epidermal keratinocytes (NHEKs). The bases significantly enhanced the de novo ceramide synthesis and gene expression in NHEKs for proteins, such as serine-palmitoyltransferase and ceramide synthase. Total ceramide, GlcCer, and sphingomyelin contents increased dramatically upon ASB treatment. In particular, GlcCer bearing very-long-chain fatty acids (≥C28) exhibited a significant content increase. These ASB-induced enhancements on de novo ceramide synthesis were only observed in undifferentiated NHEKs. This stimulation of the de novo sphingolipid synthesis may improve skin barrier functions.

Hair Follicle and Sebaceous Gland De Novo Regeneration With Cultured Epidermal Stem Cells and Skin-Derived Precursors.

PubMed

Wang, Xiaoxiao; Wang, Xusheng; Liu, Jianjun; Cai, Ting; Guo, Ling; Wang, Shujuan; Wang, Jinmei; Cao, Yanpei; Ge, Jianfeng; Jiang, Yuyang; Tredget, Edward E; Cao, Mengjun; Wu, Yaojiong

2016-12-01

: Stem cell-based organ regeneration is purported to enable the replacement of impaired organs in the foreseeable future. Here, we demonstrated that a combination of cultured epidermal stem cells (Epi-SCs) derived from the epidermis and skin-derived precursors (SKPs) was capable of reconstituting functional hair follicles and sebaceous glands (SG). When Epi-SCs and SKPs were mixed in a hydrogel and implanted into an excisional wound in nude mice, the Epi-SCs formed de novo epidermis along with hair follicles, and SKPs contributed to dermal papilla in the neogenic hair follicles. Notably, a combination of culture-expanded Epi-SCs and SKPs derived from the adult human scalp were sufficient to generate hair follicles and hair. Bone morphogenetic protein 4, but not Wnts, sustained the expression of alkaline phosphatase in SKPs in vitro and the hair follicle-inductive property in vivo when SKPs were engrafted with neonatal epidermal cells into excisional wounds. In addition, Epi-SCs were capable of differentiating into sebocytes and formed de novo SGs, which excreted lipids as do normal SGs. Thus our results indicate that cultured Epi-SCs and SKPs are sufficient to generate de novo hair follicles and SGs, implying great potential to develop novel bioengineered skin substitutes with appendage genesis capacity. In postpartum humans, skin appendages lost in injury are not regenerated, despite the considerable achievement made in skin bioengineering. In this study, transplantation of a combination of culture-expanded epidermal stem cells and skin-derived progenitors from mice and adult humans led to de novo regeneration of functional hair follicles and sebaceous glands. The data provide transferable knowledge for the development of novel bioengineered skin substitutes with epidermal appendage regeneration capacity. ©AlphaMed Press.

Hair Follicle and Sebaceous Gland De Novo Regeneration With Cultured Epidermal Stem Cells and Skin-Derived Precursors

PubMed Central

Wang, Xiaoxiao; Wang, Xusheng; Liu, Jianjun; Cai, Ting; Guo, Ling; Wang, Shujuan; Wang, Jinmei; Cao, Yanpei; Ge, Jianfeng; Jiang, Yuyang; Tredget, Edward E.; Cao, Mengjun

2016-01-01

Stem cell-based organ regeneration is purported to enable the replacement of impaired organs in the foreseeable future. Here, we demonstrated that a combination of cultured epidermal stem cells (Epi-SCs) derived from the epidermis and skin-derived precursors (SKPs) was capable of reconstituting functional hair follicles and sebaceous glands (SG). When Epi-SCs and SKPs were mixed in a hydrogel and implanted into an excisional wound in nude mice, the Epi-SCs formed de novo epidermis along with hair follicles, and SKPs contributed to dermal papilla in the neogenic hair follicles. Notably, a combination of culture-expanded Epi-SCs and SKPs derived from the adult human scalp were sufficient to generate hair follicles and hair. Bone morphogenetic protein 4, but not Wnts, sustained the expression of alkaline phosphatase in SKPs in vitro and the hair follicle-inductive property in vivo when SKPs were engrafted with neonatal epidermal cells into excisional wounds. In addition, Epi-SCs were capable of differentiating into sebocytes and formed de novo SGs, which excreted lipids as do normal SGs. Thus our results indicate that cultured Epi-SCs and SKPs are sufficient to generate de novo hair follicles and SGs, implying great potential to develop novel bioengineered skin substitutes with appendage genesis capacity. Significance In postpartum humans, skin appendages lost in injury are not regenerated, despite the considerable achievement made in skin bioengineering. In this study, transplantation of a combination of culture-expanded epidermal stem cells and skin-derived progenitors from mice and adult humans led to de novo regeneration of functional hair follicles and sebaceous glands. The data provide transferable knowledge for the development of novel bioengineered skin substitutes with epidermal appendage regeneration capacity. PMID:27458264

c-Jun/AP-1 pathway-mediated cyclin D1 expression participates in low dose arsenite-induced transformation in mouse epidermal JB6 Cl41 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang Dongyun; Li Jingxia; Gao Jimin

2009-02-15

Arsenic is a well-documented human carcinogen associated with skin carcinogenesis. Our previous work reveals that arsenite exposure is able to induce cell transformation in mouse epidermal cell JB6 Cl41 through the activation of ERK, rather than JNK pathway. Our current studies further evaluate downstream pathway in low dose arsenite-induced cell transformation in JB6 Cl41 cells. Our results showed that treatment of cells with low dose arsenite induced activation of c-Jun/AP-1 pathway, and ectopic expression of dominant negative mutant of c-Jun (TAM67) blocked arsenite-induced transformation. Furthermore, our data indicated that cyclin D1 was an important downstream molecule involved in c-Jun/AP-1-mediated cellmore » transformation upon low dose arsenite exposure, because inhibition of cyclin D1 expression by its specific siRNA in the JB6 Cl41 cells resulted in impairment of anchorage-independent growth of cells induced by low dose arsenite. Collectively, our results demonstrate that c-Jun/AP-1-mediated cyclin D1 expression is at least one of the key events implicated in cell transformation upon low dose arsenite exposure.« less

Down-regulation of microRNA-146a is associated with high-risk human papillomavirus infection and epidermal growth factor receptor overexpression in penile squamous cell carcinoma.

PubMed

Peta, Elektra; Cappellesso, Rocco; Masi, Giulia; Sinigaglia, Alessandro; Trevisan, Marta; Grassi, Angela; Di Camillo, Barbara; Vassarotto, Elisa; Fassina, Ambrogio; Palù, Giorgio; Barzon, Luisa

2017-03-01

Dysregulation of host microRNA expression has been involved in the development and progression of human papillomavirus (HPV)-related tumors. Analysis of miR-146a expression in a series of 59 penile squamous cell carcinomas (PSCCs) showed that its levels were lower in high-risk HPV-positive than in HPV-negative PSCCs and inversely correlated with expression of epidermal growth factor receptor (EGFR), a known target for miR-146a. Analysis of genotype distribution for rs2910164, a common functional polymorphism of miR-146a, did not identify correlations with miR-146a levels and EGFR expression in PSCCs. In vitro experiments demonstrated that E6 of HPV type 16, but not low-risk HPV-6, down-regulated miR-146a in human foreskin keratinocytes and up-regulated EGFR. Ectopic expression of miR-146a decreased expression of EGFR and inhibited proliferation of keratinocytes and cervical carcinoma cells. EGFR is commonly overexpressed in penile cancer and in other squamous cell carcinomas. Molecular mechanisms leading to EGFR overexpression and activation are known for HPV-negative cancers and include amplification or mutations of the EGFR gene. The results of this study indicate that down-regulation of miR-146a may represent another mechanism of EGFR overexpression in PSCCs, which can be mediated by high-risk HPV E6 in HPV-related tumors. Copyright © 2016 Elsevier Inc. All rights reserved.

Genomic determinants of epidermal appendage patterning and structure in domestic birds

PubMed Central

Boer, Elena F.; Van Hollebeke, Hannah F.; Shapiro, Michael D.

2017-01-01

Variation in regional identity, patterning, and structure of epidermal appendages contributes to skin diversity among many vertebrate groups, and is perhaps most striking in birds. In pioneering work on epidermal appendage patterning, John Saunders and his contemporaries took advantage of epidermal appendage diversity within and among domestic chicken breeds to establish the importance of mesoderm-ectoderm signaling in determining skin patterning. Diversity in chickens and other domestic birds, including pigeons, is driving a new wave of research to dissect the molecular genetic basis of epidermal appendage patterning. Domestic birds are not only outstanding models for embryonic manipulations, as Saunders recognized, but they are also ideal genetic models for discovering the specific genes that control normal development and the mutations that contribute to skin diversity. Here, we review recent genetic and genomic approaches to uncover the basis of epidermal macropatterning, micropatterning, and structural variation. We also present new results that confirm expression changes in two limb identity genes in feather-footed pigeons, a case of variation in appendage structure and identity. PMID:28347644

Genomic determinants of epidermal appendage patterning and structure in domestic birds.

PubMed

Boer, Elena F; Van Hollebeke, Hannah F; Shapiro, Michael D

2017-09-15

Variation in regional identity, patterning, and structure of epidermal appendages contributes to skin diversity among many vertebrate groups, and is perhaps most striking in birds. In pioneering work on epidermal appendage patterning, John Saunders and his contemporaries took advantage of epidermal appendage diversity within and among domestic chicken breeds to establish the importance of mesoderm-ectoderm signaling in determining skin patterning. Diversity in chickens and other domestic birds, including pigeons, is driving a new wave of research to dissect the molecular genetic basis of epidermal appendage patterning. Domestic birds are not only outstanding models for embryonic manipulations, as Saunders recognized, but they are also ideal genetic models for discovering the specific genes that control normal development and the mutations that contribute to skin diversity. Here, we review recent genetic and genomic approaches to uncover the basis of epidermal macropatterning, micropatterning, and structural variation. We also present new results that confirm expression changes in two limb identity genes in feather-footed pigeons, a case of variation in appendage structure and identity. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Evaluation of dermal-epidermal skin equivalents ('composite-skin') of human keratinocytes in a collagen-glycosaminoglycan matrix(Integra artificial skin).

PubMed

Kremer, M; Lang, E; Berger, A C

2000-09-01

Integra artificial skin (Integra LifeSciences Corp., Plainsboro, NJ, USA) is a dermal template consisting of bovine collagen, chondroitin-6-sulphate and a silastic membrane manufactured as Integra. This product has gained widespread use in the clinical treatment of third degree burn wounds and full thickness skin defects of different aetiologies. The product was designed to significantly reduce the time needed to achieve final wound closure in the treatment of major burn wounds, to optimise the sparse autologous donor skin resources and to improve the durable mechanical quality of the skin substitute. The clinical procedure requires two stages. The first step creates a self neodermis, the second creates a self epidermis on the neodermis. However, it is desirable to cover major burn wounds early in a single step by a skin substitute consisting of a dermal equivalent seeded in vitro with autologous keratinocytes ('composite-skin') out of which a full thickness skin develops in vivo.The goal of this experimental study was to develop a method to integrate human keratinocytes in homogeneous distribution and depth into Integra Artificial Skin. The seeded cell-matrix composites were grafted onto athymic mice in order to evaluate their potential to reconstitute a human epidermis in vivo. We were able to demonstrate that the inoculated human keratinocytes reproducibly displayed a homogeneous pattern of distribution, adherence, proliferation and confluence. The cell-matrix composites grafted in this model exhibited good wound adherence, complete healing, minor wound contraction and had the potential to reconstitute an elastic, functional and durable human skin. Histologically we were able to show that the inoculated human keratinocytes in vivo colonised the matrix in a histomorphologically characteristic epidermal pattern (keratomorula, keratinocyte bubbling) and developed a persisting, stratified, keratinising epidermis which immunohistologically proved to be of human

Thy-1+ dendritic epidermal cells express T3 antigen and the T-cell receptor gamma chain.

PubMed Central

Stingl, G; Koning, F; Yamada, H; Yokoyama, W M; Tschachler, E; Bluestone, J A; Steiner, G; Samelson, L E; Lew, A M; Coligan, J E

1987-01-01

The murine epidermis is a heterogeneous epithelium composed of keratinocytes, melanocytes, Langerhans cells, and a recently described subpopulation (2-3%) of bone-marrow-derived leukocytes with a dendritic morphology and the cell surface phenotype Thy-1+, L3T4-, Lyt-2-. Previous studies have demonstrated that cell lines derived from freshly explanted Thy-1+ dendritic epidermal cells (DEC) have abundant mRNA for rearranged T-cell receptor (TCR) gamma-chain genes. Analysis of Thy-1+ DEC in situ, freshly isolated cell suspensions of Thy-1+ DEC, and long-term Thy-1+ DEC lines demonstrated that 100% of the Thy-1+ DEC reacted with a monoclonal antibody to the epsilon chain of the murine T3 complex and that 40-60% of resident Thy-1+ DEC were also reactive with an antiserum to the TCR gamma chain. Two Thy-1+ DEC lines expressed a disulfide-linked 70-kDa molecule that could be precipitated with an anti-gamma-chain antiserum and could be coprecipitated with an antiserum to the T3 delta chain; the molecule appeared as a single 34-kDa band under reducing conditions. The phenotype of Thy-1+ DEC (T3+, L3T4-, Lyt-2-, TCR gamma chain+) thus resembles that of the recently described subpopulation of murine and human lymphocytes that have been identified in the thymus, peripheral blood, and fetal blood. Images PMID:2885839

Psoriasiform skin disease in transgenic pigs with high-copy ectopic expression of human integrins α2 and β1.

PubMed

Staunstrup, Nicklas Heine; Stenderup, Karin; Mortensen, Sidsel; Primo, Maria Nascimento; Rosada, Cecilia; Steiniche, Torben; Liu, Ying; Li, Rong; Schmidt, Mette; Purup, Stig; Dagnæs-Hansen, Frederik; Schrøder, Lisbeth Dahl; Svensson, Lars; Petersen, Thomas Kongstad; Callesen, Henrik; Bolund, Lars; Mikkelsen, Jacob Giehm

2017-07-01

Psoriasis is a complex human-specific disease characterized by perturbed keratinocyte proliferation and a pro-inflammatory environment in the skin. Porcine skin architecture and immunity are very similar to that in humans, rendering the pig a suitable animal model for studying the biology and treatment of psoriasis. Expression of integrins, which is normally confined to the basal layer of the epidermis, is maintained in suprabasal keratinocytes in psoriatic skin, modulating proliferation and differentiation as well as leukocyte infiltration. Here, we generated minipigs co-expressing integrins α2 and β1 in suprabasal epidermal layers. Integrin-transgenic minipigs born into the project displayed skin phenotypes that correlated with the number of inserted transgenes. Molecular analyses were in good concordance with histological observations of psoriatic hallmarks, including hypogranulosis and T-lymphocyte infiltration. These findings mark the first creation of minipigs with a psoriasiform phenotype resembling human psoriasis and demonstrate that integrin signaling plays a key role in psoriasis pathology. © 2017. Published by The Company of Biologists Ltd.

Psoriasiform skin disease in transgenic pigs with high-copy ectopic expression of human integrins α2 and β1

PubMed Central

Staunstrup, Nicklas Heine; Stenderup, Karin; Mortensen, Sidsel; Primo, Maria Nascimento; Steiniche, Torben; Liu, Ying; Li, Rong; Schmidt, Mette; Purup, Stig; Dagnæs-Hansen, Frederik; Schrøder, Lisbeth Dahl; Svensson, Lars; Petersen, Thomas Kongstad; Callesen, Henrik; Bolund, Lars

2017-01-01

ABSTRACT Psoriasis is a complex human-specific disease characterized by perturbed keratinocyte proliferation and a pro-inflammatory environment in the skin. Porcine skin architecture and immunity are very similar to that in humans, rendering the pig a suitable animal model for studying the biology and treatment of psoriasis. Expression of integrins, which is normally confined to the basal layer of the epidermis, is maintained in suprabasal keratinocytes in psoriatic skin, modulating proliferation and differentiation as well as leukocyte infiltration. Here, we generated minipigs co-expressing integrins α2 and β1 in suprabasal epidermal layers. Integrin-transgenic minipigs born into the project displayed skin phenotypes that correlated with the number of inserted transgenes. Molecular analyses were in good concordance with histological observations of psoriatic hallmarks, including hypogranulosis and T-lymphocyte infiltration. These findings mark the first creation of minipigs with a psoriasiform phenotype resembling human psoriasis and demonstrate that integrin signaling plays a key role in psoriasis pathology. PMID:28679670

Studies of thermostability in Camelus bactrianus (Bactrian camel) single-domain antibody specific for the mutant epidermal-growth-factor receptor expressed by Pichia.

PubMed

Omidfar, Kobra; Rasaee, Mohhamad Javad; Kashanian, Soheila; Paknejad, Malieheh; Bathaie, Zahra

2007-01-01

Camelids have a unique immune system capable of producing heavy-chain antibodies lacking the light chains and CH1 (constant heavy-chain domain 1). It has been shown that, in contrast with conventional antibody fragments, the variable domains of these heavy-chain antibodies are functional at or after exposure to high temperatures. In the present study, the VHH (variable domain of heavy-chain antibody) camel antibody was subcloned into vector Ppiczc and expressed in Pichia pastoris. ORB1-83 VHH antibody recognizes the external domain of the mutant EGFR [EGF (epidermal growth factor) receptor], EGFR VIII. This tumour-specific antigen is ligand-independent, contains a constitutively active tyrosine kinase domain and has been shown to be present in a number of human malignancies. We report here that, although expression from P. pastoris resulted in a significantly increased level of expression of the anti-EGFR VIII VHH antibodies compared with Escherichia coli [Omidfar, Rasaee, Modjtahedi, Forouzandeh, Taghikhani, Bakhtiari, Paknejad and Kashanian (2004) Tumor Biol. 25, 179-187; Omidfar, Rasaee, Modjtahedi, Forouzandeh, Taghikhani and Golmakany (2004) Tumor Biol. 25, 296-305], this antibody selectively bound to the EGFR VIII peptide and reacted specifically with the immunoaffinity-purified antigen from non-small-cell lung cancer. Furthermore, thermal denaturation stability and CD spectra analysis of the Camelus bactrianus (Bactrian camel) VHH and heavy-chain antibodies at different temperature proved reversibility and binding activity after heat denaturation. Our results indicate that the P. pastoris expression system may be useful for the expression of camel single domain antibody and the ability of the expressed protein to reversibly melt without aggregation, allowing it to regain binding activity after heat denaturation.

Steroids are required for epidermal cell fate establishment in Arabidopsis roots.

PubMed

Kuppusamy, Kavitha T; Chen, Andrew Y; Nemhauser, Jennifer L

2009-05-12

The simple structure of Arabidopsis roots provides an excellent model system to study epidermal cell fate specification. Epidermal cells in contact with 2 underlying cortical cells differentiate into hair cells (H cells; trichoblasts), whereas cells that contact only a single cortical cell differentiate into mature hairless cells (N cells; atrichoblasts). This position-dependent patterning, in combination with the constrained orientation of cell divisions, results in hair and nonhair cell files running longitudinally along the root epidermis. Here, we present strong evidence that steroid hormones called brassinosteroids (BRs) are required to maintain position-dependent fate specification in roots. We show that BRs are required for normal expression levels and patterns of WEREWOLF (WER) and GLABRA2 (GL2), master regulators of epidermal patterning. Loss of BR signaling results in loss of hair cells in H positions, likely as a consequence of reduced expression of CAPRICE (CPC), a direct downstream target of WER. Our observations demonstrate that in addition to their well-known role in cell expansion, BRs play an essential role in directing cell fate.

Steroids are required for epidermal cell fate establishment in Arabidopsis roots

PubMed Central

Kuppusamy, Kavitha T.; Chen, Andrew Y.; Nemhauser, Jennifer L.

2009-01-01

The simple structure of Arabidopsis roots provides an excellent model system to study epidermal cell fate specification. Epidermal cells in contact with 2 underlying cortical cells differentiate into hair cells (H cells; trichoblasts), whereas cells that contact only a single cortical cell differentiate into mature hairless cells (N cells; atrichoblasts). This position-dependent patterning, in combination with the constrained orientation of cell divisions, results in hair and nonhair cell files running longitudinally along the root epidermis. Here, we present strong evidence that steroid hormones called brassinosteroids (BRs) are required to maintain position-dependent fate specification in roots. We show that BRs are required for normal expression levels and patterns of WEREWOLF (WER) and GLABRA2 (GL2), master regulators of epidermal patterning. Loss of BR signaling results in loss of hair cells in H positions, likely as a consequence of reduced expression of CAPRICE (CPC), a direct downstream target of WER. Our observations demonstrate that in addition to their well-known role in cell expansion, BRs play an essential role in directing cell fate. PMID:19416891

Impact of epidermal growth factor receptor gene expression level on clinical outcomes in epidermal growth factor receptor mutant lung adenocarcinoma patients taking first-line epidermal growth factor receptor-tyrosine kinase inhibitors.

PubMed

Chang, Huang-Chih; Chen, Yu-Mu; Tseng, Chia-Cheng; Huang, Kuo-Tung; Wang, Chin-Chou; Chen, Yung-Che; Lai, Chien-Hao; Fang, Wen-Feng; Kao, Hsu-Ching; Lin, Meng-Chih

2017-03-01

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are first-choice treatments for advanced non-small-cell lung cancer patients harboring EGFR mutations. Although EGFR mutations are strongly predictive of patients' outcomes and their response to treatment with EGFR-TKIs, early failure of first-line therapy with EGFR-TKIs in patients with EGFR mutations is not rare. Besides several clinical factors influencing EGFR-TKI efficacies studied earlier such as the Eastern Cooperative Oncology Group performance status or uncommon mutation, we would like to see whether semi-quantify EGFR mutation gene expression calculated by 2 -ΔΔct was a prognostic factor in EGFR-mutant non-small cell lung cancer patients receiving first-line EGFR-TKIs. This retrospective study reviews 926 lung cancer patients diagnosed from January 2011 to October 2013 at the Kaohsiung Chang Gung Memorial Hospital in Taiwan. Of 224 EGFR-mutant adenocarcinoma patients, 148 patients who had 2 -ΔΔct data were included. The best cutoff values of 2 -ΔΔct for in-frame deletions in exon 19 (19 deletion) and a position 858 substituted from leucine (L) to an arginine (R) in exon 21 (L858R) were determined using receiver operating characteristic curves. Patients were divided into high and low 2 -ΔΔct expression based on the above cutoff level. The best cutoff point of 2 -ΔΔct value of 19 deletion and L858R was 31.1 and 104.7, respectively. In all, 92 (62.1%) patients showed high 2 -ΔΔct expression and 56 patients (37.9%) low 2 -ΔΔct expression. The mean age was 65.6 years. Progression-free survival of 19 deletion mutant patients with low versus high expression level was 17.07 versus 12.04 months (P = 0.004), respectively. Progression-free survival of L858 mutant patients was 13.75 and 7.96 months (P = 0.008), respectively. EGFR-mutant lung adenocarcinoma patients with lower EGFR gene expression had longer progression-free survival duration without interfering

Innate and adaptive immunity gene expression of human keratinocytes cultured of severe burn injury.

PubMed

Noronha, Silvana Aparecida Alves Corrêa de; Noronha, Samuel Marcos Ribeiro de; Lanziani, Larissa Elias; Ferreira, Lydia Masako; Gragnani, Alfredo

2014-01-01

Evaluate the expression profile of genes related to Innate and Adaptive Immune System (IAIS) of human Primary Epidermal keratinocytes (hPEKP) of patients with severe burns. After obtaining viable fragments of skin with and without burning, culture hKEP was initiated by the enzymatic method using Dispase (Sigma-Aldrich). These cells were treated with Trizol(r) (Life Technologies) for extraction of total RNA. This was quantified and analyzed for purity for obtaining cDNA for the analysis of gene expression using specific IAIS PCR Arrays plates (SA Biosciences). After the analysis of gene expression we found that 63% of these genes were differentially expressed, of which 77% were repressed and 23% were hyper-regulated. Among these, the following genes (fold increase or decrease): IL8 (41), IL6 (32), TNF (-92), HLA-E (-86), LYS (-74), CCR6 (- 73), CD86 (-41) and HLA-A (-35). This study contributes to the understanding of the molecular mechanisms underlying wound infection caused by the burn. Furthermore, it may provide new strategies to restore normal expression of these genes and thereby change the healing process and improve clinical outcome.

Epidermal ADAM17 maintains the skin barrier by regulating EGFR ligand–dependent terminal keratinocyte differentiation

PubMed Central

Cobzaru, Cristina; Triantafyllopoulou, Antigoni; Löffek, Stefanie; Horiuchi, Keisuke; Threadgill, David W.; Kurz, Thomas; van Rooijen, Nico; Bruckner-Tuderman, Leena

2012-01-01

ADAM17 (a disintegrin and metalloproteinase 17) is ubiquitously expressed and cleaves membrane proteins, such as epidermal growth factor receptor (EGFR) ligands, l-selectin, and TNF, from the cell surface, thus regulating responses to tissue injury and inflammation. However, little is currently known about its role in skin homeostasis. We show that mice lacking ADAM17 in keratinocytes (A17ΔKC) have a normal epidermal barrier and skin architecture at birth but develop pronounced defects in epidermal barrier integrity soon after birth and develop chronic dermatitis as adults. The dysregulated expression of epidermal differentiation proteins becomes evident 2 d after birth, followed by reduced transglutaminase (TGM) activity, transepidermal water loss, up-regulation of the proinflammatory cytokine IL-36α, and inflammatory immune cell infiltration. Activation of the EGFR was strongly reduced in A17ΔKC skin, and topical treatment of A17ΔKC mice with recombinant TGF-α significantly improved TGM activity and decreased skin inflammation. Finally, we show that mice lacking the EGFR in keratinocytes (EgfrΔKC) closely resembled A17ΔKC mice. Collectively, these results identify a previously unappreciated critical role of the ADAM17–EGFR signaling axis in maintaining the homeostasis of the postnatal epidermal barrier and suggest that this pathway could represent a good target for treatment of epidermal barrier defects. PMID:22565824

Real-time three-dimensional imaging of epidermal splitting and removal by high-definition optical coherence tomography.

PubMed

Boone, Marc; Draye, Jean Pierre; Verween, Gunther; Pirnay, Jean-Paul; Verbeken, Gilbert; De Vos, Daniel; Rose, Thomas; Jennes, Serge; Jemec, Gregor B E; Del Marmol, Véronique

2014-10-01

While real-time 3-D evaluation of human skin constructs is needed, only 2-D non-invasive imaging techniques are available. The aim of this paper is to evaluate the potential of high-definition optical coherence tomography (HD-OCT) for real-time 3-D assessment of the epidermal splitting and decellularization. Human skin samples were incubated with four different agents: Dispase II, NaCl 1 M, sodium dodecyl sulphate (SDS) and Triton X-100. Epidermal splitting, dermo-epidermal junction, acellularity and 3-D architecture of dermal matrices were evaluated by High-definition optical coherence tomography before and after incubation. Real-time 3-D HD-OCT assessment was compared with 2-D en face assessment by reflectance confocal microscopy (RCM). (Immuno) histopathology was used as control. HD-OCT imaging allowed real-time 3-D visualization of the impact of selected agents on epidermal splitting, dermo-epidermal junction, dermal architecture, vascular spaces and cellularity. RCM has a better resolution (1 μm) than HD-OCT (3 μm), permitting differentiation of different collagen fibres, but HD-OCT imaging has deeper penetration (570 μm) than RCM imaging (200 μm). Dispase II and NaCl treatments were found to be equally efficient in the removal of the epidermis from human split-thickness skin allografts. However, a different epidermal splitting level at the dermo-epidermal junction could be observed and confirmed by immunolabelling of collagen type IV and type VII. Epidermal splitting occurred at the level of the lamina densa with dispase II and above the lamina densa (in the lamina lucida) with NaCl. The 3-D architecture of dermal papillae and dermis was more affected by Dispase II on HD-OCT which corresponded with histopathologic (orcein staining) fragmentation of elastic fibres. With SDS treatment, the epidermal removal was incomplete as remnants of the epidermal basal cell layer remained attached to the basement membrane on the dermis. With Triton X-100 treatment

Glucose transporter member 1 is involved in UVB-induced epidermal hyperplasia by enhancing proliferation in epidermal keratinocytes.

PubMed

Tochio, Takumi; Tanaka, Hiroshi; Nakata, Satoru

2013-03-01

Glucose transporter member 1 (GLUT-1) is one of the major facilitated glucose transporters and contributes to the promotion of keratinocyte proliferation in psoriasis and carcinogenic lesions. In this study, we postulate that GLUT-1 is involved in ultraviolet B (UVB)-induced epidermal hyperplasia. The purpose of this study is to investigate the possible role of GLUT-1 in UVB-induced hyperplasia. The effects of UVB on GLUT-1 expression levels were investigated in in vitro and in vivo studies. In addition, the involvement of epidermal growth factor (EGF) and hypoxia inducible factor-1 alpha (HIF-1α), transcriptional factors for GLUT-1, in GLUT-1-related events were investigated. GLUT-1 mRNA and its protein levels were markedly increased by UVB irradiation in HaCaT cells. In in vivo studies, a strong immunofluorescence signal of GLUT-1 was clearly observed around the basal layer of the epidermis, which proliferated excessively by UVB irradiation. In HaCaT cells, EGF mRNA and its protein levels were markedly increased by UVB irradiation, and then the GLUT-1 mRNA level was significantly increased by treatment with EGF. Additionally, the upregulation of GLUT-1 by both UVB irradiation and treatment with EGF was significantly suppressed by transfection with HIF-1α siRNA. We conclude that GLUT-1 is involved in UVB-induced epidermal hyperplasia by enhancing proliferation of epidermal basal cells, and the GLUT-1-related event might be regulated by an increase in HIF-1α stimulated by EGF. © 2013 The International Society of Dermatology.

Epidermal Phytochrome B Inhibits Hypocotyl Negative Gravitropism Non-Cell-Autonomously.

PubMed

Kim, Jaewook; Song, Kijong; Park, Eunae; Kim, Keunhwa; Bae, Gabyong; Choi, Giltsu

2016-11-01

Seedling hypocotyls display negative gravitropism in the dark but agravitropism in the light. The Arabidopsis thaliana pif quadruple mutant (pifQ), which lacks four PHYTOCHROME-INTERACTING FACTORS (PIFs), is agravitropic in the dark. Endodermis-specific expression of PIF1 rescues gravitropism in pifQ mutant seedlings. Since phytochromes induce light responses by inhibiting PIFs and the COP1-SPA ubiquitin E3 ligase complex in the nucleus, we asked whether phyB can cell autonomously inhibit hypocotyl negative gravitropism in the endodermis. We found that while epidermis-specific expression of PHYB rescues hypocotyl negative gravitropism and all other phyB mutant phenotypes, endodermis-specific expression of PHYB does not. Epidermal phyB induces the phosphorylation and degradation of endodermal PIFs in response to red light. This induces a global gene expression pattern similar to that induced by red light treatment of seedlings expressing PHYB under the control of its own endogenous promoter. Our results imply that epidermal phyB generates an unidentified mobile signal that travels to the endodermis where it promotes PIF degradation and inhibits hypocotyl negative gravitropism. © 2016 American Society of Plant Biologists. All rights reserved.

Egr-5 is a post-mitotic regulator of planarian epidermal differentiation

PubMed Central

Tu, Kimberly C; Cheng, Li-Chun; TK Vu, Hanh; Lange, Jeffrey J; McKinney, Sean A; Seidel, Chris W; Sánchez Alvarado, Alejandro

2015-01-01

Neoblasts are an abundant, heterogeneous population of adult stem cells (ASCs) that facilitate the maintenance of planarian tissues and organs, providing a powerful system to study ASC self-renewal and differentiation dynamics. It is unknown how the collective output of neoblasts transit through differentiation pathways to produce specific cell types. The planarian epidermis is a simple tissue that undergoes rapid turnover. We found that as epidermal progeny differentiate, they progress through multiple spatiotemporal transition states with distinct gene expression profiles. We also identified a conserved early growth response family transcription factor, egr-5, that is essential for epidermal differentiation. Disruption of epidermal integrity by egr-5 RNAi triggers a global stress response that induces the proliferation of neoblasts and the concomitant expansion of not only epidermal, but also multiple progenitor cell populations. Our results further establish the planarian epidermis as a novel paradigm to uncover the molecular mechanisms regulating ASC specification in vivo. DOI: http://dx.doi.org/10.7554/eLife.10501.001 PMID:26457503

Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

PubMed

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Chang, Yao-Wen; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2017-12-01

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-119 (IgG 1 , kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H 2 Mab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although H 2 Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with H 2 Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that H 2 Mab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.

Identification of human leukemia antigen A*0201-restricted epitopes derived from epidermal growth factor pathway substrate number 8.

PubMed

Tang, Baishan; Zhou, Weijun; Du, Jingwen; He, Yanjie; Li, Yuhua

2015-08-01

T-cell-mediated immunotherapy of hematological malignancies requires selection of targeted tumor-associated antigens and T-cell epitopes contained in these tumor proteins. Epidermal growth factor receptor pathway substrate 8 (EPS8), whose function is pivotal for tumor proliferation, progression and metastasis, has been found to be overexpressed in most human tumor types, while its expression in normal tissue is low. The aim of the present study was to identify human leukemia antigen (HLA)-A*0201-restricted epitopes of EPS8 by using a reverse immunology approach. To achieve this, computer algorithms were used to predict HLA-A*0201 molecular binding, proteasome cleavage patterns as well as translocation of transporters associated with antigen processing. Candidate peptides were experimentally validated by T2 binding affinity assay and brefeldin-A decay assay. The functional avidity of peptide-specific cytotoxic T lymphocytes (CTLs) induced from peripheral blood mononuclear cells of healthy volunteers were evaluated by using an enzyme-linked immunosorbent spot assay and a cytotoxicity assay. Four peptides, designated as P455, P92, P276 and P360, had high affinity and stability of binding towards the HLA-A*0201 molecule, and specific CTLs induced by them significantly responded to the corresponding peptides and secreted IFN-γ. At the same time, the CTLs were able to specifically lyse EPS8-expressing cell lines in an HLA-A*0201-restricted manner. The present study demonstrated that P455, P92, P276 and P360 were CTL epitopes of EPS8, and were able to be used for epitope-defined adoptive T-cell transfer and multi-epitope-based vaccine design.

Age-related changes in expression and function of Toll-like receptors in human skin

PubMed Central

Iram, Nousheen; Mildner, Michael; Prior, Marion; Petzelbauer, Peter; Fiala, Christian; Hacker, Stefan; Schöppl, Alice; Tschachler, Erwin; Elbe-Bürger, Adelheid

2012-01-01

Toll-like receptors (TLRs) initiate innate immune responses and direct subsequent adaptive immunity. They play a major role in cutaneous host defense against micro-organisms and in the pathophysiology of several inflammatory skin diseases. To understand the role of TLRs in the acquisition of immunological competence, we conducted a comprehensive study to evaluate TLR expression and function in the developing human skin before and after birth and compared it with adults. We found that prenatal skin already expresses the same spectrum of TLRs as adult skin. Strikingly, many TLRs were significantly higher expressed in prenatal (TLRs 1-5) and infant and child (TLRs 1 and 3) skin than in adult skin. Surprisingly, neither dendritic cell precursors in prenatal skin nor epidermal Langerhans cells and dermal dendritic cells in adult skin expressed TLRs 3 and 6, whereas the staining pattern and intensity of both TLRs in fetal basal keratinocytes was almost comparable to those of adults. Stimulation of primary human keratinocytes from fetal, neonatal and adult donors with selected TLR agonists revealed that the synthetic TLR3 ligand poly (I:C) specifically, mimicking viral double-stranded RNA, induced a significantly enhanced secretion of CXCL8/IL8, CXCL10/IP-10 and TNFα in fetal and neonatal keratinocytes compared with adult keratinocytes. This study demonstrates quantitative age-specific modifications in TLR expression and innate skin immune reactivity in response to TLR activation. Thus, antiviral innate immunity already in prenatal skin may contribute to protect the developing human body from viral infections in utero in a scenario where the adaptive immune system is not yet fully functional. PMID:23034637

64Cu-Labeled Repebody Molecules for Imaging of Epidermal Growth Factor Receptor-Expressing Tumors.

PubMed

Pyo, Ayoung; Yun, Misun; Kim, Hyeon Sik; Kim, Tae-Yoon; Lee, Joong-Jae; Kim, Jung Young; Lee, Sunwoo; Kwon, Seong Young; Bom, Hee-Seung; Kim, Hak-Sung; Kim, Dong-Yeon; Min, Jung-Joon

2018-02-01

The epidermal growth factor receptor (EGFR) is a member of the erbB family of receptors and is overexpressed in many tumor types. A repebody is a newly designed nonantibody protein scaffold for tumor targeting that contains leucine-rich repeat modules. In this study, 3 64 Cu-labeled anti-EGFR repebodies with different chelators were synthesized, and their biologic characteristics were assessed in cultured cells and tumor-bearing mice. Methods: Repebodies were synthesized with the chelators 2-( p -isothiocyanatobenzyl)-1,4,7-triazacyclononane- N,N',N,″- triacetic acid trihydrochloride ([ p -SCN-Bn]-NOTA), 2,2',2″-(10-(2-(2,5-dioxopyrrolidin-1-yloxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (DOTA- N -hydroxysuccinimide ester), or 1-( p -isothiocyanatobenzyl)diethylenetriamine pentaacetic acid trihydrochloride ([ p -SCN-Bn]-DTPA) in 1.0 M NaHCO 3 buffer (pH 9.2) for 24 h. Purified NOTA-, DOTA-, and DTPA-conjugated repebody were radiolabeled with 64 Cu in 0.1 M NH 4 OAc buffer (pH 5.5). To compare the EGFR-binding affinities of the repebodies, cellular uptake studies were performed with the human non-small cell lung cancer cell line H1650 (high expression of EGFR) and the human colon adenocarcinoma cell line SW620 (low expression of EGFR). Biodistribution and small-animal PET imaging studies were performed using H1650 tumor-bearing mice. Results: Radiochemical yields of the 64 Cu-labeled repebodies were approximately 70%-80%. Cellular uptake of the NOTA-, DOTA-, and DTPA-repebodies was over 4-fold higher in H1650 cells than in SW620 cells at 1 h. The 3 repebodies had accumulated specifically in H1650 tumor-bearing nude mice by 1 h after intravenous injection and were retained for over 24 h, as measured by the percentage injected dose per gram of tissue (%ID/g). Tumor uptake of all repebodies increased from 1 to 6 h (at 1 h, 6.28, 8.46, and 6.91 %ID/g for NOTA-, DOTA-, and DTPA-repebody, respectively; at 6 h, 9.4, 8.28, and 10.1 %ID

MATRIX METALLOPROTEINS (MMP)-MEDIATED PHOSPHORYLATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZINC (ZN)

EPA Science Inventory

Matrix Metalloproteinase (MMP)-Mediated Phosphorylation of The Epidermal Growth Factor Receptor (EGFR) in Human Airway Epithelial Cells (HAEC) Exposed to Zinc (Zn)
Weidong Wu, James M. Samet, Robert Silbajoris, Lisa A. Dailey, Lee M. Graves, and Philip A. Bromberg
Center fo...

Epidermal Th22 and Tc17 cells form a localized disease memory in clinically healed psoriasis.

PubMed

Cheuk, Stanley; Wikén, Maria; Blomqvist, Lennart; Nylén, Susanne; Talme, Toomas; Ståhle, Mona; Eidsmo, Liv

2014-04-01

Psoriasis is a common and chronic inflammatory skin disease in which T cells play a key role. Effective treatment heals the skin without scarring, but typically psoriasis recurs in previously affected areas. A pathogenic memory within the skin has been proposed, but the nature of such site-specific disease memory is unknown. Tissue-resident memory T (TRM) cells have been ascribed a role in immunity after resolved viral skin infections. Because of their localization in the epidermal compartment of the skin, TRM may contribute to tissue pathology during psoriasis. In this study, we investigated whether resolved psoriasis lesions contain TRM cells with the ability to maintain and potentially drive recurrent disease. Three common and effective therapies, narrowband-UVB treatment and long-term biologic treatment systemically inhibiting TNF-α or IL-12/23 signaling were studied. Epidermal T cells were highly activated in psoriasis and a high proportion of CD8 T cells expressed TRM markers. In resolved psoriasis, a population of cutaneous lymphocyte-associated Ag, CCR6, CD103, and IL-23R expressing epidermal CD8 T cells was highly enriched. Epidermal CD8 T cells expressing the TRM marker CD103 responded to ex vivo stimulation with IL-17A production and epidermal CD4 T cells responded with IL-22 production after as long as 6 y of TNF-α inhibition. Our data suggest that epidermal TRM cells are retained in resolved psoriasis and that these cells are capable of producing cytokines with a critical role in psoriasis pathogenesis. We provide a potential mechanism for a site-specific T cell-driven disease memory in psoriasis.

Blockade of epidermal growth factor receptor signaling in tumor cells and tumor-associated endothelial cells for therapy of androgen-independent human prostate cancer growing in the bone of nude mice.

PubMed

Kim, Sun-Jin; Uehara, Hisanori; Karashima, Takashi; Shepherd, David L; Killion, Jerald J; Fidler, Isaiah J

2003-03-01

We determined whether blockade of the epidermal growth factor receptor (EGF-R) signaling pathway by oral administration of the EGF-R tyrosine kinase inhibitor (PKI 166) alone or in combination with injectable Taxol inhibits the growth of PC-3MM2 human prostate cancer cells in the bone of nude mice. Male nude mice implanted with PC-3MM2 cells in the tibia were treated with oral administrations of PKI 166 or PKI 166 plus injectable Taxol beginning 3 days after implantation. The incidence and size of bone tumors and destruction of bone were determined by digitalized radiography. Expression of epidermal growth factor (EGF), EGF-R, and activated EGF-R in tumor cells and tumor-associated endothelial cells was determined by immunohistochemistry. Oral administration of PKI 166 or PKI 166 plus injectable Taxol reduced the incidence and size of bone tumors and destruction of bone. Immunohistochemical analysis revealed that PC-3MM2 cells growing adjacent to the bone expressed high levels of EGF and activated EGF-R, whereas tumor cells in the adjacent musculature did not. Moreover, endothelial cells within the bone tumor lesions, but not in uninvolved bone or tumors in the muscle, expressed high levels of activated EGF-R. Treatment with PKI 166 and more so with PKI 166 plus Taxol significantly inhibited phosphorylation of EGF-R on tumor and endothelial cells and induced significant apoptosis and endothelial cells within tumor lesions. These data indicate that endothelial cells exposed to EGF produced by tumor cells express activated EGF-R and that targeting EGF-R can produce significant therapeutic effects against prostate cancer bone metastasis.

Topical or oral treatment of peach flower extract attenuates UV-induced epidermal thickening, matrix metalloproteinase-13 expression and pro-inflammatory cytokine production in hairless mice skin

PubMed Central

Yang, Jiwon; Shin, Chang-Yup; Chung, Jin Ho

2018-01-01

BACKGROUND/OBJECTIVES Ultraviolet radiation (UV) is a major cause of skin photoaging. Previous studies reported that ethanol extract (PET) of Prunus persica (L.) Batsch flowers (PPF, peach flowers) and its subfractions, particularly the ethylacetate (PEA) and n-butanol extracts (PBT), have potent antioxidant activity and attenuate the UV-induced matrix metalloproteinase (MMP) expression in human skin cells. In this study, we investigated the protective activity of PPF extract against UV-induced photoaging in a mouse model. MATERIALS/METHODS Hairless mice were treated with PET or a mixture of PEA and PBT either topically or orally along with UV irradiation. Histological changes and biochemical alterations of mouse skin were examined. Major phenolic compounds in PPF extract were analyzed using an ACQUITY UPLC system. RESULTS The overall effects of topical and oral treatments with PPF extract on the UV-induced skin responses exhibited similar patterns. In both experiments, the mixture of PEA and PBT significantly inhibited the UV-induced skin and epidermal thickening, while PET inhibited only the UV-induced epidermal thickening. Treatment of PET or the mixture of PEA and PBT significantly inhibited the UV-induced MMP-13 expression, but not typeⅠ collagen expression. Topical treatment of the mixture of PEA and PBT with UV irradiation significantly elevated catalase, superoxide dismutase (SOD) and glutathione-peroxidase (GPx) activities in the skin compared to those in the UV irradiated control group, while oral treatment of the mixture of PEA and PBT or PET elevated only catalase and SOD activities, but not GPx. Thirteen phytochemical compounds including 4-O-caffeoylquinic acid, cimicifugic acid E and B, quercetin-3-O-rhamnoside and kaempferol glycoside derivatives were identified in the PPF extract. CONCLUSIONS These results demonstrate that treatment with PET or the mixture of PEA and PBT, both topically or orally, attenuates UV-induced photoaging via the

Cellular processes involved in human epidermal cells exposed to extremely low frequency electric fields.

PubMed

Collard, J-F; Hinsenkamp, M

2015-05-01

We observed on different tissues and organisms a biological response after exposure to pulsed low frequency and low amplitude electric or electromagnetic fields but the precise mechanism of cell response remains unknown. The aim of this publication is to understand, using bioinformatics, the biological relevance of processes involved in the modification of gene expression. The list of genes analyzed was obtained after microarray protocol realized on cultures of human epidermal explants growing on deepidermized human skin exposed to a pulsed low frequency electric field. The directed acyclic graph on a WebGestalt Gene Ontology module shows six categories under the biological process root: "biological regulation", "cellular process", "cell proliferation", "death", "metabolic process" and "response to stimulus". Enriched derived categories are coherent with the type of in vitro culture, the stimulation protocol or with the previous results showing a decrease of cell proliferation and an increase of differentiation. The Kegg module on WebGestalt has highlighted "cell cycle" and "p53 signaling pathway" as significantly involved. The Kegg website brings out interactions between FoxO, MAPK, JNK, p53, p38, PI3K/Akt, Wnt, mTor or NF-KappaB. Some genes expressed by the stimulation are known to have an exclusive function on these pathways. Analyses performed with Pathway Studio linked cell proliferation, cell differentiation, apoptosis, cell cycle, mitosis, cell death etc. with our microarrays results. Medline citation generated by the software and the fold change variation confirms a diminution of the proliferation, activation of the differentiation and a less well-defined role of apoptosis or wound healing. Wnt and DKK functional classes, DKK1, MACF1, ATF3, MME, TXNRD1, and BMP-2 genes proposed in previous publications after a manual analysis are also highlighted with other genes after Pathway Studio automatic procedure. Finally, an analysis conducted on a list of genes

The expression of epidermal growth factor (EGF) and its receptor (EGFR) during post-natal testes development in the yak.

PubMed

Pan, Y; Cui, Y; Yu, S; Zhang, Q; Fan, J; Abdul Rasheed, B; Yang, K

2014-12-01

Growth factors play critical role in cell proliferation, regulate tissue differentiation and modulate organogenesis. Several growth factors have been identified in the testes of various mammalian species in last few years. In present investigation, the objective was to determine the expression of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in yak testicular tissue by relative quantitative real time polymerase chain reaction (RT-PCR), Western blot (WB) and immunohistochemistry (IHC) from mRNA and protein levels. The testicular tissues were collected from male yak at 6 and 24 months old. Results of RT-PCR and WB showed that the expression quantity of EGF and EGFR at 24 months of age was higher than at 6 months, and the increase rate of EGFR on mRNA and protein levels was higher than the increase rate EGF during post-natal testes development. Positive staining for EGF and EGFR was very low and mainly localized to Leydig cells testes at 6 months of age with immunohistochemistry, and seminiferous tubules were not observed. At 24 month of age, both the EGF and EGFR could be detected in Leydig cells, peritubular myoid cells, sertoli cells and germ cells of the yak testes. However, EGF and EGFR were localized to preferential adluminal compartment and basal compartment in the seminiferous tubules, respectively. In conclusion, the findings in present studies suggest that EGF and EGFR as important paracrine and/or autocrine regulators in yak testes development and spermatogenesis. © 2014 Blackwell Verlag GmbH.

TGM5 mutations impact epidermal differentiation in acral peeling skin syndrome.

PubMed

Pigors, Manuela; Kiritsi, Dimitra; Cobzaru, Cristina; Schwieger-Briel, Agnes; Suárez, Jose; Faletra, Flavio; Aho, Heikki; Mäkelä, Leeni; Kern, Johannes S; Bruckner-Tuderman, Leena; Has, Cristina

2012-10-01

Acral peeling skin syndrome (APSS) is an autosomal recessive skin disorder characterized by acral blistering and peeling of the outermost layers of the epidermis. It is caused by mutations in the gene for transglutaminase 5, TGM5. Here, we report on clinical and molecular findings in 11 patients and extend the TGM5 mutation database by four, to our knowledge, previously unreported mutations: p.M1T, p.L41P, p.L214CfsX15, and p.S604IfsX9. The recurrent mutation p.G113C was found in 9 patients, but also in 3 of 100 control individuals in a heterozygous state, indicating that APSS might be more widespread than hitherto expected. Using quantitative real-time PCR, immunoblotting, and immunofluorescence analysis, we demonstrate that expression and distribution of several epidermal differentiation markers and corneodesmosin (CDSN) is altered in APSS keratinocytes and skin. Although the expression of transglutaminases 1 and 3 was not changed, we found an upregulation of keratin 1, keratin 10, involucrin, loricrin, and CDSN, probably as compensatory mechanisms for stabilization of the epidermal barrier. Our results give insights into the consequences of TGM5 mutations on terminal epidermal differentiation.

Epidermal Dysfunction Leads to an Age-Associated Increase in Levels of Serum Inflammatory Cytokines.

PubMed

Hu, Lizhi; Mauro, Theodora M; Dang, Erle; Man, George; Zhang, Jing; Lee, Dale; Wang, Gang; Feingold, Kenneth R; Elias, Peter M; Man, Mao-Qiang

2017-06-01

Even though elderly populations lack visible or other clinical signs of inflammation, their serum cytokine and C-reactive protein levels typically are elevated. However, the origin of age-associated systemic inflammation is unknown. Our previous studies showed that abnormalities in epidermal function provoke cutaneous inflammation, and because intrinsically aged skin displays compromised permeability barrier homeostasis and reduced stratum corneum hydration, we hypothesized here that epidermal dysfunction could contribute to the elevations in serum cytokines in the elderly. Our results show first that acute disruption of the epidermal permeability barrier in young mice leads not only to a rapid increase in cutaneous cytokine mRNA expression but also an increase in serum cytokine levels. Second, cytokine levels in both the skin and serum increase in otherwise normal, aged mice (>12 months). Third, expression of tumor necrosis factor-α and amyloid A mRNA levels increased in the epidermis, but not in the liver, in parallel with a significant elevation in serum levels of cytokines. Fourth, disruption of the permeability barrier induced similar elevations in epidermal and serum cytokine levels in normal and athymic mice, suggesting that T cells play a negligible role in the elevations in cutaneous and serum inflammatory cytokines induced by epidermal dysfunction. Fifth, correction of epidermal function significantly reduced cytokine levels not only in the skin but also in the serum of aged mice. Together, these results indicate that the sustained abnormalities in epidermal function in chronologically aged skin contribute to the elevated serum levels of inflammatory cytokines, potentially predisposing the elderly to the subsequent development or exacerbation of chronic inflammatory disorders. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

[Progress in epidermal stem cells].

PubMed

Wang, Li-Juan; Wang, You-Liang; Yang, Xiao

2010-03-01

Mammalian skin epidermis contains different epidermal stem cell pools which contribute to the homeostasis and repair of skin epithelium. Epidermal stem cells possess two essential features common to all stem cells: self-renewal and differentiation. Disturbing the balance between self-renewal and differentiation of epidermal stem cell often causes tumors or other skin diseases. Epidermal stem cell niches provide a special microenvironment that maintains a balance of stem cell quiescence and activity. This review primarily concentrates on the following points of the epidermal stem cells: the existing evidences, the self-renewal and differentiation, the division pattern, the signal pathways regulating self-renewal and differentiation, and the microenvironment (niche) and macroenvironment maintaining the homeostasis of stem cells.

Epidermal patterning genes are active during embryogenesis in Arabidopsis.

PubMed

Costa, Silvia; Dolan, Liam

2003-07-01

Epidermal cells in the root of Arabidopsis seedling differentiate either as hair or non-hair cells, while in the hypocotyl they become either stomatal or elongated cells. WEREWOLF (WER) and GLABRA2 (GL2) are positive regulators of non-hair and elongated cell development. CAPRICE (CPC) is a positive regulator of hair cell development in the root. We show that WER, GL2 and CPC are expressed and active during the stages of embryogenesis when the pattern of cells in the epidermis of the root-hypocotyl axis forms. GL2 is first expressed in the future epidermis in the heart stage embryo and its expression is progressively restricted to those cells that will acquire a non-hair identity in the transition between torpedo and mature stage. The expression of GL2 at the heart stage requires WER function. WER and CPC are transiently expressed throughout the root epidermal layer in the torpedo stage embryo when the cell-specific pattern of GL2 expression is being established in the epidermis. We also show that WER positively regulates CPC transcription and GL2 negatively regulates WER transcription in the mature embryo. We propose that the restriction of GL2 to the future non-hair cells in the root epidermis can be correlated with the activities of WER and CPC during torpedo stage. In the embryonic hypocotyl we show that WER controls GL2 expression. We also provide evidence indicating that CPC may also regulate GL2 expression in the hypocotyl.

P311 Accelerates Skin Wound Reepithelialization by Promoting Epidermal Stem Cell Migration Through RhoA and Rac1 Activation.

PubMed

Yao, Zhihui; Li, Haisheng; He, Weifeng; Yang, Sisi; Zhang, Xiaorong; Zhan, Rixing; Xu, Rui; Tan, Jianglin; Zhou, Junyi; Wu, Jun; Luo, Gaoxing

2017-03-15

P311 is a newly discovered functional gene, and it has been proved to play a key role in blood pressure homeostasis, glioblastoma invasion, renal fibrosis, hypertrophic scar formation, and others. In this study, for the first time, we found that P311 could enhance reepithelialization during wound healing via promoting epidermal stem cell (EpSC) migration through Rho GTPases. P311 expression was highly increased in neo-epidermal cells during human and mouse skin wound healing, and P311was co-localized with 5-bromo-2'-deoxyuridine positive label-retaining cells in a mouse superficial second-degree burn wound model. Furthermore, transfection of human EpSCs with adenovirus encoding P311 significantly accelerated the cell migration in vitro. Moreover, highly expressed P311 could enhance the activities of the Rho GTPases (RhoA, Rac1, and Cdc42) in cultured human EpSCs. P311-knockout mouse EpSCs showed dramatically decreased cell migration and activities of Rho GTPases (RhoA, Rac1, and Cdc42). Besides, both the RhoA-specific inhibitor and the Rac1 inhibitor, not the Cdc42 inhibitor, could significantly suppress P311-induced human EpSC migration. In vivo, the reepithelialization was markedly impaired during wound healing after P311 was knocked out. Together, our results suggested that P311 could accelerate skin wound reepithelialization by promoting the migration of EpSCs through RhoA and Rac1 activation. P311 could serve as a novel target for regulation of EpSC migration during cutaneous wound healing.

Role of epidermal growth factor and transforming growth factor α in the developing stomach

PubMed Central

Kelly, E; Newell, S; Brownlee, K; Farmery, S; Cullinane, C; Reid, W; Jackson, P; Gray, S; Primrose, J; Lagopoulos, M

1997-01-01

AIMS—To determine whether epidermal growth factor (EGF) or the related transforming growth factor α (TGFα) may have a role in the developing human stomach; to substantiate the presence of EGF in human liquor in the non-stressed infant and whether EGF in amniotic fluid is maternally or fetally derived.
METHODS—The temporal expression and localisation of EGF, TGFα, and their receptors during fetal and neonatal life were examined in 20 fetal and five infant stomachs. Simultaneously, samples of amniotic fluid and fetal urine from 10 newborn infants were collected and assayed for EGF by radioimmunoassay.
RESULTS—EGF immunoreactivity was not noted in any of the specimens examined. In contrast, TGFα immunoreactivity was shown in mucous cells from 18 weeks of gestation onwards. EGF receptor immunoreactivity was seen on superficial mucous cells in gastric mucosa from 18 weeks of gestation onwards. The median concentration of EGF was 30 and 8.5 pg/ml in amniotic fluid and fetal urine, respectively, suggesting that EGF is not produced by the fetus.
CONCLUSIONS—This study adds weight to the hypothesis that swallowed EGF, probably produced by the amniotic membranes, and locally produced TGFα, may have a role in the growth and maturation of the human stomach.

 Keywords: epidermal growth factor; transforming growth factor α; EGF receptors; stomach PMID:9175944

Anti-Epidermal Growth Factor Receptor Gene Therapy for Glioblastoma

PubMed Central

Hicks, Martin J.; Chiuchiolo, Maria J.; Ballon, Douglas; Dyke, Jonathan P.; Aronowitz, Eric; Funato, Kosuke; Tabar, Viviane; Havlicek, David; Fan, Fan; Sondhi, Dolan; Kaminsky, Stephen M.; Crystal, Ronald G.

2016-01-01

Glioblastoma multiforme (GBM) is the most common and aggressive primary intracranial brain tumor in adults with a mean survival of 14 to 15 months. Aberrant activation of the epidermal growth factor receptor (EGFR) plays a significant role in GBM progression, with amplification or overexpression of EGFR in 60% of GBM tumors. To target EGFR expressed by GBM, we have developed a strategy to deliver the coding sequence for cetuximab, an anti-EGFR antibody, directly to the CNS using an adeno-associated virus serotype rh.10 gene transfer vector. The data demonstrates that single, local delivery of an anti-EGFR antibody by an AAVrh.10 vector coding for cetuximab (AAVrh.10Cetmab) reduces GBM tumor growth and increases survival in xenograft mouse models of a human GBM EGFR-expressing cell line and patient-derived GBM. AAVrh10.CetMab-treated mice displayed a reduction in cachexia, a significant decrease in tumor volume and a prolonged survival following therapy. Adeno-associated-directed delivery of a gene encoding a therapeutic anti-EGFR monoclonal antibody may be an effective strategy to treat GBM. PMID:27711187

The relationship between quantitative human epidermal growth factor receptor 2 gene expression by the 21-gene reverse transcriptase polymerase chain reaction assay and adjuvant trastuzumab benefit in Alliance N9831.

PubMed

Perez, Edith A; Baehner, Frederick L; Butler, Steven M; Thompson, E Aubrey; Dueck, Amylou C; Jamshidian, Farid; Cherbavaz, Diana; Yoshizawa, Carl; Shak, Steven; Kaufman, Peter A; Davidson, Nancy E; Gralow, Julie; Asmann, Yan W; Ballman, Karla V

2015-10-01

The N9831 trial demonstrated the efficacy of adjuvant trastuzumab for patients with human epidermal growth factor receptor 2 (HER2) locally positive tumors by protein or gene analysis. We used the 21-gene assay to examine the association of quantitative HER2 messenger RNA (mRNA) gene expression and benefit from trastuzumab. N9831 tested the addition of trastuzumab to chemotherapy in stage I-III HER2-positive breast cancer. For two of the arms of the trial, doxorubicin and cyclophosphamide followed by paclitaxel (AC-T) and doxorubicin and cyclophosphamide followed by paclitaxel and trastuzumab concurrent chemotherapy-trastuzumab (AC-TH), recurrence score (RS) and HER2 mRNA expression were determined by the 21-gene assay (Oncotype DX®) (negative <10.7, equivocal 10.7 to <11.5, and positive ≥11.5 log2 expression units). Cox regression was used to assess the association of HER2 expression with trastuzumab benefit in preventing distant recurrence. Median follow-up was 7.4 years. Of 1,940 total patients, 901 had consent and sufficient tissue. HER2 by reverse transcriptase polymerase chain reaction (RT-PCR) was negative in 130 (14 %), equivocal in 85 (9 %), and positive in 686 (76 %) patients. Concordance between HER2 assessments was 95 % for RT-PCR versus central immunohistochemistry (IHC) (>10 % positive cells = positive), 91 % for RT-PCR versus central fluorescence in situ hybridization (FISH) (≥2.0 = positive) and 94 % for central IHC versus central FISH. In the primary analysis, the association of HER2 expression by 21-gene assay with trastuzumab benefit was marginally nonsignificant (nonlinear p = 0.057). In hormone receptor-positive patients (local IHC) the association was significant (p = 0.002). The association was nonlinear with the greatest estimated benefit at lower and higher HER2 expression levels. Concordance among HER2 assessments by central IHC, FISH, and RT-PCR were similar and high. Association of HER2 mRNA expression with

Patritumab plus trastuzumab and paclitaxel in human epidermal growth factor receptor 2-overexpressing metastatic breast cancer.

PubMed

Mukai, Hirofumi; Saeki, Toshiaki; Aogi, Kenjiro; Naito, Yoichi; Matsubara, Nobuaki; Shigekawa, Takashi; Ueda, Shigeto; Takashima, Seiki; Hara, Fumikata; Yamashita, Tomonari; Ohwada, Shoichi; Sasaki, Yasutsuna

2016-10-01

Human epidermal growth factor receptor 3 (HER3) expression in lung and breast cancers has a negative impact on survival. Patritumab, a human anti-HER3 mAb, has shown anticancer activity in preclinical models. This study examined the safety and pharmacokinetics of patritumab in combination with trastuzumab and paclitaxel in patients with HER2-overexpressing metastatic breast cancer. In this open-label, multicenter, dose-escalation, phase Ib study, patients received patritumab 9 or 18 mg/kg plus trastuzumab and paclitaxel at known tolerated doses. Safety and tolerability were assessed based on dose-limiting toxicities and other non-life threatening adverse events. The pharmacokinetic profile for patritumab was determined based on the target trough level. Clinical efficacy was evaluated based on the overall response rate and progression-free survival. Six patients received patritumab 9 mg/kg and 12 received 18 mg/kg. The most common adverse events were diarrhea, alopecia, leukopenia, neutropenia, and maculopapular rash. No dose-limiting toxicities were observed. The target trough serum concentration was achieved in all patients at a dose of 18 mg/kg. Overall response rate was 38.9% and median progression-free survival was 274 days. In conclusion, patritumab plus trastuzumab and paclitaxel was tolerable and efficacious at both doses. We recommend the dose level of 18 mg/kg for future phase II studies. (Clinical trial registration: JapicCTI-121772.). © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Epidermal growth factor-like growth factors prevent apoptosis of alcohol-exposed human placental cytotrophoblast cells.

PubMed

Wolff, Garen S; Chiang, Po Jen; Smith, Susan M; Romero, Roberto; Armant, D Randall

2007-07-01

Maternal alcohol abuse during pregnancy can produce an array of birth defects comprising fetal alcohol syndrome. A hallmark of fetal alcohol syndrome is intrauterine growth retardation, which is associated with elevated apoptosis of placental cytotrophoblast cells. Using a human first trimester cytotrophoblast cell line, we examined the relationship between exposure to ethanol and cytotrophoblast survival, as well as the ameliorating effects of epidermal growth factor (EGF)-like growth factors produced by human cytotrophoblast cells. After exposure to 0-100 mM ethanol, cell death was quantified by the TUNEL method, and expression of the nuclear proliferation marker, Ki67, was measured by immunohistochemistry. The mode of cell death was determined by assessing annexin V binding, caspase 3 activation, pyknotic nuclear morphology, reduction of TUNEL by caspase inhibition, and cellular release of lactate dehydrogenase. Ethanol significantly reduced proliferation and increased cell death approximately 2.5-fold through the apoptotic pathway within 1-2 h of exposure to 50 mM alcohol. Exposure to 25-50 mM ethanol significantly increased transforming growth factor alpha (TGFA) and heparin-binding EGF-like growth factor (HBEGF), but not EGF or amphiregulin (AREG). When cytotrophoblasts were exposed concurrently to 100 mM ethanol and 1 nM HBEGF or TGFA, the increase in apoptosis was prevented, while EGF ameliorated at 10 nM and AREG was weakly effective. HBEGF survival-promoting activity required ligation of either of its cognate receptors, HER1 or HER4. These findings reveal the potential for ethanol to rapidly induce cytotrophoblast apoptosis. However, survival factor induction could provide cytotrophoblasts with an endogenous cytoprotective mechanism.

Human papillomavirus oncogenic E6 protein regulates human β-defensin 3 (hBD3) expression via the tumor suppressor protein p53

PubMed Central

Yue, Hong; Wang, Liming; Jin, Jessica; Ghosh, Santosh K.; Kawsar, Hameem I.; Zender, Chad; Androphy, Elliot J.; Weinberg, Aaron; McCormick, Thomas S.; Jin, Ge

2016-01-01

Human β-defensin-3 (hBD3) is an epithelial cell-derived innate immune regulatory molecule overexpressed in oral dysplastic lesions and fosters a tumor-promoting microenvironment. Expression of hBD3 is induced by the epidermal growth factor receptor signaling pathway. Here we describe a novel pathway through which the high-risk human papillomavirus type-16 (HPV-16) oncoprotein E6 induces hBD3 expression in mucosal keratinocytes. Ablation of E6 by siRNA induces the tumor suppressor p53 and diminishes hBD3 in HPV-16 positive CaSki cervical cancer cells and UM-SCC-104 head and neck cancer cells. Malignant cells in HPV-16-associated oropharyngeal cancer overexpress hBD3. HPV-16 E6 induces hBD3 mRNA expression, peptide production and gene promoter activity in mucosal keratinocytes. Reduction of cellular levels of p53 stimulates hBD3 expression, while activation of p53 by doxorubicin inhibits its expression in primary oral keratinocytes and CaSki cells, suggesting that p53 represses hBD3 expression. A p53 binding site in the hBD3 gene promoter has been identified by using electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP). In addition, the p63 protein isoform ΔNp63α, but not TAp63, stimulated transactivation of the hBD3 gene and was co-expressed with hBD3 in head and neck cancer specimens. Therefore, high-risk HPV E6 oncoproteins may stimulate hBD3 expression in tumor cells to facilitate tumorigenesis of HPV-associated head and neck cancer. PMID:27034006

Mitochondrial tolerance to single and repeat exposure to simulated sunlight in human epidermal and dermal skin cells.

PubMed

Kelly, J; Murphy, J E J

2016-12-01

Sunlight represents the primary threat to mitochondrial integrity in skin given the unique nature of the mitochondrial genome and its proximity to the electron transport chain. The accumulation of mitochondrial DNA (mtDNA) mutations is a key factor in many human pathologies and this is linked to key roles of mitochondrial function in terms of energy production and cell regulation. The main objective of this study was to evaluate solar radiation induced changes in mitochondrial integrity, function and dynamics in human skin cells using a Q-Sun solar simulator to deliver a close match to the intensity of summer sunlight. Spontaneously immortalised human skin epidermal keratinocytes (HaCaT) and Human Dermal Fibroblasts (HDFn) were divided into two groups. Group A were irradiated once and Group B twice 7days apart and evaluated using cell survival, viability and mitochondrial membrane potential (MMP) and mass at 1, 4 and 7days post one exposure for Group A and 1, 4, 7 and 14days post second exposure for Group B. Viability and survival of HaCaT and HDFn cells decreased after repeat exposure to Simulated Sunlight Irradiation (SSI) with no recovery. HDFn cells showed no loss in MMP after one or two exposures to SSI compared to HaCaT cells which showed a periodic loss of MMP after one exposure with a repeat exposure causing a dramatic decrease from which cells did not recover. Mitochondrial Mass in exposed HDFn cells was consistent with control after one or two exposures to SSI; however mitochondrial mass was significantly decreased in HaCaT cells. Data presented here suggests that mitochondria in epidermal cells are more sensitive to sunlight damage compared to mitochondria in dermal cells, despite their origin, confirming a skin layer specific sensitivity to sunlight, but not as expected. Copyright © 2016 Elsevier B.V. All rights reserved.

Secreted HoxA3 Promotes Epidermal Proliferation and Angiogenesis in Genetically Modified Three-Dimensional Composite Skin Constructs

PubMed Central

Kuo, Jennifer H.; Cuevas, Ileana; Chen, Amy; Dunn, Ashley; Kuri, Mauricio; Boudreau, Nancy

2014-01-01

Objective: Homeobox (HOX) transcription factors coordinate gene expression in wound repair and angiogenesis. Previous studies have shown that gene transfer of HoxA3 to wounds of diabetic mice accelerates wound healing, increasing angiogenesis and keratinocyte migration. In this study, we examined whether HoxA3 can also improve angiogenesis, epidermal integrity, and viability of composite skin grafts. Approach: To determine the effects of HoxA3 on composite skin grafts, we constructed bilayered composite grafts incorporating fibroblasts engineered to constitutively secrete HoxA3. We then transplanted these composite grafts in vivo. Results: The composite grafts produced a stratified epidermal layer after seventeen days in culture and following transplantation in vivo, these grafts exhibit normal epidermal differentiation and reduced contraction compared to controls. In addition, HoxA3 grafts showed increased angiogenesis. Quantitative polymerase chain reaction (PCR) analyses of HoxA3 graft tissue reveal an increase in the downstream HoxA3 target genes MMP-14 and uPAR expression, as well as a reduction in CCL-2 and CxCl-12. Innovation: Expression of secreted HoxA3 in composite grafts represents a comprehensive approach that targets both keratinocytes and endothelial cells to promote epidermal proliferation and angiogenesis. Conclusion: Secreted HoxA3 improves angiogenesis, reduces expression of inflammatory mediators, and prolongs composite skin graft integrity. PMID:25302136

micro RNA 172 (miR172) signals epidermal infection and is expressed in cells primed for bacterial invasion in Lotus japonicus roots and nodules.

PubMed

Holt, Dennis B; Gupta, Vikas; Meyer, Dörte; Abel, Nikolaj B; Andersen, Stig U; Stougaard, Jens; Markmann, Katharina

2015-10-01

Legumes interact with rhizobial bacteria to form nitrogen-fixing root nodules. Host signalling following mutual recognition ensures a specific response, but is only partially understood. Focusing on the stage of epidermal infection with Mesorhizobium loti, we analysed endogenous small RNAs (sRNAs) of the model legume Lotus japonicus to investigate their involvement in host response regulation. We used Illumina sequencing to annotate the L. japonicus sRNA-ome and isolate infection-responsive sRNAs, followed by candidate-based functional characterization. Sequences from four libraries revealed 219 novel L. japonicus micro RNAs (miRNAs) from 114 newly assigned families, and 76 infection-responsive sRNAs. Unlike infection-associated coding genes such as NODULE INCEPTION (NIN), a micro RNA 172 (miR172) isoform showed strong accumulation in dependency of both Nodulation (Nod) factor and compatible rhizobia. The genetics of miR172 induction support the existence of distinct epidermal and cortical signalling events. MIR172a promoter activity followed a previously unseen pattern preceding infection thread progression in epidermal and cortical cells. Nodule-associated miR172a expression was infection-independent, representing the second of two genetically separable activity waves. The combined data provide a valuable resource for further study, and identify miR172 as an sRNA marking successful epidermal infection. We show that miR172 acts upstream of several APETALA2-type (AP2) transcription factors, and suggest that it has a role in fine-tuning AP2 levels during bacterial symbiosis. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Eradication of damaged keratinocytes in cutaneous lichen planus forms demonstrated by evaluation of epidermal and follicular expression of CK15, indices of apoptosis and regulatory protein S100.

PubMed

Upeniece, Ilze; Groma, Valerie; Skuja, Sandra; Cauce, Vinita

The study of cytoskeleton arrangement and its contribution to survival of cell-to-cell contacts appears to be essential for understanding of numerous cellular and tissue processes. Applying CK15, S100 labeling and TUNEL reaction to cutaneous lichen planus subtypes, we found CK15 expression in the outer and inner root sheath of hair follicles, the basal epidermal layer, and eccrine glands. Its follicular expression was decreased in nearby inflammatory infiltrates. The CK15 immunopositivity was mostly described as weak (92.3%) for lichen planus but equally subdivided into weak, moderate and strong in lichen planopilaris (2 = 32.514; df = 4; p < 0.001). The greatly varying apoptotic index was the highest in the lichen planopilaris involving the scalp: 81.2 ±10.7; 87.8 ±10.7 and 88.0 ±10.5 for the basal, spinous and upper epidermal layers, respectively. S100 positive epidermal and follicular cells did not differ in the lesions demonstrated in the study groups; still immunoreactivity was more pronounced in the scalp region of lichen planopilaris. Damage of cell-to-cell contacts was confirmed by electron microscopy. Apart from immunocyte-mediated keratinocyte death, cytoskeleton-based injury and loss of cell-to-cell and matrix contacts may be of great importance, leading to eradication of degrading cells and thus contributing to the pathogenesis of lichen planus.

Expression of the transforming growth factor alpha protooncogene in differentiating human promyelocytic leukemia (HL-60) cells.

PubMed

Walz, T M; Malm, C; Wasteson, A

1993-01-01

The process of myeloid differentiation in human promyelocytic leukemia cells (HL-60) is accompanied by the coordinate expression of numerous protooncogenes. To investigate the expression of transforming growth factor alpha (TGF-alpha) in myeloid differentiation, HL-60 cells were induced to differentiate into granulocytes with 1.25% dimethyl sulfoxide, 0.2 microM all-trans retinoic acid, or 500 microM N6,O2-dibutyryladenosine-3'5'-cyclic monophosphate or differentiated along the monocyte/macrophage pathway with 0.1 microM phorbol-12-myristate-13-acetate. Using Northern blot analyses, TGF-alpha transcripts were detected within 24 h of treatment in cells differentiating toward granulocytes; maximal levels of gene expression were reached after 3 days or later and remained essentially constant throughout the observation period. These cells released TGF-alpha protein, as demonstrated by analysis of the incubation medium. In contrast, no TGF-alpha RNA or protein was detectable in HL-60 cell cultures when induced with phorbol-12-myristate-13-acetate. Epidermal growth factor receptor transcripts could not be detected either in undifferentiated or in differentiated HL-60 cells; therefore it appears as if an autocrine loop involving TGF-alpha in HL-60 cells is unlikely. In conclusion, the results demonstrate, for the first time, the expression of TGF-alpha in human granulocyte precursor cells. Our findings may indicate novel regulatory pathways in hematopoiesis.

Topical Human Epidermal Growth Factor in the Treatment of Senile Purpura and the Prevention of Dermatoporosis.

PubMed

McKnight, Braden; Seidel, Rachel; Moy, Ron

2015-10-01

Senile purpura presents itself as a largely unexplored challenge as it has been long thought of as a benign condition without long-term health sequelae. It is becoming increasingly accepted that skin aging not only results in cosmetic disturbances, but as a functional ones. With modern increases in lifespan, skin atrophy associated with solar damage is presenting as a clinically significant inability to mechanically protect patients. This chronic cutaneous insufficiency/fragility syndrome was recently termed dermatoporosis and senile purpura appears to be a visible marker of early stage dysfunction. To examine the effects of topically human epidermal growth factor on the clinical presence of senile purpura and its effect on skin thickness as measured via cutaneous ultrasound. Six subjects applied human epidermal growth factor morning and night for six weeks. Clinical outcomes were evaluated by comparing initial clinical photos to 6-week photos and performing a blinded investigator's global assessment (IGA). Skin thickness was evaluated via cutaneous ultrasound measurement. Ultrasound measurements indicated a mean skin thickening of 195.2 ± 35.7 um (SEM) over 6 weeks. The average number of purpuric lesions decreased from 15 ± 4.6 (SEM) to 2.3 ± 0.7 (SEM) over that same period. Senile purpura presents itself as a cosmetic disturbance posing significant psychological distress and serves as a marker of the severity of skin thinning. In this study, we demonstrate that topical h-EGF diminishes the appearance of senile purpura by thickening skin and may help prevent the development of late stage dermatoporosis.

FBXO11 promotes ubiquitination of the Snail family of transcription factors in cancer progression and epidermal development.

PubMed

Jin, Yue; Shenoy, Anitha K; Doernberg, Samuel; Chen, Hao; Luo, Huacheng; Shen, Huangxuan; Lin, Tong; Tarrash, Miriam; Cai, Qingsong; Hu, Xin; Fiske, Ryan; Chen, Ting; Wu, Lizi; Mohammed, Kamal A; Rottiers, Veerle; Lee, Siu Sylvia; Lu, Jianrong

2015-06-28

The Snail family of transcription factors are core inducers of epithelial-to-mesenchymal transition (EMT). Here we show that the F-box protein FBXO11 recognizes and promotes ubiquitin-mediated degradation of multiple Snail family members including Scratch. The association between FBXO11 and Snai1 in vitro is independent of Snai1 phosphorylation. Overexpression of FBXO11 in mesenchymal cells reduces Snail protein abundance and cellular invasiveness. Conversely, depletion of endogenous FBXO11 in epithelial cancer cells causes Snail protein accumulation, EMT, and tumor invasion, as well as loss of estrogen receptor expression in breast cancer cells. Expression of FBXO11 is downregulated by EMT-inducing signals TGFβ and nickel. In human cancer, high FBXO11 levels correlate with expression of epithelial markers and favorable prognosis. The results suggest that FBXO11 sustains the epithelial state and inhibits cancer progression. Inactivation of FBXO11 in mice leads to neonatal lethality, epidermal thickening, and increased Snail protein levels in epidermis, validating that FBXO11 is a physiological ubiquitin ligase of Snail. Moreover, in C. elegans, the FBXO11 mutant phenotype is attributed to the Snail factors as it is suppressed by inactivation/depletion of Snail homologs. Collectively, these findings suggest that the FBXO11-Snail regulatory axis is evolutionarily conserved and critically governs carcinoma progression and mammalian epidermal development. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Ultra-weak photon emission as a non-invasive tool for monitoring of oxidative processes in the epidermal cells of human skin: comparative study on the dorsal and the palm side of the hand.

PubMed

Rastogi, Anshu; Pospísil, Pavel

2010-08-01

All living organisms emit spontaneous ultra-weak photon emission as a result of cellular metabolic processes. Exposure of living organisms to exogenous factors results in oxidative processes and enhancement in ultra-weak photon emission. Here, hydrogen peroxide (H(2)O(2)), as a strongly oxidizing molecule, was used to induce oxidative processes and enhance ultra-weak photon emission in human hand skin. The presented work intends to compare both spontaneous and peroxide-induced ultra-weak photon emission from the epidermal cells on the dorsal and the palm side of the hand. A highly sensitive photomultiplier tube and a charge-coupled device camera were used to detect ultra-weak photon emission from human hand skin. Spontaneous ultra-weak photon emission from the epidermal cells on the dorsal side of the hand was 4 counts/s. Topical application of 500 mM H(2)O(2) to the dorsal side of the hand caused enhancement in ultra-weak photon emission to 40 counts/s. Interestingly, both spontaneous and peroxide-induced ultra-weak photon emission from the epidermal cells on the palm side of the hand were observed to increase twice their values, i.e. 8 and 80 counts/s, respectively. Similarly, the two-dimensional image of ultra-weak photon emission observed after topical application of H(2)O(2) to human skin reveals that photon emission from the palm side exceeds the photon emission from the dorsal side of the hand. The results presented indicate that the ultra-weak photon emission originating from the epidermal cells on the dorsal and the palm side of the hand is related to the histological structure of the human hand skin. Ultra-weak photon emission is shown as a non-destructive technique for monitoring of oxidative processes in the epidermal cells of the human hand skin and as a diagnostic tool for skin diseases.

Induction of a chloracne phenotype in an epidermal equivalent model by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent on aryl hydrocarbon receptor activation and is not reproduced by aryl hydrocarbon receptor knock down.

PubMed

Forrester, Alison R; Elias, Martina S; Woodward, Emma L; Graham, Mark; Williams, Faith M; Reynolds, Nick J

2014-01-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent activator of the aryl hydrocarbon receptor (AhR) and causes chloracne in humans. The pathogenesis and role of AhR in chloracne remains incompletely understood. To elucidate the mechanisms contributing to the development of the chloracne-like phenotype in a human epidermal equivalent model and identify potential biomarkers. Using primary normal human epidermal keratinocytes (NHEK), we studied AhR activation by XRE-luciferase, AhR degradation and CYP1A1 induction. We treated epidermal equivalents with high affinity TCDD or two non-chloracnegens: β-naphthoflavone (β-NF) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Using Western blotting and immunochemistry for filaggrin (FLG), involucrin (INV) and transglutaminase-1 (TGM-1), we compared the effects of the ligands on keratinocyte differentiation and development of the chloracne-like phenotype by H&E. In NHEKs, activation of an XRE-luciferase and CYP1A1 protein induction correlated with ligand binding affinity: TCDD>β-NF>ITE. AhR degradation was induced by all ligands. In epidermal equivalents, TCDD induced a chloracne-like phenotype, whereas β-NF or ITE did not. All three ligands induced involucrin and TGM-1 protein expression in epidermal equivalents whereas FLG protein expression decreased following treatment with TCDD and β-NF. Inhibition of AhR by α-NF blocked TCDD-induced AhR activation in NHEKs and blocked phenotypic changes in epidermal equivalents; however, AhR knock down did not reproduce the phenotype. Ligand-induced CYP1A1 and AhR degradation did not correlate with their chloracnegenic potential, indicating that neither CYP1A1 nor AhR are suitable biomarkers. Mechanistic studies showed that the TCDD-induced chloracne-like phenotype depends on AhR activation whereas AhR knock down did not appear sufficient to induce the phenotype. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier

Induction of a chloracne phenotype in an epidermal equivalent model by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent on aryl hydrocarbon receptor activation and is not reproduced by aryl hydrocarbon receptor knock down

PubMed Central

Forrester, Alison R.; Elias, Martina S.; Woodward, Emma L.; Graham, Mark; Williams, Faith M.; Reynolds, Nick J.

2014-01-01

Background 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent activator of the aryl hydrocarbon receptor (AhR) and causes chloracne in humans. The pathogenesis and role of AhR in chloracne remains incompletely understood. Objective To elucidate the mechanisms contributing to the development of the chloracne-like phenotype in a human epidermal equivalent model and identify potential biomarkers. Methods Using primary normal human epidermal keratinocytes (NHEK), we studied AhR activation by XRE-luciferase, AhR degradation and CYP1A1 induction. We treated epidermal equivalents with high affinity TCDD or two non-chloracnegens: β-naphthoflavone (β-NF) and 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Using Western blotting and immunochemistry for filaggrin (FLG), involucrin (INV) and transglutaminase-1 (TGM-1), we compared the effects of the ligands on keratinocyte differentiation and development of the chloracne-like phenotype by H&E. Results In NHEKs, activation of an XRE-luciferase and CYP1A1 protein induction correlated with ligand binding affinity: TCDD > β-NF > ITE. AhR degradation was induced by all ligands. In epidermal equivalents, TCDD induced a chloracne-like phenotype, whereas β-NF or ITE did not. All three ligands induced involucrin and TGM-1 protein expression in epidermal equivalents whereas FLG protein expression decreased following treatment with TCDD and β-NF. Inhibition of AhR by α-NF blocked TCDD-induced AhR activation in NHEKs and blocked phenotypic changes in epidermal equivalents; however, AhR knock down did not reproduce the phenotype. Conclusion Ligand-induced CYP1A1 and AhR degradation did not correlate with their chloracnegenic potential, indicating that neither CYP1A1 nor AhR are suitable biomarkers. Mechanistic studies showed that the TCDD-induced chloracne-like phenotype depends on AhR activation whereas AhR knock down did not appear sufficient to induce the phenotype. PMID:24161567

Epidermal growth in the bottlenose dolphin, Tursiops truncatus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hicks, B.D.; St. Aubin, D.J.; Geraci, J.R.

1985-07-01

Epidermal growth in two mature female bottlenose dolphins, Tursiops truncatus, was investigated by following the movement of a cohort of tritiated thymidine-labeled epidermal cells for 59 days. The majority of the cells migrated in a cluster which was estimated to reach the skin surface in 73 days. The authors calculate that the outermost cell layer is sloughed 12 times per day. Turnover time and sloughing rate are estimated to be 1.7 times longer and 8.5 times faster than the respective values for epidermal cell kinetics in humans. This apparent inconsistency of slow transit time and rapid sloughing rate is reconciledmore » by the convoluted structure of the stratum germinativum in the dolphin which results in a ratio of germinatival to superficial cells of 876:1. The stratum germinativum of dolphin epidermis appears to lack morphologically distinct, spatially segregated subpopulations of anchoring and stem cells. Dolphin epidermis has a large capacity for cell population, relatively long turnover time, and rapid sloughing rate. The adaptive advantages of these characteristics are discussed.« less

Autophagy participates in isoliquiritigenin-induced melanin degradation in human epidermal keratinocytes through PI3K/AKT/mTOR signaling.

PubMed

Yang, Zhibo; Zeng, Biyun; Pan, Yi; Huang, Pan; Wang, Chang

2018-01-01

Melanin is the pigment responsible for the color of human skin and hair. Melanin serves as a double-edge sword which can exert both protective and spot-causing effects on skin. Although melanin has an important role in protecting the skin against UV damage, an excessive or uneven melanin production can lead to the formation of freckles and age spots. Isoliquiritigenin (ISL) has been reported to inhibit melanin synthesis; however, its role in melanin degradation remains unclear. In the present study, we evaluated the detailed function of ISL in melanin degradation in human epidermal keratinocytes. Since autophagy has been reported to be related to melanin degradation, we also examined the activation of autophagy by ISL treatment in keratinocytes by measurement of autophagy-related proteins, ATG7, LC3 and p62. Moreover, si-ATG7-induced ATG7 knockdown and autophagy inhibitor 3-MA decreased LC3 II protein levels and increased PMEL17, p62 and melanin levels in HaCaT cells, which could be partially reversed by ISL treatment, indicating that autophagy participated in melanin degradation. The decreased p-AKT and p-mTOR proteins upon ISL treatment indicated the involvement of PI3K/AKT/mTOR signaling in ISL-induced melanin degradation. Taken together, we demonstrated that autophagy participates in ISL-induced melanin degradation in human epidermal keratinocytes through PI3K/AKT/mTOR signaling. Copyright © 2017. Published by Elsevier Masson SAS.

Langerhans cell precursors acquire RANK/CD265 in prenatal human skin

PubMed Central

Schöppl, Alice; Botta, Albert; Prior, Marion; Akgün, Johnnie; Schuster, Christopher; Elbe-Bürger, Adelheid

2015-01-01

The skin is the first barrier against foreign pathogens and the prenatal formation of a strong network of various innate and adaptive cells is required to protect the newborn from perinatal infections. While many studies about the immune system in healthy and diseased adult human skin exist, our knowledge about the cutaneous prenatal/developing immune system and especially about the phenotype and function of antigen-presenting cells such as epidermal Langerhans cells (LCs) in human skin is still scarce. It has been shown previously that LCs in healthy adult human skin express receptor activator of NF-κB (RANK), an important molecule prolonging their survival. In this study, we investigated at which developmental stage LCs acquire this important molecule. Immunofluorescence double-labeling of cryostat sections revealed that LC precursors in prenatal human skin either do not yet [10–11 weeks of estimated gestational age (EGA)] or only faintly (13–15 weeks EGA) express RANK. LCs express RANK at levels comparable to adult LCs by the end of the second trimester. Comparable with adult skin, dermal antigen-presenting cells at no gestational age express this marker. These findings indicate that epidermal leukocytes gradually acquire RANK during gestation – a phenomenon previously observed also for other markers on LCs in prenatal human skin. PMID:25722033

Restoring E-cadherin expression increases sensitivity to epidermal growth factor receptor inhibitors in lung cancer cell lines.

PubMed

Witta, Samir E; Gemmill, Robert M; Hirsch, Fred R; Coldren, Christopher D; Hedman, Karla; Ravdel, Larisa; Helfrich, Barbara; Dziadziuszko, Rafal; Chan, Daniel C; Sugita, Michio; Chan, Zeng; Baron, Anna; Franklin, Wilbur; Drabkin, Harry A; Girard, Luc; Gazdar, Adi F; Minna, John D; Bunn, Paul A

2006-01-15

The epidermal growth factor receptor (EGFR) is overexpressed in the majority of non-small cell lung cancers (NSCLC). EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib, produce 9% to 27% response rates in NSCLC patients. E-Cadherin, a calcium-dependent adhesion molecule, plays an important role in NSCLC prognosis and progression, and interacts with EGFR. The zinc finger transcriptional repressor, ZEB1, inhibits E-cadherin expression by recruiting histone deacetylases (HDAC). We identified a significant correlation between sensitivity to gefitinib and expression of E-cadherin, and ZEB1, suggesting their predictive value for responsiveness to EGFR-tyrosine kinase inhibitors. E-Cadherin transfection into a gefitinib-resistant line increased its sensitivity to gefitinib. Pretreating resistant cell lines with the HDAC inhibitor, MS-275, induced E-cadherin along with EGFR and led to a growth-inhibitory and apoptotic effect of gefitinib similar to that in gefitinib-sensitive NSCLC cell lines including those harboring EGFR mutations. Thus, combined HDAC inhibitor and gefitinib treatment represents a novel pharmacologic strategy for overcoming resistance to EGFR inhibitors in patients with lung cancer.

Differentiated epidermal cells regain the ability to regenerate a skin equivalent by increasing the level of β-catenin in the cells.

PubMed

Zhao, Zhili; Zhang, Cuiping; Fu, Xiaobing; Yang, Rongya; Peng, Chen; Gu, Tingmin; Sui, Zhifu; Wang, Congmin; Liu, Chang

2012-01-01

Epidermal stem cells are of major importance for skin regeneration and tissue engineering, but differentiated epidermal cells lost their proliferative capacity and are no longer able to regenerate a skin equivalent. Here, we investigated the role of β-catenin in regulating regenerative functions of differentiated epidermal cells. Lithium chloride and a highly specific glycogen synthase kinase (GSK)-3β inhibitor were applied to induce the expression of β-catenin in differentiated epidermal cells. After a 6-day induction, the large flat-shaped cells with a small nuclear-cytoplasmic ratio had changed into small round-shaped cells with a large nuclear-cytoplasmic ratio. Phenotypic assays showed a remarkably higher expression of CK19, β(1)-integrin, Oct4 and Nanog in induced cells than in the control group (p < 0.01). In addition, the results of growth and functional investigations demonstrated that the induced epidermal cells exhibited a high colony-forming ability, a long-term proliferative potential and the ability to regenerate a skin equivalent, which were regarded as the most important features of epidermal stem cells. These results suggest that the activation of β-catenin favors the reversion or dedifferentiation of differentiated epidermal cells to an immature or a less differentiated state. This study may also offer a new approach to yield enough epidermal stem cells for skin regeneration and tissue engineering. Copyright © 2012 S. Karger AG, Basel.

The Antiaging Properties of Andrographis paniculata by Activation Epidermal Cell Stemness.

PubMed

You, Jiyoung; Roh, Kyung-Baeg; Li, Zidan; Liu, Guangrong; Tang, Jian; Shin, Seoungwoo; Park, Deokhoon; Jung, Eunsun

2015-09-22

Andrographis paniculata (A. paniculata, Chuanxinlian), a medicinal herb with an extremely bitter taste that is native to China and other parts of Southeast Asia, possesses immense therapeutic value; however, its therapeutic properties have rarely been applied in the field of skin care. In this study, we investigated the effect of an A. paniculata extract (APE) on human epidermal stem cells (EpSCs), and confirmed its anti-aging effect through in vitro, ex vivo, and in vivo study. An MTT assay was used to determine cell proliferation. A flow cytometric analysis, with propidium iodide, was used to evaluate the cell cycle. The expression of integrin β1 (CD29), the stem cell marker, was detected with antibodies, using flow cytometry in vitro, and immunohistochemical assays in ex vivo. Type 1 collagen and VEGF (vascular endothelial growth factor) were measured using an enzyme-linked immunosorbent assay (ELISA). During the clinical study, skin hydration, elasticity, wrinkling, sagging, and dermal density were evaluated before treatment and at four and eight weeks after the treatment with the test product (containing the APE) on the face. The proliferation of the EpSCs, treated with the APE, increased significantly. In the cell cycle analysis, the APE increased the G2/M and S stages in a dose-dependent manner. The expression of integrin β1, which is related to epidermal progenitor cell expansion, was up-regulated in the APE-treated EpSCs and skin explants. In addition, the production of VEGF in the EpSCs increased significantly in response to the APE treatment. Consistent with these results, the VEGF and APE-treated EpSCs conditioned medium enhanced the Type 1 collagen production in normal human fibroblasts (NHFs). In the clinical study, the APE improved skin hydration, dermal density, wrinkling, and sagging significantly. Our findings revealed that the APE promotes a proliferation of EpSCs, through the up-regulation of the integrin β1 and VEGF expression. The VEGF

Differences in expression of the cancer stem cell marker aldehyde dehydrogenase 1 among estrogen receptor-positive/human epidermal growth factor receptor type 2-negative breast cancer cases with early, late, and no recurrence.

PubMed

Miyoshi, Yuichiro; Shien, Tadahiko; Ogiya, Akiko; Ishida, Naoko; Yamazaki, Kieko; Horii, Rie; Horimoto, Yoshiya; Masuda, Norikazu; Yasojima, Hiroyuki; Inao, Touko; Osako, Tomofumi; Takahashi, Masato; Tomioka, Nobumoto; Endo, Yumi; Hosoda, Mitsuchika; Doihara, Hiroyoshi; Miyoshi, Shinichiro; Yamashita, Hiroko

2016-07-02

The significance of the expression of aldehyde dehydrogenase 1 (ALDH1), a cancer stem cell marker, for predicting the recurrence of estrogen receptor (ER)-positive/human epidermal growth factor receptor type 2 (HER2)-negative breast cancer is still poorly understood. The value of ALDH1 in predicting the time of recurrence remains unknown. In total, 184 patients with early distant recurrence, 134 patients with late distant recurrence, and 321 control patients without recurrence for more than 10 years after starting initial treatment for ER-positive/HER2-negative breast cancer, registered in 9 institutions, were analyzed. We assessed relationships between ALDH1 and other clinicopathological features, and ALDH1 expression was compared among the three groups. The relationship between ALDH1 expression and overall survival after recurrence was also evaluated in each group. The rates of ALDH1 expression positivity (more than 1 %) in the early, late, and no recurrence groups were 18.4 %, 13.4 %, and 8.4 %, respectively. ALDH1 expression correlated significantly with lymph node metastases (p = 0.048) and the Ki-67 labeling index (p < 0.001) in the early recurrence group. Multivariate analysis revealed ALDH1 expression to be significantly higher in the early recurrence group than in the no recurrence group (adjusted OR 2.140, 95 % CI 1.144-4.003, p = 0.016). Moreover, there was a significant difference in ALDH1 expression between the early and no recurrence groups receiving adjuvant endocrine therapy and chemotherapy (adjusted OR 4.625, 95 % CI 1.881-12.474, p < 0.001). However, there was no difference in ALDH1 expression between the late and no recurrence groups in univariate analysis (OR 1.507, 95 % CI 0.738-2.998, p = 0.253). In multivariate analysis, ALDH1 was not a factor independently predicting overall survival after the detection of recurrence (adjusted OR 1.451, 95 % CI 0.985-2.085, p = 0.059). Among patients with ER-positive/HER2

High-level expression and purification of heparin-binding epidermal growth factor (HB-EGF) with SUMO fusion.

PubMed

Lu, Wuguang; Cao, Peng; Lei, Huangzong; Zhang, Shuangquan

2010-03-01

Heparin-binding epidermal growth factor (HB-EGF) can stimulate the division of various cell types and has potential clinical applications that stimulate growth and differentiation. HB-EGF has an EGF-like domain typical of all members of the EGF family. The high expression of active HB-EGF in Escherichia coli has not been successful as the protein contains three intra-molecular disulfide bonds, the same as other members of the EGF super family that are difficult to form correctly in the bacterial intracellular environment. This work fused the non-glycosylated HB-EGF gene with a small ubiquitin-related modifier gene (SUMO) by over-lap PCR. The resulting fusion gene SUMO-HBEGF was highly expressed in BL21(DE3) that the soluble SUMO-HBEGF was up to 30% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease Ulp1 to obtain the native HB-EGF, which was further purified by Ni-NTA affinity chromatography. MTT assays indicated the purified HB-EGF, as well as SUMO-HBEGF, had mitogenic activity in a dose-dependent manner.

Using scale and feather traits for module construction provides a functional approach to chicken epidermal development.

PubMed

Bao, Weier; Greenwold, Matthew J; Sawyer, Roger H

2017-11-01

Gene co-expression network analysis has been a research method widely used in systematically exploring gene function and interaction. Using the Weighted Gene Co-expression Network Analysis (WGCNA) approach to construct a gene co-expression network using data from a customized 44K microarray transcriptome of chicken epidermal embryogenesis, we have identified two distinct modules that are highly correlated with scale or feather development traits. Signaling pathways related to feather development were enriched in the traditional KEGG pathway analysis and functional terms relating specifically to embryonic epidermal development were also enriched in the Gene Ontology analysis. Significant enrichment annotations were discovered from customized enrichment tools such as Modular Single-Set Enrichment Test (MSET) and Medical Subject Headings (MeSH). Hub genes in both trait-correlated modules showed strong specific functional enrichment toward epidermal development. Also, regulatory elements, such as transcription factors and miRNAs, were targeted in the significant enrichment result. This work highlights the advantage of this methodology for functional prediction of genes not previously associated with scale- and feather trait-related modules.

Skn-1a/Oct-11 and {Delta}Np63{alpha} exert antagonizing effects on human keratin expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lena, Anna Maria; Cipollone, Rita; Amelio, Ivano

2010-10-29

Research highlights: {yields} Skn-1a markedly downregulates {Delta}Np63-driven K14 expression. {yields} {Delta}Np63 inhibits Skn-1a-mediated K10 expression. {yields} {Delta}Np63, mutated in SAM domain, is less effecting in K10 downregulation. {yields} Immunolocalization in human skin of the two transcription factors is partially overlapping. {yields} The antagonistic effects of Skn-1a and p63 is through competition for overlapping responsive elements or through an indirect interaction. -- Abstract: The formation of a stratified epidermis requires a carefully controlled balance between keratinocyte proliferation and differentiation. Here, we report the reciprocal effect on keratin expression of {Delta}Np63, pivotal in normal epidermal morphogenesis and maintenance, and Skn-1a/Oct-11, a POUmore » transcription factor that triggers and regulates the differentiation of keratinocytes. The expression of Skn-1a markedly downregulated {Delta}Np63-driven K14 expression in luciferase reporter assays. The extent of downregulation was comparable to the inhibition of Skn-1a-mediated K10 expression upon expression of {Delta}Np63. {Delta}Np63, mutated in the protein-protein interaction domain (SAM domain; mutated in human ectodermal dysplasia syndrome), was significantly less effecting in downregulating K10, raising the possibility of a direct interaction among Skn-1a and {Delta}Np63. Immunolocalization in human skin biopsies revealed that the expression of the two transcription factors is partially overlapping. Co-immunoprecipitation experiments did not, however, demonstrate a direct interaction between {Delta}Np63 and Skn-1a, suggesting that the antagonistic effects of Skn-1a and p63 on keratin promoter transactivation is probably through competition for overlapping binding sites on target gene promoter or through an indirect interaction.« less

Deletion of epidermal Rac1 inhibits HPV-8 induced skin papilloma formation and facilitates HPV-8- and UV-light induced skin carcinogenesis

PubMed Central

Deshmukh, Jayesh; Pofahl, Ruth; Pfister, Herbert; Haase, Ingo

2016-01-01

Overexpression and increased activity of the small Rho GTPase Rac1 has been linked to squamous cell carcinoma of the epidermis and mucosa in humans. Targeted deletion of Rac1 or inhibition of Rac1 activity in epidermal keratinocytes reduced papilloma formation in a chemical skin carcinogenesis mouse model. However, a potential role of Rac1 in HPV- and UV-light induced skin carcinogenesis has not been investigated so far, solar UV radiation being an important carcinogen to the skin. To investigate this, we deleted Rac1 or modulated its activity in mice with transgenic expression of Human papilloma virus type-8 (HPV-8) in epidermal keratinocytes. Our data show that inhibition or deletion of Rac1 results in reduced papilloma formation upon UV-irradiation with a single dose, whereas constitutive activation of Rac1 strongly increases papilloma frequency in these mice. Surprisingly, we observed that, upon chronic UV-irradiation, the majority of mice with transgenic expression of HPV-8 and epidermis specific Rac1 deletion developed squamous cell carcinomas. Taken together, our data show that Rac1 exerts a dual role in skin carcinogenesis: its activation is, on one hand, required for HPV-8- and UV-light induced papilloma formation but, on the other, suppresses the development of squamous cell carcinomas. PMID:27506937

Deletion of epidermal Rac1 inhibits HPV-8 induced skin papilloma formation and facilitates HPV-8- and UV-light induced skin carcinogenesis.

PubMed

Deshmukh, Jayesh; Pofahl, Ruth; Pfister, Herbert; Haase, Ingo

2016-09-06

Overexpression and increased activity of the small Rho GTPase Rac1 has been linked to squamous cell carcinoma of the epidermis and mucosa in humans. Targeted deletion of Rac1 or inhibition of Rac1 activity in epidermal keratinocytes reduced papilloma formation in a chemical skin carcinogenesis mouse model. However, a potential role of Rac1 in HPV- and UV-light induced skin carcinogenesis has not been investigated so far, solar UV radiation being an important carcinogen to the skin.To investigate this, we deleted Rac1 or modulated its activity in mice with transgenic expression of Human papilloma virus type-8 (HPV-8) in epidermal keratinocytes. Our data show that inhibition or deletion of Rac1 results in reduced papilloma formation upon UV-irradiation with a single dose, whereas constitutive activation of Rac1 strongly increases papilloma frequency in these mice. Surprisingly, we observed that, upon chronic UV-irradiation, the majority of mice with transgenic expression of HPV-8 and epidermis specific Rac1 deletion developed squamous cell carcinomas. Taken together, our data show that Rac1 exerts a dual role in skin carcinogenesis: its activation is, on one hand, required for HPV-8- and UV-light induced papilloma formation but, on the other, suppresses the development of squamous cell carcinomas.

Effect of low-level laser irradiation and epidermal growth factor on adult human adipose-derived stem cells.

PubMed

Mvula, B; Moore, T J; Abrahamse, H

2010-01-01

The study investigated the effects of low-level laser radiation and epidermal growth factor (EGF) on adult adipose-derived stem cells (ADSCs) isolated from human adipose tissue. Isolated cells were cultured to semi-confluence, and the monolayers of ADSCs were exposed to low-level laser at 5 J/cm(2) using 636 nm diode laser. Cell viability and proliferation were monitored using adenosine triphosphate (ATP) luminescence and optical density at 0 h, 24 h and 48 h after irradiation. Application of low-level laser irradiation at 5 J/cm(2) on human ADSCs cultured with EGF increased the viability and proliferation of these cells. The results indicate that low-level laser irradiation in combination with EGF enhances the proliferation and maintenance of ADSCs in vitro.

Guidelines for human epidermal growth factor receptor 2 testing: biologic and methodologic considerations.

PubMed

Sauter, Guido; Lee, James; Bartlett, John M S; Slamon, Dennis J; Press, Michael F

2009-03-10

The goal of this review is to systematically address a number of issues raised in the American Society of Clinical Oncology-College of American Pathologists (ASCO-CAP) guidelines on testing for the human epidermal growth factor receptor 2 (HER-2) alteration. A group of investigators who are experienced in the conduct and interpretation of HER-2 assay methods reviewed the ASCO-CAP guidelines and address several areas of the HER-2 testing guidelines with a particular emphasis on biologic and methodologic considerations. Although HER-2 status determined by immunohistochemistry (IHC) and the status determined by fluorescent in situ hybridization (FISH) are significantly correlated, we feel that standard considerations of laboratory testing, including test accuracy, reproducibility, and precision, as well as the current data favor FISH over IHC assay methods for determining HER-2 status. These considerations are clearly important in clinical practice because HER2 amplification is directly linked to protein expression levels in breast cancer. However, this protein is not consistently analyzed in formalin-fixed tissues as a result of variability in fixation methods and times and the impact of fixation on HER-2 protein antigenicity. Conversely, gene amplification and FISH are significantly less dependent on tissue fixation methods, making this assay more reproducible between central and peripheral laboratories than IHC. Moreover, review of the existing data demonstrate that FISH is more strongly correlated with responsiveness to either trastuzumab or lapatinib treatment. Until other methods achieve similar test accuracy, reproducibility, and predictive value, we suggest FISH as the primary HER-2 testing modality for women with breast cancer who are candidates for HER-2-targeted therapies.

Virus-Induced Gene Silencing of the Eggplant Chalcone Synthase Gene during Fruit Ripening Modifies Epidermal Cells and Gravitropism.

PubMed

Wang, Cuicui; Fu, Daqi

2018-03-21

Eggplant ( Solanum melongena L.) fruits accumulate flavonoids in their cuticle and epidermal cells during ripening. Although many mutants available in model plant species, such as Arabidopsis thaliana and Medicago truncatula, are enabling the intricacies of flavonoid-related physiology to be deduced, the mechanisms whereby flavonoids influence eggplant fruit physiology are unknown. Virus-induced gene silencing (VIGS) is a reliable tool for the study of flavonoid function in fruit, and in this study, we successfully applied this technique to downregulate S. melongena chalcone synthase gene ( SmCHS) expression during eggplant fruit ripening. In addition to the expected change in fruit color attributable to a lack of anthocyanins, several other modifications, including differences in epidermal cell size and shape, were observed in the different sectors. We also found that silencing of CHS gene expression was associated with a negative gravitropic response in eggplant fruits. These observations indicate that epidermal cell expansion during ripening is dependent upon CHS expression and that there may be a relationship between CHS expression and gravitropism during eggplant fruit ripening.

Phosphorylation of hepatocyte growth factor receptor and epidermal growth factor receptor of human hepatocytes can be maintained in a (3D) collagen sandwich culture system.

PubMed

Engl, Tobias; Boost, Kim A; Leckel, Kerstin; Beecken, Wolf-Dietrich; Jonas, Dietger; Oppermann, Elsie; Auth, Marcus K H; Schaudt, André; Bechstein, Wolf-Otto; Blaheta, Roman A

2004-08-01

In vitro culture models that employ human liver cells could be potent tools for predictive studies on drug toxicity and metabolism in the pharmaceutical industry. However, an adequate receptor responsiveness is necessary to allow intracellular signalling and metabolic activity. We tested the ability of three-dimensionally arranged human hepatocytes to respond to the growth factors hepatocyte growth factor (HGF) or epidermal growth factor (EGF). Isolated adult human hepatocytes were cultivated within a three-dimensional collagen gel (sandwich) or on a two-dimensional collagen matrix. Cells were treated with HGF or EGF and expression and phosphorylative activity of HGF receptors (HGFr, c-met) or EGF receptors (EGFr) were measured by flow cytometry and Western blot. Increasing HGFr and EGFr levels were detected in hepatocytes growing two-dimensionally. However, both receptors were not activated in presence of growth factors. In contrast, when hepatocytes were plated within a three-dimensional matrix, HGFr and EGFr levels remained constantly low. However, both receptors became strongly phosphorylated by soluble HGF or EGF. We conclude that cultivation of human hepatocytes in a three-dimensionally arranged in vitro system allows the maintenance of specific functional activities. The necessity of cell dimensionality for HGFr and EGFr function should be considered when an adequate in vitro system has to be introduced for drug testing.

Expression of Filaggrin and its Degradation Products in Human Skin Following Erythemal Doses of Ultraviolet B Irradiation.

PubMed

Simonsen, Stine; Thyssen, Jacob P; Heegaard, Steffen; Kezic, Sanja; Skov, Lone

2017-07-06

Epidermal filaggrin level is affected by ultraviolet B irradiation in animal and experimental models. This study examined the effect of ultraviolet B irradiation on epidermal filaggrin and natural moisturizing factors in vivo in healthy adults (n = 22). Participants were irradiated with 2 minimal erythema doses of ultraviolet B on the skin. Biopsies and tape strips were collected from skin irradiated 24 and 72 h earlier and from non-irradiated skin (control). Real-time quantitative PCR on skin biopsies showed significantly reduced profilaggrin mRNA expression 24 h after irradiation (mean relative mRNA expression ± standard deviation: control, 3.86 ± 2.06 vs. 24 h, 1.52 ± 0.640; p = 0.02; n = 8). Immunohistochemistry showed aberrant spatial distribution of filaggrin protein 72 h after irradiation (n = 3). High-pressure liquid chromatography of tape extracts showed no change in mean total natural moisturizing factor levels after irradiation, but mean trans-urocanic acid was significantly reduced, as expected (n = 8). In conclusion, erythemal doses of ultraviolet B exert acute effects on profilaggrin mRNA and filaggrin protein in human skin in vivo.

Optimization for the blockade of epidermal growth factor receptor signaling for therapy of human pancreatic carcinoma.

PubMed

Solorzano, C C; Baker, C H; Tsan, R; Traxler, P; Cohen, P; Buchdunger, E; Killion, J J; Fidler, I J

2001-08-01

We determined the optimal administration schedule of a novel epidermal growth factor receptor (EGFR) protein tyrosine kinase inhibitor (PKI), PKI 166 (4-(R)-phenethylamino-6-(hydroxyl)phenyl-7H-pyrrolo[2.3-d]-pyrimidine), alone or in combination with gemcitabine (administered i.p.) for therapy of L3.6pl human pancreatic carcinoma growing in the pancreas of nude mice. Seven days after orthotopic implantation of L3.6pl cells, the mice received daily oral doses of PKI 166. PKI 166 therapy significantly inhibited phosphorylation of the EGFR without affecting EGFR expression. EGFR phosphorylation was restored 72 h after cessation of therapy. Seven days after orthotopic injection of L3.6pl cells, groups of mice received daily or thrice weekly oral doses of PKI 166 alone or in combination with gemcitabine. Treatment with PKI 166 (daily), PKI 166 (3 times/week), or gemcitabine alone produced a 72%, 69%, or 70% reduction in the volume of pancreatic tumors in mice, respectively. Daily oral PKI 166 or thrice weekly oral PKI 166 in combination with injected gemcitabine produced 97% and 95% decreases in volume of pancreatic cancers and significant inhibition of lymph node and liver metastasis. Daily oral PKI 166 produced a 20% decrease in body weight, whereas treatment 3 times/week did not. Decreased microvessel density, decreased proliferating cell nuclear antigen staining, and increased tumor cell and endothelial cell apoptosis correlated with therapeutic success. Collectively, our results demonstrate that three weekly oral administrations of an EGFR tyrosine kinase inhibitor in combination with gemcitabine are sufficient to significantly inhibit primary and metastatic human pancreatic carcinoma.

Recurrent exposure to nicotine differentiates human bronchial epithelial cells via epidermal growth factor receptor activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinez-Garcia, Eva; Irigoyen, Marta; Anso, Elena

Cigarette smoking is the major preventable cause of lung cancer in developed countries. Nicotine (3-(1-methyl-2-pyrrolidinyl)-pyridine) is one of the major alkaloids present in tobacco. Besides its addictive properties, its effects have been described in panoply of cell types. In fact, recent studies have shown that nicotine behaves as a tumor promoter in transformed epithelial cells. This research focuses on the effects of acute repetitive nicotine exposure on normal human bronchial epithelial cells (NHBE cells). Here we show that treatment of NHBE cells with recurrent doses of nicotine up to 500 {mu}M triggered cell differentiation towards a neuronal-like phenotype: cells emittedmore » filopodia and expressed neuronal markers such as neuronal cell adhesion molecule, neurofilament-M and the transcription factors neuronal N and Pax-3. We also demonstrate that nicotine treatment induced NF-kB translocation to the nucleus, phosphorylation of the epidermal growth factor receptor (EGFR), and accumulation of heparin binding-EGF in the extracellular medium. Moreover, addition of AG1478, an inhibitor of EGFR tyrosine phosphorylation, or cetuximab, a monoclonal antibody that precludes ligand binding to the same receptor, prevented cell differentiation by nicotine. Lastly, we show that differentiated cells increased their adhesion to the extracellular matrix and their protease activity. Given that several lung pathologies are strongly related to tobacco consumption, these results may help to better understand the damaging consequences of nicotine exposure.« less

Isolation and characterization of a novel human scFv inhibiting EGFR vIII expressing cancers.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Dariushnejad, Hassan; Hosseini, Mohammad Kazem

2016-12-01

EGFRvIII, a mutant form of epidermal growth factor receptor is highly expressed in glioblastoma, carcinoma of the breast, ovary, and lung but not in normal cells. This tumor specific antigen has emerged as a promising candidate for antibody based therapy of several cancers. The aim of the present study was isolation and characterization of a human single chain antibody against EGFRvIII as a promising target for cancer therapy. For this, a synthetic peptide corresponding to EGFRvIII protein was used for screening the naive human scFv phage library. Selection was performed using a novel screening strategy for enrichment of rare specific clones. After five rounds of screening, six positive scFv clones against EGFRvIII were selected using monoclonal phage ELISA, among them, a clone with an amber mutation in VH CDR2 coding sequence showed higher reactivity. The mutation was corrected through site directed mutagenesis and then scFv fragment was expressed after subcloning into the bacterial expression vector. Expression in BL21 pLysS resulted in a highly soluble scFv appeared in soluble fraction of E. coli lysate. Bioinformatic in silico analysis between scFv and EGFRvIII sequences confirmed specific binding of desired scFv to EGFRvIII in CDR regions. The specific reactivity of the purified scFv with native EGFRvIII was confirmed by cell based ELISA and western blot. In conclusion, human anti- EGFRvIII scFv isolated from a scFv phage library displayed high reactivity with EGFRvIII. The scFv isolated in this study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFRvIII expressing cancers. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis.

PubMed

Li, Jingting; Sen, George L

2015-12-14

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

Flow Cytometry of Human Primary Epidermal and Follicular Keratinocytes

PubMed Central

Gragnani, Alfredo; Ipolito, Michelle Zampieri; Sobral, Christiane S; Brunialti, Milena Karina Coló; Salomão, Reinaldo; Ferreira, Lydia Masako

2008-01-01

Objective: The aim of this study was to characterize using flow cytometry cultured human primary keratinocytes isolated from the epidermis and hair follicles by different methods. Methods: Human keratinocytes derived from discarded fragments of total skin and scalp hair follicles from patients who underwent plastic surgery in the Plastic Surgery Division at UNIFESP were used. The epidermal keratinocytes were isolated by using 3 different methods: the standard method, upon exposure to trypsin for 30 minutes; the second, by treatment with dispase for 18 hours and with trypsin for 10 minutes; and the third, by treatment with dispase for 18 hours and with trypsin for 30 minutes. Follicular keratinocytes were isolated using the standard method. Results: On comparing the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with dispase for 18 hours and with trypsin for 30 minutes, it was observed that the first group presented the largest number of viable cells, the smallest number of cells in late apoptosis and necrosis with statistical significance, and no difference in apoptosis. When we compared the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with trypsin, the first group presented the largest number of viable cells, the smallest number of cells in apoptosis with statistical significance, and no difference in late apoptosis and necrosis. When we compared the results of the group treated with dispase for 18 hours and with trypsin for 10 minutes with the results for follical isolation, there was a statistical difference in apoptosis and viable cells. Conclusion: The isolation method of treatment with dispase for 18 hours and with trypsin for 10 minutes produced the largest number of viable cells and the smallest number of cells in apoptosis/necrosis. PMID:18350110

Flow cytometry of human primary epidermal and follicular keratinocytes.

PubMed

Gragnani, Alfredo; Ipolito, Michelle Zampieri; Sobral, Christiane S; Brunialti, Milena Karina Coló; Salomão, Reinaldo; Ferreira, Lydia Masako

2008-02-19

The aim of this study was to characterize using flow cytometry cultured human primary keratinocytes isolated from the epidermis and hair follicles by different methods. Human keratinocytes derived from discarded fragments of total skin and scalp hair follicles from patients who underwent plastic surgery in the Plastic Surgery Division at UNIFESP were used. The epidermal keratinocytes were isolated by using 3 different methods: the standard method, upon exposure to trypsin for 30 minutes; the second, by treatment with dispase for 18 hours and with trypsin for 10 minutes; and the third, by treatment with dispase for 18 hours and with trypsin for 30 minutes. Follicular keratinocytes were isolated using the standard method. On comparing the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with dispase for 18 hours and with trypsin for 30 minutes, it was observed that the first group presented the largest number of viable cells, the smallest number of cells in late apoptosis and necrosis with statistical significance, and no difference in apoptosis. When we compared the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with trypsin, the first group presented the largest number of viable cells, the smallest number of cells in apoptosis with statistical significance, and no difference in late apoptosis and necrosis. When we compared the results of the group treated with dispase for 18 hours and with trypsin for 10 minutes with the results for follical isolation, there was a statistical difference in apoptosis and viable cells. The isolation method of treatment with dispase for 18 hours and with trypsin for 10 minutes produced the largest number of viable cells and the smallest number of cells in apoptosis/necrosis.

Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I.

PubMed

Matsumoto, M; Imagawa, M; Aoki, Y

2000-07-01

Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGF alpha induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression.

Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I.

PubMed Central

Matsumoto, M; Imagawa, M; Aoki, Y

2000-01-01

Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGF alpha induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression. PMID:10861232

Expression of cancer-related carbonic anhydrases IX and XII in normal skin and skin neoplasms.

PubMed

Syrjänen, Leo; Luukkaala, Tiina; Leppilampi, Mari; Kallioinen, Matti; Pastorekova, Silvia; Pastorek, Jaromir; Waheed, Abdul; Sly, William S; Parkkila, Seppo; Karttunen, Tuomo

2014-09-01

Purpose of the study was to evaluate the presence of hypoxia-inducible, tumour-associated carbonic anhydrases IX and XII in normal skin and a series of cutaneous tumours. Human tumour samples were taken during surgical operations performed on 245 patients and were immunohistochemically stained. A histological score value was calculated for statistical analyses which were performed using SPSS for Windows, versions 17.0 and 20.0. In normal skin, the highest expression of CA IX was detected in hair follicles, sebaceous glands, and basal parts of epidermis. CA XII was detected in all epithelial components of skin. Both CA IX and CA XII expression levels were significantly different in epidermal, appendigeal, and melanocytic tumour categories. Both CA IX and XII showed the most intense immunostaining in epidermal tumours, whereas virtually all melanocytic tumours were devoid of CA IX and XII immunostaining. In premalignant lesions, CA IX expression significantly increased when the tumours progressed to more severe dysplasia forms. Both CA IX and XII are highly expressed in different epithelial components of skin. They are also highly expressed in epidermal tumours, in which CA IX expression levels also correlate with the dysplasia grade. Interestingly, both isozymes are absent in melanocytic tumours. © 2014 APMIS. Published by John Wiley & Sons Ltd.

Human epidermal growth factor receptor 2 overexpression in breast cancer of patients with anti-Yo--associated paraneoplastic cerebellar degeneration.

PubMed

Rojas-Marcos, Iñigo; Picard, Geraldine; Chinchón, David; Gelpi, Ellen; Psimaras, Dimitri; Giometto, Bruno; Delattre, J Y; Honnorat, J; Graus, F

2012-04-01

Isolated case reports suggest that breast tumors from patients with paraneoplastic cerebellar degeneration (PCD) and Yo antibodies overexpress human epidermal growth factor receptor 2 (HER2). HER2 overexpression is present in 15%-25% of breast cancers and is associated with poor prognosis. We retrospectively analyzed the status of HER2 in breast tumors of 27 patients with anti-Yo-associated PCD to evaluate whether HER2 overexpression in this group of patients is higher than expected. In addition, we analyzed HER2 status of 19 breast tumors from patients with paraneoplastic neurological syndromes and Ri antibodies to see whether HER2 was specifically related to anti-Yo-associated PCD. We also assessed cdr2 expression (the onconeural antigen recognized by Yo antibodies) in 21 HER2-positive breast tumors from patients without paraneoplastic neurological syndromes. HER2 was overexpressed in 26 patients (96.3%) with anti-Yo-associated PCD but only in 2 patients (10.5%) with paraneoplastic neurological syndromes associated with Ri antibodies (P< .0001). Only 5 (23.8%) of the 21 HER2-positive breast tumors showed cdr2 immunoreactivity. This study shows a very high frequency of HER2 overexpression in breast cancers in patients with anti-Yo-associated PCD but not in those from patients with Ri antibodies. Although the expression of cdr2 onconeural antigen is not high in HER2-positive breast cancers, HER2 overexpression seems to be an important requirement to develop an anti-Yo-associated PCD.

Gene Expression of Normal Human Epidermal Keratinocytes Modulated by Trivalent Arsenicals

EPA Science Inventory

Chronic exposure to inorganic arsenic (iAs) is associated with the development of benign and malignant human skin lesions including nonmelanoma skin cancers. The precise arsenical form(s) responsible for this carcinogenic effect are unknown, although trivalent inorganic arsenic (...

Toxic epidermal necrolysis

PubMed Central

Hoetzenecker, Wolfram; Mehra, Tarun; Saulite, Ieva; Glatz, Martin; Schmid-Grendelmeier, Peter; Guenova, Emmanuella; Cozzio, Antonio; French, Lars E.

2016-01-01

Toxic epidermal necrolysis (TEN) is a rare, life-threatening drug-induced skin disease with a mortality rate of approximately 30%. The clinical hallmark of TEN is a marked skin detachment caused by extensive keratinocyte cell death associated with mucosal involvement. The exact pathogenic mechanism of TEN is still uncertain. Recent advances in this field have led to the identification of several factors that might contribute to the induction of excessive apoptosis of keratinocytes. In addition, specific human leukocyte antigen types seem to be associated with certain drugs and the development of TEN. As well-controlled studies are lacking, patients are treated with various immunomodulators (e.g. intravenous immunoglobulin) in addition to the best supportive care. PMID:27239294

Protective Role of Mitochondrial Peroxiredoxin III against UVB-Induced Apoptosis of Epidermal Keratinocytes.

PubMed

Baek, Jin Young; Park, Sujin; Park, Jiyoung; Jang, Ji Yong; Wang, Su Bin; Kim, Sin Ri; Woo, Hyun Ae; Lim, Kyung Min; Chang, Tong-Shin

2017-06-01

UVB light induces generation of reactive oxygen species, ultimately leading to skin cell damage. Mitochondria are a major source of reactive oxygen species in UVB-irradiated skin cells, with increased levels of mitochondrial reactive oxygen species having been implicated in keratinocyte apoptosis. Peroxiredoxin III (PrxIII) is the most abundant and potent H 2 O 2 -removing enzyme in the mitochondria of most cell types. Here, the protective role of PrxIII against UVB-induced apoptosis of epidermal keratinocytes was investigated. Mitochondrial H 2 O 2 levels were differentiated from other types of ROS using mitochondria-specific fluorescent H 2 O 2 indicators. Upon UVB irradiation, PrxIII-knockdown HaCaT human keratinocytes and PrxIII-deficient (PrxIII -/- ) mouse primary keratinocytes exhibited enhanced accumulation of mitochondrial H 2 O 2 compared with PrxIII-expressing controls. Keratinocytes lacking PrxIII were subsequently sensitized to apoptosis through mitochondrial membrane potential loss, cardiolipin oxidation, cytochrome c release, and caspase activation. Increased UVB-induced epidermal tissue damage in PrxIII -/- mice was attributable to increased caspase-dependent keratinocyte apoptosis. Our findings show that mitochondrial H 2 O 2 is a key mediator in UVB-induced apoptosis of keratinocytes and that PrxIII plays a critical role in protecting epidermal keratinocytes against UVB-induced apoptosis through eliminating mitochondrial H 2 O 2 . These findings support the concept that reinforcing mitochondrial PrxIII defenses may help prevent UVB-induced skin damage such as inflammation, sunburn, and photoaging. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Characterization of the efficacies of osimertinib and nazartinib against cells expressing clinically relevant epidermal growth factor receptor mutations.

PubMed

Masuzawa, Keita; Yasuda, Hiroyuki; Hamamoto, Junko; Nukaga, Shigenari; Hirano, Toshiyuki; Kawada, Ichiro; Naoki, Katsuhiko; Soejima, Kenzo; Betsuyaku, Tomoko

2017-12-01

Third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) were developed to overcome EGFR T790M-mediated resistance to first- and second-generation EGFR-TKIs. Third-generation EGFR-TKIs, such as osimertinib and nazartinib, are effective for patients with the EGFR T790M mutation. However, there are no direct comparison data to guide the selection of a third-generation EGFR-TKI for patients with different EGFR mutations. We previously established an in vitro model to estimate the therapeutic windows of EGFR-TKIs by comparing their relative efficacies against cells expressing mutant or wild type EGFRs. The present study used this approach to characterize the efficacy of third-generation EGFR-TKIs and compare them with that of other EGFR-TKIs. Treatment efficacy was examined using human lung cancer-derived cell lines and Ba/F3 cells, which were transduced with clinically relevant mutant EGFRs. Interestingly, mutation-related differences in EGFR-TKI sensitivity were observed. For classic EGFR mutations (exon 19 deletion and L858R, with or without T790M), osimertinib showed lower IC50 values and wider therapeutic windows than nazartinib. For less common EGFR mutations (G719S or L861Q), afatinib showed the lowest IC50 values. For G719S+T790M or L861Q+T790M, the IC50 values of osimertinib and nazartinib were around 100 nM, which was 10- to 100-fold higher than those for classic+T790M mutations. On the contrary, osimertinib and nazartinib showed similar efficacies in cells expressing EGFR exon 20 insertions. The findings highlight the diverse mutation-related sensitivity pattern of EGFR-TKIs. These data may help in the selection of EGFR-TKIs for non-small cell lung cancer patients harboring EGFR mutations.

Characterization of the efficacies of osimertinib and nazartinib against cells expressing clinically relevant epidermal growth factor receptor mutations

PubMed Central

Masuzawa, Keita; Yasuda, Hiroyuki; Hamamoto, Junko; Nukaga, Shigenari; Hirano, Toshiyuki; Kawada, Ichiro; Naoki, Katsuhiko; Soejima, Kenzo; Betsuyaku, Tomoko

2017-01-01

Third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) were developed to overcome EGFR T790M-mediated resistance to first- and second-generation EGFR-TKIs. Third-generation EGFR-TKIs, such as osimertinib and nazartinib, are effective for patients with the EGFR T790M mutation. However, there are no direct comparison data to guide the selection of a third-generation EGFR-TKI for patients with different EGFR mutations. We previously established an in vitro model to estimate the therapeutic windows of EGFR-TKIs by comparing their relative efficacies against cells expressing mutant or wild type EGFRs. The present study used this approach to characterize the efficacy of third-generation EGFR-TKIs and compare them with that of other EGFR-TKIs. Treatment efficacy was examined using human lung cancer-derived cell lines and Ba/F3 cells, which were transduced with clinically relevant mutant EGFRs. Interestingly, mutation-related differences in EGFR-TKI sensitivity were observed. For classic EGFR mutations (exon 19 deletion and L858R, with or without T790M), osimertinib showed lower IC50 values and wider therapeutic windows than nazartinib. For less common EGFR mutations (G719S or L861Q), afatinib showed the lowest IC50 values. For G719S+T790M or L861Q+T790M, the IC50 values of osimertinib and nazartinib were around 100 nM, which was 10- to 100-fold higher than those for classic+T790M mutations. On the contrary, osimertinib and nazartinib showed similar efficacies in cells expressing EGFR exon 20 insertions. The findings highlight the diverse mutation-related sensitivity pattern of EGFR-TKIs. These data may help in the selection of EGFR-TKIs for non-small cell lung cancer patients harboring EGFR mutations. PMID:29285266

Expression, purification, and refolding of active recombinant human E-selectin lectin and EGF domains in Escherichia coli.

PubMed

Kawano, Susumu; Iyaguchi, Daisuke; Okada, Chiaki; Sasaki, Yusuke; Toyota, Eiko

2013-06-01

Attempts to obtain active E-selectin from Escherichia coli (E. coli) have not yet been successful. In this study, we succeeded in expressing the recombinant lectin and epidermal growth factor domain fragments of human E-selectin (rh-ESLE) in E. coli on a large-scale. The rh-ESLE protein was expressed as an inactive form in the inclusion bodies. The inactive form of rh-ESLE was denatured and solubilized by 6 M guanidine hydrochloride and then purified by Ni(2+) affinity chromatography under denaturing conditions. Denatured rh-ESLE was then refolded by a rapid-dilution method using a large amount of refolding buffer, which contained arginine and cysteine/cystine. The refolded rh-ESLE showed binding affinity for sLe(X) (K(d) = 321 nM, B(max) = 1.9 pmol/μg protein). This result suggests that the refolded rh-ESLE recovered its native and functional structure.

Cutaneous gene expression of plasmid DNA in excised human skin following delivery via microchannels created by radio frequency ablation.

PubMed

Birchall, James; Coulman, Sion; Anstey, Alexander; Gateley, Chris; Sweetland, Helen; Gershonowitz, Amikam; Neville, Lewis; Levin, Galit

2006-04-07

The skin is a valuable organ for the development and exploitation of gene medicines. Delivering genes to skin is restricted however by the physico-chemical properties of DNA and the stratum corneum (SC) barrier. In this study, we demonstrate the utility of an innovative technology that creates transient microconduits in human skin, allowing DNA delivery and resultant gene expression within the epidermis and dermis layers. The radio frequency (RF)-generated microchannels were of sufficient morphology and depth to permit the epidermal delivery of 100 nm diameter nanoparticles. Model fluorescent nanoparticles were used to confirm the capacity of the channels for augmenting diffusion of macromolecules through the SC. An ex vivo human organ culture model was used to establish the gene expression efficiency of a beta-galactosidase reporter plasmid DNA applied to ViaDerm treated skin. Skin treated with ViaDerm using 50 microm electrode arrays promoted intense levels of gene expression in the viable epidermis. The intensity and extent of gene expression was superior when ViaDerm was used following a prior surface application of the DNA formulation. In conclusion, the RF-microchannel generator (ViaDerm) creates microchannels amenable for delivery of nanoparticles and gene therapy vectors to the viable region of skin.

The Expression of the Endogenous mTORC1 Inhibitor Sestrin 2 Is Induced by UVB and Balanced with the Expression Level of Sestrin 1.

PubMed

Mlitz, Veronika; Gendronneau, Gaelle; Berlin, Irina; Buchberger, Maria; Eckhart, Leopold; Tschachler, Erwin

2016-01-01

Sestrin 2 (SESN2) is an evolutionarily conserved regulator of mechanistic target of rapamycin complex 1 (mTORC1) which controls central cellular processes such as protein translation and autophagy. Previous studies have suggested that SESN2 itself is subjected to regulation at multiple levels. Here, we investigated the expression of SESN2 in the skin and in isolated skin cells. SESN2 was detected by immunofluorescence analysis in fibroblasts and keratinocytes of human skin. Differentiation of epidermal keratinocytes was not associated with altered SESN2 expression and siRNA-mediated knockdown of SESN2 did not impair stratum corneum formation in vitro. However, SESN2 was increased in both cell types when the expression of its paralog SESN1 was blocked by siRNA-mediated knock down, indicating a compensatory mechanism for the control of expression. Irradiation with UVB but not with UVA significantly increased SESN2 expression in both keratinocytes and fibroblasts. Upregulation of SESN2 expression could be completely blocked by suppression of p53. These results suggest that SESN2 is dispensable for normal epidermal keratinization but involved in the UVB stress response of skin cells.

TORNADO1 regulates root epidermal patterning through the WEREWOLF pathway in Arabidopsis thaliana.

PubMed

Kwak, Su-Hwan; Song, Sang-Kee; Lee, Myeong Min; Schiefelbein, John

2015-01-01

Cell fate in the root epidermis of Arabidopsis thaliana is determined in a position-dependent manner. SCRAMBLED (SCM), an atypical leucine-rich repeat receptor-like kinase, mediates this positional regulation via its effect on WEREWOLF (WER) expression, and subsequently, its downstream transcription factor, GLABRA2 (GL2), which are required for nonhair cell development. Previously, TORNADO1 (TRN1), a plant-specific protein with a leucine-rich repeat ribonuclease inhibitor-like domain, was shown to be required for proper epidermal patterning in Arabidopsis roots. In this work, we analyzed the possible involvement of TRN1 in the known root epidermal gene network. We discovered that the trn1 mutant caused the ectopic expression of WER and the randomized expression of GL2 and EGL3. This suggests that TRN1 regulates the position-dependent cell fate determination by affecting WER expression in Arabidopsis root epidermis. Additionally, the distinct phenotypes of the aerial parts of the trn1-t and scm-2 mutant suggest that TRN1 and SCM might have different functions in the development of aerial parts.

TORNADO1 regulates root epidermal patterning through the WEREWOLF pathway in Arabidopsis thaliana

PubMed Central

Kwak, Su-Hwan; Song, Sang-Kee; Lee, Myeong Min; Schiefelbein, John

2015-01-01

Cell fate in the root epidermis of Arabidopsis thaliana is determined in a position-dependent manner. SCRAMBLED (SCM), an atypical leucine-rich repeat receptor-like kinase, mediates this positional regulation via its effect on WEREWOLF (WER) expression, and subsequently, its downstream transcription factor, GLABRA2 (GL2), which are required for nonhair cell development. Previously, TORNADO1 (TRN1), a plant-specific protein with a leucine-rich repeat ribonuclease inhibitor-like domain, was shown to be required for proper epidermal patterning in Arabidopsis roots. In this work, we analyzed the possible involvement of TRN1 in the known root epidermal gene network. We discovered that the trn1 mutant caused the ectopic expression of WER and the randomized expression of GL2 and EGL3. This suggests that TRN1 regulates the position-dependent cell fate determination by affecting WER expression in Arabidopsis root epidermis. Additionally, the distinct phenotypes of the aerial parts of the trn1-t and scm-2 mutant suggest that TRN1 and SCM might have different functions in the development of aerial parts. PMID:26451798

Cost-Effectiveness of Pertuzumab in Human Epidermal Growth Factor Receptor 2–Positive Metastatic Breast Cancer

PubMed Central

Qian, Yushen; Pollom, Erqi L.; King, Martin T.; Dudley, Sara A.; Shaffer, Jenny L.; Chang, Daniel T.; Gibbs, Iris C.; Goldhaber-Fiebert, Jeremy D.; Horst, Kathleen C.

2016-01-01

Purpose The Clinical Evaluation of Pertuzumab and Trastuzumab (CLEOPATRA) study showed a 15.7-month survival benefit with the addition of pertuzumab to docetaxel and trastuzumab (THP) as first-line treatment for patients with human epidermal growth factor receptor 2 (HER2) –overexpressing metastatic breast cancer. We performed a cost-effectiveness analysis to assess the value of adding pertuzumab. Patient and Methods We developed a decision-analytic Markov model to evaluate the cost effectiveness of docetaxel plus trastuzumab (TH) with or without pertuzumab in US patients with metastatic breast cancer. The model followed patients weekly over their remaining lifetimes. Health states included stable disease, progressing disease, hospice, and death. Transition probabilities were based on the CLEOPATRA study. Costs reflected the 2014 Medicare rates. Health state utilities were the same as those used in other recent cost-effectiveness studies of trastuzumab and pertuzumab. Outcomes included health benefits expressed as discounted quality-adjusted life-years (QALYs), costs in US dollars, and cost effectiveness expressed as an incremental cost-effectiveness ratio. One- and multiway deterministic and probabilistic sensitivity analyses explored the effects of specific assumptions. Results Modeled median survival was 39.4 months for TH and 56.9 months for THP. The addition of pertuzumab resulted in an additional 1.81 life-years gained, or 0.62 QALYs, at a cost of $472,668 per QALY gained. Deterministic sensitivity analysis showed that THP is unlikely to be cost effective even under the most favorable assumptions, and probabilistic sensitivity analysis predicted 0% chance of cost effectiveness at a willingness to pay of $100,000 per QALY gained. Conclusion THP in patients with metastatic HER2-positive breast cancer is unlikely to be cost effective in the United States. PMID:26351332

Epidermal Growth Factor Receptor Expression As Prognostic Marker in Patients With Anal Carcinoma Treated With Concurrent Chemoradiation Therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fraunholz, Ingeborg, E-mail: inge.fraunholz@kgu.de; Rödel, Franz; Kohler, Daniela

Purpose: To investigate the prognostic value of epidermal growth factor receptor (EGFR) expression in pretreatment tumor biopsy specimens of patients with anal cancer treated with concurrent 5-fluorouracil and mitomycin C-based chemoradiation therapy (CRT). Methods and Materials: Immunohistochemical staining for EGFR was performed in pretreatment biopsy specimens of 103 patients with anal carcinoma. EGFR expression was correlated with clinical and histopathologic characteristics and with clinical endpoints, including local failure-free survival (LFFS), colostomy-free survival (CFS), distant metastases-free survival (DMFS), cancer-specific survival (CSS), and overall survival (OS). Results: EGFR staining intensity was absent in 3%, weak in 23%, intermediate in 36% and intensemore » in 38% of the patients. In univariate analysis, the level of EGFR staining was significantly correlated with CSS (absent/weak vs intermediate/intense expression: 5-year CSS, 70% vs 86%, P=.03). As a trend, this was also observed for DMFS (70% vs 86%, P=.06) and LFFS (70% vs 87%, P=.16). In multivariate analysis, N stage, tumor differentiation, and patients’ sex were independent prognostic factors for CSS, whereas EGFR expression only reached borderline significance (hazard ratio 2.75; P=.08). Conclusion: Our results suggest that elevated levels of pretreatment EGFR expression could be correlated with favorable clinical outcome in anal cancer patients treated with CRT. Further studies are warranted to elucidate how EGFR is involved in the response to CRT.« less

Neutrophil extracellular trap formation is increased in psoriasis and induces human β-defensin-2 production in epidermal keratinocytes

PubMed Central

Hu, Stephen Chu-Sung; Yu, Hsin-Su; Yen, Feng-Lin; Lin, Chi-Ling; Chen, Gwo-Shing; Lan, Cheng-Che E.

2016-01-01

Neutrophil extracellular traps (NETs) have been implicated in the development of certain immune-mediated diseases, but their role in psoriasis has not been clearly defined. Human β-defensin-2 (HBD-2) is an important antimicrobial peptide overexpressed in psoriasis epidermis. We evaluated whether the amount of NETs is increased in psoriasis and determined the effect of NETs on HBD-2 production in epidermal keratinocytes. Using fluorescent microscopy, we found that patients with psoriasis (n = 48) had higher amount of NETotic cells in their peripheral blood compared to healthy controls (n = 48) and patients with eczema (n = 35). Psoriasis sera showed increased ability to induce NET formation in control neutrophils but normal NET degradation ability. The amount of NETs in the peripheral blood correlated with psoriasis disease severity. NETosis was also observed in the majority (18 of 20) of psoriasis skin specimens. Furthermore, NETs induced HBD-2 mRNA and protein production in keratinocytes, and immunohistochemical analysis confirmed strong expression of HBD-2 in psoriasis lesional skin. In summary, NET formation is increased in peripheral blood and lesional skin of psoriasis patients and correlates with disease severity. Additionally, NET-induced HBD-2 production may provide a novel mechanism for the decreased susceptibility of psoriasis plaques to microbial infections. PMID:27493143

Epidermal cyst mimicking incision line metastasis.

PubMed

Gündoğdu, Ramazan; Ayhan, Erhan; Çolak, Tahsin

2017-01-01

Epidermal cysts are cystic tumors lined with keratinized squamous layer and filled with keratin debris. Epidermal cysts may develop by implantation of surface epidermal layer into the dermis or subcutaneous tissue after trauma or surgical procedures. Cervix cancer spreads either directly or via the vascular and lymphatic systems. Distant skin metastasis of endometrium or cervix cancer is very rare. In this case report, a patient who had a history of cervix cancer operation 11 years ago and presented with a mass that mimicked incision line metastasis and was histopathologically diagnosed with epidermal cyst is presented.

Effects of non-ablative fractional erbium glass laser treatment on gene regulation in human three-dimensional skin models.

PubMed

Amann, Philipp M; Marquardt, Yvonne; Steiner, Timm; Hölzle, Frank; Skazik-Voogt, Claudia; Heise, Ruth; Baron, Jens M

2016-04-01

Clinical experiences with non-ablative fractional erbium glass laser therapy have demonstrated promising results for dermal remodelling and for the indications of striae, surgical scars and acne scars. So far, molecular effects on human skin following treatment with these laser systems have not been elucidated. Our aim was to investigate laser-induced effects on skin morphology and to analyse molecular effects on gene regulation. Therefore, human three-dimensional (3D) organotypic skin models were irradiated with non-ablative fractional erbium glass laser systems enabling qRT-PCR, microarray and histological studies at same and different time points. A decreased mRNA expression of matrix metalloproteinases (MMPs) 3 and 9 was observed 3 days after treatment. MMP3 also remained downregulated on protein level, whereas the expression of other MMPs like MMP9 was recovered or even upregulated 5 days after irradiation. Inflammatory gene regulatory responses measured by the expression of chemokine (C-X-C motif) ligands (CXCL1, 2, 5, 6) and interleukin expression (IL8) were predominantly reduced. Epidermal differentiation markers such as loricrin, filaggrin-1 and filaggrin-2 were upregulated by both tested laser optics, indicating a potential epidermal involvement. These effects were also shown on protein level in the immunofluorescence analysis. This novel standardised laser-treated human 3D skin model proves useful for monitoring time-dependent ex vivo effects of various laser systems on gene expression and human skin morphology. Our study reveals erbium glass laser-induced regulations of MMP and interleukin expression. We speculate that these alterations on gene expression level could play a role for dermal remodelling, anti-inflammatory effects and increased epidermal differentiation. Our finding may have implications for further understanding of the molecular mechanism of erbium glass laser-induced effects on human skin.

Expression of Epidermal Growth Factor Receptor and Transforming Growth Factor Alpha in Cancer Bladder: Schistosomal and Non-Schistosomal

PubMed Central

Badawy, Afkar A.; El-Hindawi, Ali; Hammam, Olfat; Moussa, Mona; Helal, Noha S.; Kamel, Amira

2017-01-01

Introduction Overexpression of epidermal growth factor receptor (EGFR) has been described in several solid tumors including bladder cancer. Transforming growth factor alpha (TGFα) is frequently deregulated in neoplastic cells and plays a role in the development of bladder cancer. TGFα-EGFR ligand-receptor combination constitutes an important event in multistep tumorigenesis. Methods This study was done on 30 bladder biopsies from patients with urothelial carcinoma, 15 with squamous cell carcinoma, 10 with cystitis and 5 normal control bladder specimens. All were immuohistochemically stained with EGFR and TGFα antibodies. Results EGFR and TGFα were over-expressed in higher grades and late stages of bladder cancer. Moreover, they show higher expression in squamous cell carcinoma compared to urothelial carcinoma and in schistosomal associated lesions than in non-schistosomal associated lesions. Conclusion EGFR and TGFα could be used as prognostic predictors in early stage and grade of bladder cancer cases, especially those with schistosomal association. In addition they can help in selecting patients who can get benefit from anti-EGFR molecular targeted therapy. PMID:28413380

Expression of IL-22 in the Skin Causes Th2-Biased Immunity, Epidermal Barrier Dysfunction and Pruritus via Stimulating Epithelial Th2 Cytokines and the GRP Pathway

PubMed Central

Lou, Hongfei; Lu, Jingning; Choi, Eun Byul; Oh, Min Hee; Jeong, Mingeum; Barmettler, Sara; Zhu, Zhou; Zheng, Tao

2017-01-01

Increased expression of Th22 cytokine IL-22 is a characteristic finding in atopic dermatitis (AD). However, the specific role of IL-22 in the pathogenesis of AD in vivo has yet to be elucidated. Consistent with observations in human AD, IL-22 was significantly increased in the AD skin of mice after epicutaneous sensitization to house dust mite allergen. Utilizing a skin-specific inducible transgenic system, we show here that expression of IL-22 in the skin of mice caused an AD-like phenotype characterized by chronic pruritic dermatitis associated with Th2-biased local and systemic immune responses, down-regulation of Epidermal Differentiation Complex genes and enhanced dermatitis upon epicutaneous allergen exposure. IL-22 potently induced the expression of gastrin-releasing peptide (GRP), a neuropeptide pruritogen, in dermal immune cells and sensory afferents and in their skin-innervating sensory neurons. IL-22 also differentially up-regulated the expression of GRP receptor (GRPR) on keratinocytes of AD skin. The number of GRP+ cells in the skin correlated with the AD severity and the intensity of pruritus. IL-22 directly upregulated the expression of epithelial-derived type 2 cytokines (TSLP and IL-33) and GRP in primary keratinocytes. Furthermore, GRP not only strongly induced TSLP but also increased the expression IL-33 and GRPR synergistically with IL-22. Importantly, we found that the expression of GRP was strikingly increased in the skin of patients with AD. These results indicate that IL-22 plays important pathogenic roles in the initiation and development of AD, in part through inducing keratinocyte production of type 2 cytokines and activation of the GRP/GRPR pathway. PMID:28228560

Human Facial Expressions as Adaptations:Evolutionary Questions in Facial Expression Research

PubMed Central

SCHMIDT, KAREN L.; COHN, JEFFREY F.

2007-01-01

The importance of the face in social interaction and social intelligence is widely recognized in anthropology. Yet the adaptive functions of human facial expression remain largely unknown. An evolutionary model of human facial expression as behavioral adaptation can be constructed, given the current knowledge of the phenotypic variation, ecological contexts, and fitness consequences of facial behavior. Studies of facial expression are available, but results are not typically framed in an evolutionary perspective. This review identifies the relevant physical phenomena of facial expression and integrates the study of this behavior with the anthropological study of communication and sociality in general. Anthropological issues with relevance to the evolutionary study of facial expression include: facial expressions as coordinated, stereotyped behavioral phenotypes, the unique contexts and functions of different facial expressions, the relationship of facial expression to speech, the value of facial expressions as signals, and the relationship of facial expression to social intelligence in humans and in nonhuman primates. Human smiling is used as an example of adaptation, and testable hypotheses concerning the human smile, as well as other expressions, are proposed. PMID:11786989

Langerhans cell precursors acquire RANK/CD265 in prenatal human skin.

PubMed

Schöppl, Alice; Botta, Albert; Prior, Marion; Akgün, Johnnie; Schuster, Christopher; Elbe-Bürger, Adelheid

2015-01-01

The skin is the first barrier against foreign pathogens and the prenatal formation of a strong network of various innate and adaptive cells is required to protect the newborn from perinatal infections. While many studies about the immune system in healthy and diseased adult human skin exist, our knowledge about the cutaneous prenatal/developing immune system and especially about the phenotype and function of antigen-presenting cells such as epidermal Langerhans cells (LCs) in human skin is still scarce. It has been shown previously that LCs in healthy adult human skin express receptor activator of NF-κB (RANK), an important molecule prolonging their survival. In this study, we investigated at which developmental stage LCs acquire this important molecule. Immunofluorescence double-labeling of cryostat sections revealed that LC precursors in prenatal human skin either do not yet [10-11 weeks of estimated gestational age (EGA)] or only faintly (13-15 weeks EGA) express RANK. LCs express RANK at levels comparable to adult LCs by the end of the second trimester. Comparable with adult skin, dermal antigen-presenting cells at no gestational age express this marker. These findings indicate that epidermal leukocytes gradually acquire RANK during gestation - a phenomenon previously observed also for other markers on LCs in prenatal human skin. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.

Blunted epidermal L-tryptophan metabolism in vitiligo affects immune response and ROS scavenging by Fenton chemistry, part 1: Epidermal H2O2/ONOO(-)-mediated stress abrogates tryptophan hydroxylase and dopa decarboxylase activities, leading to low serotonin and melatonin levels.

PubMed

Schallreuter, Karin U; Salem, Mohamed A E L; Gibbons, Nick C J; Martinez, Aurora; Slominski, Radomir; Lüdemann, Jürgen; Rokos, Hartmut

2012-06-01

Vitiligo is characterized by a progressive loss of inherited skin color. The cause of the disease is still unknown. To date, there is accumulating in vivo and in vitro evidence for massive oxidative stress via hydrogen peroxide (H(2)O(2)) and peroxynitrite (ONOO(-)) in the skin of affected individuals. Autoimmune etiology is the favored theory. Since depletion of the essential amino acid L-tryptophan (Trp) affects immune response mechanisms, we here looked at epidermal Trp metabolism via tryptophan hydroxylase (TPH) with its downstream cascade, including serotonin and melatonin. Our in situ immunofluorescence and Western blot data reveal significantly lower TPH1 expression in patients with vitiligo. Expression is also low in melanocytes and keratinocytes under in vitro conditions. Although in vivo Fourier transform-Raman spectroscopy proves the presence of 5-hydroxytryptophan, epidermal TPH activity is completely absent. Regulation of TPH via microphthalmia-associated transcription factor and L-type calcium channels is severely affected. Moreover, dopa decarboxylase (DDC) expression is significantly lower, in association with decreased serotonin and melatonin levels. Computer simulation supports H(2)O(2)/ONOO(-)-mediated oxidation/nitration of TPH1 and DDC, affecting, in turn, enzyme functionality. Taken together, our data point to depletion of epidermal Trp by Fenton chemistry and exclude melatonin as a relevant contributor to epidermal redox balance and immune response in vitiligo.

JACKDAW controls epidermal patterning in the Arabidopsis root meristem through a non-cell-autonomous mechanism.

PubMed

Hassan, Hala; Scheres, Ben; Blilou, Ikram

2010-05-01

In Arabidopsis, specification of the hair and non-hair epidermal cell types is position dependent, in that hair cells arise over clefts in the underlying cortical cell layer. Epidermal patterning is determined by a network of transcriptional regulators that respond to an as yet unknown cue from underlying tissues. Previously, we showed that JACKDAW (JKD), a zinc finger protein, localizes in the quiescent centre and the ground tissue, and regulates tissue boundaries and asymmetric cell division by delimiting SHORT-ROOT movement. Here, we provide evidence that JKD controls position-dependent signals that regulate epidermal-cell-type patterning. JKD is required for appropriately patterned expression of the epidermal cell fate regulators GLABRA2, CAPRICE and WEREWOLF. Genetic interaction studies indicate that JKD operates upstream of the epidermal patterning network in a SCRAMBLED (SCM)-dependent fashion after embryogenesis, but acts independent of SCM in embryogenesis. Tissue-specific induction experiments indicate non-cell-autonomous action of JKD from the underlying cortex cell layer to specify epidermal cell fate. Our findings are consistent with a model where JKD induces a signal in every cortex cell that is more abundant in the hair cell position owing to the larger surface contact of cells located over a cleft.

Epidermal cyst mimicking incision line metastasis

PubMed Central

Gündoğdu, Ramazan; Ayhan, Erhan; Çolak, Tahsin

2017-01-01

Epidermal cysts are cystic tumors lined with keratinized squamous layer and filled with keratin debris. Epidermal cysts may develop by implantation of surface epidermal layer into the dermis or subcutaneous tissue after trauma or surgical procedures. Cervix cancer spreads either directly or via the vascular and lymphatic systems. Distant skin metastasis of endometrium or cervix cancer is very rare. In this case report, a patient who had a history of cervix cancer operation 11 years ago and presented with a mass that mimicked incision line metastasis and was histopathologically diagnosed with epidermal cyst is presented. PMID:28740968

Phase I study of bortezomib and cetuximab in patients with solid tumours expressing epidermal growth factor receptor

PubMed Central

Dudek, A Z; Lesniewski-Kmak, K; Shehadeh, N J; Pandey, O N; Franklin, M; Kratzke, R A; Greeno, E W; Kumar, P

2009-01-01

Bortezomib inhibits nuclear factor-κB (NF-κB). Cetuximab is a chimeric mouse–human antibody targeted against epidermal growth factor receptor (EGFR). We hypothesised that concomitant blockade of NF-κB and EGFR signalling would overcome EGFR-mediated resistance to single-agent bortezomib and induce apoptosis through two molecular pathways. The aim of this phase I trial was to establish the maximum tolerated dose (MTD) for bortezomib plus cetuximab in patients with EGFR-expressing epithelial tumours. The 21-day treatment cycle consisted of bortezomib administered on days 1 and 8 through dose escalation (1.3–2 mg m−2). Cetuximab was delivered at a dose of 250 mg m−2 on days 1, 8 and 15 (400 mg m−2 day 1 cycle 1). A total of 37 patients were enroled and given a total 91 cycles. No grade ⩾3 haematological toxicity was noted. Non-hematological grade ⩾3 toxicities included fatigue (22% of patients), dyspnoea (16%) and infection (11%). The MTD was not reached at the highest tested bortezomib dose (2.0 mg m−2). Efficacy outcomes included disease progression in 21 patients (56.7%) and stable disease (SD) at 6 weeks in 16 patients (43.3%). Five of the six patients with SD at 12 weeks were diagnosed with cancers of the lungs or head and neck. This combination therapy was moderately effective in extensively pretreated patients with non-small cell lung or head and neck cancers and warrants further investigation. PMID:19401697

Endothelial network formed with human dermal microvascular endothelial cells in autologous multicellular skin substitutes.

PubMed

Ponec, Maria; El Ghalbzouri, Abdoelwaheb; Dijkman, Remco; Kempenaar, Johanna; van der Pluijm, Gabri; Koolwijk, Pieter

2004-01-01

A human skin equivalent from a single skin biopsy harboring keratinocytes and melanocytes in the epidermal compartment, and fibroblasts and microvascular dermal endothelial cells in the dermal compartment was developed. The results of the study revealed that the nature of the extracellular matrix of the dermal compartments plays an important role in establishment of endothelial network in vitro. With rat-tail type I collagen matrices only lateral but not vertical expansion of endothelial networks was observed. In contrast, the presence of extracellular matrix of entirely human origin facilitated proper spatial organization of the endothelial network. Namely, when human dermal fibroblasts and microvascular endothelial cells were seeded on the bottom of an inert filter and subsequently epidermal cells were seeded on top of it, fibroblasts produced extracellular matrix throughout which numerous branched tubes were spreading three-dimensionally. Fibroblasts also facilitated the formation of basement membrane at the epidermal/matrix interface. Under all culture conditions, fully differentiated epidermis was formed with numerous melanocytes present in the basal epidermal cell layer. The results of the competitive RT-PCR revealed that both keratinocytes and fibroblasts expressed VEGF-A, -B, -C, aFGF and bFGF mRNA, whereas fibroblasts also expressed VEGF-D mRNA. At protein level, keratinocytes produced 10 times higher amounts of VEGF-A than fibroblasts did. The generation of multicellular skin equivalent from a single human skin biopsy will stimulate further developments for its application in the treatment of full-thickness skin defects. The potential development of biodegradable, biocompatible material suitable for these purposes is a great challenge for future research.

Morphological and functional studies on the epidermal cells of amphioxus ( Branchiostoma belcheri tsingtauense) at different developmental stages

NASA Astrophysics Data System (ADS)

Mao, Bing-Yu; Sun, Xiao-Yang; Zhang, Hong-Wei; Zhang, Shi-Cui; Wu, Xian-Han

1997-09-01

Epidermal cells of amphioxus at different developmental stages were investigated by electron microscopy and colloidal carbon tracing experiments. Amphioxus epidermal cells showed different ultrastructural characteristics at larval and adult stages. The epidermal cells at all larval stages studied (24 96 h) had numerous vesicles containing electron dense materials in their apical cytoplasm. In tracing experiments, carbon particles were found in apical vesicles and interoellular spaces. Under scanning electron microscope, many crater-like protrusions were observed on the surface of the cells. These results indicated that amphioxus larval epidermal cells may be capable of endocytosis. The epidermal cells of 3-month and adult amphioxus were obviously secretory ones characterized by well-developed peripheral filaments, a prominent Golgi apparatus and abundant apical secretory vesicles. This study also showed that adult amphioxus body surface mucus contained lectin that could agglutinate human red blood cells. The authors propose that the epidermal cells of amphioxus larva and adult may contribute to the immune defense of the amimal by different means.

The Herbal Bitter Drug Gentiana lutea Modulates Lipid Synthesis in Human Keratinocytes In Vitro and In Vivo

PubMed Central

Haarhaus, Birgit; Seiwerth, Jasmin; Cawelius, Anja; Schwabe, Kay; Quirin, Karl-Werner; Schempp, Christoph M.

2017-01-01

Gentiana lutea is a herbal bitter drug that is used to enhance gastrointestinal motility and secretion. Recently we have shown that amarogentin, a characteristic bitter compound of Gentiana lutea extract (GE), binds to the bitter taste receptors TAS2R1 and TAS2R38 in human keratinocytes, and stimulates the synthesis of epidermal barrier proteins. Here, we wondered if GE also modulates lipid synthesis in human keratinocytes. To address this issue, human primary keratinocytes were incubated for 6 days with GE. Nile Red labeling revealed that GE significantly increased lipid synthesis in keratinocytes. Similarly, gas chromatography with flame ionization detector indicated that GE increases the amount of triglycerides in keratinocytes. GE induced the expression of epidermal ceramide synthase 3, but not sphingomyelinase. Lipid synthesis, as well as ceramide synthase 3 expression, could be specifically blocked by inhibitors of the p38 MAPK and PPARγ signaling pathway. To assess if GE also modulates lipid synthesis in vivo, we performed a proof of concept half side comparison on the volar forearms of 33 volunteers. In comparison to placebo, GE significantly increased the lipid content of the treated skin areas, as measured with a sebumeter. Thus, GE enhances lipid synthesis in human keratinocytes that is essential for building an intact epidermal barrier. Therefore, GE might be used to improve skin disorders with an impaired epidermal barrier, e.g., very dry skin and atopic eczema. PMID:28829355

The Herbal Bitter Drug Gentiana lutea Modulates Lipid Synthesis in Human Keratinocytes In Vitro and In Vivo.

PubMed

Wölfle, Ute; Haarhaus, Birgit; Seiwerth, Jasmin; Cawelius, Anja; Schwabe, Kay; Quirin, Karl-Werner; Schempp, Christoph M

2017-08-22

Gentiana lutea is a herbal bitter drug that is used to enhance gastrointestinal motility and secretion. Recently we have shown that amarogentin, a characteristic bitter compound of Gentiana lutea extract (GE), binds to the bitter taste receptors TAS2R1 and TAS2R38 in human keratinocytes, and stimulates the synthesis of epidermal barrier proteins. Here, we wondered if GE also modulates lipid synthesis in human keratinocytes. To address this issue, human primary keratinocytes were incubated for 6 days with GE. Nile Red labeling revealed that GE significantly increased lipid synthesis in keratinocytes. Similarly, gas chromatography with flame ionization detector indicated that GE increases the amount of triglycerides in keratinocytes. GE induced the expression of epidermal ceramide synthase 3, but not sphingomyelinase. Lipid synthesis, as well as ceramide synthase 3 expression, could be specifically blocked by inhibitors of the p38 MAPK and PPARγ signaling pathway. To assess if GE also modulates lipid synthesis in vivo, we performed a proof of concept half side comparison on the volar forearms of 33 volunteers. In comparison to placebo, GE significantly increased the lipid content of the treated skin areas, as measured with a sebumeter. Thus, GE enhances lipid synthesis in human keratinocytes that is essential for building an intact epidermal barrier. Therefore, GE might be used to improve skin disorders with an impaired epidermal barrier, e.g., very dry skin and atopic eczema.

3D pharmacophore-based virtual screening, docking and density functional theory approach towards the discovery of novel human epidermal growth factor receptor-2 (HER2) inhibitors.

PubMed

Gogoi, Dhrubajyoti; Baruah, Vishwa Jyoti; Chaliha, Amrita Kashyap; Kakoti, Bibhuti Bhushan; Sarma, Diganta; Buragohain, Alak Kumar

2016-12-21

Human epidermal growth factor receptor 2 (HER2) is one of the four members of the epidermal growth factor receptor (EGFR) family and is expressed to facilitate cellular proliferation across various tissue types. Therapies targeting HER2, which is a transmembrane glycoprotein with tyrosine kinase activity, offer promising prospects especially in breast and gastric/gastroesophageal cancer patients. Persistence of both primary and acquired resistance to various routine drugs/antibodies is a disappointing outcome in the treatment of many HER2 positive cancer patients and is a challenge that requires formulation of new and improved strategies to overcome the same. Identification of novel HER2 inhibitors with improved therapeutics index was performed with a highly correlating (r=0.975) ligand-based pharmacophore model (Hypo1) in this study. Hypo1 was generated from a training set of 22 compounds with HER2 inhibitory activity and this well-validated hypothesis was subsequently used as a 3D query to screen compounds in a total of four databases of which two were natural product databases. Further, these compounds were analyzed for compliance with Veber's drug-likeness rule and optimum ADMET parameters. The selected compounds were then subjected to molecular docking and Density Functional Theory (DFT) analysis to discern their molecular interactions at the active site of HER2. The findings thus presented would be an important starting point towards the development of novel HER2 inhibitors using well-validated computational techniques. Copyright © 2016 Elsevier Ltd. All rights reserved.

Aloe-emodin inhibits HER-2 expression through the downregulation of Y-box binding protein-1 in HER-2-overexpressing human breast cancer cells.

PubMed

Ma, Jui-Wen; Hung, Chao-Ming; Lin, Ying-Chao; Ho, Chi-Tang; Kao, Jung-Yie; Way, Tzong-Der

2016-09-13

Human epidermal growth factor receptor-2 (HER-2)-positive breast cancer tends to be aggressive, highly metastatic, and drug resistant and spreads rapidly. Studies have indicated that emodin inhibits HER-2 expression. This study compared the HER-2-inhibitory effects of two compounds extracted from rhubarb roots: aloe-emodin (AE) and rhein. Our results indicated that AE exerted the most potent inhibitory effect on HER-2 expression. Treatment of HER-2-overexpressing breast cancer cells with AE reduced tumor initiation, cell migration, and cell invasion. AE was able to suppress YB-1 expression, further suppressing downstream HER-2 expression. AE suppressed YB-1 expression through the inhibition of Twist in HER-2-overexpressing breast cancer cells. Our data also found that AE inhibited cancer metastasis and cancer stem cells through the inhibition of EMT. Interestingly, AE suppressed YB-1 expression through the downregulation of the intracellular integrin-linked kinase (ILK)/protein kinase B (Akt)/mTOR signaling pathway in HER-2-overexpressing breast cancer cells. In vivo study showed the positive result of antitumor activity of AE in nude mice injected with human HER-2-overexpressing breast cancer cells. These findings suggest the possible application of AE in the treatment of HER-2-positive breast cancer.

Thermal analysis of epidermal electronic devices integrated with human skin considering the effects of interfacial thermal resistance

NASA Astrophysics Data System (ADS)

Li, Yuhang; Zhang, Jianpeng; Xing, Yufeng; Song, Jizhou

2018-05-01

Epidermal electronic devices (EEDs) have similar mechanical properties as those of human skin such that they can be integrated with human skin for potential applications in monitoring of human vital signs for diagnostic, therapeutic or surgical functions. Thermal management is critical for EEDs in these applications since excessive heating may cause discomfort. Comprehensive analytical studies, finite element analysis and experiments are carried out to study the effects of interfacial thermal resistance between EEDs and human skin on thermal properties of the EED/skin system in this paper. The coupling between the Fourier heat transfer in EEDs and the bio-heat transfer in human skin is accounted in the analytical model based on the transfer matrix method to give accurate predictions on temperatures, which agree well with finite element analysis and experimental measurements. It is shown that the maximum temperature increase of the EED for the case of imperfect bonding between EED and skin is much higher than that of perfect bonding. These results may help the design of EEDs in bi-integrated applications and suggest a valuable route to evaluate the bonding condition between EEDs and biological tissues.

Interrogation of Multidrug Resistance (MDR1) P-glycoprotein (Pgp, ABCB1) Expression in Human Pancreatic Carcinoma Cells: Correlation of 99mTc-Sestamibi Uptake with Western Blot Analysis

PubMed Central

Harpstrite, Scott E.; Gu, Hannah; Natarajan, Radhika; Sharma, Vijay

2014-01-01

Objective Histopathological studies indicate approximately 63% of pancreatic tumors express MDR1 Pgp and its polymorphic variants. However, Pgp expression detected at the messenger RNA or protein level does not always correlate with functional transport activity. Because Pgp transport activity is affected by specific mutations as well as the phosphorylation state of the protein, altered or less active forms of Pgp may also be detected by PCR or immunohistochemistry, which do not accurately reflect the status of tumor cell resistance. To interrogate status of functional expression of MDR1 Pgp in MiaPaCa-2 and PANC-1 cells, cellular transport studies using 99mTc-Sestamibi were performed and correlated with western blot analysis. Methods Biochemical transport assays in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells, human epidermal carcinoma drug sensitive KB-3-1 cells and human breast carcinoma MCF-7 cells (negative controls), and human epidermal carcinoma drug resistant KB-8-5 cells, human breast carcinoma stably transfected with Pgp MCF-7/MDR1Pgp cells, and liver carcinoma HepG2 cells (positive controls) were performed. Protein levels were determined using a monoclonal antibody C219. Results 99mTc-Sestamibi demonstrates accumulation in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells. Uptake profiles are not affected by treatment with LY335979, a Pgp-inhibitor, and correlate to Western blot analysis. Conclusions These cellular transport studies indicate an absence of Pgp at a functional level in MiaPaCa-2 and PANC-1 cells. Because major pancreatic tumors originate from pancreatic duct and 99mTc-Sestamibi undergoes a dominant hepatobiliary mode of excretion, it would not be a sensitive probe for imaging pancreatic adenocarcinomas. Following interrogation of the functional status of Pgp in other pancreatic carcinoma cells, chemotherapeutic drugs that are also MDR1 substrates could offer alternative therapeutics for treating pancreatic adenocarcinomas

Epidermal ablation of Dlx3 is linked to IL-17–associated skin inflammation

PubMed Central

Hwang, Joonsung; Kita, Ryosuke; Kwon, Hyouk-Soo; Choi, Eung Ho; Lee, Seung Hun; Udey, Mark C.; Morasso, Maria I.

2011-01-01

In an effort to understand the role of Distal-less 3 (Dlx3) in cutaneous biology and pathophysiology, we generated and characterized a mouse model with epidermal ablation of Dlx3. K14cre;Dlx3Kin/f mice exhibited epidermal hyperproliferation and abnormal differentiation of keratinocytes. Results from subsequent analyses revealed cutaneous inflammation that featured accumulation of IL-17–producing CD4+ T, CD8+ T, and γδ T cells in the skin and lymph nodes of K14cre;Dlx3Kin/f mice. The gene expression signature of K14cre;Dlx3Kin/f skin shared features with lesional psoriatic skin, and Dlx3 expression was markedly and selectively decreased in psoriatic skin. Interestingly, cultured Dlx3 null keratinocytes triggered cytokine production that is potentially linked to inflammatory responses in K14cre;Dlx3Kin/f mice. Thus, Dlx3 ablation in epidermis is linked to altered epidermal differentiation, barrier development, and IL-17–associated skin inflammation. This model provides a platform that will allow the systematic exploration of the contributions of keratinocytes to cutaneous inflammation. PMID:21709238

Punicalagin and (-)-Epigallocatechin-3-Gallate Rescue Cell Viability and Attenuate Inflammatory Responses of Human Epidermal Keratinocytes Exposed to Airborne Particulate Matter PM10.

PubMed

Seok, Jin Kyung; Lee, Jeong-Won; Kim, Young Mi; Boo, Yong Chool

2018-01-01

Airborne particulate matter with a diameter of < 10 µm (PM10) causes oxidative damage, inflammation, and premature skin aging. In this study, we evaluated whether polyphenolic antioxidants attenuate the inflammatory responses of PM10-exposed keratinocytes. Primary human epidermal keratinocytes were exposed in vitro to PM10 in the absence or presence of punicalagin and (-)-epigallocatechin-3-gallate (EGCG), which are the major polyphenolic antioxidants found in pomegranate and green tea, respectively. Assays were performed to determine cell viability, production of reactive oxygen species (ROS), and expression of NADPH oxidases (NOX), proinflammatory cytokines, and matrix metalloproteinase (MMP)-1. PM10 decreased cell viability and increased ROS production in a dose-dependent manner. It also increased the expression levels of NOX-1, NOX-2, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, and MMP-1. Punicalagin was not cytotoxic up to 300 μM, and (-)-EGCG was cytotoxic above 30 μM, respectively. Further, punicalagin (3-30 μM) and EGCG (3-10 μM) rescued the viability of PM10-exposed cells. They also attenuated ROS production and the expression of NOX-1, NOX-2, TNF-α, IL-1β, IL-6, IL-8, and MMP-1 stimulated by PM10. This study demonstrates that polyphenolic antioxidants, such as punicalagin and (-)-EGCG, rescue keratinocyte viability and attenuate the inflammatory responses of these cells due to airborne particles. © 2018 S. Karger AG, Basel.

Phthalocyanine-Peptide Conjugates for Epidermal Growth Factor Receptor Targeting1

PubMed Central

Ongarora, Benson G.; Fontenot, Krystal R.; Hu, Xiaoke; Sehgal, Inder; Satyanarayana-Jois, Seetharama D.; Vicente, M. Graça H.

2012-01-01

Four phthalocyanine (Pc)-peptide conjugates designed to target the epidermal growth factor receptor (EGFR) were synthesized and evaluated in vitro using four cell lines: human carcinoma A431 and HEp2, human colorectal HT-29, and kidney Vero (negative control) cells. Two peptide ligands for EGFR were investigated: EGFR-L1 and -L2, bearing 6 and 13 amino acid residues, respectively. The peptides and Pc-conjugates were shown to bind to EGFR using both theoretical (Autodock) and experimental (SPR) investigations. The Pc-EGFR-L1 conjugates 5a and 5b efficiently targeted EGFR and were internalized, in part due to their cationic charge, whereas the uncharged Pc-EGFR-L2 conjugates 4b and 6a poorly targeted EGFR maybe due to their low aqueous solubility. All conjugates were non-toxic (IC50 > 100 µM) to HT-29 cells, both in the dark and upon light activation (1 J/cm2). Intravenous (iv) administration of conjugate 5b into nude mice bearing A431 and HT-29 human tumor xenografts resulted in a near-IR fluorescence signal at ca. 700 nm, 24 h after administration. Our studies show that Pc-EGFR-L1 conjugates are promising near-IR fluorescent contrast agents for CRC, and potentially other EGFR over-expressing cancers. PMID:22468711

Elevated gene expression levels distinguish human from non-human primate brains

PubMed Central

Cáceres, Mario; Lachuer, Joel; Zapala, Matthew A.; Redmond, John C.; Kudo, Lili; Geschwind, Daniel H.; Lockhart, David J.; Preuss, Todd M.; Barlow, Carrolee

2003-01-01

Little is known about how the human brain differs from that of our closest relatives. To investigate the genetic basis of human specializations in brain organization and cognition, we compared gene expression profiles for the cerebral cortex of humans, chimpanzees, and rhesus macaques by using several independent techniques. We identified 169 genes that exhibited expression differences between human and chimpanzee cortex, and 91 were ascribed to the human lineage by using macaques as an outgroup. Surprisingly, most differences between the brains of humans and non-human primates involved up-regulation, with ≈90% of the genes being more highly expressed in humans. By contrast, in the comparison of human and chimpanzee heart and liver, the numbers of up- and down-regulated genes were nearly identical. Our results indicate that the human brain displays a distinctive pattern of gene expression relative to non-human primates, with higher expression levels for many genes belonging to a wide variety of functional classes. The increased expression of these genes could provide the basis for extensive modifications of cerebral physiology and function in humans and suggests that the human brain is characterized by elevated levels of neuronal activity. PMID:14557539

[Effect of Tribulus terrestris extract on melanocyte-stimulating hormone expression in mouse hair follicles].

PubMed

Yang, Liu; Lu, Jian-wei; An, Jing; Jiang, Xuan

2006-12-01

To observe the effect of Tribulus terrestris extract on melanocyte stimulating hormone (MSH) expression in C57BL/6J mouse hair follicles, and investigate the role of Tribulus terrestris extract in activation, proliferation, epidermal migration of dormant hair follicle melanocytes. The aqueous extract of Tribulus terrestris was administered orally in specific pathogen-free C57BL/6J mouse at the daily dose equivalent to 1 g/1 kg in adult human, and the expression and distribution of MSH in the mouse hair follicles was observed with immunohistochemistry. The positivity rate of MSH expression in the hair follicle melanocytes was 75% in mice treated with the extract, significantly higher than the rate of only 18.75% in the control group (P<0.01). The aqueous extract of Tribulus terrestris can significantly increase MSH expression in the hair follicle melanocytes by activating tyrosinase activity and promoting melanocyte proliferation, melanine synthesis, and epidermal migration of dormant melanocytes.

Reconstruction of living bilayer human skin equivalent utilizing human fibrin as a scaffold.

PubMed

Mazlyzam, A L; Aminuddin, B S; Fuzina, N H; Norhayati, M M; Fauziah, O; Isa, M R; Saim, L; Ruszymah, B H I

2007-05-01

Our aim of this study was to develop a new methodology for constructing a bilayer human skin equivalent to create a more clinical compliance skin graft composite for the treatment of various skin defects. We utilized human plasma derived fibrin as the scaffold for the development of a living bilayer human skin equivalent: fibrin-fibroblast and fibrin-keratinocyte (B-FF/FK SE). Skin cells from six consented patients were culture-expanded to passage 1. For B-FF/FK SE formation, human fibroblasts were embedded in human fibrin matrix and subsequently another layer of human keratinocytes in human fibrin matrix was stacked on top. The B-FF/FK SE was then transplanted to athymic mice model for 4 weeks to evaluate its regeneration and clinical performance. The in vivo B-FF/FK SE has similar properties as native human skin by histological analysis and expression of basal Keratin 14 gene in the epidermal layer and Collagen type I gene in the dermal layer. Electron microscopy analysis of in vivo B-FF/FK SE showed well-formed and continuous epidermal-dermal junction. We have successfully developed a technique to engineer living bilayer human skin equivalent using human fibrin matrix. The utilization of culture-expanded human skin cells and fibrin matrix from human blood will allow a fully autologous human skin equivalent construction.

Problem-Solving Test: The Role of Ubiquitination in Epidermal Growth Factor Receptor Trafficking

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2012-01-01

Terms to be familiar with before you start to solve the test: growth factor signaling, epidermal growth factor, tyrosine protein kinase, tyrosine phosphorylation, ubiquitin, monoubiquitination, polyubiquitination, site-directed mutagenesis, transfection, expression vector, cDNA, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, Western…

Effect of Standardized Boesenbergia pandurata Extract and Its Active Compound Panduratin A on Skin Hydration and Barrier Function in Human Epidermal Keratinocytes.

PubMed

Woo, Seon Wook; Rhim, Dong-Bin; Kim, Changhee; Hwang, Jae-Kwan

2015-03-01

The skin plays a key role in protecting the body from the environment and from water loss. Cornified envelope (CE) and natural moisturizing factor (NMF) are considered as the primary regulators of skin hydration and barrier function. The CE prevents loss of water from the body and is formed by cross-linking of several proteins. Among these proteins, filaggrin is an important protein because NMF is produced by the degradation of filaggrin. Proteases, including matriptase and prostasin, stimulate the generation of filaggrin from profilaggrin and caspase-14 plays a role in the degradation of filaggrin. This study elucidated the effects of an ethanol extract of Boesenbergia pandurata (Roxb.) Schltr., known as fingerroot, and its active compound panduratin A on CE formation and filaggrin processing in HaCaT, human epidermal keratinocytes. B. pandurata extract (BPE) and panduratin A significantly stimulated not only CE formation but also the expression of CE proteins, such as loricrin, involucrin, and transglutaminase, which were associated with PPARα expression. The mRNA and protein levels of filaggrin and filaggrin-related enzymes, such as matriptase, prostasin, and caspase-14 were also up-regulated by BPE and panduratin A treatment. These results suggest that BPE and panduratin A are potential nutraceuticals which can enhance skin hydration and barrier function based on their CE formation and filaggrin processing.

ΔNp63 is an ectodermal gatekeeper of epidermal morphogenesis

PubMed Central

Shalom-Feuerstein, R; Lena, A M; Zhou, H; De La Forest Divonne, S; Van Bokhoven, H; Candi, E; Melino, G; Aberdam, D

2011-01-01

p63, a member of p53 family, has a significant role in the development and maintenance of stratified epithelia. However, a persistent dispute remained over the last decade concerning the interpretation of the severe failure of p63-null embryos to develop stratified epithelia. In this study, by investigating both p63-deficient strains, we demonstrated that p63-deficient epithelia failed to develop beyond ectodermal stage as they remained a monolayer of non-proliferating cells expressing K8/K18. Importantly, in the absence of p63, corneal-epithelial commitment (which occurs at embryonic day 12.5 of mouse embryogenesis) was hampered 3 weeks before corneal stem cell renewal (that begins at P14). Taken together, these data illustrate the significant role of p63 in epithelial embryogenesis, before and independently of other functions of p63 in adult stem cells regulation. Transcriptome analysis of laser captured-embryonic tissues confirmed the latter hypothesis, demonstrating that a battery of epidermal genes that were activated in wild-type epidermis remained silent in p63-null tissues. Furthermore, we defined a subset of novel bona fide p63-induced genes orchestrating first epidermal stratification and a subset of p63-repressed mesodermal-specific genes. These data highlight the earliest recognized action of ΔNp63 in the induction epidermal morphogenesis at E11.5. In the absence of p63, a mesodermal program is activated while epidermal morphogenesis does not initiate. PMID:21127502

Topical stabilized retinol treatment induces the expression of HAS genes and HA production in human skin in vitro and in vivo.

PubMed

Li, Wen-Hwa; Wong, Heng-Kuan; Serrano, José; Randhawa, Manpreet; Kaur, Simarna; Southall, Michael D; Parsa, Ramine

2017-05-01

Skin Aging manifests primarily with wrinkles, dyspigmentations, texture changes, and loss of elasticity. During the skin aging process, there is a loss of moisture and elasticity in skin resulting in loss of firmness finally leading to skin sagging. The key molecule involved in skin moisture is hyaluronic acid (HA), which has a significant water-binding capacity. HA levels in skin decline with age resulting in decrease in skin moisture, which may contribute to loss of firmness. Clinical trials have shown that topically applied ROL effectively reduces wrinkles and helps retain youthful appearance. In the current study, ROL was shown to induce HA production and stimulates the gene expression of all three forms of hyaluronic acid synthases (HAS) in normal human epidermal keratinocytes monolayer cultures. Moreover, in human skin equivalent tissues and in human skin explants, topical treatment of tissues with a stabilized-ROL formulation significantly induced the gene expression of HAS mRNA concomitant with an increased HA production. Finally, in a vehicle-controlled human clinical study, histochemical analysis confirmed increased HA accumulation in the epidermis in ROL-treated human skin as compared to vehicle. These results show that ROL increases skin expression of HA, a significant contributing factor responsible for wrinkle formation and skin moisture, which decrease during aging. Taken together with the activity to increase collagen, elastin, and cell proliferation, these studies establish that retinol provides multi-functional activity for photodamaged skin.

Highly stretchable and ultrathin nanopaper composites for epidermal strain sensors.

PubMed

Sun, Jingyao; Zhao, Yanan; Yang, Zhaogang; Shen, Jingjing; Cabrera, Eusebio; Lertola, Matthew J; Yang, Willie; Zhang, Dan; Benatar, Avi; Castro, Jose M; Wu, Daming; Lee, L James

2018-08-31

Multifunctional electronics are attracting great interest with the increasing demand and fast development of wearable electronic devices. Here, we describe an epidermal strain sensor based on an all-carbon conductive network made from multi-walled carbon nanotubes (MWCNTs) impregnated with poly(dimethyl siloxane) (PDMS) matrix through a vacuum filtration process. An ultrasonication treatment was performed to complete the penetration of PDMS resin in seconds. The entangled and overlapped MWCNT network largely enhances the electrical conductivity (1430 S m -1 ), uniformity (remaining stable on different layers), reliable sensing range (up to 80% strain), and cyclic stability of the strain sensor. The homogeneous dispersion of MWCNTs within the PDMS matrix leads to a strong interaction between the two phases and greatly improves the mechanical stability (ca. 160% strain at fracture). The flexible, reversible and ultrathin (<100 μm) film can be directly attached on human skin as epidermal strain sensors for high accuracy and real-time human motion detection.

Suppression of the Epidermal Growth Factor-like Domain 7 and Inhibition of Migration and Epithelial-Mesenchymal Transition in Human Pancreatic Cancer PANC-1 Cells.

PubMed

Wang, Yun-Liang; Dong, Feng-Lin; Yang, Jian; Li, Zhi; Zhi, Qiao-Ming; Zhao, Xin; Yang, Yong; Li, De-Chun; Shen, Xiao-Chun; Zhou, Jin

2015-01-01

Epidermal growth factor-like domain multiple 7 (EGFL7), a secreted protein specifically expressed by endothelial cells during embryogenesis, recently was identified as a critical gene in tumor metastasis. Epithelial-mesenchymal transition (EMT) was found to be closely related with tumor progression. Accordingly, it is important to investigate the migration and EMT change after knock-down of EGFL7 gene expression in human pancreatic cancer cells. EGFL7 expression was firstly testified in 4 pancreatic cancer cell lines by real-time polymerase chain reaction (Real-time PCR) and western blot, and the highest expression of EGFL7 was found in PANC-1 cell line. Then, PANC-1 cells transfected with small interference RNA (siRNA) of EGFL7 using plasmid vector were named si-PANC-1, while transfected with negative control plasmid vector were called NC-PANC-1. Transwell assay was used to analyze the migration of PANC-1 cells. Real-time PCR and western blotting were used to detect the expression change of EGFL7 gene, EMT markers like E-Cadherin, N-Cadherin, Vimentin, Fibronectin and transcription factors like snail, slug in PANC-1, NC- PANC-1, and si-PANC-1 cells, respectively. After successful plasmid transfection, EGFL7 gene were dramatically knock-down by RNA interference in si-PANC-1 group. Meanwhile, migration ability decreased significantly, compared with PANC-1 and NC-PANC-1 group. Meanwhile, the expression of epithelial phenotype marker E-Cadherin increased and that of mesenchymal phenotype markers N-Cadherin, Vimentin, Fibronectin dramatically decreased in si-PANC-1 group, indicating a reversion of EMT. Also, transcription factors snail and slug decreased significantly after RNA interference. Current study suggested that highly-expressed EGFL7 promotes migration of PANC-1 cells and acts through transcription factors snail and slug to induce EMT, and further study is needed to confirm this issue.

The Epidermal Growth Factor Receptor Critically Regulates Endometrial Function during Early Pregnancy

PubMed Central

Large, Michael J.; Wetendorf, Margeaux; Lanz, Rainer B.; Hartig, Sean M.; Creighton, Chad J.; Mancini, Michael A.; Kovanci, Ertug; Lee, Kuo-Fen; Threadgill, David W.; Lydon, John P.; Jeong, Jae-Wook; DeMayo, Francesco J.

2014-01-01

Infertility and adverse gynecological outcomes such as preeclampsia and miscarriage represent significant female reproductive health concerns. The spatiotemporal expression of growth factors indicates that they play an important role in pregnancy. The goal of this study is to define the role of the ERBB family of growth factor receptors in endometrial function. Using conditional ablation in mice and siRNA in primary human endometrial stromal cells, we identified the epidermal growth factor receptor (Egfr) to be critical for endometrial function during early pregnancy. While ablation of Her2 or Erbb3 led to only a modest reduction in litter size, mice lacking Egfr expression are severely subfertile. Pregnancy demise occurred shortly after blastocyst implantation due to defects in decidualization including decreased proliferation, cell survival, differentiation and target gene expression. To place Egfr in a genetic regulatory hierarchy, transcriptome analyses was used to compare the gene signatures from mice with conditional ablation of Egfr, wingless-related MMTV integration site 4 (Wnt4) or boneless morphogenic protein 2 (Bmp2); revealing that not only are Bmp2 and Wnt4 key downstream effectors of Egfr, but they also regulate distinct physiological functions. In primary human endometrial stromal cells, marker gene expression, a novel high content image-based approach and phosphokinase array analysis were used to demonstrate that EGFR is a critical regulator of human decidualization. Furthermore, inhibition of EGFR signaling intermediaries WNK1 and AKT1S1, members identified in the kinase array and previously unreported to play a role in the endometrium, also attenuate decidualization. These results demonstrate that EGFR plays an integral role in establishing the cellular context necessary for successful pregnancy via the activation of intricate signaling and transcriptional networks, thereby providing valuable insight into potential therapeutic targets. PMID

Epidermal wound repair is regulated by the planar cell polarity signaling pathway.

PubMed

Caddy, Jacinta; Wilanowski, Tomasz; Darido, Charbel; Dworkin, Sebastian; Ting, Stephen B; Zhao, Quan; Rank, Gerhard; Auden, Alana; Srivastava, Seema; Papenfuss, Tony A; Murdoch, Jennifer N; Humbert, Patrick O; Parekh, Vishwas; Boulos, Nidal; Weber, Thomas; Zuo, Jian; Cunningham, John M; Jane, Stephen M

2010-07-20

The mammalian PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, including neural tube closure and cochlear stereociliary orientation. Here, we show that epidermal wound repair is regulated by PCP signaling. Mice carrying mutant alleles of PCP genes Vangl2, Celsr1, PTK7, and Scrb1, and the transcription factor Grhl3, interact genetically, exhibiting failed wound healing, neural tube defects, and disordered cochlear polarity. Using phylogenetic analysis, ChIP, and gene expression in Grhl3(-)(/-) mice, we identified RhoGEF19, a homolog of a RhoA activator involved in PCP signaling in Xenopus, as a direct target of GRHL3. Knockdown of Grhl3 or RhoGEF19 in keratinocytes induced defects in actin polymerization, cellular polarity, and wound healing, and re-expression of RhoGEF19 rescued these defects in Grhl3-kd cells. These results define a role for Grhl3 in PCP signaling and broadly implicate this pathway in epidermal repair. (c) 2010 Elsevier Inc. All rights reserved.

Epidermal wound repair is regulated by the planar cell polarity signaling pathway

PubMed Central

Caddy, Jacinta; Wilanowski, Tomasz; Darido, Charbel; Dworkin, Sebastian; Ting, Stephen B.; Zhao, Quan; Rank, Gerhard; Auden, Alana; Srivastava, Seema; Papenfuss, Tony A.; Murdoch, Jennifer N.; Humbert, Patrick O.; Boulos, Nidal; Weber, Thomas; Zuo, Jian; Cunningham, John M.; Jane, Stephen M.

2010-01-01

SUMMARY The mammalian PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, including neural tube closure and cochlear stereociliary orientation. Here, we show that epidermal wound repair is regulated by PCP signaling. Mice carrying mutant alleles of PCP genes Vangl2, Celsr1, PTK7, and Scrb1, and the transcription factor Grhl3, interact genetically, exhibiting failed wound healing, neural tube defects and disordered cochlear polarity. Using phylogenetic analysis, ChIP, and gene expression in Grhl3−/− mice, we identified RhoGEF19, a homologue of a RhoA activator involved in PCP signaling in Xenopus, as a direct target of GRHL3. Knockdown of Grhl3 or RhoGEF19 in keratinocytes induced defects in actin polymerisation, cellular polarity and wound healing, and re-expression of RhoGEF19 rescued these defects in Grhl3-kd cells. These results define a role for Grhl3 in PCP signaling, and broadly implicate this pathway in epidermal repair. PMID:20643356

Epidermal growth factor receptor expression in gastric tumors and its relationship with the germline polymorphisms - 216 G>T, -191 C>A, (CA) n IVS1, and R521K.

PubMed

Torres-Jasso, J H; Bustos-Carpinteyro, A R; Garcia-Gonzalez, J R; Peregrina-Sandoval, J; Cruz-Ramos, J A; Santiago-Luna, E; Sanchez-Lopez, J Y

2016-01-01

Gastric cancer (GC) is the third worldwide leading cause of cancer-related death affecting both sexes. The aberrant expression of epidermal growth factor receptor (EGFR) gene has been detected in many human epithelial malignancies and linked to advanced disease, more aggressive phenotype, and poor prognosis. To analyze the relation that the expression of EGFR in gastric tumors holds with pathological characteristics and with the germline polymorphisms -216 G>T, -191 C>A, (CA) n IVS1, and R521K. We studied 22 biopsies from gastric tumors obtained by endoscopy. EGFR expression was determined by relative quantification real-time polymerase chain reaction with the glyceraldehyde-3-phosphate dehydrogenase reference gene (as for messenger RNA [mRNA]) and by immunohistochemistry (IHC) (as for protein). EGFR germline polymorphisms were analyzed by sequencing, GeneScan, and restriction fragment length polymorphisms. EGFR mRNA expression was increased (>2-fold) in 13.6% of GC cases, decreased (<0.5-fold) in 68.2%, and normal in 18.2%; overexpression was related to well-differentiated gastric tumors, whereas underexpression was linked to moderate or poorly differentiated gastric tumors (P < 0.001). EGFR protein expression was high (IHC 2+ and 3+) in 29.4% of gastric tumors and was normal or low (score 0 to 1+) in 70.6% cases. EGFR expression, in both mRNA and protein, was not related to any EGFR polymorphism (P > 0.05). Most gastric tumors showed low EGFR expression (mRNA and protein), whereas EGFR overexpression was related to well-differentiated gastric tumors. Furthermore, germinal polymorphisms -216, -191, (CA) n IVS1, and R521K were not related to EGFR expression (mRNA or protein).

Grhl3 and Lmo4 play coordinate roles in epidermal migration.

PubMed

Hislop, Nikki R; Caddy, Jacinta; Ting, Stephen B; Auden, Alana; Vasudevan, Sumitha; King, Sarah L; Lindeman, Geoffrey J; Visvader, Jane E; Cunningham, John M; Jane, Stephen M

2008-09-01

In addition to its role in formation of the epidermal barrier, the mammalian transcription factor Grainy head-like 3 (Grhl3) is also essential for neural tube closure and wound repair, processes that are dependent in part on epidermal migration. Here, we demonstrate that the LIM-only domain protein, LMO4 serves as a functional partner of GRHL3 in its established roles, and define a new cooperative role for these factors in another developmental epidermal migration event, eyelid fusion. GRHL3 and LMO4 interact biochemically and genetically, with mutant mice exhibiting fully penetrant exencephaly, thoraco-lumbo-sacral spina bifida, defective skin barrier formation, and a co-incident eyes-open-at-birth (EOB) phenotype, which is not observed in the original individual null lines. The two genes are co-expressed in the surface ectoderm of the migrating eyelid root, and electron microscopy of Grhl3/Lmo4-null eyes reveals a failure in epithelial extension and a lack of peridermal clump formation at the eyelid margins. Accumulation of actin fibers is also absent in the circumference of these eyelids, and ERK1/2 phosphorylation is lost in the epidermis and eyelids of Grhl3(-/-)/Lmo4(-/-) embryos. Keratinocytes from mutant mice fail to "heal" in in vitro scratch assays, consistent with a general epidermal migratory defect that is dependent on ERK activation and actin cable formation.

Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

PubMed

Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG 1 , kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

Inhibitory effect of Paeonia lactiflora Pallas extract (PE) on poly (I:C)-induced immune response of epidermal keratinocytes.

PubMed

Choi, Mi-Ra; Choi, Dae-Kyoung; Sohn, Kyung-Cheol; Lim, Seul Ki; Kim, Dong-Il; Lee, Young Ho; Im, Myung; Lee, Young; Seo, Young-Joon; Kim, Chang Deok; Lee, Jeung-Hoon

2015-01-01

Epidermal keratinocytes provide protective role against external stimuli by barrier formation. In addition, kertinocytes exerts their role as the defense cells via activation of innate immunity. Disturbance of keratinocyte functions is related with skin disorders. Psoriasis is a common skin disease related with inflammatory reaction in epidermal cells. We attempted to find therapeutics for psoriasis, and found that Paeonia lactiflora Pallas extract (PE) has an inhibitory potential on poly (I:C)-induced inflammation of keratinocytes. PE significantly inhibited poly (I:C)-induced expression of crucial psoriatic cytokines, such as IL-6, IL-8, CCL20 and TNF-α, via down-regulation of NF-κB signaling pathway in human keratinocytes. In addition, PE significantly inhibited poly (I:C)-induced inflammasome activation, in terms of IL-1β and caspase-1 secretion. Finally, PE markedly inhibited poly (I:C)-increased NLRP3, an important component of inflammasome. These results indicate that PE has an inhibitory effect on poly (I:C)-induced inflammatory reaction of keratinocytes, suggesting that PE can be developed for the treatment of psoriasis.

Ultraviolet-B radiation induces modulation of antigen presentation of herpes simplex virus by human epidermal cells.

PubMed

van der Molen, R G; Out-Luiting, C; Claas, F H; Norval, M; Koerten, H K; Mommaas, A M

2001-06-01

Although ultraviolet (UV) B radiation is known to be immunosuppressive, there is little information regarding a relevant immunological endpoint to assess human subjects in vivo. Therefore, we have examined the effect of in vivo UV radiation on the ability of human epidermal cells (EC) to present herpes simplex virus (HSV) antigens to memory T cells. Human volunteers, who were seropositive for HSV, were exposed to one minimal erythemal dose (MED) for four consecutive days. EC, prepared from suction blister roofs, were co-cultured with autologous T cells in the presence of HSV. HSV antigen presentation by UV-exposed EC was increased compared with control, nonexposed EC. This up-regulation correlated with an influx of macrophages into the epidermis, which are considered to be associated with UV-induced tolerance. Altering the UV protocol to a sub-erythemal UV dose for four consecutive days or to a single high dose of 2 MED, resulted in suppressed HSV antigen presentation, without the influx of the UV-macrophages. One of the goals of the present study was to eventually use this HSV system to investigate sunscreen immunoprotection. A pilot study with a TiO2-containing sunscreen suggested that the endpoint for UV-induced immunosuppression presented here is promising to be used for human in vivo sunscreen immunoprotection studies.

Double-Blind, Placebo-Controlled, Randomized Phase III Trial Evaluating Pertuzumab Combined With Chemotherapy for Low Tumor Human Epidermal Growth Factor Receptor 3 mRNA-Expressing Platinum-Resistant Ovarian Cancer (PENELOPE).

PubMed

Kurzeder, Christian; Bover, Isabel; Marmé, Frederik; Rau, Joern; Pautier, Patricia; Colombo, Nicoletta; Lorusso, Domenica; Ottevanger, Petronella; Bjurberg, Maria; Marth, Christian; Barretina-Ginesta, Pilar; Vergote, Ignace; Floquet, Anne; Del Campo, Josep M; Mahner, Sven; Bastière-Truchot, Lydie; Martin, Nicolas; Oestergaard, Mikkel Z; Kiermaier, Astrid; Schade-Brittinger, Carmen; Polleis, Sandra; du Bois, Andreas; Gonzalez-Martin, Antonio

2016-07-20

The AGO-OVAR 2.29/ENGOT-ov14/PENELOPE prospectively randomized phase III trial evaluated the addition of pertuzumab to chemotherapy in patients with platinum-resistant ovarian carcinoma with low tumor human epidermal growth factor receptor 3 (HER3) mRNA expression. We report the results of the primary efficacy analysis. Eligible patients had ovarian carcinoma that progressed during or within 6 months of completing four or more platinum cycles, centrally tested low tumor HER3 mRNA expression (concentration ratio ≤ 2.81 by quantitative reverse transcriptase polymerase chain reaction on cobas z480 [Roche Molecular Diagnostics, Pleasanton, CA]), and no more than two prior lines of chemotherapy. After investigators' selection of the chemotherapy backbone (single-agent topotecan, weekly paclitaxel, or gemcitabine), patients were randomly assigned to also receive either placebo or pertuzumab (840-mg loading dose followed by 420 mg every 3 weeks). Stratification factors were selected chemotherapy, prior antiangiogenic therapy, and platinum-free interval. The primary end point was independent review committee-assessed progression-free survival (PFS). Additional end points included overall survival, investigator-assessed PFS, objective response rate, safety, patient-reported outcomes, and translational research. Overall, 156 patients were randomly assigned. Adding pertuzumab to chemotherapy did not significantly improve independent review committee-assessed PFS for the primary analysis (stratified hazard ratio, 0.74; 95% CI, 0.50 to 1.11; P = .14; median PFS, 4.3 months for pertuzumab plus chemotherapy v 2.6 months for placebo plus chemotherapy). Sensitivity analyses and secondary efficacy end point results were consistent with the primary analysis. The effect on PFS favoring pertuzumab was more pronounced in the gemcitabine and paclitaxel cohorts. No new safety signals were seen. Although the primary objective was not met, subgroup analyses showed trends in PFS favoring

The tryptophan-derived endogenous arylhydrocarbon receptor ligand 6-formylindolo[3,2-b]carbazole (FICZ) is a nanomolar UVA-photosensitizer in epidermal keratinocytes

PubMed Central

Williams, Joshua D.; Cabello, Christopher M.; Qiao, Shuxi; Wondrak, Georg T.

2014-01-01

Endogenous UVA-chromophores may act as sensitizers of oxidative stress underlying cutaneous photoaging and photocarcinogenesis, but the molecular identity of non-DNA key chromophores displaying UVA-driven photodyamic activity in human skin remains largely undefined. Here we report that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct and endogenous high affinity aryl hydrocarbon receptor (AhR) agonist, acts as a nanomolar photosensitizer potentiating UVA-induced oxidative stress irrespective of AhR ligand activity. In human HaCaT and primary epidermal keratinocytes, photodynamic induction of apoptosis was elicited by the combined action of solar simulated UVA and FICZ, whereas exposure to the isolated action of UVA or FICZ did not impair viability. In a human epidermal tissue reconstruct, FICZ/UVA-cotreatment caused pronounced phototoxicity inducing keratinocyte cell death, and FICZ photodynamic activity was also substantiated in a murine skin exposure model. Array analysis revealed pronounced potentiation of cellular heat shock, ER stress, and oxidative stress response gene expression observed only upon FICZ/UVA-cotreatment. FICZ photosensitization caused intracellular oxidative stress, and comet analysis revealed introduction of formamidopyrimidine-DNA glycosylase (FPG)-sensitive oxidative DNA lesions suppressible by antioxidant cotreatment. Taken together, our data demonstrate that the endogenous AhR ligand FICZ displays nanomolar photodynamic activity representing a molecular mechanism of UVA-induced photooxidative stress potentially operative in human skin. PMID:25431849

Transcriptional effect of an Aframomum angustifolium seed extract on human cutaneous cells using low-density DNA chips.

PubMed

Bonnet-Duquennoy, Mathilde; Dumas, Marc; Debacker, Adeline; Lazou, Kristell; Talbourdet, Sylvie; Franchi, Jocelyne; Heusèle, Catherine; André, Patrice; Schnebert, Sylvianne; Bonté, Frédéric; Kurfürst, Robin

2007-06-01

Studying photoexposed and photoprotected skin biopsies from young and aged women, it has been found that a specific zone, composed of the basal layers of the epidermis, the dermal epidermal junction, and the superficial dermis, is major target of aging and reactive oxygen species. We showed that this zone is characterized by significant variations at a transcriptional and/or protein levels. Using low-density DNA chip technology, we evaluated the effect of a natural mixture of Aframomum angustifolium seed extract containing labdane diterpenoids on these aging markers. Expression profiles of normal human fibroblasts (NHF) were studied using a customized cDNA macroarray system containing genes covering dermal structure, inflammatory responses, and oxidative stress defense mechanisms. For normal human keratinocyte (NHK) investigations, we chose OLISA technique, a sensitive and quantitative method developed by BioMérieux specifically designed to investigate cell death, proliferation, epidermal structure, differentiation, and oxidative stress defense response. We observed that this extract strongly modified gene expression profiles of treated NHK, but weakly for NHF. This extract regulated antioxidant defenses, dermal-epidermal junction components, and epidermal renewal-related genes. Using low-density DNA chip technology, we identified new potential actions of A. angustifolium seed extract on skin aging.

Linking γ-aminobutyric acid A receptor to epidermal growth factor receptor pathways activation in human prostate cancer.

PubMed

Wu, Weijuan; Yang, Qing; Fung, Kar-Ming; Humphreys, Mitchell R; Brame, Lacy S; Cao, Amy; Fang, Yu-Ting; Shih, Pin-Tsen; Kropp, Bradley P; Lin, Hsueh-Kung

2014-03-05

Neuroendocrine (NE) differentiation has been attributed to the progression of castration-resistant prostate cancer (CRPC). Growth factor pathways including the epidermal growth factor receptor (EGFR) signaling have been implicated in the development of NE features and progression to a castration-resistant phenotype. However, upstream molecules that regulate the growth factor pathway remain largely unknown. Using androgen-insensitive bone metastasis PC-3 cells and androgen-sensitive lymph node metastasis LNCaP cells derived from human prostate cancer (PCa) patients, we demonstrated that γ-aminobutyric acid A receptor (GABA(A)R) ligand (GABA) and agonist (isoguvacine) stimulate cell proliferation, enhance EGF family members expression, and activate EGFR and a downstream signaling molecule, Src, in both PC-3 and LNCaP cells. Inclusion of a GABA(A)R antagonist, picrotoxin, or an EGFR tyrosine kinase inhibitor, Gefitinib (ZD1839 or Iressa), blocked isoguvacine and GABA-stimulated cell growth, trans-phospohorylation of EGFR, and tyrosyl phosphorylation of Src in both PCa cell lines. Spatial distributions of GABAAR α₁ and phosphorylated Src (Tyr416) were studied in human prostate tissues by immunohistochemistry. In contrast to extremely low or absence of GABA(A)R α₁-positive immunoreactivity in normal prostate epithelium, elevated GABA(A)R α₁ immunoreactivity was detected in prostate carcinomatous glands. Similarly, immunoreactivity of phospho-Src (Tyr416) was specifically localized and limited to the nucleoli of all invasive prostate carcinoma cells, but negative in normal tissues. Strong GABAAR α₁ immunoreactivity was spatially adjacent to the neoplastic glands where strong phospho-Src (Tyr416)-positive immunoreactivity was demonstrated, but not in adjacent to normal glands. These results suggest that the GABA signaling is linked to the EGFR pathway and may work through autocrine or paracine mechanism to promote CRPC progression. Copyright © 2013 Elsevier

Photoprotective Properties of Isothiocyanate and Nitrile Glucosinolate Derivatives From Meadowfoam (Limnanthes alba) Against UVB Irradiation in Human Skin Equivalent.

PubMed

Carpenter, Evan L; Le, Mai N; Miranda, Cristobal L; Reed, Ralph L; Stevens, Jan F; Indra, Arup K; Ganguli-Indra, Gitali

2018-01-01

Exposure to ultraviolet B (UVB) irradiation of the skin leads to numerous dermatological concerns including skin cancer and accelerated aging. Natural product glucosinolate derivatives, like sulforaphane, have been shown to exhibit chemopreventive and photoprotective properties. In this study, we examined meadowfoam ( Limnanthes alba ) glucosinolate derivatives, 3-methoxybenzyl isothiocyanate (MBITC) and 3-methoxyphenyl acetonitrile (MPACN), for their activity in protecting against the consequences of UV exposure. To that end, we have exposed human primary epidermal keratinocytes (HPEKs) and 3D human skin reconstructed in vitro (EpiDerm TM FT-400) to UVB insult and investigated whether MBITC and MPACN treatment ameliorated the harmful effects of UVB damage. Activity was determined by the compounds' efficacy in counteracting UVB-induced DNA damage, matrix-metalloproteinase (MMP) expression, and proliferation. We found that in monolayer cultures of HPEK, MBITC and MPACN did not protect against a UVB-induced loss in proliferation and MBITC itself inhibited cell proliferation. However, in human reconstructed skin-equivalents, MBITC and MPACN decrease epidermal cyclobutane pyrimidine dimers (CPDs) and significantly reduce total phosphorylated γH2A.X levels. Both MBITC and MPACN inhibit UVB-induced MMP-1 and MMP-3 expression indicating their role to prevent photoaging. Both compounds, and MPACN in particular, showed activity against UVB-induced proliferation as indicated by fewer epidermal PCNA+ cells and prevented UVB-induced hyperplasia as determined by a reduction in reconstructed skin epidermal thickness (ET). These data demonstrate that MBITC and MPACN exhibit promising anti-photocarcinogenic and anti-photoaging properties in the skin microenvironment and could be used for therapeutic interventions.

Keratin K15 as a Biomarker of Epidermal Stem Cells

PubMed Central

Bose, Amrita; Teh, Muy-Teck; Mackenzie, Ian C.; Waseem, Ahmad

2013-01-01

Keratin 15 (K15) is type I keratin protein co-expressed with the K5/K14 pair present in the basal keratinocytes of all stratified epithelia. Although it is a minor component of the cytoskeleton with a variable expression pattern, nonetheless its expression has been reported as a stem cell marker in the bulge of hair follicles. Conversely, suprabasal expression of K15 has also been reported in both normal and diseased tissues, which is inconsistent with its role as a stem cell marker. Our recently published work has given evidence of the molecular pathways that seem to control the expression of K15 in undifferentiated and differentiated cells. In this article, we have critically reviewed the published work to establish the reliability of K15 as an epidermal stem cell marker. PMID:24071939

Ultrathin epidermal strain sensor based on an elastomer nanosheet with an inkjet-printed conductive polymer

NASA Astrophysics Data System (ADS)

Tetsu, Yuma; Yamagishi, Kento; Kato, Akira; Matsumoto, Yuya; Tsukune, Mariko; Kobayashi, Yo; Fujie, Masakatsu G.; Takeoka, Shinji; Fujie, Toshinori

2017-08-01

To minimize the interference that skin-contact strain sensors cause natural skin deformation, physical conformability to the epidermal structure is critical. Here, we developed an ultrathin strain sensor made from poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) inkjet-printed on a polystyrene-polybutadiene-polystyrene (SBS) nanosheet. The sensor, whose total thickness and gauge factor were ˜1 µm and 0.73 ± 0.10, respectively, deeply conformed to the epidermal structure and successfully detected the small skin strain (˜2%) while interfering minimally with the natural deformation of the skin. Such an epidermal strain sensor will open a new avenue for precisely detecting the motion of human skin and artificial soft-robotic skin.

WEREWOLF, a MYB-related protein in Arabidopsis, is a position-dependent regulator of epidermal cell patterning.

PubMed

Lee, M M; Schiefelbein, J

1999-11-24

The formation of the root epidermis of Arabidopsis provides a simple and elegant model for the analysis of cell patterning. A novel gene, WEREWOLF (WER), is described here that is required for position-dependent patterning of the epidermal cell types. The WER gene encodes a MYB-type protein and is preferentially expressed within cells destined to adopt the non-hair fate. Furthermore, WER is shown to regulate the position-dependent expression of the GLABRA2 homeobox gene, to interact with a bHLH protein, and to act in opposition to the CAPRICE MYB. These results suggest a simple model to explain the specification of the two root epidermal cell types, and they provide insight into the molecular mechanisms used to control cell patterning.

SRC-mediated EGF Receptor Activation Regulates Ozone-induced Interleukin 8 Expression in Human Bronchial Epithelial Cells

EPA Science Inventory

BACKGROUND: Human exposure to ozone (03) results in pulmonary function decrements and airway inflammation. The mechanisms underlying these adverse effects remain unclear. Epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of lung inflammation. ...

Selenoprotein W controls epidermal growth factor receptor surface expression, activation and degradation via receptor ubiquitination

USDA-ARS?s Scientific Manuscript database

Epidermal growth factor (EGF) receptor (EGFR) is the founding member of the ErbB family of growth factor receptors that modulate a complex network of intracellular signaling pathways controlling growth, proliferation and differentiation. Selenoprotein W (SEPW1) is a diet-regulated, highly conserved...

Protective Effect of Tropical Highland Blackberry Juice (Rubus adenotrichos Schltdl.) Against UVB-Mediated Damage in Human Epidermal Keratinocytes and in a Reconstituted Skin Equivalent Model

PubMed Central

Calvo-Castro, Laura; Syed, Deeba N.; Chamcheu, Jean C.; Vilela, Fernanda M. P.; Pérez, Ana M.; Vaillant, Fabrice; Rojas, Miguel; Mukhtar, Hasan

2014-01-01

Solar ultraviolet (UV) radiation, particularly its UVB (280–320 nm) spectrum, is the primary environmental stimulus leading to skin carcinogenesis. Several botanical species with antioxidant properties have shown photochemopreventive effects against UVB damage. Costa Rica’s tropical highland blackberry (Rubus adenotrichos) contains important levels of phenolic compounds, mainly ellagitannins and anthocyanins, with strong antioxidant properties. In this study, we examined the photochemopreventive effect of R. adenotrichos blackberry juice (BBJ) on UVB-mediated responses in human epidermal keratinocytes and in a three-dimensional (3D) reconstituted normal human skin equivalent (SE). Pretreatment (2 h) and posttreatment (24 h) of normal human epidermal keratinocytes (NHEKs) with BBJ reduced UVB (25 mJ cm−2)-mediated (1) cyclobutane pyrimidine dimers (CPDs) and (2) 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) formation. Furthermore, treatment of NHEKs with BBJ increased UVB-mediated (1) poly(ADP-ribose) polymerase cleavage and (2) activation of caspases 3, 8 and 9. Thus, BBJ seems to alleviate UVB-induced effects by reducing DNA damage and increasing apoptosis of damaged cells. To establish the in vivo significance of these findings to human skin, immunohistochemistry studies were performed in a 3D SE model, where BBJ was also found to decrease CPDs formation. These data suggest that BBJ may be developed as an agent to ameliorate UV-induced skin damage. PMID:23711186

Protective effect of tropical highland blackberry juice (Rubus adenotrichos Schltdl.) against UVB-mediated damage in human epidermal keratinocytes and in a reconstituted skin equivalent model.

PubMed

Calvo-Castro, Laura; Syed, Deeba N; Chamcheu, Jean C; Vilela, Fernanda M P; Pérez, Ana M; Vaillant, Fabrice; Rojas, Miguel; Mukhtar, Hasan

2013-01-01

Solar ultraviolet (UV) radiation, particularly its UVB (280-320 nm) spectrum, is the primary environmental stimulus leading to skin carcinogenesis. Several botanical species with antioxidant properties have shown photochemopreventive effects against UVB damage. Costa Rica's tropical highland blackberry (Rubus adenotrichos) contains important levels of phenolic compounds, mainly ellagitannins and anthocyanins, with strong antioxidant properties. In this study, we examined the photochemopreventive effect of R. adenotrichos blackberry juice (BBJ) on UVB-mediated responses in human epidermal keratinocytes and in a three-dimensional (3D) reconstituted normal human skin equivalent (SE). Pretreatment (2 h) and posttreatment (24 h) of normal human epidermal keratinocytes (NHEKs) with BBJ reduced UVB (25 mJ cm(-2))-mediated (1) cyclobutane pyrimidine dimers (CPDs) and (2) 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation. Furthermore, treatment of NHEKs with BBJ increased UVB-mediated (1) poly(ADP-ribose) polymerase cleavage and (2) activation of caspases 3, 8 and 9. Thus, BBJ seems to alleviate UVB-induced effects by reducing DNA damage and increasing apoptosis of damaged cells. To establish the in vivo significance of these findings to human skin, immunohistochemistry studies were performed in a 3D SE model, where BBJ was also found to decrease CPDs formation. These data suggest that BBJ may be developed as an agent to ameliorate UV-induced skin damage. © 2013 The American Society of Photobiology.

Suppression of the epidermal growth factor receptor inhibits epithelial-mesenchymal transition in human pancreatic cancer PANC-1 cells.

PubMed

Chang, Zhi-Gang; Wei, Jun-Min; Qin, Chang-Fu; Hao, Kun; Tian, Xiao-Dong; Xie, Kun; Xie, Xue-Hai; Yang, Yin-Mo

2012-05-01

Aberrant expression of epidermal growth factor receptor (EGFR) has been detected in pancreatic cancer; however, the mechanisms of EGFR in inducing pancreatic cancer development have not been adequately elucidated. The objective of this study was to determine the role of EGFR in mediating epithelial-mesenchymal transition (EMT) in pancreatic cancer cells. Pancreatic cancer cell line PANC-1 was transfected with small interfering RNA of EGFR by use of a lentiviral expression vector to establish an EGFR-knockdown cell line (si-PANC-1). PANC-1 cells transfected with lentiviral vector expressing negative control sequence were used as negative control (NC-PANC-1). Scratch assay and transwell study were used to analyze cell migration and invasion. Real-time PCR and Western blotting were used to detect the expression of EMT markers E-cadherin, N-cadherin, vimentin, and fibronectin and transcription factors snail, slug, twist1, and sip1 in PANC-1, NC-PANC-1, and si-PANC-1 cells. Immunofluorescent staining with these antibodies and confocal microscopy were used to observe their cellular location and morphologic changes. After RNA interference of EGFR, the migration and invasion ability of si-PANC-1 cells decreased significantly. The expression of epithelial phenotype marker E-cadherin increased and the expression of mesenchymal phenotype markers N-cadherin, vimentin, and fibronectin decreased, indicating reversion of EMT. We also observed intracellular translocation of E-cadherin. Expression of transcription factors snail and slug in si-PANC-1 cells decreased significantly. Suppression of EGFR expression can significantly inhibit EMT of pancreatic cancer PANC-1 cells. The mechanism may be related with the down-regulation of the expression of transcription factors snail and slug.

Keratin 17 is overexpressed and predicts poor survival in estrogen receptor-negative/human epidermal growth factor receptor-2-negative breast cancer.

PubMed

Merkin, Ross D; Vanner, Elizabeth A; Romeiser, Jamie L; Shroyer, A Laurie W; Escobar-Hoyos, Luisa F; Li, Jinyu; Powers, Robert S; Burke, Stephanie; Shroyer, Kenneth R

2017-04-01

Clinicopathological features of breast cancer have limited accuracy to predict survival. By immunohistochemistry (IHC), keratin 17 (K17) expression has been correlated with triple-negative status (estrogen receptor [ER]/progesterone receptor/human epidermal growth factor receptor-2 [HER2] negative) and decreased survival, but K17 messenger RNA (mRNA) expression has not been evaluated in breast cancer. K17 is a potential prognostic cancer biomarker, targeting p27, and driving cell cycle progression. This study compared K17 protein and mRNA expression to ER/progesterone receptor/HER2 receptor status and event-free survival. K17 IHC was performed on 164 invasive breast cancers and K17 mRNA was evaluated in 1097 breast cancers. The mRNA status of other keratins (16/14/9) was evaluated in 113 ER - /HER2 - ductal carcinomas. IHC demonstrated intense cytoplasmic and membranous K17 localization in myoepithelial cells of benign ducts and lobules and tumor cells of ductal carcinoma in situ. In ductal carcinomas, K17 protein was detected in most triple-negative tumors (28/34, 82%), some non-triple-negative tumors (52/112, 46%), but never in lobular carcinomas (0/15). In ductal carcinomas, high K17 mRNA was associated with reduced 5-year event-free survival in advanced tumor stage (n = 149, hazard ratio [HR] = 3.68, P = .018), and large (n = 73, HR = 3.95, P = .047), triple-negative (n = 103, HR = 2.73, P = .073), and ER - /HER2 - (n = 113, HR = 2.99, P = .049) tumors. There were significant correlations among keratins 17, 16, 14, and 9 mRNA levels suggesting these keratins (all encoded on chromosome 17) could be coordinately expressed in breast cancer. Thus, K17 is expressed in a subset of triple-negative breast cancers, and is a marker of poor prognosis in patients with advanced stage and ER - /HER2 - breast cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Immunohistochemical expression of AQP-3 in vitiligo: a new potential guide for disease activity.

PubMed

Hodeib, Abeer; Hegab, Doaa; Rizk, Omnia; Mohammed, Shahdan

2017-08-01

Vitiligo is a depigmenting skin disorder, with disappearance of functioning epidermal melanocytes. Aquaporin-3 (AQP-3) is an aquaglyceroporin expressed in epidermal keratinocytes, where it shares in regulating their proliferation and differentiation, and so it might affect melanocytes indirectly. So far, little is known regarding its possible role in vitiligo. This work aimed to study the changes in immunohistochemical expression of AQP-3 protein in vitiligo to detect its possible role in disease pathogenesis. Skin biopsies were taken from lesional skin of 30 vitiligo patients in addition to 20 normal controls. Epidermal immunohistochemical expression of AQP-3 was assessed as: +3 = strong expression, +2 = moderate, +1= weak and 0= negative expression. AQP-3 was significantly less expressed in vitiligo epidermis than control (P<0.001) with an inverse correlation with Vitiligo Index of Disease Activity (r =-0.505, P=0.004). Reduced epidermal AQP-3 may have a role in impaired melanocyte survival in vitiligo, and might be a potential negative biological marker for vitiligo activity. Larger trials should further elucidate the effect of changes in epidermal AQP-3 expression in development of vitiligo, and that might pave the road for discovering new therapeutic modalities for the disease.

Sulfur mustard induces the formation of keratin aggregates in human epidermal keratinocytes.

PubMed

Dillman, James F; McGary, Kriston L; Schlager, John J

2003-12-01

The vesicant sulfur mustard is an alkylating agent that has the capacity to cross-link biological molecules. We are interested in identifying specific proteins that are altered upon sulfur mustard exposure. Keratins are particularly important for the structural integrity of skin, and several genetically inherited blistering diseases have been linked to mutations in keratin 5 and keratin 14. We examined whether sulfur mustard exposure alters keratin biochemistry in cultured human epidermal keratinocytes. Western blotting with specific monoclonal antibodies revealed the formation of stable high-molecular-weight "aggregates" containing keratin 14 and/or keratin 5. These aggregates begin to form within 15 min after sulfur mustard exposure. These aggregates display a complex gel electrophoresis pattern between approximately 100 and approximately 200 kDa. Purification and analysis of these aggregates by one- and two-dimensional gel electrophoresis and mass spectrometry confirmed the presence of keratin 14 and keratin 5 and indicate that at least some of the aggregates are composed of keratin 14-keratin 14, keratin 14-keratin 5, or keratin 5-keratin 5 dimers. These studies demonstrate that sulfur mustard induces keratin aggregation in keratinocytes and support further investigation into the role of keratin aggregation in sulfur mustard-induced vesication.

Interaction of recombinant human epidermal growth factor with phospholipid vesicles. A steady-state and time-resolved fluorescence study of the bis-tryptophan sequence (Trp49-Trp50).

PubMed

Li De La Sierra, I M; Vincent, M; Padron, G; Gallay, J

1992-01-01

The interaction of recombinant human epidermal growth factor with small unilamellar phospholipid vesicles was studied by steady-state and time-resolved fluorescence of the bis-tryptophan sequence (Trp49-Trp50). Steady-state anisotropy measurements demonstrate that strong binding occurred with small unilamellar vesicles made up of acidic phospholipids at acidic pH only (pH < or = 4.7). An apparent stoichiometry for 1,2-dimyristoyl-sn-phosphoglycerol of about 12 phospholipid molecules per molecule of human epidermal growth factor was estimated. The binding appears to be more efficient at temperatures above the gel to liquid-crystalline phase transition. The conformation and the environment of the Trp-Trp sequence are not greatly modified after binding, as judged from the invariance of the excited state lifetime distribution and from that of the fast processes affecting the anisotropy decay. This suggests that the Trp-Trp sequence is not embedded within the bilayer, in contrast to the situation in surfactant micelles (Mayo et al. 1987; Kohda and Inigaki 1992).

Epidermal growth factor (EGF) receptor-ligand based molecular staging predicts prognosis in head and neck squamous cell carcinoma partly due to deregulated EGF- induced amphiregulin expression.

PubMed

Gao, Jian; Ulekleiv, Camilla H; Halstensen, Trond S

2016-09-26

Increased expression of epidermal growth factor receptor (EGFR) and its ligands is associated with poor prognosis and chemoresistance in many carcinoma types, but its role in head and neck squamous cell carcinoma (HNSCC) is unclear. Our aim was to clarify whether mRNA expression of EGFR-ligands was linked to prognosis and cisplatin resistance, and if so, which ligand was most important and how was the expression regulated. To examine the prognostic effect of EGFR-ligand expression, we analyzed tumorous mRNA expression in 399 HNSCC patients. The intracellular signaling pathways controlling epidermal growth factor (EGF)-induced amphiregulin (AREG) expression were examined in three oral squamous cell carcinoma (OSCC) cell lines. Effect of AREG on cisplatin resistance was examined by viability assays in four-, and by association in 11 OSCC cell lines. The patients were divided into five groups according to the median mRNA expression levels of four EGFR ligands, i.e. AREG, EGF, heparin-binding EGF-like growth factor (HBEGF) and beta-cellulin (BTC). The number of increased-expressed EGFR-ligands were progressively correlated to five-year survival, even in advanced TNM-stage IV patients, where five-year mortality increased from 26 % if tumor expressed none to one EGFR-ligand, to 45 % in three to four ligand expressing tumors. Thus, staging the tumor according to these EGFR-ligand mRNA expression pattern completely out performed TNM staging in predicting prognosis. Multivariate analysis identified AREG as the dominating predictor, and AREG was overexpressed in OSCC compared to tumors from other sites. Both EGF and HBEGF stimulation induced strong AREG increase in OSCC cell lines, which was partially mediated by the extracellular signal-regulated kinase 1/2 pathway, and negatively regulated by p38, c-Jun N-terminal kinase, and phosphoinositide-3 kinase. Although increased AREG mRNA expression predicted unfavorable prognosis in platinum treated HNSCC patients, AREG did

Altered epidermal lipid processing and calcium distribution in the KID syndrome mouse model Cx26S17F.

PubMed

Bosen, Felicitas; Celli, Anna; Crumrine, Debra; vom Dorp, Katharina; Ebel, Philipp; Jastrow, Holger; Dörmann, Peter; Winterhager, Elke; Mauro, Theodora; Willecke, Klaus

2015-07-08

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. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Expression of the ERBB Family of Ligands and Receptors in Gastric Cancer.

PubMed

Byeon, Sun-Ju; Lee, Hye Seung; Kim, Min-A; Lee, Byung Lan; Kim, Woo Ho

2017-01-01

Gastric cancer (GC) is the second most common cancer and the third leading cause of cancer-related death in Korea. Alterations in the ERBB (homology to the erythroblastoma viral gene product, v-erbB) receptor family and ERBB-related signaling pathways are frequently observed in GC. However, the roles of the ERBB receptors and their ligands in GC are not well established. We evaluated the expression levels of various ERBB receptor ligands (i.e., heparin-binding epidermal growth factor-like growth factor [HBEGF], transforming growth factor-α [TGFA], amphiregulin [AREG], epiregulin [EREG], epidermal growth factor [EGF], and betacellulin [BTC]) and 3 ERBB family receptors (i.e., epidermal growth factor receptor [EGFR], human EGFR2 [HER2], and ERBB3) in 313 cases of GC using immunohistochemistry, fluorescence in situ hybridization, and mRNA in situ hybridization. A high expression of EGFR, HER2, and ERBB3 was observed in 30, 32, and 27 cases, respectively. A high expression of HBEGF, TGFA, AREG, EREG, EGF, and BTC was observed in 91, 97, 151, 74, 26, and 37 cases, respectively. A high expression of TGFA was associated with better survival, while a high expression of BTC was associated with worse survival. These results were confirmed using Cox proportional hazards analysis. HBEGF, TGFA, AREG, tumor-node-metastasis classification, Lauren's classification, and ERBB3 were significant survival parameters in multivariate analysis. Among the ERBB family receptors and ligands examined, 3 ligands (i.e., TGFA, HBEGF, and AREG) and ERBB3 had a prognostic impact. © 2017 S. Karger AG, Basel.

Batch and fed-batch cultivation for excretive production of human epidermal growth factor (hEGF) with recombinant E. Coli K12 system.

PubMed

Wang, J; Chen, J; Xu, R; Xu, Z

2008-01-01

Batch and fed-batch production of recombinant human epidermal growth factor (hEGF) was studied in an E. coli secretary expression system. By using MMBL medium containing 5 g/L glucose, controlling the temperature at 32 degrees C and maintaining the dissolved oxgen level over 20% saturation, a high yield of hEGF (32 mg/L) was obtained after an 18 hr batch cultivation with 0.2 mM IPTG induction at mid-log phase. Three different glucose feeding strategies were employed to further improve hEGF productivity in a bench top fermentor. Compared with the batch results, hEGF yield was improved up to 25.5% or 28.1%, respectively by intermittent or pH-stat glucose feeding, and up to 150% improvement of hEGF production was achieved by constant feeding of 200 g/L glucose solution at a rate of 0.11 mL/min. The effects of further combined feeding with other medium components and inducer on hEGF yield were also examined in the benchtop fermentor. This work is very helpful to further improve the productivity of extracellular hEGF in the recombinant E. coli system.

Defining the cellular precursors to human breast cancer

PubMed Central

Keller, Patricia J.; Arendt, Lisa M.; Skibinski, Adam; Logvinenko, Tanya; Klebba, Ina; Dong, Shumin; Smith, Avi E.; Prat, Aleix; Perou, Charles M.; Gilmore, Hannah; Schnitt, Stuart; Naber, Stephen P.; Garlick, Jonathan A.; Kuperwasser, Charlotte

2012-01-01

Human breast cancers are broadly classified based on their gene-expression profiles into luminal- and basal-type tumors. These two major tumor subtypes express markers corresponding to the major differentiation states of epithelial cells in the breast: luminal (EpCAM+) and basal/myoepithelial (CD10+). However, there are also rare types of breast cancers, such as metaplastic carcinomas, where tumor cells exhibit features of alternate cell types that no longer resemble breast epithelium. Until now, it has been difficult to identify the cell type(s) in the human breast that gives rise to these various forms of breast cancer. Here we report that transformation of EpCAM+ epithelial cells results in the formation of common forms of human breast cancer, including estrogen receptor-positive and estrogen receptor-negative tumors with luminal and basal-like characteristics, respectively, whereas transformation of CD10+ cells results in the development of rare metaplastic tumors reminiscent of the claudin-low subtype. We also demonstrate the existence of CD10+ breast cells with metaplastic traits that can give rise to skin and epidermal tissues. Furthermore, we show that the development of metaplastic breast cancer is attributable, in part, to the transformation of these metaplastic breast epithelial cells. These findings identify normal cellular precursors to human breast cancers and reveal the existence of a population of cells with epidermal progenitor activity within adult human breast tissues. PMID:21940501

Overexpression of cyclin D1 induces the reprogramming of differentiated epidermal cells into stem cell-like cells.

PubMed

Zhao, Along; Yang, Leilei; Ma, Kui; Sun, Mengli; Li, Lei; Huang, Jin; Li, Yang; Zhang, Cuiping; Li, Haihong; Fu, Xiaobing

2016-01-01

It has been reported that Wnt/β-catenin is critical for dedifferentiation of differentiated epidermal cells. Cyclin D1 (CCND1) is a β-catenin target gene. In this study, we provide evidence that overexpression of CCND1 induces reprogramming of epidermal cells into stem cell-like cells. After introducing CCND1 gene into differentiated epidermal cells, we found that the large flat-shaped cells with a small nuclear-cytoplasmic ratio changed into small round-shaped cells with a large nuclear-cytoplasmic ratio. The expressions of CK10, β1-integrin, Oct4 and Nanog in CCND1 induced cells were remarkably higher than those in the control group (P < 0.01). In addition, the induced cells exhibited a high colony-forming ability and a long-term proliferative potential. When the induced cells were implanted into a wound of laboratory animal model, the wound healing was accelerated. These results suggested that overexpression of CCND1 induced the reprogramming of differentiated epidermal cells into stem cell-like cells. This study may also offer a new approach to yield epidermal stem cells for wound repair and regeneration.

Structural Model for the Interaction of a Designed Ankyrin Repeat Protein with the Human Epidermal Growth Factor Receptor 2

PubMed Central

Epa, V. Chandana; Dolezal, Olan; Doughty, Larissa; Xiao, Xiaowen; Jost, Christian; Plückthun, Andreas; Adams, Timothy E.

2013-01-01

Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2). HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84–1.14 Ångstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions. PMID:23527120

Evolution of petal epidermal micromorphology in Leguminosae and its use as a marker of petal identity

PubMed Central

Ojeda, Isidro; Francisco-Ortega, Javier; Cronk, Quentin C. B.

2009-01-01

Background and Aims The legume flower is highly variable in symmetry and differentiation of petal types. Most papilionoid flowers are zygomorphic with three types of petals: one dorsal, two lateral and two ventral petals. Mimosoids have radial flowers with reduced petals while caesalpinioids display a range from strongly zygomorphic to nearly radial symmetry. The aims are to characterize the petal micromorphology relative to flower morphology and evolution within the family and assess its use as a marker of petal identity (whether dorsal, lateral or ventral) as determined by the expression of developmental genes. Methods Petals were analysed using the scanning electron microscope and light microscope. A total of 175 species were studied representing 26 tribes and 89 genera in all three subfamilies of the Leguminosae. Key Results The papilionoids have the highest degree of variation of epidermal types along the dorsiventral axis within the flower. In Loteae and genistoids, in particular, it is common for each petal type to have a different major epidermal micromorphology. Papillose conical cells are mainly found on dorsal and lateral petals. Tabular rugose cells are mainly found on lateral petals and tabular flat cells are found only in ventral petals. Caesalpinioids lack strong micromorphological variation along this axis and usually have only a single major epidermal type within a flower, although the type maybe either tabular rugose cells, papillose conical cells or papillose knobby rugose cells, depending on the species. Conclusions Strong micromorphological variation between different petals in the flower is exclusive to the subfamily Papilionoideae. Both major and minor epidermal types can be used as micromorphological markers of petal identity, at least in papilionoids, and they are important characters of flower evolution in the whole family. The molecular developmental pathway between specific epidermal micromorphology and the expression of petal identity

Evolution of petal epidermal micromorphology in Leguminosae and its use as a marker of petal identity.

PubMed

Ojeda, Isidro; Francisco-Ortega, Javier; Cronk, Quentin C B

2009-11-01

The legume flower is highly variable in symmetry and differentiation of petal types. Most papilionoid flowers are zygomorphic with three types of petals: one dorsal, two lateral and two ventral petals. Mimosoids have radial flowers with reduced petals while caesalpinioids display a range from strongly zygomorphic to nearly radial symmetry. The aims are to characterize the petal micromorphology relative to flower morphology and evolution within the family and assess its use as a marker of petal identity (whether dorsal, lateral or ventral) as determined by the expression of developmental genes. Petals were analysed using the scanning electron microscope and light microscope. A total of 175 species were studied representing 26 tribes and 89 genera in all three subfamilies of the Leguminosae. The papilionoids have the highest degree of variation of epidermal types along the dorsiventral axis within the flower. In Loteae and genistoids, in particular, it is common for each petal type to have a different major epidermal micromorphology. Papillose conical cells are mainly found on dorsal and lateral petals. Tabular rugose cells are mainly found on lateral petals and tabular flat cells are found only in ventral petals. Caesalpinioids lack strong micromorphological variation along this axis and usually have only a single major epidermal type within a flower, although the type maybe either tabular rugose cells, papillose conical cells or papillose knobby rugose cells, depending on the species. Strong micromorphological variation between different petals in the flower is exclusive to the subfamily Papilionoideae. Both major and minor epidermal types can be used as micromorphological markers of petal identity, at least in papilionoids, and they are important characters of flower evolution in the whole family. The molecular developmental pathway between specific epidermal micromorphology and the expression of petal identity genes has yet to be established.

Expression pattern of G protein‑coupled estrogen receptor 1 (GPER) in human cumulus granulosa cells (CGCs) of patients with PCOS.

PubMed

Zang, Lili; Zhang, Quan; Zhou, Yi; Zhao, Yan; Lu, Linlin; Jiang, Zhou; Peng, Zhen; Zou, Shuhua

2016-06-01

Estradiol mediates its actions by binding to classical nuclear receptors, estrogen receptor α (ER-α) and estrogen receptor β (ER-β), and the non-classical G protein-coupled estrogen receptor 1(GPER). Several gene knockdown models have shown the importance of the receptors for growth of the oocyte and for ovulation. The aim of our study was to identify the pattern of GPER expression in human cumulus granulosa cells (CGCs) from ovarian follicles at different stages of oocyte maturation, and the differences of GPER expression between polycystic ovary syndrome (PCOS) patients and non-PCOS women. Thirty-eight cases of PCOS patients and a control group of thirty-two infertile women without PCOS were used in this study. GPER's location in CGCs was investigated by immunohistochemistry. Quantitative RT-PCR and western blot were used to identify the quantify GPER expression. Here we demonstrated that GPER was expressed in CGCs of both PCOS patients and non-PCOS women, and the expression of GPER was decreased significantly during oocyte maturation. But the expression levels of GPER in CGCs of PCOS patients and non-PCOS women were not significantly different. The data indicate that GPER may play a role during human oocyte maturation through its action in cumulus granulosa cells. AMHRIIs: anti-Mullerian hormone type II receptors; BMI: body mass index; CGCs: cumulus granulosa cells; COH: controlled ovarian hyperstimulation; E2: estradiol; EGFR: epidermal growth factor receptor; ER-α: estrogen receptor; ER-β: estrogen receptor β; FF: follicular fluid; FSH: follicle-stimulating hormone; GCs: granulosa cells; GPER: G protein-coupled estrogen receptor 1; GV: germinal vesicle; GVBD: germinal vesicle breakdown; HCG: human chorionic gonadotropin; IRS: immunoreactive score; IVF-ET: in vitro fertilization and embryo transfer; MI: metaphase I; MII: metaphase II; MAPK: mitogen-activated protein kinase; OCCCs: oocyte corona cumulus complexes; PCOS: polycystic ovarian syndrome; q

Reduced expression of the epidermal growth factor signaling system in preeclampsia.

PubMed

Armant, D R; Fritz, R; Kilburn, B A; Kim, Y M; Nien, J K; Maihle, N J; Romero, R; Leach, R E

2015-03-01

The epidermal growth factor (EGF) signaling system regulates trophoblast differentiation, and its disruption could contribute to perinatal disease. We hypothesized that this pathway is altered in preeclampsia, a disorder associated with trophoblast apoptosis and failure to invade and remodel the uterine spiral arteries. Six EGF family peptides and a truncated EGF receptor splice variant (p110/EGFR) were examined using immunohistochemistry in the trophoblast of placentas (N = 76) from women with preeclampsia, and compared to placentas from women of similar gestational age (GA) with preterm labor (PTL) or small for gestational age (SGA) fetuses, as well as normal term placentas. EGF, transforming growth factor-α (TGFA), and heparin-binding EGF-like growth factor (HBEGF) were evaluated using ELISA in maternal plasma from another 20 pregnancies with or without preeclampsia. Cell death was evaluated in the HTR-8/SVneo human cytotrophoblast cell line using TUNEL to evaluate the protective effects of EGF peptides. Trophoblast HBEGF, TGFA, and EGF were significantly reduced in preeclampsia compared to PTL and SGA, while p110/EGFR accumulated significantly on the surface of the chorionic villi (p < 0.05). Plasma EGF levels were significantly decreased in preeclamptic patients, compared to non-preeclamptic patients (p < 0.05). HBEGF, EGF, TGFA, epiregulin, and betacellulin each blocked cytotrophoblast cell death in vitro (p < 0.05). Three members of the EGF family are dysregulated in placentas with preeclampsia, whereas p110/EGFR, a potential EGF receptor antagonist, is overexpressed. These findings are consistent with the concept that disruption of the EGF signaling system contributes to aberrant trophoblast development associated with preeclampsia. Copyright © 2014 Elsevier Ltd. All rights reserved.

Reduced Expression of the Epidermal Growth Factor Signaling System in Preeclampsia

PubMed Central

Armant, D. Randall; FRITZ, Rani; KILBURN, Brian A.; KIM, Yeon Mee; NIEN, Jyh Kae; MAIHLE, Nita J.; ROMERO, Roberto; LEACH, Richard E.

2014-01-01

Introduction The epidermal growth factor (EGF) signaling system regulates trophoblast differentiation, and its disruption could contribute to perinatal disease. We hypothesized that this pathway is altered in preeclampsia, a disorder associated with trophoblast apoptosis and failure to invade and remodel the uterine spiral arteries. Methods Six EGF family peptides and a truncated EGF receptor splice variant (p110/EGFR) were examined using immunocytochemistry in the trophoblast of placentas (N=76) from women with preeclampsia, and compared to placentas from women of similar gestational age (GA) with preterm labor (PTL) or small for gestational age (SGA) fetuses, as well as normal term placentas. EGF, transforming growth factor-α (TGFA), and heparin-binding EGF-like growth factor (HBEGF) were evaluated using ELISA in maternal plasma from another 20 pregnancies with or without preeclampsia. Cell death was evaluated in the HTR-8/SVneo human cytotrophoblast cell line using TUNEL to evaluate the protective effects of EGF peptides. Results Trophoblast HBEGF, TGFA, and EGF were significantly reduced in preeclampsia compared to PTL and SGA, while p110/EGFR accumulated significantly on the surface of the chorionic villi (p<0.05). Plasma EGF levels were significantly decreased in preeclamptic patients, compared to non-preeclamptic patients (p<0.05). HBEGF, EGF, TGFA, epiregulin, and betacellulin each blocked cytotrophoblast cell death in vitro (p< 0.05). Discussion Three members of the EGF family are dysregulated in placentas with preeclampsia, whereas p110/EGFR, a potential EGF receptor antagonist, is overexpressed. These findings are consistent with the concept that disruption of the EGF signaling system contributes to aberrant trophoblast development associated with preeclampsia. PMID:25589361

Epidermal protection with cryogen spray cooling during high fluence pulsed dye laser irradiation: an ex vivo study.

PubMed

Tunnell, J W; Nelson, J S; Torres, J H; Anvari, B

2000-01-01

Higher laser fluences than currently used in therapy (5-10 J/cm(2)) are expected to result in more effective treatment of port wine stain (PWS) birthmarks. However, higher incident fluences increase the risk of epidermal damage caused by absorption of light by melanin. Cryogen spray cooling offers an effective method to reduce epidermal injury during laser irradiation. The objective of this study was to determine whether high laser incident fluences (15-30 J/cm(2)) could be used while still protecting the epidermis in ex vivo human skin samples. Non-PWS skin from a human cadaver was irradiated with a Candela ScleroPlus Laser (lambda = 585 nm; pulse duration = 1.5 msec) by using various incident fluences (8-30 J/cm(2)) without and with cryogen spray cooling (refrigerant R-134a; spurt durations: 40-250 msec). Assessment of epidermal damage was based on histologic analysis. Relatively short spurt durations (40-100 msec) protected the epidermis for laser incident fluences comparable to current therapeutic levels (8-10 J/cm(2)). However, longer spurt durations (100-250 msec) increased the fluence threshold for epidermal damage by a factor of three (up to 30 J/cm(2)) in these ex vivo samples. Results of this ex vivo study show that epidermal protection from high laser incident fluences can be achieved by increasing the cryogen spurt duration immediately before pulsed laser exposure. Copyright 2000 Wiley-Liss, Inc.

Rat epidermal keratinocyte organotypic culture (ROC) compared to human cadaver skin: the effect of skin permeation enhancers.

PubMed

Pappinen, Sari; Tikkinen, Sanna; Pasonen-Seppänen, Sanna; Murtomäki, Lasse; Suhonen, Marjukka; Urtti, Arto

2007-03-01

The objective of this study was to evaluate the response of the rat epidermal keratinocyte organotypic culture (ROC) to permeation enhancers, and to compare these responses to those in human cadaver skin. Different concentrations of two mixtures for enhancing permeation were investigated, sodium dodecyl sulfate:phenyl piperazine and methyl pyrrolidone:dodecyl pyridinium chloride, using skin impedance spectroscopy and two experimental compounds, the lipophilic corticosterone and the hydrophilic sucrose. The chemical irritation effects of the formulations were evaluated based on leakage of lactate dehydrogenase enzyme (LDH) and cellular morphological perturbation. This study provides evidence for direct correlations of permeation/permeation, impedance/impedance and permation/impedance between the culture model and human skin. The only exception was the enhancer induced permeation of sucrose which was 1-40-fold higher in ROC compared to human skin, reflecting the more disordered lipid organization in stratum corneum and consequently the greater number of polar pathways. LDH leakage and cellular morphology indicated that it was possible to differentiate between safe permeation enhancers from irritating agents. This is not only the first study to have compared the enhancer effects on a cultured skin model with human skin, but also it has demonstrated enhancer induced irritation using an artificial skin model.

Caenorhabditis elegans anillin (ani-1) regulates neuroblast cytokinesis and epidermal morphogenesis during embryonic development.

PubMed

Fotopoulos, N; Wernike, D; Chen, Y; Makil, N; Marte, A; Piekny, A

2013-11-01

The formation of tissues is essential for metazoan development. During Caenorhabditis elegans embryogenesis, ventral epidermal cells migrate to encase the ventral surface of the embryo in a layer of epidermis by a process known as ventral enclosure. This process is regulated by guidance cues secreted by the underlying neuroblasts. However, since the cues and their receptors are differentially expressed in multiple cell types, the role of the neuroblasts in ventral enclosure is not fully understood. Furthermore, although F-actin is required for epidermal cell migration, it is not known if nonmuscle myosin is also required. Anillin (ANI-1) is an actin and myosin-binding protein that coordinates actin-myosin contractility in the early embryo. Here, we show that ANI-1 localizes to the cleavage furrows of dividing neuroblasts during mid-embryogenesis and is required for their division. Embryos depleted of ani-1 display a range of ventral enclosure phenotypes, where ventral epidermal cells migrate with similar speeds to control embryos, but contralateral neighbors often fail to meet and are misaligned. The ventral enclosure phenotypes in ani-1 RNAi embryos suggest that the position or shape of neuroblasts is important for directing ventral epidermal cell migration, although does not rule out an autonomous requirement for ani-1 in the epidermal cells. Furthermore, we show that rho-1 and other regulators of nonmuscle myosin activity are required for ventral epidermal cell migration. Interestingly, altering nonmuscle myosin contractility alleviates or strengthens ani-1's ventral enclosure phenotypes. Our findings suggest that ventral enclosure is a complex process that likely relies on inputs from multiple tissues. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

mRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma.

PubMed

Bache, Matthias; Rot, Swetlana; Keßler, Jacqueline; Güttler, Antje; Wichmann, Henri; Greither, Thomas; Wach, Sven; Taubert, Helge; Söling, Ariane; Bilkenroth, Udo; Kappler, Matthias; Vordermark, Dirk

2015-06-01

The roles of hypoxia-induced and stem cell-associated genes in the development of malignancy and tumour progression are well known. However, there are a limited number of studies analysing the impact of mRNA expression levels of hypoxia-induced and stem cell-associated genes in the tissues of brain tumours and glioblastoma patients. In this study, tumour tissues from patients with glioblastoma multiforme and tumour adjacent tissues were analysed. We investigated mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1) and osteopontin (OPN), and stem cell-associated genes survivin, epidermal growth factor receptor (EGFR), human telomerase reverse transcriptase (hTERT), Nanog and octamer binding transcription factor 4 (OCT4) using quantitative real-time polymerase chain reaction (qRT-PCR). Our data revealed higher mRNA expression levels of hypoxia-induced and stem cell-associated genes in tumour tissue than levels in the tumour adjacent tissues in patients with glioblastoma multiforme. A strong positive correlation between the mRNA expression levels of HIF-2α, CA9, VEGF, GLUT-1 and OPN suggests a specific hypoxia-associated profile of mRNA expression in glioblastoma multiforme. Additionally, the results indicate the role of stem-cell-related genes in tumour hypoxia. Kaplan-Maier analysis revealed that high mRNA expression levels of hypoxia-induced markers showed a trend towards shorter overall survival in glioblastoma patients (P=0.061). Our data suggest that mRNA expression levels of hypoxia-induced genes are important tumour markers in patients with glioblastoma multiforme.

Use of cultured human epidermal keratinocytes for allografting burns and conditions for temporary banking of the cultured allografts.

PubMed

Bolívar-Flores, J; Poumian, E; Marsch-Moreno, M; Montes de Oca, G; Kuri-Harcuch, W

1990-02-01

Five children who suffered burns clinically regarded as full skin thickness loss were grafted with cultured allogeneic skin from newborn prepuce. The wounds had remained open and infected without healing for about 20 days before the patients were received in the burn unit. To avoid losing surviving deep epidermal cells the wounds were débrided but not deeply excised and, a few days before allografting, they were washed with isodine solution and sterile water, and treated with silvadene cream application. All children received 76 cultured allografts of about 60 cm2 each. After allografting, the wounds were epithelized in 7-10 days and the allogeneic grafted skin began desquamation suggesting that the allograft did not 'take' permanently but was replaced by the newly formed skin. On the other hand, since allografting is an adequate therapy to provide early temporary coverage in extensively burned patients, we developed conditions for banking cultured skin to make it available for immediate use. The conditions described allow banking of the cultured grafts for 15-20 days with retention of clonal growth ability similar to that of unstored epithelia. The results show that cultured epidermal cells obtained from human newborn foreskin, when used as allografts for coverage of full skin or deep partial skin thickness burns, allow rapid epithelization of the burn wounds.

Functions of Vγ4 T Cells and Dendritic Epidermal T Cells on Skin Wound Healing

PubMed Central

Li, Yashu; Wu, Jun; Luo, Gaoxing; He, Weifeng

2018-01-01

Wound healing is a complex and dynamic process that progresses through the distinct phases of hemostasis, inflammation, proliferation, and remodeling. Both inflammation and re-epithelialization, in which skin γδ T cells are heavily involved, are required for efficient skin wound healing. Dendritic epidermal T cells (DETCs), which reside in murine epidermis, are activated to secrete epidermal cell growth factors, such as IGF-1 and KGF-1/2, to promote re-epithelialization after skin injury. Epidermal IL-15 is not only required for DETC homeostasis in the intact epidermis but it also facilitates the activation and IGF-1 production of DETC after skin injury. Further, the epidermal expression of IL-15 and IGF-1 constitutes a feedback regulatory loop to promote wound repair. Dermis-resident Vγ4 T cells infiltrate into the epidermis at the wound edges through the CCR6-CCL20 pathway after skin injury and provide a major source of IL-17A, which enhances the production of IL-1β and IL-23 in the epidermis to form a positive feedback loop for the initiation and amplification of local inflammation at the early stages of wound healing. IL-1β and IL-23 suppress the production of IGF-1 by DETCs and, therefore, impede wound healing. A functional loop may exist among Vγ4 T cells, epidermal cells, and DETCs to regulate wound repair.

Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bae, Ok-Nam; Ahn, Seyeon; Jin, Sun Hee

2015-03-01

Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture.more » VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD. - Highlights: • Pro-inflammatory cytokines induced VEGF production in normal

Inhibitory effect of indigo naturalis on tumor necrosis factor-α-induced vascular cell adhesion molecule-1 expression in human umbilical vein endothelial cells.

PubMed

Chang, Hsin-Ning; Pang, Jong-Hwei Su; Yang, Sien-Hung; Hung, Chi-Feng; Chiang, Chi-Hsin; Lin, Tung-Yi; Lin, Yin-Ku

2010-09-14

The use of indigo naturalis to treat psoriasis has proved effective in our previous clinical studies. The present study was designed to examine the anti-inflammatory effect of indigo naturalis in primary cultured human umbilical vein endothelial cells (HUVECs). Pretreatment of cells with indigo naturalis extract attenuated TNF-α-induced increase in Jurkat T cell adhesion to HUVECs as well as decreased the protein and messenger (m)RNA expression levels of vascular cell adhesion molecule-1 (VCAM-1) on HUVECs. Indigo naturalis extract also inhibited the protein expression of activator protein-1 (AP-1)/c-Jun, a critical transcription factor for the activation of VCAM-1 gene expression. Since the reduction of lymphocyte adhesion to vascular cells by indigo naturalis extract could subsequently reduce the inflammatory reactions caused by lymphocyte infiltration in the epidermal layer and help to improve psoriasis, this study provides a potential mechanism for the anti-inflammatory therapeutic effect of indigo naturalis extract in psoriasis.

Stromal cell-derived factor 1 (SDF-1) accelerated skin wound healing by promoting the migration and proliferation of epidermal stem cells.

PubMed

Guo, Rui; Chai, Linlin; Chen, Liang; Chen, Wenguang; Ge, Liangpeng; Li, Xiaoge; Li, Hongli; Li, Shirong; Cao, Chuan

2015-06-01

Epidermal stem cells could contribute to skin repair through the migration of cells from the neighboring uninjured epidermis, infundibulum, hair follicle, or sebaceous gland. However, little is known about the factors responsible for the complex biological processes in wound healing. Herein, we will show that the attracting chemokine, SDF-1/CXCR4, is a major regulator involved in the migration of epidermal stem cells during wound repair. We found that the SDF-1 levels were markedly increased at the wound margins following injury and CXCR4 expressed in epidermal stem cells and proliferating epithelial cells. Blocking the SDF-1/CXCR4 axis resulted in a significant reduction in epidermal stem cell migration toward SDF-1 in vitro and delayed wound healing in vivo, while an SDF-1 treatment enhanced epidermal stem cell migration and proliferation and accelerated wound healing. These results provide direct evidence that SDF-1 promotes epidermal stem cell migration, accelerates skin regeneration, and makes the development of new regenerative therapeutic strategies for wound healing possible.

Induction of hyaluronan cables and monocyte adherence in epidermal keratinocytes.

PubMed

Jokela, Tiina A; Lindgren, Antti; Rilla, Kirsi; Maytin, Edward; Hascall, Vincent C; Tammi, Raija H; Tammi, Markku I

2008-01-01

Hyaluronan attached to cell surface can form at least two very different structures; a pericellular coat close to plasma membrane and hyaluronan chains coalesced into "cables" that can span several cell lengths. The hyaluronan in cables, induced by many inflammatory agents, can bind leukocytes, whereas that in the pericellular coat does not contribute to leukocyte binding. Therefore, this structural change seems to have a major role in inflammation. In the present study we checked whether cells of squamous epithelium, like epidermal keratinocytes, can form hyaluronan cables and bind leukocytes. In addition, we checked whether hyaluronan synthesis is affected during the induction of cables. Control keratinocytes expressed pericellular hyaluronan as small patches on plasma membrane. But when treated with inflammatory agents or stressful conditions (tunicamycin, interleukin-1beta, tumor necrosis factor-alpha, and high glucose concentration), hyaluronan organization changed into cable-like structures that avidly bound monocytes. Simultaneously, the total amount of secreted hyaluronan was slightly decreased, and the expression levels of hyaluronan synthases (Has1-3) and CD44 were not significantly changed. The results show that epidermal keratinocytes can form cables and bind leukocytes under inflammatory provocation and that these effects are not dependent on stimulation of hyaluronan secretion.

Effects of adenosine 5'-monophosphate on epidermal turnover.

PubMed

Furukawa, Fukumi; Kanehara, Shoko; Harano, Fumiki; Shinohara, Shigeo; Kamimura, Junko; Kawabata, Shigekatsu; Igarashi, Sachiyo; Kawamura, Mitsuaki; Yamamoto, Yuki; Miyachi, Yoshiki

2008-10-01

The structure and function of the epidermis is maintained by cell renewal based on epidermal turnover. Epidermal turnover is delayed by aging, and it is thought that the delay of the epidermal turnover is a cause of aging alternation of skin. The epidermal turnover is related to the energy metabolism of epidermal basal cells. Adenosine 5'-triphosphate (ATP) is needed for cell renewal: cell division, and adenosine 5'-monophosphate (AMP) increases the amount of intracellular ATP. These findings suggest that AMP accelerates the epidermal turnover delayed by aging. This study investigated whether AMP and adenosine 5'-monophosphate disodium salt (AMP2Na) accelerates the epidermal turnover. An effect of AMP2Na on cell proliferation was examined by our counting of keratinocytes. An effect of AMP2Na on cell cycle was examined by our counting of basal cells in DNA synthetic period of hairless rats. The effects of AMP2Na (or AMP) on the epidermal turnover were examined by our measuring stratum corneum transit time by use of guinea pigs, and by our measuring stratum corneum surface area by use of hairless rats and in a clinical pharmacological study. The AMP2Na showed two different profiles on the proliferation of primary cultured keratinocytes. At a low concentration it induced cell growth, whereas at a high concentration it inhibited cell growth. The number of basal cells in the DNA synthetic period of AMP2Na was significantly higher than that of the vehicle in hairless rats. The stratum corneum transit time of AMP2Na was significantly shorter than that of the vehicle in guinea pigs. The corneocyte surface area of emulsion containing AMP2Na was significantly smaller than that of the vehicle in volunteers. We conclude that AMP promotes the cell proliferation and the cell cycle progression of epidermal basal cells and accelerates epidermal turnover safely. In addition, AMP is useful for skin rejuvenation in dermatology and aesthetic dermatology.

Suppressors for Human Epidermal Growth Factor Receptor 2/4 (HER2/4): A New Family of Anti-Toxoplasmic Agents in ARPE-19 Cells

PubMed Central

Kim, Yeong Hoon; Bhatt, Lokraj; Ahn, Hye-Jin; Yang, Zhaoshou; Lee, Won-Kyu; Nam, Ho-Woo

2017-01-01

The effects of tyrosine kinase inhibitors (TKIs) were evaluated on growth inhibition of intracellular Toxoplasma gondii in host ARPE-19 cells. The number of tachyzoites per parasitophorous vacuolar membrane (PVM) was counted after treatment with TKIs. T. gondii protein expression was assessed by western blot. Immunofluorescence assay was performed using Programmed Cell Death 4 (PDCD4) and T. gondii GRA3 antibodies. The TKIs were divided into 3 groups; non-epidermal growth factor receptor (non-EGFR), anti-human EGFR 2 (anti-HER2), and anti-HER2/4 TKIs, respectively. Group I TKIs (nintedanib, AZD9291, and sunitinib) were unable to inhibit proliferation without destroying host cells. Group II TKIs (lapatinib, gefitinib, erlotinib, and AG1478) inhibited proliferation up to 98% equivalent to control pyrimethamine (5 μM) at 20 μM and higher, without affecting host cells. Group III TKIs (neratinib, dacomitinib, afatinib, and pelitinib) inhibited proliferation up to 98% equivalent to pyrimethamine at 1–5 μM, but host cells were destroyed at 10–20 μM. In Group I, TgHSP90 and SAG1 inhibitions were weak, and GRA3 expression was moderately inhibited. In Group II, TgHSP90 and SAG1 expressions seemed to be slightly enhanced, while GRA3 showed none to mild inhibition; however, AG1478 inhibited all proteins moderately. Protein expression was blocked in Group III, comparable to pyrimethamine. PDCD4 and GRA3 were well localized inside the nuclei in Group I, mildly disrupted in Group II, and were completely disrupted in Group III. This study suggests the possibility of a vital T. gondii TK having potential HER2/4 properties, thus anti-HER2/4 TKIs may inhibit intracellular parasite proliferation with minimal adverse effects on host cells. PMID:29103264

Suppressors for Human Epidermal Growth Factor Receptor 2/4 (HER2/4): A New Family of Anti-Toxoplasmic Agents in ARPE-19 Cells.

PubMed

Kim, Yeong Hoon; Bhatt, Lokraj; Ahn, Hye-Jin; Yang, Zhaoshou; Lee, Won-Kyu; Nam, Ho-Woo

2017-10-01

The effects of tyrosine kinase inhibitors (TKIs) were evaluated on growth inhibition of intracellular Toxoplasma gondii in host ARPE-19 cells. The number of tachyzoites per parasitophorous vacuolar membrane (PVM) was counted after treatment with TKIs. T. gondii protein expression was assessed by western blot. Immunofluorescence assay was performed using Programmed Cell Death 4 (PDCD4) and T. gondii GRA3 antibodies. The TKIs were divided into 3 groups; non-epidermal growth factor receptor (non-EGFR), anti-human EGFR 2 (anti-HER2), and anti-HER2/4 TKIs, respectively. Group I TKIs (nintedanib, AZD9291, and sunitinib) were unable to inhibit proliferation without destroying host cells. Group II TKIs (lapatinib, gefitinib, erlotinib, and AG1478) inhibited proliferation up to 98% equivalent to control pyrimethamine (5 μM) at 20 μM and higher, without affecting host cells. Group III TKIs (neratinib, dacomitinib, afatinib, and pelitinib) inhibited proliferation up to 98% equivalent to pyrimethamine at 1-5 μM, but host cells were destroyed at 10-20 μM. In Group I, TgHSP90 and SAG1 inhibitions were weak, and GRA3 expression was moderately inhibited. In Group II, TgHSP90 and SAG1 expressions seemed to be slightly enhanced, while GRA3 showed none to mild inhibition; however, AG1478 inhibited all proteins moderately. Protein expression was blocked in Group III, comparable to pyrimethamine. PDCD4 and GRA3 were well localized inside the nuclei in Group I, mildly disrupted in Group II, and were completely disrupted in Group III. This study suggests the possibility of a vital T. gondii TK having potential HER2/4 properties, thus anti-HER2/4 TKIs may inhibit intracellular parasite proliferation with minimal adverse effects on host cells.

Genetics Home Reference: Stevens-Johnson syndrome/toxic epidermal necrolysis

MedlinePlus

... Conditions Stevens-Johnson syndrome/toxic epidermal necrolysis Stevens-Johnson syndrome/toxic epidermal necrolysis Printable PDF Open All ... to view the expand/collapse boxes. Description Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) is a ...

Bradykinin-induced growth inhibition of normal rat kidney (NRK) cells is paralleled by a decrease in epidermal-growth-factor receptor expression.

PubMed Central

Van Zoelen, E J; Peters, P H; Afink, G B; Van Genesen, S; De Roos, D G; Van Rotterdam, W; Theuvenet, A P

1994-01-01

Normal rat kidney fibroblasts, grown to density arrest in the presence of epidermal growth factor (EGF), can be induced to undergo phenotypic transformation by treatment with transforming growth factor beta or retinoic acid. Here we show that bradykinin blocks this growth-stimulus-induced loss of density-dependent growth arrest by a specific receptor-mediated mechanism. The effects of bradykinin are specific, and are not mimicked by other phosphoinositide-mobilizing agents such as prostaglandin F2 alpha. Northern-blot analysis and receptor-binding studies demonstrate that bradykinin also inhibits the retinoic acid-induced increase in EGF receptor levels in these cells. These studies provide additional evidence that EGF receptor levels modulate EGF-induced expression of the transformed phenotype in these cells. Images Figure 5 PMID:8135739

Burn Injury Alters Epidermal Cholinergic Mediators and Increases HMGB1 and Caspase 3 in Autologous Donor Skin and Burn Margin

PubMed Central

Holmes, Casey J.; Plichta, Jennifer K.; Gamelli, Richard L.; Radek, Katherine A.

2016-01-01

Burn wound healing complications, such as graft failure or infection, are a major source of morbidity and mortality in burn patients. The mechanisms by which local burn injury alters epidermal barrier function in autologous donor skin and surrounding burn margin are largely undefined. We hypothesized that defects in the epidermal cholinergic system may impair epidermal barrier function and innate immune responses. The objective was to identify alterations in the epidermal cholinergic pathway, and their downstream targets, associated with inflammation and cell death. We established that protein levels, but not gene expression, of the α7 nicotinic acetylcholine receptor (CHRNA7) were significantly reduced in both donor and burn margin skin. Furthermore, the gene and protein levels of an endogenous allosteric modulator of CHRNA7, secreted mammalian Ly-6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP1) and acetylcholine were significantly elevated in donor and burn margin skin. As downstream proteins of inflammatory and cell death targets of nAChR activation, we found significant elevations in epidermal High Mobility Group Box Protein 1 (HMGB1) and caspase 3 in donor and burn margin skin. Lastly, we employed a novel in vitro keratinocyte burn model to establish that burn injury influences the gene expression of these cholinergic mediators and their downstream targets. These results indicate that defects in cholinergic mediators and inflammatory/apoptotic molecules in donor and burn margin skin may directly contribute to graft failure or infection in burn patients. PMID:27648692

Bystander killing effect of DS-8201a, a novel anti-human epidermal growth factor receptor 2 antibody-drug conjugate, in tumors with human epidermal growth factor receptor 2 heterogeneity.

PubMed

Ogitani, Yusuke; Hagihara, Katsunobu; Oitate, Masataka; Naito, Hiroyuki; Agatsuma, Toshinori

2016-07-01

Antibody-drug conjugates deliver anticancer agents selectively and efficiently to tumor tissue and have significant antitumor efficacy with a wide therapeutic window. DS-8201a is a human epidermal growth factor receptor 2 (HER2)-targeting antibody-drug conjugate prepared using a novel linker-payload system with a potent topoisomerase I inhibitor, exatecan derivative (DX-8951 derivative, DXd). It was effective against trastuzumab emtansine (T-DM1)-insensitive patient-derived xenograft models with both high and low HER2 expression. In this study, the bystander killing effect of DS-8201a was evaluated and compared with that of T-DM1. We confirmed that the payload of DS-8201a, DXd (1), was highly membrane-permeable whereas that of T-DM1, Lys-SMCC-DM1, had a low level of permeability. Under a coculture condition of HER2-positive KPL-4 cells and negative MDA-MB-468 cells in vitro, DS-8201a killed both cells, whereas T-DM1 and an antibody-drug conjugate with a low permeable payload, anti-HER2-DXd (2), did not. In vivo evaluation was carried out using mice inoculated with a mixture of HER2-positive NCI-N87 cells and HER2-negative MDA-MB-468-Luc cells by using an in vivo imaging system. In vivo, DS-8201a reduced the luciferase signal of the mice, indicating suppression of the MDA-MB-468-Luc population; however, T-DM1 and anti-HER2-DXd (2) did not. Furthermore, it was confirmed that DS-8201a was not effective against MDA-MB-468-Luc tumors inoculated at the opposite side of the NCI-N87 tumor, suggesting that the bystander killing effect of DS-8201a is observed only in cells neighboring HER2-positive cells, indicating low concern in terms of systemic toxicity. These results indicated that DS-8201a has a potent bystander effect due to a highly membrane-permeable payload and is beneficial in treating tumors with HER2 heterogeneity that are unresponsive to T-DM1. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer

Blockade of vascular endothelial growth factor receptor and epidermal growth factor receptor signaling for therapy of metastatic human pancreatic cancer.

PubMed

Baker, Cheryl H; Solorzano, Carmen C; Fidler, Isaiah J

2002-04-01

We determined whether concurrent blockage of vascular endothelial growth factor (VEGF) receptor and epidermal growth factor (EGF) receptor signaling by two novel tyrosine kinase inhibitors, PTK 787 and PKI 166, respectively, can inhibit angiogenesis and, hence, the growth and metastasis of human pancreatic carcinoma in nude mice. Highly metastatic human pancreatic carcinoma L3.6pl cells were injected into the pancreas of nude mice. Seven days later, groups of mice began receiving oral doses of PTK 787 and PKI 166 three times weekly. Some groups of mice also received i.p. injections of gemcitabine twice a week. The mice were necropsied when the control mice became moribund. Treatment with PTK 787 and PKI 166, with gemcitabine alone, or with the combination of PTK 787, PKI 166, and gemcitabine produced 69, 50, and 97% reduction in the volume of pancreatic tumors, respectively. Administration of protein tyrosine kinase inhibitors and gemcitabine also significantly decreased the incidence of lymph node and liver metastasis. The therapeutic efficacy directly correlated with a decrease in circulating proangiogenic molecules (VEGF, interleukin-8), a decrease in microvessel density, a decrease in proliferating cell nuclear antigen staining, and an increase in apoptosis of tumor cells and endothelial cells. Therapies produced by combining gemcitabine with either PKI 166 or PTK 787 were similar to those produced by combining gemcitabine with both PKI 166 and PTK 787. These results suggest that blockade of either epidermal growth factor receptor or VEGF receptor signaling combined with chemotherapy provides an effective approach to the therapy of pancreatic cancer.

Comparative prognostic value of epidermal growth factor quantitative protein expression compared with FISH for head and neck squamous cell carcinoma.

PubMed

Pectasides, Eirini; Rampias, Theodore; Kountourakis, Panteleimon; Sasaki, Clarence; Kowalski, Diane; Fountzilas, George; Zaramboukas, Thomas; Rimm, David; Burtness, Barbara; Psyrri, Amanda

2011-05-01

Epidermal growth factor receptor (EGFR) overexpression correlates with recurrence and with treatment resistance in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to evaluate the relationship of EGFR gene copy number utilizing FISH and protein expression with automated quantitative analysis (AQUA) and to correlate those with patient outcome. A tissue microarray composed of 102 HNSCC treated with (chemo)radiation was constructed and analyzed for EGFR copy number by FISH (Vysis; Abbott Laboratories) and EGFR protein expression using AQUA analysis of EGFR staining scored on a scale of 0 to 255. We evaluated associations of EGFR FISH status and AQUA score with clinicopathologic parameters and survival prognosis. Eleven (17.2%) of 64 tumors with FISH results showed EGFR high polysomy and/or gene amplification (FISH positive). Protein levels assessed by AQUA in FISH-positive cases were significantly higher (P = 0.04) than in FISH-negative cases. Using the continuous AQUA scores for EGFR expression, AQUA and FISH showed significant agreement (Pearson's ρ = 0.353, P = 0.04). Patients with high tumor EGFR protein expression had inferior 5-year overall survival (27.7%) compared with those with low tumor EGFR expression (54%; P = 0.029). There was no significant association between EGFR FISH status and overall survival (P = 0.201). In the multivariate model, high tumor EGFR protein expression status remained an independent prognostic factor for overall survival (P = 0.047). EGFR protein content correlates with gene copy number if protein content is quantitated and automatically analyzed, as with AQUA. EGFR protein levels assessed by AQUA strongly predict for patient outcome in HNSCC, whereas EGFR FISH status does not provide prognostic information. ©2011 AACR.

Epidermal nevus syndrome and didymosis aplasticosebacea.

PubMed

Demerdjieva, Zdravka; Kavaklieva, Svetlana; Tsankov, Nikolay

2007-01-01

The epidermal nevus syndrome is a disease complex consisting of the association of an epidermal nevus with various developmental abnormalities of the skin, eyes, nervous, skeletal, cardiovascular, and urogenital systems. The epidermal nevi are classified according to their predominant component; nevus sebaceus (sebaceous glands), nevus comedonicus (hair follicles), and nevus verrucosus (keratinocytes). We report a neonate who presented with a nevus sebaceus on the scalp and face as well as a coloboma and dermoid on his left eye. Within the sebaceous nevus on the scalp, circumscribed lesions of aplasia cutis congenita were detected, which is consistent with the recently proposed term in the literature didymosis aplasticosebacea.

Posttraumatic epidermal inclusion cyst of the deep infratemporal fossa.

PubMed

Acarturk, T O; Stofman, G M

2001-01-01

The authors report a case of an epidermal inclusion cyst found in the deep infratemporal fossa 12 years after the patient sustained blunt trauma to that region. Posttraumatic epidermal inclusion cysts are rare and occur mainly in the fingers, palms, and soles. Introduction of the epidermal elements into the dermis during the trauma is thought to be the cause. This case is rare in presentation, with few reports in the English literature that describe an epidermal inclusion cyst in the deep infratemporal fossa. Review of the English literature disclosed no other cases of epidermal inclusion cyst after blunt trauma involving the deep infratemporal region.

Human Epidermal Langerhans Cells Maintain Immune Homeostasis in Skin by Activating Skin Resident Regulatory T Cells

PubMed Central

Seneschal, Julien; Clark, Rachael A.; Gehad, Ahmed; Baecher-Allan, Clare M.; Kupper, Thomas S.

2013-01-01

Recent discoveries indicate that the skin of a normal individual contains 10-20 billion resident memory T cells ( which include various T helper, T cytotoxic, and T regulatory subsets, that are poised to respond to environmental antigens. Using only autologous human tissues, we report that both in vitro and in vivo, resting epidermal Langerhan cells (LC) selectively and specifically induced the activation and proliferation of skin resident regulatory T cells (Treg), a minor subset of skin resident memory T cells. In the presence of foreign pathogen, however, the same LC activated and induced proliferation of effector memory T (Tem) cells and limited Treg cells activation. These underappreciated properties of LC: namely maintenance of tolerance in normal skin, and activation of protective skin resident memory T cells upon infectious challenge, help clarify the role of LC in skin. PMID:22560445

Epidermal growth factor induces G protein-coupled receptor 30 expression in estrogen receptor-negative breast cancer cells.