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Sample records for epidermal cell area

  1. Epidermal Stem Cells in Orthopaedic Regenerative Medicine

    PubMed Central

    Li, Jin; Zhen, Gehua; Tsai, Shin-Yi; Jia, Xiaofeng

    2013-01-01

    In the last decade, great advances have been made in epidermal stem cell studies at the cellular and molecular level. These studies reported various subpopulations and differentiations existing in the epidermal stem cell. Although controversies and unknown issues remain, epidermal stem cells possess an immune-privileged property in transplantation together with easy accessibility, which is favorable for future clinical application. In this review, we will summarize the biological characteristics of epidermal stem cells, and their potential in orthopedic regenerative medicine. Epidermal stem cells play a critical role via cell replacement, and demonstrate significant translational potential in the treatment of orthopedic injuries and diseases, including treatment for wound healing, peripheral nerve and spinal cord injury, and even muscle and bone remodeling. PMID:23727934

  2. Mechanotransduction in epidermal Merkel cells

    PubMed Central

    Nakatani, Masashi; Maksimovic, Srdjan; Baba, Yoshichika; Lumpkin, Ellen A.

    2014-01-01

    The cellular and molecular basis of vertebrate touch reception remains least understood among the traditional five senses. Somatosensory afferents that innervate the skin encode distinct tactile qualities, such as flutter, slip and pressure. Gentle touch is thought to be transduced by somatosensory afferents whose tactile end organs selectively filter mechanical stimuli. These tactile end organs comprise afferent terminals in association with non-neuronal cell types such as Merkel cells, keratinocytes and Schwann cells. An open question is whether these non-neuronal cells serve primarily as passive mechanical filters or whether they actively participate in mechanosensory transduction. This question has been most extensively studied in Merkel cells, which are epidermal cells that complex with sensory afferents in regions of high tactile acuity such as fingertips, whisker follicles, and touch domes. Merkel cell-neurite complexes mediate slowly adapting type I (SAI) responses, which encode sustained pressure and represent object features with high fidelity. How Merkel cells contribute to unique SAI firing patterns has been debated for decades; however, three recent studies in rodent models provide some direct answers. First, whole-cell recordings demonstrate that Merkel cells are touch-sensitive cells with fast, mechanically activated currents that require Piezo2. Second, optogenetics and intact recordings show that Merkel cells mediate sustained SAI firing. Finally, loss-of-function studies in transgenic mouse models reveal that SAI afferents are also touch sensitive. Together, these studies identify molecular mechanisms of mechanotransduction in Merkel cells, reveal unexpected functions for these cells in touch and support a revised, two-receptor site model of mechanosensory transduction. PMID:25053537

  3. 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

  4. Jarid2 regulates mouse epidermal stem cell activation and differentiation.

    PubMed

    Mejetta, Stefania; Morey, Lluis; Pascual, Gloria; Kuebler, Bernd; Mysliwiec, Matthew R; Lee, Youngsook; Shiekhattar, Ramin; Di Croce, Luciano; Benitah, Salvador Aznar

    2011-08-02

    Jarid2 is required for the genomic recruitment of the polycomb repressive complex-2 (PRC2) in embryonic stem cells. However, its specific role during late development and adult tissues remains largely uncharacterized. Here, we show that deletion of Jarid2 in mouse epidermis reduces the proliferation and potentiates the differentiation of postnatal epidermal progenitors, without affecting epidermal development. In neonatal epidermis, Jarid2 deficiency reduces H3K27 trimethylation, a chromatin repressive mark, in epidermal differentiation genes previously shown to be targets of the PRC2. However, in adult epidermis Jarid2 depletion does not affect interfollicular epidermal differentiation but results in delayed hair follicle (HF) cycling as a consequence of decreased proliferation of HF stem cells and their progeny. We conclude that Jarid2 is required for the scheduled proliferation of epidermal stem and progenitor cells necessary to maintain epidermal homeostasis.

  5. Metabolic profiling of Arabidopsis thaliana epidermal cells

    PubMed Central

    Ebert, Berit; Zöller, Daniela; Erban, Alexander; Fehrle, Ines; Hartmann, Jürgen; Niehl, Annette; Kopka, Joachim; Fisahn, Joachim

    2010-01-01

    Metabolic phenotyping at cellular resolution may be considered one of the challenges in current plant physiology. A method is described which enables the cell type-specific metabolic analysis of epidermal cell types in Arabidopsis thaliana pavement, basal, and trichome cells. To achieve the required high spatial resolution, single cell sampling using microcapillaries was combined with routine gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) based metabolite profiling. The identification and relative quantification of 117 mostly primary metabolites has been demonstrated. The majority, namely 90 compounds, were accessible without analytical background correction. Analyses were performed using cell type-specific pools of 200 microsampled individual cells. Moreover, among these identified metabolites, 38 exhibited differential pool sizes in trichomes, basal or pavement cells. The application of an independent component analysis confirmed the cell type-specific metabolic phenotypes. Significant pool size changes between individual cells were detectable within several classes of metabolites, namely amino acids, fatty acids and alcohols, alkanes, lipids, N-compounds, organic acids and polyhydroxy acids, polyols, sugars, sugar conjugates and phenylpropanoids. It is demonstrated here that the combination of microsampling and GC-MS based metabolite profiling provides a method to investigate the cellular metabolism of fully differentiated plant cell types in vivo. PMID:20150518

  6. Immunohistochemical analyses point to epidermal origin of human Merkel cells.

    PubMed

    Tilling, Thomas; Wladykowski, Ewa; Failla, Antonio Virgilio; Houdek, Pia; Brandner, Johanna M; Moll, Ingrid

    2014-04-01

    Merkel cells, the neurosecretory cells of skin, are essential for light-touch responses and may probably fulfill additional functions. Whether these cells derive from an epidermal or a neural lineage has been a matter of dispute for a long time. In mice, recent studies have clearly demonstrated an epidermal origin of Merkel cells. Given the differences in Merkel cell distribution between human and murine skin, it is, however, unclear whether the same holds true for human Merkel cells. We therefore attempted to gain insight into the human Merkel cell lineage by co-immunodetection of the Merkel cell marker protein cytokeratin 20 (CK20) with various proteins known to be expressed either in epidermal or in neural stem cells of the skin. Neither Sox10 nor Pax3, both established markers of the neural crest lineage, exhibited any cell co-labeling with CK20. By contrast, β1 integrin, known to be enriched in epidermal stem cells, was found in nearly 70 % of interfollicular epidermal and 25 % of follicular Merkel cells. Moreover, LRIG1, also enriched in epidermal stem cells, displayed significant co-immunolabeling with CK20 as well (approximately 20 % in the interfollicular epidermis and 7 % in the hair follicle, respectively). Further epidermal markers were detected in sporadic Merkel cells. Cells co-expressing CK20 with epidermal markers may represent a transitory state between stem cells and differentiated cells. β1 integrin is probably also synthesized by a large subset of mature Merkel cells. Summarizing, our data suggest that human Merkel cells may originate from epidermal rather than neural progenitors.

  7. Optimal allocation of leaf epidermal area for gas exchange.

    PubMed

    de Boer, Hugo J; Price, Charles A; Wagner-Cremer, Friederike; Dekker, Stefan C; Franks, Peter J; Veneklaas, Erik J

    2016-06-01

    A long-standing research focus in phytology has been to understand how plants allocate leaf epidermal space to stomata in order to achieve an economic balance between the plant's carbon needs and water use. Here, we present a quantitative theoretical framework to predict allometric relationships between morphological stomatal traits in relation to leaf gas exchange and the required allocation of epidermal area to stomata. Our theoretical framework was derived from first principles of diffusion and geometry based on the hypothesis that selection for higher anatomical maximum stomatal conductance (gsmax ) involves a trade-off to minimize the fraction of the epidermis that is allocated to stomata. Predicted allometric relationships between stomatal traits were tested with a comprehensive compilation of published and unpublished data on 1057 species from all major clades. In support of our theoretical framework, stomatal traits of this phylogenetically diverse sample reflect spatially optimal allometry that minimizes investment in the allocation of epidermal area when plants evolve towards higher gsmax . Our results specifically highlight that the stomatal morphology of angiosperms evolved along spatially optimal allometric relationships. We propose that the resulting wide range of viable stomatal trait combinations equips angiosperms with developmental and evolutionary flexibility in leaf gas exchange unrivalled by gymnosperms and pteridophytes. PMID:26991124

  8. Specification of epidermal cell fate in plant shoots.

    PubMed

    Takada, Shinobu; Iida, Hiroyuki

    2014-01-01

    Land plants have evolved a single layer of epidermal cells, which are characterized by mostly anticlinal cell division patterns, formation of a waterproof coat called cuticle, and unique cell types such as stomatal guard cells and trichomes. The shoot epidermis plays important roles not only to protect plants from dehydration and pathogens but also to ensure their proper organogenesis and growth control. Extensive molecular genetic studies in Arabidopsis and maize have identified a number of genes that are required for epidermal cell differentiation. However, the mechanism that specifies shoot epidermal cell fate during plant organogenesis remains largely unknown. Particularly, little is known regarding positional information that should restrict epidermal cell fate to the outermost cell layer of the developing organs. Recent studies suggested that certain members of the HD-ZIP class IV homeobox genes are possible master regulators of shoot epidermal cell fate. Here, we summarize the roles of the regulatory genes that are involved in epidermal cell fate specification and discuss the possible mechanisms that limit the expression and/or activity of the master transcriptional regulators to the outermost cell layer in plant shoots. PMID:24616724

  9. Integrity of the permeability barrier regulates epidermal Langerhans cell density.

    PubMed

    Proksch, E; Brasch, J; Sterry, W

    1996-04-01

    Previous studies have shown that barrier requirements regulate epidermal liquid and DNA synthesis. In the present study, we examined the possibility that the integrity of the permeability barrier influences epidermal Langerhans cells involved with the immune response. Barrier disruption was achieved by treatment of human skin with acetone, sodium dodecylsulphate (SDS), or tape stripping, until a 10-20-fold increase in transepidermal water loss was achieved. Serial biopsies were performed 6-168 h after treatment, and Langerhans cells were complexed with anti-CD1a (Leu6) or S-100 antibodies, and visualized with an immunoperoxidase technique. Acetone treatment resulted in an increase in epidermal Langerhans cell density, reaching a maximum of 94% over control (P < 0.01) by 24 and 48 h post-treatment. Following SDS treatment or tape stripping, epidermal Langerhans cell density was increased by 100 and 175% (P < 0.01), respectively. There was a linear correlation between the degree of barrier disruption and the increase in epidermal Langerhans cell density. Studies with the Ki-S3 proliferation-associated nuclear antigen revealed a two- to threefold increase in epidermal proliferation after barrier disruption. The time curves of the increase in Langerhans cell density and the increase in epidermal proliferation were similar, suggesting that there was a coordinate regulation. In contrast with our previous studies employing patch test reactions to allergens or irritants, disruption of barrier function neither resulted in an increased dermal Langerhans cell density, nor influenced T lymphocytes (CD3+, Leu4+), macrophages (KiM8+), ICAM-1 or ELAM-1 expression in the skin. In addition, barrier disruption did not result in either dermal inflammation or epidermal spongiosis. In summary, these findings support our hypothesis that the permeability barrier influences epidermal Langerhans cell density, which is involved in maintaining an immunological barrier.

  10. Markers of epidermal stem cell subpopulations in adult mammalian skin.

    PubMed

    Kretzschmar, Kai; Watt, Fiona M

    2014-10-01

    The epidermis is the outermost layer of mammalian skin and comprises a multilayered epithelium, the interfollicular epidermis, with associated hair follicles, sebaceous glands, and eccrine sweat glands. As in other epithelia, adult stem cells within the epidermis maintain tissue homeostasis and contribute to repair of tissue damage. The bulge of hair follicles, where DNA-label-retaining cells reside, was traditionally regarded as the sole epidermal stem cell compartment. However, in recent years multiple stem cell populations have been identified. In this review, we discuss the different stem cell compartments of adult murine and human epidermis, the markers that they express, and the assays that are used to characterize epidermal stem cell properties.

  11. Do epidermal lens cells facilitate the absorptance of diffuse light?

    PubMed

    Brodersen, Craig R; Vogelmann, Thomas C

    2007-07-01

    Many understory plants rely on diffuse light for photosynthesis because direct light is usually scattered by upper canopy layers before it strikes the forest floor. There is a considerable gap in the literature concerning the interaction of direct and diffuse light with leaves. Some understory plants have well-developed lens-shaped epidermal cells, which have long been thought to increase the absorption of diffuse light. To assess the role of epidermal cell shape in capturing direct vs. diffuse light, we measured leaf reflectance and transmittance with an integrating sphere system using leaves with flat (Begonia erythrophylla, Citrus reticulata, and Ficus benjamina) and lens-shaped epidermal cells (B. bowerae, Colocasia esculenta, and Impatiens velvetea). In all species examined, more light was absorbed when leaves were irradiated with direct as opposed to diffuse light. When leaves were irradiated with diffuse light, more light was transmitted and more was reflected in both leaf types, resulting in absorptance values 2-3% lower than in leaves irradiated with direct light. These data suggest that lens-shaped epidermal cells do not aid the capture of diffuse light. Palisade and mesophyll cell anatomy and leaf thickness appear to have more influence in the capture and absorption of light than does epidermal cell shape.

  12. 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.

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

    PubMed

    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

  14. Cell Fate Determination and the Switch from Diffuse Growth to Planar Polarity in Arabidopsis Root Epidermal Cells

    PubMed Central

    Balcerowicz, Daria; Schoenaers, Sébastjen; Vissenberg, Kris

    2015-01-01

    Plant roots fulfill important functions as they serve in water and nutrient uptake, provide anchorage of the plant body in the soil and in some species form the site of symbiotic interactions with soil-living biota. Root hairs, tubular-shaped outgrowths of specific epidermal cells, significantly increase the root’s surface area and aid in these processes. In this review we focus on the molecular mechanisms that determine the hair and non-hair cell fate of epidermal cells and that define the site on the epidermal cell where the root hair will be initiated (=planar polarity determination). In the model plant Arabidopsis, trichoblast and atrichoblast cell fate results from intra- and intercellular position-dependent signaling and from complex feedback loops that ultimately regulate GL2 expressing and non-expressing cells. When epidermal cells reach the end of the root expansion zone, root hair promoting transcription factors dictate the establishment of polarity within epidermal cells followed by the selection of the root hair initiation site at the more basal part of the trichoblast. Molecular players in the abovementioned processes as well as the role of phytohormones are discussed, and open areas for future experiments are identified. PMID:26779192

  15. The interfollicular epidermal stem cell saga: sensationalism versus reality check.

    PubMed

    Kaur, Pritinder; Potten, Christopher S

    2011-09-01

    Adult stem cells in rapidly renewing tissues have been classically defined as rare, relatively quiescent cells with the unique capacity to constantly self-renew and regenerate tissues during homeostasis. Although this view remains firmly embedded in the skin field, particularly in the area of hair follicle stem cell biology, it has been challenged by a number of notable publications in 2007. These papers leave an uncomfortable feeling with the reader if one believes that stem cells and transit amplifying cells are two polar opposites and 'never the twain shall meet.' Even if you do not subscribe to this extreme view, the implications appear to be far-reaching given that the majority of techniques devised for stem cell identification have used the fundamental tenet that the proliferating compartment is comprised of two distinct, mutually exclusive compartments, i.e. a minor proportion of long-lived quiescent stem cells with unlimited self-renewal and a large pool of rapidly cycling, short-lived transient amplifying cells with limited or no self-renewal capacity in normal steady-state conditions. However, these recent findings have resulted in papers that could be described as sensationalistic because they make little or no attempt to reconcile their observations with the large bulk of historical data with direct bearing on the interpretation of stem cell activity in normal steady-state conditions. Here, we offer some explanations that may help to integrate all of the data while presenting a case that both quiescent stem cells and cycling 'transit amplifying' cells contribute to epidermal replacement. PMID:21834906

  16. Markers of Epidermal Stem Cell Subpopulations in Adult Mammalian Skin

    PubMed Central

    Kretzschmar, Kai; Watt, Fiona M.

    2014-01-01

    The epidermis is the outermost layer of mammalian skin and comprises a multilayered epithelium, the interfollicular epidermis, with associated hair follicles, sebaceous glands, and eccrine sweat glands. As in other epithelia, adult stem cells within the epidermis maintain tissue homeostasis and contribute to repair of tissue damage. The bulge of hair follicles, where DNA-label-retaining cells reside, was traditionally regarded as the sole epidermal stem cell compartment. However, in recent years multiple stem cell populations have been identified. In this review, we discuss the different stem cell compartments of adult murine and human epidermis, the markers that they express, and the assays that are used to characterize epidermal stem cell properties. PMID:24993676

  17. Familial papular epidermal nevus with "skyline" basal cell layer.

    PubMed

    Brena, Michela; Besagni, Francesca; Boneschi, Vinicio; Tadini, Gianluca

    2014-01-01

    Papular epidermal nevus with "skyline" basal cell layer (PENS), a novel keratinocytic nevus, has recently been described as a mosaic condition with varying presentations. We herein describe typical PENS lesions, which usually occur sporadically, affecting two members of the same family. The concept of paradominant inheritance is proposed to explain the paradox of occasional transmission of normally sporadically occurring traits.

  18. The circadian molecular clock creates epidermal stem cell heterogeneity.

    PubMed

    Janich, Peggy; Pascual, Gloria; Merlos-Suárez, Anna; Batlle, Eduard; Ripperger, Jürgen; Albrecht, Urs; Cheng, Hai-Ying M; Obrietan, Karl; Di Croce, Luciano; Benitah, Salvador Aznar

    2011-11-09

    Murine epidermal stem cells undergo alternate cycles of dormancy and activation, fuelling tissue renewal. However, only a subset of stem cells becomes active during each round of morphogenesis, indicating that stem cells coexist in heterogeneous responsive states. Using a circadian-clock reporter-mouse model, here we show that the dormant hair-follicle stem cell niche contains coexisting populations of cells at opposite phases of the clock, which are differentially predisposed to respond to homeostatic cues. The core clock protein Bmal1 modulates the expression of stem cell regulatory genes in an oscillatory manner, to create populations that are either predisposed, or less prone, to activation. Disrupting this clock equilibrium, through deletion of Bmal1 (also known as Arntl) or Per1/2, resulted in a progressive accumulation or depletion of dormant stem cells, respectively. Stem cell arrhythmia also led to premature epidermal ageing, and a reduction in the development of squamous tumours. Our results indicate that the circadian clock fine-tunes the temporal behaviour of epidermal stem cells, and that its perturbation affects homeostasis and the predisposition to tumorigenesis.

  19. Why do so many petals have conical epidermal cells?

    PubMed Central

    Whitney, Heather M.; Bennett, K. M. Veronica; Dorling, Matthew; Sandbach, Lucy; Prince, David; Chittka, Lars; Glover, Beverley J.

    2011-01-01

    Background The conical epidermal cells found on the petals of most Angiosperm species are so widespread that they have been used as markers of petal identity, but their function has only been analysed in recent years. This review brings together diverse data on the role of these cells in pollination biology. Scope The published effects of conical cells on petal colour, petal reflexing, scent production, petal wettability and pollinator grip on the flower surface are considered. Of these factors, pollinator grip has been shown to be of most significance in the well-studied Antirrhinum majus/bumble-bee system. Published data on the relationship between epidermal cell morphology and floral temperature were limited, so an analysis of the effects of cell shape on floral temperature in Antirrhinum is presented here. Statistically significant warming by conical cells was not detected, although insignificant trends towards faster warming at dawn were found, and it was also found that flat-celled flowers could be warmer on warm days. The warming observed is less significant than that achieved by varying pigment content. However, the possibility that the effect of conical cells on temperature might be biologically significant in certain specific instances such as marginal habitats or weather conditions cannot be ruled out. Conclusions Conical epidermal cells can influence a diverse set of petal properties. The fitness benefits they provide to plants are likely to vary with pollinator and habitat, and models are now required to understand how these different factors interact. PMID:21470973

  20. Estimating the Size of Onion Epidermal Cells from Diffraction Patterns

    NASA Astrophysics Data System (ADS)

    Groff, Jeffrey R.

    2012-10-01

    Bioscience and premedical profession students are a major demographic served by introductory physics courses at many colleges and universities. Exposing these students to biological applications of physical principles will help them to appreciate physics as a useful tool for their future professions. Here I describe an experiment suitable for introductory physics where principles of wave optics are applied to probe the size of onion epidermal cells. The epidermis tissue is composed of cells of relatively uniform size and shape (Fig. 1) so the tissue acts like a one-dimensional transmission diffraction grating. The diffraction patterns generated when a laser beam passes through the tissue (Fig. 2) are analyzed and an estimate of the average width of individual onion epidermal cells is calculated. The results are compared to direct measurements taken using a light microscope. The use of microscopes and plant-cell tissue slides creates opportunities for cross-discipline collaboration between physics and biology instructors.

  1. Spatiotemporal coordination of stem cell commitment during epidermal homeostasis

    PubMed Central

    Rompolas, Panteleimon; Mesa, Kailin R.; Kawaguchi, Kyogo; Park, Sangbum; Gonzalez, David; Brown, Samara; Boucher, Jonathan; Klein, Allon M.; Greco, Valentina

    2016-01-01

    Adult tissues replace lost cells via pools of stem cells. However, the mechanisms of cell self-renewal, commitment, and functional integration into the tissue remain unsolved. Using imaging techniques in live mice, we captured the lifetime of individual cells in the ear and paw epidermis. Our data suggest that epidermal stem cells have equal potential to either divide or directly differentiate. Tracking stem cells over multiple generations reveals that cell behavior is not coordinated between generations. However, sibling cell fate and lifetimes are coupled. We did not observe regulated asymmetric cell divisions. Lastly, we demonstrated that differentiating stem cells integrate into preexisting ordered spatial units of the epidermis. This study elucidates how a tissue is maintained by both temporal and spatial coordination of stem cell behaviors. PMID:27229141

  2. EGFR-Ras-Raf Signaling in Epidermal Stem Cells: Roles in Hair Follicle Development, Regeneration, Tissue Remodeling and Epidermal Cancers

    PubMed Central

    Doma, Eszter; Rupp, Christian; Baccarini, Manuela

    2013-01-01

    The mammalian skin is the largest organ of the body and its outermost layer, the epidermis, undergoes dynamic lifetime renewal through the activity of somatic stem cell populations. The EGFR-Ras-Raf pathway has a well-described role in skin development and tumor formation. While research mainly focuses on its role in cutaneous tumor initiation and maintenance, much less is known about Ras signaling in the epidermal stem cells, which are the main targets of skin carcinogenesis. In this review, we briefly discuss the properties of the epidermal stem cells and review the role of EGFR-Ras-Raf signaling in keratinocyte stem cells during homeostatic and pathological conditions. PMID:24071938

  3. 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

  4. Human dermal stem cells differentiate into functional epidermal melanocytes.

    PubMed

    Li, Ling; Fukunaga-Kalabis, Mizuho; Yu, Hong; Xu, Xiaowei; Kong, Jun; Lee, John T; Herlyn, Meenhard

    2010-03-15

    Melanocytes sustain a lifelong proliferative potential, but a stem cell reservoir in glabrous skin has not yet been found. Here, we show that multipotent dermal stem cells isolated from human foreskins lacking hair follicles are able to home to the epidermis to differentiate into melanocytes. These dermal stem cells, grown as three-dimensional spheres, displayed a capacity for self-renewal and expressed NGFRp75, nestin and OCT4, but not melanocyte markers. In addition, cells derived from single-cell clones were able to differentiate into multiple lineages including melanocytes. In a three-dimensional skin equivalent model, sphere-forming cells differentiated into HMB45-positive melanocytes, which migrated from the dermis to the epidermis and aligned singly among the basal layer keratinocytes in a similar fashion to pigmented melanocytes isolated from the epidermis. The dermal stem cells were negative for E-cadherin and N-cadherin, whereas they acquired E-cadherin expression and lost NGFRp75 expression upon contact with epidermal keratinocytes. These results demonstrate that stem cells in the dermis of human skin with neural-crest-like characteristics can become mature epidermal melanocytes. This finding could significantly change our understanding of the etiological factors in melanocyte transformation and pigmentation disorders; specifically, that early epigenetic or genetic alterations leading to transformation may take place in the dermis rather than in the epidermis.

  5. Short communication: Initial evidence supporting existence of potential rumen epidermal stem and progenitor cells.

    PubMed

    Yohe, T T; Tucker, H L M; Parsons, C L M; Geiger, A J; Akers, R M; Daniels, K M

    2016-09-01

    The bovine rumen epidermis is a keratinized multilayered tissue that experiences persistent cell turnover. Because of this constant cell turnover, epidermal stem cells and their slightly more differentiated daughter cells, epidermal progenitor cells, must exist in the stratum basale of rumen epidermis. To date, these 2 epidermal cell populations and any unique cellular markers they may possess remain completely uncharacterized in the bovine rumen. An important first step in this new research area is the demonstration of the relative abundance and existence of markers for these cells in rumen tissue. A related second step is to document rumen epidermal proliferative responses to an extrinsic signal such as nutrient concentration within the rumen. The objectives of this experiment were to evaluate the extrinsic effect of diet on (1) gene expression of 6 potential rumen epidermal stem or progenitor cell markers and (2) rumen epidermal cell proliferation within the stratum basale. Twelve preweaned Holstein heifers were fed either a restricted diet (R) or an enhanced diet (EH). Animals on R received a milk replacer (MR) diet fed at 0.44kg of powder dry matter (DM)/d (20.9% crude protein, 29.8% fat, DM basis) and EH received MR at 1.08kg of powder dry matter/d (28.9% crude protein, 26.2% fat, DM basis). All calves had access to a 20% crude protein starter and were weaned during wk 7 of the experiment. Lifetime DM intake was 0.73kg of DM/calf per day for R (5.88 Mcal of net energy/calf per day) and 1.26kg of DM/calf per day for EH (10.68 Mcal of net energy/calf per day). Twenty-four hours before slaughter heifers received an intravenous dose of 5-bromo-2'-deoxyuridine to label proliferating cells. Heifers were slaughtered at 8 wk of age, and rumen samples from the ventral sac region were obtained and stored in RNA preservative and processed for routine histology. Quantitative real-time reverse transcriptase PCR was used to analyze relative abundance of genes. Candidate

  6. Heterogeneity of epidermal growth factor binding kinetics on individual cells.

    PubMed Central

    Chung, J C; Sciaky, N; Gross, D J

    1997-01-01

    Binding of fluorescein-conjugated epidermal growth factor (EGF) to individual A431 cells at 4 degrees C is measured by a quantitative fluorescence imaging technique. After background fluorescence and cell autofluorescence photobleaching corrections, the kinetic data are fit to simple models of one monovalent site and two independent monovalent sites, both of which include a first-order dye photobleaching process. Model simulations and the results from data analysis indicate that the one-monovalent-site model does not describe EGF binding kinetics at the single-cell level, whereas the two-site model is consistent with, but not proved by, the single-cell binding data. In addition, the kinetics of binding of fluorescein-EGF to different cells from the same coverslip often differ significantly from each other, indicating cell-to-cell variations in the binding properties of the EGF receptor. PMID:9251825

  7. Effects of Nitrogen on Mesophyll Cell Division and Epidermal Cell Elongation in Tall Fescue Leaf Blades 1

    PubMed Central

    MacAdam, Jennifer W.; Volenec, Jeffrey J.; Nelson, Curtis J.

    1989-01-01

    Leaf elongation rate (LER) in grasses is dependent on epidermal cell supply (number) and on rate and duration of epidermal cell elongation. Nitrogen (N) fertilization increases LER. Longitudinal sections from two genotypes of tall fescue (Festuca arundinacea Schreb.), which differ by 50% in LER, were used to quantify the effects of N on the components of epidermal cell elongation and on mesophyll cell division. Rate and duration of epidermal cell elongation were determined by using a relationship between cell length and displacement velocity derived from the continuity equation. Rate of epidermal cell elongation was exponential. Relative rates of epidermal cell elongation increased by 9% with high N, even though high N increased LER by 89%. Duration of cell elongation was approximately 20 h longer in the high- than in the low-LER genotype regardless of N treatment. The percentage of mesophyll cells in division was greater in the high- than in the low-LER genotype. This increased with high N in both genotypes, indicating that LER increased with cell supply. Division of mesophyll cells adjacent to abaxial epidermal cells continued after epidermal cell division stopped, until epidermal cells had elongated to a mean length of 40 micrometers in the high-LER and a mean length of 50 micrometers in the low-LER genotype. The cell cycle length for mesophyll cells was calculated to be 12 to 13 hours. Nitrogen increased mesophyll cell number more than epidermal cell number: in both genotypes, the final number of mesophyll cells adjacent to each abaxial epidermal cell was 10 with low N and 14 with high N. A spatial model is used to describe three cell development processes relevant to leaf growth. It illustrates the overlap of mesophyll cell division and epidermal cell elongation, and the transition from epidermal cell elongation to secondary cell wall deposition. PMID:16666581

  8. Streptococcus induces circulating CLA(+) memory T-cell-dependent epidermal cell activation in psoriasis.

    PubMed

    Ferran, Marta; Galván, Ana B; Rincón, Catalina; Romeu, Ester R; Sacrista, Marc; Barboza, Erika; Giménez-Arnau, Ana; Celada, Antonio; Pujol, Ramon M; Santamaria-Babí, Luis F

    2013-04-01

    Streptococcal throat infection is associated with a specific variant of psoriasis and with HLA-Cw6 expression. In this study, activation of circulating psoriatic cutaneous lymphocyte-associated antigen (CLA)(+) memory T cells cultured together with epidermal cells occurred only when streptococcal throat extracts were added. This triggered the production of Th1, Th17, and Th22 cytokines, as well as epidermal cell mediators (CXCL8, CXCL9, CXCL10, and CXCL11). Streptococcal extracts (SEs) did not induce any activation with either CLA(-) cells or memory T cells cultured together with epidermal cells from healthy subjects. Intradermal injection of activated culture supernatants into mouse skin induced epidermal hyperplasia. SEs also induced activation when we used epidermal cells from nonlesional skin of psoriatic patients with CLA(+) memory T cells. Significant correlations were found between SE induced upregulation of mRNA expression for ifn-γ, il-17, il-22, ip-10, and serum level of antistreptolysin O in psoriatic patients. This study demonstrates the direct involvement of streptococcal infection in pathological mechanisms of psoriasis, such as IL-17 production and epidermal cell activation.

  9. Measurement of relative phase distribution of onion epidermal cells by using the polarization microscope

    NASA Astrophysics Data System (ADS)

    Shin, In Hee; Lee, Ji Yong; Lee, Seungrag; Lee, Dong Ju; Kim, Dug Young

    2007-02-01

    Bio-cells and tissues have intrinsic polarization characteristics, which are changed by external stimulus and internal metamorphosis in cells and tissues and some of the bio-cells and tissues have intrinsic birefringence characteristics, which are also changed by external stimulus and internal metamorphosis in cells and tissues. In this paper, we have developed the polarization microscope for measurement of relative phase which results from birefringence characteristics of materials with improved linear polarizing method and have measured relative phase distribution of onion epidermal cells. From the measurement of the relative phase distribution of onion epidermal cells, decrease of relative phase distribution of onion epidermal cells was investigated as the elapse of time. In decrease of relative phase distribution, relative phase of cell membrane in onion epidermal cells decreased radically as compared with that of cytoplasm because decline of function in cell membrane that takes charge of matter transfer in onion epidermal cells has occurred.

  10. Growth of melanocytes in human epidermal cell cultures

    SciTech Connect

    Staiano-Coico, L.; Hefton, J.M.; Amadeo, C.; Pagan-Charry, I.; Madden, M.R.; Cardon-Cardo, C. )

    1990-08-01

    Epidermal cell cultures were grown in keratinocyte-conditioned medium for use as burn wound grafts; the melanocyte composition of the grafts was studied under a variety of conditions. Melanocytes were identified by immunohistochemistry based on a monoclonal antibody (MEL-5) that has previously been shown to react specifically with melanocytes. During the first 7 days of growth in primary culture, the total number of melanocytes in the epidermal cultures decreased to 10% of the number present in normal skin. Beginning on day 2 of culture, bipolar melanocytes were present at a mean cell density of 116 +/- 2/mm2; the keratinocyte to melanocyte ratio was preserved during further primary culture and through three subpassages. Moreover, exposure of cultures to mild UVB irradiation stimulated the melanocytes to proliferate, suggesting that the melanocytes growing in culture maintained their responsiveness to external stimuli. When the sheets of cultured cells were enzymatically detached from the plastic culture flasks before grafting, melanocytes remained in the basal layer of cells as part of the graft applied to the patient.

  11. Dedifferentiation derived cells exhibit phenotypic and functional characteristics of epidermal stem cells.

    PubMed

    Zhang, Cuiping; Fu, Xiaobing; Chen, Peng; Bao, Xiaoxia; Li, Fu; Sun, Xiaoyan; Lei, Yonghong; Cai, Sa; Sun, Tongzhu; Sheng, Zhiyong

    2010-05-01

    Differentiated epidermal cells can dedifferentiate into stem cells or stem cell-like cells in vivo. In this study, we report the isolation and characterization of dedifferentiation-derived cells. Epidermal sheets eliminated of basal stem cells were transplanted onto the skin wounds in 47 nude athymic (BALB/c-nu/nu) mice. After 5 days, cells negative for CK10 but positive for CK19 and beta1-integrin emerged at the wound-neighbouring side of the epidermal sheets. Furthermore, the percentages of CK19 and beta1-integrin+ cells detected by flow cytometric analysis were increased after grafting (P < 0.01) and CK10+ cells in grafted sheets decreased (P < 0.01). Then we isolated these cells on the basis of rapid adhesion to type IV collagen and found that there were 4.56% adhering cells (dedifferentiation-derived cells) in the grafting group within 10 min. The in vitro phenotypic assays showed that the expressions of CK19, beta1-integrin, Oct4 and Nanog in dedifferentiation-derived cells were remarkably higher than those in the control group (differentiated epidermal cells) (P < 0.01). In addition, the results of the functional investigation of dedifferentiation-derived cells demonstrated: (1) the numbers of colonies consisting of 5-10 cells and greater than 10 cells were increased 5.9-fold and 6.7-fold, respectively, as compared with that in the control (P < 0.01); (2) more cells were in S phase and G2/M phase of the cell cycle (proliferation index values were 21.02% in control group, 45.08% in group of dedifferentiation); (3) the total days of culture (28 days versus 130 days), the passage number of cells (3 passages versus 20 passages) and assumptive total cell output (1 x 10(5) cells versus 1 x 10(12) cells) were all significantly increased and (4) dedifferentiation-derived cells, as well as epidermal stem cells, were capable of regenerating a skin equivalent, but differentiated epidermal cells could not. These results suggested that the characteristics of

  12. Programmed Cell Death Progresses Differentially in Epidermal and Mesophyll Cells of Lily Petals

    PubMed Central

    Mochizuki-Kawai, Hiroko; Niki, Tomoko; Shibuya, Kenichi; Ichimura, Kazuo

    2015-01-01

    In the petals of some species of flowers, programmed cell death (PCD) begins earlier in mesophyll cells than in epidermal cells. However, PCD progression in each cell type has not been characterized in detail. We separately constructed a time course of biochemical signs and expression patterns of PCD-associated genes in epidermal and mesophyll cells in Lilium cv. Yelloween petals. Before visible signs of senescence could be observed, we found signs of PCD, including DNA degradation and decreased protein content in mesophyll cells only. In these cells, the total proteinase activity increased on the day after anthesis. Within 3 days after anthesis, the protein content decreased by 61.8%, and 22.8% of mesophyll cells was lost. A second peak of proteinase activity was observed on day 6, and the number of mesophyll cells decreased again from days 4 to 7. These biochemical and morphological results suggest that PCD progressed in steps during flower life in the mesophyll cells. PCD began in epidermal cells on day 5, in temporal synchrony with the time course of visible senescence. In the mesophyll cells, the KDEL-tailed cysteine proteinase (LoCYP) and S1/P1 nuclease (LoNUC) genes were upregulated before petal wilting, earlier than in epidermal cells. In contrast, relative to that in the mesophyll cells, the expression of the SAG12 cysteine proteinase homolog (LoSAG12) drastically increased in epidermal cells in the final stage of senescence. These results suggest that multiple PCD-associated genes differentially contribute to the time lag of PCD progression between epidermal and mesophyll cells of lily petals. PMID:26605547

  13. Epidermal Development in Mammals: Key Regulators, Signals from Beneath, and Stem Cells

    PubMed Central

    Liu, Shuang; Zhang, Huishan; Duan, Enkui

    2013-01-01

    Epidermis is one of the best-studied tissues in mammals that contain types of stem cells. Outstanding works in recent years have shed great light on behaviors of different epidermal stem cell populations in the homeostasis and regeneration of the epidermis as well as hair follicles. Also, the molecular mechanisms governing these stem cells are being elucidated, from genetic to epigenetic levels. Compared with the explicit knowledge about adult skin, embryonic development of the epidermis, especially the early period, still needs exploration. Furthermore, stem cells in the embryonic epidermis are largely unstudied or ambiguously depicted. In this review, we will summarize and discuss the process of embryonic epidermal development, with focuses on some key molecular regulators and the role of the sub-epidermal mesenchyme. We will also try to trace adult epidermal stem cell populations back to embryonic development. In addition, we will comment on in vitro derivation of epidermal lineages from ES cells and iPS cells. PMID:23708093

  14. Role of DNA endoreduplication, lipotubuloids, and gibberellic acid in epidermal cell growth during fruit development of Ornithogalum umbellatum.

    PubMed

    Kwiatkowska, Maria; Poplonska, Katarzyna; Kazmierczak, Andrzej; Stepinski, Dariusz; Rogala, Katarzyna; Polewczyk, Katarzyna

    2007-01-01

    Cytophotometry of individual nuclei was used to examine the level of endoreduplication in epidermal cells from the upper and lower parts of the ovary during Ornithogalum umbellatum flower and fruit development. An increase in DNA content from 2-4C to 2-8C in both parts of the ovary was observed, while the epidermal cell surface area grew about 6-fold and 15-fold in the lower and upper parts of the ovary, respectively. However, the correlation between mean epidermal cell size and ploidy was distinct during epidermis growth. Lipotubuloids became bigger in the upper than in the lower part during ovary and fruit development. In addition, more dynamic growth of the epidermal cells of the upper than of the lower part of the ovary was connected to the higher content of gibberellic acid. A hypothesis has been put forward that the role of DNA endoreduplication in epidermal cell growth was modulated by the function of lipotubuloids and the gradient of gibberellin.

  15. Effects of Wnt3a on proliferation and differentiation of human epidermal stem cells

    SciTech Connect

    Jia Liwei; Zhou Jiaxi; Peng Sha; Li Juxue; Cao Yujing; Duan Enkui

    2008-04-11

    Epidermal stem cells maintain development and homeostasis of mammalian epidermis throughout life. However, the molecular mechanisms involved in the proliferation and differentiation of epidermal stem cells are far from clear. In this study, we investigated the effects of Wnt3a and Wnt/{beta}-catenin signaling on proliferation and differentiation of human fetal epidermal stem cells. We found both Wnt3a and active {beta}-catenin, two key members of the Wnt/{beta}-catenin signaling, were expressed in human fetal epidermis and epidermal stem cells. In addition, Wnt3a protein can promote proliferation and inhibit differentiation of epidermal stem cells in vitro culture. Our results suggest that Wnt/{beta}-catenin signaling plays important roles in human fetal skin development and homeostasis, which also provide new insights on the molecular mechanisms of oncogenesis in human epidermis.

  16. Whole organ, venation and epidermal cell morphological variations are correlated in the leaves of Arabidopsis mutants.

    PubMed

    Pérez-Pérez, José Manuel; Rubio-Díaz, Silvia; Dhondt, Stijn; Hernández-Romero, Diana; Sánchez-Soriano, Joaquín; Beemster, Gerrit T S; Ponce, María Rosa; Micol, José Luis

    2011-12-01

    Despite the large number of genes known to affect leaf shape or size, we still have a relatively poor understanding of how leaf morphology is established. For example, little is known about how cell division and cell expansion are controlled and coordinated within a growing leaf to eventually develop into a laminar organ of a definite size. To obtain a global perspective of the cellular basis of variations in leaf morphology at the organ, tissue and cell levels, we studied a collection of 111 non-allelic mutants with abnormally shaped and/or sized leaves, which broadly represent the mutational variations in Arabidopsis thaliana leaf morphology not associated with lethality. We used image-processing techniques on these mutants to quantify morphological parameters running the gamut from the palisade mesophyll and epidermal cells to the venation, whole leaf and rosette levels. We found positive correlations between epidermal cell size and leaf area, which is consistent with long-standing Avery's hypothesis that the epidermis drives leaf growth. In addition, venation parameters were positively correlated with leaf area, suggesting that leaf growth and vein patterning share some genetic controls. Positional cloning of the genes affected by the studied mutations will eventually establish functional links between genotypes, molecular functions, cellular parameters and leaf phenotypes.

  17. Langerhans Cells Facilitate UVB-induced Epidermal Carcinogenesis

    PubMed Central

    Lewis, Julia M.; Bürgler, Christina D.; Freudzon, Marianna; Golubets, Kseniya; Gibson, Juliet F.; Filler, Renata B.; Girardi, Michael

    2015-01-01

    Ultraviolet B (UVB) light is considered the major environmental inducer of human keratinocyte DNA mutations, including within the tumor-suppressor gene p53, and chronic exposure is associated with cutaneous squamous cell carcinoma (SCC) formation. Langerhans cells (LC) comprise a dendritic network within the suprabasilar epidermis, yet the role of LC in UVB-induced carcinogenesis is largely unknown. Herein, we show that LC-intact epidermis develops UVB-induced tumors more readily than LC-deficient epidermis. While levels of epidermal cyclopyrimidine dimers (CPD) following acute UVB exposure are equivalent in the presence or absence of LC, chronic UVB-induced p53 mutant clonal islands expand more readily in association with LC which remain largely intact and are preferentially found in proximity to the expanding mutant keratinocyte populations. The observed LC facilitation of mutant p53 clonal expansion is completely αβ and γδ T-cell independent, and is associated with increased intraepidermal expression of interleukin (IL)-22 and the presence of group 3 innate lymphoid cells (ILC3). These data demonstrate that LC play a key role in UVB-induced cutaneous carcinogenesis, and suggest that LC locally stimulate keratinocyte proliferation and innate immune cells that provoke tumor outgrowth. PMID:26053049

  18. Retinoid-mediated transcriptional regulaton of keratin genes in human epidermal and squamous cell carcinoma cells

    SciTech Connect

    Stellmach, V.; Leask, A.; Fuchs, E. )

    1991-06-01

    Vitamin A and other retinoids profoundly inhibit morphological and biochemical heatures of epidermal differentiation in vivo and in vitro. To elucidate the molecular mechanisms underlying the differential expression of epidermal keratins and their regulation by retinoids, the authors retinoid-mediated changes in total protein expression, protein synthesis, mRNA expression, and transcription in cultured human keratinocytes and in squamous cell carcinoma (SCC-13) cells of epidermal origin. The studies revealed that the epidermal keratins, K5, K6, K14, and K16, their mRNAs, and their transcripts were diminished relative to actin as a consequence of retinoic acid (RA) treatment. The effects were most pronounced in SCC-13 and were detected as early as 6 hr post-RA treatment, with enhancement over an additional 24-48 hr. Repression was also observed when 5{prime} upstream sequences of K14 or K5 genes were used to drive expression of a chloramphenicol acetyltransferase reporter gene in SCC-13 keratinocytes. Both cell types were found to express mRNAs for the RA receptors {alpha} and {gamma}, which may be involved in the RA-mediated transcriptional changes in these cells. The rapid transcriptional changes in epidermal keratin genes were in striking contrast to the previously reported slow transcriptional changes in simple epithelial keratin genes.

  19. Arsenite maintains germinative state in cultured human epidermal cells

    SciTech Connect

    Patterson, Timothy J.; Reznikova, Tatiana V.; Phillips, Marjorie A.; Rice, Robert H. . E-mail: rhrice@ucdavis.edu

    2005-08-22

    Arsenic is a well-known carcinogen for human skin, but its mechanism of action and proximal macromolecular targets remain to be elucidated. In the present study, low micromolar concentrations of sodium arsenite maintained the proliferative potential of epidermal keratinocytes, decreasing their exit from the germinative compartment under conditions that promote differentiation of untreated cells. This effect was observed in suspension and in post-confluent surface cultures as measured by colony-forming ability and by proportion of rapidly adhering colony-forming cells. Arsenite-treated cultures exhibited elevated levels of {beta}1-integrin and {beta}-catenin, two proteins enriched in cells with high proliferative potential. Levels of phosphorylated (inactive) glycogen synthase kinase 3{beta} were higher in the treated cultures, likely accounting for the increased levels of transcriptionally available {beta}-catenin. These findings suggest that arsenic could have co-carcinogenic and tumor co-promoting activities in the epidermis as a result of increasing the population and persistence of germinative cells targeted by tumor initiators and promoters. These findings also identify a critical signal transduction pathway meriting further exploration in pursuit of this phenomenon.

  20. Targeting epidermal growth factor receptor for head and neck squamous cell carcinoma: still lost in translation?

    PubMed Central

    Chapman, Christopher H.; Saba, Nabil F.

    2016-01-01

    The epidermal growth factor receptor (EGFR) is preferentially expressed in head and neck squamous cell carcinoma (HNSCC), and is a promising therapeutic target. Yet other than cetuximab, no agent targeting EGFR has been approved for this disease, and none has shown benefit over the standard of care. Several randomized trials of antibody and small molecule agents have found no new indication for these agents, despite their initial promise. In this review, we examine the major clinical evidence and discuss potential future developments of translational science in this area, including use of these agents in risk-stratified subgroups, inhibition of downstream/parallel targets, and combination with immunotherapy. PMID:27004227

  1. Epigenetic Regulation of Epidermal Stem Cell Biomarkers and Their Role in Wound Healing

    PubMed Central

    Saldanha, Sabita N.; Royston, Kendra J.; Udayakumar, Neha; Tollefsbol, Trygve O.

    2015-01-01

    As an actively renewable tissue, changes in skin architecture are subjected to the regulation of stem cells that maintain the population of cells responsible for the formation of epidermal layers. Stems cells retain their self-renewal property and express biomarkers that are unique to this population. However, differential regulation of the biomarkers can initiate the pathway of terminal cell differentiation. Although, pockets of non-clarity in stem cell maintenance and differentiation in skin still exist, the influence of epigenetics in epidermal stem cell functions and differentiation in skin homeostasis and wound healing is clearly evident. The focus of this review is to discuss the epigenetic regulation of confirmed and probable epidermal stem cell biomarkers in epidermal stratification of normal skin and in diseased states. The role of epigenetics in wound healing, especially in diseased states of diabetes and cancer, will also be conveyed. PMID:26712738

  2. Epigenetic Regulation of Epidermal Stem Cell Biomarkers and Their Role in Wound Healing.

    PubMed

    Saldanha, Sabita N; Royston, Kendra J; Udayakumar, Neha; Tollefsbol, Trygve O

    2015-12-24

    As an actively renewable tissue, changes in skin architecture are subjected to the regulation of stem cells that maintain the population of cells responsible for the formation of epidermal layers. Stems cells retain their self-renewal property and express biomarkers that are unique to this population. However, differential regulation of the biomarkers can initiate the pathway of terminal cell differentiation. Although, pockets of non-clarity in stem cell maintenance and differentiation in skin still exist, the influence of epigenetics in epidermal stem cell functions and differentiation in skin homeostasis and wound healing is clearly evident. The focus of this review is to discuss the epigenetic regulation of confirmed and probable epidermal stem cell biomarkers in epidermal stratification of normal skin and in diseased states. The role of epigenetics in wound healing, especially in diseased states of diabetes and cancer, will also be conveyed.

  3. 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.

  4. Gloss, colour and grip: multifunctional epidermal cell shapes in bee- and bird-pollinated flowers.

    PubMed

    Papiorek, Sarah; Junker, Robert R; Lunau, Klaus

    2014-01-01

    Flowers bear the function of filters supporting the attraction of pollinators as well as the deterrence of floral antagonists. The effect of epidermal cell shape on the visual display and tactile properties of flowers has been evaluated only recently. In this study we quantitatively measured epidermal cell shape, gloss and spectral reflectance of flowers pollinated by either bees or birds testing three hypotheses: The first two hypotheses imply that bee-pollinated flowers might benefit from rough surfaces on visually-active parts produced by conical epidermal cells, as they may enhance the colour signal of flowers as well as the grip on flowers for bees. In contrast, bird-pollinated flowers might benefit from flat surfaces produced by flat epidermal cells, by avoiding frequent visitation from non-pollinating bees due to a reduced colour signal, as birds do not rely on specific colour parameters while foraging. Moreover, flat petal surfaces in bird-pollinated flowers may hamper grip for bees that do not touch anthers and stigmas while consuming nectar and thus, are considered as nectar thieves. Beside this, the third hypothesis implies that those flower parts which are vulnerable to nectar robbing of bee- as well as bird-pollinated flowers benefit from flat epidermal cells, hampering grip for nectar robbing bees. Our comparative data show in fact that conical epidermal cells are restricted to visually-active parts of bee-pollinated flowers, whereas robbing-sensitive parts of bee-pollinated as well as the entire floral surface of bird-pollinated flowers possess on average flat epidermal cells. However, direct correlations between epidermal cell shape and colour parameters have not been found. Our results together with published experimental studies show that epidermal cell shape as a largely neglected flower trait might act as an important feature in pollinator attraction and avoidance of antagonists, and thus may contribute to the partitioning of flower-visitors.

  5. Basal Cell Carcinoma Arising on a Verrucous Epidermal Nevus: A Case Report

    PubMed Central

    Viana, Analia; Aguinaga, Felipe; Marinho, Flauberto; Rodrigues, Rosangela; Cuzzi, Tullia; Ramos-e-Silva, Marcia

    2015-01-01

    We report a case of basal cell carcinoma that appeared from an epidermal verrucous nevus in a 61-year-old patient. The onset of basal cell carcinoma in sebaceous nevi, basal cell nevi and dysplastic nevi is relatively common, but it is rarely associated with epidermal verrucous nevi. There is no consensus on whether the two lesions have a common cellular origin or whether they merely represent a collision of two distinct tumors. Since this association – as with other malignant tumors – is rare, there is no need for prophylactic removal of epidermal verrucous nevi. PMID:25848348

  6. Pleiotropic age-dependent effects of mitochondrial dysfunction on epidermal stem cells.

    PubMed

    Velarde, Michael C; Demaria, Marco; Melov, Simon; Campisi, Judith

    2015-08-18

    Tissue homeostasis declines with age partly because stem/progenitor cells fail to self-renew or differentiate. Because mitochondrial damage can accelerate aging, we tested the hypothesis that mitochondrial dysfunction impairs stem cell renewal or function. We developed a mouse model, Tg(KRT14-cre/Esr1) (20Efu/J) × Sod2 (tm1Smel) , that generates mitochondrial oxidative stress in keratin 14-expressing epidermal stem/progenitor cells in a temporally controlled manner owing to deletion of Sod2, a nuclear gene that encodes the mitochondrial antioxidant enzyme superoxide dismutase 2 (Sod2). Epidermal Sod2 loss induced cellular senescence, which irreversibly arrested proliferation in a fraction of keratinocytes. Surprisingly, in young mice, Sod2 deficiency accelerated wound closure, increasing epidermal differentiation and reepithelialization, despite the reduced proliferation. In contrast, at older ages, Sod2 deficiency delayed wound closure and reduced epidermal thickness, accompanied by epidermal stem cell exhaustion. In young mice, Sod2 deficiency accelerated epidermal thinning in response to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, phenocopying the reduced regeneration of older Sod2-deficient skin. Our results show a surprising beneficial effect of mitochondrial dysfunction at young ages, provide a potential mechanism for the decline in epidermal regeneration at older ages, and identify a previously unidentified age-dependent role for mitochondria in skin quality and wound closure.

  7. Epidermal Viral Immunity Induced by CD8α+ Dendritic Cells But Not by Langerhans Cells

    NASA Astrophysics Data System (ADS)

    Allan, Rhys S.; Smith, Chris M.; Belz, Gabrielle T.; van Lint, Allison L.; Wakim, Linda M.; Heath, William R.; Carbone, Francis R.

    2003-09-01

    The classical paradigm for dendritic cell function derives from the study of Langerhans cells, which predominate within skin epidermis. After an encounter with foreign agents, Langerhans cells are thought to migrate to draining lymph nodes, where they initiate T cell priming. Contrary to this, we show here that infection of murine epidermis by herpes simplex virus did not result in the priming of virus-specific cytotoxic T lymphocytes by Langerhans cells. Rather, the priming response required a distinct CD8α+ dendritic cell subset. Thus, the traditional view of Langerhans cells in epidermal immunity needs to be revisited to accommodate a requirement for other dendritic cells in this response.

  8. Isolation and cultivation of dermal stem cells that differentiate into functional epidermal melanocytes.

    PubMed

    Li, Ling; Fukunaga-Kalabis, Mizuho; Herlyn, Meenhard

    2012-01-01

    Human melanocytes have been extensively studied, but a melanocyte stem cell reservoir in glabrous skin has not yet been found. Human dermis contains cells that are nonpigmented but can differentiate to several different cell types. We have recently shown that multipotent dermal stem cells isolated from human neonatal foreskins are able to differentiate to multiple cell lineages, including pigmented melanocytes. The dermal stem cells grow as three-dimensional spheres in human embryonic stem cell medium and express some neural crest stem cell and embryonic stem cell markers. Melanocytes derived from dermal stem cells express melanocytic markers and act the same way as mature epidermal melanocytes. Dermal spheres, embedded in the reconstructed dermis consisting of collagen with fibroblasts, can migrate to the basement membrane, where they become pigmented in the same way as epidermal melanocytes suggesting that dermal stem cells can give rise to epidermal melanocytes.

  9. In vitro suppression of the epidermal Langerhans' cells in necro split skin.

    PubMed

    Alsbjörn, B F; Nielsen, S L; Jensen, M G

    1987-01-01

    The effect of ultraviolet light B irradiation and glucocorticosteroid incubation on the epidermal Langerhans' cell density and tissue viability was investigated, in vitro, on human thin necro split skin.

  10. Mechanosensory calcium-selective cation channels in epidermal cells

    NASA Technical Reports Server (NTRS)

    Ding, J. P.; Pickard, B. G.

    1993-01-01

    This paper explores the properties and likely functions of an epidermal Ca(2+)-selective cation channel complex activated by tension. As many as eight or nine linked or linkable equivalent conductance units or co-channels can open together. Open time for co-channel quadruplets and quintuplets tends to be relatively long with millimolar Mg2+ (but not millimolar Ca2+) at the cytosolic face of excised plasma membrane. Sensitivity to tension is regulated by transmembrane voltage and temperature. Under some circumstances channel activity is sychronized in rhythmic pulses. Certain lanthanides and a cytoskeleton-disturbing herbicide that inhibit gravitropic reception act on the channel system at low concentrations. Specifically, ethyl-N-phenylcarbamate promotes tension-dependent activity at micromolar levels. With moderate suction, Gd3+ provided at about 0.5 micromole at the extracellular face of the membrane promotes for several seconds but may then become inhibitory. Provision at 1-2 micromoles promotes and subsequently inhibits more vigorously (often abruptly and totally), and at high levels inhibits immediately. La3+, a poor gravitropic inhibitor, acts similarly but much more gradually and only at much higher concentrations. These properties, particularly these susceptibilities to modulation, indicate that in vivo the mechanosensitive channel must be mechanosensory and mechanoregulatory. It could serve to transduce the shear forces generated in the integrated wall-membrane-cytoskeleton system during turgor changes and cell expansion as well as transducing the stresses induced by gravity, touch and flexure. In so far as such transduction is modulated by voltage and temperature, the channels would also be sensors for these modalities as long as the wall-membrane-cytoskeleton system experiences mechanical stress.

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

    SciTech Connect

    Han, Chang Yeob; Hien, Tran Thi; Lim, Sung Chul; Kang, Keon Wook

    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, we 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.

  12. Glucose and ethylene signalling pathways converge to regulate trans-differentiation of epidermal transfer cells in Vicia narbonensis cotyledons.

    PubMed

    Andriunas, Felicity A; Zhang, Hui-Ming; Weber, Hans; McCurdy, David W; Offler, Christina E; Patrick, John W

    2011-12-01

    Transfer cells are specialized transport cells containing invaginated wall ingrowths that provide an amplified plasma membrane surface area with high densities of transporter proteins. They trans-differentiate from differentiated cells at sites where enhanced rates of nutrient transport occur across apo/symplasmic boundaries. Despite their physiological importance, the signal(s) and signalling cascades responsible for initiating their trans-differentiation are poorly understood. In culture, adaxial epidermal cells of Vicia narbonensis cotyledons were induced to trans-differentiate to a transfer cell morphology. Manipulating their intracellular glucose concentrations by transgenic knock-down of ADP-glucose pyrophosphorylase expression and/or culture on a high-glucose medium demonstrated that glucose functioned as a negative regulator of wall ingrowth induction. In contrast, glucose had no detectable effect on wall ingrowth morphology. The effect on wall ingrowth induction of culture on media containing glucose analogues suggested that glucose acts through a hexokinase-dependent signalling pathway. Elevation of an epidermal cell-specific ethylene signal alone, or in combination with glucose analogues, countered the negative effect of glucose on wall ingrowth induction. Glucose modulated the amplitude of ethylene-stimulated wall ingrowth induction by down-regulating the expression of ethylene biosynthetic genes and an ethylene insensitive 3 (EIN3)-like gene (EIL) encoding a key transcription factor in the ethylene signalling cascade. A model is presented describing the interaction between glucose and ethylene signalling pathways regulating the induction of wall ingrowth formation in adaxial epidermal cells.

  13. In situ localization of epidermal stem cells using a novel multi epitope ligand cartography approach.

    PubMed

    Ruetze, Martin; Gallinat, Stefan; Wenck, Horst; Deppert, Wolfgang; Knott, Anja

    2010-06-01

    Precise knowledge of the frequency and localization of epidermal stem cells within skin tissue would further our understanding of their role in maintaining skin homeostasis. As a novel approach we used the recently developed method of multi epitope ligand cartography, applying a set of described putative epidermal stem cell markers. Bioinformatic evaluation of the data led to the identification of several discrete basal keratinocyte populations, but none of them displayed the complete stem cell marker set. The distribution of the keratinocyte populations within the tissue was remarkably heterogeneous, but determination of distance relationships revealed a population of quiescent cells highly expressing p63 and the integrins alpha(6)/beta(1) that represent origins of a gradual differentiation lineage. This population comprises about 6% of all basal cells, shows a scattered distribution pattern and could also be found in keratinocyte holoclone colonies. The data suggest that this population identifies interfollicular epidermal stem cells.

  14. Epidermal Merkel Cells are Mechanosensory Cells that Tune Mammalian Touch Receptors

    PubMed Central

    Maksimovic, Srdjan; Nakatani, Masashi; Baba, Yoshichika; Nelson, Aislyn M.; Marshall, Kara L.; Wellnitz, Scott A.; Firozi, Pervez; Woo, Seung-Hyun; Ranade, Sanjeev; Patapoutian, Ardem; Lumpkin, Ellen A.

    2014-01-01

    Touch submodalities, such as flutter and pressure, are mediated by somatosensory afferents whose terminal specializations extract tactile features and encode them as action potential trains with unique activity patterns1. Whether non-neuronal cells tune touch receptors through active or passive mechanisms is debated. Terminal specializations are thought to function as passive mechanical filters analogous to the cochlea’s basilar membrane, which deconstructs complex sounds into tones that are transduced by mechanosensory hair cells. The model that cutaneous specializations are merely passive has been recently challenged because epidermal cells express sensory ion channels and neurotransmitters2,3; however, direct evidence that epidermal cells excite tactile afferents is lacking. Epidermal Merkel cells display features of sensory receptor cells4,5 and make “synapse-like” contacts5,6 with slowly adapting type I (SAI) afferents7–9. These complexes, which encode spatial features such as edges and texture1, localize to skin regions with high tactile acuity, including whisker follicles, fingertips and touch domes. Here, we show that Merkel cells actively participate in touch reception in mice. First, Merkel cells display fast, touch-evoked mechanotransduction currents. Second, optogenetic approaches in intact skin show that Merkel cells are both necessary and sufficient for sustained action-potential firing in tactile afferents. Third, recordings from touch-dome afferents lacking Merkel cells demonstrate that Merkel cells confer high-frequency responses to dynamic stimuli and enable sustained firing. These data are the first to directly demonstrate a functional, excitatory connection between epidermal cells and sensory neurons. Together, these findings indicate that Merkel cells actively tune mechanosensory responses to facilitate high spatio-temporal acuity. Moreover, our results suggest a division of labour in the Merkel cell-neurite complex: Merkel cells signal

  15. Enrichment of epidermal stem cells of rats by Vario magnetic activated cell sorting system.

    PubMed

    Chen, Wei; Zhang, Wei-wei; Shi, Chunying; Lian, Xiaohua; Yi, Shanghong; Yang, Tian

    2013-09-01

    Epidermal stem cells (ESCs) play an important role in skin homeostasis, wound repair, and tumorigensis which have great potential in scientific research and clinical application. So, the efficient isolation of these infrequent stem cells is very important for researchers to solve the problem of low purity and insufficient quantity of stem cells in vitro. The aim of this study was to investigate a method for the enrichment of ESCs by magnetic activated cell sorting system. The isolation strategy was CD71 depletion followed by α6-integrin positive selection. The percentage of α6(bri)CD71(dim) cells in isolated cells was 94.59%. Transmission electron microscopy results revealed that α6(bri) CD71(dim) cells exhibited some typical characteristics like progenitor cells, such as big nucleus, obvious nucleolus, large nuclear-cytoplasm ratio, and few organelles in cytoplasm. When cultured in vitro, the α6(bri)CD71(dim) cells had greater proliferating potential and higher colony-forming ability, and high levels of epidermal stem cell markers were expressed in our positive cells. ESCs have been successfully isolated from neonatal epidermis using Vario MACS and cultured in vitro. This isolation method is simple, fast, and inexpensive, providing an important tool for tissue engineering and cell transplantation studies.

  16. Homologs of SCAR/WAVE complex components are required for epidermal cell morphogenesis in rice.

    PubMed

    Zhou, Wenqi; Wang, Yuchuan; Wu, Zhongliang; Luo, Liang; Liu, Ping; Yan, Longfeng; Hou, Suiwen

    2016-07-01

    Filamentous actins (F-actins) play a vital role in epidermal cell morphogenesis. However, a limited number of studies have examined actin-dependent leaf epidermal cell morphogenesis events in rice. In this study, two recessive mutants were isolated: less pronounced lobe epidermal cell2-1 (lpl2-1) and lpl3-1, whose leaf and stem epidermis developed a smooth surface, with fewer serrated pavement cell (PC) lobes, and decreased papillae. The lpl2-1 also exhibited irregular stomata patterns, reduced plant height, and short panicles and roots. Molecular genetic studies demonstrated that LPL2 and LPL3 encode the PIROGI/Specifically Rac1-associated protein 1 (PIR/SRA1)-like and NCK-associated protein 1 (NAP1)-like proteins, respectively, two components of the suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (SCAR/WAVE) regulatory complex involved in actin nucleation and function. Epidermal cells exhibited abnormal arrangement of F-actins in both lpl2 and lpl3 expanding leaves. Moreover, the distorted trichomes of Arabidopsis pir could be partially restored by an overexpression of LPL2 A yeast two-hybrid assay revealed that LPL2 can directly interact with LPL3 in vitro Collectively, the results indicate that LPL2 and LPL3 are two functionally conserved homologs of the SCAR/WAVE complex components, and that they play an important role in controlling epidermal cell morphogenesis in rice by organising F-actin.

  17. 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.

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

    PubMed

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

    2016-01-01

    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.

  19. Abscisic acid induces ectopic outgrowth in epidermal cells through cortical microtubule reorganization in Arabidopsis thaliana

    PubMed Central

    Takatani, Shogo; Hirayama, Takashi; Hashimoto, Takashi; Takahashi, Taku; Motose, Hiroyasu

    2015-01-01

    Abscisic acid (ABA) regulates seed maturation, germination and various stress responses in plants. The roles of ABA in cellular growth and morphogenesis, however, remain to be explored. Here, we report that ABA induces the ectopic outgrowth of epidermal cells in Arabidopsis thaliana. Seedlings of A. thaliana germinated and grown in the presence of ABA developed ectopic protrusions in the epidermal cells of hypocotyls, petioles and cotyledons. One protrusion was formed in the middle of each epidermal cell. In the hypocotyl epidermis, two types of cell files are arranged alternately into non-stoma cell files and stoma cell files, ectopic protrusions being restricted to the non-stoma cell files. This suggests the presence of a difference in the degree of sensitivity to ABA or in the capacity of cells to form protrusions between the two cell files. The ectopic outgrowth was suppressed in ABA insensitive mutants, whereas it was enhanced in ABA hypersensitive mutants. Interestingly, ABA-induced ectopic outgrowth was also suppressed in mutants in which microtubule organization was compromised. Furthermore, cortical microtubules were disorganized and depolymerized by the ABA treatment. These results suggest that ABA signaling induces ectopic outgrowth in epidermal cells through microtubule reorganization. PMID:26068445

  20. Single-cell gene expression profiling reveals functional heterogeneity of undifferentiated human epidermal cells

    PubMed Central

    Tan, David W. M.; Jensen, Kim B.; Trotter, Matthew W. B.; Connelly, John T.; Broad, Simon; Watt, Fiona M.

    2013-01-01

    Human epidermal stem cells express high levels of β1 integrins, delta-like 1 (DLL1) and the EGFR antagonist LRIG1. However, there is cell-to-cell variation in the relative abundance of DLL1 and LRIG1 mRNA transcripts. Single-cell global gene expression profiling showed that undifferentiated cells fell into two clusters delineated by expression of DLL1 and its binding partner syntenin. The DLL1+ cluster had elevated expression of genes associated with endocytosis, integrin-mediated adhesion and receptor tyrosine kinase signalling. Differentially expressed genes were not independently regulated, as overexpression of DLL1 alone or together with LRIG1 led to the upregulation of other genes in the DLL1+ cluster. Overexpression of DLL1 and LRIG1 resulted in enhanced extracellular matrix adhesion and increased caveolin-dependent EGFR endocytosis. Further characterisation of CD46, one of the genes upregulated in the DLL1+ cluster, revealed it to be a novel cell surface marker of human epidermal stem cells. Cells with high endogenous levels of CD46 expressed high levels of β1 integrin and DLL1 and were highly adhesive and clonogenic. Knockdown of CD46 decreased proliferative potential and β1 integrin-mediated adhesion. Thus, the previously unknown heterogeneity revealed by our studies results in differences in the interaction of undifferentiated basal keratinocytes with their environment. PMID:23482486

  1. p63 and Brg1 control developmentally regulated higher-order chromatin remodelling at the epidermal differentiation complex locus in epidermal progenitor cells

    PubMed Central

    Mardaryev, Andrei N.; Gdula, Michal R.; Yarker, Joanne L.; Emelianov, Vladimir N.; Poterlowicz, Krzysztof; Sharov, Andrey A.; Sharova, Tatyana Y.; Scarpa, Julie A.; Chambon, Pierre; Botchkarev, Vladimir A.; Fessing, Michael Y.

    2014-01-01

    Chromatin structural states and their remodelling, including higher-order chromatin folding and three-dimensional (3D) genome organisation, play an important role in the control of gene expression. The role of 3D genome organisation in the control and execution of lineage-specific transcription programmes during the development and differentiation of multipotent stem cells into specialised cell types remains poorly understood. Here, we show that substantial remodelling of the higher-order chromatin structure of the epidermal differentiation complex (EDC), a keratinocyte lineage-specific gene locus on mouse chromosome 3, occurs during epidermal morphogenesis. During epidermal development, the locus relocates away from the nuclear periphery towards the nuclear interior into a compartment enriched in SC35-positive nuclear speckles. Relocation of the EDC locus occurs prior to the full activation of EDC genes involved in controlling terminal keratinocyte differentiation and is a lineage-specific, developmentally regulated event controlled by transcription factor p63, a master regulator of epidermal development. We also show that, in epidermal progenitor cells, p63 directly regulates the expression of the ATP-dependent chromatin remodeller Brg1, which binds to distinct domains within the EDC and is required for relocation of the EDC towards the nuclear interior. Furthermore, Brg1 also regulates gene expression within the EDC locus during epidermal morphogenesis. Thus, p63 and its direct target Brg1 play an essential role in remodelling the higher-order chromatin structure of the EDC and in the specific positioning of this locus within the landscape of the 3D nuclear space, as required for the efficient expression of EDC genes in epidermal progenitor cells during skin development. PMID:24346698

  2. New coal tar extract and coal tar shampoos. Evaluation by epidermal cell DNA synthesis suppression assay.

    PubMed

    Lowe, N J; Breeding, J H; Wortzman, M S

    1982-07-01

    Coal tar therapy has been used for many years in the treatment of scaling skin diseases, including psoriasis and eczema. Previous studies of the potential effectiveness of tar have utilized phototoxic erythema assays with long-wave ultraviolet light (UV-A). However, in clinical use, coal tar is rarely used with UV-A, particularly for scalp disease. Therefore, we investigated a nonphototoxic approach to evaluate different coal tar products. Coal tar was found to suppress epidermal cell DNA synthesis in the hairless mouse model, and this is the basis for the assay presented. Using the epidermal cell DNA synthesis suppression assay, we observed that crude coal tar and a new extract of crude coal tar were equally effective and that a concentration gradient effect was achieved. In addition, four commercial coal tar shampoos assayed varied greatly in their ability to suppress epidermal cell DNA synthesis. One shampoo was washed after ten minutes and no significant alteration of suppressive effect was seen.

  3. Persistence of skin-resident memory T cells within an epidermal niche

    PubMed Central

    Zaid, Ali; Mackay, Laura K.; Rahimpour, Azad; Braun, Asolina; Veldhoen, Marc; Carbone, Francis R.; Manton, Jonathan H.; Heath, William R.; Mueller, Scott N.

    2014-01-01

    Barrier tissues such as the skin contain various populations of immune cells that contribute to protection from infections. These include recently identified tissue-resident memory T cells (TRM). In the skin, these memory CD8+ T cells reside in the epidermis after being recruited to this site by infection or inflammation. In this study, we demonstrate prolonged persistence of epidermal TRM preferentially at the site of prior infection despite sustained migration. Computational simulation of TRM migration within the skin over long periods revealed that the slow rate of random migration effectively constrains these memory cells within the region of skin in which they form. Notably, formation of TRM involved a concomitant local reduction in dendritic epidermal γδ T-cell numbers in the epidermis, indicating that these populations persist in mutual exclusion and may compete for local survival signals. Accordingly, we show that expression of the aryl hydrocarbon receptor, a transcription factor important for dendritic epidermal γδ T-cell maintenance in skin, also contributes to the persistence of skin TRM. Together, these data suggest that skin tissue-resident memory T cells persist within a tightly regulated epidermal T-cell niche. PMID:24706879

  4. Persistence of skin-resident memory T cells within an epidermal niche.

    PubMed

    Zaid, Ali; Mackay, Laura K; Rahimpour, Azad; Braun, Asolina; Veldhoen, Marc; Carbone, Francis R; Manton, Jonathan H; Heath, William R; Mueller, Scott N

    2014-04-01

    Barrier tissues such as the skin contain various populations of immune cells that contribute to protection from infections. These include recently identified tissue-resident memory T cells (TRM). In the skin, these memory CD8(+) T cells reside in the epidermis after being recruited to this site by infection or inflammation. In this study, we demonstrate prolonged persistence of epidermal TRM preferentially at the site of prior infection despite sustained migration. Computational simulation of TRM migration within the skin over long periods revealed that the slow rate of random migration effectively constrains these memory cells within the region of skin in which they form. Notably, formation of TRM involved a concomitant local reduction in dendritic epidermal γδ T-cell numbers in the epidermis, indicating that these populations persist in mutual exclusion and may compete for local survival signals. Accordingly, we show that expression of the aryl hydrocarbon receptor, a transcription factor important for dendritic epidermal γδ T-cell maintenance in skin, also contributes to the persistence of skin TRM. Together, these data suggest that skin tissue-resident memory T cells persist within a tightly regulated epidermal T-cell niche.

  5. Engineered Microenvironments to Direct Epidermal Stem Cell Behavior at Single-Cell Resolution.

    PubMed

    Watt, Fiona M

    2016-09-26

    Mammalian epidermis is maintained through proliferation of stem cells and differentiation of their progeny. The balance between self-renewal and differentiation is controlled by a variety of interacting intrinsic and extrinsic factors. Although the nature of these interactions is complex, they can be modeled in a reductionist fashion by capturing single epidermal stem cells on micropatterned substrates and exposing them to individual stimuli, alone or in combination, over defined time points. These studies have shown that different extrinsic stimuli trigger a common outcome-initiation of terminal differentiation-by activating different signaling pathways and eliciting different transcriptional responses. PMID:27676433

  6. Genetics Home Reference: epidermal nevus

    MedlinePlus

    ... primarily of a specific cell type called a keratinocyte. One group of epidermal nevi, called keratinocytic or nonorganoid epidermal nevi, includes nevi that involve only keratinocytes. Keratinocytic epidermal nevi are typically found on the ...

  7. 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.

  8. Association between vascular-poor area of primary tumors and epidermal growth factor receptor gene status in advanced lung adenocarcinoma.

    PubMed

    Togashi, Yosuke; Masago, Katsuhiro; Kubo, Takeshi; Fujimoto, Daichi; Sakamori, Yuichi; Nagai, Hiroki; Kim, Young Hak; Togashi, Kaori; Mishima, Michiaki

    2012-12-01

    Mutation of the epidermal growth factor receptor gene (EGFR mutation) is a very important marker in the treatment for non-small cell lung cancer. Since signaling from this receptor induces tumor-associated angiogenesis, we hypothesized that lung cancers with EGFR mutations tend to develop locally with increased angiogenesis. Thus, the association between vascular-poor area of primary tumors and EGFR status was retrospectively investigated in advanced lung adenocarcinomas. To assess vascular-poor area, contrast-enhanced computed tomography scans taken before initial treatment for lung cancer were analyzed, together with primary tumor location (peripheral or central) and size. We analyzed 178 patients with advanced lung adenocarcinoma. EGFR mutations were detected in 95 of the 178 patients (53.4 %). EGFR mutation was found to be significantly related to women (P = 0.0070), never-smokers (P < 0.0001), and tumors without vascular-poor area (P < 0.0001). Based on a multivariate analysis, presence of EGFR mutations was independently associated with never-smokers (P = 0.0046), lack of vascular-poor area (P = 0.0001), and tumor size >30 mm (P = 0.0080). EGFR mutations were found in 41 of 51 never-smokers without vascular-poor area (80.4 %), 19 of 36 never-smokers with vascular-poor area (52.8 %), 19 of 37 current or former-smokers without vascular-poor area (51.4 %), and 16 of 54 current or former-smokers with vascular-poor area (29.6 %). This study showed an association between vascular-poor area of primary tumors and EGFR status. As a consequence, evaluation using a combination of smoking status and vascular-poor area allows us to predict presence of EGFR mutations at a high frequency.

  9. A Theoretical Model of Jigsaw-Puzzle Pattern Formation by Plant Leaf Epidermal Cells

    PubMed Central

    Higaki, Takumi; Kutsuna, Natsumaro; Akita, Kae; Takigawa-Imamura, Hisako; Yoshimura, Kenji; Miura, Takashi

    2016-01-01

    Plant leaf epidermal cells exhibit a jigsaw puzzle–like pattern that is generated by interdigitation of the cell wall during leaf development. The contribution of two ROP GTPases, ROP2 and ROP6, to the cytoskeletal dynamics that regulate epidermal cell wall interdigitation has already been examined; however, how interactions between these molecules result in pattern formation remains to be elucidated. Here, we propose a simple interface equation model that incorporates both the cell wall remodeling activity of ROP GTPases and the diffusible signaling molecules by which they are regulated. This model successfully reproduces pattern formation observed in vivo, and explains the counterintuitive experimental results of decreased cellulose production and increased thickness. Our model also reproduces the dynamics of three-way cell wall junctions. Therefore, this model provides a possible mechanism for cell wall interdigitation formation in vivo. PMID:27054467

  10. A Theoretical Model of Jigsaw-Puzzle Pattern Formation by Plant Leaf Epidermal Cells.

    PubMed

    Higaki, Takumi; Kutsuna, Natsumaro; Akita, Kae; Takigawa-Imamura, Hisako; Yoshimura, Kenji; Miura, Takashi

    2016-04-01

    Plant leaf epidermal cells exhibit a jigsaw puzzle-like pattern that is generated by interdigitation of the cell wall during leaf development. The contribution of two ROP GTPases, ROP2 and ROP6, to the cytoskeletal dynamics that regulate epidermal cell wall interdigitation has already been examined; however, how interactions between these molecules result in pattern formation remains to be elucidated. Here, we propose a simple interface equation model that incorporates both the cell wall remodeling activity of ROP GTPases and the diffusible signaling molecules by which they are regulated. This model successfully reproduces pattern formation observed in vivo, and explains the counterintuitive experimental results of decreased cellulose production and increased thickness. Our model also reproduces the dynamics of three-way cell wall junctions. Therefore, this model provides a possible mechanism for cell wall interdigitation formation in vivo.

  11. Proteins deposited in the dermis are rapidly captured and presented by epidermal Langerhans cells

    PubMed Central

    Flacher, Vincent; Tripp, Christoph H.; Stoitzner, Patrizia; Haid, Bernhard; Ebner, Susanne; Koch, Franz; Park, Chae Gyu; Steinman, Ralph M.; Idoyaga, Juliana; Romani, Nikolaus

    2010-01-01

    Antigen-presenting cells can capture antigens that are deposited in the skin, including vaccines given subcutaneously. These include different dendritic cells (DC) such as epidermal Langerhans cells (LC), dermal DC and dermal langerin+ DC. To evaluate access of dermal antigens to skin DC, we used mAb to two C-type lectin endocytic receptors, DEC-205/CD205 and langerin/CD207. When applied to murine and human skin explant cultures, these mAb were efficiently taken up by epidermal LC. Additionally, anti-DEC-205 targeted langerin+ CD103+ and langerin− CD103− mouse dermal DC. Unexpectedly, intradermal injection of either mAb, but not isotype control, resulted in strong and rapid labelling of LC in situ, implying that large molecules can diffuse through the basement membrane into the epidermis. Epidermal LC targeted in vivo by ovalbumin-coupled anti-DEC-205 potently presented antigen to CD4+ and CD8+ T cells. Thus, epidermal LC play a major role in uptake of lectin-binding ligands under standard vaccination conditions. PMID:19890348

  12. 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

  13. Homologs of SCAR/WAVE complex components are required for epidermal cell morphogenesis in rice.

    PubMed

    Zhou, Wenqi; Wang, Yuchuan; Wu, Zhongliang; Luo, Liang; Liu, Ping; Yan, Longfeng; Hou, Suiwen

    2016-07-01

    Filamentous actins (F-actins) play a vital role in epidermal cell morphogenesis. However, a limited number of studies have examined actin-dependent leaf epidermal cell morphogenesis events in rice. In this study, two recessive mutants were isolated: less pronounced lobe epidermal cell2-1 (lpl2-1) and lpl3-1, whose leaf and stem epidermis developed a smooth surface, with fewer serrated pavement cell (PC) lobes, and decreased papillae. The lpl2-1 also exhibited irregular stomata patterns, reduced plant height, and short panicles and roots. Molecular genetic studies demonstrated that LPL2 and LPL3 encode the PIROGI/Specifically Rac1-associated protein 1 (PIR/SRA1)-like and NCK-associated protein 1 (NAP1)-like proteins, respectively, two components of the suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (SCAR/WAVE) regulatory complex involved in actin nucleation and function. Epidermal cells exhibited abnormal arrangement of F-actins in both lpl2 and lpl3 expanding leaves. Moreover, the distorted trichomes of Arabidopsis pir could be partially restored by an overexpression of LPL2 A yeast two-hybrid assay revealed that LPL2 can directly interact with LPL3 in vitro Collectively, the results indicate that LPL2 and LPL3 are two functionally conserved homologs of the SCAR/WAVE complex components, and that they play an important role in controlling epidermal cell morphogenesis in rice by organising F-actin. PMID:27252469

  14. The effect of ruby laser light on cellular proliferation of epidermal cells.

    PubMed

    Liew, S H; Grobbelaar, A O; Gault, D T; Green, C J; Linge, C

    1999-11-01

    In ruby laser-assisted hair removal, microscopic damage is often seen in the basal epidermal cells, where melanosomes are concentrated. It is not known whether this treatment leads to cellular hyperproliferation. It was the aim of this study to investigate this. Ten white patients were treated with the Chromos 694-nm Depilation Ruby Laser, and biopsies taken before and after treatments to assess the presence of cell hyperproliferation, which normally accompanies epidermal damage, with immunohistochemical staining of keratin 16 and Ki67. No evidence of cell hyperproliferation was seen in all specimens examined after ruby laser irradiation. The authors conclude that despite the possible microscopic damages seen in the basal epidermis after laser hair removal, there is no evidence of cellular hyperproliferation. This is in contrast to ultraviolet-irradiated cell damage, in which increased basal cell turnover is seen.

  15. Dual mechanisms of green tea extract (EGCG)-induced cell survival in human epidermal keratinocytes.

    PubMed

    Chung, Jin Ho; Han, Ji Hyun; Hwang, Eun Ju; Seo, Jin Young; Cho, Kwang Hyun; Kim, Kyu Han; Youn, Jai Il; Eun, Hee Chul

    2003-10-01

    Beneficial effects attributed to green tea, such as its anticancer and antioxidant properties, may be mediated by (-)-epigallocatechin-3-gallate (EGCG). In this study, the effects of EGCG on cell proliferation and UV-induced apoptosis were investigated in normal epidermal keratinocytes. When topically applied to aged human skin, EGCG stimulated the proliferation of epidermal keratinocytes, which increased the epidermal thickness. In addition, this topical application also inhibited the UV-induced apoptosis of epidermal keratinocytes. EGCG was found to increase the phosphorylation of Bad protein at the Ser112 and Ser136. Moreover, EGCG-induced Erk phosphorylation was found to be critical for the phosphorylation of Ser112 in Bad protein, and the EGCG-induced activation of the Akt pathway was found to be involved in the phosphorylation of Ser136. Furthermore, EGCG increased Bcl-2 expression but decreased Bax expression, causing an increase in the Bcl-2-to-Bax ratio. In addition, we demonstrate the differential growth inhibitory effects of EGCG on cancer cells. In conclusion, this study demonstrates that EGCG promotes keratinocyte survival and inhibits the UV-induced apoptosis via two mechanisms: by phosphorylating Ser112 and Ser136 of Bad protein through Erk and Akt pathways, respectively, and by increasing the Bcl-2-to-Bax ratio. Moreover, these two proposed mechanisms of EGCG-induced cell proliferation may differ kinetically to promote keratinocyte survival.

  16. Epidermal stem cells: markers, patterning and the control of stem cell fate.

    PubMed Central

    Watt, F M

    1998-01-01

    Within the epidermis, proliferation takes place in the basal layer of keratinocytes that are attached to an underlying basement membrane. Cells that leave the basal layer undergo terminal differentiation as they move towards the tissue surface. The basal layer contains two types of proliferative keratinocyte: stem cells, which have unlimited self-renewal capacity, and transit amplifying cells, those daughters of stem cells that are destined to withdraw from the cell cycle and terminally differentiate after a few rounds of division. Stem cells express higher levels of the beta 1-integrin family of extracellular matrix receptors than transit amplifying cells and this can be used to isolate each subpopulation of keratinocyte and to determine its location within the epidermis. Variation in the levels of E-cadherin, beta-catenin and plakoglobin within the basal layer suggests that stem cells may also differ from transit amplifying cells in intercellular adhesiveness. Stem cells have a patterned distribution within the epidermal basal layer and patterning is subject to autoregulation. Constitutive expression of the transcription factor c-Myc promotes terminal differentiation by driving keratinocytes from the stem cell compartment into the transit amplifying compartment. PMID:9684280

  17. Effects of Telomerase and Telomere Length on Epidermal Stem Cell Behavior

    NASA Astrophysics Data System (ADS)

    Flores, Ignacio; Cayuela, María L.; Blasco, María A.

    2005-08-01

    A key process in organ homeostasis is the mobilization of stem cells out of their niches. We show through analysis of mouse models that telomere length, as well as the catalytic component of telomerase, Tert, are critical determinants in the mobilization of epidermal stem cells. Telomere shortening inhibited mobilization of stem cells out of their niche, impaired hair growth, and resulted in suppression of stem cell proliferative capacity in vitro. In contrast, Tert overexpression in the absence of changes in telomere length promoted stem cell mobilization, hair growth, and stem cell proliferation in vitro. The effects of telomeres and telomerase on stem cell biology anticipate their role in cancer and aging.

  18. Epidermal Cells Expressing Putative Cell Markers in Nonglabrous Skin Existing in Direct Proximity with the Distal End of the Arrector Pili Muscle

    PubMed Central

    Rufaut, N. W.; Jones, L.; Sinclair, R.

    2016-01-01

    Inconsistent with the view that epidermal stem cells reside randomly spread along the basal layer of the epidermal rete ridges, we found that epidermal cells expressing stem cell markers in nonglabrous skin exist in direct connection with the distal end of the arrector pili muscle. The epidermal cells that express stem cell markers consist of a subpopulation of basal keratinocytes located in a niche at the lowermost portion of the rete ridges at the distal arrector pili muscle attachment site. Keratinocytes in the epidermal stem cell niche express K15, MCSP, and α6 integrin. α5 integrin marks the distal end of the APM colocalized with basal keratinocytes expressing stem cell markers located in a well-protected and nourished environment at the lowermost point of the epidermis; these cells are hypothesized to participate directly in epidermal renewal and homeostasis and also indirectly in wound healing through communication with the hair follicle bulge epithelial stem cell population through the APM. Our findings, plus a reevaluation of the literature, support the hierarchical model of interfollicular epidermal stem cell units of Fitzpatrick. This new view provides insights into epidermal control and the possible involvement of epidermal stem cells in nonmelanoma skin carcinogenesis. PMID:27375744

  19. A liquid film model of tetrakaidecahedral packing to account for the establishment of epidermal cell columns.

    PubMed

    Menton, D N

    1976-05-01

    The possiblity that the organization of cells into columns in the mammalian epidermis may be a result of the close packing of these cells has been investigated in a model system involving the association of randomly produced soap bubbles into a stable froth. Upon floating to the surface of a liquid, soap bubbles have been found to spontaneously assemble into precise columns of interdigitating bubbles. The tetrakaidecahedral shape and the spatial configuration of these bubbles closely resemble those of stacked epidermal cells, although the columns of a froth were oriented at a 60degrees angle to their substratum rather than at right angles as occurs in the epidermal cell columns. These observations lend support to the theory that the organization of the cells in the epidermis into columns is due to the assumption of the keratocytes of a minimum surface-close packing array. Such an organizing mechanism would be independent of both positional control of the underlying mitoses and active guidance of the cells as they become superficially displaced within the epidermis. The observation that a high rate of cell turnover is incompatible with the epidermal column structure may be related to the finding that rapidly produced soap bubbles do not, at least initially, assemble into a columnar array. PMID:1270835

  20. Three-dimensional culture of epidermal cells on ordered cellulose scaffolds.

    PubMed

    Seyama, Tomoko; Suh, Eun Young; Kondo, Tetsuo

    2013-06-01

    An ordered cellulose film scaffold, termed a nematic ordered cellulose (NOC) template, had unique surface properties and successfully induced the establishment of a three-dimensional (3D), hierarchical structure of epidermal cells by cell attachment and subsequent culture. Initially, the scaffold surface properties were characterized through contact angle measurements and atomic force microscopy to evaluate appropriate hydrophobicity and orientation of molecular chains for 3D culture. The template surfaces exhibited higher hydrophobicity, in the range of 70-75°, than usual cellulose films and appeared suitable for surface cell adhesion. In fact, epidermal cells successfully attached and proliferated favorably on the NOC templates, similar to development in normal culture flasks. Furthermore, the NOC film, as a semipermeable template, was also employed to allow 3D proliferation of epidermal cell layers in the perpendicular direction. The template proved to be suitable as a 3D cell culture device, resulting in the proposal that the construction processes of these 3D cell layers followed the basic concept of skin formation. PMID:23624420

  1. Single cell-type comparative metabolomics of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum

    PubMed Central

    Barkla, Bronwyn J.; Vera-Estrella, Rosario

    2015-01-01

    One of the remarkable adaptive features of the halophyte Mesembryanthemum crystallinum are the specialized modified trichomes called epidermal bladder cells (EBC) which cover the leaves, stems, and peduncle of the plant. They are present from an early developmental stage but upon salt stress rapidly expand due to the accumulation of water and sodium. This particular plant feature makes it an attractive system for single cell type studies, with recent proteomics and transcriptomics studies of the EBC establishing that these cells are metabolically active and have roles other than sodium sequestration. To continue our investigation into the function of these unusual cells we carried out a comprehensive global analysis of the metabolites present in the EBC extract by gas chromatography Time-of-Flight mass spectrometry (GC-TOF) and identified 194 known and 722 total molecular features. Statistical analysis of the metabolic changes between control and salt-treated samples identified 352 significantly differing metabolites (268 after correction for FDR). Principal components analysis provided an unbiased evaluation of the data variance structure. Biochemical pathway enrichment analysis suggested significant perturbations in 13 biochemical pathways as defined in KEGG. More than 50% of the metabolites that show significant changes in the EBC, can be classified as compatible solutes and include sugars, sugar alcohols, protein and non-protein amino acids, and organic acids, highlighting the need to maintain osmotic homeostasis to balance the accumulation of Na+ and Cl− ions. Overall, the comparison of metabolic changes in salt treated relative to control samples suggests large alterations in M. crystallinum epidermal bladder cells. PMID:26113856

  2. Differential Requirements of TCR Signaling in Homeostatic Maintenance and Function of Dendritic Epidermal T Cells.

    PubMed

    Zhang, Baojun; Wu, Jianxuan; Jiao, Yiqun; Bock, Cheryl; Dai, Meifang; Chen, Benny; Chao, Nelson; Zhang, Weiguo; Zhuang, Yuan

    2015-11-01

    Dendritic epidermal T cells (DETCs) are generated exclusively in the fetal thymus and maintained in the skin epithelium throughout postnatal life of the mouse. DETCs have restricted antigenic specificity as a result of their exclusive usage of a canonical TCR. Although the importance of the TCR in DETC development has been well established, the exact role of TCR signaling in DETC homeostasis and function remains incompletely defined. In this study, we investigated TCR signaling in fully matured DETCs by lineage-restricted deletion of the Lat gene, an essential signaling molecule downstream of the TCR. We found that Lat deletion impaired TCR-dependent cytokine gene activation and the ability of DETCs to undergo proliferative expansion. However, linker for activation of T cells-deficient DETCs were able to maintain long-term population homeostasis, although with a reduced proliferation rate. Mice with Lat deletion in DETCs exhibited delayed wound healing accompanied by impaired clonal expansion within the wound area. Our study revealed differential requirements for TCR signaling in homeostatic maintenance of DETCs and in their effector function during wound healing. PMID:26408667

  3. Transcriptional down-regulation of epidermal growth factor receptors by nerve growth factor treatment of PC12 cells.

    PubMed

    Shibutani, M; Lazarovici, P; Johnson, A C; Katagiri, Y; Guroff, G

    1998-03-20

    Treatment of PC12 cells with nerve growth factor leads to a decrease in the number of epidermal growth factor receptors on the cell membrane. The mRNA for the epidermal growth factor receptor decreases in a comparable fashion. This decrease appears due to a decrease in the transcription of the epidermal growth factor receptor gene because first, there is no difference in the stability of the epidermal growth factor receptor mRNA, second, newly transcribed epidermal growth factor receptor mRNA is decreased in nerve growth factor-differentiated cells, and third, constructs containing the promoter region of the epidermal growth factor receptor gene are transcribed much less readily in nerve growth factor-differentiated cells than in untreated cells. The decreases in mRNA are not seen in the p140(trk)-deficient variant PC12nnr5 cells nor in cells containing either dominant-negative Ras or dominant-negative Src. Treatment with nerve growth factor also increases the cellular content of GCF2, a putative transcription factor inhibitory for the transcription of the epidermal growth factor receptor gene. The increase in GCF2, like the decrease in the epidermal growth factor receptor mRNA, is not seen in PC12nnr5 cells nor in cells expressing either dominant-negative Ras or dominant-negative Src. The results suggest that nerve growth factor-induced down-regulation of the epidermal growth factor receptor is under transcriptional control, is p140(trk)-, Ras-, and Src-dependent, and may involve transcriptional repression by GCF2.

  4. Patterning as a signature of human epidermal stem cell regulation

    PubMed Central

    Klein, Allon M.; Nikolaidou-Neokosmidou, Varvara; Doupé, David P.; Jones, Philip H.; Simons, Benjamin D.

    2011-01-01

    Understanding how stem cells are regulated in adult tissues is a major challenge in cell biology. In the basal layer of human epidermis, clusters of almost quiescent stem cells are interspersed with proliferating and differentiating cells. Previous studies have shown that the proliferating cells follow a pattern of balanced stochastic cell fate. This behaviour enables them to maintain homeostasis, while stem cells remain confined to their quiescent clusters. Intriguingly, these clusters reappear spontaneously in culture, suggesting that they may play a functional role in stem cell auto-regulation. We propose a model of pattern formation that explains how clustering could regulate stem cell activity in homeostatic tissue through contact inhibition and stem cell aggregation. PMID:21632613

  5. Proliferative and toxic effects of ultraviolet light and inflammation on epidermal pigment cells

    SciTech Connect

    Nordlund, J.J.; Ackles, A.E.; Traynor, F.F.

    1981-10-01

    The ear of the mouse is useful for studying the effects of ultraviolet light on epidermal pigment cells. The quantity of light penetrating into the skin causing an inflammatory response can be assessed easily by measuring with an engineering calipers the swelling of the ear. The inflammatory response of the ear exhibits a linear relationship to the dose of light delivered. We observed that doses of shortwave ultraviolet light which are noninflammatory when repeated at daily intervals induce moderate to severe inflammation. Small doses of psoralen and prolonged exposure to UVA (PUVA) were more inflammatory than larger amounts of psoralen and short exposure to light. Doses of shortwave ultraviolet light and PUVA which produce only a minimal inflammation of the skin stimulate the proliferation of epidermal melanocytes. In contrast, PUVA in doses sufficiently large to cause a marked inflammatory reaction in the skin seems injurious to pigment cells and kills them or causes only a minimal proliferative response. The inflammatory reaction itself does not seem to stimulate or inhibit the proliferation of melanocytes. Prostaglandins A, E, and F2 alpha have no effect on the proliferation of epidermal pigment cells. In contrast, dimethyl sulfoxide (DMSO) and allergic contact dermatitis increase the numerical density of pigment cells. Steroids may block the function of the enzyme tyrosinase. Our experiments indicate that pigment cells, like many other varieties of cells, are susceptible to injury and can be killed at least by large doses of PUVA.

  6. Genetically induced cell death in bulge stem cells reveals their redundancy for hair and epidermal regeneration.

    PubMed

    Driskell, Iwona; Oeztuerk-Winder, Feride; Humphreys, Peter; Frye, Michaela

    2015-03-01

    Adult mammalian epidermis contains multiple stem cell populations in which quiescent and more proliferative stem and progenitor populations coexist. However, the precise interrelation of these populations in homeostasis remains unclear. Here, we blocked the contribution of quiescent keratin 19 (K19)-expressing bulge stem cells to hair follicle formation through genetic ablation of the essential histone methyltransferase Setd8 that is required for the maintenance of adult skin. Deletion of Setd8 eliminated the contribution of bulge cells to hair follicle regeneration through inhibition of cell division and induction of cell death, but the growth and morphology of hair follicles were unaffected. Furthermore, ablation of Setd8 in the hair follicle bulge blocked the contribution of K19-postive stem cells to wounded epidermis, but the wound healing process was unaltered. Our data indicate that quiescent bulge stem cells are dispensable for hair follicle regeneration and epidermal injury in the short term and support the hypothesis that quiescent and cycling stem cell populations are equipotent.

  7. Epidermal surface lipids

    PubMed Central

    2009-01-01

    A layer of lipids, which are of both sebaceous and keratinocyte origin, covers the surface of the skin. The apparent composition of surface lipids varies depending on the selected method of sampling. Lipids produced by the epidermal cells are an insignificant fraction of the total extractable surface lipid on areas rich in sebaceous glands. Due to the holocrine activity of the sebaceous gland, its product of secretion (sebum) is eventually released to the surface of the skin and coats the fur as well. Lipids of epidermal origin fill the spaces between the cells, like mortar or cement. The sebaceous lipids are primarily non polar lipids as triglycerides, wax esters and squalene, while epidermal lipids are a mixture of ceramides, free fatty acids and cholesterol. The composition of the sebaceous lipids is unique and intriguing and elevated sebum excretion is a major factor involved in the pathophysiology of acne. Recent studies have elucidated the roles that epidermal surface lipids have on normal skin functions and acne. PMID:20224687

  8. Human epidermal neural crest stem cells as a source of Schwann cells

    PubMed Central

    Sakaue, Motoharu; Sieber-Blum, Maya

    2015-01-01

    We show that highly pure populations of human Schwann cells can be derived rapidly and in a straightforward way, without the need for genetic manipulation, from human epidermal neural crest stem cells [hEPI-NCSC(s)] present in the bulge of hair follicles. These human Schwann cells promise to be a useful tool for cell-based therapies, disease modelling and drug discovery. Schwann cells are glia that support axons of peripheral nerves and are direct descendants of the embryonic neural crest. Peripheral nerves are damaged in various conditions, including through trauma or tumour-related surgery, and Schwann cells are required for their repair and regeneration. Schwann cells also promise to be useful for treating spinal cord injuries. Ex vivo expansion of hEPI-NCSC isolated from hair bulge explants, manipulating the WNT, sonic hedgehog and TGFβ signalling pathways, and exposure of the cells to pertinent growth factors led to the expression of the Schwann cell markers SOX10, KROX20 (EGR2), p75NTR (NGFR), MBP and S100B by day 4 in virtually all cells, and maturation was completed by 2 weeks of differentiation. Gene expression profiling demonstrated expression of transcripts for neurotrophic and angiogenic factors, as well as JUN, all of which are essential for nerve regeneration. Co-culture of hEPI-NCSC-derived human Schwann cells with rodent dorsal root ganglia showed interaction of the Schwann cells with axons, providing evidence of Schwann cell functionality. We conclude that hEPI-NCSCs are a biologically relevant source for generating large and highly pure populations of human Schwann cells. PMID:26251357

  9. Prevention of skin tumorigenesis and impairment of epidermal cell proliferation by targeted aquaporin-3 gene disruption.

    PubMed

    Hara-Chikuma, Mariko; Verkman, A S

    2008-01-01

    Aquaporin-3 (AQP3) is a water/glycerol-transporting protein expressed strongly at the plasma membranes of basal epidermal cells in skin. We found that human skin squamous cell carcinoma strongly overexpresses AQP3. A novel role for AQP3 in skin tumorigenesis was discovered using mice with targeted AQP3 gene disruption. We found that AQP3-null mice were remarkably resistant to the development of skin tumors following exposure to a tumor initiator and phorbol ester promoter. Though tumor initiator challenge produced comparable apoptotic responses in wild-type and AQP3-null mice, promoter-induced cell proliferation was greatly impaired in the AQP3-null epidermis. Reductions of epidermal cell glycerol, its metabolite glycerol-3-phosphate, and ATP were found in AQP3 deficiency without impairment of mitochondrial function. Glycerol supplementation corrected the reduced proliferation and ATP content in AQP3 deficiency, with cellular glycerol, ATP, and proliferative ability being closely correlated. Our data suggest involvement of AQP3-facilitated glycerol transport in epidermal cell proliferation and tumorigenesis by a novel mechanism implicating cellular glycerol as a key determinant of cellular ATP energy. AQP3 may thus be an important determinant in skin tumorigenesis and hence a novel target for tumor prevention and therapy. PMID:17967887

  10. Epidermal Notch1 recruits RORγ+ group 3 innate lymphoid cells to orchestrate normal skin repair

    PubMed Central

    Li, Zhi; Hodgkinson, Tom; Gothard, Elizabeth J.; Boroumand, Soulmaz; Lamb, Rebecca; Cummins, Ian; Narang, Priyanka; Sawtell, Amy; Coles, Jenny; Leonov, German; Reboldi, Andrea; Buckley, Christopher D.; Cupedo, Tom; Siebel, Christian; Bayat, Ardeshir; Coles, Mark C.; Ambler, Carrie A.

    2016-01-01

    Notch has a well-defined role in controlling cell fate decisions in the embryo and the adult epidermis and immune systems, yet emerging evidence suggests Notch also directs non-cell-autonomous signalling in adult tissues. Here, we show that Notch1 works as a damage response signal. Epidermal Notch induces recruitment of immune cell subsets including RORγ+ ILC3s into wounded dermis; RORγ+ ILC3s are potent sources of IL17F in wounds and control immunological and epidermal cell responses. Mice deficient for RORγ+ ILC3s heal wounds poorly resulting from delayed epidermal proliferation and macrophage recruitment in a CCL3-dependent process. Notch1 upregulates TNFα and the ILC3 recruitment chemokines CCL20 and CXCL13. TNFα, as a Notch1 effector, directs ILC3 localization and rates of wound healing. Altogether these findings suggest that Notch is a key stress/injury signal in skin epithelium driving innate immune cell recruitment and normal skin tissue repair. PMID:27099134

  11. Epidermal Notch1 recruits RORγ(+) group 3 innate lymphoid cells to orchestrate normal skin repair.

    PubMed

    Li, Zhi; Hodgkinson, Tom; Gothard, Elizabeth J; Boroumand, Soulmaz; Lamb, Rebecca; Cummins, Ian; Narang, Priyanka; Sawtell, Amy; Coles, Jenny; Leonov, German; Reboldi, Andrea; Buckley, Christopher D; Cupedo, Tom; Siebel, Christian; Bayat, Ardeshir; Coles, Mark C; Ambler, Carrie A

    2016-01-01

    Notch has a well-defined role in controlling cell fate decisions in the embryo and the adult epidermis and immune systems, yet emerging evidence suggests Notch also directs non-cell-autonomous signalling in adult tissues. Here, we show that Notch1 works as a damage response signal. Epidermal Notch induces recruitment of immune cell subsets including RORγ(+) ILC3s into wounded dermis; RORγ(+) ILC3s are potent sources of IL17F in wounds and control immunological and epidermal cell responses. Mice deficient for RORγ(+) ILC3s heal wounds poorly resulting from delayed epidermal proliferation and macrophage recruitment in a CCL3-dependent process. Notch1 upregulates TNFα and the ILC3 recruitment chemokines CCL20 and CXCL13. TNFα, as a Notch1 effector, directs ILC3 localization and rates of wound healing. Altogether these findings suggest that Notch is a key stress/injury signal in skin epithelium driving innate immune cell recruitment and normal skin tissue repair. PMID:27099134

  12. Epidermal cells are the primary phagocytes in the fragmentation and clearance of degenerating dendrites in Drosophila

    PubMed Central

    Xiao, Hui; Wang, Denan; Franc, Nathalie C.; Jan, Lily Yeh; Jan, Yuh-Nung

    2014-01-01

    SUMMARY During developmental remodeling, neurites destined for pruning often degenerate on-site. Physical injury also induces degeneration of neurites distal to the injury site. Prompt clearance of degenerating neurites is important for maintaining tissue homeostasis and preventing inflammatory responses. Here we show that in both dendrite pruning and dendrite injury of Drosophila sensory neurons, epidermal cells rather than hemocytes are the primary phagocytes in clearing degenerating dendrites. Epidermal cells act via Draper-mediated recognition to facilitate dendrite degeneration and to engulf and degrade degenerating dendrites. Using multiple dendritic membrane markers to trace phagocytosis, we show that two members of the CD36 family, croquemort (crq) and debris buster (dsb), act at distinct stages of phagosome maturation for dendrite clearance. Our finding reveals the physiological importance of coordination between neurons and their surrounding epidermis, for both dendrite fragmentation and clearance. PMID:24412417

  13. 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.

  14. Indomethacin interferes with epidermal growth factor binding and proliferative response of gastric KATO III cells.

    PubMed

    Fujiwara, Y; Schmassmann, A; Arakawa, T; Halter, F; Tarnawski, A

    1995-01-01

    Indomethacin induces gastric ulcerations and decreases cell proliferation in the gastric ulcer margin. Since epithelial cell proliferation is under control of epidermal growth factor (EGF), we studied whether indomethacin may affect specific binding of [125I]-EGF to its receptors in cultured human gastric KATO III cells. To assess effects of EGF, indomethacin and their combination on cell proliferation, KATO III cells were incubated for 24 h with either (a) vehicle (b) indomethacin (doses from 10(-5) to 10(-3) M), EGF (doses 0.01, 0.05 or 0.1 microgram/ml) or (d) a combination of b and c, and the bromodeoxyuridine labeling index was determined. Indomethacin in a dose which did not affect cell viability significantly (by 21.5%) decreased [125I]-EGF binding to the KATO III cells and decreased the bromodeoxyuridine labeling index. Epidermal growth factor significantly increased cell proliferation and increased the labeling index from 28.9 +/- 0.6% in the vehicle group to 36.2 +/- 0.5%. Co-treatment with indomethacin significantly reduced the proliferative response of KATO III cells to EGF. In conclusion, indomethacin, in a dose which does not affect cell viability, decreased binding of EGF to cultured gastric KATO III cells and decreased their proliferative response to EGF. PMID:8549878

  15. Skin Stem Cells: At the Frontier Between the Laboratory and Clinical Practice. Part 1: Epidermal Stem Cells.

    PubMed

    Pastushenko, I; Prieto-Torres, L; Gilaberte, Y; Blanpain, C

    2015-11-01

    Stem cells are characterized by their ability to self-renew and differentiate into the different cell lineages of their tissue of origin. The discovery of stem cells in adult tissues, together with the description of specific markers for their isolation, has opened up new lines of investigation, expanding the horizons of biomedical research and raising new hope in the treatment of many diseases. In this article, we review in detail the main characteristics of the stem cells that produce the specialized cells of the skin (epidermal, mesenchymal, and melanocyte stem cells) and their potential implications and applications in diseases affecting the skin. Part I deals with the principal characteristics and potential applications of epidermal stem cells in dermatology.

  16. Skin Stem Cells: At the Frontier Between the Laboratory and Clinical Practice. Part 1: Epidermal Stem Cells.

    PubMed

    Pastushenko, I; Prieto-Torres, L; Gilaberte, Y; Blanpain, C

    2015-11-01

    Stem cells are characterized by their ability to self-renew and differentiate into the different cell lineages of their tissue of origin. The discovery of stem cells in adult tissues, together with the description of specific markers for their isolation, has opened up new lines of investigation, expanding the horizons of biomedical research and raising new hope in the treatment of many diseases. In this article, we review in detail the main characteristics of the stem cells that produce the specialized cells of the skin (epidermal, mesenchymal, and melanocyte stem cells) and their potential implications and applications in diseases affecting the skin. Part I deals with the principal characteristics and potential applications of epidermal stem cells in dermatology. PMID:26189363

  17. Birbeck Granules Are Subdomains of Endosomal Recycling Compartment in Human Epidermal Langerhans Cells, Which Form Where Langerin Accumulates

    PubMed Central

    Mc Dermott, Ray; Ziylan, Umit; Spehner, Danièle; Bausinger, Huguette; Lipsker, Dan; Mommaas, Mieke; Cazenave, Jean-Pierre; Raposo, Graça; Goud, Bruno; de la Salle, Henri; Salamero, Jean; Hanau, Daniel

    2002-01-01

    Birbeck granules are unusual rod-shaped structures specific to epidermal Langerhans cells, whose origin and function remain undetermined. We investigated the intracellular location and fate of Langerin, a protein implicated in Birbeck granule biogenesis, in human epidermal Langerhans cells. In the steady state, Langerin is predominantly found in the endosomal recycling compartment and in Birbeck granules. Langerin internalizes by classical receptor-mediated endocytosis and the first Birbeck granules accessible to endocytosed Langerin are those connected to recycling endosomes in the pericentriolar area, where Langerin accumulates. Drug-induced inhibition of endocytosis results in the appearance of abundant open-ended Birbeck granule-like structures appended to the plasma membrane, whereas inhibition of recycling induces Birbeck granules to merge with a tubular endosomal network. In mature Langerhans cells, Langerin traffic is abolished and the loss of internal Langerin is associated with a concomitant depletion of Birbeck granules. Our results demonstrate an exchange of Langerin between early endosomal compartments and the plasma membrane, with dynamic retention in the endosomal recycling compartment. They show that Birbeck granules are not endocytotic structures, rather they are subdomains of the endosomal recycling compartment that form where Langerin accumulates. Finally, our results implicate ADP-ribosylation factor proteins in Langerin trafficking and the exchange between Birbeck granules and other endosomal membranes. PMID:11809842

  18. Substance P combined with epidermal stem cells promotes wound healing and nerve regeneration in diabetes mellitus.

    PubMed

    Zhu, Fei-Bin; Fang, Xiang-Jing; Liu, De-Wu; Shao, Ying; Zhang, Hong-Yan; Peng, Yan; Zhong, Qing-Ling; Li, Yong-Tie; Liu, De-Ming

    2016-03-01

    Exogenous substance P accelerates wound healing in diabetes, but the mechanism remains poorly understood. Here, we established a rat model by intraperitoneally injecting streptozotocin. Four wounds (1.8 cm diameter) were drilled using a self-made punch onto the back, bilateral to the vertebral column, and then treated using amniotic membrane with epidermal stem cells and/or substance P around and in the middle of the wounds. With the combined treatment the wound-healing rate was 100% at 14 days. With prolonged time, type I collagen content gradually increased, yet type III collagen content gradually diminished. Abundant protein gene product 9.5- and substance P-immunoreactive nerve fibers regenerated. Partial nerve fiber endings extended to the epidermis. The therapeutic effects of combined substance P and epidermal stem cells were better than with amniotic membrane and either factor alone. Our results suggest that the combination of substance P and epidermal stem cells effectively contributes to nerve regeneration and wound healing in diabetic rats.

  19. Substance P combined with epidermal stem cells promotes wound healing and nerve regeneration in diabetes mellitus

    PubMed Central

    Zhu, Fei-bin; Fang, Xiang-jing; Liu, De-wu; Shao, Ying; Zhang, Hong-yan; Peng, Yan; Zhong, Qing-ling; Li, Yong-tie; Liu, De-ming

    2016-01-01

    Exogenous substance P accelerates wound healing in diabetes, but the mechanism remains poorly understood. Here, we established a rat model by intraperitoneally injecting streptozotocin. Four wounds (1.8 cm diameter) were drilled using a self-made punch onto the back, bilateral to the vertebral column, and then treated using amniotic membrane with epidermal stem cells and/or substance P around and in the middle of the wounds. With the combined treatment the wound-healing rate was 100% at 14 days. With prolonged time, type I collagen content gradually increased, yet type III collagen content gradually diminished. Abundant protein gene product 9.5- and substance P-immunoreactive nerve fibers regenerated. Partial nerve fiber endings extended to the epidermis. The therapeutic effects of combined substance P and epidermal stem cells were better than with amniotic membrane and either factor alone. Our results suggest that the combination of substance P and epidermal stem cells effectively contributes to nerve regeneration and wound healing in diabetic rats. PMID:27127492

  20. Nucleus-associated microtubules help determine the division plane of plant epidermal cells: avoidance of four-way junctions and the role of cell geometry.

    PubMed

    Flanders, D J; Rawlins, D J; Shaw, P J; Lloyd, C W

    1990-04-01

    To investigate the spatial relationship between the nucleus and the cortical division site, epidermal cells were selected in which the separation between these two areas is large. Avoiding enzyme treatment and air drying, Datura stramonium cells were labeled with antitubulin antibodies and the three-dimensional aspect of the cytoskeletons was reconstructed using computer-aided optical sectioning. In vacuolated cells preparing for division, the nucleus migrates into the center of the cell, suspended by transvacuolar strands. These strands are now shown to contain continuous bundles of microtubules which bridge the nucleus to the cortex. These nucleus-radiating microtubules adopt different configurations in cells of different shape. In elongated cells with more or less parallel side walls, oblique strands radiating from the nucleus to the long side walls are presumably unstable, for they are progressively realigned into a transverse disc (the phragmosome) as broad, cortical, preprophase bands (PPBs) become tighter. The phragmosome and the PPB are both known predictors of the division plane and our observations indicate that they align simultaneously in elongated epidermal cells. These observations suggest another hypothesis: that the PPB may contain microtubules polymerized from the nuclear surface. In elongated cells, the majority of the radiating microtubules, therefore, come to anchor the nucleus in the transverse plane, consistent with the observed tendency of such cells to divide perpendicular to the long axis. In nonrectangular isodiametric epidermal cells, which approximate regular hexagons in section, the radial microtubular strands emanating from the nucleus tend to remain associated with the middle of each subtending cell wall. The strands are not reorganized into a single dominant transverse bar, but remain as a starlike array until mitosis. PPBs in these cells are not as tight; they may only be a sparse accumulation of microtubules, even forming along non

  1. Asymmetric growth of root epidermal cells is related to the differentiation of root hair cells in Hordeum vulgare (L.)

    PubMed Central

    Marzec, Marek

    2013-01-01

    The root epidermis of most vascular plants harbours two cell types, namely trichoblasts (capable of producing a root hair) and atrichoblasts. Here, in vivo analysis, confocal laser-scanning microscopy, transmission electron microscopy, histological analysis, and three-dimensional reconstruction were used to characterize the cell types present in the barley root epidermis and their distribution in the tissue. Both trichoblasts and atrichoblasts were present in the wild-type cultivars and could be distinguished from one another at an early stage. Trichoblast/atrichoblast differentiation depended on asymmetric cell expansion after a period of symmetrical cell division. After asymmetric growth, only the shorter epidermal cells could produce root hairs, whereas the longer cells became atrichoblasts. Moreover, the root epidermis did not develop root hairs at all if the epidermal cells did not differentiate into two asymmetric cell types. The root hairless phenotype of bald root barley (brb) and root hairless 1.b (rhl1.b) mutants was caused by a mutation in a gene related to the asymmetric expansion of the root epidermal cells. Additionally, the results showed that the mechanism of trichoblast/atrichoblast differentiation is not evolutionally conserved across the subfamilies of the Poaceae; in the Pooideae subfamily, both asymmetric division and asymmetric cell expansion have been observed. PMID:24043851

  2. Identifying subcellular protein localization with fluorescent protein fusions after transient expression in onion epidermal cells.

    PubMed

    Nebenführ, Andreas

    2014-01-01

    Most biochemical functions of plant cells are carried out by proteins which act at very specific places within these cells, for example, within different organelles. Identifying the subcellular localization of proteins is therefore a useful tool to narrow down the possible functions that a novel or unknown protein may carry out. The discovery of genetically encoded fluorescent markers has made it possible to tag specific proteins and visualize them in vivo under a variety of conditions. This chapter describes a simple method to use transient expression of such fluorescently tagged proteins in onion epidermal cells to determine their subcellular localization relative to known markers.

  3. Effects of epidermal growth factor on neural crest cells in tissue culture

    SciTech Connect

    Erickson, C.A.; Turley, E.A.

    1987-04-01

    Epidermal growth factor (EGF) stimulates the release of hyaluronic acid (HA) and chondroitin sulfate proteoglycan (CSPG) from quail trunk neural crest cultures in a dose-dependent fashion. It also promotes the expression of cell-associated heparan sulfate proteoglycan (HSPG) as detected by immunofluorescence and immunoprecipitation of the /sup 3/H-labeled proteoglycan. Furthermore, EGF stimulates (/sup 3/H)thymidine incorporation into total cell DNA. These results raise the possibility that EGF or an analogous growth factor is involved in regulation of neural crest cell morphogenesis.

  4. Mutual induction of growth factor gene expression by epidermal-dermal cell interaction

    PubMed Central

    1993-01-01

    Epithelial-mesenchymal interactions control epidermal growth and differentiation, but little is known about the mechanisms of this interaction. We have examined the effects of human dermal microvascular endothelial cells (DMEC) and fibroblasts on keratinocytes in conventional (feeder layer) and organotypic cocultures (lifted collagen gels) and demonstrated the induction of paracrine growth factor gene expression. Clonal keratinocyte growth was similarly stimulated in cocultures with irradiated DMEC and fibroblasts as feeder cells. This effect is most probably caused by induction of growth factor expression in cocultured dermal cells. Keratinocytes stimulated mRNA levels for KGF and IL-6 in both mesenchymal cell types and GM-CSF in fibroblasts. The feeder effect could not be replaced by conditioned media or addition of isolated growth factors. In organotypic cocultures with keratinocytes growing on collagen gels (repopulated with dermal cells), a virtually normal epidermis was formed within 7 to 10 d. Keratinocyte proliferation was drastically stimulated by dermal cells (histone 3 mRNA expression and BrdU labeling) which continued to proliferate as well in the gel. Expression of all typical differentiation markers was provoked in the reconstituted epithelium, though with different localization as compared to normal epidermis. Keratins K1 and K10 appeared coexpressed but delayed, reflecting conditions in epidermal hyperplasia. Keratin localization and proliferation were normalized under in vivo conditions, i.e., in surface transplants on nude mice. From these data it is concluded that epidermal homeostasis is in part controlled by complex reciprocally induced paracrine acting factors in concert with cell-cell interactions and extracellular matrix influences. PMID:8320264

  5. Corrective transduction of human epidermal stem cells in laminin-5-dependent junctional epidermolysis bullosa.

    PubMed

    Dellambra, E; Vailly, J; Pellegrini, G; Bondanza, S; Golisano, O; Macchia, C; Zambruno, G; Meneguzzi, G; De Luca, M

    1998-06-10

    Laminin-5 is composed of three distinct polypeptides, alpha3, beta3, and gamma2, which are encoded by three different genes, LAMA3, LAMB3, and LAMC2, respectively. We have isolated epidermal keratinocytes from a patient presenting with a lethal form of junctional epidermolysis bullosa characterized by a homozygous mutation of the LAMB3 gene, which led to complete absence of the beta3 polypeptide. In vitro, beta3-null keratinocytes were unable to synthesize laminin-5 and to assemble hemidesmosomes, maintained the impairment of their adhesive properties, and displayed a decrease of their colony-forming ability. A retroviral construct expressing a human beta3 cDNA was used to transduce primary beta3-null keratinocytes. Clonogenic beta3-null keratinocytes were transduced with an efficiency of 100%. Beta3-transduced keratinocytes were able to synthesize and secrete mature heterotrimeric laminin-5. Gene correction fully restored the keratinocyte adhesion machinery, including the capacity of proper hemidesmosomal assembly, and prevented the loss of the colony-forming ability, suggesting a direct link between adhesion to laminin-5 and keratinocyte proliferative capacity. Clonal analysis demonstrated that holoclones expressed the transgene permanently, suggesting stable correction of epidermal stem cells. Because cultured keratinocytes are used routinely to make autologous grafts for patients suffering from large skin or mucosal defects, the full phenotypic reversion of primary human epidermal stem cells defective for a structural protein opens new perspectives in the long-term treatment of genodermatoses. PMID:9650620

  6. Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

    SciTech Connect

    Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

    2011-08-26

    Highlights: {yields} Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. {yields} The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. {yields} Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

  7. Model system for plant cell biology: GFP imaging in living onion epidermal cells

    NASA Technical Reports Server (NTRS)

    Scott, A.; Wyatt, S.; Tsou, P. L.; Robertson, D.; Allen, N. S.

    1999-01-01

    The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

  8. Communication is key: Reducing DEK1 activity reveals a link between cell-cell contacts and epidermal cell differentiation status.

    PubMed

    Galletti, Roberta; Ingram, Gwyneth C

    2015-01-01

    Plant epidermis development requires not only the initial acquisition of tissue identity, but also the ability to differentiate specific cell types over time and to maintain these differentiated states throughout the plant life. To set-up and maintain differentiation, plants activate specific transcriptional programs. Interfering with these programs can prevent differentiation and/or force differentiated cells to lose their identity and re-enter a proliferative state. We have recently shown that the Arabidopsis Defective Kernel 1 (DEK1) protein is required both for the differentiation of epidermal cells and for the maintenance of their fully differentiated state. Defects in DEK1 activity lead to a deregulation of the expression of epidermis-specific differentiation-promoting HD-ZIP IV transcription factors. Here we propose a working model in which DEK1, by maintaining cell-cell contacts, and thus communication between neighboring cells, influences HD-ZIP IV gene expression and epidermis differentiation. PMID:27064205

  9. A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains.

    PubMed Central

    Partanen, J; Armstrong, E; Mäkelä, T P; Korhonen, J; Sandberg, M; Renkonen, R; Knuutila, S; Huebner, K; Alitalo, K

    1992-01-01

    Endothelial cell surfaces play key roles in several important physiological and pathological processes such as blood clotting, angiogenic responses, and inflammation. Here we describe the cloning and characterization of tie, a novel type of human endothelial cell surface receptor tyrosine kinase. The extracellular domain of the predicted tie protein product has an exceptional multidomain structure consisting of a cluster of three epidermal growth factor homology motifs embedded between two immunoglobulinlike loops, which are followed by three fibronectin type III repeats next to the transmembrane region. Additionally, a cDNA form lacking the first of the three epidermal growth factor homology domains was isolated, suggesting that alternative splicing creates different tie-type receptors. Cells transfected with tie cDNA expression vector produce glycosylated polypeptides of 117 kDa which are reactive to antisera raised against the tie carboxy terminus. The tie gene was located in chromosomal region 1p33 to 1p34. Expression of the tie gene appeared to be restricted in some cell lines; large amounts of tie mRNA were detected in endothelial cell lines and in some myeloid leukemia cell lines with erythroid and megakaryoblastoid characteristics. In addition, mRNA in situ studies further indicated the endothelial expression of the tie gene. The tie receptor tyrosine kinase may have evolved for multiple protein-protein interactions, possibly including cell adhesion to the vascular endothelium. Images PMID:1312667

  10. On the mechanism of callose synthesis induction by metal ions in onion epidermal cells.

    PubMed

    Kartusch, R

    2003-03-01

    Metal ions induce the synthesis of callose in Allium cepa epidermal cells. Callose is deposited as single knoblike local accumulations, aggregates of knobs, or furrowed clusters tightly attached to the cell wall. The most effective metal is copper, it induces callose formation at micromolar concentrations. Agents acting on inositolphosphate metabolism, phospholipase inhibitors, calcium channel inhibitors, modulators of cytoplasmic calcium, or receptor antagonists influence callose synthesis. It is concluded that metal ions, especially Cu(2+), initiate a signal transduction chain by activation of phospholipases and generation of inositol 1,4,5-trisphosphate, and that callose synthesis is a cellular defence reaction caused by the disturbance of intracellular calcium homeostasis. PMID:12664286

  11. Cell Surface Epidermal Growth Factor Receptors Increase Src and c-Cbl Activity and Receptor Ubiquitylation*

    PubMed Central

    Parks, Eileen E.; Ceresa, Brian P.

    2014-01-01

    There is an established role for the endocytic pathway in regulation of epidermal growth factor receptor (EGFR) signaling to downstream effectors. However, because ligand-mediated EGFR endocytosis utilizes multiple “moving parts,” dissecting the spatial versus temporal contributions has been challenging. Blocking all endocytic trafficking can have unintended effects on other receptors as well as give rise to compensatory mechanisms, both of which impact interpretation of EGFR signaling. To overcome these limitations, we used epidermal growth factor (EGF) conjugated to polystyrene beads (EGF beads). EGF beads simultaneously activate the EGFR while blocking its endocytosis and allow analysis of EGFR signaling from the plasma membrane. Human telomerase immortalized corneal epithelial (hTCEpi) cells were used to model normal epithelial cell biology. In hTCEpi cells, both cell surface and intracellular EGFRs exhibited dose-dependent increases in effector activity after 15 min of ligand stimulation, but only the serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) was statistically significant when accounting for receptor phosphorylation. However, over time with physiological levels of receptor phosphorylation, cell surface receptors produced either enhanced or sustained mitogen-activated protein kinase kinase (MEK), Casitas B-lineage lymphoma (c-Cbl), and the pro-oncogene Src activity. These increases in effector communication by cell surface receptors resulted in an increase in EGFR ubiquitylation with sustained ligand incubation. Together, these data indicate that spatial regulation of EGFR signaling may be an important regulatory mechanism in receptor down-regulation. PMID:25074934

  12. Vanadate induces apoptosis in epidermal JB6 P+ cells via hydrogen peroxide-mediated reactions.

    PubMed

    Ye, J; Ding, M; Leonard, S S; Robinson, V A; Millecchia, L; Zhang, X; Castranova, V; Vallyathan, V; Shi, X

    1999-12-01

    Apoptosis is a physiological mechanism for the control of DNA integrity in mammalian cells. Vanadium induces both DNA damage and apoptosis. It is suggested that vanadium-induced apoptosis serves to eliminate DNA-damaged cells. This study is designed to clarify a role of reactive oxygen species in the mechanism of apoptosis induced by vanadium. We established apoptosis model with murine epidermal JB6 P+ cells in the response to vanadium stimulation. Apoptosis was detected by a cell death ELISA assay and morphological analysis. The result shows that apoptosis induced by vanadate is dose-dependent, reaching its saturation level at a concentration of 100 microM vanadate. Vanadyl (IV) can also induce apoptosis albeit with lesser potency. A role of reactive oxygen species was analyzed by multiple reagents including specific scavengers of different reactive oxygen species. The result shows that vanadate-induced apoptosis is enhanced by NADPH, superoxide dismutase and sodium formate, but was inhibited by catalase and deferoxamine. Cells exposed to vanadium consume more molecular oxygen and at the same time, produce more H2O2 as measured by the change in fluorescence of scopoletin in the presence of horseradish peroxidase. This change in oxygen consumption and H2O2 production is enhanced by NADPH. Taken together, these results show that vanadate induces apoptosis in epidermal cells and H2O2 induced by vanadate plays a major role in this process. PMID:10705990

  13. Ultrastructure of the Epidermal Cell Wall and Cuticle of Tomato Fruit (Solanum lycopersicum L.) during Development1[OPEN

    PubMed Central

    Segado, Patricia; Domínguez, Eva

    2016-01-01

    The epidermis plays a pivotal role in plant development and interaction with the environment. However, it is still poorly understood, especially its outer epidermal wall: a singular wall covered by a cuticle. Changes in the cuticle and cell wall structures are important to fully understand their functions. In this work, an ultrastructure and immunocytochemical approach was taken to identify changes in the cuticle and the main components of the epidermal cell wall during tomato fruit development. A thin and uniform procuticle was already present before fruit set. During cell division, the inner side of the procuticle showed a globular structure with vesicle-like particles in the cell wall close to the cuticle. Transition between cell division and elongation was accompanied by a dramatic increase in cuticle thickness, which represented more than half of the outer epidermal wall, and the lamellate arrangement of the non-cutinized cell wall. Changes in this non-cutinized outer wall during development showed specific features not shared with other cell walls. The coordinated nature of the changes observed in the cuticle and the epidermal cell wall indicate a deep interaction between these two supramolecular structures. Hence, the cuticle should be interpreted within the context of the outer epidermal wall. PMID:26668335

  14. Epidermal growth factor increases LRF/Pokemon expression in human prostate cancer cells.

    PubMed

    Aggarwal, Himanshu; Aggarwal, Anshu; Agrawal, Devendra K

    2011-10-01

    Leukemia/lymphoma related factor/POK erythroid myeloid ontogenic factor (LRF/Pokemon) is a member of the POK family of proteins that promotes oncogenesis in several forms of cancer. Recently, we found higher LRF expression in human breast and prostate carcinomas compared to the corresponding normal tissues. The aim of this study was to examine the regulation of LRF expression in human prostate cells. Epidermal growth factor (EGF) and its receptors mediate several tumorigenic cascades that regulate cell differentiation, proliferation, migration and survival of prostate cancer cells. There was significantly higher level of LRF expression in the nucleus of LNCaP and PC-3 cells than RWPE-1 cells. A significant increase in LRF expression was observed with increasing doses of EGF in more aggressive and androgen-sensitive prostate cancer cells suggesting that EGF signaling pathway is critical in upregulating the expression of LRF/Pokemon to promote oncogenesis. PMID:21640721

  15. Recycling of epidermal growth factor in a human pancreatic carcinoma cell line

    SciTech Connect

    Korc, M.; Magun, B.E.

    1985-09-01

    PANC-1 human pancreatic carcinoma cells readily bound and internalized /sup 125/I-labeled epidermal growth factor (EGF). Bound /sup 125/I-labeled EGF was then partially processed to a number of high molecular weight acidic species. Percoll gradient centrifugation of cell homogenates indicated that the majority of /sup 125/I activity localized to several intracellular vesicular compartments. Both intact EGF and its processed species were subsequently released into the incubation medium. A major portion of the released radioactivity was capable of rebinding to the cell. Only a small amount of bound /sup 125/I-labeled EGF was degraded to low molecular weight products, and this degradation was completely blocked by methylamine. These findings suggest that in PANC-1 cells, bound EGF undergoes only limited processing. Both intact EGF and its major processed species bypass the cellular degradative pathways, are slowly released from the cell, and then rebind to the cell.

  16. Basic helix-loop-helix transcription factors and epidermal cell fate determination in Arabidopsis

    PubMed Central

    Zhao, Hongtao; Li, Xia; Ma, Ligeng

    2012-01-01

    Cell fate determination is an important process in multicellular organisms. Plant epidermis is a readily-accessible, well-used model for the study of cell fate determination. Our knowledge of cell fate determination is growing steadily due to genetic and molecular analyses of root hairs, trichomes, and stomata, which are derived from the epidermal cells of roots and aerial tissues. Studies have shown that a large number of factors are involved in the establishment of these cell types, especially members of the basic helix-loop-helix (bHLH) superfamily, which is an important family of transcription factors. In this mini-review, we focus on the role of bHLH transcription factors in cell fate determination in Arabidopsis. PMID:23073001

  17. Evidence that the epidermal growth factor receptor on host cells confers reovirus infection efficiency.

    PubMed

    Strong, J E; Tang, D; Lee, P W

    1993-11-01

    Reovirus binds to multiple sialoglycoproteins on the host cell surface. In an attempt to probe additional specific determinants that dictate host cell susceptibility to reovirus infection, we found that two mouse cell lines (NR6 and B82) previously shown to express no endogenous epidermal growth factor (EGF) receptors were relatively resistant to reovirus infection, whereas the same cell lines transfected with the gene encoding the EGF receptor manifested significantly higher susceptibility as determined by induction of cytopathic effects, viral protein synthesis, and plaque titration. This enhancement of infection efficiency requires a functional EGF receptor since it was not observed in cells expressing a mutated (kinase-inactive) EGF receptor. The observed difference in infection efficiency is not due to differences in virus binding or internalization. These studies suggest that the reovirus infection process is closely coupled to the EGF receptor-mediated cell signal transduction pathway.

  18. Epidermal growth factor increases LRF/Pokemon expression in human prostate cancer cells.

    PubMed

    Aggarwal, Himanshu; Aggarwal, Anshu; Agrawal, Devendra K

    2011-10-01

    Leukemia/lymphoma related factor/POK erythroid myeloid ontogenic factor (LRF/Pokemon) is a member of the POK family of proteins that promotes oncogenesis in several forms of cancer. Recently, we found higher LRF expression in human breast and prostate carcinomas compared to the corresponding normal tissues. The aim of this study was to examine the regulation of LRF expression in human prostate cells. Epidermal growth factor (EGF) and its receptors mediate several tumorigenic cascades that regulate cell differentiation, proliferation, migration and survival of prostate cancer cells. There was significantly higher level of LRF expression in the nucleus of LNCaP and PC-3 cells than RWPE-1 cells. A significant increase in LRF expression was observed with increasing doses of EGF in more aggressive and androgen-sensitive prostate cancer cells suggesting that EGF signaling pathway is critical in upregulating the expression of LRF/Pokemon to promote oncogenesis.

  19. Surface position, not signaling from surrounding maternal tissues, specifies aleurone epidermal cell fate in maize.

    PubMed

    Gruis, Darren Fred; Guo, Hena; Selinger, David; Tian, Qing; Olsen, Odd-Arne

    2006-07-01

    Maize (Zea mays) endosperm consists of an epidermal-like surface layer of aleurone cells, an underlying body of starchy endosperm cells, and a basal layer of transfer cells. To determine whether surrounding maternal tissues perform a role in specifying endosperm cell fates, a maize endosperm organ culture technique was established whereby the developing endosperm is completely removed from surrounding maternal tissues. Using cell type-specific fluorescence markers, we show that aleurone cell fate specification occurs exclusively in response to surface position and does not require specific, continued maternal signal input. The starchy endosperm and aleurone cell fates are freely interchangeable throughout the lifespan of the endosperm, with internalized aleurone cells converting to starchy endosperm cells and with starchy endosperm cells that become positioned at the surface converting to aleurone cells. In contrast to aleurone and starchy endosperm cells, transfer cells fail to develop in in vitro-grown endosperm, supporting earlier indications that maternal tissue interaction is required to fully differentiate this cell type. Several parameters confirm that the maize endosperm organ cultures described herein retain the main developmental features of in planta endosperm, including fidelity of aleurone mutant phenotypes, temporal and spatial control of cell type-specific fluorescent markers, specificity of cell type transcripts, and control of mitotic cell divisions.

  20. Effect of Ca2+ on programmed death of guard and epidermal cells of pea leaves.

    PubMed

    Kiselevsky, D B; Kuznetsova, Yu E; Vasil'ev, L A; Lobysheva, N V; Zinovkin, R A; Nesov, A V; Shestak, A A; Samuilov, V D

    2010-05-01

    The effect of Ca2+ on programmed death of guard cells (GC) and epidermal cells (EC) determined from destruction of the cell nucleus was investigated in epidermis of pea leaves. Ca2+ at concentrations of 1-100 microM increased and at a concentration of 1 mM prevented the CN(-)-induced destruction of the nucleus in GC, disrupting the permeability barrier of GC plasma membrane for propidium iodide (PI). Ca2+ at concentrations of 0.1-1 mM enhanced drastically the number of EC nuclei stained by PI in epidermis treated with chitosan, an inducer of programmed cell death. The internucleosomal DNA fragmentation caused by CN(-) was suppressed by 2 mM Ca2+ on 6 h incubation, but fragmentation was stimulated on more prolonged treatment (16 h). Presumably, the disruption of the permeability barrier of plasma membrane for PI is not a sign of necrosis in plant cells. Quinacrine and diphenylene iodonium at 50 microM concentration prevented GC death induced by CN(-) or CN(-) + 0.1 mM Ca2+ but had no influence on respiration and photosynthetic O2 evolution in pea leaf slices. The generation of reactive oxygen species determined from 2',7'-dichlorofluorescein fluorescence was promoted by Ca2+ in epidermal peels from pea leaves.

  1. Response of mouse epidermal cells to single doses of heavy-particles

    NASA Technical Reports Server (NTRS)

    Leith, J. T.; Schilling, W. A.; Welch, G. P.

    1972-01-01

    The survival of mouse epidermal cells to heavy-particles has been studied In Vivo by the Withers clone technique. Experiments with accelerated helium, lithium and carbon ions were performed. The survival curve for the helium ion irradiations used a modified Bragg curve method with a maximum tissue penetration of 465 microns, and indicated that the dose needed to reduce the original cell number to 1 surviving cell/square centimeters was 1525 rads with a D sub o of 95 rads. The LET at the basal cell layer was 28.6 keV per micron. Preliminary experiments with lithium and carbon used treatment doses of 1250 rads with LET's at the surface of the skin of 56 and 193 keV per micron respectively. Penetration depths in skin were 350 and 530 microns for the carbon and lithium ions whose Bragg curves were unmodified. Results indicate a maximum RBE for skin of about 2 using the skin cloning technique. An attempt has been made to relate the epidermal cell survival curve to mortality of the whole animal for helium ions.

  2. A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

    PubMed

    Marc; Granger; Brincat; Fisher; Kao; McCubbin; Cyr

    1998-11-01

    Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation. PMID:9811799

  3. Epidermal growth factor receptor subunit locations determined in hydrated cells with environmental scanning electron microscopy

    PubMed Central

    Peckys, Diana B.; Baudoin, Jean-Pierre; Eder, Magdalena; Werner, Ulf; de Jonge, Niels

    2013-01-01

    Imaging single epidermal growth factor receptors (EGFR) in intact cells is presently limited by the available microscopy methods. Environmental scanning electron microscopy (ESEM) of whole cells in hydrated state in combination with specific labeling with gold nanoparticles was used to localize activated EGFRs in the plasma membranes of COS7 and A549 cells. The use of a scanning transmission electron microscopy (STEM) detector yielded a spatial resolution of 3 nm, sufficient to identify the locations of individual EGFR dimer subunits. The sizes and distribution of dimers and higher order clusters of EGFRs were determined. The distance between labels bound to dimers amounted to 19 nm, consistent with a molecular model. A fraction of the EGFRs was found in higher order clusters with sizes ranging from 32–56 nm. ESEM can be used for quantitative whole cell screening studies of membrane receptors, and for the study of nanoparticle-cell interactions in general. PMID:24022088

  4. Epidermal growth factor receptor subunit locations determined in hydrated cells with environmental scanning electron microscopy.

    PubMed

    Peckys, Diana B; Baudoin, Jean-Pierre; Eder, Magdalena; Werner, Ulf; de Jonge, Niels

    2013-01-01

    Imaging single epidermal growth factor receptors (EGFR) in intact cells is presently limited by the available microscopy methods. Environmental scanning electron microscopy (ESEM) of whole cells in hydrated state in combination with specific labeling with gold nanoparticles was used to localize activated EGFRs in the plasma membranes of COS7 and A549 cells. The use of a scanning transmission electron microscopy (STEM) detector yielded a spatial resolution of 3 nm, sufficient to identify the locations of individual EGFR dimer subunits. The sizes and distribution of dimers and higher order clusters of EGFRs were determined. The distance between labels bound to dimers amounted to 19 nm, consistent with a molecular model. A fraction of the EGFRs was found in higher order clusters with sizes ranging from 32-56 nm. ESEM can be used for quantitative whole cell screening studies of membrane receptors, and for the study of nanoparticle-cell interactions in general.

  5. Transdifferentiation of Umbilical Cord-Derived Mesenchymal Stem Cells Into Epidermal-Like Cells by the Mimicking Skin Microenvironment.

    PubMed

    Chen, Deyun; Hao, Haojie; Tong, Chuan; Liu, Jiejie; Dong, Liang; Ti, Dongdong; Hou, Qian; Liu, Huiling; Han, Weidong; Fu, Xiaobing

    2015-06-01

    Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are multipotent, primitive, and have been widely used for skin tissue engineering. Their transdifferentiation is determined by the local microenvironment. In this study, we investigated the potential epidermal differentiation of UC-MSCs and the formation of epidermis substitutes in a 3-dimensional (3D) microenvironment, which was fabricated by UC-MSCs embedded into collagen-chitosan scaffolds (CCSs) combined with an air-liquid interface (ALI) culture system. Using fluorescence microscope, we observed that UC-MSCs were spindle-shaped and evenly distributed in the scaffold. Methyl thiazolyl blue tetrazolium bromide assay and Live/Dead assay indicated that the CCSs have good biocompatibility with UC-MSCs. Immunohistochemistry and western blotting assay showed that UC-MSCs on the surface of the CCSs were positive for the epidermal markers cytokeratin 19 and involucrin at 14 days. In addition, hematoxylin-eosin staining indicated that multilayered epidermis substitutes were established. The constructed epidermis substitutes were applied to treat full-thickness wounds in rats and proved to promote wound healing. In conclusion, manipulating the 3D microenvironment is a novel method for inducing the epidermal differentiation of MSCs to engineer epidermal substitutes, which provides an alternative strategy for skin tissue engineering. PMID:25700709

  6. IL-31-Driven Skin Remodeling Involves Epidermal Cell Proliferation and Thickening That Lead to Impaired Skin-Barrier Function

    PubMed Central

    Singh, Brijendra; Jegga, Anil G.; Shanmukhappa, Kumar S.; Edukulla, Ramakrishna; Khurana, Gurjit H.; Medvedovic, Mario; Dillon, Stacey R.; Madala, Satish K.

    2016-01-01

    Interleukin-31 (IL-31) is a type 2 helper T-cell-derived cytokine that has recently been shown to cause severe inflammation and tissue remodeling in multiple chronic diseases of the skin and lungs. IL-31 is upregulated in allergic and inflammatory diseases, including atopic dermatitis, asthma, cutaneous T-cell lymphomas, and allergic rhinitis, as well as autoimmune diseases such as systemic erythematosus. Overexpression of IL-31 in T cells causes severe inflammation, with histological features similar to skin lesions of patients with atopic dermatitis. However, the molecular mechanisms involved in IL31-driven pathological remodeling in skin diseases remain largely unknown. Here, we studied the role of IL-31 in skin damage as a result of intradermal administration of recombinant IL-31 into mice. Notably, IL-31 was sufficient to increase epidermal basal-cell proliferation and thickening of the epidermal skin layer. Our findings demonstrate a progressive increase in transepidermal water loss with chronic administration of IL-31 into the skin. Further, analysis of the skin transcriptome indicates a significant increase in the transcripts involved in epidermal-cell proliferation, epidermal thickening, and mechanical integrity. In summary, our findings demonstrate an important role for IL-31 signaling in epidermal cell proliferation and thickening that together may lead to impaired skin-barrier function in pathological remodeling of the skin. PMID:27556734

  7. IL-31-Driven Skin Remodeling Involves Epidermal Cell Proliferation and Thickening That Lead to Impaired Skin-Barrier Function.

    PubMed

    Singh, Brijendra; Jegga, Anil G; Shanmukhappa, Kumar S; Edukulla, Ramakrishna; Khurana, Gurjit H; Medvedovic, Mario; Dillon, Stacey R; Madala, Satish K

    2016-01-01

    Interleukin-31 (IL-31) is a type 2 helper T-cell-derived cytokine that has recently been shown to cause severe inflammation and tissue remodeling in multiple chronic diseases of the skin and lungs. IL-31 is upregulated in allergic and inflammatory diseases, including atopic dermatitis, asthma, cutaneous T-cell lymphomas, and allergic rhinitis, as well as autoimmune diseases such as systemic erythematosus. Overexpression of IL-31 in T cells causes severe inflammation, with histological features similar to skin lesions of patients with atopic dermatitis. However, the molecular mechanisms involved in IL31-driven pathological remodeling in skin diseases remain largely unknown. Here, we studied the role of IL-31 in skin damage as a result of intradermal administration of recombinant IL-31 into mice. Notably, IL-31 was sufficient to increase epidermal basal-cell proliferation and thickening of the epidermal skin layer. Our findings demonstrate a progressive increase in transepidermal water loss with chronic administration of IL-31 into the skin. Further, analysis of the skin transcriptome indicates a significant increase in the transcripts involved in epidermal-cell proliferation, epidermal thickening, and mechanical integrity. In summary, our findings demonstrate an important role for IL-31 signaling in epidermal cell proliferation and thickening that together may lead to impaired skin-barrier function in pathological remodeling of the skin. PMID:27556734

  8. The expression of peripheral benzodiazepine receptors in human skin: the relationship with epidermal cell differentiation.

    PubMed

    Stoebner, P E; Carayon, P; Penarier, G; Fréchin, N; Barnéon, G; Casellas, P; Cano, J P; Meynadier, J; Meunier, L

    1999-06-01

    The peripheral benzodiazepine receptor (PBR) is a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands. PBR is part of a heteromeric receptor complex involved in the formation of mitochondrial permeability transition pores and in the early events of apoptosis. PBR may function as an oxygen-dependent signal generator; recent data indicate that these receptors may preserve the mitochondria of haematopoietic cell lines from damage caused by oxygen radicals. To identify PBRs in human skin, we used a specific monoclonal antibody directed against the C-terminus fragment of the human receptor. PBR immunoreactivity was found in keratinocytes, Langerhans cells, hair follicles and dermal vascular endothelial cells. Interestingly, confocal microscopic examination of skin sections revealed that PBR expression was strongly upregulated in the superficial differentiated layers of the epidermis. Ultrastructurally, PBRs were distributed throughout the cytoplasm but were selectively expressed on the mitochondrial membranes of epidermal cells. The elevated level of PBRs in the spinous layer was not associated with an increased number of mitochondria nor with an increased amount of mRNA as assessed by in situ hybridization on microautoradiographed skin sections. The present work provides, for the first time, evidence of PBR immunoreactivity in human skin. This mitochondrial receptor may modulate apoptosis in the epidermis; its increased expression in differentiated epidermal layers may represent a novel mechanism of natural skin protection against free radical damage generated by ultraviolet exposure. PMID:10354064

  9. S179D prolactin diminishes the effects of UV light on epidermal gamma delta T cells

    PubMed Central

    Guzmán, Esther A.; Langowski, John L.; De Guzman, Ariel; Konrad Muller, H.; Walker, Ameae M.; Owen, Laurie B.

    2008-01-01

    Epidermal gamma delta T cells (γδ T) and Langerhans cells (LC) are immune cells altered by exposure to ultraviolet radiation (UVB), a powerful stressor resulting in immune suppression. Prolactin (PRL) has been characterized as an immunomodulator, particularly during stress. In this study, we have asked whether separate administration of the two major forms of prolactin, unmodified and phosphorylated, to groups of 15 mice (3 experiments, each with 5 mice per treatment group) affected the number and morphology of these epidermal immune cells under control conditions, and following UV irradiation. Under control conditions, both PRLs reduced the number of γδ T, but a molecular mimic of phosphorylated PRL (S179D PRL) was more effective, resulting in a 30% reduction. In the irradiated group, however, S179D PRL was protective against a UV-induced reduction in γδ T number and change in morphology (halved the reduction and normalized the morphology). In addition, S179D PRL, but not unmodified (U-PRL), maintained a normal LC: γδ T ratio and sustained the dendritic morphology of LC after UV exposure. These findings suggest that S179D PRL may have an overall protective effect, countering UV-induced cellular alterations in the epidermis. PMID:17945411

  10. Three-Dimensional Analysis of the Effect of Epidermal Growth Factor on Cell-Cell Adhesion in Epithelial Cell Clusters

    PubMed Central

    Notbohm, J.; Kim, J.-H.; Asthagiri, A.R.; Ravichandran, G.

    2012-01-01

    The effect that growth factors such as epidermal growth factor (EGF) have on cell-cell adhesion is of interest in the study of cellular processes such as epithelial-mesenchymal transition. Because cell-cell adhesions cannot be measured directly, we use three-dimensional traction force microscopy to measure the tractions applied by clusters of MCF-10A cells to a compliant substrate beneath them before and after stimulating the cells with EGF. To better interpret the results, a finite element model, which simulates a cluster of individual cells adhered to one another and to the substrate with linear springs, is developed to better understand the mechanical interaction between the cells in the experiments. The experiments and simulations show that the cluster of cells acts collectively as a single unit, indicating that cell-cell adhesion remains strong before and after stimulation with EGF. In addition, the experiments and model emphasize the importance of three-dimensional measurements and analysis in these experiments. PMID:22455915

  11. In Situ Measurement of Epidermal Cell Turgor, Leaf Water Potential, and Gas Exchange in Tradescantia virginiana L. 1

    PubMed Central

    Shackel, Kenneth A.; Brinckmann, Enno

    1985-01-01

    A combined system has been developed in which epidermal cell turgor, leaf water potential, and gas exchange were determined for transpiring leaves of Tradescantia virginiana L. Uniform and stable values of turgor were observed in epidermal cells (stomatal complex cells were not studied) under stable environmental conditions for both upper and lower epidermises. The changes in epidermal cell turgor that were associated with changes in leaf transpiration were larger than the changes in leaf water potential, indicating the presence of transpirationally induced within-leaf water potential gradients. Estimates of 3 to 5 millimoles per square meter per second per megapascal were obtained for the value of within-leaf hydraulic conductivity. Step changes in atmospheric humidity caused rapid changes in epidermal cell turgor with little or no initial change in stomatal conductance, indicating little direct relation between stomatal humidity response and epidermal water status. The significance of within-leaf water potential gradients to measurements of plant water potential and to current hypotheses regarding stomatal response to humidity is discussed. PMID:16664210

  12. Loss of epidermal integrity by T cell-mediated attack induces long-term local resistance to subsequent attack. I. Induction of resistance correlates with increases in Thy-1+ epidermal cell numbers

    PubMed Central

    1990-01-01

    The cutaneous graft-versus-host disease (GVHD) lesions induced by intradermal injection of cloned autoreactive T cells have been shown to subside rapidly and the epidermis returns to normal 2 wk after injection. Those mice that had spontaneously recovered from the cutaneous GVHD became resistant to subsequent attempts to induce the cutaneous GVHD by the T cells while maintaining their activity to mount delayed-type hypersensitivity (DTH) responses and to induce the enlargement of the popliteal lymph nodes (PLN). The resistance appeared to be restricted to the epidermal structures of the injection sites, suggesting the involvement of locally acting suppression mechanisms. This local resistance was not specific for the clonotype used for the induction of the resistance. A loss of the epidermal integrity by an attack of T cells capable of producing cutaneous GVHD was a prerequisite for the induction of the resistance. By up to at least 8 mo after injection of the T cells, no mice became susceptible to the cutaneous GVHD again, provided that the T cells were injected into the same footpad sites that had initially received the T cells. This resistance correlated well with the great increase (20-30-fold) in Thy- 1+ EC number. The great increase in the number of Thy-1+ EC following destruction of epidermal structures may be important in protecting the epidermal integrity from an additional attack by T cells. PMID:1969918

  13. [Microtubules in epidermal and cortical root cells of Brassica rapa during clinorotation].

    PubMed

    Kalinina, Ia M

    2006-01-01

    Using confocal microscopy the organization of tubulin cytoskeleton including endoplasmic and cortical microtubules (CMTs) has been studied in epidermal and cortical cells of the different growth zones of main root of Brassica rapa L. 6-days-old seedlings in control conditions and under clinorotation. It was shown that changes in CMTs orientation occured only in the distal elongation zone (DEZ). In the control, CMT arrays oriented transversely to the root long axis. Under clinorotation appearance of the shorter randomly organized CMTs was observed. Simultaneously, a significant decrease in the cell length in the central elongation zone (CEZ) under clinorotation was detected. It is suggested that the decline of anisotropic growth typical for CEZ cells is connected with CMTs disorientation under clinorotation.

  14. Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy.

    PubMed

    Peckys, Diana B; de Jonge, Niels

    2015-09-11

    This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e., one emitting at 655 nm and at 800 revealed similar characteristic results.

  15. Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

    PubMed Central

    Peckys, Diana B.; de Jonge, Niels

    2015-01-01

    This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e., one emitting at 655 nm and at 800 revealed similar characteristic results. PMID:26383083

  16. Epidermal growth factor-mediated effects on equine vascular smooth muscle cells

    SciTech Connect

    Grosenbaugh, D.A.; Amoss, M.S.; Hood, D.M.; Morgan, S.J.; Williams, J.D. )

    1988-10-01

    Epidermal growth factor (EGF) receptor binding kinetics and EGF-mediated stimulation of DNA synthesis and cellular proliferation were studied in cultured vascular smooth muscle cells (VSMC) from the equine thoracic aorta. Binding studies, using murine {sup 125}I-labeled EGF, indicate the presence of a single class of high-affinity binding sites, with an estimated maximal binding capacity of 5,800 sites/cells. EGF stimulated ({sup 3}H)thymidine uptake in confluent quiescent monolayers in a dose-dependent fashion, half-maximal stimulation occurring at 7.5 {times} 10{sup {minus}11} M. Likewise, EGF-mediated cellular proliferation was dose dependent under reduced serum concentrations. Equine VSMC contain specific receptors for EGF, and EGF can stimulate DNA synthesis and proliferation in these cultured cells, which suggests that EGF may participate in the proliferative changes observed in equine distal digital peripheral vascular disease.

  17. Regulation of human epidermal stem cell proliferation and senescence requires polycomb- dependent and -independent functions of Cbx4.

    PubMed

    Luis, Nuno Miguel; Morey, Lluis; Mejetta, Stefania; Pascual, Gloria; Janich, Peggy; Kuebler, Bernd; Cozutto, Luca; Roma, Guglielmo; Nascimento, Elisabete; Frye, Michaela; Di Croce, Luciano; Benitah, Salvador Aznar

    2011-09-01

    Human epidermal stem cells transit from a slow cycling to an actively proliferating state to contribute to homeostasis. Both stem cell states differ in their cell cycle profiles but must remain guarded from differentiation and senescence. Here we show that Cbx4, a Polycomb Repressive Complex 1 (PRC1)-associated protein, maintains human epidermal stem cells as slow-cycling and undifferentiated, while protecting them from senescence. Interestingly, abrogating the polycomb activity of Cbx4 impairs its antisenescent function without affecting stem cell differentiation, indicating that differentiation and senescence are independent processes in human epidermis. Conversely, Cbx4 inhibits stem cell activation and differentiation through its SUMO ligase activity. Global transcriptome and chromatin occupancy analyses indicate that Cbx4 regulates modulators of epidermal homeostasis and represses factors such as Ezh2, Dnmt1, and Bmi1 to prevent the active stem cell state. Our results suggest that distinct Polycomb complexes balance epidermal stem cell dormancy and activation, while continually preventing senescence and differentiation. PMID:21885019

  18. Cell culture from lizard skin: a tool for the study of epidermal differentiation.

    PubMed

    Polazzi, Elisabetta; Alibardi, Lorenzo

    2011-12-01

    An in vitro system of isolated skin cells has been developed in order to address the understanding on the factors that control the shedding cycle and differentiation of lizard epidermis. The skin from the regenerating lizard tail has been separated in epidermis and dermis, cells have been dissociated, cultivated in vitro, and studied ultrastructurally after 1-30 days of culture condition. Dissociated keratinocytes after 12 days in culture show numerous cell elongations and contain bundles of keratin or sparse keratin filaments. These cells often contain one to three 0.5-3 μm large and dense "keratinaceous bodies", an organelle representing tonofilament disassembling. Most keratinocytes have sparse tonofilaments in the cytoplasm and form shorter bundles of keratin in the cell periphery. The dissociated dermis mainly consists of mesenchymal cells containing sparse bundles of intermediate filaments. These cells proliferate and form multi-stratified layers and a dermal pellicle in about 2-3 weeks in vitro in our basic medium. Conversely, cultures of keratinocytes do not expand but eventually reduce to few viable cells within 2-3 weeks of in vitro condition. It is suggested that dermal cells sustain themselves through the production of growth factors but that epidermal cells requires specific growth factors still to be identified before setting-up an in vitro system that allows analyzing the control of the shedding cycle in lizards.

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

    SciTech Connect

    Martinez-Garcia, Eva; Irigoyen, Marta; Anso, Elena; Martinez-Irujo, Juan Jose; Rouzaut, Ana

    2008-05-01

    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 emitted 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.

  20. The effect of the state of differentiation on labeling of epidermal cell surface glycoproteins

    SciTech Connect

    Brysk, M.M.; Snider, J.M.

    1982-05-01

    Epidermal cells were grown in a medium in which the Ca++ concentration controlled the stage of differentiation. Cell surface molecules of differentiated and undifferentiated cells were compared by lactoperoxidase-catalyzed iodination, by the interaction with /sup 125/I-lectins, and by the metabolic incorporation of L-(/sup 3/H)-fucose. Molecular weights of the labeled components were determined by SDS-PAGE and autoradiography. After lactoperoxidase iodination, most of the radioactivity was found in polypeptide bands of 79,000, 65,000 and 56,000 daltons. The 79,000 band is the most intense for undifferentiated cells but disappears as differentiation proceeds. The 56,000 band is present in normal cells at all stages of differentiation but is absent from neoplastic cells. Glycoproteins reacted with /sup 125/I-lectins were found at 180,000, 130,000 and 85,000 daltons. The 130,000 band was the most prominent for differentiated cells labeled with wheat germ agglutinin but was essentially absent from the undifferentiated cells. With Ricinus communis agglutinin, this band was weaker for undifferentiated than for differentiated cells but was the most intense for both. After metabolic incorporation of tritiated fucose, radioactive glycoproteins were found at 130,000 and 85,000 daltons, with comparable intensities for differentiated and undifferentiated cells.

  1. Delivery of Epidermal Neural Crest Stem Cells (EPI-NCSC) to hippocamp in Alzheimer's Disease Rat Model

    PubMed Central

    Esmaeilzade, Banafshe; Nobakht, Maliheh; Joghataei, Mohammad Taghi; Rahbar Roshandel, Nahid; Rasouli, Homa; Samadi Kuchaksaraei, Ali; Hosseini, Seyed Mohammad; Najafzade, Nowruz; Asalgoo, Sara; Hejazian, Leila Beygom; Ghoroghi, Fatima Moghani

    2012-01-01

    Background: Alzheimer’s disease (AD) is characterized by progressive neuronal loss in hippocamp. Epidermal neural crest stem cells (EPI-NCSC) can differentiate into neurons, astrocytes and oligodendrocytes. The purpose of this study was to evaluate the effects of transplanting EPI-NCSC into AD rat model. Methods: Two weeks after induction of AD by injection of Amyloid-β 1-40 into CA1 area of rat hippocamp, Y-maze and single-trial passive avoidance tests were used to show deficit of learning and memory abilities. EPI-NCSC were obtained from the vibrissa hair follicle of rat, cultured and labeled with bromodeoxyuridine. When Alzheimer was proved by behavioral tests, EPI-NCSC was transplanted into CA3 area of hippocamp in AD rat model. The staining of EPI-NCSC markers (nestin and SOX10) was done in vitro. Double-labeling immunofluorescence was performed to study survival and differentiation of the grafted cells. Results: We showed that transplanted EPI-NCSC survive and produce many neurons and a few glial cells, presenting glial fibrillary acidic protein. Total number of granule cells in hippocamp was estimated to be more in the AD rat model with transplanted cells as compared to AD control group. We observed that rats with hippocampal damage made more errors than control rats on the Y-maze, when reward locations were reversed. Conclusion: Transplanted cells were migrated to all areas of hippocamp and the total number of granule cell in treatment group was equal compared to control group. Transplantation of EPI-NCSC into hippocamp might differentiate into cholinergic neurons and could cure impairment of memory in AD rat model. PMID:22562026

  2. Studies on the relationship between epidermal cell turnover kinetics and permeability of hairless mouse skin

    SciTech Connect

    Han, S.R.

    1988-01-01

    The primary aim of this study was to develop non-invasive, physical means to quantitatively assess the epidermal turnover kinetics and barrier properties of the skin and relate these to the cutaneous irritation which results from ultraviolet light irradiation and mold thermal burns. After systematically injecting radiolabeled glycine, the appearance of radioactivity at the skin's surface indicated the transit time of radiolabeled cells through the skin. By plotting the data as the cumulative specific activity against time and then fitting them with a third order polynomial equation, it is possible to estimate the turnover time of the stratum corneum. The skin turnover was coordinated with non-invasive transepidermal water loss (TEWL) studies determined with an evaporimeter. In vitro diffusion studies of the permeability of hydrocortisone through UVB irradiated and thermally burned skin were also performed. The studies indicated that irritated skin offers a relatively low diffusional resistance to hydrocortisone. Depending on the severity of the trauma, the increases in hydrocortisone's permeability coefficient through irritated skin ranged from a low of about 2 times normal to a high of about 210 times normal. Trauma-induced changes in hydrocortisone permeability parallel changes in TEWL, proving that the barrier deficient state resulting from rapid epidermal turnover is a general phenomenon.

  3. Epidermal growth factor mediated healing in stem cell-derived vocal fold mucosa

    PubMed Central

    Palencia, Liliana; Das, Amritava; Palecek, Sean P; Thibeault, Susan; Leydon, Ciara

    2015-01-01

    Background The goal of vocal fold wound healing is the reconstitution of functional tissue, including a structurally and functionally intact epithelium. Mechanisms underlying reepithelialization in vocal folds are not known, although it is suspected that healing involves the interplay between several growth factors. We used a three-dimensional human embryonic stem cell-derived model of vocal fold mucosa to examine the effects of one growth factor, exogenous epidermal growth factor (EGF), on wound healing. Materials and methods A scratch wound was created in the in vitro model. Rate of wound healing, epidermal growth factor receptor (EGFR) activation, and cell proliferation post-injury were analyzed with and without application of both exogenous EGF and an EGFR inhibitor, Gefitinib. Results Wound repair after injury was significantly hastened by application of exogenous EGF (13.3 µm/hour, ±2.63) compared to absence of exogenous EGF (7.1 µm/hour ±2.84), but inhibited with concurrent addition of Gefitinib (5.2 µm/hour, ±2.23), indicating that EGF mediates wound healing in an EGFR-dependent manner. Immunohistochemistry revealed that EGFR activation occurred only in the presence of exogenous EGF. While not statistically significant, increased density of Ki67 staining in epithelium adjacent to the scratch wound was observed following treatment with EGF, suggesting a tendency for exogenous EGF to increase epithelial cell proliferation. Conclusions Exogenous EGF increases the rate of wound healing in an EGFR-dependent manner in a three-dimensional stem cell-derived model of vocal fold mucosa. This model of wound healing can be used to gain insight into the mechanisms that regulate vocal fold epithelial repair following injury. PMID:25818979

  4. Three-Dimensional Analysis of Cell Division Orientation in Epidermal Basal Layer Using Intravital Two-Photon Microscopy

    PubMed Central

    Nemoto, Tomomi

    2016-01-01

    Epidermal structures are different among body sites, and proliferative keratinocytes in the epidermis play an important role in the maintenance of the epidermal structures. In recent years, intravital skin imaging has been used in mammalian skin research for the investigation of cell behaviors, but most of these experiments were performed with rodent ears. Here, we established a non-invasive intravital imaging approach for dorsal, ear, hind paw, or tail skin using R26H2BEGFP hairless mice. Using four-dimensional (x, y, z, and time) imaging, we successfully visualized mitotic cell division in epidermal basal cells. A comparison of cell division orientation relative to the basement membrane in each body site revealed that most divisions in dorsal and ear epidermis occurred in parallel, whereas the cell divisions in hind paw and tail epidermis occurred both in parallel and oblique orientations. Based on the quantitative analysis of the four-dimensional images, we showed that the epidermal thickness correlated with the basal cell density and the rate of the oblique divisions. PMID:27657513

  5. Does salinity reduce growth in maize root epidermal cells by inhibiting their capacity for cell wall acidification?

    PubMed

    Zidan, I; Azaizeh, H; Neumann, P M

    1990-05-01

    The reduction in growth of maize (Zea mays L.) seedling primary roots induced by salinization of the nutrient medium with 100 millimolar NaCl was accompanied by reductions in the length of the root tip elongation zone, the length of fully elongated epidermal cells, and the apparent rate of cell production: Each was partially restored when calcium levels in the salinized growth medium were increased from 0.5 to 10.0 millimolar. We investigated the possibility that the inhibition of elongation growth by salinity might be associated with an inhibition of cell wall acidification, such as that which occurs when root growth is inhibited by IAA. A qualitative assay of root surface acidification, using bromocresol purple pH indicator in agar, showed that salinized roots, with and without extra calcium, produced a zone of surface acidification which was similar to that produced by control roots. The zone of acidification began 1 to 2 millimeters behind the tip and coincided with the zone of cell elongation. The remainder of the root alkalinized its surface. Kinetics of surface acidification were assayed quantitatively by placing a flat tipped pH electrode in contact with the elongation zone. The pH at the epidermal surfaces of roots grown either with 100 millimolar NaCl (growth inhibitory), or with 10 millimolar calcium +/- NaCl (little growth inhibition), declined from 6.0 to 5.1 over 30 minutes. We conclude that NaCl did not inhibit growth by reducing the capacity of epidermal cells to acidify their walls. PMID:16667468

  6. 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

  7. Growth and differentiation in cultured human thyroid cells: effects of epidermal growth factor and thyrotropin.

    PubMed

    Errick, J E; Ing, K W; Eggo, M C; Burrow, G N

    1986-01-01

    Human thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture conditions on cell growth were measured by changes in cell numbers and by stimulation of [3H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of insulin (10 micrograms/ml), transferrin (5 micrograms/ml), hydrocortisone (10 micrograms/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine (10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present at 10(-9) M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system provides a means to study the hormonal control of growth and differentiation in human thyroid cells. PMID:3511027

  8. 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.

  9. Regulation of actin-dependent cytoplasmic motility by type II phytochrome occurs within seconds in Vallisneria gigantea epidermal cells.

    PubMed

    Takagi, Shingo; Kong, Sam-Geun; Mineyuki, Yoshinobu; Furuya, Masaki

    2003-02-01

    The effects of light on actin-dependent cytoplasmic motility in epidermal cells of green leaves of the aquatic angiosperm Vallisneria gigantea were investigated quantitatively using a custom-made dynamic image analyzer. Cytoplasmic motility was measured by monitoring changes in the brightness of individual pixels on digitized images taken sequentially under infrared light. Acceleration and deceleration of cytoplasmic motility were regulated photoreversibly by type II phytochrome(s). This phytochrome-dependent induction of cytoplasmic motility did not occur uniformly in cytoplasm but took place as scattered patches in which no particular organelles, including nucleus, existed. The induction became detectable at 2.5 s after the start of irradiation with pulsed red light. In cells exposed to microbeam irradiation, cytoplasmic motility was induced only in sites in the cytoplasm that were irradiated directly, whereas nonirradiated neighboring areas were unaffected. The effect was short-lived, disappearing within a few minutes, and no signal was transmitted from an irradiated cell to its neighbors. Anti-phytochrome antibody-responsive protein(s) was detectable in the leaf extract by immunoblot and zinc blot analyses and in cryosections of the epidermis by immunocytochemistry. Although the phytochrome-dependent cytoplasmic motility was blocked by exogenously applied latrunculin B or cytochalasins, treatment of the dark-adapted cells with Ca(2+)-chelating reagents induced the cytoplasmic motility. We have proposed a model for the phytochrome regulation of cytoplasmic motility as one of the earliest responses to a light stimulus. PMID:12566576

  10. Regulation of Actin-Dependent Cytoplasmic Motility by Type II Phytochrome Occurs within Seconds in Vallisneria gigantea Epidermal Cells

    PubMed Central

    Takagi, Shingo; Kong, Sam-Geun; Mineyuki, Yoshinobu; Furuya, Masaki

    2003-01-01

    The effects of light on actin-dependent cytoplasmic motility in epidermal cells of green leaves of the aquatic angiosperm Vallisneria gigantea were investigated quantitatively using a custom-made dynamic image analyzer. Cytoplasmic motility was measured by monitoring changes in the brightness of individual pixels on digitized images taken sequentially under infrared light. Acceleration and deceleration of cytoplasmic motility were regulated photoreversibly by type II phytochrome(s). This phytochrome-dependent induction of cytoplasmic motility did not occur uniformly in cytoplasm but took place as scattered patches in which no particular organelles, including nucleus, existed. The induction became detectable at 2.5 s after the start of irradiation with pulsed red light. In cells exposed to microbeam irradiation, cytoplasmic motility was induced only in sites in the cytoplasm that were irradiated directly, whereas nonirradiated neighboring areas were unaffected. The effect was short-lived, disappearing within a few minutes, and no signal was transmitted from an irradiated cell to its neighbors. Anti-phytochrome antibody–responsive protein(s) was detectable in the leaf extract by immunoblot and zinc blot analyses and in cryosections of the epidermis by immunocytochemistry. Although the phytochrome-dependent cytoplasmic motility was blocked by exogenously applied latrunculin B or cytochalasins, treatment of the dark-adapted cells with Ca2+-chelating reagents induced the cytoplasmic motility. We have proposed a model for the phytochrome regulation of cytoplasmic motility as one of the earliest responses to a light stimulus. PMID:12566576

  11. High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display

    PubMed Central

    Salema, Valencio; Mañas, Carmen; Cerdán, Lidia; Piñero-Lambea, Carlos; Marín, Elvira; Roovers, Rob C.; Van Bergen en Henegouwen, Paul M.P.; Fernández, Luis Ángel

    2016-01-01

    ABSTRACT Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens. PMID:27472381

  12. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation.

    PubMed

    Nagata, Yosuke; Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

    2014-08-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor.

  13. Parabens and Human Epidermal Growth Factor Receptor Ligand Cross-Talk in Breast Cancer Cells

    PubMed Central

    Pan, Shawn; Yuan, Chaoshen; Tagmount, Abderrahmane; Rudel, Ruthann A.; Ackerman, Janet M.; Yaswen, Paul; Vulpe, Chris D.; Leitman, Dale C.

    2015-01-01

    Background: Xenoestrogens are synthetic compounds that mimic endogenous estrogens by binding to and activating estrogen receptors. Exposure to estrogens and to some xenoestrogens has been associated with cell proliferation and an increased risk of breast cancer. Despite evidence of estrogenicity, parabens are among the most widely used xenoestrogens in cosmetics and personal-care products and are generally considered safe. However, previous cell-based studies with parabens do not take into account the signaling cross-talk between estrogen receptor α (ERα) and the human epidermal growth factor receptor (HER) family. Objectives: We investigated the hypothesis that the potency of parabens can be increased with HER ligands, such as heregulin (HRG). Methods: The effects of HER ligands on paraben activation of c-Myc expression and cell proliferation were determined by real-time polymerase chain reaction, Western blots, flow cytometry, and chromatin immunoprecipitation assays in ERα- and HER2-positive human BT-474 breast cancer cells. Results: Butylparaben (BP) and HRG produced a synergistic increase in c-Myc mRNA and protein levels in BT-474 cells. Estrogen receptor antagonists blocked the synergistic increase in c-Myc protein levels. The combination of BP and HRG also stimulated proliferation of BT-474 cells compared with the effects of BP alone. HRG decreased the dose required for BP-mediated stimulation of c-Myc mRNA expression and cell proliferation. HRG caused the phosphorylation of serine 167 in ERα. BP and HRG produced a synergistic increase in ERα recruitment to the c-Myc gene. Conclusion: Our results show that HER ligands enhanced the potency of BP to stimulate oncogene expression and breast cancer cell proliferation in vitro via ERα, suggesting that parabens might be active at exposure levels not previously considered toxicologically relevant from studies testing their effects in isolation. Citation: Pan S, Yuan C, Tagmount A, Rudel RA, Ackerman JM

  14. IL-4 Downregulates IL-1β and IL-6 and Induces GATA3 in Psoriatic Epidermal Cells: Route of Action of a Th2 Cytokine.

    PubMed

    Onderdijk, Armanda J; Baerveldt, Ewout M; Kurek, Dorota; Kant, Marius; Florencia, Edwin F; Debets, Reno; Prens, Errol P

    2015-08-15

    Clinical improvement of psoriasis induced by IL-4 treatment has been ascribed to changes in dermal inflammatory cells, such as activation of Th2 cells and tolerization of dendritic cells by suppressing IL-23 production. The pathologic epidermal alterations in psoriatic lesional skin include increased epidermal expression of IL-1β, IL-6, S100A7, and human β-defensin 2 (hBD2) and a downregulated expression of the epidermal transcription factor GATA3. Effects of IL-4 on the epidermal compartment of psoriasis lesions were not previously investigated. Therefore, we investigated whether IL-4 directly affects abovementioned psoriatic markers in the epidermal compartment. We cultured freshly isolated psoriatic epidermal cells, whole psoriatic and healthy skin biopsies, human keratinocytes and Langerhans cells with IL-4. The secretion of IL-1β and IL-6 by psoriatic epidermal cells was inhibited by IL-4 via transcriptional and posttranscriptional mechanisms, respectively. In normal skin, IL-4 inhibited IL-1β- and IL-17A-induced hBD2 expression in vitro. In addition, IL-4 reduced the protein expression of hBD2 in psoriatic skin biopsies and induced phospho-STAT6 protein. Epidermal GATA3 mRNA and protein were significantly upregulated by IL-4 in epidermal cells and keratinocytes. Our data argue that IL-4 improves psoriasis not only via modification/induction of Th2 cells and type II dendritic cells, but also via direct inhibition of inflammatory cytokines in resident IL-4R-expressing epidermal cells and thereby alters the psoriatic skin phenotype toward a healthy skin phenotype.

  15. Gefitinib in the treatment of nonsmall cell lung cancer with activating epidermal growth factor receptor mutation

    PubMed Central

    Nurwidya, Fariz; Takahashi, Fumiyuki; Takahashi, Kazuhisa

    2016-01-01

    Lung cancer is still the main cause of cancer-related deaths worldwide, with most patients present with advanced disease and poor long-term prognosis. The aim of lung cancer treatment is to slow down the progression of the disease, to relieve the patients from the lung cancer symptoms and whenever possible, to increase the overall survival. The discovery of small molecule targeting tyrosine kinase of epidermal growth factor receptor opens a new way in the management of advanced nonsmall cell lung cancer (NSCLC). This review will discuss several Phase II and III trials evaluated the clinical efficacy of gefitinib as monotherapy in pretreated patients with advanced NSCLC, as well as both monotherapy and combined with chemotherapy in chemotherapy-naive patients. PMID:27433059

  16. A tale of the epidermal growth factor receptor: The quest for structural resolution on cells.

    PubMed

    Tynan, Christopher J; Lo Schiavo, Valentina; Zanetti-Domingues, Laura; Needham, Sarah R; Roberts, Selene K; Hirsch, Michael; Rolfe, Daniel J; Korovesis, Dimitrios; Clarke, David T; Martin-Fernandez, Marisa L

    2016-02-15

    The challenge of determining the architecture and geometry of oligomers of the epidermal growth factor receptor (EGFR) on the cell surface has been approached using a variety of biochemical and biophysical methods. This review is intended to provide a narrative of how key concepts in the field of EGFR research have evolved over the years, from the origins of the prevalent EGFR signalling dimer hypothesis through to the development and implementation of methods that are now challenging the conventional view. The synergy between X-ray crystallography and cellular fluorescence microscopy has become particularly important, precisely because the results from these two methods diverged and highlighted the complexity of the challenge. We illustrate how developments in super-resolution microscopy are now bridging this gap. Exciting times lie ahead where knowledge of the nature of the complexes can assist with the development of a new generation of anti-cancer drugs. PMID:26484734

  17. INSULIN INDUCED EPIDERMAL GROWTH FACTOR ACTIVATION IN VASCULAR SMOOTH MUSCLE CELLS IS ADAM-DEPENDENT

    PubMed Central

    Roztocil, Elisa; Nicholl, Suzanne M.; Davies, Mark G.

    2008-01-01

    Background With the rise in metabolic syndrome, understanding the role of insulin signaling within the cells of vasculature has become more important but yet remains poorly defined. The study examines the role of insulin actions on a pivotal cross-talk receptor, Epidermal Growth Factor Receptor (EGFR). EGFR is transactivated by both G-protein-coupled receptors and receptor linked tyrosine kinases and is key to many of their responses. Objective To determine the pathway of EGFR transactivation by insulin in human coronary smooth muscle cells (VSMC) Methods VSMC were cultured in vitro. Assays of EGFR phosphorylation were examined in response to insulin in the presence and absence of the plasmin inhibitors (e-aminocaproic acid and aprotinin) matrix metalloprotease (MMP) inhibitor GM6001, the ADAM (A Disintegrin And Metalloproteinase Domain) inhibitors TAPI-0 and TAPI-1, Heparin binding epidermal growth factor (HB-EGF) inhibitor, CRM197, HB-EGF inhibitory antibodies, EGF inhibitory antibodies and the EGFR inhibitor AG1478. Results Insulin induced time-dependent EGFR phosphorylation, which was inhibited by AG1478 in a concentration dependent manner. Application of the plasmin inhibitors did not block the response. EGFR phosphorylation by insulin was blocked by inhibition of MMP activity and the ligand HB-EGF. The presence of the ADAM inhibitors, TAPI-0 and TAPI-1 significantly decreased EGFR activation. EGFR phosphorylation by EGF was not interrupted by inhibition of plasmin, MMPs TAPIs, or HB-EGF. Direct blockade of the EGFR prevented activation by both insulin and EGF. Conclusion Insulin can induce transactivation of EGFR by an ADAM-mediated, HB-EGF dependent process. This is the first description of crosstalk via ADAM between insulin and EGFR in vascular SMC. Targeting a pivotal cross-talk receptor such as EGFR, which can be transactivated by both G-protein-coupled receptors and receptor tyrosine kinases is an attractive molecular target. PMID:18656632

  18. Effects of Epidermal Cell Shape and Pigmentation on Optical Properties of Antirrhinum Petals at Visible and Ultraviolet Wavelengths.

    PubMed Central

    Gorton, H. L.; Vogelmann, T. C.

    1996-01-01

    We used the Mixta+ and mixta- lines of Antirrhinum majus as a model system to investigate the effects of epidermal cell shape and pigmentation on tissue optical properties in the visible and ultraviolet (UV) spectral regions. Adaxial epidermal cells of Mixta+ flowers have a conical-papillate shape; in the mixta- line the cells are slightly domed. Mixta+ cells contained significantly more anthocyanin and other flavonoids than mixta- cells when plants were grown under either high- or low-UV conditions. Mixta+ cells focused light (3.5-4.7 times incident) within their pigmented interiors, whereas mixta- cells focused light (2.1-2.7 times incident) in the unpigmented mesophyll. UV light penetrated the epidermis (commonly 20-50% transmittance at 312 nm) mainly through the unpigmented peripheral regions of the cells that were similar for the two lines, so that overall penetration through Mixta+ and mixta- epidermises was equal. However, maximum UV absorption in the central region of epidermal cells was slightly greater in Mixta+ than mixta-, and intact Mixta+ flowers reflected less light in the spectral regions with intermediate flavonoid absorbance. In both cases, about 50 to 75% of the difference could be attributed to cell shape and resulting changes in the optical pathlength or focusing. PMID:12226425

  19. Model-based analysis of Arabidopsis leaf epidermal cells reveals distinct division and expansion patterns for pavement and guard cells.

    PubMed

    Asl, Leila Kheibarshekan; Dhondt, Stijn; Boudolf, Véronique; Beemster, Gerrit T S; Beeckman, Tom; Inzé, Dirk; Govaerts, Willy; De Veylder, Lieven

    2011-08-01

    To efficiently capture sunlight for photosynthesis, leaves typically develop into a flat and thin structure. This development is driven by cell division and expansion, but the individual contribution of these processes is currently unknown, mainly because of the experimental difficulties to disentangle them in a developing organ, due to their tight interconnection. To circumvent this problem, we built a mathematic model that describes the possible division patterns and expansion rates for individual epidermal cells. This model was used to fit experimental data on cell numbers and sizes obtained over time intervals of 1 d throughout the development of the first leaf pair of Arabidopsis (Arabidopsis thaliana). The parameters were obtained by a derivative-free optimization method that minimizes the differences between the predicted and experimentally observed cell size distributions. The model allowed us to calculate probabilities for a cell to divide into guard or pavement cells, the maximum size at which it can divide, and its average cell division and expansion rates at each point during the leaf developmental process. Surprisingly, average cell cycle duration remained constant throughout leaf development, whereas no evidence for a maximum cell size threshold for cell division of pavement cells was found. Furthermore, the model predicted that neighboring cells of different sizes within the epidermis expand at distinctly different relative rates, which could be verified by direct observations. We conclude that cell division seems to occur independently from the status of cell expansion, whereas the cell cycle might act as a timer rather than as a size-regulated machinery.

  20. New dynamics in an old friend: dynamic tubular vacuoles radiate through the cortical cytoplasm of red onion epidermal cells.

    PubMed

    Wiltshire, Elizabeth J; Collings, David A

    2009-10-01

    The textbook image of the plant vacuole sitting passively in the centre of the cell is not always correct. We observed vacuole dynamics in the epidermal cells of red onion (Allium cepa) bulbs, using confocal microscopy to detect autofluorescence from the pigment anthocyanin. The central vacuole was penetrated by highly mobile transvacuolar strands of cytoplasm, which were also visible in concurrent transmitted light images. Tubular vacuoles also extended from the large central vacuole and radiated through the cortical cytoplasm. These tubules were thin, having a diameter of about 1.5 microm, and were connected to the central vacuole as shown by fluorescence recovery after photobleaching (FRAP) experiments. The tubules were bounded by the tonoplast, as revealed by transient expression of green fluorescent protein (GFP) targeted to the vacuolar membrane and through labeling with the dye MDY-64. Expression of endoplasmic reticulum-targeted GFP demonstrated that the vacuolar tubules were distinct from the cortical endoplasmic reticulum. Movement of the tubular vacuoles depended on actin microfilaments, as microfilament disruption blocked tubule movement and caused their collapse into minivacuoles. The close association of the tubules with GFP-tagged actin microfilaments suggests that the tubules are associated with myosin, and that tubules likely move along microfilaments. Tubular vacuoles do not require anthocyanin for their formation, as tubules were also present in white onion cells that lack anthocyanin. The function of these tubular vacuoles remains unknown, but as they greatly increase the surface area of the tonoplast, they might increase transport rates between the cytoplasm and vacuole.

  1. Phosphatidylinositol kinase is activated in membranes derived from cells treated with epidermal growth factor.

    PubMed Central

    Walker, D H; Pike, L J

    1987-01-01

    The ability of epidermal growth factor (EGF) to stimulate phosphatidylinositol (PtdIns) kinase activity in A431 cells was examined. The incorporation of 32P from [gamma-32P]ATP into PtdIns by A431 membranes was increased in membranes prepared from cells that had been pretreated with EGF. Demonstration of a stimulation of the PtdIns kinase activity by EGF required the use of subconfluent cultures and was dependent on the inclusion of protease inhibitors in the buffers used to prepare the membranes. Stimulation of the PtdIns kinase activity was rapid. The activation peaked 2 min after the addition of EGF and declined slowly thereafter. Half-maximal stimulation of the PtdIns kinase occurred at 7 nM EGF. Kinetic analyses of the reaction indicated that treatment of the cells with EGF resulted in a decrease in the Km for PtdIns with no change in the Vmax. The kinetic parameters for the utilization of ATP were unchanged in the EGF-treated membranes compared to the control membranes. Pretreatment of the cells with the phorbol ester phorbol 12-myristate 13-acetate blocked the ability of EGF to stimulate PtdIns kinase activity. These findings demonstrate that a PtdIns kinase activity in A431 cells is regulated by EGF and provide a good system for examining the mechanism by which EGF stimulates the activity of this intracellular enzyme. PMID:2823265

  2. Epidermal growth factor promotes proliferation of dermal papilla cells via Notch signaling pathway.

    PubMed

    Zhang, Haihua; Nan, Weixiao; Wang, Shiyong; Zhang, Tietao; Si, Huazhe; Wang, Datao; Yang, Fuhe; Li, Guangyu

    2016-08-01

    The effect of epidermal growth factor (EGF) on the development and growth of hair follicle is controversial. In the present study, 2-20 ng/ml EGF promoted the growth of mink hair follicles in vitro, whereas 200 ng/ml EGF inhibited follicle growth. Further, dermal papilla (DP) cells, a group of mesenchymal cells that govern hair follicle development and growth, were isolated and cultured in vitro. Treatment with or forced expression of EGF accelerated proliferation and induced G1/S transition in DP cells. Moreover, EGF upregulated the expression of DP mesenchymal genes, such as alkaline phosphatase (ALP) and insulin-like growth factor (IGF-1), as well as the Notch pathway molecules including Notch1, Jagged1, Hes1 and Hes5. In addition, inhibition of Notch signaling pathway by DAPT significantly reduced the basal and EGF-enhanced proliferation rate, and also suppressed cell cycle progression. We also show that the expression of several follicle-regulatory genes, such as Survivin and Msx2, were upregulated by EGF, and was inhibited by DAPT. In summary, our study demonstrates that the concentration of EGF is critical for the switch between hair follicle growth and inhibition, and EGF promotes DP cell proliferation via Notch signaling pathway.

  3. Oak ellagitannins suppress the phosphorylation of the epidermal growth factor receptor in human colon carcinoma cells.

    PubMed

    Fridrich, Diana; Glabasnia, Arne; Fritz, Jessica; Esselen, Melanie; Pahlke, Gudrun; Hofmann, Thomas; Marko, Doris

    2008-05-14

    The ellagitannins castalagin and vescalagin, and the C-glycosides grandinin and roburin E as well as ellagic acid were found to potently inhibit the growth of human colon carcinoma cells (HT29) in vitro. In a cell-free system these compounds were identified as potent inhibitors of the protein tyrosine kinase activity of the epidermal growth factor receptor (EGFR) with IC 50 values in the low nanomolar range. To address the question of whether the interference with the activity of the isolated EGFR also plays a role within intact cells, effects on the phosphorylation status of the EGFR, as a measure for its activity, were determined in HT29 cells. As exemplified for castalagin and grandinin, both the nonglycosylated and the glycosylated ellagitannins effectively suppressed EGFR phosphorylation, but only at concentrations > or =10 microM, thus, in a concentration range where growth inhibition was observed. These results indicate that the suppression of EGFR-mediated signaling might contribute to the growth inhibitory effects of these compounds present in oak-matured wines and spirits such as whiskey. In contrast, despite substantial growth inhibitory properties, ellagic acid did not significantly affect EGFR phosphorylation in HT29 cells up to 100 microM. PMID:18419129

  4. Intracellular processing of epidermal growth factor by early wound healing cells

    SciTech Connect

    Seyfer, A.E.; Nassaux, P.; Emory, R.; Wray, H.L.; Schaudies, R.P. )

    1990-01-01

    Epidermal growth factor (EGF) is a potent 53-amino-acid residue polypeptide that has been implicated in normal wound healing. Although past studies have shown that locally applied EGF accelerates wound healing, these studies have not examined intracellular events related to the processing of the growth factor. The objective of this study was to characterize both initial and later postbinding intracellular processing of EGF by a responsive cell line (osteoblasts) that is important in the healing of wounds. Cloned mouse calvarial osteoblasts (MC-3TC-E1) were incubated with radiolabeled EGF, with and without preincubation with nonlabeled EGF, for specific time intervals. Cell-associated radioactivity was characterized by nondenaturing polyacrylamide gel electrophoresis. Results showed that EGF is processed as three distinct species and that the relative proportions of these species are altered at later time periods when compared with initial processing. The patterns, similar to those reported for human fibroblasts, indicate a possible common pathway for the mitogenic signal in cells associated with the early events of wound healing. In addition, these data represent the first direct evidence that preexposure of cells to nonlabeled EGF alters the processing of radiolabeled EGF. This is significant, because cells must be exposed to EGF for 5 to 8 hours to elicit a growth response. Such data may help to explain the lag phase of wound healing.

  5. 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

    2015-01-01

    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

  6. 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.

  7. A single epidermal stem cell strategy for safe ex vivo gene therapy.

    PubMed

    Droz-Georget Lathion, Stéphanie; Rochat, Ariane; Knott, Graham; Recchia, Alessandra; Martinet, Danielle; Benmohammed, Sara; Grasset, Nicolas; Zaffalon, Andrea; Besuchet Schmutz, Nathalie; Savioz-Dayer, Emmanuelle; Beckmann, Jacques Samuel; Rougemont, Jacques; Mavilio, Fulvio; Barrandon, Yann

    2015-02-27

    There is a widespread agreement from patient and professional organisations alike that the safety of stem cell therapeutics is of paramount importance, particularly for ex vivo autologous gene therapy. Yet current technology makes it difficult to thoroughly evaluate the behaviour of genetically corrected stem cells before they are transplanted. To address this, we have developed a strategy that permits transplantation of a clonal population of genetically corrected autologous stem cells that meet stringent selection criteria and the principle of precaution. As a proof of concept, we have stably transduced epidermal stem cells (holoclones) obtained from a patient suffering from recessive dystrophic epidermolysis bullosa. Holoclones were infected with self-inactivating retroviruses bearing a COL7A1 cDNA and cloned before the progeny of individual stem cells were characterised using a number of criteria. Clonal analysis revealed a great deal of heterogeneity among transduced stem cells in their capacity to produce functional type VII collagen (COLVII). Selected transduced stem cells transplanted onto immunodeficient mice regenerated a non-blistering epidermis for months and produced a functional COLVII. Safety was assessed by determining the sites of proviral integration, rearrangements and hit genes and by whole-genome sequencing. The progeny of the selected stem cells also had a diploid karyotype, was not tumorigenic and did not disseminate after long-term transplantation onto immunodeficient mice. In conclusion, a clonal strategy is a powerful and efficient means of by-passing the heterogeneity of a transduced stem cell population. It guarantees a safe and homogenous medicinal product, fulfilling the principle of precaution and the requirements of regulatory affairs. Furthermore, a clonal strategy makes it possible to envision exciting gene-editing technologies like zinc finger nucleases, TALENs and homologous recombination for next-generation gene therapy.

  8. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

    SciTech Connect

    Nagata, Yosuke Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

    2014-08-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its

  9. An Essential Role for CD44 Variant Isoforms in Epidermal Langerhans Cell and Blood Dendritic Cell Function

    PubMed Central

    Weiss, Johannes M.; Sleeman, Jonathan; Renkl, Andreas C.; Dittmar, Henning; Termeer, Christian C.; Taxis, Sabine; Howells, Norma; Hofmann, Martin; Köhler, Gabriele; Schöpf, Erwin; Ponta, Helmut; Herrlich, Peter; Simon, Jan C.

    1997-01-01

    Upon antigen contact, epidermal Langerhans cells (LC) and dendritic cells (DC) leave peripheral organs and home to lymph nodes via the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Since splice variants of CD44 promote metastasis of certain tumors to lymph nodes, we explored the expression of CD44 proteins on migrating LC and DC. We show that upon antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6, and v9. Antibodies against CD44 epitopes inhibit the emigration of LC from the epidermis, prevent binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC. PMID:9166413

  10. Epidermal growth factor receptors on PC12 cells: alteration of binding properties by lectins

    SciTech Connect

    Vale, R.D.; Shooter, E.M.

    1983-01-01

    The PC12 cell line displays cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). It has been previously shown that the lectin wheat germ agglutinin (WGA) alters the properties of NGF receptors on these cells. We now report that preincubations with either WGA or concanavalin A (Con A) decrease the binding of /sup 125/I-EGF to PC12 cells by greater than 50%. The inhibition of binding occurred at 37 degrees C and 4 degrees C and could be blocked or reversed by the addition of sugars which bind specifically to WGA or Con A. Scatchard analysis revealed that these lectins decreased binding primarily by lowering the affinity of the receptor and to a lesser extent by decreasing receptor number. Succinylation of Con A (sCon A) produced a derivative that was less effective than the native lectin in decreasing EGF binding; however, addition of an antibody against Con A restored the ability of sCon A to decrease binding. Similar to results obtained with /sup 125/I-NGF binding, WGA but not Con A was found to increase, by severalfold, the proportion of /sup 125/I-EGF binding that is resistant to solubilization by Triton X-100 detergent. A potential association of the EGF receptor with cytoskeletal elements is discussed which could account for such results.

  11. Epidermal growth factor induces tyrosine hydroxylase in a clonal pheochromocytoma cell line, PC-G2

    SciTech Connect

    Goodman, R.; Slater, E.; Herschman, H.R.

    1980-03-01

    We have previously described the isolation of a clonal cell line (PC-G2) in which the level of tyrosine hydroxylase (TH), the rate-limiting step in the synthesis of the catecholamine neurotransmitters, is induced by nerve growth factor (NGF). We now report that epidermal growth factor (EGF) also induces TH in the PC-G2 cell line. Although EFG has been shown to be mitogenic for many cultured cells, no neuronal function has been previously reported for this protein. The TH response to EGF is elicited in a dose-dependent fashion at concentrations as low as 0.1 ng/ml and is maximal at 10 ng/ml EGF. The maximal response is observed after 3 to 4 d of exposure to 10 ng/ml EGF. The induction by NGF and EGF is inhibited by their respective antisera. Dexamethasone, a synthetic glucocorticoid which we have previously shown modulates the response of PC-G2 cells to NGF, also modulates the TH induction elicited by EGF.

  12. UV Radiation Induces the Epidermal Recruitment of Dendritic Cells that Compensate for the Depletion of Langerhans Cells in Human Skin.

    PubMed

    Achachi, Amine; Vocanson, Marc; Bastien, Philippe; Péguet-Navarro, Josette; Grande, Sophie; Goujon, Catherine; Breton, Lionel; Castiel-Higounenc, Isabelle; Nicolas, Jean-François; Gueniche, Audrey

    2015-08-01

    UVR causes skin injury and inflammation, resulting in impaired immune function and increased skin cancer risk. Langerhans cells (LCs), the immune sentinels of the epidermis, are depleted for several days following a single UVR exposure and can be reconstituted from circulating monocytes. However, the differentiation pathways leading to the recovery of a normal pool of LCs is still unclear. To study the dynamic changes in human skin with UV injury, we exposed a cohort of 29 healthy human volunteers to a clinically relevant dose of UVR and analyzed sequential epidermal biopsies for changes in leukocyte and dendritic cell (DC) subsets. UV-induced depletion of CD1a(high) LC was compensated by sequential appearance of various epidermal leukocytes. CD14(+) monocytes were recruited as early as D1 post exposure, followed by recruitment of two inflammatory DC subsets that may represent precursors of LCs. These CD1a(low) CD207(-) and the heretofore unknown CD1a(low) CD207(+) DCs appeared at day 1 and day 4 post UVR, respectively, and were endowed with T-cell-activating properties similar to those of LCs. We conclude that recruitment of monocytes and inflammatory DCs appear as a physiological response of the epidermis in order to repair UVR-induced LC depletion associated with immune suppression. PMID:25806853

  13. Heterogeneity of silica and glycan-epitope distribution in epidermal idioblast cell walls in Adiantum raddianum laminae.

    PubMed

    Leroux, Olivier; Leroux, Frederic; Mastroberti, Alexandra Antunes; Santos-Silva, Fernanda; Van Loo, Denis; Bagniewska-Zadworna, Agnieszka; Van Hoorebeke, Luc; Bals, Sara; Popper, Zoë A; de Araujo Mariath, Jorge Ernesto

    2013-06-01

    Laminae of Adiantum raddianum Presl., a fern belonging to the family Pteridaceae, are characterised by the presence of epidermal fibre-like cells under the vascular bundles. These cells were thought to contain silica bodies, but their thickened walls leave no space for intracellular silica suggesting it may actually be deposited within their walls. Using advanced electron microscopy in conjunction with energy dispersive X-ray microanalysis we showed the presence of silica in the cell walls of the fibre-like idioblasts. However, it was specifically localised to the outer layers of the periclinal wall facing the leaf surface, with the thick secondary wall being devoid of silica. Immunocytochemical experiments were performed to ascertain the respective localisation of silica deposition and glycan polymers. Epitopes characteristic for pectic homogalacturonan and the hemicelluloses xyloglucan and mannan were detected in most epidermal walls, including the silica-rich cell wall layers. The monoclonal antibody, LM6, raised against pectic arabinan, labelled the silica-rich primary wall of the epidermal fibre-like cells and the guard cell walls, which were also shown to contain silica. We hypothesise that the silicified outer wall layers of the epidermal fibre-like cells support the lamina during cell expansion prior to secondary wall formation. This implies that silicification does not impede cell elongation. Although our results suggest that pectic arabinan may be implicated in silica deposition, further detailed analyses are needed to confirm this. The combinatorial approach presented here, which allows correlative screening and in situ localisation of silicon and cell wall polysaccharide distribution, shows great potential for future studies.

  14. Synergistic action of auxin and ethylene on root elongation inhibition is caused by a reduction of epidermal cell length

    PubMed Central

    Alarcón, M Victoria; Lloret, Pedro G; Salguero, Julio

    2014-01-01

    Auxin and ethylene have been largely reported to reduce root elongation in maize primary root. However the effects of auxin are greater than those caused by ethylene. Although auxin stimulates ethylene biosynthesis through the specific increase of ACC synthase, the auxin inhibitory effect on root elongation is not mediated by the auxin-induced increase of ethylene production. Recently it has been demonstrated that root inhibition by the application of the synthetic auxin NAA (1-naphtalenacetic acid) is increased if combined with the ethylene precursor ACC (1-aminocyclopropane-1-carboxilic acid) when both compounds are applied at very low concentrations. Root elongation is basically the result of two processes: a) cell divisions in the meristem where meristematic cells continuously generate new cells and b) subsequently polarized growth by elongation along the root axis as cells leave the meristem and enter the root elongation zone. Our results indicate that exogenous auxin reduced both root elongation and epidermal cell length. In a different way, ethylene at very low concentrations only inhibited root elongation without affecting significantly epidermal cell length. However, these concentrations of ethylene increased the inhibitory effect of auxin on root elongation and cell length. Consequently the results support the hypothesis that ethylene acts synergistically with auxin in the regulation of root elongation and that inhibition by both hormones is due, at least partially, to the reduction of cell length in the epidermal layer. PMID:24598313

  15. Synergistic action of auxin and ethylene on root elongation inhibition is caused by a reduction of epidermal cell length.

    PubMed

    Alarcón, M Victoria; Lloret, Pedro G; Salguero, Julio

    2014-01-01

    Auxin and ethylene have been largely reported to reduce root elongation in maize primary root. However the effects of auxin are greater than those caused by ethylene. Although auxin stimulates ethylene biosynthesis through the specific increase of ACC synthase, the auxin inhibitory effect on root elongation is not mediated by the auxin-induced increase of ethylene production. Recently it has been demonstrated that root inhibition by the application of the synthetic auxin NAA (1-naphtalenacetic acid) is increased if combined with the ethylene precursor ACC (1-aminocyclopropane-1-carboxilic acid) when both compounds are applied at very low concentrations.   Root elongation is basically the result of two processes: a) cell divisions in the meristem where meristematic cells continuously generate new cells and b) subsequently polarized growth by elongation along the root axis as cells leave the meristem and enter the root elongation zone. Our results indicate that exogenous auxin reduced both root elongation and epidermal cell length. In a different way, ethylene at very low concentrations only inhibited root elongation without affecting significantly epidermal cell length. However, these concentrations of ethylene increased the inhibitory effect of auxin on root elongation and cell length. Consequently the results support the hypothesis that ethylene acts synergistically with auxin in the regulation of root elongation and that inhibition by both hormones is due, at least partially, to the reduction of cell length in the epidermal layer.

  16. Gene silencing for epidermal growth factor receptor variant III induces cell-specific cytotoxicity

    PubMed Central

    Yamoutpour, Farnaz; Bodempudi, Vidya; Park, Shay E.; Pan, Weihong; Mauzy, Mary Jean; Kratzke, Robert A.; Dudek, Arkadiusz; Potter, David A.; Woo, Richard A.; O’Rourke, Donald M.; Tindall, Donald J.; Farassati, Faris

    2009-01-01

    Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively active mutant form of EGFR that is expressed in 40% to 50% of gliomas and several other malignancies. Here, we describe the therapeutic effects of silencing EGFRvIII on glioma cell lines in vitro and in vivo. A small interfering RNA molecule against EGFRvIII was introduced into EGFRvIII-expressing glioma cells (U87Δ) by electroporation resulting in complete inhibition of expression of EGFRvIII as early as 48 h post-treatment. During EGFRvIII silencing, a decrease in the proliferation and invasiveness of U87Δ cells was accompanied by an increase in apoptosis (P < 0.05). Notably, EGFRvIII silencing inhibited the signal transduction machinery downstream of EGFRvIII as evidenced by decreases in the activated levels of Ras and extracellular signal-regulated kinase. A lentivirus capable of expressing anti-EGFRvIII short hairpin RNA was also able to achieve progressive silencing of EGFRvIII in U87Δ cells in addition to inhibiting cell proliferation, invasiveness, and colony formation in a significant manner (P < 0.05). Silencing EGFRvIII in U87Δ cultures with this virus reduced the expression of factors involved in epithelial-mesenchymal transition including N-cadherin, β-catenin, Snail, Slug, and paxillin but not E-cadherin. The anti-EGFRvIII lentivirus also affected the cell cycle progression of U87Δ cells with a decrease in G1 and increase in S and G2 fractions. In an in vivo model, tumor growth was completely inhibited in severe combined immunodeficient mice (n = 10) injected s.c. with U87Δ cells treated with the anti-EGFRvIII lentivirus (P = 0.005). We conclude that gene specific silencing of EGFRvIII is a promising strategy for treating cancers that contain this mutated receptor. PMID:19001441

  17. Modulation of cultured porcine granulosa cell responsiveness to follicle stimulating hormone and epidermal growth factor

    SciTech Connect

    Buck, P.A.

    1986-01-01

    Ovarian follicular development is dependent upon the coordinated growth and differentiation of the granulosa cells which line the follicle. Follicle stimulating hormone (FSH) induces granulosa cell differentiation both in vivo and in vitro. Epidermal growth factor (EGF) stimulates granulosa cell proliferation in vitro. The interaction of these two effectors upon selected parameters of growth and differentiation was examined in monolayer cultures of porcine granulose cells. Analysis of the EGF receptor by /sup 125/I-EGF binding revealed that the receptor was of high affinity with an apparent dissociation constant of 4-6 x 10/sup -10/ M. The average number of receptors per cell varied with the state of differentiation both in vivo and in vitro; highly differentiated cells bound two-fold less /sup 125/I-EGF and this effect was at least partially induced by FSH in vitro. EGF receptor function was examined by assessing EGF effects on cell number and /sup 3/H-thymidine incorporation. EGF stimulated thymidine incorporation in both serum-free and serum-supplemented culture, but only in serum-supplemented conditions was cell number increased. EGF receptor function was inversely related to the state of differentiation and was attenuated by FSH. The FSH receptor was examined by /sup 125/I-FSH binding. EGF increased FSH receptor number, and lowered the affinity of the receptor. The function of these receptors was assessed by /sup 125/I-hCG binding and progesterone radioimmunoassay. If EGF was present continuously in the cultures. FSH receptor function was attenuated regardless of FSH receptor number. A preliminary effort to examine the mechanism of this interaction was performed by analyzing hormonally controlled protein synthesis with /sup 35/S-methionine labeling, SDS polyacrylamide gel electrophoresis and fluorography. FSH promoted the expression of a 27,000 dalton protein. This effect was attenuated by EGF.

  18. Rapid and dynamic subcellular reorganization following mechanical stimulation of Arabidopsis epidermal cells mimics responses to fungal and oomycete attack

    PubMed Central

    Hardham, Adrienne R; Takemoto, Daigo; White, Rosemary G

    2008-01-01

    Background Plant cells respond to the presence of potential fungal or oomycete pathogens by mounting a basal defence response that involves aggregation of cytoplasm, reorganization of cytoskeletal, endomembrane and other cell components and development of cell wall appositions beneath the infection site. This response is induced by non-adapted, avirulent and virulent pathogens alike, and in the majority of cases achieves penetration resistance against the microorganism on the plant surface. To explore the nature of signals that trigger this subcellular response and to determine the timing of its induction, we have monitored the reorganization of GFP-tagged actin, microtubules, endoplasmic reticulum (ER) and peroxisomes in Arabidopsis plants – after touching the epidermal surface with a microneedle. Results Within 3 to 5 minutes of touching the surface of Arabidopsis cotyledon epidermal cells with fine glass or tungsten needles, actin microfilaments, ER and peroxisomes began to accumulate beneath the point of contact with the needle. Formation of a dense patch of actin was followed by focusing of actin cables on the site of contact. Touching the cell surface induced localized depolymerization of microtubules to form a microtubule-depleted zone surrounding a dense patch of GFP-tubulin beneath the needle tip. The concentration of actin, GFP-tubulin, ER and peroxisomes remained focused on the contact site as the needle moved across the cell surface and quickly dispersed when the needle was removed. Conclusion Our results show that plant cells can detect the gentle pressure of a microneedle on the epidermal cell surface and respond by reorganizing subcellular components in a manner similar to that induced during attack by potential fungal or oomycete pathogens. The results of our study indicate that during plant-pathogen interactions, the basal defence response may be induced by the plant's perception of the physical force exerted by the pathogen as it attempts to

  19. Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG.

    PubMed

    Kyakumoto, S; Kurokawa, R; Ota, M

    1990-07-12

    Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor

  20. An epidermal stem cells niche microenvironment created by engineered human amniotic membrane.

    PubMed

    Ji, Shi-zhao; Xiao, Shi-chu; Luo, Peng-fei; Huang, Guo-feng; Wang, Guang-yi; Zhu, Shi-hui; Wu, Min-juan; Xia, Zhao-fan

    2011-11-01

    How to amplify epidermal stem cells (ESCs) rapidly is a challenging crux in skin tissue engineering research. The present study describes the preparation of 3D micronized (300-600 μm) amniotic membrane (mAM) by means of repeated freeze-thawing cycles to deplete cell components and homogenized with a macrohomogenizer in liquid nitrogen. This newly prepared mAM not only possessed the characteristics of a microcarrier but completely retained the basement membrane structure and abundant active substances such as NGF, HGF, KGF, bFGF, TGF-β1 and EGF in the AM matrix. The result showed that mAM combined with rotary cell culture system (RCCS) was able to amplify ESCs quickly. The relative cell viability at day 7 and 14 was significantly higher than that of the conventional 2D plate culture (326 ± 28% and 535 ± 47% versus 232 ± 21% and 307 ± 32%, P < 0.05). In addition, the new method was able to prevent cell differentiation effectively and retain the characteristics of stem cells. When mAM loaded with ESCs (ESC-mAM) was further transplanted to full-thickness skin defects in nude mice, ESCs survived well and formed a new epidermis. Four weeks after transplantation, papilla-like structures were observed, and collagen fibers were well and regularly arranged in the newly formed dermal layer. In conclusion, the mAM as a novel natural microcarrier possesses an intact basement membrane structure and bioactivities. It not only provides the microenvironment similar to the stem cell niche within the human body favorable for ex vivo culture and amplification of ESCs but can be used as the dermal scaffold in constructing a skin substitute containing ESCs for the repair of full-thickness skin defects.

  1. Epidermal growth factor precursor in mouse lactating mammary gland alveolar cells

    SciTech Connect

    Brown, C.F.; Teng, C.T.; Pentecost, B.T.; DiAugustine, R.P. )

    1989-07-01

    Previous studies have demonstrated that high levels of epidermal growth factor (EGF) occur in human and rodent milk and that oral administration of this polypeptide stimulates rodent gastrointestinal development. It is not known whether EGF in milk originates from cells of the lactating mammary gland or is sequestered from an extramammary source. In the present study, prepro-EGF mRNA (approximately 4.7 kilobases) was detected in the CD-1 mouse mammary gland throughout the period of lactation; by comparison, negligible levels of this EGF transcript were found in the gland during pregnancy. Low levels of EGF immunoreactivity (4-5 ng/g wet wt tissue) were extracted from lactating (day 18) mammary glands with dilute acetic acid. Immunolocalization was evident with antisera to either EGF or two other regions of the EGF precursor in essentially all alveolar cells of the lactating gland. The most prominent staining with antiserum to EGF was observed along the luminal borders of cells; this pattern of cellular staining required proteolytic pretreatment of tissue sections. Western blot analyses of cell membranes isolated from the day 16 lactating mammary gland revealed an EGF-immunoreactive band at about 145K, which was equivalent in size to the EGF precursor found in mouse kidney cell membranes. Despite these findings, labeling of lactating mammary gland mince with L-(35S)methionine and cysteine for up to 4 h did not reveal any specific bands in immunoprecipitates. These cumulative findings suggest that the precursor form of EGF occurs in alveolar cells of lactating mammary gland and that this protein is translocated to the cell membrane.

  2. An epidermal stem cells niche microenvironment created by engineered human amniotic membrane.

    PubMed

    Ji, Shi-zhao; Xiao, Shi-chu; Luo, Peng-fei; Huang, Guo-feng; Wang, Guang-yi; Zhu, Shi-hui; Wu, Min-juan; Xia, Zhao-fan

    2011-11-01

    How to amplify epidermal stem cells (ESCs) rapidly is a challenging crux in skin tissue engineering research. The present study describes the preparation of 3D micronized (300-600 μm) amniotic membrane (mAM) by means of repeated freeze-thawing cycles to deplete cell components and homogenized with a macrohomogenizer in liquid nitrogen. This newly prepared mAM not only possessed the characteristics of a microcarrier but completely retained the basement membrane structure and abundant active substances such as NGF, HGF, KGF, bFGF, TGF-β1 and EGF in the AM matrix. The result showed that mAM combined with rotary cell culture system (RCCS) was able to amplify ESCs quickly. The relative cell viability at day 7 and 14 was significantly higher than that of the conventional 2D plate culture (326 ± 28% and 535 ± 47% versus 232 ± 21% and 307 ± 32%, P < 0.05). In addition, the new method was able to prevent cell differentiation effectively and retain the characteristics of stem cells. When mAM loaded with ESCs (ESC-mAM) was further transplanted to full-thickness skin defects in nude mice, ESCs survived well and formed a new epidermis. Four weeks after transplantation, papilla-like structures were observed, and collagen fibers were well and regularly arranged in the newly formed dermal layer. In conclusion, the mAM as a novel natural microcarrier possesses an intact basement membrane structure and bioactivities. It not only provides the microenvironment similar to the stem cell niche within the human body favorable for ex vivo culture and amplification of ESCs but can be used as the dermal scaffold in constructing a skin substitute containing ESCs for the repair of full-thickness skin defects. PMID:21803416

  3. Effects of Narrow Band UVB (311 nm) Irradiation on Epidermal Cells

    PubMed Central

    Reich, Adam; Mędrek, Karolina

    2013-01-01

    Ultraviolet radiation (UVR) is known to be one of the most important environmental hazards acting on the skin. It was revealed that chronic exposure to UVR accelerates skin aging, induces immunosuppression and may lead to the development of skin cancers. On the other hand, UVR has been shown to be effective in the treatment of numerous skin diseases and thus, various phototherapy modalities have been developed to date. Narrow-band ultraviolet B (NB-UVB) emitting a light with a peak around 311 nm has been demonstrated to be effective in the treatment of various skin disorders; currently it is one of the most commonly used phototherapy devices. Despite NB-UVB has been developed more than 30 years ago, the exact mechanism of its therapeutic action remains poorly understood. To date, most of NB-UVB effects were attributed to its influence on immune cells; however, nearly 90% of NB-UVB irradiation is absorbed by epidermis and keratinocytes seem to be important players in mediating NB-UVB biological activity. Here, we have reviewed the current data about the influence of NB-UVB on epidermal cells, with a special emphasis on cell proliferation and death. PMID:23594996

  4. Autoradiographic localization of epidermal growth factor receptors to all major uterine cell types

    SciTech Connect

    Lin, T.H.; Mukku, V.R.; Verner, G.; Kirkland, J.L.; Stancel, G.M.

    1988-03-01

    We have recently studied the structure and function of the uterine epidermal growth factor (EGF) receptor, its hormonal regulation, and its possible role in estrogen-induced uterine DNA synthesis. Since the uterus is composed of multiple cell types, we sought, in the work reported here, to localize EGF binding in this organ by autoradiography. Prior to the actual autoradiography, we performed a companion series of experiments to insure that EGF binding to uterine tissue in situ represented a true receptor interaction. Uteri from immature female rats were incubated in vitro with 125I-EGF at 25 degrees C. Tissue binding was maximal within 120 min and remained constant for at least an additional 120 min. This binding of labeled EGF was largely abolished by excess unlabeled EGF but not by other growth factors, indicating that binding was to specific receptors. The binding of 125I-EGF was saturable and reached a plateau at 4-8 nM; specific binding was half-maximal at 1-2 nM EGF. In situ cross-linking studies revealed that 125I-EGF was bound predominantly to a 170,000 MW EGF receptor similar to that seen in isolated uterine membranes. Incubation of uteri with 125I-EGF followed by autoradiography revealed binding to epithelial cells, stroma, and myometrium. These results provide evidence for the presence of specific EGF receptors in all major uterine cell types of the immature rat.

  5. The Potential of Menstrual Blood-Derived Stem Cells in Differentiation to Epidermal Lineage: A Preliminary Report

    PubMed Central

    Faramarzi, Hossein; Mehrabani, Davood; Fard, Maryam; Akhavan, Maryam; Zare, Sona; Bakhshalizadeh, Shabnam; Manafi, Amir; Kazemnejad, Somaieh; Shirazi, Reza

    2016-01-01

    BACKGROUND Menstrual blood-derived stem cells (MenSCs) are a novel source of stem cells that can be easily isolated non-invasively from female volunteered donor without ethical consideration. These mesenchymal-like stem cells have high rate of proliferation and possess multi lineage differentiation potency. This study was undertaken to isolate the MenSCs and assess their potential in differentiation into epidermal lineage. METHODS About 5-10 ml of menstrual blood (MB) was collected using sterile Diva cups inserted into vagina during menstruation from volunteered healthy fertile women aged between 22-30 years. MB was transferred into Falcon tubes containing phosphate buffered saline (PBS) without Ca2+ or Mg2+ supplemented with 2.5 µg/ml fungizone, 100 µg/mL streptomycin, 100 U/mL penicillin and 0.5 mM EDTA. Mononuclear cells were separated using Ficoll-Hypaque density gradient centrifugation and washed out in PBS. The cell pellet was suspended in DMEM-F12 medium supplemented with 10% FBS and cultured in tissue culture plates. The isolated cells were co-cultured with keratinocytes derived from the foreskin of healthy newborn male aged 2-10 months who was a candidate for circumcision for differentiation into epidermal lineage. RESULTS The isolated MenSCs were adhered to the plate and exhibited spindle-shaped morphology. Flow cytometric analysis revealed the expression of mesenchymal markers of CD10, CD29, CD73, and CD105 and lack of hematopoietic stem cells markers. An early success in derivation of epidermal lineage from MenSCs was visible. CONCLUSION The MenSCs are a real source to design differentiation to epidermal cells that can be used non-invasively in various dermatological lesions and diseases. PMID:27308237

  6. Differentiation of ionic currents in CNS progenitor cells: dependence upon substrate attachment and epidermal growth factor.

    PubMed

    Feldman, D H; Thinschmidt, J S; Peel, A L; Papke, R L; Reier, P J

    1996-08-01

    Multipotential progenitor cells grown from central nervous system (CNS) tissues in defined media supplemented with epidermal growth factor (EGF), when attached to a suitable substratum, differentiate to express neural and glial histochemical markers and morphologies. To assess the functional characteristics of such cells, expression of voltage-gated Na+ and K+ currents (INa, IK) was studied by whole-cell patch clamp methods in progenitors raised from postnatal rat forebrain. Undifferentiated cells were acutely dissociated from proliferative "spheres," and differentiated cells were studied 1-25 days after plating spheres onto polylysine/laminin-treated coverslips. INa and IK were detected together in 58%, INa alone in 11%, and IK alone in 19% of differentiated cells recorded with K(+)-containing pipettes. With internal Cs+ (to isolate INa), INa up to 45 pA/pF was observed in some cells within 1 day after plating. I Na ranged up to 150 pA/pF subsequently. Overall, 84% of cells expressed I Na, with an average of 38 pA/pF. INa had fast kinetics, as in neurons, but steadystate inactivation curves were strongly negative, resembling those of glial INa. Inward tail currents sensitive to [K+]out were observed upon repolarization after the 10-ms test pulse with internal Cs+, indicating the expression of K+ channels in 82% of cells. In contrast to the substantial currents observed in differentiating cells, little or no INa or Ik-tail currents were detected in recordings from cells acutely dissociated from spheres. Thus, in the presence of EGF, ionic currents develop early during differentiation induced by attachment to an appropriate substratum. Cells switched from EGF to basic fibroblast growth factor (bFGF) when plated onto coverslips showed greatly reduced proliferation and developed less neuron-like morphologies than cells plated in the presence of EGF. INa was observed in only 53% of bFGF-treated cells, with an average of 9 pA/pF. Thus, in contrast to reports that b

  7. The carboxy-terminus of p63 links cell cycle control and the proliferative potential of epidermal progenitor cells

    PubMed Central

    Suzuki, Daisuke; Sahu, Raju; Leu, N. Adrian; Senoo, Makoto

    2015-01-01

    The transcription factor p63 (Trp63) plays a key role in homeostasis and regeneration of the skin. The p63 gene is transcribed from dual promoters, generating TAp63 isoforms with growth suppressive functions and dominant-negative ΔNp63 isoforms with opposing properties. p63 also encodes multiple carboxy (C)-terminal variants. Although mutations of C-terminal variants have been linked to the pathogenesis of p63-associated ectodermal disorders, the physiological role of the p63 C-terminus is poorly understood. We report here that deletion of the p63 C-terminus in mice leads to ectodermal malformation and hypoplasia, accompanied by a reduced proliferative capacity of epidermal progenitor cells. Notably, unlike the p63-null condition, we find that p63 C-terminus deficiency promotes expression of the cyclin-dependent kinase inhibitor p21Waf1/Cip1 (Cdkn1a), a factor associated with reduced proliferative capacity of both hematopoietic and neuronal stem cells. These data suggest that the p63 C-terminus plays a key role in the cell cycle progression required to maintain the proliferative potential of stem cells of many different lineages. Mechanistically, we show that loss of Cα, the predominant C-terminal p63 variant in epithelia, promotes the transcriptional activity of TAp63 and also impairs the dominant-negative activity of ΔNp63, thereby controlling p21Waf1/Cip1 expression. We propose that the p63 C-terminus links cell cycle control and the proliferative potential of epidermal progenitor cells via mechanisms that equilibrate TAp63 and ΔNp63 isoform function. PMID:25503409

  8. The xipotl Mutant of Arabidopsis Reveals a Critical Role for Phospholipid Metabolism in Root System Development and Epidermal Cell Integrity

    PubMed Central

    Cruz-Ramírez, Alfredo; López-Bucio, José; Ramírez-Pimentel, Gabriel; Zurita-Silva, Andrés; Sánchez-Calderon, Lenin; Ramírez-Chávez, Enrique; González-Ortega, Emmanuel; Herrera-Estrella, Luis

    2004-01-01

    Phosphocholine (PCho) is an essential metabolite for plant development because it is the precursor for the biosynthesis of phosphatidylcholine, which is the major lipid component in plant cell membranes. The main step in PCho biosynthesis in Arabidopsis thaliana is the triple, sequential N-methylation of phosphoethanolamine, catalyzed by S-adenosyl-l-methionine:phosphoethanolamine N-methyltransferase (PEAMT). In screenings performed to isolate Arabidopsis mutants with altered root system architecture, a T-DNA mutagenized line showing remarkable alterations in root development was isolated. At the seedling stage, the mutant phenotype is characterized by a short primary root, a high number of lateral roots, and short epidermal cells with aberrant morphology. Genetic and biochemical characterization of this mutant showed that the T-DNA was inserted at the At3g18000 locus (XIPOTL1), which encodes PEAMT (XIPOTL1). Further analyses revealed that inhibition of PCho biosynthesis in xpl1 mutants not only alters several root developmental traits but also induces cell death in root epidermal cells. Epidermal cell death could be reversed by phosphatidic acid treatment. Taken together, our results suggest that molecules produced downstream of the PCho biosynthesis pathway play key roles in root development and act as signals for cell integrity. PMID:15295103

  9. Effect of ultraviolet B radiation on S-100 protein antigen in epidermal Langerhans cells

    SciTech Connect

    Schneider, S.A.; Fukuyama, K.; Maceira, J.; Epstein, W.L.

    1985-02-01

    Ultraviolet B (UVB) radiation has been shown to induce significant alterations in both function and surface antigen expression of epidermal Langerhans cells (ELC). In this study the effect of UVB radiation on ELC marker S-100 protein antigen (S-100 Ag) which is present in the nucleus and cytoplasm of human ELC was investigated. A total of 34 sites on 31 volunteers were exposed to 3 MED (minimal erythema dose) of UVB and biopsied at various times up to 7 days after irradiation. Skin from 9 noninjured and 7 slice-wounded subjects served as controls. The avidin-biotin-peroxidase staining technique was used to identify S-100 Ag in sections of formalin-fixed, paraffin-embedded tissue, and the numbers of stained suprabasal dendritic cells were then counted over a 200 basal cell length of interfollicular epidermis. Noninjured skin had 3.56 +/- 3.01 cells, whereas slice-wounded skin had elevated numbers (greater than 10.0 cells) at 1, 24, and 48 h after injury. Following UVB irradiation, a significant (p less than 0.001) increase in antigen-positive cells (14 +/- 3.46) was found at 1 h; this number declined to just below normal at 12 h, but by 48 h returned to and remained at preinjury levels. In contrast to previous observations of the depletion of ELC surface markers by UVB radiation, the authors demonstrate here that the numbers of S-100 Ag-positive ELC actually increase following comparable doses of radiation. Since this increase occurs so rapidly following both UVB irradiation and slice injury, S-100 Ag may be synthesized or unmasked within the ELC as a response to wounding of the epidermis.

  10. The CD44+ALDH+ Population of Human Keratinocytes Is Enriched for Epidermal Stem Cells with Long-Term Repopulating Ability

    PubMed Central

    Szabo, Akos Z.; Fong, Stephen; Yue, Lili; Zhang, Kai; Strachan, Lauren R.; Scalapino, Kenneth; Mancianti, Maria Laura; Ghadially, Ruby

    2014-01-01

    Like for other somatic tissues, isolation of a pure population of stem cells has been a primary goal in epidermal biology. We isolated discrete populations of freshly obtained human neonatal keratinocytes (HNKs) using previously untested candidate stem cell markers aldehyde dehydrogenase (ALDH) and CD44 as well as the previously studied combination of integrin α6 and CD71. An in vivo transplantation assay combined with limiting dilution analysis was used to quantify enrichment for long-term repopulating cells in the isolated populations. The ALDH+CD44+ population was enriched 12.6-fold for long-term repopulating epidermal stem cells (EpiSCs) and the integrin α6hiCD71lo population was enriched 5.6-fold, over unfractionated cells. In addition to long-term repopulation, CD44+ALDH+ keratinocytes exhibited other stem cell properties. CD44+ALDH+ keratinocytes had self-renewal ability, demonstrated by increased numbers of cells expressing nuclear Bmi-1, serial transplantation of CD44+ALDH+ cells, and holoclone formation in vitro. CD44+ALDH+ cells were multipotent, producing greater numbers of hair follicle-like structures than CD44−ALDH− cells. Furthermore, 58% ± 7% of CD44+ALDH+ cells exhibited label-retention. In vitro, CD44+ALDH+ cells showed enhanced colony formation, in both keratinocyte and embryonic stem cell growth media. In summary, the CD44+ALDH+ population exhibits stem cell properties including long-term epidermal regeneration, multipotency, label retention, and holoclone formation. This study shows that it is possible to quantify the relative number of EpiSCs in human keratinocyte populations using long-term repopulation as a functional test of stem cell nature. Future studies will combine isolation strategies as dictated by the results of quantitative transplantation assays, in order to achieve a nearly pure population of EpiSCs. PMID:23335266

  11. Preparation of Epidermal Peels and Guard Cell Protoplasts for Cellular, Electrophysiological, and -Omics Assays of Guard Cell Function.

    PubMed

    Zhu, Mengmeng; Jeon, Byeong Wook; Geng, Sisi; Yu, Yunqing; Balmant, Kelly; Chen, Sixue; Assmann, Sarah M

    2016-01-01

    Bioassays are commonly used to study stomatal phenotypes. There are multiple options in the choice of plant materials and species used for observation of stomatal and guard cell responses in vivo. Here, detailed procedures for bioassays of stomatal responses to abscisic acid (ABA) in Arabidopsis thaliana are described, including ABA promotion of stomatal closure, ABA inhibition of stomatal opening, and ABA promotion of reaction oxygen species (ROS) production in guard cells. We also include an example of a stomatal bioassay for the guard cell CO2 response using guard cell-enriched epidermal peels from Brassica napus. Highly pure preparations of guard cell protoplasts can be produced, which are also suitable for studies on guard cell signaling, as well as for studies on guard cell ion transport. Small-scale and large-scale guard cell protoplast preparations are commonly used for electrophysiological and -omics studies, respectively. We provide a procedure for small-scale guard cell protoplasting from A. thaliana. Additionally, a general protocol for large-scale preparation of guard cell protoplasts, with specifications for three different species, A. thaliana, B. napus, and Vicia faba is also provided. PMID:26577784

  12. Preparation of Epidermal Peels and Guard Cell Protoplasts for Cellular, Electrophysiological, and -Omics Assays of Guard Cell Function.

    PubMed

    Zhu, Mengmeng; Jeon, Byeong Wook; Geng, Sisi; Yu, Yunqing; Balmant, Kelly; Chen, Sixue; Assmann, Sarah M

    2016-01-01

    Bioassays are commonly used to study stomatal phenotypes. There are multiple options in the choice of plant materials and species used for observation of stomatal and guard cell responses in vivo. Here, detailed procedures for bioassays of stomatal responses to abscisic acid (ABA) in Arabidopsis thaliana are described, including ABA promotion of stomatal closure, ABA inhibition of stomatal opening, and ABA promotion of reaction oxygen species (ROS) production in guard cells. We also include an example of a stomatal bioassay for the guard cell CO2 response using guard cell-enriched epidermal peels from Brassica napus. Highly pure preparations of guard cell protoplasts can be produced, which are also suitable for studies on guard cell signaling, as well as for studies on guard cell ion transport. Small-scale and large-scale guard cell protoplast preparations are commonly used for electrophysiological and -omics studies, respectively. We provide a procedure for small-scale guard cell protoplasting from A. thaliana. Additionally, a general protocol for large-scale preparation of guard cell protoplasts, with specifications for three different species, A. thaliana, B. napus, and Vicia faba is also provided.

  13. Transient expression of minimum linear gene cassettes in onion epidermal cells via direct transformation.

    PubMed

    Cheng, Yun-Qing; Yang, Jun; Xu, Feng-Ping; An, Li-Jia; Liu, Jian-Feng; Chen, Zhi-Wen

    2009-12-01

    A new method without any special devices for direct transformation of linear gene cassettes was developed. Its feasibility was verified through 5'-fluorescent dye (fluorescein isothiocyanate, FITC)-labeled fluorescent tracing and transient expression of a gus reporter gene. Minimal linear gene cassettes, containing necessary regulation elements and a gus reporter gene, was prepared by polymerase chain reaction and dissolved in transformation buffer solution to 100 ng/mL. The basic transformation solution used was Murashige and Skoog basal salt mixture (MS) liquid medium. Hypertonic pretreatment of explants and transformation cofactors, including Ca(2+), surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated. Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h, the onion low epidermal explants were pre-cultured in darkness at 27 degrees C for 48 h and then transferred to MS solid media for 72 h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei. By GUS staining and flow-cytometry-mediated fluorescent detection, a significant increase of the ratios of fluorescent nuclei as well as expression of the gus reporter gene was observed by each designed transformation solution. This potent and feasible method showed prospective applications in plant transgenic research. PMID:19255730

  14. Transient expression of minimum linear gene cassettes in onion epidermal cells via direct transformation.

    PubMed

    Cheng, Yun-Qing; Yang, Jun; Xu, Feng-Ping; An, Li-Jia; Liu, Jian-Feng; Chen, Zhi-Wen

    2009-12-01

    A new method without any special devices for direct transformation of linear gene cassettes was developed. Its feasibility was verified through 5'-fluorescent dye (fluorescein isothiocyanate, FITC)-labeled fluorescent tracing and transient expression of a gus reporter gene. Minimal linear gene cassettes, containing necessary regulation elements and a gus reporter gene, was prepared by polymerase chain reaction and dissolved in transformation buffer solution to 100 ng/mL. The basic transformation solution used was Murashige and Skoog basal salt mixture (MS) liquid medium. Hypertonic pretreatment of explants and transformation cofactors, including Ca(2+), surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated. Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h, the onion low epidermal explants were pre-cultured in darkness at 27 degrees C for 48 h and then transferred to MS solid media for 72 h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei. By GUS staining and flow-cytometry-mediated fluorescent detection, a significant increase of the ratios of fluorescent nuclei as well as expression of the gus reporter gene was observed by each designed transformation solution. This potent and feasible method showed prospective applications in plant transgenic research.

  15. Epidermal stem cells (ESCs) accelerate diabetic wound healing via the Notch signalling pathway

    PubMed Central

    Yang, Rong-Hua; Qi, Shao-Hai; Shu, Bin; Ruan, Shu-Bin; Lin, Ze-Peng; Lin, Yan; Shen, Rui; Zhang, Feng-Gang; Chen, Xiao-Dong; Xie, Ju-Lin

    2016-01-01

    Chronic, non-healing wounds are a major complication of diabetes. Recently, various cell therapies have been reported for promotion of diabetic wound healing. Epidermal stem cells (ESCs) are considered a powerful tool for tissue therapy. However, the effect and the mechanism of the therapeutic properties of ESCs in the diabetic wound healing are unclear. Herein, to determine the ability of ESCs to diabetic wound healing, a dorsal skin defect in a streptozotocin (STZ)-induced diabetes mellitus (DM) mouse model was used. ESCs were isolated from mouse skin. We found that both the mRNA and protein levels of a Notch ligand Jagged1 (Jag1), Notch1 and Notch target gene Hairy Enhancer of Split-1 (Hes1) were significantly increased at the wound margins. In addition, we observed that Jag1 was high expressed in ESCs. Overexpression of Jag1 promotes ESCs migration, whereas knockdown Jag1 resulted in a significant reduction in ESCs migration in vitro. Importantly, Jag1 overexpression improves diabetic wound healing in vivo. These results provide evidence that ESCs accelerate diabetic wound healing via the Notch signalling pathway, and provide a promising potential for activation of the Notch pathway for the treatment of diabetic wound. PMID:27129289

  16. Epidermal stem cells (ESCs) accelerate diabetic wound healing via the Notch signalling pathway.

    PubMed

    Yang, Rong-Hua; Qi, Shao-Hai; Shu, Bin; Ruan, Shu-Bin; Lin, Ze-Peng; Lin, Yan; Shen, Rui; Zhang, Feng-Gang; Chen, Xiao-Dong; Xie, Ju-Lin

    2016-08-01

    Chronic, non-healing wounds are a major complication of diabetes. Recently, various cell therapies have been reported for promotion of diabetic wound healing. Epidermal stem cells (ESCs) are considered a powerful tool for tissue therapy. However, the effect and the mechanism of the therapeutic properties of ESCs in the diabetic wound healing are unclear. Herein, to determine the ability of ESCs to diabetic wound healing, a dorsal skin defect in a streptozotocin (STZ)-induced diabetes mellitus (DM) mouse model was used. ESCs were isolated from mouse skin. We found that both the mRNA and protein levels of a Notch ligand Jagged1 (Jag1), Notch1 and Notch target gene Hairy Enhancer of Split-1 (Hes1) were significantly increased at the wound margins. In addition, we observed that Jag1 was high expressed in ESCs. Overexpression of Jag1 promotes ESCs migration, whereas knockdown Jag1 resulted in a significant reduction in ESCs migration in vitro Importantly, Jag1 overexpression improves diabetic wound healing in vivo These results provide evidence that ESCs accelerate diabetic wound healing via the Notch signalling pathway, and provide a promising potential for activation of the Notch pathway for the treatment of diabetic wound. PMID:27129289

  17. Transient silencing of CHALCONE SYNTHASE during fruit ripening modifies tomato epidermal cells and cuticle properties.

    PubMed

    España, Laura; Heredia-Guerrero, José A; Reina-Pinto, José J; Fernández-Muñoz, Rafael; Heredia, Antonio; Domínguez, Eva

    2014-11-01

    Tomato (Solanum lycopersicum) fruit ripening is accompanied by an increase in CHALCONE SYNTHASE (CHS) activity and flavonoid biosynthesis. Flavonoids accumulate in the cuticle, giving its characteristic orange color that contributes to the eventual red color of the ripe fruit. Using virus-induced gene silencing in fruits, we have down-regulated the expression of SlCHS during ripening and compared the cuticles derived from silenced and nonsilenced regions. Silenced regions showed a pink color due to the lack of flavonoids incorporated to the cuticle. This change in color was accompanied by several other changes in the cuticle and epidermis. The epidermal cells displayed a decreased tangential cell width; a decrease in the amount of cuticle and its main components, cutin and polysaccharides, was also observed. Flavonoids dramatically altered the cuticle biomechanical properties by stiffening the elastic and viscoelastic phase and by reducing the ability of the cuticle to deform. There seemed to be a negative relation between SlCHS expression and wax accumulation during ripening that could be related to the decreased cuticle permeability to water observed in the regions silencing SlCHS. A reduction in the overall number of ester linkages present in the cutin matrix was also dependent on the presence of flavonoids.

  18. Nitric oxide promotes epidermal stem cell migration via cGMP-Rho GTPase signalling.

    PubMed

    Zhan, Rixing; He, Weifeng; Wang, Fan; Yao, Zhihui; Tan, Jianglin; Xu, Rui; Zhou, Junyi; Wang, Yuzhen; Li, Haisheng; Wu, Jun; Luo, Gaoxing

    2016-01-01

    The migration and reepithelization of epidermal stem cells (ESCs) are the most critical processes in wound healing. The gaseous messenger nitric oxide (NO) has multiple biological effects, but its actions on ESCs are poorly understood. In this study, an NO donor, S-nitroso-N-acetylpenicillamine (SNAP), was found to facilitate the in vitro migration of human ESCs (huESCs) in both live-imaging and scratch models. In addition, pull-down assays demonstrated that SNAP could activate the small GTPases RhoA and Rac1 of the Rho family, but not Cdc42. Moreover, the effects of SNAP on the migration and F-actin polymerization of ESCs could be blocked by inhibitors of cGMP, PKG, RhoA or Rac1, and by a specific siRNA of RhoA or Rac1, but not by a Cdc42 inhibitor or siRNA. Furthermore, the roles of NO in ESC migration via cGMP-Rho GTPase signalling in vivo were confirmed by tracing 5-bromo-2-deoxyuridine (BrdU)-labelled cells in a superficial, partial-thickness scald mouse model. Thus, the present study demonstrated that the NO donor SNAP could promote huESC migration in vitro. Furthermore, NO was found to induce ESC migration via cGMP-Rho GTPase RhoA and Rac1 signalling, but not Cdc42 signalling, both in vivo and in vitro. PMID:27469024

  19. Nitric oxide promotes epidermal stem cell migration via cGMP-Rho GTPase signalling

    PubMed Central

    Zhan, Rixing; He, Weifeng; Wang, Fan; Yao, Zhihui; Tan, Jianglin; Xu, Rui; Zhou, Junyi; Wang, Yuzhen; Li, Haisheng; Wu, Jun; LUO, Gaoxing

    2016-01-01

    The migration and reepithelization of epidermal stem cells (ESCs) are the most critical processes in wound healing. The gaseous messenger nitric oxide (NO) has multiple biological effects, but its actions on ESCs are poorly understood. In this study, an NO donor, S-nitroso-N-acetylpenicillamine (SNAP), was found to facilitate the in vitro migration of human ESCs (huESCs) in both live-imaging and scratch models. In addition, pull-down assays demonstrated that SNAP could activate the small GTPases RhoA and Rac1 of the Rho family, but not Cdc42. Moreover, the effects of SNAP on the migration and F-actin polymerization of ESCs could be blocked by inhibitors of cGMP, PKG, RhoA or Rac1, and by a specific siRNA of RhoA or Rac1, but not by a Cdc42 inhibitor or siRNA. Furthermore, the roles of NO in ESC migration via cGMP-Rho GTPase signalling in vivo were confirmed by tracing 5-bromo-2-deoxyuridine (BrdU)-labelled cells in a superficial, partial-thickness scald mouse model. Thus, the present study demonstrated that the NO donor SNAP could promote huESC migration in vitro. Furthermore, NO was found to induce ESC migration via cGMP-Rho GTPase RhoA and Rac1 signalling, but not Cdc42 signalling, both in vivo and in vitro. PMID:27469024

  20. Modulation of Regorafenib effects on HCC cell lines by epidermal growth factor

    PubMed Central

    Lippolis, Catia; Carella, Nicola; Messa, Caterina; Cavallini, Aldo; Carr, Brian Irving

    2015-01-01

    Purpose Blood platelet numbers are correlated to growth and aggressiveness of several tumor types, including hepatocellular carcinoma (HCC). We previously found that platelet lysates (hPLs) also stimulated growth and migration, and antagonized the growth-inhibitory and apoptotic effects of both Sorafenib and Regorafenib, two multikinase inhibitors, on three HCC cell lines. In this study, in vitro function of human epidermal growth factor (EGF) with and without Sorafenib or Regorafenib was investigated. Methods An ELISA kit was used to evaluate the EGF concentrations in hPLs. In vitro function of EGF was assessed with proliferation MTT test. Apoptosis assay, scratch assays, and Transwell assays were performed for apoptosis, invasion, and migration, respectively. MAPK Activation Kit was used to explore MAPK phosphorylation. Results EGF antagonized the growth inhibition of Regorafenib on three HCC cell lines. Regorafenib-mediated growth inhibition was blocked by 70 % when the cells were pre-treated with EGF. EGF also blocked Regorafenib-induced apoptosis, as well as Regorafenib-induced decreases in cell migration and invasion. The EGF effects were in turn antagonized by concomitant addition to the cultures of EGF receptor antagonist Erlotinib, showing that the EGF receptor was involved in the mechanisms of EGF-mediated blocking of Regorafenib effects. Erlotinib also partially blocked the effects of hPLs in antagonizing Regorafenib-mediated growth inhibition, showing that EGF was an important component of hPL actions. Conclusions All these results show that EGF antagonized Regorafenib-mediated growth and migration inhibition and apoptosis induction in HCC cells and reinforce the idea that microenvironment can influence cancer drug actions. PMID:25907508

  1. Circulating T cells of patients with active psoriasis respond to streptococcal M-peptides sharing sequences with human epidermal keratins.

    PubMed

    Sigmundsdottir, H; Sigurgeirsson, B; Troye-Blomberg, M; Good, M F; Valdimarsson, H; Jonsdottir, I

    1997-06-01

    Psoriasis is a T-cell mediated inflammatory skin disease which has been associated with group A, beta-haemolytic streptococcal infections. Four 20 a.a. long M6-peptides sharing 5-6 a.a. sequences with human epidermal keratins were identified. To investigate the role of potentially cross-reactive T cells in the pathogenesis of psoriasis, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) responses of circulating T cells to these peptides were analysed by ELISPOT and RT-PCR in 14 psoriatic patients, 12 healthy individuals and six patients with atopic dermatitis (AD). Untreated psoriatic patients' responses were significantly higher to these peptides than healthy and AD controls, while responses to a control M6-peptide, not sharing sequences with keratin, were negligible in all groups. No difference was found in response to streptokinase/streptodornase (SK/SD). M6-protein and peptides exclusively elicited IFN-gamma production, with little IL-4 production, even in AD patients. Interferon-gamma responses to all the M6-peptides were abolished after successful treatment of psoriatic patients, but responses to SK/SD were unaffected. The results indicate that active psoriasis is associated with Th1-like cells responding to streptococcal M6-peptides sharing sequences with human epidermal keratin. This is consistent with the hypothesis that psoriasis may be induced and exacerbated in susceptible individuals by M-protein specific Th1-like cells that cross-react with human epidermal keratin.

  2. Optical characterization of epidermal cells and their relationship to DNA recovery from touch samples.

    PubMed

    Stanciu, Cristina E; Philpott, M Katherine; Kwon, Ye Jin; Bustamante, Eduardo E; Ehrhardt, Christopher J

    2015-01-01

    The goal of this study was to investigate the relative contributions of different cellular and genetic components to biological samples created by touch or contact with a surface - one of the most challenging forms of forensic evidence. Touch samples were generated by having individuals hold an object for five minutes and analyzed for quantity of intact epidermal cells, extracellular DNA, and DNA from pelleted cell material after elution from the collection swab. Comparisons were made between samples where individuals had washed their hands immediately prior to handling and those where hand washing was not controlled. The vast majority (84-100%) of DNA detected in these touch samples was extracellular and was uncorrelated to the number of epidermal cells detected. Although little to no extracellular or cell pellet-associated DNA was detected when individuals washed their hands prior to substrate handling, we found that a significant number of epidermal cells (between ~5x10 (3) and ~1x10 (5)) could still be recovered from these samples, suggesting that other types of biological information may be present even when no amplifiable nuclear DNA is present. These results help to elucidate the biological context for touch samples and characterize factors that may contribute to patterns of transfer and persistence of genetic material in forensic evidence. PMID:26870321

  3. The influence of stromal cells on the pigmentation of tissue-engineered dermo-epidermal skin grafts.

    PubMed

    Biedermann, Thomas; Böttcher-Haberzeth, Sophie; Klar, Agnieszka S; Widmer, Daniel S; Pontiggia, Luca; Weber, Andreas D; Weber, Daniel M; Schiestl, Clemens; Meuli, Martin; Reichmann, Ernst

    2015-03-01

    It has been shown in vitro that melanocyte proliferation and function in palmoplantar skin is regulated by mesenchymal factors derived from fibroblasts. In this study, we investigated in vivo the influence of mesenchymal-epithelial interactions in human tissue-engineered skin substitutes reconstructed from palmar- and nonpalmoplantar-derived fibroblasts. Tissue-engineered dermo-epidermal analogs based on collagen type I hydrogels were populated with either human palmar or nonpalmoplantar fibroblasts and seeded with human nonpalmoplantar-derived melanocytes and keratinocytes. These skin substitutes were transplanted onto full-thickness skin wounds of immunoincompetent rats. Four weeks after transplantation the development of skin color was measured and grafts were excised and analyzed with regard to epidermal characteristics, in particular melanocyte number and function. Skin substitutes containing palmar-derived fibroblasts in comparison to nonpalmoplantar-derived fibroblasts showed (a) a significantly lighter pigmentation; (b) a reduced amount of epidermal melanin granules; and (c) a distinct melanosome expression. However, the number of melanocytes in the basal layer remained similar in both transplantation groups. These findings demonstrate that human palmar fibroblasts regulate the function of melanocytes in human pigmented dermo-epidermal skin substitutes after transplantation, whereas the number of melanocytes remains constant. This underscores the influence of site-specific stromal cells and their importance when constructing skin substitutes for clinical application. PMID:25300246

  4. The Influence of Stromal Cells on the Pigmentation of Tissue-Engineered Dermo-Epidermal Skin Grafts

    PubMed Central

    Biedermann, Thomas; Böttcher-Haberzeth, Sophie; Klar, Agnieszka S.; Widmer, Daniel S.; Pontiggia, Luca; Weber, Andreas D.; Weber, Daniel M.; Schiestl, Clemens; Meuli, Martin

    2015-01-01

    It has been shown in vitro that melanocyte proliferation and function in palmoplantar skin is regulated by mesenchymal factors derived from fibroblasts. In this study, we investigated in vivo the influence of mesenchymal–epithelial interactions in human tissue-engineered skin substitutes reconstructed from palmar- and nonpalmoplantar-derived fibroblasts. Tissue-engineered dermo-epidermal analogs based on collagen type I hydrogels were populated with either human palmar or nonpalmoplantar fibroblasts and seeded with human nonpalmoplantar-derived melanocytes and keratinocytes. These skin substitutes were transplanted onto full-thickness skin wounds of immunoincompetent rats. Four weeks after transplantation the development of skin color was measured and grafts were excised and analyzed with regard to epidermal characteristics, in particular melanocyte number and function. Skin substitutes containing palmar-derived fibroblasts in comparison to nonpalmoplantar-derived fibroblasts showed (a) a significantly lighter pigmentation; (b) a reduced amount of epidermal melanin granules; and (c) a distinct melanosome expression. However, the number of melanocytes in the basal layer remained similar in both transplantation groups. These findings demonstrate that human palmar fibroblasts regulate the function of melanocytes in human pigmented dermo-epidermal skin substitutes after transplantation, whereas the number of melanocytes remains constant. This underscores the influence of site-specific stromal cells and their importance when constructing skin substitutes for clinical application. PMID:25300246

  5. Mediated exodus of L-dopa from human epidermal Langerhans cells.

    PubMed

    Falck, B; Bendsoe, N; Ronquist, G

    2004-03-01

    L-3,4-dihydroxyphenylalanine (L-dopa) is not metabolized within human epidermal Langerhans cells (LC); yet they can take up substantial amounts of this amino acid which subsequently can be released into the extracellular space. We recently reported that human epidermal energy metabolism is predominantly anaerobic and that the influx mechanism is a unidirectional L-dopa/proton counter-transport system and now we describe conditions for the mediated transport of L-dopa out of the LC. It is demonstrated that certain amino acids and one dipeptide can effectively trigger the efflux of L-dopa taken up by the LC.Thus, alpha-methyl-dopa (alpha-m-dopa), D-dopa and the dipeptide, met-ala at the outside of the plasma membrane stimulated the efflux of L-dopa from L-dopa loaded LC. Similar effects were achieved by a variety of other amino acids in the extracellular fluid while some other amino acids were inactive. The time required for 50% D-methionine-induced exodus of L-dopa from L-dopa loaded LC was in the range of 5-7 min and a complete exodus of L-dopa was attained at about 20 min of incubation. This dislocation of L-dopa to the extracellular fluid is interpreted as an expression of trans-stimulation. In the case of alpha-m-dopa, D-dopa and met-ala, which admittedly were not able to penetrate the plasma membrane of LC, the concept of trans-stimulation was given a new purport, since none of them were able to participate in an exchange reaction. Finally, it could be concluded that L-dopa escaped by a route different from the one responsible for L-dopa uptake in LC.Thus, while the influx of L-dopa supports extrusion of protons deriving from anaerobic glycolysis in the LC, L-dopa efflux can provide the cells with useful amino acids in an energy-saving way, altogether a remarkable biological process. From this follows that L-dopa has a biological function of its own, besides being a precursor in the catecholamine and pigment syntheses.

  6. In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor.

    PubMed Central

    Damstrup, L.; Rude Voldborg, B.; Spang-Thomsen, M.; Brünner, N.; Skovgaard Poulsen, H.

    1998-01-01

    Formation of metastasis is a multistep process involving attachment to the basement membrane, local proteolysis and migration into surrounding tissues, lymph or bloodstream. In the present study, we have analysed the correlation between in vitro invasion and presence of the epidermal growth factor receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labelling assays. In 11 small-cell lung cancer (SCLC) cell lines, EGFR mRNA was detected by Northern blot analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16% of the cells added to the upper chamber were able to traverse the Matrigel membrane. Expression of several matrix metalloproteases (MMP), of tissue inhibitor of MMP (TIMP) and of cathepsin B was evaluated by immunoprecipitation, Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition of this antibody resulted in a significant reduction of the in vitro invasion in three selected EGFR-positive cell lines. Our results show that only EGFR-positive SCLC cell lines had the in vitro invasive phenotype, and it is therefore suggested that the EGFR might play an important role for the invasion potential of SCLC cell lines. Images Figure 1 Figure 3 Figure 4 PMID:9744504

  7. Ectodomain of Coxsackievirus and Adenovirus Receptor Genetically Fused to Epidermal Growth Factor Mediates Adenovirus Targeting to Epidermal Growth Factor Receptor-Positive Cells

    PubMed Central

    Dmitriev, Igor; Kashentseva, Elena; Rogers, Buck E.; Krasnykh, Victor; Curiel, David T.

    2000-01-01

    Human adenovirus (Ad) is extensively used for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to target cells expressing marginal levels of the Ad fiber receptor. Therefore, the present generation of Ad vectors could potentially be improved by modification of Ad tropism to target the virus to specific organs and tissues. The fact that coxsackievirus and adenovirus receptor (CAR) does not play any role in virus internalization, but functions merely as the virus attachment site, suggests that the extracellular part of CAR might be utilized to block the receptor recognition site on the Ad fiber knob domain. We proposed to design bispecific fusion proteins formed by a recombinant soluble form of truncated CAR (sCAR) and a targeting ligand. In this study, we derived sCAR genetically fused with human epidermal growth factor (EGF) and investigated its ability to target Ad infection to the EGF receptor (EGFR) overexpressed on cancer cell lines. We have demonstrated that sCAR-EGF protein is capable of binding to Ad virions and directing them to EGFR, thereby achieving targeted delivery of reporter gene. These results show that sCAR-EGF protein possesses the ability to effectively retarget Ad via a non-CAR pathway, with enhancement of gene transfer efficiency. PMID:10888627

  8. Stringent control of cytoplasmic Ca2+ in guard cells of intact plants compared to their counterparts in epidermal strips or guard cell protoplasts.

    PubMed

    Levchenko, V; Guinot, D R; Klein, M; Roelfsema, M R G; Hedrich, R; Dietrich, P

    2008-01-01

    Cytoplasmic calcium elevations, transients, and oscillations are thought to encode information that triggers a variety of physiological responses in plant cells. Yet Ca(2+) signals induced by a single stimulus vary, depending on the physiological state of the cell and experimental conditions. We compared Ca(2+) homeostasis and stimulus-induced Ca(2+) signals in guard cells of intact plants, epidermal strips, and isolated protoplasts. Single-cell ratiometric imaging with the Ca(2+)-sensitive dye Fura 2 was applied in combination with electrophysiological recordings. Guard cell protoplasts were loaded with Fura 2 via a patch pipette, revealing a cytoplasmic free Ca(2+) concentration of around 80 nM at -47 mV. Upon hyperpolarization of the plasma membrane to -107 mV, the Ca(2+) concentration increased to levels exceeding 400 nM. Intact guard cells were able to maintain much lower cytoplasmic free Ca(2+) concentrations at hyperpolarized potentials, the average concentration at -100 mV was 183 and 90 nM in epidermal strips and intact plants, respectively. Further hyperpolarization of the plasma membrane to -160 mV induced a sustained rise of the guard cell cytoplasmic Ca(2+) concentration, which slowly returned to the prestimulus level in intact plants but not in epidermal strips. Our results show that cytoplasmic Ca(2+) concentrations are stringently controlled in guard cells of intact plants but become increasingly more sensitive to changes in the plasma membrane potential in epidermal strips and isolated protoplasts.

  9. Multiple Mechanisms Are Responsible for Transactivation of the Epidermal Growth Factor Receptor in Mammary Epithelial Cells*

    PubMed Central

    Rodland, Karin D.; Bollinger, Nikki; Ippolito, Danielle; Opresko, Lee K.; Coffey, Robert J.; Zangar, Richard; Wiley, H. Steven

    2008-01-01

    The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-α, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR. EGFR transactivation also correlated with an increase in cell proliferation and could be inhibited with metalloprotease inhibitors. However, there were significant differences with respect to transactivation kinetics and sensitivity to different inhibitors. In particular, IGF-1 displayed relatively slow transactivation kinetics and was resistant to inhibition by the selective ADAM-17 inhibitor WAY-022 compared with LPA-induced transactivation. Studies using anti-ligand antibodies showed that IGF-1 transactivation required amphiregulin production, whereas LPA was dependent on multiple ligands. Direct measurement of ligand shedding confirmed that LPA treatment stimulated shedding of multiple EGFR ligands, but paradoxically, IGF-1 had little effect on the shedding rate of any ligand, including amphiregulin. Instead, IGF-1 appeared to work by enhancing EGFR activation of Ras in response to constitutively produced amphiregulin. This enhancement of EGFR signaling was independent of both receptor phosphorylation and PI-3-kinase activity, suggestive of a novel mechanism. Our studies demonstrate that within a single cell type, the EGFR autocrine system can couple multiple signaling pathways to ERK activation and that this modulation of EGFR autocrine signaling can be accomplished at multiple regulatory steps. PMID:18782770

  10. Selective Targeting of Antibody Conjugated Multifunctional Nanoclusters (Nanoroses) to Epidermal Growth Factor Receptors in Cancer Cells

    PubMed Central

    Ma, Li Leo; Tam, Justina O.; Willsey, Brian W.; Rigdon, Daniel; Ramesh, Rajagopal; Sokolov, Konstantin; Johnston, Keith P.

    2011-01-01

    The ability of smaller than 100 nm antibody (Ab) nanoparticle conjugates to target and modulate the biology of specific cell types may enable major advancements in cellular imaging and therapy in cancer. A key challenge is to load a high degree of targeting, imaging, and therapeutic functionality into small, yet stable particles. A versatile method called thin autocatalytic growth on substrate (TAGs) has been developed in our previous study to form ultra-thin and asymmetric gold coatings on iron oxide nanocluster cores producing exceptional near infrared (NIR) absorbance. AlexaFluor 488 labeled Abs were used to correlate the number of Abs conjugated to iron oxide/gold nanoclusters (nanoroses) with the hydrodynamic size. A transition from sub-monolayer to multilayer aggregates of Abs on the nanorose surface was observed for 54 Abs and an overall particle diameter of ~60 to 65 nm. The hydrodynamic diameter indicated coverage of a monolayer of 54 Abs, in agreement with the prediction of a geometric model, by assuming a circular footprint of 16.9 nm diameter per Ab molecule. The targeting efficacy of nanoclusters conjugated with monoclonal Abs specific for epidermal growth factor receptor (EGFR) was evaluated in A431 cancer cells using dark field microscopy and atomic absorbance spectrometry (AAS) analysis. Intense NIR scattering was achieved from both high uptake of nanoclusters in cells and high intrinsic NIR absorbance of individual nanoclusters. Dual mode imaging with dark field reflectance microscopy and fluorescence microscopy indicates the Abs remained attached to the Au surfaces upon the uptake by the cancer cells. The ability to load intense multifunctionality, specifically strong NIR absorbance, conjugation of an Ab monolayer in addition to a strong r2 MRI contrast that was previously demonstrated in a total particle size of only 63 nm, is an important step forward in development of theranostic agents for combined molecular specific imaging and therapy. PMID

  11. Attenuation fluctuations and local dermal reflectivity are indicators of immune cell infiltrate and epidermal hyperplasia in skin inflammation

    NASA Astrophysics Data System (ADS)

    Phillips, Kevin G.; Wang, Yun; Choudhury, Niloy; Levitz, David; Swanzey, Emily; Lagowski, James; Kulesz-Martin, Molly; Jacques, Steven

    2012-02-01

    Psoriasis is a common inflammatory skin disease resulting from genetic and environmental alterations of cutaneous immune responses responsible for skin homeostasis. While numerous therapeutic targets involved in the immunopathogenesis of psoriasis have been identified, the in vivo dynamics of psoriasis remains under investigated. To elucidate the spatial-temporal morphological evolution of psoriasis we undertook in vivo time course focus-tracked optical coherence tomography (OCT) imaging to non-invasively document dermal alterations due to immune cell infiltration and epidermal hyperplasia in an Imiquimod (IMQ) induced model of psoriasis-like inflammation in DBA2/C57Bl6 hybrid mice. Quantitative appraisal of dermal architectural changes was achieved through a three parameter fit of OCT axial scans in the dermis of the form A(z) = ρ exp(-mu;z +ɛ(z)). Ensemble averaging of the fit parameters over 2000 axial scans per mouse in each treatment arm revealed that the local dermal reflectivity ρ, decreased significantly in response to 6 day IMQ treatment (p = 0.0001), as did the standard deviation of the attenuation fluctuation std(ɛ(z)), (p = 0.04), in comparison to cream controls and day 1 treatments. No significant changes were observed in the average dermal attenuation rate, μ. Our results suggest these label-free OCT-based metrics can be deployed to investigate new therapeutic targets in animal models as well as aid in clinical staging of psoriasis in conjunction with the psoriasis area and severity index.

  12. Mesoderm induction in Xenopus laevis: responding cells must be in contact for mesoderm formation but suppression of epidermal differentiation can occur in single cells.

    PubMed

    Symes, K; Yaqoob, M; Smith, J C

    1988-12-01

    When Xenopus embryos are cultured in calcium- and magnesium-free medium (CMFM), the blastomeres lose adhesion but continue dividing to form a loose heap of cells. If divalent cations are restored at the early gastrula stage the cells re-adhere and eventually form muscle (a mesodermal cell type) as well as epidermis. If, however, the cells are dispersed during culture in CMFM, muscle does not form following reaggregation although epidermis does. This suggests that culturing blastomeres in a heap allows the transmission of mesoderm-induction signals from cell to cell while dispersion effectively dilutes the signal. In this paper, we have attempted to substitute for cell proximity by culturing dispersed blastomeres in XTC mesoderm-inducing factor (MIF). We find that dispersed cells do not respond to XTC-MIF by forming mesodermal cell types after reaggregation, but the factor does inhibit epidermal differentiation. One interpretation of this observation is that an early stage in mesoderm induction is the suppression of epidermal differentiation and that formation of mesoderm may require contact-mediated signals that are produced in response to XTC-MIF. We have gone on to study the suppression of epidermal differentiation in more detail. We find that this is a dose-dependent phenomenon that can occur in single cells in the absence of cell division. Animal pole blastomeres become more difficult to divert from epidermal differentiation at later stages of development and by stage 12 they are 'determined' to this fate. Fibroblast growth factor (FGF) also suppresses epidermal differentiation in isolated animal pole blastomeres and transforming growth factor-beta 1 acts synergistically with FGF in doing so.

  13. Blue-light-induced rapid chloroplast de-anchoring in Vallisneria epidermal cells.

    PubMed

    Sakai, Yuuki; Inoue, Shin-ichiro; Harada, Akiko; Shimazaki, Ken-Ichiro; Takagi, Shingo

    2015-01-01

    In the outer periclinal cytoplasm of leaf epidermal cells of an aquatic angiosperm Vallisneria, blue light induces "chloroplast de-anchoring", a rapid decline in the resistance of chloroplasts against centrifugal force. Chloroplast de-anchoring is known induced within 1 min of irradiation with high-fluence-rate blue light specifically, preceding the commencement of chloroplasts migration toward the anticlinal cytoplasm. However, its regulatory mechanism has remained elusive, although pharmacological analysis suggested that a calcium release from intracellular calcium stores is necessary for the response. In search of the responsible photoreceptors, immunoblotting analysis using antibodies against phototropins demonstrated that cross-reactive polypeptides of 120-kDa exist in the plasma-membrane fraction prepared from the leaves. In vitro phosphorylation analysis revealed that 120-kDa polypeptides were phosphorylated by exposure to blue light in a fluence-dependent manner. The blue-light-induced phosphorylation activity was sensitive to a Ser/Thr kinase inhibitor, staurosporine, and unusually was retained at a high level for a long time in darkness. Furthermore, phototropin gene homologs (Vallisneria PHOTOTROPIN1 and PHOTOTROPIN2) expressed in leaves were isolated. We propose that calcium-regulated chloroplast de-anchoring, possibly mediated by phototropins, is an initial process of the blue-light-induced avoidance response of chloroplasts in Vallisneria.

  14. Blue-light-induced rapid chloroplast de-anchoring in Vallisneria epidermal cells.

    PubMed

    Sakai, Yuuki; Inoue, Shin-ichiro; Harada, Akiko; Shimazaki, Ken-Ichiro; Takagi, Shingo

    2015-01-01

    In the outer periclinal cytoplasm of leaf epidermal cells of an aquatic angiosperm Vallisneria, blue light induces "chloroplast de-anchoring", a rapid decline in the resistance of chloroplasts against centrifugal force. Chloroplast de-anchoring is known induced within 1 min of irradiation with high-fluence-rate blue light specifically, preceding the commencement of chloroplasts migration toward the anticlinal cytoplasm. However, its regulatory mechanism has remained elusive, although pharmacological analysis suggested that a calcium release from intracellular calcium stores is necessary for the response. In search of the responsible photoreceptors, immunoblotting analysis using antibodies against phototropins demonstrated that cross-reactive polypeptides of 120-kDa exist in the plasma-membrane fraction prepared from the leaves. In vitro phosphorylation analysis revealed that 120-kDa polypeptides were phosphorylated by exposure to blue light in a fluence-dependent manner. The blue-light-induced phosphorylation activity was sensitive to a Ser/Thr kinase inhibitor, staurosporine, and unusually was retained at a high level for a long time in darkness. Furthermore, phototropin gene homologs (Vallisneria PHOTOTROPIN1 and PHOTOTROPIN2) expressed in leaves were isolated. We propose that calcium-regulated chloroplast de-anchoring, possibly mediated by phototropins, is an initial process of the blue-light-induced avoidance response of chloroplasts in Vallisneria. PMID:25231366

  15. Transient Gene Expression in Epidermal Cells of Plant Leaves by Biolistic DNA Delivery

    PubMed Central

    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. PMID:23104330

  16. Multiple requirements for SHPTP2 in epidermal growth factor-mediated cell cycle progression.

    PubMed Central

    Bennett, A M; Hausdorff, S F; O'Reilly, A M; Freeman, R M; Neel, B G

    1996-01-01

    Using transient overexpression and microinjection approaches, we examined SHPTP2's function in growth factor signaling. Overexpression of catalytically inactive SHPTP2 (PTP2CS) but not catalytically inactive SHPTP1, inhibited mitogen-activated protein (MAP) kinase activation and Elk-1 transactivation following epidermal growth factor (EGF) stimulation of 293 cells. An SHPTP2 mutant with both C-terminal tyrosyl phosphorylation sites converted to phenylalanine (PTP2YF) was also without effect; moreover, PTP2YF rescued PTP2CS-induced inhibition of EGF-induced Elk-1 transactivation. PTP2CS did not inhibit transactivation by activated Ras, suggesting that SHPTP2 acts upstream of or parallel to Ras. Neither PTP2CS nor PTP2YF inhibited platelet-derived growth factor (PDGF)-induced Elk-1 transactivation. Thus, protein-tyrosine phosphatase activity, but not tyrosyl phosphorylation of SHPTP2, is required for the immediate-early responses to EGF but not to PDGF. To determine whether SHPTP2 is required later in the cell cycle, we assessed S-phase entry in NIH 3T3 cells microinjected with anti-SHPTP2 antibodies or with a glutathione S-transferase (GST) fusion protein encoding both SH2 domains (GST-SH2). Microinjection of anti-SHPTP2 antibodies prior to stimulation inhibited EGF- but no PDGF- or serum-induced S-phase entry. Anti-SHPTP2 antibodies or GST-SH2 fusion protein could inhibit EGF-induced S-phase entry for up to 8 h after EGF addition. Although MAP kinase activation was detected shortly after EGF stimulation, no MAP kinase activation was detected around the restriction point. Therefore, SHPTP2 is absolutely required for immediate-early and late events induced by some, but not all, growth factors, and the immediate-early and late signal transduction pathways regulated by SHPTP2 are distinguishable. PMID:8622663

  17. Aging affects epidermal Langerhans cell development and function and alters their miRNA gene expression profile.

    PubMed

    Xu, Ying-Ping; Qi, Rui-Qun; Chen, Wenbin; Shi, Yuling; Cui, Zhi-Zhong; Gao, Xing-Hua; Chen, Hong-Duo; Zhou, Li; Mi, Qing-Sheng

    2012-11-01

    Immunosenescence is a result of progressive decline in immune system function with advancing age. Epidermal Langerhans cells (LCs), belonging to the dendritic cell (DC) family, act as sentinels to play key roles in the skin immune responses. However, it has not been fully elucidated how aging affects development and function of LCs. Here, we systemically analyzed LC development and function during the aging process in C57BL/6J mice, and performed global microRNA (miRNA) gene expression profiles in aged and young LCs. We found that the frequency and maturation of epidermal LCs were significantly reduced in aged mice starting at 12 months of age, while the Langerin expression and ability to phagocytose Dextran in aged LCs were increased compared to LCs from < 6 month old mice. The migration of LCs to draining lymph nodes was comparable between aged and young mice. Functionally, aged LCs were impaired in their capacity to induce OVA-specific CD4+ and CD8+ T cell proliferation. Furthermore, the expression of miRNAs in aged epidermal LCs showed a distinct profile compared to young LCs. Most interestingly, aging-regulated miRNAs potentially target TGF-β-dependent and non- TGF-β-dependent signal pathways related to LCs. Overall, our data suggests that aging affects LCs development and function, and that age-regulated miRNAs may contribute to the LC developmental and functional changes in aging.

  18. Chalcone synthase localization in early stages of plant development. I. Immunohistochemical use of plasmolysis for localizing the enzyme in epidermal cell cytoplasm of illuminated buckwheat hypocotyls.

    PubMed

    Zobel, A M; Hrazdina, G

    1995-01-01

    Immunohistochemical methods combined with progressive plasmolysis were used to localize chalcone synthase (CHS), an important enzyme for plant metabolism of aromatics in hypocotyls of illuminated buckwheat (Fagopyrum esculentum M.) seedlings. Illumination of etiolated seedlings with white light results in anthocyanin synthesis in the epidermal layer of the hypocotyl. Anthocyanin-containing epidermal peels, after fixation for 30 min in 4% paraformaldehyde, 2.5% glutaraldehyde, 0.1% caffeine, were treated with a specific rabbit anti-buckwheat CHS antibody and a 20 nm goat anti-rabbit IgG gold conjugate. CHS is specifically shown in epidermal cells as pink to dark red deposits. Progressive plasmolysis combined with our immunohistochemical method showed that CHS was located exclusively in the cytoplasm of the epidermal cells of buckwheat hypocotyls except for the guard cells, which contained no detectable CHS.

  19. Althaea rosea Cavanil and Plantago major L. suppress neoplastic cell transformation through the inhibition of epidermal growth factor receptor kinase.

    PubMed

    Choi, Eun-Sun; Cho, Sung-Dae; Shin, Ji-Ae; Kwon, Ki Han; Cho, Nam-Pyo; Shim, Jung-Hyun

    2012-10-01

    For thousands of years in Asia, Althaea rosea Cavanil (ARC) and Plantago major L. (PML) have been used as powerful non-toxic therapeutic agents that inhibit inflammation. However, the anticancer mechanisms and molecular targets of ARC and PML are poorly understood, particularly in epidermal growth factor (EGF)-induced neoplastic cell transformation. The aim of this study was to evaluate the chemopreventive effects and mechanisms of the methanol extracts from ARC (MARC) and PML (MPML) in EGF-induced neoplastic cell transformation of JB6 P+ mouse epidermal cells using an MTS assay, anchorage-independent cell transformation assay and western blotting. Our results showed that MARC and MPML significantly suppressed neoplastic cell transformation by inhibiting the kinase activity of the EGF receptor (EGFR). The activation of EGFR by EGF was suppressed by MARC and MPML treatment in EGFR(+/+) cells, but not in EGFR(-/-) cells. In addition, MARC and MPML inhibited EGF-induced cell proliferation in EGFR-expressing murine embryonic fibroblasts (EGFR(+/+)). These results strongly indicate that EGFR targeting by MARC and MPML may be a good strategy for chemopreventive or chemotherapeutic applications. PMID:22767187

  20. Althaea rosea Cavanil and Plantago major L. suppress neoplastic cell transformation through the inhibition of epidermal growth factor receptor kinase.

    PubMed

    Choi, Eun-Sun; Cho, Sung-Dae; Shin, Ji-Ae; Kwon, Ki Han; Cho, Nam-Pyo; Shim, Jung-Hyun

    2012-10-01

    For thousands of years in Asia, Althaea rosea Cavanil (ARC) and Plantago major L. (PML) have been used as powerful non-toxic therapeutic agents that inhibit inflammation. However, the anticancer mechanisms and molecular targets of ARC and PML are poorly understood, particularly in epidermal growth factor (EGF)-induced neoplastic cell transformation. The aim of this study was to evaluate the chemopreventive effects and mechanisms of the methanol extracts from ARC (MARC) and PML (MPML) in EGF-induced neoplastic cell transformation of JB6 P+ mouse epidermal cells using an MTS assay, anchorage-independent cell transformation assay and western blotting. Our results showed that MARC and MPML significantly suppressed neoplastic cell transformation by inhibiting the kinase activity of the EGF receptor (EGFR). The activation of EGFR by EGF was suppressed by MARC and MPML treatment in EGFR(+/+) cells, but not in EGFR(-/-) cells. In addition, MARC and MPML inhibited EGF-induced cell proliferation in EGFR-expressing murine embryonic fibroblasts (EGFR(+/+)). These results strongly indicate that EGFR targeting by MARC and MPML may be a good strategy for chemopreventive or chemotherapeutic applications.

  1. Purification and characterization of the human epidermal fatty acid-binding protein: localization during epidermal cell differentiation in vivo and in vitro.

    PubMed

    Siegenthaler, G; Hotz, R; Chatellard-Gruaz, D; Didierjean, L; Hellman, U; Saurat, J H

    1994-09-01

    Epidermal fatty acid-binding protein (E-FABP) was isolated from human skin and purified to homogeneity. Its molecular mass was estimated to be 15 kDa and the pI of non-denaturing protein was 5.6. Scatchard-plot analysis revealed one class of binding site for oleic acid with a Kd of 0.46 microM. Structure-binding relation experiments revealed a high affinity of E-FABP for stearic acid which decreased on reduction of the number of carbon atoms or introduction of double bonds into the fatty acid chain. Squalene, cholesterol and retinoic acid isomers showed no affinity, suggesting that E-FABP displays high specificity for fatty acids. E-FABP is a scarce cytosolic protein (0.065% of total protein). Only trace amounts could be detected in normal human skin but up to 42.5 +/- 3.4 pmol/mg of protein was found in a non-malignant defect of keratinocyte differentiation (psoriatic lesions). E-FABP levels were low in cultured human keratinocytes grown under proliferation-stimulating conditions but increased about 2-fold on induction of differentiation by Ca2+. Immunohistochemical localization showed cytosolic staining in differentiated cells of normal and psoriatic skin, suggesting a link between E-FABP and keratinocyte differentiation. The presence of E-FABP in tissues other than skin (heart, intestine and adipose tissue) excludes its specific role in fatty acid metabolism in epithelial cells or its involvement in skin lipid-barrier function.

  2. Plasma membrane-cell wall connections: roles in mitosis and cytokinesis revealed by plasmolysis of Tradescantia virginiana leaf epidermal cells.

    PubMed

    Cleary, A L

    2001-01-01

    Tradescantia virginiana leaf epidermal cells were plasmolysed by sequential treatment with 0.8 M and 0.3 M sucrose. Plasmolysis revealed adhesion of the plasma membrane to the cell wall at sites coinciding with cytoskeletal arrays involved in the polarisation of cells undergoing asymmetric divisions--cortical actin patch--and in the establishment and maintenance of the division site--preprophase band of microtubules and filamentous (F) actin. The majority of cells retained adhesions at the actin patch throughout mitosis. However, only approximately 13% of cells formed or retained attachments at the site of the preprophase band. After the breakdown of the nuclear envelope, plasmolysis had a dramatic effect on spindle orientation, cell plate formation, and the plane of cytokinesis. Spindles were rotated at abnormal angles including tilted into the plane of the epidermis. Cell plates formed but were quickly replaced by vacuole-like intercellular compartments containing no Tinopal-stainable cell wall material. This compartment usually opened to the apoplast at one side, and cytokinesis was completed by the furrow extending across the protoplast. This atypical cytokinesis was facilitated by a phragmoplast containing microtubules and F-actin. Progression of the furrow was unaffected by 25 micrograms of cytochalasin B per ml but inhibited by 10 microM oryzalin. Phragmoplasts were contorted and misguided and cytokinesis prolonged, indicating severe disruption to the guidance mechanisms controlling phragmoplast expansion. These results are discussed in terms of cytoskeleton-plasma membrane-cell wall connections that could be important to the localisation of plasma membrane molecules defining the cortical division site and hence providing positional information to the cytokinetic apparatus, and/or for providing an anchor for cytoplasmic F-actin necessary to generate tension on the phragmoplast and facilitate its directed, planar expansion.

  3. Ultraviolet Radiation-Induced Skin Aging: The Role of DNA Damage and Oxidative Stress in Epidermal Stem Cell Damage Mediated Skin Aging

    PubMed Central

    Panich, Uraiwan; Sittithumcharee, Gunya; Rathviboon, Natwarath

    2016-01-01

    Skin is the largest human organ. Skin continually reconstructs itself to ensure its viability, integrity, and ability to provide protection for the body. Some areas of skin are continuously exposed to a variety of environmental stressors that can inflict direct and indirect damage to skin cell DNA. Skin homeostasis is maintained by mesenchymal stem cells in inner layer dermis and epidermal stem cells (ESCs) in the outer layer epidermis. Reduction of skin stem cell number and function has been linked to impaired skin homeostasis (e.g., skin premature aging and skin cancers). Skin stem cells, with self-renewal capability and multipotency, are frequently affected by environment. Ultraviolet radiation (UVR), a major cause of stem cell DNA damage, can contribute to depletion of stem cells (ESCs and mesenchymal stem cells) and damage of stem cell niche, eventually leading to photoinduced skin aging. In this review, we discuss the role of UV-induced DNA damage and oxidative stress in the skin stem cell aging in order to gain insights into the pathogenesis and develop a way to reduce photoaging of skin cells. PMID:27148370

  4. Stomatal Spacing Safeguards Stomatal Dynamics by Facilitating Guard Cell Ion Transport Independent of the Epidermal Solute Reservoir.

    PubMed

    Papanatsiou, Maria; Amtmann, Anna; Blatt, Michael R

    2016-09-01

    Stomata enable gaseous exchange between the interior of the leaf and the atmosphere through the stomatal pore. Control of the pore aperture depends on osmotic solute accumulation by, and its loss from the guard cells surrounding the pore. Stomata in most plants are separated by at least one epidermal cell, and this spacing is thought to enhance stomatal function, although there are several genera that exhibit stomata in clusters. We made use of Arabidopsis (Arabidopsis thaliana) stomatal patterning mutants to explore the impact of clustering on guard cell dynamics, gas exchange, and ion transport of guard cells. These studies showed that stomatal clustering in the Arabidopsis too many mouths (tmm1) mutant suppressed stomatal movements and affected CO2 assimilation and transpiration differentially between dark and light conditions and were associated with alterations in K(+) channel gating. These changes were consistent with the impaired dynamics of tmm1 stomata and were accompanied by a reduced accumulation of K(+) ions in the guard cells. Our findings underline the significance of spacing for stomatal dynamics. While stomatal spacing may be important as a reservoir for K(+) and other ions to facilitate stomatal movements, the effects on channel gating, and by inference on K(+) accumulation, cannot be explained on the basis of a reduced number of epidermal cells facilitating ion supply to the guard cells. PMID:27406168

  5. Stomatal Spacing Safeguards Stomatal Dynamics by Facilitating Guard Cell Ion Transport Independent of the Epidermal Solute Reservoir12[OPEN

    PubMed Central

    Papanatsiou, Maria; Amtmann, Anna

    2016-01-01

    Stomata enable gaseous exchange between the interior of the leaf and the atmosphere through the stomatal pore. Control of the pore aperture depends on osmotic solute accumulation by, and its loss from the guard cells surrounding the pore. Stomata in most plants are separated by at least one epidermal cell, and this spacing is thought to enhance stomatal function, although there are several genera that exhibit stomata in clusters. We made use of Arabidopsis (Arabidopsis thaliana) stomatal patterning mutants to explore the impact of clustering on guard cell dynamics, gas exchange, and ion transport of guard cells. These studies showed that stomatal clustering in the Arabidopsis too many mouths (tmm1) mutant suppressed stomatal movements and affected CO2 assimilation and transpiration differentially between dark and light conditions and were associated with alterations in K+ channel gating. These changes were consistent with the impaired dynamics of tmm1 stomata and were accompanied by a reduced accumulation of K+ ions in the guard cells. Our findings underline the significance of spacing for stomatal dynamics. While stomatal spacing may be important as a reservoir for K+ and other ions to facilitate stomatal movements, the effects on channel gating, and by inference on K+ accumulation, cannot be explained on the basis of a reduced number of epidermal cells facilitating ion supply to the guard cells. PMID:27406168

  6. Stomatal Spacing Safeguards Stomatal Dynamics by Facilitating Guard Cell Ion Transport Independent of the Epidermal Solute Reservoir.

    PubMed

    Papanatsiou, Maria; Amtmann, Anna; Blatt, Michael R

    2016-09-01

    Stomata enable gaseous exchange between the interior of the leaf and the atmosphere through the stomatal pore. Control of the pore aperture depends on osmotic solute accumulation by, and its loss from the guard cells surrounding the pore. Stomata in most plants are separated by at least one epidermal cell, and this spacing is thought to enhance stomatal function, although there are several genera that exhibit stomata in clusters. We made use of Arabidopsis (Arabidopsis thaliana) stomatal patterning mutants to explore the impact of clustering on guard cell dynamics, gas exchange, and ion transport of guard cells. These studies showed that stomatal clustering in the Arabidopsis too many mouths (tmm1) mutant suppressed stomatal movements and affected CO2 assimilation and transpiration differentially between dark and light conditions and were associated with alterations in K(+) channel gating. These changes were consistent with the impaired dynamics of tmm1 stomata and were accompanied by a reduced accumulation of K(+) ions in the guard cells. Our findings underline the significance of spacing for stomatal dynamics. While stomatal spacing may be important as a reservoir for K(+) and other ions to facilitate stomatal movements, the effects on channel gating, and by inference on K(+) accumulation, cannot be explained on the basis of a reduced number of epidermal cells facilitating ion supply to the guard cells.

  7. The regeneration of epidermal cells of Saintpaulia leaves as a new plant-tissue system for cellular radiation biology.

    PubMed

    Engels, F M; van der Laan, F M; Leenhouts, H P; Chadwick, K H

    1980-09-01

    Investigation of the nucleus of epidermal cells of the petioles of Saintpaulia leaves by cytofluorimetry revealed that all cells are in a non-cycling pre DNA synthesis phase. Cultivation of dissected leaves results in a synchronous regeneration process of a defined number of cells. Five days after onset of cultivation the cells reach the first mitosis. The nuclear development during the regeneration process is described. Irradiation of the leaves results in a directly visible inhibition of this regenerating capability which is used to quantify cell survival in a tissue. The data show that the radiation response has a similar shape to that of the survival of single cells in culture. This response can be observed before the first mitosis of the cells and its application as a new plant tissue system for cellular radiation research is discussed. PMID:7012060

  8. Effects of immunomodulatory drugs on TNF-α and IL-12 production by purified epidermal langerhans cells and peritoneal macrophages

    PubMed Central

    2011-01-01

    Background Langerhans cells constitute a special subset of immature dendritic cells localized in the epidermis that play a key role in the skin's immune response. The production of cytokines is a key event in both the initiation and the regulation of immune responses, and different drugs can be used to remove or modify their production by DC and, therefore, alter immune responses in a broad spectrum of diseases, mainly in human inflammatory and autoimmune diseases. In the present study, we examined the effects of prednisone, thalidomide, cyclosporine A, and amitriptyline, drugs used in a variety of clinical conditions, on the production of TNF-α, IL-10, and IL-12 by purified epidermal Langerhans cells and peritoneal macrophages in BALB/c mice. Findings All drugs inhibited TNF-α production by Langerhans cells after 36 hours of treatment at two different concentrations, while prednisone and thalidomide decreased IL-12 secretion significantly, amitriptyline caused a less pronounced reduction and cyclosporine A had no effect. Additionally, TNF-α and IL-12 production by macrophages decreased, but IL-10 levels were unchanged after all treatments. Conclusions Our results demonstrate that these drugs modulate the immune response by regulating pro-inflammatory cytokine production by purified epidermal Langerhans cells and peritoneal macrophages, indicating that these cells are important targets for immunosuppression in various clinical settings. PMID:21276247

  9. BAG-1 enhances cell-cell adhesion, reduces proliferation and induces chaperone-independent suppression of hepatocyte growth factor-induced epidermal keratinocyte migration

    SciTech Connect

    Hinitt, C.A.M.; Wood, J.; Lee, S.S.; Williams, A.C.; Howarth, J.L.; Glover, C.P.; Uney, J.B.; Hague, A.

    2010-08-01

    Cell motility is important in maintaining tissue homeostasis, facilitating epithelial wound repair and in tumour formation and progression. The aim of this study was to determine whether BAG-1 isoforms regulate epidermal cell migration in in vitro models of wound healing. In the human epidermal cell line HaCaT, endogenous BAG-1 is primarily nuclear and increases with confluence. Both transient and stable p36-Bag-1 overexpression resulted in increased cellular cohesion. Stable transfection of either of the three human BAG-1 isoforms p36-Bag-1 (BAG-1S), p46-Bag-1 (BAG-1M) and p50-Bag-1 (BAG-1L) inhibited growth and wound closure in serum-containing medium. However, in response to hepatocyte growth factor (HGF) in serum-free medium, BAG-1S/M reduced communal motility and colony scattering, but BAG-1L did not. In the presence of HGF, p36-Bag-1 transfectants retained proliferative response to HGF with no change in ERK1/2 activation. However, the cells retained E-cadherin localisation at cell-cell junctions and exhibited pronounced cortical actin. Point mutations in the BAG domain showed that BAG-1 inhibition of motility is independent of its function as a chaperone regulator. These findings are the first to suggest that BAG-1 plays a role in regulating cell-cell adhesion and suggest an important function in epidermal cohesion.

  10. Surface Position, Not Signaling from Surrounding Maternal Tissues, Specifies Aleurone Epidermal Cell Fate in Maize[OA

    PubMed Central

    Gruis, Darren (Fred); Guo, Hena; Selinger, David; Tian, Qing; Olsen, Odd-Arne

    2006-01-01

    Maize (Zea mays) endosperm consists of an epidermal-like surface layer of aleurone cells, an underlying body of starchy endosperm cells, and a basal layer of transfer cells. To determine whether surrounding maternal tissues perform a role in specifying endosperm cell fates, a maize endosperm organ culture technique was established whereby the developing endosperm is completely removed from surrounding maternal tissues. Using cell type-specific fluorescence markers, we show that aleurone cell fate specification occurs exclusively in response to surface position and does not require specific, continued maternal signal input. The starchy endosperm and aleurone cell fates are freely interchangeable throughout the lifespan of the endosperm, with internalized aleurone cells converting to starchy endosperm cells and with starchy endosperm cells that become positioned at the surface converting to aleurone cells. In contrast to aleurone and starchy endosperm cells, transfer cells fail to develop in in vitro-grown endosperm, supporting earlier indications that maternal tissue interaction is required to fully differentiate this cell type. Several parameters confirm that the maize endosperm organ cultures described herein retain the main developmental features of in planta endosperm, including fidelity of aleurone mutant phenotypes, temporal and spatial control of cell type-specific fluorescent markers, specificity of cell type transcripts, and control of mitotic cell divisions. PMID:16698897

  11. Characterization of a Putative Receptor Binding Surface on Skint-1, a Critical Determinant of Dendritic Epidermal T Cell Selection*

    PubMed Central

    Salim, Mahboob; Knowles, Timothy J.; Hart, Rosie; Mohammed, Fiyaz; Woodward, Martin J.; Willcox, Carrie R.; Overduin, Michael; Hayday, Adrian C.; Willcox, Benjamin E.

    2016-01-01

    Dendritic epidermal T cells (DETC) form a skin-resident γδ T cell population that makes key contributions to cutaneous immune stress surveillance, including non-redundant contributions to protection from cutaneous carcinogens. How DETC become uniquely associated with the epidermis was in large part solved by the identification of Skint-1, the prototypic member of a novel B7-related multigene family. Expressed only by thymic epithelial cells and epidermal keratinocytes, Skint-1 drives specifically the development of DETC progenitors, making it the first clear candidate for a selecting ligand for non-MHC/CD1-restricted T cells. However, the molecular mechanisms underpinning Skint-1 activity are unresolved. Here, we provide evidence that DETC selection requires Skint-1 expression on the surface of thymic epithelial cells, and depends upon specific residues on the CDR3-like loop within the membrane-distal variable domain of Skint-1 (Skint-1 DV). Nuclear magnetic resonance of Skint-1 DV revealed a core tertiary structure conserved across the Skint family, but a highly distinct surface charge distribution, possibly explaining its unique function. Crucially, the CDR3-like loop formed an electrostatically distinct surface, featuring key charged and hydrophobic solvent-exposed residues, at the membrane-distal tip of DV. These results provide the first structural insights into the Skint family, identifying a putative receptor binding surface that directly implicates Skint-1 in receptor-ligand interactions crucial for DETC selection. PMID:26917727

  12. Accumulation and activation of epidermal γδ T cells in a mouse model of chronic dermatitis is not required for the inflammatory phenotype.

    PubMed

    Sulcova, Jitka; Maddaluno, Luigi; Meyer, Michael; Werner, Sabine

    2015-09-01

    Chronic skin inflammation resulting from a defective epidermal barrier is a hallmark of atopic dermatitis (AD). We previously demonstrated that mice lacking FGF receptors 1 and 2 in keratinocytes (K5-R1/R2 mice) develop an AD-like chronic dermatitis as a result of an impaired epidermal barrier. Here, we show that γδ T cells, which rapidly respond to various insults, accumulate in the epidermis of K5-R1/R2 mice before the development of histological abnormalities. Their number and activation further increase as the phenotype progresses, most likely as a consequence of increased expression of Il-2 and Il-7 and the stress-induced proteins Rae-1, H60c, Mult1, PlexinB2, and Skint1. To determine the role of γδ T cells in the skin phenotype, we generated quadruple mutant K5-R1/-R2 mice lacking γδ T cells. Surprisingly, loss of γδ T cells did not or only marginally affect keratinocyte proliferation, epidermal thickness, epidermal barrier function, and accumulation and activation of different immune cells in the skin of K5-R1/R2 mice, possibly due to partial compensation by αβ T cells. These results demonstrate that γδ T cells do not contribute to the development or maintenance of chronic inflammation in response to a defect in the epidermal barrier.

  13. Neuropilin 1 expression correlates with differentiation status of epidermal cells and cutaneous squamous cell carcinomas

    PubMed Central

    Shahrabi-Farahani, Shokoufeh; Wang, Lili; Zwaans, Bernadette M. M.; Santana, Jeans M.; Shimizu, Akio; Takashima, Seiji; Kreuter, Michael; Coultas, Leigh; D'Amore, Patricia A.; Arbeit, Jeffrey M.; Akslen, Lars A.; Bielenberg, Diane R.

    2014-01-01

    Neuropilins (NRP) are cell surface receptors for VEGF and SEMA3 family members. The role of NRP in neurons and endothelial cells has been investigated, but the expression and role of NRP in epithelial cells is much less clear. Herein, the expression and localization of neuropilin 1 (NRP1) was investigated in human and mouse skin and squamous cell carcinomas (SCC). Results indicated that NRP1 mRNA and protein was expressed in the suprabasal epithelial layers of skin sections. NRP1 staining did not overlap with that of keratin 14 (K14) or proliferating cell nuclear antigen, but did colocalize with staining for keratin 1, indicating that differentiated keratinocytes express NRP1. Similar to the expression of NRP1, VEGF-A was expressed in suprabasal epithelial cells, whereas Nrp2 and VEGFR2 were not detectable in the epidermis. The expression of NRP1 correlated with a high degree of differentiation in human SCC specimens, human SCC xenografts, and mouse K14-HPV16 transgenic SCC. UVB irradiation of mouse skin induced Nrp1 upregulation. In vitro, Nrp1 was upregulated in primary keratinocytes in response to differentiating media or EGF-family growth factors. In conclusion, the expression of NRP1 is regulated in the skin and is selectively produced in differentiated epithelial cells. NRP1 may function as a reservoir to sequester VEGF ligand within the epithelial compartment, thereby modulating its bioactivity. PMID:24791743

  14. Mdm2-p53 signaling regulates epidermal stem cell senescence and premature aging phenotypes in mouse skin.

    PubMed

    Gannon, Hugh S; Donehower, Lawrence A; Lyle, Stephen; Jones, Stephen N

    2011-05-01

    The p53 transcription factor is activated by various types of cell stress or DNA damage and induces the expression of genes that control cell growth and inhibit tumor formation. Analysis of mice that express mutant forms of p53 suggest that inappropriate p53 activation can alter tissue homeostasis and life span, connecting p53 tumor suppressor functions with accelerated aging. However, other mouse models that display increased levels of wildtype p53 in various tissues fail to corroborate a link between p53 and aging phenotypes, possibly due to the retention of signaling pathways that negatively regulate p53 activity in these models. In this present study, we have generated mice lacking Mdm2 in the epidermis. Deletion of Mdm2, the chief negative regulator of p53, induced an aging phenotype in the skin of mice, including thinning of the epidermis, reduced wound healing, and a progressive loss of fur. These phenotypes arise due to an induction of p53-mediated senescence in epidermal stem cells and a gradual loss of epidermal stem cell function. These results reveal that activation of endogenous p53 by ablation of Mdm2 can induce accelerated aging phenotypes in mice.

  15. In situ expression of the costimulatory molecules CD80 and CD86 on langerhans cells and inflammatory dendritic epidermal cells (IDEC) in atopic dermatitis.

    PubMed

    Schuller, E; Teichmann, B; Haberstok, J; Moderer, M; Bieber, T; Wollenberg, A

    2001-09-01

    The functional expression of costimulatory molecules on antigen-presenting cells may be a key event in the pathogenesis of atopic dermatitis (AD). Recently, the expression of CD86 (B7-2/B70) has been demonstrated on CD1a+ epidermal dendritic cells (DC) in AD lesions by immunohistological and functional analysis. Therefore, we sought to further characterize the in situ expression of costimulatory molecules on these cells, considering the two subpopulations of (1) CD1a+++/CD11b- Langerhans cells (LC) containing Birbeck granules and (2) CD1a+/CD11b+++ inflammatory dendritic epidermal cells (IDEC), devoid of Birbeck granules, from AD and other inflammatory skin diseases. Flow cytometry, skin mixed lymphocyte reactions (SMLR) and immunohistological analysis were performed, and showed that IDEC and not LC are the relevant cells expressing the costimulatory molecules CD80 and CD86 in situ. This expression varied with the underlying diagnosis, with AD showing the highest expression of both CD80 and CD86 in situ. Furthermore, the expression of CD80, CD86 and CD36 were significantly correlated. With short-term culture, both CD80 and CD86 were further upregulated on LC and IDEC. Finally, anti-CD86 antibody reduced the stimulatory activity of epidermal DC. These results indicate that costimulatory molecules on LC and IDEC might play a role in the pathogenesis of AD.

  16. Involvement of aquaporin-3 in epidermal growth factor receptor signaling via hydrogen peroxide transport in cancer cells.

    PubMed

    Hara-Chikuma, Mariko; Watanabe, Sachiko; Satooka, Hiroki

    2016-03-18

    Aquaporin 3 (AQP3), a water/glycerol channel protein, is capable of transporting hydrogen peroxide (H2O2). Here, we show that AQP3-mediated intracellular H2O2 is involved in epidermal growth factor (EGF)-induced cell signaling and its dependent cell function in the EGF receptor (EGFR)-positive cancer cell lines A431 and H1666. AQP3 knockdown suppressed the transport into the cells of extracellular H2O2 produced in response to EGF in A431 and H1666 cells. EGF-induced Erk and Akt activation, which occurred through SHP2 and/or PTEN modulation, was impaired by AQP3 knockdown. Cell growth and migration induced by EGF stimulation were attenuated in AQP3 knockdown cells compared with those in control cells. Coincidentally, tumor growth of A431 cell xenografts in immunodeficient mice was decreased by AQP3 knockdown. Accordingly, a xenograft with AQP3 knockdown A431 cells significantly enhanced the survival of recipient mice compared with the transplantation with control cells. In addition, AQP3 associated with EGFR and NADPH oxidase 2, which we propose is linked to AQP3 producing a localized increase in intracellular H2O2 to function as a second messenger during EGFR cell signaling. Therefore, our findings suggest that AQP3 is required for EGF-EGFR cell signaling in cancer cells and is a therapeutic target for cancer progression.

  17. Variable area fuel cell cooling

    DOEpatents

    Kothmann, Richard E.

    1982-01-01

    A fuel cell arrangement having cooling fluid flow passages which vary in surface area from the inlet to the outlet of the passages. A smaller surface area is provided at the passage inlet, which increases toward the passage outlet, so as to provide more uniform cooling of the entire fuel cell. The cooling passages can also be spaced from one another in an uneven fashion.

  18. (−)-Epigallocatechin gallate causes internalization of the epidermal growth factor receptor in human colon cancer cells

    PubMed Central

    Adachi, Seiji; Nagao, Tomokazu; To, Satoshi; Joe, Andrew K.; Shimizu, Masahito; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Moriwaki, Hisataka; Maxfield, Frederick R.; Weinstein, I.Bernard

    2008-01-01

    We recently found that the inhibitory effect of (−)-epigallocatechin gallate (EGCG) on epidermal growth factor (EGF) binding to the epidermal growth factor receptor (EGFR) is associated with alterations in lipid organization in the plasma membrane of colon cancer cells. Since changes in lipid organizations are thought to play a role in the trafficking of several membrane proteins, in this study we examined the effects of EGCG on cellular localization of the EGFR in SW480 cells. Treatment of the cells for 30 min with as little as 1 μg/ml of EGCG caused a decrease in cell surface-associated EGFRs and this was associated with internalization of EGFRs into endosomal vesicles. Similar effects were seen with a green fluorescent protein (GFP)–EGFR fusion protein. As expected, the EGFR protein was phosphorylated at tyrosine residues, ubiquitinated and partially degraded when the cells were treated with EGF, but treatment with EGCG caused none of these effects. The loss of EGFRs from the cell surface induced by treating the cells with EGF for 30 min persisted for at least 2 h. However, the loss of EGFRs from the cell surface induced by temporary exposure to EGCG was partially restored within 1–2 h. These studies provide the first evidence that EGCG can induce internalization of EGFRs into endosomes, which can recycle back to the cell surface. This sequestrating of inactivated EGFRs into endosomes may explain, at least in part, the ability of EGCG to inhibit activation of the EGFR and thereby exert anticancer effects. PMID:18586691

  19. Starting to Gel: How Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls

    PubMed Central

    Voiniciuc, Cătălin; Yang, Bo; Schmidt, Maximilian Heinrich-Wilhelm; Günl, Markus; Usadel, Björn

    2015-01-01

    For more than a decade, the Arabidopsis seed coat epidermis (SCE) has been used as a model system to study the synthesis, secretion and modification of cell wall polysaccharides, particularly pectin. Our detailed re-evaluation of available biochemical data highlights that Arabidopsis seed mucilage is more than just pectin. Typical secondary wall polymers such as xylans and heteromannans are also present in mucilage. Despite their low abundance, these components appear to play essential roles in controlling mucilage properties, and should be further investigated. We also provide a comprehensive community resource by re-assessing the mucilage phenotypes of almost 20 mutants using the same conditions. We conduct an in-depth functional evaluation of all the SCE genes described in the literature and propose a revised model for mucilage production. Further investigation of SCE cells will improve our understanding of plant cell walls. PMID:25658798

  20. Starting to gel: how Arabidopsis seed coat epidermal cells produce specialized secondary cell walls.

    PubMed

    Voiniciuc, Cătălin; Yang, Bo; Schmidt, Maximilian Heinrich-Wilhelm; Günl, Markus; Usadel, Björn

    2015-02-04

    For more than a decade, the Arabidopsis seed coat epidermis (SCE) has been used as a model system to study the synthesis, secretion and modification of cell wall polysaccharides, particularly pectin. Our detailed re-evaluation of available biochemical data highlights that Arabidopsis seed mucilage is more than just pectin. Typical secondary wall polymers such as xylans and heteromannans are also present in mucilage. Despite their low abundance, these components appear to play essential roles in controlling mucilage properties, and should be further investigated. We also provide a comprehensive community resource by re-assessing the mucilage phenotypes of almost 20 mutants using the same conditions. We conduct an in-depth functional evaluation of all the SCE genes described in the literature and propose a revised model for mucilage production. Further investigation of SCE cells will improve our understanding of plant cell walls.

  1. Binding, sequestration, and processing of epidermal growth factor and nerve growth factor by PC12 cells. [Rats

    SciTech Connect

    Chandler, C.E.; Herschman, H.R.

    1983-03-01

    Th rat PC12 pheochromocytoma cell line exhibits biological responses to both nerve growth factor (NGF) and epidermal growth factor (EGF). The existence of receptors and biological responses on a common cell for these two well-characterized polypeptide growth factors makes this an attractive system for comparison of ligand binding and processing. Both NGF and EGF are bound to PC12 cells in a competable form at 4/sup 0/C. At 37/sup 0/C both ligands are ''sequestered,'' but at different rates and to different extents. While sequestration happens rapidly and nearly quantitatively for bound EGF, the dissociation reaction appears to compete favorably with NFG sequestration. Both EGF and NGF are degraded by PC12 cells. Sequestered EGF, however, is degraded to a greater extent than sequestered NGF.

  2. Epidermal growth factor induces biphasic activation of ornithine decarboxylase in human stomach-derived KATO-III cells.

    PubMed

    Ishikawa, T; Mitsuhashi, M; Ichikawa, Y; Tarnawski, A

    1994-01-01

    Effect of epidermal growth factor (EGF) on ornithine decarboxylase (ODC) was examined in human gastric cancer-derived KATO-III cells, because 125I-EGF binding studies indicated a presence of specific binding sites for EGF on these cells. Upon stimulation with EGF, both ODC mRNA expression and ODC enzyme activity were significantly increased in KATO-III cells. However, unlike in other cellular systems, both EGF-induced ODC mRNA expression and ODC enzyme activation were biphasic with the peaks at 15 +/- 10 min and 2.1 +/- 1.5 hrs (mean +/- SE) for mRNA, and 3.1 +/- 1.5 and 7.7 +/- 1.8 hrs (mean +/- SE) for enzyme activity, respectively. Therefore, KATO-III cell line may provide a unique model for the biochemical analysis of EGF action on ODC activation. PMID:8190004

  3. Cadmium induces autophagy through ROS-dependent activation of the LKB1-AMPK signaling in skin epidermal cells

    SciTech Connect

    Son, Young-Ok; Wang Xin; Hitron, John Andrew; Zhang Zhuo; Cheng Senping; Budhraja, Amit; Ding Songze; Lee, Jeong-Chae; Shi Xianglin

    2011-09-15

    Cadmium is a toxic heavy metal which is environmentally and occupationally relevant. The mechanisms underlying cadmium-induced autophagy are not yet completely understood. The present study shows that cadmium induces autophagy, as demonstrated by the increase of LC3-II formation and the GFP-LC3 puncta cells. The induction of autophagosomes was directly visualized by electron microscopy in cadmium-exposed skin epidermal cells. Blockage of LKB1 or AMPK by siRNA transfection suppressed cadmium-induced autophagy. Cadmium-induced autophagy was inhibited in dominant-negative AMPK-transfected cells, whereas it was accelerated in cells transfected with the constitutively active form of AMPK. mTOR signaling, a negative regulator of autophagy, was downregulated in cadmium-exposed cells. In addition, cadmium generated reactive oxygen species (ROS) at relatively low levels, and caused poly(ADP-ribose) polymerase-1 (PARP) activation and ATP depletion. Inhibition of PARP by pharmacological inhibitors or its siRNA transfection suppressed ATP reduction and autophagy in cadmium-exposed cells. Furthermore, cadmium-induced autophagy signaling was attenuated by either exogenous addition of catalase and superoxide dismutase, or by overexpression of these enzymes. Consequently, these results suggest that cadmium-mediated ROS generation causes PARP activation and energy depletion, and eventually induces autophagy through the activation of LKB1-AMPK signaling and the down-regulation of mTOR in skin epidermal cells. - Highlights: > Cadmium, a toxic heavy metal, induces autophagic cell death through ROS-dependent activation of the LKB1-AMPK signaling. > Cadmium generates intracellular ROS at low levels and this leads to severe DNA damage and PARP activation, resulting in ATP depletion, which are the upstream events of LKB1-AMPK-mediated autophagy. > This novel finding may contribute to further understanding of cadmium-mediated diseases.

  4. Differentiated epidermal outgrowths in the planarian Dugesia gonocephala: a model for studying cell renewal and patterning in single-layered epithelial tissue.

    PubMed

    Chandebois, R

    1985-01-01

    Large deep wounds on the ventral side of a flatworm (Planaria) will not heal. Instead, the damage to the parenchyma in the wound's roof will result in a differentiated swelling in the dorsal epidermis, above the wound which will eventually disappear with the disintegration of the underlying damaged tissue and a ventrodorsal hole appears in place of the wound. The dorsal epidermal outgrowth is formed by a number of excrescences, the development of which involves four successive stages. Their analysis suggests that epidermal cells are continuously produced by their own stem cells which remain unnoticed because their nuclei are hardly stainable. The daughter cells differentiate without information from either the underlying tissues or the basal epithelial membrane. During the first stage of this differentiation the cells become ciliated and motile, with some embryonic features. They then produce rhabdites and take up a columnar shape as they may become attached to the basal membrane. After wound setting the production of epidermal cells increases and the overcrowding of the basal membrane results in (1) detachment of stem cells and motile ciliated cells from the basal tissues, i.e. outgrowths; (2) stretching of columnar cells at the base of the outgrowths. When in the process of tissue disintegration the basal membrane of the epithelium also disappears, the cells remain in a single-layered epithelial configuration and retain their original polarity. These results are at variance with the generally accepted hypothesis that, in planarians, epidermal cells originate from the parenchyma and the epidermis is not an autonomous tissue.

  5. CPK3-phosphorylated RhoGDI1 is essential in the development of Arabidopsis seedlings and leaf epidermal cells.

    PubMed

    Wu, Yuxuan; Zhao, Shujuan; Tian, Han; He, Yuqing; Xiong, Wei; Guo, Lin; Wu, Yan

    2013-08-01

    The regulation of Rho of plants (ROP) in morphogenesis of leaf epidermal cells has been well studied, but the roles concerning regulators of ROPs such as RhoGDIs are poorly understood. This study reports that AtRhoGDI1 (GDI1) acts as a versatile regulator to modulate development of seedlings and leaf pavement cells. In mutant gdi1, leaf pavement cells showed shorter lobes in comparison with those in wild type. In GDI1-14 seedlings (GDI1-overexpression line) the growth of lobes in pavement cells was severely suppressed and the development of seedlings was altered. These results indicate that GDI1 plays an essential role in morphogenesis of epidermal pavement cells through modulating the ROP signalling pathways. The interaction between GDI1 and ROP2 or ROP6 was detected in the leaf pavement cells using FRET analysis. Dominant negative, not constitutively active, DN-rop6 could weaken the effect caused by overexpression of GDI1; because the pleiotropic phenotype of GDI1-14 plants was eliminated in the hybrid line GDI1-14 DN-rop6. GDI1 could be phosphorylated by CPK3. Three conserved Ser/Thr residues in GDI1 were determined as targeted amino acids for CPK3. Overexpression of GDI1(3D), not GDI1(3A), could rescue the abnormal growth phenotypes of gdi1-1 seedlings, demonstrating the impact of GDI1 phosphorylation in the development of Arabidopsis. In summary, these results suggest that GDI1 regulation in morphogenesis of seedlings and leaf pavement cells could be undergone through modulating the ROP signalling pathways and the phosphorylation of GDI1 by CPK3 was required for the developmental modulation in Arabidopsis.

  6. Effects of epidermal Langerhans cell's conditioned medium on keratinocytes: a role of Langerhans cells in cholesteatoma.

    PubMed

    Kamide, Y; Sasaki, H; Abramson, M; Huang, C C

    1991-01-01

    Langerhans cells (LCs) are known to play an important role in the immunosurveillance system. In this study, as in others, numerous LCs were detected in the epithelial layer of acquired cholesteatoma by immunohistochemical staining. This finding suggests that cell-mediated immune responses are initiated by LCs in cholesteatoma; however, documentation concerning the microenvironment of LCs-keratinocytes in cholesteatoma is limited. Therefore, we investigated the effects of LCs on keratinocytes in vitro. To study these effects it was necessary to isolate and purify LCs. Our present study revealed that good enrichment and a high degree of purity (95%) of LCs could be obtained from neonatal rat skin using the immunomagnetic beads (Dynabeads M-450) sorting technique. These isolated LCs have the biologic activity of LCs, and Langerhans cells' conditioned medium (LCCM) stimulates DNA synthesis in thymocytes. The effect of LCCM on keratinocytes was then studied. We found that (1) LCCM stimulated DNA synthesis in keratinocytes was then studied. We found that (1) LCCM stimulated DNA synthesis in keratinocytes, but not protein synthesis, and (2) LCCM stimulated the incorporation of 3H-putrescine into keratinocytes by the activation of transglutaminase. Transglutaminase is a known marker of terminal differentiation in keratinocytes. By Western blot analysis, we identified a 17-kd immunoreactive mouse interleukin-1 alpha in LCCM. Our results imply that LCs found in cholesteatoma tissue may play an important role in stimulating both hyper-proliferation and cornification of keratinocytes; two characteristic features of cholesteatoma formation. These stimulatory effects may be due to the release of interleukin-1 or other factors by LCs.

  7. Investigation of macromolecule orientation in dry and hydrated walls of single onion epidermal cells by FTIR microspectroscopy

    NASA Astrophysics Data System (ADS)

    Chen, Limei; Wilson, Reginald H.; McCann, Maureen C.

    1997-06-01

    Polarised infrared spectra from the wall of a single epidermal onion cell were obtained using a Fourier transform infrared (FTIR) microscope. The use of a newly constructed hydration cell allowed studies of both composition and architecture of intact walls of single hydrated plant cells. By comparing spectra taken with infrared light polarised perpendicular, or parallel, to the long axis of the cell, orientations of macromolecules in dry and hydrated cell walls were investigated. It was observed that bands associated with pectin were stronger with polarisation perpendicular to the direction of the cell elongation. On the other hand, bands associated with cellulose were more intense with polarisation parallel to the direction of cell elongation. These results show that in dry and hydrated cell walls, not only was there a net orientation of cellulose, but also of pectin. The implication of this is that pectin, which was previously thought to play no structural role in cell walls may, in fact, contribute to the mechanical and structural properties of the cell network. Such results are likely to have a tremendous impact on the formulation of definitive models for the static and growing cell wall.

  8. Differential protein phosphorylation in induction of thyroid cell proliferation by thyrotropin, epidermal growth factor, or phorbol ester.

    PubMed Central

    Contor, L; Lamy, F; Lecocq, R; Roger, P P; Dumont, J E

    1988-01-01

    Protein phosphorylation was studied in primary cultures of thyroid epithelial cells after the addition of different mitogens: thyrotropin (TSH) acting through cyclic AMP, epidermal growth factor (EGF), or 12-O-tetradecanoylphorbol-13-acetate (TPA). EGF or TPA increased the phosphorylation of five common polypeptides. Among these, two 42-kilodalton proteins contained phosphotyrosine and phosphoserine with or without phosphothreonine. Their characteristics suggested that they are similar to the two 42-kilodalton target proteins for tyrosine protein phosphorylation demonstrated in fibroblasts in response to mitogens. No common phosphorylated proteins were detected in TSH-treated cells and in EGF- or TPA-treated cells. The differences in the protein phosphorylation patterns in response to TSH, EGF, and TPA suggested that the newly emerging cyclic AMP-mediated mitogenic pathway is distinct from the better known growth factor- and tumor promoter-induced pathways. Images PMID:3261388

  9. DNA vaccination strategy targets epidermal dendritic cells, initiating their migration and induction of a host immune response

    PubMed Central

    Smith, Trevor RF; Schultheis, Katherine; Kiosses, William B; Amante, Dinah H; Mendoza, Janess M; Stone, John C; McCoy, Jay R; Sardesai, Niranjan Y; Broderick, Kate E

    2014-01-01

    The immunocompetence and clinical accessibility of dermal tissue offers an appropriate and attractive target for vaccination. We previously demonstrated that pDNA injection into the skin in combination with surface electroporation (SEP), results in rapid and robust expression of the encoded antigen in the epidermis. Here, we demonstrate that intradermally EP-enhanced pDNA vaccination results in the rapid induction of a host humoral immune response. In the dermally relevant guinea pig model, we used high-resolution laser scanning confocal microscopy to observe direct dendritic cell (DC) transfections in the epidermis, to determine the migration kinetics of these cells from the epidermal layer into the dermis, and to follow them sequentially to the immediate draining lymph nodes. Furthermore, we delineate the relationship between the migration of directly transfected epidermal DCs and the generation of the host immune response. In summary, these data indicate that direct presentation of antigen to the immune system by DCs through SEP-based in vivo transfection in the epidermis, is related to the generation of a humoral immune response. PMID:26052522

  10. Tumor-Preferential Induction of Immune Responses and Epidermal Cell Death in Actinic Keratoses by Ingenol Mebutate

    PubMed Central

    Zibert, John R.; Schön, Margarete; Hald, Andreas; Hansen, Maria H.; Litman, Thomas; Schön, Michael P.

    2016-01-01

    The rapid and strong clinical efficacy of the first-in-class, ingenol mebutate, against actinic keratosis (AK) has resulted in its recent approval. We conducted the first comprehensive analysis of the cellular and molecular mode of action of topical ingenol mebutate 0.05% gel in both AK and uninvolved skin of 26 patients in a phase I, single-center, open-label, within-patient comparison. As early as 1 day after application, ingenol mebutate induced profound epidermal cell death, along with a strong infiltrate of CD4+ and CD8+ T-cells, neutrophils, and macrophages. Endothelial ICAM-1 activation became evident after 2 days. The reaction pattern was significantly more pronounced in AK compared with uninvolved skin, suggesting a tumor-preferential mode of action. Extensive molecular analyses and transcriptomic profiling of mRNAs and microRNAs demonstrated alterations in gene clusters functionally associated with epidermal development, inflammation, innate immunity, and response to wounding. Ingenol mebutate reveals a unique mode of action linking directly to anti-tumoral effects. Trial Registration: ClinicalTrials.gov NCT01387711 PMID:27612149

  11. Mechanical properties of epidermal cells of whole living roots of Arabidopsis thaliana: An atomic force microscopy study

    NASA Astrophysics Data System (ADS)

    Fernandes, Anwesha N.; Chen, Xinyong; Scotchford, Colin A.; Walker, James; Wells, Darren M.; Roberts, Clive J.; Everitt, Nicola M.

    2012-02-01

    The knowledge of mechanical properties of root cell walls is vital to understand how these properties interact with relevant genetic and physiological processes to bring about growth. Expansion of cell walls is an essential component of growth, and the regulation of cell wall expansion is one of the ways in which the mechanics of growth is controlled, managed and directed. In this study, the inherent surface mechanical properties of living Arabidopsis thaliana whole-root epidermal cells were studied at the nanoscale using the technique of atomic force microscopy (AFM). A novel methodology was successfully developed to adapt AFM to live plant roots. Force-Indentation (F-I) experiments were conducted to investigate the mechanical properties along the length of the root. F-I curves for epidermal cells of roots were also generated by varying turgor pressure. The F-I curves displayed a variety of features due to the heterogeneity of the surface. Hysteresis is observed. Application of conventional models to living biological systems such as cell walls in nanometer regimes tends to increase error margins to a large extent. Hence information from the F-I curves were used in a preliminary semiquantitative analysis to infer material properties and calculate two parameters. The work done in the loading and unloading phases (hysteresis) of the force measurements were determined separately and were expressed in terms of “Index of Plasticity” (η), which characterized the elasticity properties of roots as a viscoelastic response. Scaling approaches were used to find the ratio of hardness to reduced modulus ((H)/(E*)).

  12. Leptin stimulates endothelin-1 expression via extracellular signal-regulated kinase by epidermal growth factor receptor transactivation in rat aortic smooth muscle cells.

    PubMed

    Chao, Hung-Hsing; Hong, Hong-Jye; Liu, Ju-Chi; Lin, Jia-Wei; Chen, Yen-Ling; Chiu, Wen-Ta; Wu, Chieh-Hsi; Shyu, Kou-Gi; Cheng, Tzu-Hurng

    2007-11-14

    Obesity is a major risk factor for the development of hypertension. Recent studies have suggested that leptin, a 167-amino acid peptide hormone produced by white adipose tissue, is related to the pathogenesis of obesity-related hypertension. However, the signaling mechanisms underlying the effects of leptin remain to be extensively examined. In this study, we found that leptin induced extracellular signal-regulated kinase phosphorylation and endothelin-1 expression in rat aortic smooth muscle cells. Both PD98059 and U0126, inhibitors of the upstream activator of mitogen-activated protein kinase kinase, inhibited augmentation of endothelin-1 expression stimulated with leptin. Leptin induced significant tyrosine phosphorylation of epidermal growth factor receptor, which was significantly attenuated by two inhibitors, an epidermal growth factor receptor tyrosine kinase inhibitor, AG1478, and a broad-spectrum matrix metalloproteinase inhibitor, GM6001. This indicates that the pathway of epidermal growth factor receptor transactivation induced by leptin is dependent on proteolytically released epidermal growth factor receptor ligands. Pretreatment of cells with AG1478 significantly reduced the degree of phosphorylation of extracellular signal-regulated kinase and endothelin-1 expression. Our results reveal that epidermal growth factor receptor transactivation is involved in the leptin signaling pathway in vascular smooth muscle cells, which may be related to the increased risk of hypertension and other cardiovascular diseases in obese subjects. PMID:17678888

  13. Combining xanthan and chitosan membranes to multipotent mesenchymal stromal cells as bioactive dressings for dermo-epidermal wounds.

    PubMed

    Bellini, Márcia Z; Caliari-Oliveira, Carolina; Mizukami, Amanda; Swiech, Kamilla; Covas, Dimas T; Donadi, Eduardo A; Oliva-Neto, Pedro; Moraes, Ângela M

    2015-03-01

    The association between tridimensional scaffolds to cells of interest has provided excellent perspectives for obtaining viable complex tissues in vitro, such as skin, resulting in impressive advances in the field of tissue engineering applied to regenerative therapies. The use of multipotent mesenchymal stromal cells in the treatment of dermo-epidermal wounds is particularly promising due to several relevant properties of these cells, such as high capacity of proliferation in culture, potential of differentiation in multiple skin cell types, important paracrine and immunomodulatory effects, among others. Membranes of chitosan complexed with xanthan may be potentially useful as scaffolds for multipotent mesenchymal stromal cells, given that they present suitable physico-chemical characteristics and have adequate tridimensional structure for the adhesion, growth, and maintenance of cell function. Therefore, the purpose of this work was to assess the applicability of bioactive dressings associating dense and porous chitosan-xanthan membranes to multipotent mesenchymal stromal cells for the treatment of skin wounds. The membranes showed to be non-mutagenic and allowed efficient adhesion and proliferation of the mesenchymal stromal cells in vitro. In vivo assays performed with mesenchymal stromal cells grown on the surface of the dense membranes showed acceleration of wound healing in Wistar rats, thus indicating that the use of this cell-scaffold association for tissue engineering purposes is feasible and attractive.

  14. Epidermal mosaicism and Blaschko's lines.

    PubMed Central

    Moss, C; Larkins, S; Stacey, M; Blight, A; Farndon, P A; Davison, E V

    1993-01-01

    To test the hypothesis that epidermal rather than dermal mosaicism determines Blaschko's lines in hypomelanosis of Ito (HI), we studied the distribution of chromosomal mosaicism in four patients. In two, mosaicism had not been detected in lymphocytes or dermal fibroblasts, but was clearly shown in epidermal keratinocytes; furthermore, the abnormal cell line was confirmed to the hypopigmented epidermis and the normal epidermis contained only normal cells. Negative findings in the other two patients might be because of mosaicism which was undetected either because it was submicroscopic or because it was present in melanocytes, which have not yet been studied. These preliminary results support the ideas that (1) Blaschko's lines represent single clones of epidermal cells; (2) in patients with HI and severe neurological involvement mosaicism, if detectable, is best shown in keratinocytes; and (3) the cytogenetic defect in epidermal cells may be directly responsible for the failure of pigmentation in HI. Images PMID:8411070

  15. Toxic Epidermal Necrolysis.

    PubMed

    Castelain, Florence; Humbert, Phillip

    2013-02-01

    Toxic epidermal necrolysis (TEN) is a severe mucocutaneous drug-induced syndrome that causes massive keratinocyte apoptosis and therefore hydro-electrolytic disorders and systemic infection. TEN approximately affects one to two cases per million per year. Mortality rate may reach thirty percent of cases. Thus, TEN constitute a therapeutic emergency at diagnosis. Typically, clinical examination shows a mucocutaneous detachment involving more than thirty percent of body area. Definitive diagnosis is made on cutaneous biopsy with histological exam that shows the blister of necrotic keratinocytes. Main differential diagnosis are acute staphylococcus epidermis, acute generalized exanthematous pustulosis, linear IgA bullous dermatosis, paraneoplastic pemphigus, bullous fixed pigmented erythema, acute lupus erythematosus. In the early days, SCORTEN gives a good estimation and is now widely used as prognostic score. Drugs are generally considered as the main etiology of TEN but in some cases bacterial or viral infections could be involved. Physiopathology remains unclear even if recent advances have reported the possible implication of immune pathways based on activation of T and NK cells. Treatment of TEN requires to be instituted as soon as the diagnosis is made and the patient is preferentially referred to a specialized unit. Supportive care consist in covering areas of cutaneous detachment. No other therapy have demonstrated its efficiency, but high-dose intravenous immunoglobulin might improve the prognosis. PMID:23373551

  16. Toxic epidermal necrolysis.

    PubMed

    Castelain, Florence; Humbert, Philippe

    2012-11-01

    Toxic epidermal necrolysis (TEN) is a severe mucocutaneous drug-induced syndrome that causes massive keratinocyte apoptosis and therefore hydro-electrolytic disorders and systemic infection. TEN approximately affects one to two cases per million per year. Mortality rate may reach thirty percent of cases. Thus, TEN constitutes a therapeutic emergency at diagnosis. Typically, clinical examination shows a mucocutaneous detachment involving more than thirty percent of body area. Definitive diagnosis is made on cutaneous biopsy with histological exam that shows the blister of necrotic keratinocytes. Main differential diagnosis are acute staphylococcus epidermis, acute generalized exanthematous pustulosis, linear IgA bullous dermatosis, paraneoplastic pemphigus, bullous fixed pigmented erythema, acute lupus erythematosus. In the early days, SCORTEN gives a good estimation and is now widely used as prognostic score. Drugs are generally considered as the main etiology of TEN but in some cases bacterial or viral infections could be involved. Physiopathology remains unclear even if recent advances have reported the possible implication of immune pathways based on activation of T and NK cells. Treatment of TEN requires to be instituted as soon as the diagnosis is made and the patient is preferentially referred to a specialized unit. Supportive care consist of covering areas of cutaneous detachment. No other therapy has demonstrated its efficiency, but high-dose intravenous immunoglobulin might improve the prognosis. PMID:23441982

  17. CELLULOSE SYNTHASE9 Serves a Nonredundant Role in Secondary Cell Wall Synthesis in Arabidopsis Epidermal Testa Cells1[C][W][OA

    PubMed Central

    Stork, Jozsef; Harris, Darby; Griffiths, Jonathan; Williams, Brian; Beisson, Fred; Li-Beisson, Yonghua; Mendu, Venugopal; Haughn, George; DeBolt, Seth

    2010-01-01

    Herein, we sought to explore the contribution of cellulose biosynthesis to the shape and morphogenesis of hexagonal seed coat cells in Arabidopsis (Arabidopsis thaliana). Consistent with seed preferential expression of CELLULOSE SYNTHASE9 (CESA9), null mutations in CESA9 caused no change in cellulose content in leaves or stems, but caused a 25% reduction in seeds. Compositional studies of cesa9 seeds uncovered substantial proportional increases in cell wall neutral sugars and in several monomers of cell wall-associated polyesters. Despite these metabolic compensations, cesa9 seeds were permeable to tetrazolium salt, implying that cellulose biosynthesis, via CESA9, is required for correct barrier function of the seed coat. A syndrome of depleted radial wall, altered seed coat cell size, shape, and internal angle uniformity was quantified using scanning electron micrographs in cesa9 epidermal cells. By contrast, morphological defects were absent in cesa9 embryos, visually inspected from torpedo to bent cotyledon, consistent with no reduction in postgermination radical or hypocotyl elongation. These data implied that CESA9 was seed coat specific or functionally redundant in other tissues. Assessment of sections from glutaraldehyde fixed wild-type and cesa9 mature seeds supported results of scanning electron micrographs and quantitatively showed depletion of secondary cell wall synthesis in the radial cell wall. Herein, we show a nonredundant role for CESA9 in secondary cell wall biosynthesis in radial cell walls of epidermal seed coats and document its importance for cell morphogenesis and barrier function of the seed coat. PMID:20335403

  18. Potent endogenous allelopathic compounds in Lepidium sativum seed exudate: effects on epidermal cell growth in Amaranthus caudatus seedlings.

    PubMed

    Iqbal, Amjad; Fry, Stephen C

    2012-04-01

    Many plants exude allelochemicals--compounds that affect the growth of neighbouring plants. This study reports further studies of the reported effect of cress (Lepidium sativum) seed(ling) exudates on seedling growth in Amaranthus caudatus and Lactuca sativa. In the presence of live cress seedlings, both species grew longer hypocotyls and shorter roots than cress-free controls. The effects of cress seedlings were allelopathic and not due to competition for resources. Amaranthus seedlings grown in the presence of cress allelochemical(s) had longer, thinner hypocotyls and shorter, thicker roots--effects previously attributed to lepidimoide. The active principle was more abundant in cress seed exudate than in seedling (root) exudates. It was present in non-imbibed seeds and releasable from heat-killed seeds. Release from live seeds was biphasic, starting rapidly but then continuing gradually for 24 h. The active principle was generated by aseptic cress tissue and was not a microbial digestion product or seed-treatment chemical. Crude seed exudate affected hypocotyl and root growth at ~25 and ~450 μg ml(-1) respectively. The exudate slightly (28%) increased epidermal cell number along the length of the Amaranthus hypocotyl but increased total hypocotyl elongation by 129%; it resulted in a 26% smaller hypocotyl circumference but a 55% greater epidermal cell number counted round the circumference. Therefore, the effect of the allelochemical(s) on organ morphology was imposed primarily by regulation of cell expansion, not cell division. It is concluded that cress seeds exude endogenous substances, probably including lepidimoide, that principally regulate cell expansion in receiver plants.

  19. Study of lung-metastasized prostate cancer cell line chemotaxis to epidermal growth factor with a BIOMEMS device

    NASA Astrophysics Data System (ADS)

    Tata, Uday; Rao, Smitha M. N.; Sharma, Akash; Pabba, Krishna; Pokhrel, Kushal; Adhikari, Bandita; Lin, Victor K.; Chiao, J.-C.

    2012-09-01

    Understanding the effects of different growth factors on cancer metastasis will enable researchers to develop effective post-surgery therapeutic strategies to stop the spread of cancer. Conventional Boyden chamber assays to evaluate cell motility in metastasis studies require high volumes of reagents and are impractical for high-throughput analysis. A microfluidic device was designed for arrayed assaying of prostate cancer cell migration towards different growth factors. The device was created with polydimethylsiloxane (PDMS) and featured two wells connected by 10 micro channels. One well was for cell seeding and the other well for specific growth factors. Each channel has a width of 20 μm, a length of 1 mm and a depth of 10 μm. The device was placed on a culture dish and primed with growth media. Lung-metastasized cells in suspension of RPMI 1640 media1 supplemented with 2% of fetal bovine serum (FBS) were seeded in the cell wells. Cell culture media with epidermal growth factor (EGF) of 25, 50, 75, 100 and 125 ng ml-1 concentrations were individually added in the respective growth factor wells. A 5-day time-lapsed study of cell migration towards the chemoattractant was performed. The average numbers of cells per device in the microchannels were obtained for each attractant condition. The results indicated migration of cells increased from 50 to 100 ng ml-1 of EGF and significantly decreased at 125 ng ml-1 of EGF, as compared to control.

  20. Study of lung-metastasized prostate cancer cell line chemotaxis to epidermal growth factor with a BIOMEMS device

    NASA Astrophysics Data System (ADS)

    Tata, Uday; Rao, Smitha M. N.; Sharma, Akash; Pabba, Krishna; Pokhrel, Kushal; Adhikari, Bandita; Lin, Victor K.; Chiao, J.-C.

    2012-09-01

    Understanding the effects of different growth factors on cancer metastasis will enable researchers to develop effective post-surgery therapeutic strategies to stop the spread of cancer. Conventional Boyden chamber assays to evaluate cell motility in metastasis studies require high volumes of reagents and are impractical for high-throughput analysis. A microfluidic device was designed for arrayed assaying of prostate cancer cell migration towards different growth factors. The device was created with polydimethylsiloxane (PDMS) and featured two wells connected by 10 micro channels. One well was for cell seeding and the other well for specific growth factors. Each channel has a width of 20 μm, a length of 1 mm and a depth of 10 μm. The device was placed on a culture dish and primed with growth media. Lung-metastasized cells in suspension of RPMI 1640 media1 supplemented with 2% of fetal bovine serum (FBS) were seeded in the cell wells. Cell culture media with epidermal growth factor (EGF) of 25, 50, 75, 100 and 125 ng ml‑1 concentrations were individually added in the respective growth factor wells. A 5-day time-lapsed study of cell migration towards the chemoattractant was performed. The average numbers of cells per device in the microchannels were obtained for each attractant condition. The results indicated migration of cells increased from 50 to 100 ng ml‑1 of EGF and significantly decreased at 125 ng ml‑1 of EGF, as compared to control.

  1. Blue-light-induced reorganization of the actin cytoskeleton and the avoidance response of chloroplasts in epidermal cells of Vallisneria gigantea.

    PubMed

    Sakurai, Nami; Domoto, Kikuko; Takagi, Shingo

    2005-04-01

    In leaf epidermal cells of the aquatic angiosperm Vallisneria gigantea Graebner, high-intensity blue light induces the actin-dependent avoidance response of chloroplasts. By semi-quantitative motion analysis and phalloidin staining, time courses of the blue-light-induced changes in the mode of movement of individual chloroplasts and in the configuration of actin filaments were examined in the presence and absence of a flavoprotein inhibitor, diphenylene iodonium. In dark-adapted cells, short, thick actin bundles seemed to surround each chloroplast, which was kept motionless in the outer periclinal cytoplasm of the cells. After 10 min of irradiation with high-intensity blue light, a rapid, unidirectional movement of chloroplasts was induced, concomitant with the appearance of aggregated, straight actin bundles stretched over the outer periclinal cytoplasm. Diphenylene iodonium inhibited the avoidance response of chloroplasts, apparently by delaying a change in the mode of chloroplast movement from random sway to unidirectional migration, by suppressing the appearance of aggregated, straight actin bundles. In partially irradiated individual cells, redistribution of chloroplasts and reorganization of actin filaments occurred only in the areas exposed to blue light. From the results, we propose that the short, thick actin bundles in the vicinity of chloroplasts function to anchor the chloroplasts in dark-adapted cells, and that the aggregated, straight actin bundles organized under blue-light irradiation provide tracks for unidirectional movement of chloroplasts. PMID:15843965

  2. miR-143 suppresses the proliferation of NSCLC cells by inhibiting the epidermal growth factor receptor

    PubMed Central

    Zhang, Hong-Bo; Sun, Li-Chao; Ling, Lan; Cong, Lu-Hong; Lian, Rui

    2016-01-01

    MicroRNAs (miRs) regulate the proliferation and metastasis of numerous cancer cell types. It was previously reported that miR-143 levels were downregulated in non-small cell lung cancer (NSCLC) tissues and cell lines, and that the migration and invasion of NSCLC cells was inhibited upon suppression of cell proliferation and colony formation by the upregulation of miR-143. Epidermal growth factor receptor (EGFR), which is a vital factor in the promotion of cancer cell proliferation and has been investigated as a potential focus in cancer therapy, has been reported to be a possible target of miR-143. The present study aimed to investigate the role of miR-143 in NSCLC using NSCLC cell lines and primary cells from NSCLC patients. NSCLC cells were co-transfected with EGFR and miR-143, and the mRNA and protein expression of EGFR were analyzed. Furthermore, the activity of the transfected cancer cells with regard to colony formation, migration, invasion and apoptosis were evaluated. The levels of miR-143 were decreased in the NSCLC cell lines and primary cells from patients with NSCLC compared with the controls. Following transfection with miR-143, the ability of NSCLC cells to proliferate, form colonies, migrate and invade was inhibited. Similarly, knockdown of EGFR led to the suppression of NSCLC cell proliferation. The mRNA and protein expression levels of EGFR were significantly reduced following miR-143 overexpression, and the level of miR-143 was inversely correlated with that of EGFR in NSCLC cells. The results of the present study demonstrated that miR-143 was able to suppress NSCLC cell proliferation and invasion by inhibiting the effects of EGFR, suggesting that EGFR may be considered a potential target for NSCLC therapy. PMID:27602093

  3. miR-143 suppresses the proliferation of NSCLC cells by inhibiting the epidermal growth factor receptor

    PubMed Central

    Zhang, Hong-Bo; Sun, Li-Chao; Ling, Lan; Cong, Lu-Hong; Lian, Rui

    2016-01-01

    MicroRNAs (miRs) regulate the proliferation and metastasis of numerous cancer cell types. It was previously reported that miR-143 levels were downregulated in non-small cell lung cancer (NSCLC) tissues and cell lines, and that the migration and invasion of NSCLC cells was inhibited upon suppression of cell proliferation and colony formation by the upregulation of miR-143. Epidermal growth factor receptor (EGFR), which is a vital factor in the promotion of cancer cell proliferation and has been investigated as a potential focus in cancer therapy, has been reported to be a possible target of miR-143. The present study aimed to investigate the role of miR-143 in NSCLC using NSCLC cell lines and primary cells from NSCLC patients. NSCLC cells were co-transfected with EGFR and miR-143, and the mRNA and protein expression of EGFR were analyzed. Furthermore, the activity of the transfected cancer cells with regard to colony formation, migration, invasion and apoptosis were evaluated. The levels of miR-143 were decreased in the NSCLC cell lines and primary cells from patients with NSCLC compared with the controls. Following transfection with miR-143, the ability of NSCLC cells to proliferate, form colonies, migrate and invade was inhibited. Similarly, knockdown of EGFR led to the suppression of NSCLC cell proliferation. The mRNA and protein expression levels of EGFR were significantly reduced following miR-143 overexpression, and the level of miR-143 was inversely correlated with that of EGFR in NSCLC cells. The results of the present study demonstrated that miR-143 was able to suppress NSCLC cell proliferation and invasion by inhibiting the effects of EGFR, suggesting that EGFR may be considered a potential target for NSCLC therapy.

  4. The Effect of MCP-1/CCR2 on the Proliferation and Senescence of Epidermal Constituent Cells in Solar Lentigo

    PubMed Central

    Lee, Woo Jin; Jo, Soo Youn; Lee, Mi Hye; Won, Chong Hyun; Lee, Mi Woo; Choi, Jee Ho; Chang, Sung Eun

    2016-01-01

    Solar lentigo (SL) is a representative photoaging skin disorder. Alteration of the main epidermal constituent cells—keratinocytes and melanocytes—in relation to the photoaged dermal environment or chemokine/cytokine network is suggested as its pathogenesis. Among these, we focused on monocyte chemoattractant protein-1 (MCP-1), as it is known to be associated with tissue aging. For the first time, we report that the MCP-1 receptor, CCR2, is expressed in normal human melanocytes. In SL tissue, there was an increase of CCR2+Melan A+ melanocytes with positivity to Rb protein compared to peri-lesional normal skin. MCP-1 induced the proliferation of normal human melanocytes without a significant change in the melanin content. MCP-1 treatment in normal human keratinocytes showed an increase in senescence-associated β-galactosidase staining and p53 and p21 protein expressions. In summary, MCP-1 may participate in the development of SL by affecting epidermal constituent cells, for example, by inducing melanocyte proliferation and keratinocyte senescence. PMID:27314341

  5. An Autopsy Case of Two Distinct, Acquired Drug Resistance Mechanisms in Epidermal Growth Factor Receptor-mutant Lung Adenocarcinoma: Small Cell Carcinoma Transformation and Epidermal Growth Factor Receptor T790M Mutation.

    PubMed

    Furugen, Makoto; Uechi, Kayoko; Hirai, Jun; Aoyama, Hajime; Saio, Masanao; Yoshimi, Naoki; Kinjo, Takeshi; Miyagi, Kazuya; Haranaga, Shusaku; Higa, Futoshi; Tateyama, Masao; Fujita, Jiro

    2015-01-01

    We herein describe the case of a 63-year-old man who died from relapsed epidermal growth factor receptor gene (EGFR) exon 19 deletion lung adenocarcinoma treated with erlotinib. According to the autopsy results, he was confirmed to have small cell carcinoma without the EGFR T790M mutation in his pancreas and left kidney metastatic specimens, while the adenocarcinoma metastatic lesion in his right kidney had the EGFR T790M mutation; both retained the somatic EGFR exon 19 deletion. We herein report an autopsy case of resistance to an EGFR tyrosine kinase inhibitor via small cell carcinoma transformation and the EGFRT790M mutation in separate metastatic organs. PMID:26424310

  6. Endogenous p53 protein generated from wild-type alternatively spliced p53 RNA in mouse epidermal cells.

    PubMed Central

    Kulesz-Martin, M F; Lisafeld, B; Huang, H; Kisiel, N D; Lee, L

    1994-01-01

    We previously demonstrated that a wild-type alternatively spliced p53 (p53as) RNA exists in mouse cultured cells and normal mouse tissues at approximately 25 to 33% of the level of the major p53 RNA form. The alternative RNA transcript is 96 nucleotides longer than the major transcript as a result of alternative splicing of intron 10 sequences. The protein expected to be generated from the p53as transcript is 9 amino acids shorter than the major p53 protein and has 17 different amino acids at the carboxyl terminus. We report here that p53as protein exists in nontransformed and malignant epidermal cells and is localized to the nucleus. In addition, p53as protein is preferentially expressed during the G2 phase of the cell cycle and in cells with greater than G2 DNA content compared with the major p53 protein, which is preferentially expressed in G1. The p53as immunoreactivity is elevated and shifted to the G1 phase of the cell cycle following actinomycin D treatment of nontransformed cells but not malignant cells. In view of the dimerization and tetramerization of p53 protein which may be necessary for its DNA binding and transcriptional activation activities, the presence of p53as protein in cells has important implications for understanding the physiological function(s) of the p53 gene. Images PMID:8114705

  7. Expression of human epidermal growth factor precursor cDNA in transfected mouse NIH 3T3 cells.

    PubMed Central

    Mroczkowski, B; Reich, M; Whittaker, J; Bell, G I; Cohen, S

    1988-01-01

    Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 microM ZnCl2. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is approximately 150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with 125I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions of the EGF precursor itself. Images PMID:3257563

  8. Honokiol inhibits the growth of head and neck squamous cell carcinoma by targeting epidermal growth factor receptor.

    PubMed

    Singh, Tripti; Gupta, Nirzari A; Xu, Su; Prasad, Ram; Velu, Sadanandan E; Katiyar, Santosh K

    2015-08-28

    Here, we report the chemotherapeutic effect of honokiol, a phytochemical from Magnolia plant, on human head and neck squamous cell carcinoma (HNSCC). Treatment of HNSCC cell lines from different sub-sites, SCC-1 (oral cavity), SCC-5 (larynx), OSC-19 (tongue) and FaDu (pharynx) with honokiol inhibited their cell viability, which was associated with the: (i) induction of apoptosis, (ii) correction of dysregulatory cell cycle proteins of G0/G1 phase. Honokiol decreased the expression levels of epidermal growth factor receptor (EGFR), mTOR and their downstream signaling molecules. Treatment of FaDu and SCC-1 cell lines with rapamycin, an inhibitor of mTOR pathway, also reduced cell viability of HNSCC cells. Administration of honokiol by oral gavage (100 mg/kg body weight) significantly (P < 0.01-0.001) inhibited the growth of SCC-1 and FaDu xenografts in athymic nude mice, which was associated with: (i) inhibition of tumor cell proliferation, (ii) induction of apoptosis, (iii) reduced expressions of cyclins and Cdks, and (iv) inhibition of EGFR signaling pathway. Molecular docking analysis of honokiol in EGFR binding site indicated that the chemotherapeutic effect of honokiol against HNSCC is mediated through its firm binding with EGFR, which is better than that of gefitinib, a commonly used drug for HNSCC treatment.

  9. Expression of human epidermal growth factor pressures cDNA in transfected mouse NIH 3T3 cells

    SciTech Connect

    Mroczkowski, B.; Reich, M.; Whittaker, J.; Bell, G.I.; Cohen, S.

    1988-01-01

    Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 ..mu..M ZnCl/sub 2/. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is approx. = 150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with /sup 125/I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions of the EGF precursor itself.

  10. 3D In Vitro Model of a Functional Epidermal Permeability Barrier from Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

    PubMed Central

    Petrova, Anastasia; Celli, Anna; Jacquet, Laureen; Dafou, Dimitra; Crumrine, Debra; Hupe, Melanie; Arno, Matthew; Hobbs, Carl; Cvoro, Aleksandra; Karagiannis, Panagiotis; Devito, Liani; Sun, Richard; Adame, Lillian C.; Vaughan, Robert; McGrath, John A.; Mauro, Theodora M.; Ilic, Dusko

    2014-01-01

    Summary Cornification and epidermal barrier defects are associated with a number of clinically diverse skin disorders. However, a suitable in vitro model for studying normal barrier function and barrier defects is still lacking. Here, we demonstrate the generation of human epidermal equivalents (HEEs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). HEEs are structurally similar to native epidermis, with a functional permeability barrier. We exposed a pure population of hESC/iPSC-derived keratinocytes, whose transcriptome corresponds to the gene signature of normal primary human keratinocytes (NHKs), to a sequential high-to-low humidity environment in an air/liquid interface culture. The resulting HEEs had all of the cellular strata of the human epidermis, with skin barrier properties similar to those of normal skin. Such HEEs generated from disease-specific iPSCs will be an invaluable tool not only for dissecting molecular mechanisms that lead to epidermal barrier defects but also for drug development and screening. PMID:24936454

  11. [The pathogenesis of psoriasis. Autoradiographic in vitro studies on cell proliferation in psoriasis vulgaris and other normal and hyperproliferative states of epidermal and dermal human cells].

    PubMed

    Pullman, H

    1978-07-01

    In the epidermal cells of patients suffering from psoriasis we found a significant prolongation of DNA-synthesis time (ts) in uninvolved skin, very early lesions, and fully developed plaques. In uninvolved psoriatic skin ts in addition increased significantly within 6 hours after stripping of the horny layer. In normal epidermis and in other states of epidermal inflammation and hyperproliferation (akanthosis by petrolatum, toxic dermatitis, chronic allergic ekzema, neurodermitis, allergic patch test reaction) a comparable prolongation of ts was not ascertainable. This prolongation is most distinct in the early lesions and proceeds the development of hyperproliferation and akanthosis. A dermal infiltrate with increased proliferative activity seems to be a stimulus, in the sense of a Koebner-phenomenon. The abnormal psoriatic epidermis, with disturbed DNA-synthesis, reacts to this infiltrate as well as to other irritants not with a limited hyperproliferation but with the development of psoriatic plaque. PMID:149749

  12. Hydrogel Microencapsulated Insulin-Secreting Cells Increase Keratinocyte Migration, Epidermal Thickness, Collagen Fiber Density, and Wound Closure in a Diabetic Mouse Model of Wound Healing

    PubMed Central

    Faulknor, Renea; Berthiaume, François; Olabisi, Ronke M.

    2015-01-01

    Wound healing is a hierarchical process of intracellular and intercellular signaling. Insulin is a potent chemoattractant and mitogen for cells involved in wound healing. Insulin's potential to promote keratinocyte growth and stimulate collagen synthesis in fibroblasts is well described. However, there currently lacks an appropriate delivery mechanism capable of consistently supplying a wound environment with insulin; current approaches require repeated applications of insulin, which increase the chances of infecting the wound. In this study, we present a novel cell-based therapy that delivers insulin to the wound area in a constant or glucose-dependent manner by encapsulating insulin-secreting cells in nonimmunogenic poly(ethylene glycol) diacrylate (PEGDA) hydrogel microspheres. We evaluated cell viability and insulin secretory characteristics of microencapsulated cells. Glucose stimulation studies verified free diffusion of glucose and insulin through the microspheres, while no statistical difference in insulin secretion was observed between cells in microspheres and cells in monolayers. Scratch assays demonstrated accelerated keratinocyte migration in vitro when treated with microencapsulated cells. In excisional wounds on the dorsa of diabetic mice, microencapsulated RIN-m cells accelerated wound closure by postoperative day 7; a statistically significant increase over AtT-20ins-treated and control groups. Histological results indicated significantly greater epidermal thickness in both microencapsulated RIN-m and AtT-20ins-treated wounds. The results suggest that microencapsulation enables insulin-secreting cells to persist long enough at the wound site for a therapeutic effect and thereby functions as an effective delivery vehicle to accelerate wound healing. PMID:26239745

  13. Epidermal growth factor receptor-dependent stimulation of amphiregulin expression in androgen-stimulated human prostate cancer cells.

    PubMed Central

    Sehgal, I; Bailey, J; Hitzemann, K; Pittelkow, M R; Maihle, N J

    1994-01-01

    Amphiregulin is a heparin-binding epidermal growth factor (EGF)-related peptide that binds to the EGF receptor (EGF-R) with high affinity. In this study, we report a role for amphiregulin in androgen-stimulated regulation of prostate cancer cell growth. Androgen is known to enhance EGF-R expression in the androgen-sensitive LNCaP human prostate carcinoma cell line, and it has been suggested that androgenic stimuli may regulate proliferation, in part, through autocrine mechanisms involving the EGF-R. In this study, we demonstrate that LNCaP cells express amphiregulin mRNA and peptide and that this expression is elevated by androgenic stimulation. We also show that ligand-dependent EGF-R stimulation induces amphiregulin expression and that androgenic effects on amphiregulin synthesis are mediated through this EGF-R pathway. Parallel studies using the estrogen-responsive breast carcinoma cell line, MCF-7, suggest that regulation of amphiregulin by estrogen may also be mediated via an EGF-R pathway. In addition, heparin treatment of LNCaP cells inhibits androgen-stimulated cell growth further suggesting that amphiregulin can mediate androgen-stimulated LNCaP proliferation. Together, these results implicate an androgen-regulated autocrine loop composed of amphiregulin and its receptor in prostate cancer cell growth and suggest that the mechanism of steroid hormone regulation of amphiregulin synthesis may occur through androgen upregulation of the EGF-R and subsequent receptor-dependent pathways. Images PMID:8049525

  14. Tropism of varicella-zoster virus for human CD4+ and CD8+ T lymphocytes and epidermal cells in SCID-hu mice.

    PubMed Central

    Moffat, J F; Stein, M D; Kaneshima, H; Arvin, A M

    1995-01-01

    To investigate the cell tropism and pathogenicity of varicella-zoster virus (VZV) strains, we analyzed VZV replication by using SCID-hu mice that carry human fetal thymus/liver implants under the kidney capsule or as subcutaneous fetal skin implants. MRC-5 cells infected with wild-type VZV or the Oka strain, used in the live attenuated varicella vaccine, were injected into the implants. The implants were surgically removed 2, 7, 14, and 21 days postinfection. The VZV titer from infected thymus/liver implants peaked on day 7 for the wild-type strain and on day 14 for the Oka strain. Histological analysis showed necrotic areas characterized by thymocyte depletion and fibrosis. VZV protein synthesis was detectable by immunohistochemical staining in the necrotic areas and in distant regions that did not show cytopathic changes, and VZV DNA was detected by in situ hybridization in the same distribution. Fluorescence-activated cell sorting analysis of thymocytes harvested at day 7 postinfection showed that VZV proteins were expressed in CD4+, CD8+, and CD4+ CD8+ T cells; VZV was cultured from each T-cell subpopulation. The Oka strain had tropism for human cell types similar to that of wild-type VZV. T lymphocytes released infectious VZV, which is a novel and important observation about the replication of this otherwise highly cell associated virus. VZV-infected skin implants exhibited microscopic epidermal lesions that were indistinguishable histologically from the characteristic lesions of varicella. These experiments demonstrate a unique tropism of VZV for human T lymphocytes, explaining its capacity to cause viremia in natural disease, and demonstrate the value of the SCID-hu model for studies of VZV pathogenesis. PMID:7636965

  15. Reorganized actin filaments anchor chloroplasts along the anticlinal walls of Vallisneria epidermal cells under high-intensity blue light.

    PubMed

    Sakai, Yuuki; Takagi, Shingo

    2005-08-01

    In epidermal cells of the aquatic angiosperm Vallisneria gigantea Graebner, high-intensity blue light (BL) induces the avoidance response of chloroplasts. We examined simultaneous BL-induced changes in the configuration of actin filaments in the cytoplasmic layers that face the outer periclinal wall (P side) and the anticlinal wall (A side). The results clearly showed that dynamic reorganization of the actin cytoskeleton occurs on both sides. Upon BL irradiation, thick, long bundles of actin filaments appeared, concomitant with the directed migration of chloroplasts from the P side to the A side. After 15-20 min of BL irradiation, fine actin bundles on only the A side appeared to associate with chloroplasts that had migrated from the P side. To examine the role of the fine actin bundles, we evaluated the anchorage of chloroplasts by centrifuging living cells. Upon BL irradiation, the resistance of chloroplasts on both the P and A sides to the centrifugal force decreased remarkably. After 20 min of BL irradiation, the resistance of chloroplasts on the A side increased again, but chloroplasts on the P side could still be displaced. The BL-induced recovery of resistance of chloroplasts on the A side was sensitive to photosynthesis inhibitors but insensitive to an inhibitor of flavoproteins. The photosynthesis inhibitors also prevented the fine actin bundles from appearing on the A side under BL irradiation. These results strongly suggest that the BL-induced avoidance response of chloroplasts includes photosynthesis-dependent and actin-dependent anchorage of chloroplasts on the A side of epidermal cells. PMID:15809866

  16. Divergent effects of epidermal growth factor and transforming growth factors on a human endometrial carcinoma cell line.

    PubMed

    Korc, M; Haussler, C A; Trookman, N S

    1987-09-15

    Epidermal growth factor (EGF), at concentrations ranging from 0.83 to 4.98 nM, markedly inhibited the proliferation of RL95-2 cells that were seeded at low plating densities (4.7 X 10(3) cells/cm2). Under the same incubation conditions, 16.6 pM EGF enhanced cell proliferation. At high plating densities (2.5 X 10(4) cells/cm2) 0.83 nM EGF also stimulated cell proliferation. Both the inhibitory and stimulatory effects of EGF were mimicked by transforming growth factor-alpha (TGF-alpha). However, the inhibitory action of TGF-alpha was always greater that of EGF. Binding studies with 125I-labeled TGF-alpha indicated that maximal cell surface binding of TGF-alpha occurred at 15 min, whereas maximal internalization occurred at 45 min. Both cell surface and internalized radioactivity declined sharply thereafter. Analysis of radioactivity released into the incubation medium during pulse-chase experiments indicated that RL95-2 cells extensively degraded both TGF-alpha and EGF. The lysosomotropic compound methylamine arrested the generation of low-molecular-weight degradation products of EGF, but not of TGF-alpha. In contrast to EGF and TGF-alpha, transforming growth factor-beta (TGF-beta) inhibited the proliferation of RL95-2 cells that were seeded at either low or high plating densities. Further, transforming growth factor-beta induced the appearance of large cuboidal cells that were readily distinguished from cells treated with either EGF or TGF-alpha. These findings point to complex regulatory actions of growth factors on the proliferation of RL95-2 cells and suggest that the processing of TGF-alpha following EGF receptor activation is distinct from the processing of EGF. PMID:3497713

  17. Differential regulation of human Eag1 channel expression by serum and epidermal growth factor in lung and breast cancer cells

    PubMed Central

    Acuña-Macías, Isabel; Vera, Eunice; Vázquez-Sánchez, Alma Yolanda; Mendoza-Garrido, María Eugenia; Camacho, Javier

    2015-01-01

    Oncogenic ether à-go-go-1 (Eag1) potassium channels are overexpressed in most primary human solid tumors. Low oxygen and nutrient/growth factor concentrations play critical roles in tumorigenesis. However, the mechanisms by which tumor cells survive and proliferate under growth factor-depleted conditions remain elusive. Here, we investigated whether serum-deprived conditions and epidermal growth factor (EGF) regulate Eag1 expression in human lung and breast cancer cells. The human cancer cell lines A549 and MCF-7 (from the lungs and breast, respectively) were obtained from the American Type Culture Collection and cultured following the manufacturer’s recommendations. Eag1 gene and protein expression were studied by real-time PCR and immunocytochemistry, respectively. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and ERK1/2 phosphorylation was investigated by Western blot. Serum-deprived conditions increased Eag1 mRNA and protein expression in both cell lines. This Eag1 upregulation was prevented by EGF and the ERK1/2 inhibitor U0126 in only lung cancer cells; vascular endothelial growth factor did not prevent Eag1 upregulation. Our results suggest that Eag1 may act as a survival and mitogenic factor under low-serum and nutrient conditions and may be a clinical target during the early stages of tumor development. PMID:26527881

  18. Helicobacter pylori culture supernatant interferes with epidermal growth factor-activated signal transduction in human gastric KATO III cells.

    PubMed Central

    Pai, R.; Wyle, F. A.; Cover, T. L.; Itani, R. M.; Domek, M. J.; Tarnawski, A. S.

    1998-01-01

    The mechanisms by which Helicobacter pylori infection leads to gastroduodenal ulceration remain poorly understood. Previous studies have shown that H. pylori vacuolating cytotoxin (VacA) inhibits proliferation of gastric epithelial cells, which suggests that H pylori may interfere with gastric mucosal repair mechanisms. In this study, we investigated the effects of H. pylori broth culture supernatants on epidermal growth factor (EGF)-mediated signal transduction pathways in a gastric carcinoma cell line (KATO III). Exposure of these cells to EGF resulted in increased expression and phosphorylation of the EGF receptor (EGF-R), increased ERK2 activity and phosphorylation, and increased c-fos protein levels. Preincubation of cells with broth culture supernatant from VacA (+) H. pylori strain 60190 inhibited the capacity of EGF to induce each of these effects. In contrast, preincubation of cells with broth culture supernatant from an isogenic VacA-mutant strain (H. pylori 60190-v1) failed to inhibit the effects of EGF. These results suggest that the H. pylori vacuolating cytotoxin interferes with EGF-activated signal transduction pathways, which are known to be essential for cell proliferation and ulcer healing. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:9626065

  19. Helicobacter pylori culture supernatant interferes with epidermal growth factor-activated signal transduction in human gastric KATO III cells.

    PubMed

    Pai, R; Wyle, F A; Cover, T L; Itani, R M; Domek, M J; Tarnawski, A S

    1998-06-01

    The mechanisms by which Helicobacter pylori infection leads to gastroduodenal ulceration remain poorly understood. Previous studies have shown that H. pylori vacuolating cytotoxin (VacA) inhibits proliferation of gastric epithelial cells, which suggests that H pylori may interfere with gastric mucosal repair mechanisms. In this study, we investigated the effects of H. pylori broth culture supernatants on epidermal growth factor (EGF)-mediated signal transduction pathways in a gastric carcinoma cell line (KATO III). Exposure of these cells to EGF resulted in increased expression and phosphorylation of the EGF receptor (EGF-R), increased ERK2 activity and phosphorylation, and increased c-fos protein levels. Preincubation of cells with broth culture supernatant from VacA (+) H. pylori strain 60190 inhibited the capacity of EGF to induce each of these effects. In contrast, preincubation of cells with broth culture supernatant from an isogenic VacA-mutant strain (H. pylori 60190-v1) failed to inhibit the effects of EGF. These results suggest that the H. pylori vacuolating cytotoxin interferes with EGF-activated signal transduction pathways, which are known to be essential for cell proliferation and ulcer healing. PMID:9626065

  20. Acquired resistance of non-small cell lung cancer to epidermal growth factor receptor tyrosine kinase inhibitors.

    PubMed

    Nurwidya, Fariz; Takahashi, Fumiyuki; Murakami, Akiko; Kobayashi, Isao; Kato, Motoyasu; Shukuya, Takehito; Tajima, Ken; Shimada, Naoko; Takahashi, Kazuhisa

    2014-03-01

    Activation of epidermal growth factor receptor (EGFR) triggers anti-apoptotic signaling, proliferation, angiogenesis, invasion, metastasis, and drug resistance, which leads to development and progression of human epithelial cancers, including non-small cell lung cancer (NSCLC). Inhibition of EGFR by tyrosine kinase inhibitors such as gefitinib and erlotinib has provided a new hope for the cure of NSCLC patients. However, acquired resistance to gefitinib and erlotinib via EGFR-mutant NSCLC has occurred through various molecular mechanisms such as T790M secondary mutation, MET amplification, hepatocyte growth factor (HGF) overexpression, PTEN downregulation, epithelial-mesenchymal transition (EMT), and other mechanisms. This review will discuss the biology of receptor tyrosine kinase inhibition and focus on the molecular mechanisms of acquired resistance to tyrosine kinase inhibitors of EGFR-mutant NSCLC.

  1. Increase in epidermal planar cell density accompanies decreased russeting of “Golden Delicious” apples treated with gibberellin A4+7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A two-year study was conducted in a “Golden Delicious” (Malus Xdomestica Borkh.) orchard having a high historical incidence of physiological fruit russeting, to examine the effect of gibberellin A4+7 (GA4+7) on apple epidermal cell size. Beginning at petal fall, four sequential applications of GA4+7...

  2. 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...

  3. Effect of epidermal growth factor on follicle-stimulating hormone-induced proliferation of granulosa cells from chicken prehierarchical follicles.

    PubMed

    Lin, Jin-xing; Jia, Yu-dong; Zhang, Cai-qiao

    2011-11-01

    The development of ovarian follicular cells is controlled by multiple circulating and local hormones and factors, including follicle-stimulating hormone (FSH) and epidermal growth factor (EGF). In this study, the stage-specific effect of EGF on FSH-induced proliferation of granulosa cells was evaluated in the ovarian follicles of egg-laying chickens. Results showed that EGF and its receptor (EGFR) mRNAs displayed a high expression in granulosa cells from the prehierarchical follicles, including the large white follicle (LWF) and small yellow follicle (SYF), and thereafter the expression decreased markedly to the stage of the largest preovulatory follicle. SYF represents a turning point of EGF/EGFR mRNA expression during follicle selection. Subsequently the granulosa cells from SYF were cultured to reveal the mediation of EGF in FSH action. Cell proliferation was remarkably increased by treatment with either EGF or FSH (0.1-100 ng/ml). This result was confirmed by elevated proliferating cell nuclear antigen (PCNA) expression and decreased cell apoptosis. Furthermore, EGF-induced cell proliferation was accompanied by increased mRNA expressions of EGFR, FSH receptor, and the cell cycle-regulating genes (cyclins D1 and E1, cyclin-dependent kinases 2 and 6) as well as decreased expression of luteinizing hormone receptor mRNA. However, the EGF or FSH-elicited effect was reversed by simultaneous treatment with an EGFR inhibitor AG1478. In conclusion, EGF and EGFR expressions manifested stage-specific changes during follicular development and EGF mediated FSH-induced cell proliferation and retarded cell differentiation in the prehierarchical follicles. These expressions thus stimulated follicular growth before selection in the egg-laying chicken.

  4. Tunable interplay between epidermal growth factor and cell–cell contact governs the spatial dynamics of epithelial growth

    PubMed Central

    Kim, Jin-Hong; Kushiro, Keiichiro; Graham, Nicholas A.; Asthagiri, Anand R.

    2009-01-01

    Contact-inhibition of proliferation constrains epithelial tissue growth, and the loss of contact-inhibition is a hallmark of cancer cells. In most physiological scenarios, cell–cell contact inhibits proliferation in the presence of other growth-promoting cues, such as soluble growth factors (GFs). How cells quantitatively reconcile the opposing effects of cell–cell contact and GFs, such as epidermal growth factor (EGF), remains unclear. Here, using quantitative analysis of single cells within multicellular clusters, we show that contact is not a “master switch” that overrides EGF. Only when EGF recedes below a threshold level, contact inhibits proliferation, causing spatial patterns in cell cycle activity within epithelial cell clusters. Furthermore, we demonstrate that the onset of contact-inhibition and the timing of spatial patterns in proliferation may be reengineered. Using micropatterned surfaces to amplify cell–cell interactions, we induce contact-inhibition at a higher threshold level of EGF. Using a complementary molecular genetics approach to enhance cell–cell interactions by overexpressing E-cadherin also increases the threshold level of EGF at which contact-inhibition is triggered. These results lead us to propose a state diagram in which epithelial cells transition from a contact-uninhibited state to a contact-inhibited state at a critical threshold level of EGF, a property that may be tuned by modulating the extent of cell–cell contacts. This quantitative model of contact-inhibition has direct implications for how tissue size may be determined and deregulated during development and tumor formation, respectively, and provides design principles for engineering epithelial tissue growth in applications such as tissue engineering. PMID:19549816

  5. Benzyladenine and gibberellin treatment of developing "Pink Lady" apples results in mature fruits with a thicker cuticle comprising clusters of epidermal cells.

    PubMed

    Fogelman, Edna; Stern, Raphael A; Ginzberg, Idit

    2015-07-01

    A mixture of 6-benzyladenine (BA) and gibberellins GA4 plus GA7 applied to "Pink Lady" apple at early phenological stages was previously shown to result in an immediate increase in epidermal cell density and associated reduction in calyx-end cracking disorder in the mature fruit, implying a long-term effect of the BA + GA4+7 mixture. Here, we analyzed the anatomical changes in the mature peel at the calyx end 210 days after full bloom (DAFB), following application of the plant growth regulators (PGRs) at the cell-division phase of fruit development, 21-50 DAFB. Experiments were conducted in northern Israel, and the PGRs were applied as the commercial formulation Superlon™ (Fine Agrochemicals Ltd.), composed of 19 g l(-1) BA and 19 g l(-1) GA4+7. Trees were sprayed with 0.025, 0.1, or 0.2 % (v/v) Superlon™. The most obvious phenomenon was the presence of epidermal cell clusters within the cuticular matrix that were detached from the native epidermal layer located at the bottom of the cuticle and which could not be detected in the untreated control fruits. Treatment with 20 mg l(-1) BA + GA4+7 (0.1 % Superlon™) resulted in a markedly thicker cuticle, a higher percentage of detached epidermal cells within the cuticular membrane and a significant reduction in calyx-end cracking at harvest. The presence of cuticle-embedded epidermal cell clusters may have contributed to strengthening the peel by adding more cell-wall components, thickening the cuticle layer and possibly enhancing crack repair.

  6. Extracellular hydrogen peroxide, produced through a respiratory burst oxidase/superoxide dismutase pathway, directs ingrowth wall formation in epidermal transfer cells of Vicia faba cotyledons.

    PubMed

    Xia, Xue; Zhang, Hui-Ming; Andriunas, Felicity A; Offler, Christina E; Patrick, John W

    2012-09-01

    The intricate, and often polarized, ingrowth walls of transfer cells (TCs) amplify their plasma membrane surface areas to confer a transport function of supporting high rates of nutrient exchange across apo-/symplasmic interfaces. The TC ingrowth wall comprises a uniform wall layer on which wall ingrowths are deposited. Signals and signal cascades inducing trans-differentiation events leading to formation of TC ingrowth walls are poorly understood. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, when placed into culture, their adaxial epidermal cells rapidly (h) and synchronously form polarized ingrowth walls accessible for experimental observations. Using this model, we recently reported findings consistent with extracellular hydrogen peroxide, produced through a respiratory burst oxidase homolog/superoxide dismutase pathway, initiating cell wall biosynthetic activity and providing directional information guiding deposition of the polarized uniform wall. Our conclusions rested on observations derived from pharmacological manipulations of hydrogen peroxide production and correlative gene expression data sets. A series of additional studies were undertaken, the results of which verify that extracellular hydrogen peroxide contributes to regulating ingrowth wall formation and is generated by a respiratory burst oxidase homolog/superoxide dismutase pathway.

  7. Wounding Sheets of Epithelial Cells Activates the Epidermal Growth Factor Receptor through Distinct Short- and Long-Range Mechanisms

    PubMed Central

    Block, Ethan R.

    2008-01-01

    Wounding epithelia induces activation of the epidermal growth factor receptor (EGFR), which is absolutely required for induction of motility. ATP is released from cells after wounding; it binds to purinergic receptors on the cell surface, and the EGFR is subsequently activated. Exogenous ATP activates phospholipase D, and we show here that ATP activates the EGFR through the phospholipase D2 isoform. The EGFR is activated in cells far (>0.3 cm) from wounds, which is mediated by diffusion of extracellular ATP because activation at a distance from wounds is abrogated by eliminating ATP in the medium with apyrase. In sharp contrast, activation of the EGFR near wounds is not sensitive to apyrase. Time-lapse microscopy revealed that cells exhibit increased motilities near edges of wounds; this increase in motility is not sensitive to apyrase, and apyrase does not detectably inhibit healing of wounds in epithelial sheets. This novel ATP/PLD2-independent pathway activates the EGFR by a transactivation process through ligand release, and it involves signaling by a member of the Src family of kinases. We conclude that wounding activates two distinct signaling pathways that induce EGFR activation and promote healing of wounds in epithelial cells. One pathway signals at a distance from wounds through release of ATP, and another pathway acts locally and is independent on ATP signaling. PMID:18799627

  8. Enhanced Human Epidermal Growth Factor Receptor 2 Degradation in Breast Cancer Cells by Lysosome-Targeting Gold Nanoconstructs.

    PubMed

    Lee, Hyojin; Dam, Duncan Hieu M; Ha, Ji Won; Yue, Jun; Odom, Teri W

    2015-10-27

    This paper describes how gold nanoparticle nanoconstructs can enhance anticancer effects of lysosomal targeting aptamers in breast cancer cells. Nanoconstructs consisting of anti-HER2 aptamer (human epidermal growth factor receptor 2, HApt) densely grafted on gold nanostars (AuNS) first targeted HER2 and then were internalized via HER2-mediated endocytosis. As incubation time increased, the nanoconstruct complexes were found in vesicular structures, starting from early endosomes to lysosomes as visualized by confocal fluorescence and differential interference contrast microscopy. Within the target organelle, lysosomes, HER2 was degraded by enzymes at low pH, which resulted in apoptosis. At specific time points related to the doubling time of the cancer cells, we found that accumulation of HER2-HApt-AuNS complexes in lysosomes, lysosomal activity, and lysosomal degradation of HER2 were positively correlated. Increased HER2 degradation by HApt-AuNS triggered cell death and cell cycle arrest in the G0/G1 phase inhibition of cell proliferation. This work shows how a perceived disadvantage of nanoparticle-based therapeutics-the inability of nanoconstructs to escape from vesicles and thus induce a biological response-can be overcome by both targeting lysosomes and exploiting lysosomal degradation of the biomarkers.

  9. miR-134 inhibits non-small cell lung cancer growth by targeting the epidermal growth factor receptor.

    PubMed

    Qin, Qin; Wei, Furong; Zhang, Jianbo; Wang, Xingwu; Li, Baosheng

    2016-10-01

    The epidermal growth factor receptor (EGFR) is frequently activated in a wide range of solid tumours and represents an important therapeutic target. MicroRNAs (miRNAs) have recently been recognized as a rational and potential modality for anti-EGFR therapies. However, more EGFR-targeting miRNAs need to be explored. In this study, we identified a novel EGFR-targeting miRNA, miRNA-134 (miR-134), in non-small-cell lung cancer (NSCLC) cell lines. Luciferase assays confirmed that EGFR is a direct target of miR-134. In addition, the overexpression of miR-134 inhibited EGFR-related signaling and suppressed NSCLC cells proliferation by inducing cell cycle arrest and/or apoptosis, suggesting that miR-134 functions as a tumour suppressor in NSCLC. Further mechanistic investigation including RNAi and rescue experiments suggested that the down-regulation of EGFR by miR-134 partially contributes to the antiproliferative role of miR-134. Last, in vivo experiments demonstrated that miR-134 suppressed tumour growth of A549 xenograft in nude mice. Taken together, our findings suggest that miR-134 inhibits non-small cell lung cancer growth by targeting the EGFR.

  10. MIIP accelerates epidermal growth factor receptor protein turnover and attenuates proliferation in non-small cell lung cancer

    PubMed Central

    Wen, Jing; Fu, Jianhua; Ling, Yihong; Zhang, Wei

    2016-01-01

    The migration and invasion inhibitory protein (MIIP) has been discovered recently to have inhibitory functions in cell proliferation and migration. Overexpression of MIIP reduced the intracellular steady-state level of epidermal growth factor receptor (EGFR) protein in lung cancer cells with no effect on EGFR mRNA expression compared to that in the control cells. This MIIP-promoted EGFR protein degradation was reversed by proteasome and lysosome inhibitors, suggesting the involvement of both proteasomal and lysosomal pathways in this degradation. This finding was further validated by pulse-chase experiments using 35S-methionine metabolic labeling. We found that MIIP accelerates EGFR protein turnover via proteasomal degradation in the endoplasmic reticulum and then via the lysosomal pathway after its entry into endocytic trafficking. MIIP-stimulated downregulation of EGFR inhibits downstream activation of Ras and blocks the MEK signal transduction pathway, resulting in inhibition of cell proliferation. The negative correlation between MIIP and EGFR protein expression was validated in lung adenocarcinoma samples. Furthermore, the higher MIIP protein expression predicts a better overall survival of Stage IA-IIIA lung adenocarcinoma patients who underwent radical surgery. These findings reveal a new mechanism by which MIIP inhibits cell proliferation. PMID:26824318

  11. Axl mediates acquired resistance of head and neck cancer cells to the epidermal growth factor receptor inhibitor erlotinib.

    PubMed

    Giles, Keith M; Kalinowski, Felicity C; Candy, Patrick A; Epis, Michael R; Zhang, Priscilla M; Redfern, Andrew D; Stuart, Lisa M; Goodall, Gregory J; Leedman, Peter J

    2013-11-01

    Elevated expression and activity of the epidermal growth factor receptor (EGFR) is associated with development and progression of head and neck cancer (HNC) and a poor prognosis. Clinical trials with EGFR tyrosine kinase inhibitors (e.g., erlotinib) have been disappointing in HNC. To investigate the mechanisms mediating resistance to these agents, we developed an HNC cell line (HN5-ER) with acquired erlotinib resistance. In contrast to parental HN5 HNC cells, HN5-ER cells exhibited an epithelial-mesenchymal (EMT) phenotype with increased migratory potential, reduced E-cadherin and epithelial-associated microRNAs (miRNA), and elevated vimentin expression. Phosphorylated receptor tyrosine kinase profiling identified Axl activation in HN5-ER cells. Growth and migration of HN5-ER cells were blocked with a specific Axl inhibitor, R428, and R428 resensitized HN5-ER cells to erlotinib. Microarray analysis of HN5-ER cells confirmed the EMT phenotype associated with acquired erlotinib resistance, and identified activation of gene expression associated with cell migration and inflammation pathways. Moreover, increased expression and secretion of interleukin (IL)-6 and IL-8 in HN5-ER cells suggested a role for inflammatory cytokine signaling in EMT and erlotinib resistance. Expression of the tumor suppressor miR-34a was reduced in HN5-ER cells and increasing its expression abrogated Axl expression and reversed erlotinib resistance. Finally, analysis of 302 HNC patients revealed that high tumor Axl mRNA expression was associated with poorer survival (HR = 1.66, P = 0.007). In summary, our results identify Axl as a key mediator of acquired erlotinib resistance in HNC and suggest that therapeutic inhibition of Axl by small molecule drugs or specific miRNAs might overcome anti-EGFR therapy resistance. PMID:24026012

  12. MicroRNA-27a functions as a tumor suppressor in renal cell carcinoma by targeting epidermal growth factor receptor

    PubMed Central

    LI, YUEYAN; LI, JIE; SUN, XIAOLEI; CHEN, JIACUN; SUN, XIAOQING; ZHENG, JUNNIAN; CHEN, RENFU

    2016-01-01

    Numerous studies have suggested that microRNAs (miRNAs) are vital in the development of various types of human cancers, including renal cell carcinoma (RCC), and the regulation of tumor progression and invasion. However, the effect of miRNA-27a (miR-27a) on the tumorigenesis of RCC is unclear. The aim of the present study was to investigate the function of miR-27a and identify its possible target genes in RCC cells. In the present study, cell proliferation, migration and invasion and the percentage of apoptotic cells were detected by methylthiazol tetrazolium assays, Annexin V analysis, wound-healing assays and Transwell invasion assays. Western blot analysis was performed to validate the protein expression level and assess whether the epidermal growth factor receptor (EGFR) was a target gene of miR-27a. A tumor xenograft animal model was used to detect the role of miR-27a on RCC cell growth in vivo. The present study demonstrated that miR-27a significantly suppressed human RCC 786-O cell proliferation and induced cell apoptosis. Restoration of miR-27 also resulted in 786-O cell migration and invasion inhibition. Furthermore, upregulated miR-27a attenuated RCC tumor growth in the tumor xenograft animal model. The present results suggested that miR-27a functions as a tumor suppressor in RCC. The western blot analysis assay revealed that EGFR was a novel target of miR-27a. The growth suppression of RCC cells was attributed partly to the downregulation of the cell cycle by ERFR inhibition. The present findings may aid in the understanding of the molecular mechanism of miR-27a in the tumorigenesis of RCC, and may provide novel diagnostic and therapeutic options for RCC. PMID:27313769

  13. 3,3',5-Triiodothyronine-induced differences in water-insoluble protein synthesis in primary epidermal cell cultures from the hind limb of premetamorphic Rana catesbeiana tadpoles.

    PubMed

    Ketola-Pirie, C A; Atkinson, B G

    1988-02-01

    Epidermal cells from the hind limb of premetamorphic tadpoles of the American bullfrog Rana catesbeiana (stages IX-XI) were maintained in primary culture for 120 hr. Cultures, maintained for 36 hr in medium supplemented with L-triiodothyronine (T3; 3 x 10(-10) mol T3/ml medium), synthesize water-insoluble proteins with Mrs of 59, 50, 48, and 43. The 59 and 48 kDa proteins synthesized by T3-supplemented cultures are not detectably synthesized by 36-hr-old cell cultures without added T3, but they are synthesized by both hormone-supplemented and unsupplemented cultures after 120 hr. These two proteins have Mrs and pIs which correspond with keratins detected in stratifying mammalian epidermis and are immunoprecipitated by antibodies prepared against human keratins. These observations indicate that T3 induces epidermal cell cultures from the hind limb of premetamorphic tadpoles to precociously synthesize water-insoluble proteins which (1) have Mrs and pIs similar to keratins from differentiating amphibian epidermal tissue and (2) are biochemically and immunologically similar to keratins associated with the differentiation of mammalian epidermal tissue.

  14. Cyclooxygenase-2 (COX-2) mediates arsenite inhibition of UVB-induced cellular apoptosis in mouse epidermal Cl41 cells.

    PubMed

    Zuo, Z; Ouyang, W; Li, J; Costa, M; Huang, C

    2012-07-01

    Inorganic arsenic is an environmental human carcinogen, and has been shown to act as a co-carcinogen with solar ultraviolet (UV) radiation in mouse skin tumor induction even at low concentrations. However, the precise mechanism of its co-carcinogenic action is largely unknown. Apoptosis plays an essential role as a protective mechanism against neoplastic development in the organism by eliminating genetically damaged cells. Thus, suppression of apoptosis is thought to contribute to carcinogenesis. It is known that cyclooxygenase-2 (COX-2) can promote carcinogenesis by inhibiting cell apoptosis under stress conditions; and our current studies investigated the potential contribution of COX-2 to the inhibitory effect of arsenite in UV-induced cell apoptosis in mouse epidermal Cl41 cells. We found that treatment of cells with low concentration (5 μM) arsenite attenuated cellular apoptosis upon UVB radiation accompanied with a coinductive effect on COX-2 expression and nuclear factor-κB (NFκB) transactivation. Our results also showed that the COX-2 induction by arsenite and UVB depended on an NFκB pathway because COX-2 co-induction could be attenuated in either p65-deficient or p50-deficient cells. Moreover, UVB-induced cell apoptosis could be dramatically reduced by the introduction of exogenous COX-2 expression, whereas the inhibitory effect of arsenite on UVB-induced cell apoptosis could be impaired in COX-2 knockdown C141 cells. Our results indicated that COX-2 mediated the anti-apoptotic effect of arsenite in UVB radiation through an NFκB-dependent pathway. Given the importance of apoptosis evasion during carcinogenesis, we anticipated that COX-2 induction might be at least partially responsible for the co-carcinogenic effect of arsenite on UVB-induced skin carcinogenesis.

  15. Control of patterns of symmetric cell division in the epidermal and cortical tissues of the Arabidopsis root.

    PubMed

    Zhang, Yanwen; Iakovidis, Michail; Costa, Silvia

    2016-03-15

    Controlled cell division is central to the growth and development of all multicellular organisms. Within the proliferating zone of the Arabidopsis root, regular symmetric divisions give rise to patterns of parallel files of cells, the genetic basis of which remains unclear. We found that genotypes impaired in the TONNEAU1a (TON1a) gene display misoriented symmetric divisions in the epidermis and have no division defects in the underlying cortical tissue. The TON1a gene encodes a microtubule-associated protein. We show that in the ton1a mutant, epidermal and cortical cells do not form narrow, ring-like preprophase bands (PPBs), which are plant-specific, cytoskeletal structures that predict the position of the division plane before mitosis. The results indicate that in the cortex but not in the epidermis, division plane positioning and patterning can proceed correctly in the absence of both a functional TON1a and PPB formation. Differences between tissues in how they respond to the signals that guide symmetric division orientation during patterning might provide the basis for organised organ growth in the absence of cell movements. PMID:26893344

  16. Control of patterns of symmetric cell division in the epidermal and cortical tissues of the Arabidopsis root.

    PubMed

    Zhang, Yanwen; Iakovidis, Michail; Costa, Silvia

    2016-03-15

    Controlled cell division is central to the growth and development of all multicellular organisms. Within the proliferating zone of the Arabidopsis root, regular symmetric divisions give rise to patterns of parallel files of cells, the genetic basis of which remains unclear. We found that genotypes impaired in the TONNEAU1a (TON1a) gene display misoriented symmetric divisions in the epidermis and have no division defects in the underlying cortical tissue. The TON1a gene encodes a microtubule-associated protein. We show that in the ton1a mutant, epidermal and cortical cells do not form narrow, ring-like preprophase bands (PPBs), which are plant-specific, cytoskeletal structures that predict the position of the division plane before mitosis. The results indicate that in the cortex but not in the epidermis, division plane positioning and patterning can proceed correctly in the absence of both a functional TON1a and PPB formation. Differences between tissues in how they respond to the signals that guide symmetric division orientation during patterning might provide the basis for organised organ growth in the absence of cell movements.

  17. Control of patterns of symmetric cell division in the epidermal and cortical tissues of the Arabidopsis root

    PubMed Central

    Zhang, Yanwen; Iakovidis, Michail; Costa, Silvia

    2016-01-01

    Controlled cell division is central to the growth and development of all multicellular organisms. Within the proliferating zone of the Arabidopsis root, regular symmetric divisions give rise to patterns of parallel files of cells, the genetic basis of which remains unclear. We found that genotypes impaired in the TONNEAU1a (TON1a) gene display misoriented symmetric divisions in the epidermis and have no division defects in the underlying cortical tissue. The TON1a gene encodes a microtubule-associated protein. We show that in the ton1a mutant, epidermal and cortical cells do not form narrow, ring-like preprophase bands (PPBs), which are plant-specific, cytoskeletal structures that predict the position of the division plane before mitosis. The results indicate that in the cortex but not in the epidermis, division plane positioning and patterning can proceed correctly in the absence of both a functional TON1a and PPB formation. Differences between tissues in how they respond to the signals that guide symmetric division orientation during patterning might provide the basis for organised organ growth in the absence of cell movements. PMID:26893344

  18. Quantification of epidermal growth factor receptor expression level and binding kinetics on cell surfaces by surface plasmon resonance imaging.

    PubMed

    Zhang, Fenni; Wang, Shaopeng; Yin, Linliang; Yang, Yunze; Guan, Yan; Wang, Wei; Xu, Han; Tao, Nongjian

    2015-10-01

    Epidermal growth factor receptor (EGFR, also known as ErbB-1 or HER-1) is a membrane bound protein that has been associated with a variety of solid tumors and the control of cell survival, proliferation, and metabolism. Quantification of the EGFR expression level in cell membranes and the interaction kinetics with drugs are thus important for cancer diagnosis and treatment. Here we report mapping of the distribution and interaction kinetics of EGFR in their native environment with the surface plasmon resonance imaging (SPRi) technique. The monoclonal anti-EGFR antibody was used as a model drug in this study. The binding of the antibody to EGFR overexpressed A431 cells was monitored in real time, which was found to follow the first-order kinetics with an association rate constant (ka) and dissociation rate constant (kd) of (2.7 ± 0.6) × 10(5) M(-1) s(-1) and (1.4 ± 0.5) × 10(-4) s(-1), respectively. The dissociation constant (KD) was determined to be 0.53 ± 0.26 nM with up to seven-fold variation among different individual A431 cells. In addition, the averaged A431 cell surface EGFR density was found to be 636/μm(2) with an estimation of 5 × 10(5) EGFR per cell. Additional measurement also revealed that different EGFR positive cell lines (A431, HeLa, and A549) show receptor density dependent anti-EGFR binding kinetics. The results demonstrate that SPRi is a valuable tool for direct quantification of membrane protein expression level and ligand binding kinetics at single cell resolution. Our findings show that the local environment affects the drug-receptor interactions, and in situ measurement of membrane protein binding kinetics is important.

  19. Enhancing mitochondrial respiration suppresses tumor promoter TPA-induced PKM2 expression and cell transformation in skin epidermal JB6 cells.

    PubMed

    Wittwer, Jennifer A; Robbins, Delira; Wang, Fei; Codarin, Sarah; Shen, Xinggui; Kevil, Christopher G; Huang, Ting-Ting; Van Remmen, Holly; Richardson, Arlan; Zhao, Yunfeng

    2011-09-01

    Differentiated cells primarily metabolize glucose for energy via the tricarboxylic acid cycle and oxidative phosphorylation, but cancer cells thrive on a different mechanism to produce energy, characterized as the Warburg effect, which describes the increased dependence on aerobic glycolysis. The M2 isoform of pyruvate kinase (PKM2), which is responsible for catalyzing the final step of aerobic glycolysis, is highly expressed in cancer cells and may contribute to the Warburg effect. However, whether PKM2 plays a contributing role during early cancer development is unclear. In our studies, we have made an attempt to elucidate the effects of varying mitochondrial respiration substrates on skin cell transformation and expression of PKM2. Tumorigenicity in murine skin epidermal JB6 P+ (promotable) cells was measured in a soft agar assay using 12-O-tetradecanoylphorbol-13-acetate (TPA) as a tumor promoter. We observed a significant reduction in cell transformation upon pretreatment with the mitochondrial respiration substrate succinate or malate/pyruvate. We observed that increased expression and activity of PKM2 in TPA-treated JB6 P+ cells and pretreatment with succinate or malate/pyruvate suppressed the effects. In addition, TPA treatment also induced PKM2 whereas PKM1 expression was suppressed in mouse skin epidermal tissues in vivo. In comparison with JB6 P+ cells, the nonpromotable JB6 P- cells showed no increase in PKM2 expression or activity upon TPA treatment. Knockdown of PKM2 using a siRNA approach significantly reduced skin cell transformation. Thus, our results suggest that PKM2 activation could be an early event and play a contributing role in skin tumorigenesis.

  20. Role of epidermal γδ T-cell-derived interleukin 13 in the skin-whitening effect of Ginsenoside F1.

    PubMed

    Han, Jiyeon; Lee, Eunkyung; Kim, EunJoo; Yeom, Myung Hun; Kwon, Ohsang; Yoon, Tae Hong; Lee, Tae Ryong; Kim, Kwangmi

    2014-11-01

    Ginsenoside F1 (GF1) is a metabolite of ginsenoside Rg1. Although GF1 has several benefits for skin physiology, the effect of GF1 on skin pigmentation has not been reported. We found that a cream containing 0.1% GF1 showed a significant whitening effect on artificially tanned human skin after 8 weeks of application. However, GF1 did not inhibit mRNA expression of tyrosinase or dopachrome tautomerase (DCT) in normal human epidermal melanocytes (NHEMs) or cocultured NHEMs/normal human epidermal keratinocytes. Interestingly, GF1 enhanced production of interleukin 13 (IL-13) from human epidermal γδ T cells. IL-13 significantly reduced the mRNA expression and protein amount of both tyrosinase and DCT and reduced melanin synthesis activities in NHEMs, resulting in visible brightening of NHEM pellet. These results suggest that enhancement of IL-13 production by GF1 from epidermal γδ T cells might play a role in the skin-whitening effect of GF1 via the suppression of tyrosinase and DCT.

  1. Inhibitory effects of tetrandrine on epidermal growth factor-induced invasion and migration in HT29 human colorectal adenocarcinoma cells.

    PubMed

    Horng, Chi-Ting; Yang, Jai-Sing; Chiang, Jo-Hua; Lu, Chi-Cheng; Lee, Chiu-Fang; Chiang, Ni-Na; Chen, Fu-An

    2016-01-01

    Tetrandrine has been shown to reduce cancer cell proliferation and to inhibit metastatic effects in multiple cancer models in vitro and in vivo. However, the effects of tetrandrine on the underlying mechanism of HT29 human colorectal adenocarcinoma cell metastasis remain to be fully elucidated. The aim of the present study was focused on tetrandrine‑treated HT29 cells following epidermal growth factor (EGF) treatment, and Transwell, gelatin zymography, gene expression and immunoblotting assays were performed to investigate metastatic effects in vitro. Tetrandrine was observed to dose‑dependently inhibit EGF‑induced HT29 cell invasion and migration, however, no effect on cell viability occurred following exposure to tetradrine between 0.5 and 2 µM. Tetrandrine treatment inhibited the enzymatic activity of matrix metalloprotease (MMP)‑2 and MMP‑9 in a concentration‑dependent manner. The present study also found a reduction in the mRNA expression levels of MMP‑2 and MMP‑9 in the tetrandrine‑treated HT29 cells. Tetrandrine also suppressed the phosphorylation of EGF receptor (EGFR) and its downstream pathway, including phosphoinositide‑dependent kinase 1, phosphatidylinositol 3‑kinase and phosphorylated AKT, suppressing the gene expression of MMP‑2 and MMP‑9. Furthermore, tetrandrine triggered mitogen‑activated protein kinase signaling through the suppressing the activation of phosphorylated extracellular signal‑regulated protein kinase. These data suggested that targeting EGFR signaling and its downstream molecules contributed to the inhibition of EGF‑induced HT29 cell metastasis caused by tetrandrine, eventually leading to a reduction in the mRNA and gelatinase activities of MMP-2 and MMP-9, respectively. PMID:26648313

  2. Insulin-like growth factor II mediates epidermal growth factor-induced mitogenesis in cervical cancer cells.

    PubMed Central

    Steller, M A; Delgado, C H; Zou, Z

    1995-01-01

    There is increasing evidence that activation of the insulin-like growth factor I (IGF-I) receptor plays a major role in the control of cellular proliferation of many cell types. We studied the mitogenic effects of IGF-I, IGF-II, and epidermal growth factor (EGF) on growth-arrested HT-3 cells, a human cervical cancer cell line. All three growth factors promoted dose-dependent increases in cell proliferation. In untransformed cells, EGF usually requires stimulation by a "progression" factor such as IGF-I, IGF-II, or insulin (in supraphysiologic concentrations) in order to exert a mitogenic effect. Accordingly, we investigated whether an autocrine pathway involving IGF-I or IGF-II participated in the EGF-induced mitogenesis of HT-3 cells. With the RNase protection assay, IGF-I mRNA was not detected. However, IGF-II mRNA increased in a time-dependent manner following EGF stimulation. The EGF-induced mitogenesis was abrogated in a dose-dependent manner by IGF-binding protein 5 (IGFBP-5), which binds to IGF-II and neutralizes it. An antisense oligonucleotide to IGF-II also inhibited the proliferative response to EGF. In addition, prolonged, but not short-term, stimulation with EGF resulted in autophosphorylation of the IGF-I receptor, and coincubations with both EGF and IGFBP-5 attenuated this effect. These data demonstrate that autocrine secretion of IGF-II in HT-3 cervical cancer cells can participate in EGF-induced mitogenesis and suggest that autocrine signals involving the IGF-I receptor occur "downstream" of competence growth factor receptors such as the EGF receptor. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8618825

  3. Identification of bone morphogenetic protein 7 (BMP7) as an instructive factor for human epidermal Langerhans cell differentiation.

    PubMed

    Yasmin, Nighat; Bauer, Thomas; Modak, Madhura; Wagner, Karin; Schuster, Christopher; Köffel, Rene; Seyerl, Maria; Stöckl, Johannes; Elbe-Bürger, Adelheid; Graf, Daniel; Strobl, Herbert

    2013-11-18

    Human Langerhans cell (LC) precursors populate the epidermis early during prenatal development and thereafter undergo massive proliferation. The prototypic antiproliferative cytokine TGF-β1 is required for LC differentiation from human CD34(+) hematopoietic progenitor cells and blood monocytes in vitro. Similarly, TGF-β1 deficiency results in LC loss in vivo. However, immunohistology studies revealed that human LC niches in early prenatal epidermis and adult basal (germinal) keratinocyte layers lack detectable TGF-β1. Here we demonstrated that these LC niches express high levels of bone morphogenetic protein 7 (BMP7) and that Bmp7-deficient mice exhibit substantially diminished LC numbers, with the remaining cells appearing less dendritic. BMP7 induces LC differentiation and proliferation by activating the BMP type-I receptor ALK3 in the absence of canonical TGF-β1-ALK5 signaling. Conversely, TGF-β1-induced in vitro LC differentiation is mediated via ALK3; however, co-induction of ALK5 diminished TGF-β1-driven LC generation. Therefore, selective ALK3 signaling by BMP7 promotes high LC yields. Within epidermis, BMP7 shows an inverse expression pattern relative to TGF-β1, the latter induced in suprabasal layers and up-regulated in outer layers. We observed that TGF-β1 inhibits microbial activation of BMP7-generated LCs. Therefore, TGF-β1 in suprabasal/outer epidermal layers might inhibit LC activation, resulting in LC network maintenance.

  4. Blister fluid T lymphocytes during toxic epidermal necrolysis are functional cytotoxic cells which express human natural killer (NK) inhibitory receptors

    PubMed Central

    Le Cleach, L; Delaire, S; Boumsell, L; Bagot, M; Bourgault-Villada, I; Bensussan, A; Roujeau, J C

    2000-01-01

    Toxic epidermal necrolysis (TEN) is a rare life-threatening adverse drug reaction characterized by a massive destruction of the epidermis. Immunohistological studies of skin biopsies of TEN showed infiltrates of predominantly CD8+ T lymphocytes even though other authors reported a prominent involvement of cells of the monocyte-macrophage lineage. The aim of this study was to characterize phenotypically and functionally the cells present in the cutaneous blister fluid of four patients with TEN. We first determined that lymphocytes were predominant in blister fluid obtained early, while monocytes/macrophages later became the most important population. We then showed that this lymphocyte population, mainly CD3+CD8+, corresponded to a peculiar cell subset as they expressed cutaneous leucocyte antigen, killer inhibitory receptors KIR/KAR and failed to express CD28 molecule. Functionally, we determined that blister T lymphocytes had a cytotoxic T lymphocyte (CTL)- and NK-like cytotoxicity. The role of this cytotoxic lymphocyte population present at the site of lesions during TEN remains to be understood. PMID:10606987

  5. Involvement of epidermal growth factor receptor-linked signaling responses in Pseudomonas fluorescens-infected alveolar epithelial cells.

    PubMed

    Choi, Hye Jin; Seo, Chan Hee; Park, Seong Hwan; Yang, Hyun; Do, Kee Hun; Kim, Juil; Kim, Hyung-Kab; Chung, Duk-Hwa; Ahn, Jung Hoon; Moon, Yuseok

    2011-05-01

    Pseudomonas fluorescens is an opportunistic indoor pathogen that can cause severe airway proinflammatory responses. Pulmonary epithelium, like other mucosal epithelial linings of the body, constitutes the first line of defense against airway microbial pathogens. Mucosal epithelial cells can be a sentinel of pathogenic bacteria via stimulation of specific cell surface receptors, including the epidermal growth factor receptor (EGFR) and Toll-like receptor (TLR). This study addressed the involvement of EGFR in airway epithelial pathogenesis by P. fluorescens. Human A549 pneumocytes showed prolonged production of proinflammatory interleukin-8 (IL-8) in response to infection with P. fluorescens, which was via the nuclear factor-kappa B (NF-κB) signaling pathway. Production of proinflammatory cytokine IL-8 was not mediated by P. fluorescens lipopolysaccharide, a representative TLR4 agonist, but was mediated through EGFR-linked signals activated by the opportunistic bacteria. Moreover, EGFR signals were involved in NF-κB signal-mediated production of proinflammatory cytokines. Along with persistent NF-κB activation, P. fluorescens enhanced the EGFR phosphorylation and subsequent activation of downstream mediators, including protein kinase B or extracellular-signal-regulated kinases 1/2. Blocking of EGFR-linked signals increased epithelial susceptibility to pathogen-induced epithelial cell death, suggesting protective roles of EGFR signals. Thus, airway epithelial exposure to P. fluorescens can trigger antiapoptotic responses via EGFR and proinflammatory responses via TLR4-independent NF-κB signaling pathway in human pneumocytes.

  6. Targeting of epidermal growth factor receptor (EGFR)-expressing tumor cells with sterically stabilized affibody liposomes (SAL).

    PubMed

    Beuttler, Julia; Rothdiener, Miriam; Müller, Dafne; Frejd, Fredrik Y; Kontermann, Roland E

    2009-06-01

    Affibody molecules are small and stable antigen-binding molecules derived from the B domain of protein A. We applied a bivalent, high-affinity epidermal growth factor receptor (EGFR)-specific affibody molecule for the generation of targeted PEGylated liposomes. These sterically stabilized affibody liposomes (SAL) were produced by chemical coupling of the cysteine-modified affibody molecule to maleimide-PEG(2000)-DSPE and subsequent insertion into PEGylated liposomes. These SAL showed strong and selective binding to EGFR-expressing tumor cell lines. Binding was dependent on the amount of inserted affibody molecule-lipid conjugates and could be blocked by soluble EGF. Approximately 30% of binding activity was still retained after 6 days of incubation in human plasma at 37 degrees C. Binding of SAL to cells led to efficient internalization of the liposomes. Using mitoxantrone-loaded liposomes, we observed for SAL, compared to untargeted liposomes, an enhanced cytotoxicity toward EGFR-expressing cells. In summary, we show that SAL can be easily prepared from affibody molecules and thus may be suitable for the development of carrier systems for targeted delivery of drugs. PMID:19435362

  7. Patterns of epidermal growth factor receptor mutation in non-small-cell lung cancers in the Gulf region

    PubMed Central

    JAZIEH, ABDUL RAHMAN; JAAFAR, HASAN; JALOUDI, MOHAMMED; MUSTAFA, RASHA SALEH; RASUL, KAKIL; ZEKRI, JAMAL; BAMEFLEH, HANAA; GASMELSEED, AHMED

    2015-01-01

    The aim of the present study was to determine the prevalence of epidermal growth factor receptor mutations (EGFRmut) in the Gulf region (GR) and its correlation with demographic and clinical characteristics. A multisite retrospective study was conducted, including institutions from Saudi Arabia, the United Arab Emirates and Qatar. All consecutive patients with non-small-cell lung cancer tested for EGFRmut were eligible. Data collected included demographic information, disease characteristics and EGFR test results. Data on 230 patients were obtained. The median age of the patients was 61 years (range, 26–87 years); 169 patients (69.83%) were male and 204 (88.7%) were Arab. The histological subtype was adenocarcinoma in 191 (83.4%) and squamous cell carcinoma in 21 cases (9.17%). Overall, EGFRmut were detected in 66 patients (28.7%), with a prevalence of 32.46% in adenocarcinoma. No squamous cell carcinomas were found to harbor EGFRmut. The univariate and multivariate analyses revealed that female gender, non-smoking status and adenocarcinoma subtype were significant predictors for EGFRmut. There was no difference between Arabs and non-Arabs. In conclusion, to the best of our knowledge, this is the first multisite study to report the prevalence of EGFRmut in the GR population, which was found to be higher compared with that in Western, but lower compared with that in Far Eastern populations. Studies evaluating the efficacy of targeted therapy in this population are underway. PMID:26807249

  8. Progressive alopecia reveals decreasing stem cell activation probability during aging of mice with epidermal deletion of DNA methyltransferase 1.

    PubMed

    Li, Ji; Jiang, Ting-Xin; Hughes, Michael W; Wu, Ping; Yu, Juehua; Widelitz, Randall B; Fan, Guoping; Chuong, Cheng-Ming

    2012-12-01

    To examine the roles of epigenetic modulation on hair follicle regeneration, we generated mice with a K14-Cre-mediated loss of DNA methyltransferase 1 (DNMT1). The mutant shows an uneven epidermal thickness and alterations in hair follicle size. When formed, hair follicle architecture and differentiation appear normal. Hair subtypes exist but hair fibers are shorter and thinner. Hair numbers appear normal at birth but gradually decrease to <50% of control in 1-year-old mice. Sections of old mutant skin show follicles in prolonged telogen with hyperplastic sebaceous glands. Anagen follicles in mutants exhibit decreased proliferation and increased apoptosis in matrix transient-amplifying cells. Although K15-positive stem cells in the mutant bulge are comparable in number to the control, their ability to proliferate and become activated to form a hair germ is reduced. As mice age, residual DNMT activity declines further, and the probability of successful anagen reentry decreases, leading to progressive alopecia. Paradoxically, there is increased proliferation in the epidermis, which also shows aberrant differentiation. These results highlight the importance of DNA methylation in maintaining stem cell homeostasis during the development and regeneration of ectodermal organs.

  9. Role of 3'-5'-cyclic adenosine monophosphate on the epidermal growth factor dependent survival in mammary epithelial cells.

    PubMed

    Grinman, Diego Y; Romorini, Leonardo; Presman, Diego M; Rocha-Viegas, Luciana; Coso, Omar A; Davio, Carlos; Pecci, Adali

    2016-01-01

    Epidermal growth factor (EGF) has been suggested to play a key role in the maintenance of epithelial cell survival during lactation. Previously, we demonstrated that EGF dependent activation of PI3K pathway prevents apoptosis in confluent murine HC11 cells cultured under low nutrient conditions. The EGF protective effect is associated with increased levels of the antiapoptotic protein Bcl-XL. Here, we identify the EGF-dependent mechanism involved in cell survival that converges in the regulation of bcl-X expression by activated CREB. EGF induces Bcl-XL expression through activation of a unique bcl-X promoter, the P1; being not only the PI3K/AKT signaling pathway but also the increase in cAMP levels and the concomitant PKA/CREB activation necessary for both bcl-XL upregulation and apoptosis avoidance. Results presented in this work suggest the existence of a novel connection between the EGF receptor and the adenylate cyclase that would have an impact in preventing apoptosis under low nutrient conditions.

  10. Transformation and tumor promoter sensitive phosphoproteins in JB-6 mouse epidermal cells: one is also sensitive to heat stress.

    PubMed

    Gindhart, T D; Stevens, L; Copley, M P

    1984-09-01

    JB-6 mouse epidermal cells undergo irreversible transformation when exposed to tumor-promoting agents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Phosphoprotein changes related to transformation were sought in four tumor cell lines related to JB-6 cells. Two dimensional polyacrylamide gel electrophoresis showed altered abundances of five phosphoproteins in the tumor cell lines compared with five untransformed clones. The mol. wt. in Kilodaltons and isoelectric points in pH units were: 120/6.0, 80/4.5, 55/6.5, 37/5.0 and 23-25/4.5. In all four transformants pp80 was markedly decreased and the pp23-25 doublet increased. In two of the four transformants pp120 and pp55 were increased and pp37 decreased. Treatment of untransformed clones with TPA affected only one of the phosphoproteins altered in the transformants. Treatment of untransformed clones with TPA produced a 2-fold increase in pp80 after 5 h. pp80 returned to baseline levels by 24 h and changed little in the continuous presence of TPA for up to 96 h. The increase in pp80 with short term TPA treatment occurred in all of the untransformed clones but none of four transformants. Late preneoplastic (P+) JB-6 cells only require treatment with a tumor promoter to transform. Early preneoplastic (P-) JB-6 cells require prior transfection of DNA from late preneoplastic JB-6 cells to transform in response to tumor promoter treatment. Quantitative analysis of pp80 in early preneoplastic, late preneoplastic, and tumor cell lines showed an inverse relationship between the level of pp80 and degree of preneoplastic progression in these cells. pp80 represents approximately 2% of total cellular phosphoprotein in JB-6 cells, shows microheterogeneity of both mol. wt. and isoelectric point, occurs in the particulate fraction of cells and is readily solubilized by 1% Triton. pp80 is increased by heat stress and shares other properties with the recently described mammalian heat stress protein, hsp 80. pp80's decrease in

  11. c-Jun N-terminal kinase negatively regulates epidermal growth factor-induced cyclooxygenase-2 expression in oral squamous cell carcinoma cell lines.

    PubMed

    Husvik, Camilla; Bryne, Magne; Halstensen, Trond S

    2009-12-01

    Epidermal growth factor (EGF)-induced cyclooxygenase-2 (COX-2) expression in squamous cell carcinomas is mediated through the extracellular signal-regulated kinase 1/2 and p38 pathways. Examination of a basaloid and a conventional oral squamous cell carcinoma cell line revealed that inhibition of c-Jun N-terminal kinase (JNK) with SP600125 increased EGF-induced (but not basal) COX-2 transcription 1.5-1.9-fold in extracellular signal-regulated kinase 1/2 and p38 pathway-dependent manners. Although JNK may phosphorylate the cyclosporine A-sensitive transcription factor, nuclear factor of activated T cells c3, it was seemingly not involved because cyclosporine A did not reduce EGF-induced COX-2 expression. Thus, JNK negatively regulated EGF-induced extracellular signal-regulated kinase 1/2 and/or p38-mediated COX-2 transcription, presumably through activating an unidentified phosphatase. PMID:20121928

  12. [CYTOSKELETON ORIENTATION IN THE EPIDERMAL CELLS OF ROOTS FORMED DE NOVO ON LEAF EXPLANTS UNDER CLINOROTATION].

    PubMed

    Bulavin, I V

    2016-01-01

    Root anatomy, cytoskeleton orientation and cell wall thickness in cells of the roots formed de novo in vitro under clinorotation (simulated microgravity) were investigated. Structure of the embryonic roots and of the roots formed de novo in cambium cells of the leaf petiole explants was shown to be similar. Root cell differentiation in vitro under clinorotation did not differ from that in control. Changes of tubulin microtubules' orientation in the epidermis of the distal elongation zone were observed under clinorotation that seems to be associated with specific physiological properties of the cells. Under clinorotation, the tendency of cell wall thinning was detected in the root cells formed in vitro.

  13. Effect of epidermal growth factor in HLA class I and class II transcription and protein expression in human breast adenocarcinoma cell lines.

    PubMed Central

    Bernard, D. J.; Courjal, F.; Maurizis, J. C.; Bignon, Y. J.; Chollet, P.; Plagne, R.

    1992-01-01

    The spontaneous expression of HLA class I and class II molecules in two human breast carcinoma cell lines (MCF7, T47D) and their modulation during epidermal growth factor treatment are reported. Transcription was analysed by Northern blot and hybridisation with HLA class II and class I cDNA specific probes. The expression of cell surface determinants was examined by internal protein labelling with 35s-methionine, immunoprecipitation with monoclonal antibodies specific for HLA class I or class II, followed by isolation of the immune complex on protein A-Sepharose; at least a quantification of glycoprotein was performed by chromatofocusing. Glycoprotein quantification showed a significant increase of HLA class I and class II (DR) antigen expression after stimulation by epidermal growth factor (0.02 microgram ml-1) in the two cell lines, when compared with untreated cell controls. However, with epidermal growth factor treatment of MCF7 and T47D cells, low increases in the amounts of HLA class I and class II RNA were obtained. These differences between expressed antigens and correspondent RNA amounts would be explained by the fact that EGF in these two cell lines acts more in post-transcription for HLA class I and class II antigens. Images Figure 1 PMID:1637682

  14. Dynamic analysis of epidermal cell divisions identifies specific roles for COP10 in Arabidopsis stomatal lineage development.

    PubMed

    Delgado, Dolores; Ballesteros, Isabel; Torres-Contreras, Javier; Mena, Montaña; Fenoll, Carmen

    2012-08-01

    Stomatal development in Arabidopsis thaliana has been linked to photoreceptor-perceived light through several components of the photomorphogenic switch, whose lack of function is often seedling-lethal. CONSTITUTIVE PHOTOMORPHOGENIC 10 (COP10) is an important component of this switch, its loss of function producing stomatal clusters. Exploiting the reduced lethality of the cop10-1 mutant we characterized the developmental basis of its stomatal phenotype. Constitutive, light-independent stomata overproduction accounts for half of cop10-1 stomatal abundance and appears very early in development. Clusters are responsible for the remaining stomata excess and build-up progressively at later stages. Serial impressions of living cotyledon epidermis allowed a dynamic, quantitative analysis of stomatal lineage types by reconstructing their division histories. We found that COP10 adjusts the initiation frequency and extension of stomatal lineages (entry and amplifying asymmetric divisions) and represses stomatal fate in lineage cells; COP10 also supervises the orientation of spacing divisions in satellite lineages, preventing the appearance of stomata in contact. Aberrant accumulation of the proliferating stomatal lineage cell marker TMMpro::TMM-GFP showed that the abundant cop10-1 stomatal lineages maintained extended and ectopic competence for stomatal fate. Expression of stomatal development master genes suggests that the mutant does not bypass major molecular actors in this process. cop10-1 first leaf produces trichomes and apparently normal pavement cells, but functionally and morphologically aberrant stomata; COP10 operates genetically in parallel to the stomatal repressor SDD1 and does not generally affect epidermal cell differentiation, but seems to operate on stomatal lineages where it controls specific cell-lineage and cell-signaling developmental mechanisms.

  15. New enzymes involved in the mechanism of action of epidermal growth factor in a clonal strain of Leydig tumor cells.

    PubMed

    Castilla, Rocío; Gadaleta, Mariana; Castillo, Ana Fernanda; Duarte, Alejandra; Neuman, Isabel; Paz, Cristina; Cornejo Maciel, Fabiana; Podestá, Ernesto J

    2008-07-01

    The studies presented herein were designed to investigate the effect of mouse epidermal growth factor (mEGF) on arachidonic acid (AA) release in a clonal strain of cultured murine Leydig cells (designed MA-10). In MA-10 cells, mEGF promotes AA release and metabolism to lipoxygenated products to induce the steroidogenic acute regulatory (StAR) protein. However, the mechanism by which mEGF releases AA in these cells is not totally elucidated. We show that mEGF produces an increment in the mitochondrial AA content in a short-term incubation (30 min). This AA is released by the action of a mitochondrial acyl-CoA thioesterase (Acot2), as demonstrated in experiments in which Acot2 was down or overexpressed. This AA in turn regulates the StAR protein expression, indirect evidence of its metabolism to lipoxygenated products. We also show that mEGF induces the expression (mRNA and protein) of Acot2 and an acyl-CoA synthetase that provides the substrate, arachidonyl-CoA, to Acot2. This effect is also observed in another steroidogenic cell line, the adrenocortical Y1 cells. Taken together, our results show that: 1) mEGF can induce the generation of AA in a specific compartment of the cells, i.e. the mitochondria; 2) mEGF can up-regulate acyl-CoA synthetase and Acot2 mRNA and protein levels; and 3) mEGF-stimulated intramitochondrial AA release leads to StAR protein induction.

  16. Death ligand TRAIL, secreted by CD1a+ and CD14+ cells in blister fluids, is involved in killing keratinocytes in toxic epidermal necrolysis.

    PubMed

    de Araujo, Elisabeth; Dessirier, Valérie; Laprée, Geneviève; Valeyrie-Allanore, Laurence; Ortonne, Nicolas; Stathopoulos, Efstathios N; Bagot, Martine; Bensussan, Armand; Mockenhaupt, Maja; Roujeau, Jean-Claude; Tsapis, Andreas

    2011-02-01

    Toxic epidermal necrolysis (TEN) is characterized by an acute detachment and destruction of keratinocytes, affecting large areas of the skin. It is often related to adverse drug reactions. Previous studies have shown that effector CD8+ T cells, which accumulate in the blister fluid, are functionally cytotoxic and act through a classical perforin/granzyme B pathway. It has recently been shown that these cytotoxic T cells also secrete granulysin peptide, which is lethal to keratinocytes. These cytotoxic T cells exert their killer activity against autologous keratinocytes in the presence of the drug. However, they are unlikely to be the only effectors of TEN. We therefore searched for soluble death factors in the blister fluids that might kill keratinocytes. We found that the amounts of interferon-γ, TRAIL and TNF-α proteins were significantly greater in TEN blister fluids than in all controls (normal sera, TEN sera, burns and Eosinophilic pustular folliculitis blister fluids) and TNF-like weak inducer of apoptosis (TWEAK) amounts are also greater in all controls except burns. We showed that these proteins acted in synergy to induce the death of keratinocytes in vitro. We also found that TRAIL and TWEAK were secreted by CD1a+ and CD14+ cells present in the blister fluids. Thus, in addition to MHC class I-restricted cytotoxic T lymphocytes (CTLs), which lyse keratinocytes, ligands secreted by non-lymphoid cells capable of inducing keratinocyte death in an MHC class I-independent manner, also seem to be present in the blister fluids of patients with TEN.

  17. 7,3',4'-Trihydroxyisoflavone inhibits epidermal growth factor-induced proliferation and transformation of JB6 P+ mouse epidermal cells by suppressing cyclin-dependent kinases and phosphatidylinositol 3-kinase.

    PubMed

    Lee, Dong Eun; Lee, Ki Won; Song, Nu Ry; Seo, Sang Kwon; Heo, Yong-Seok; Kang, Nam Joo; Bode, Ann M; Lee, Hyong Joo; Dong, Zigang

    2010-07-01

    Numerous in vitro and in vivo studies have shown that isoflavones exhibit anti-proliferative activity against epidermal growth factor (EGF) receptor-positive malignancies of the breast, colon, skin, and prostate. 7,3',4'-Trihydroxyisoflavone (7,3',4'-THIF) is one of the metabolites of daidzein, a well known soy isoflavone, but its chemopreventive activity and the underlying molecular mechanisms are poorly understood. In this study, 7,3',4'-THIF prevented EGF-induced neoplastic transformation and proliferation of JB6 P+ mouse epidermal cells. It significantly blocked cell cycle progression of EGF-stimulated cells at the G(1) phase. As shown by Western blot, 7,3',4'-THIF suppressed the phosphorylation of retinoblastoma protein at Ser-795 and Ser-807/Ser-811, which are the specific sites of phosphorylation by cyclin-dependent kinase (CDK) 4. It also inhibited the expression of G(1) phase-regulatory proteins, including cyclin D1, CDK4, cyclin E, and CDK2. In addition to regulating the expression of cell cycle-regulatory proteins, 7,3',4'-THIF bound to CDK4 and CDK2 and strongly inhibited their kinase activities. It also bound to phosphatidylinositol 3-kinase (PI3K), strongly inhibiting its kinase activity and thereby suppressing the Akt/GSK-3beta/AP-1 pathway and subsequently attenuating the expression of cyclin D1. Collectively, these results suggest that CDKs and PI3K are the primary molecular targets of 7,3',4'-THIF in the suppression of EGF-induced cell proliferation. These insights into the biological actions of 7,3',4'-THIF provide a molecular basis for the possible development of new chemoprotective agents. PMID:20444693

  18. Correlation between hormone dependency and the regulation of epidermal growth factor receptor by tumor promoters in human mammary carcinoma cells.

    PubMed Central

    Roos, W; Fabbro, D; Küng, W; Costa, S D; Eppenberger, U

    1986-01-01

    The effects of the tumor promoter phorbol 12-tetradecanoate 13-acetate (TPA) on the epidermal growth factor (EGF) receptor levels were investigated in hormone-dependent (MCF-7, T-47-D, and ZR-75-1) and hormone-independent (MDA-MB-231, HBL-100, and BT-20) human mammary carcinoma cell lines. In the absence of TPA, hormone-independent cell lines contained high concentrations of low-affinity EGF receptors (apparent Kd = 8 X 10(-10) M), whereas hormone-dependent cell lines exhibited low concentrations of high-affinity receptors (apparent Kd = 1 X 10(-10) M). TPA causes a change of the receptor from a high- to the low-affinity state in hormone-dependent cell lines (MCF-7, T-47-D, and ZR-75-1), as well as in the hormone-independent HBL-100, whereas the affinity remained unchanged in MDA-MB-231 and BT-20 cells. In addition, progesterone receptor levels are decreased after TPA treatment in the hormone-dependent cell lines MCF-7, T-47-D, and ZR-75-1, whereas the estrogen receptor levels remained unchanged. Tumor promoters such as TPA or teleocidin inhibited the proliferation of these cell lines at concentrations above 10 microM with the exception of the T-47-D cells. The most sensitive cell line towards growth inhibition by tumor promoter was the hormone-dependent MCF-7 cell line. Evaluation of different TPA analogs indicated a positive correlation between the growth-inhibitory effects and their ability to stimulate the subcellular redistribution of protein kinase C activity in MCF-7 cells. These data suggest a protein kinase C-mediated down-regulation of the progesterone receptor concentration and of the EGF receptor affinity, which is supposed to mediate the mitogenic response. Furthermore, these results support the hypothesis that the tumor-derived growth factors induced by estradiol act via the EGF receptor in hormone-dependent mammary carcinoma cells. PMID:3006036

  19. Single microfilaments mediate the early steps of microtubule bundling during preprophase band formation in onion cotyledon epidermal cells.

    PubMed

    Takeuchi, Miyuki; Karahara, Ichirou; Kajimura, Naoko; Takaoka, Akio; Murata, Kazuyoshi; Misaki, Kazuyo; Yonemura, Shigenobu; Staehelin, L Andrew; Mineyuki, Yoshinobu

    2016-06-01

    The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MTs) in G2 and narrows to demarcate the future division site during late prophase. Studies with fluorescent probes have shown that PPBs contain F-actin during early stages of their development but become actin depleted in late prophase. Although this suggests that actins contribute to the early stages of PPB formation, how actins contribute to PPB-MT organization remains unsolved. To address this question, we used electron tomography to investigate the spatial relationship between microfilaments (MFs) and MTs at different stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the PPB actins observed by fluorescence microscopy correspond to short, single MFs. A majority of the MFs are bound to MTs, with a subset forming MT-MF-MT bridging structures. During the later stages of PPB assembly, the MF-mediated links between MTs are displaced by MT-MT linkers as the PPB MT arrays mature into tightly packed MT bundles. On the basis of these observations, we propose that the primary function of actins during PPB formation is to mediate the initial bundling of the PPB MTs.

  20. Transient Silencing of CHALCONE SYNTHASE during Fruit Ripening Modifies Tomato Epidermal Cells and Cuticle Properties1[C][W

    PubMed Central

    España, Laura; Heredia-Guerrero, José A.; Reina-Pinto, José J.; Fernández-Muñoz, Rafael; Heredia, Antonio; Domínguez, Eva

    2014-01-01

    Tomato (Solanum lycopersicum) fruit ripening is accompanied by an increase in CHALCONE SYNTHASE (CHS) activity and flavonoid biosynthesis. Flavonoids accumulate in the cuticle, giving its characteristic orange color that contributes to the eventual red color of the ripe fruit. Using virus-induced gene silencing in fruits, we have down-regulated the expression of SlCHS during ripening and compared the cuticles derived from silenced and nonsilenced regions. Silenced regions showed a pink color due to the lack of flavonoids incorporated to the cuticle. This change in color was accompanied by several other changes in the cuticle and epidermis. The epidermal cells displayed a decreased tangential cell width; a decrease in the amount of cuticle and its main components, cutin and polysaccharides, was also observed. Flavonoids dramatically altered the cuticle biomechanical properties by stiffening the elastic and viscoelastic phase and by reducing the ability of the cuticle to deform. There seemed to be a negative relation between SlCHS expression and wax accumulation during ripening that could be related to the decreased cuticle permeability to water observed in the regions silencing SlCHS. A reduction in the overall number of ester linkages present in the cutin matrix was also dependent on the presence of flavonoids. PMID:25277718

  1. AtRabF2b (Ara7) acts on the vacuolar trafficking pathway in tobacco leaf epidermal cells.

    PubMed

    Kotzer, Amanda M; Brandizzi, Federica; Neumann, Ulla; Paris, Nadine; Moore, Ian; Hawes, Chris

    2004-12-15

    Rab GTPases are universal key regulators of intracellular secretory trafficking events. In particular, Rab 5 homologues have been implicated in endocytic events and in the vacuolar pathway. In this study, we investigate the location and function of a member of this family, AtRabF2b (Ara7) in tobacco (Nicotiana tabacum) leaf epidermal cells using a live cell imaging approach. Fluorescent-tagged AtRabF2b[wt] localized to the prevacuolar compartment and Golgi apparatus, as determined by coexpression studies with fluorescent markers for these compartments. Mutations that impair AtRabF2b function also alter the subcellular location of the GTPase. In addition, coexpression studies of the protein with the vacuole-targeted aleurain-green fluorescent protein (GFP) and rescue experiments with wild-type AtRabF2b indicate that the dominant-negative mutant of AtRabF2b causes the vacuolar marker to be secreted to the apoplast. Our results indicate a clear role of AtRabF2b in the vacuolar trafficking pathway.

  2. Single microfilaments mediate the early steps of microtubule bundling during preprophase band formation in onion cotyledon epidermal cells.

    PubMed

    Takeuchi, Miyuki; Karahara, Ichirou; Kajimura, Naoko; Takaoka, Akio; Murata, Kazuyoshi; Misaki, Kazuyo; Yonemura, Shigenobu; Staehelin, L Andrew; Mineyuki, Yoshinobu

    2016-06-01

    The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MTs) in G2 and narrows to demarcate the future division site during late prophase. Studies with fluorescent probes have shown that PPBs contain F-actin during early stages of their development but become actin depleted in late prophase. Although this suggests that actins contribute to the early stages of PPB formation, how actins contribute to PPB-MT organization remains unsolved. To address this question, we used electron tomography to investigate the spatial relationship between microfilaments (MFs) and MTs at different stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the PPB actins observed by fluorescence microscopy correspond to short, single MFs. A majority of the MFs are bound to MTs, with a subset forming MT-MF-MT bridging structures. During the later stages of PPB assembly, the MF-mediated links between MTs are displaced by MT-MT linkers as the PPB MT arrays mature into tightly packed MT bundles. On the basis of these observations, we propose that the primary function of actins during PPB formation is to mediate the initial bundling of the PPB MTs. PMID:27053663

  3. Heparin-Binding Epidermal Growth Factor and Its Receptors Mediate Decidualization and Potentiate Survival of Human Endometrial Stromal Cells

    PubMed Central

    Chobotova, Katya; Karpovich, Natalia; Carver, Janet; Manek, Sanjiv; Gullick, William J.; Barlow, David H.; Mardon, Helen J.

    2006-01-01

    Heparin-binding epidermal growth factor (HB-EGF) has pleiotropic biological functions in many tissues, including those of the female reproductive tract. It facilitates embryo development and mediates implantation and is thought to have a function in endometrial receptivity and maturation. The mature HB-EGF molecule manifests its activity as either a soluble factor (sol-HB-EGF) or a transmembrane precursor (tm-HB-EGF) and can bind two receptors, EGFR and ErbB4/HER4. In this study, we identify factors that modulate expression of HB-EGF, EGFR, and ErbB4 in endometrial stromal cells in vitro. We demonstrate that levels of sol- and tm-HB-EGF, EGFR, and ErbB4 are increased by cAMP, a potent inducer of decidualization of the endometrial stroma. We also show that production of sol- and tm-HB-EGF is differentially modulated by TNFα and TGFβ. Our data suggest that HB-EGF has a function in endometrial maturation in mediating decidualization and attenuating TNFα- and TGFβ-induced apoptosis of endometrial stromal cells. PMID:15562026

  4. Single microfilaments mediate the early steps of microtubule bundling during preprophase band formation in onion cotyledon epidermal cells

    PubMed Central

    Takeuchi, Miyuki; Karahara, Ichirou; Kajimura, Naoko; Takaoka, Akio; Murata, Kazuyoshi; Misaki, Kazuyo; Yonemura, Shigenobu; Staehelin, L. Andrew; Mineyuki, Yoshinobu

    2016-01-01

    The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MTs) in G2 and narrows to demarcate the future division site during late prophase. Studies with fluorescent probes have shown that PPBs contain F-actin during early stages of their development but become actin depleted in late prophase. Although this suggests that actins contribute to the early stages of PPB formation, how actins contribute to PPB-MT organization remains unsolved. To address this question, we used electron tomography to investigate the spatial relationship between microfilaments (MFs) and MTs at different stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the PPB actins observed by fluorescence microscopy correspond to short, single MFs. A majority of the MFs are bound to MTs, with a subset forming MT-MF-MT bridging structures. During the later stages of PPB assembly, the MF-mediated links between MTs are displaced by MT-MT linkers as the PPB MT arrays mature into tightly packed MT bundles. On the basis of these observations, we propose that the primary function of actins during PPB formation is to mediate the initial bundling of the PPB MTs. PMID:27053663

  5. Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

    SciTech Connect

    Ponec, M.; Weerheim, A. ); Havekes, L. ); Boonstra, J. )

    1987-08-01

    The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.

  6. The Mediator Kinase Module Restrains Epidermal Growth Factor Receptor Signaling and Represses Vulval Cell Fate Specification in Caenorhabditis elegans.

    PubMed

    Grants, Jennifer M; Ying, Lisa T L; Yoda, Akinori; You, Charlotte C; Okano, Hideyuki; Sawa, Hitoshi; Taubert, Stefan

    2016-02-01

    Cell signaling pathways that control proliferation and determine cell fates are tightly regulated to prevent developmental anomalies and cancer. Transcription factors and coregulators are important effectors of signaling pathway output, as they regulate downstream gene programs. In Caenorhabditis elegans, several subunits of the Mediator transcriptional coregulator complex promote or inhibit vulva development, but pertinent mechanisms are poorly defined. Here, we show that Mediator's dissociable cyclin dependent kinase 8 (CDK8) module (CKM), consisting of cdk-8, cic-1/Cyclin C, mdt-12/dpy-22, and mdt-13/let-19, is required to inhibit ectopic vulval cell fates downstream of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. cdk-8 inhibits ectopic vulva formation by acting downstream of mpk-1/ERK, cell autonomously in vulval cells, and in a kinase-dependent manner. We also provide evidence that the CKM acts as a corepressor for the Ets-family transcription factor LIN-1, as cdk-8 promotes transcriptional repression by LIN-1. In addition, we find that CKM mutation alters Mediator subunit requirements in vulva development: the mdt-23/sur-2 subunit, which is required for vulva development in wild-type worms, is dispensable for ectopic vulva formation in CKM mutants, which instead display hallmarks of unrestrained Mediator tail module activity. We propose a model whereby the CKM controls EGFR-Ras-ERK transcriptional output by corepressing LIN-1 and by fine tuning Mediator specificity, thus balancing transcriptional repression vs. activation in a critical developmental signaling pathway. Collectively, these data offer an explanation for CKM repression of EGFR signaling output and ectopic vulva formation and provide the first evidence of Mediator CKM-tail module subunit crosstalk in animals. PMID:26715664

  7. Epidermal CD8+ T cells reactive with group A streptococcal antigens in chronic plaque psoriasis.

    PubMed

    Ovigne, J-M; Baker, B S; Davison, S C; Powles, A V; Fry, L

    2002-08-01

    Chronic plaque psoriasis is a T cell mediated disease associated with group A streptococci (GAS). We have previously shown the presence of a psoriasis-specific dermal Th1 subset that recognizes GAS antigens. To assess whether GAS-reactive T cells are also present in lesional epidermis, fresh cell suspensions or T cell lines isolated from lesional epidermis of 33 psoriasis patients were stained for intracellular interferon-gamma after stimulation with GAS antigens. The patients were typed by PCR for HLA-DR7 and HLA-Cw6 expression. A subset of GAS-reactive CD8+ T cells (2.4% +/- 2.4) was found in 14/21 (67%) fresh cell suspensions. A smaller subset of GAS-reactive CD4+ T cells (0.9% +/- 0.9) was found in 13/21 (62%) fresh cell suspensions, which was expanded in the T cell lines. There was a significant inverse correlation between the proportions of GAS-reactive CD4+ and CD8+ T cells in the fresh suspensions (r = -0.48, P = 0.0277). The presence of GAS-reactive CD4+ or CD8+ T cells did not correlate with HLA-DR7 or HLA-Cw6 expression, respectively. This study has demonstrated GAS-reactive CD8+, and to a lesser extent CD4+, T cell subsets in psoriatic epidermis and provides further evidence that GAS antigens may play a role in the pathogenesis of chronic plaque psoriasis.

  8. The RNA–Methyltransferase Misu (NSun2) Poises Epidermal Stem Cells to Differentiate

    PubMed Central

    Blanco, Sandra; Kurowski, Agata; Nichols, Jennifer; Watt, Fiona M.; Benitah, Salvador Aznar; Frye, Michaela

    2011-01-01

    Homeostasis of most adult tissues is maintained by balancing stem cell self-renewal and differentiation, but whether post-transcriptional mechanisms can regulate this process is unknown. Here, we identify that an RNA methyltransferase (Misu/Nsun2) is required to balance stem cell self-renewal and differentiation in skin. In the epidermis, this methyltransferase is found in a defined sub-population of hair follicle stem cells poised to undergo lineage commitment, and its depletion results in enhanced quiescence and aberrant stem cell differentiation. Our results reveal that post-transcriptional RNA methylation can play a previously unappreciated role in controlling stem cell fate. PMID:22144916

  9. Simultaneous blockade of the epidermal growth factor receptor/mammalian target of rapamycin pathway by epidermal growth factor receptor inhibitors and rapamycin results in reduced cell growth and survival in biliary tract cancer cells.

    PubMed

    Herberger, Beata; Berger, Walter; Puhalla, Harald; Schmid, Katharina; Novak, Sabine; Brandstetter, Anita; Pirker, Christine; Gruenberger, Thomas; Filipits, Martin

    2009-06-01

    The prognosis of patients with biliary tract adenocarcinomas (BTA) is still poor due to lack of effective systemic treatment options. Knowledge of the molecular mechanisms involved in the pathogenesis of this disease is of importance for the development of new treatment strategies. We determined the expression of epidermal growth factor receptor (EGFR) and activated mammalian target of rapamycin (p-mTOR) in paraffin-embedded surgical specimens of BTA (n = 89) by immunohistochemistry. Overall survival was analyzed with Cox models adjusted for clinical and pathologic factors. Combined EGFR/p-mTOR expression was significantly associated with relapse-free survival [adjusted hazard ratio for relapse, 2.20; 95% confidence interval (95% CI), 1.45-3.33; P < 0.001] and overall survival (adjusted hazard ratio for death, 2.32; 95% CI, 1.50-3.58; P < 0.001) of the patients. The effect of the EGFR inhibitors erlotinib or cetuximab and the mTOR inhibitor rapamycin on growth and survival of five BTA cell lines was tested in short-term 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and long-term colony formation assays. Simultaneous blockade of EGFR and mTOR in biliary tract cancer cell lines results in a synergistic inhibition of both phosphatidylinositol-3-kinase and mitogen-activated protein kinase pathways, leading to reduced cell growth and survival. These results suggest that combined targeted therapy with EGFR and mTOR inhibitors may potentially benefit patients with BTAs and should be further evaluated in clinical trials.

  10. Regions within a single epidermal cell of Drosophila can be planar polarised independently

    PubMed Central

    Rovira, Miguel; Saavedra, Pedro; Casal, José; Lawrence, Peter A

    2015-01-01

    Planar cell polarity (PCP), the coordinated and consistent orientation of cells in the plane of epithelial sheets, is a fundamental and conserved property of animals and plants. Up to now, the smallest unit expressing PCP has been considered to be an entire single cell. We report that, in the larval epidermis of Drosophila, different subdomains of one cell can have opposite polarities. In larvae, PCP is driven by the Dachsous/Fat system; we show that the polarity of a subdomain within one cell is its response to levels of Dachsous/Fat in the membranes of contacting cells. During larval development, cells rearrange (Saavedra et al., 2014) and when two subdomains of a single cell have different types of neighbouring cells, then these subdomains can become polarised in opposite directions. We conclude that polarisation depends on a local comparison of the amounts of Dachsous and Fat within opposing regions of a cell's membrane. DOI: http://dx.doi.org/10.7554/eLife.06303.001 PMID:25671242

  11. Basal cell carcinoma develops in contact with the epidermal basal cell layer - a three-dimensional morphological study.

    PubMed

    Pirici, Ionica; Ciurea, Marius Eugen; Mîndrilă, Ion; Avrămoiu, Ioan; Pirici, Alexandru; Nicola, Monica Georgiana; Rogoveanu, Otilia Constantina

    2016-01-01

    Basal cell carcinoma is the most common malignant tumor of the skin, and it develops most frequently on the areas of the body that make its treatment and care extremely difficult, especially in cases of neglecting or aggressive growth and invasion. Both typical mild cases as well as locally aggressive tumor types do not tend to metastasize, and it has been postulated that they should share some common biological and morphological features that might explain this behavior. In this study, we have utilized a high-resolution three-dimensional reconstruction technique on pathological samples from 15 cases of common aggressive (fibrosing and adenoid types) and mild (superficial type) basal cell carcinomas, and showed that all these types shared contact points and bridges with the underlying basal cell layer of the epidermis or with the outmost layer of the hair follicle. The connections found had in fact the highest number for fibrosing type (100%), compared to the superficial (85.71%) and adenoid (55%) types. The morphology of the connection bridges was also different, adjacent moderate to abundant inflammatory infiltrate seeming to lead to a loss of basaloid features in these areas. For the adenoid type, tumor islands seemed to be connected also to each other more strongly, forming a common "tumor lace", and while it has been showed that superficial and fibrosing types have higher recurrence risks, all together these data might iterate a connection between the number of bridging points and the biological and clinical manifestation of this skin tumor. PMID:27151694

  12. Auxin Response Gene SlARF3 Plays Multiple Roles in Tomato Development and is Involved in the Formation of Epidermal Cells and Trichomes.

    PubMed

    Zhang, Xiaolan; Yan, Fang; Tang, Yuwei; Yuan, Yujin; Deng, Wei; Li, Zhengguo

    2015-11-01

    The auxin response factor (ARF) genes encode a large family of proteins involved in auxin signaling transduction. SlARF3, a member of the ARF gene family, encodes a protein containing two conserved domains, B3 and ARF, and lacking an Aux/IAA domain. Expression analysis showed that SlARF3 has a particularly high expression level in trichomes. In situ hybridization also detected the SlARF3 transcripts in epidermal pavement cells of leaves. The physiological function of SlARF3 was studied by using the RNA interference (RNAi) strategy. SlARF3-down-regulated plants exhibited decreased density of epidermal pavement cells and obviously reduced density of type I, V and VI trichomes of leaves, which indicates the important role of SlARF3 in the formation of trichomes and epidermal cells in tomato. The number of shoot xylem cells was also decreased in SlARF3-down-regulated lines. Furthermore, RNA-sequencing (RNA-Seq) analysis identified 51 differentially expressed genes (DEGs) belonging to 14 transcription factor (TF) families, such as MYB, bHLH, WD40 and C2H2 zinc finger. Twenty-seven DEGs were involved in the metabolism and signaling transduction of phytohormones, such as auxin, ethylene and gibberellin. These results indicated the important roles of the TFs and hormones in auxin-dependent transcriptional regulation of trichome formation in tomato. Taken together, our results demonstrate that SlARF3 plays an important role in the formation of epidermal cells and trichomes and reveal novel and specific functions for ARFs in tomato developmental processes. PMID:26412778

  13. Differential regulation of epidermal growth factor receptor by hydrogen peroxide and flagellin in cultured lung alveolar epithelial cells.

    PubMed

    Nishi, Hiroyuki; Maeda, Noriko; Izumi, Shunsuke; Higa-Nakamine, Sayomi; Toku, Seikichi; Kakinohana, Manabu; Sugahara, Kazuhiro; Yamamoto, Hideyuki

    2015-02-01

    In previous studies, we found that stimulation of Toll-like receptor 5 (TLR5) by flagellin induced the activation of mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAPK-2) through activation of the p38 MAPK pathway in cultured alveolar epithelial A549 cells. Our studies strongly suggested that MAPKAPK-2 phosphorylated epidermal growth factor receptor (EGFR) at Ser1047. It has been reported that phosphorylation of Ser1047 after treatment with tumor necrosis factor α (TNFα) induced the internalization of EGFR. In the present study, we first found that treatment of A549 cells with hydrogen peroxide induced the activation of MAPKAPK-2 and phosphorylation of EGFR at Ser1047 within 30 min. This was different from flagellin treatment because hydrogen peroxide treatment induced the phosphorylation of EGFR at Tyr1173 as well as Ser1047, indicating the activation of EGFR. We also found that KN93, an inhibitor of CaM kinase II, inhibited the hydrogen peroxide-induced phosphorylation of EGFR at Ser1047 through inhibition of the activation of the p38 MAPK pathway. Furthermore, we examined the internalization of EGFR by three different methods. Flow cytometry with an antibody against the extracellular domain of EGFR and biotinylation of cell surface proteins revealed that flagellin, but not hydrogen peroxide, decreased the amount of cell-surface EGFR. In addition, activation of extracellular signal-regulated kinase by EGF treatment was reduced by flagellin pre-treatment. These results strongly suggested that hydrogen peroxide activated the p38 MAPK pathway via activation of CaM kinase II and that flagellin and hydrogen peroxide regulate the functions of EGFR by different mechanisms.

  14. IL-13 and Epidermal Growth Factor Receptor Have Critical but Distinct Roles in Epithelial Cell Mucin Production

    PubMed Central

    Zhen, Guohua; Park, Sung Woo; Nguyenvu, Louis T.; Rodriguez, Madeleine W.; Barbeau, Rebecca; Paquet, Agnes C.; Erle, David J.

    2007-01-01

    Overproduction of mucus is a central feature of asthma. The cytokine, IL-13, epidermal growth factor receptor (EGFR), and transcription factor, FOXA2, have each been implicated in mucus production, but the mechanistic relationships between these molecules are not yet well understood. To address this, we established a primary normal human bronchial epithelial cell culture system with IL-13–induced mucus production and gene transcript expression changes similar to those seen in vivo in mice. IL-13 did not stimulate release of the EGFR ligand, transforming growth factor (TGF)-α. However, there was constitutive release of TGF-α from normal human bronchial epithelial cells, and inhibition of TGF-α or EGFR reduced both constitutive and IL-13–induced mucin production. Microarray analysis revealed that IL-13 and the EGFR pathway appear to have almost completely independent effects on transcript expression. IL-13 induced a relatively small set of transcripts, including several novel transcripts that might play a role in pathogenesis of allergic airway disease. In contrast, EGFR activity had extensive effects, including altered expression of many transcripts associated with cell metabolism, survival, transcription, and differentiation. One of the few common effects of IL-13 and EGFR signaling was decreased expression of FOXA2, which is known to prevent mucus production. We conclude that the IL-13 and EGFR pathways make critical but quite distinct contributions to gene regulation in airway epithelial cells, and that both pathways affect expression of the key transcription factor, FOXA2, a known regulator of mucus production. PMID:16980555

  15. Integrin-linked kinase regulates the niche of quiescent epidermal stem cells

    PubMed Central

    Morgner, Jessica; Ghatak, Sushmita; Jakobi, Tobias; Dieterich, Christoph; Aumailley, Monique; Wickström, Sara A.

    2015-01-01

    Stem cells reside in specialized niches that are critical for their function. Quiescent hair follicle stem cells (HFSCs) are confined within the bulge niche, but how the molecular composition of the niche regulates stem cell behaviour is poorly understood. Here we show that integrin-linked kinase (ILK) is a key regulator of the bulge extracellular matrix microenvironment, thereby governing the activation and maintenance of HFSCs. ILK mediates deposition of inverse laminin (LN)-332 and LN-511 gradients within the basement membrane (BM) wrapping the hair follicles. The precise BM composition tunes activities of Wnt and transforming growth factor-β pathways and subsequently regulates HFSC activation. Notably, reconstituting an optimal LN microenvironment restores the altered signalling in ILK-deficient cells. Aberrant stem cell activation in ILK-deficient epidermis leads to increased replicative stress, predisposing the tissue to carcinogenesis. Overall, our findings uncover a critical role for the BM niche in regulating stem cell activation and thereby skin homeostasis. PMID:26349061

  16. Transformation of human epidermal cells by transfection with plasmid containing simian virus 40 DNA linked to a neomycin gene is a defined medium

    SciTech Connect

    Su, R.T.; Yen-Chu Chang )

    1989-01-01

    A human epidermal cell culture was transformed by transfection with a recombinant plasmid containing simian virus 40 DNA with a deletion at the origin and an antibiotic (neomycin or G418) marker. A calcium phosphate-mediated DNA transfection method was optimized for introducing exogenous DNA into cells maintained in a fully defined medium. The transformed cells were propagated for more than 200 population doublings and did not appear to go through a crisis period. The growth characteristics of the transformed cells were similar to those found in normal epidermal cells. Transformed cells initially transfected with the recombinant plasmid could be propagated for more than 30 passages. Actively growing cells could then be repeatedly selected from cell populations based upon their neomycin (G418)-resistant phenotype for at least another 30 passages. Simian virus 40 T-antigen and extrachromosomal DNA containing plasmid- and SV40-specific DNA sequences were detected in the transformed cells. Because of their nononcogenic phenotype and defined growth requirements, the transformed cells provide a model for examining structural changes during cell proliferation and differentiation, and for exploring the multistage carcinogenesis of human epithelial cells.

  17. Intradermal injection of an anti-Langerin-HIVGag fusion vaccine targets epidermal Langerhans cells in nonhuman primates and can be tracked in vivo.

    PubMed

    Salabert, Nina; Todorova, Biliana; Martinon, Frédéric; Boisgard, Raphaël; Zurawski, Gerard; Zurawski, Sandra; Dereuddre-Bosquet, Nathalie; Cosma, Antonio; Kortulewski, Thierry; Banchereau, Jacques; Levy, Yves; Le Grand, Roger; Chapon, Catherine

    2016-03-01

    The development of new immunization strategies requires a better understanding of early molecular and cellular events occurring at the site of injection. The skin is particularly rich in immune cells and represents an attractive site for vaccine administration. Here, we specifically targeted vaccine antigens to epidermal Langerhans cells (LCs) using a fusion protein composed of HIV antigens and a monoclonal antibody targeting Langerin. We developed a fluorescence imaging approach to visualize, in vivo, the vaccine-targeted cells. Studies were performed in nonhuman primates (NHPs) because of their relevance as a model to assess human vaccines. We directly demonstrated that in NHPs, intradermally injected anti-Langerin-HIVGag specifically targets epidermal LCs and induces rapid changes in the LC network, including LC activation and migration out of the epidermis. Vaccine targeting of LCs significantly improved anti-HIV immune response without requirement of an adjuvant. Although the co-injection of the TLR-7/8 synthetic ligand, R-848 (resiquimod), with the vaccine, did not enhance significantly the antibody response, it stimulated recruitment of HLA-DR+ inflammatory cells to the site of immunization. This study allowed us to characterize the dynamics of early local events following the injection of a vaccine-targeted epidermal LCs and R-848. PMID:26678013

  18. Mechanical forces drive neuroblast morphogenesis and are required for epidermal closure.

    PubMed

    Wernike, Denise; Chen, Yun; Mastronardi, Karina; Makil, Neetha; Piekny, Alisa

    2016-04-15

    Tissue morphogenesis requires myosin-dependent events such as cell shape changes and migration to be coordinated between cells within a tissue, and/or with cells from other tissues. However, few studies have investigated the simultaneous morphogenesis of multiple tissues in vivo. We found that during Caenorhabditis elegans ventral enclosure, when epidermal cells collectively migrate to cover the ventral surface of the embryo, the underlying neuroblasts (neuronal precursor cells) also undergo morphogenesis. We found that myosin accumulates as foci along the junction-free edges of the ventral epidermal cells to form a ring, whose closure is myosin-dependent. We also observed the accumulation of myosin foci and the adhesion junction proteins E-cadherin and α-catenin in the underlying neuroblasts. Myosin may help to reorganize a subset of neuroblasts into a rosette-like pattern, and decrease their surface area as the overlying epidermal cells constrict. Since myosin is required in the neuroblasts for ventral enclosure, we propose that mechanical forces in the neuroblasts influence constriction of the overlying epidermal cells. In support of this model, disrupting neuroblast cell division or altering their fate influences myosin localization in the overlying epidermal cells. The coordination of myosin-dependent events and forces between cells in different tissues could be a common theme for coordinating morphogenetic events during metazoan development. PMID:26923492

  19. Regulation of epidermal cell fate in Arabidopsis roots: the importance of multiple feedback loops

    PubMed Central

    Schiefelbein, John; Huang, Ling; Zheng, Xiaohua

    2014-01-01

    The specification of distinct cell types in multicellular organisms is accomplished via establishment of differential gene expression. A major question is the nature of the mechanisms that establish this differential expression in time and space. In plants, the formation of the hair and non-hair cell types in the root epidermis has been used as a model to understand regulation of cell specification. Recent findings show surprising complexity in the number and the types of regulatory interactions between the multiple transcription factor genes/proteins influencing root epidermis cell fate. Here, we describe this regulatory network and the importance of the multiple feedback loops for its establishment and maintenance. PMID:24596575

  20. Barley disease susceptibility factor RACB acts in epidermal cell polarity and positioning of the nucleus

    PubMed Central

    Scheler, Björn; Schnepf, Vera; Galgenmüller, Carolina; Ranf, Stefanie; Hückelhoven, Ralph

    2016-01-01

    RHO GTPases are regulators of cell polarity and immunity in eukaryotes. In plants, RHO-like RAC/ROP GTPases are regulators of cell shaping, hormone responses, and responses to microbial pathogens. The barley (Hordeum vulgare L.) RAC/ROP protein RACB is required for full susceptibility to penetration by Blumeria graminis f.sp. hordei (Bgh), the barley powdery mildew fungus. Disease susceptibility factors often control host immune responses. Here we show that RACB does not interfere with early microbe-associated molecular pattern-triggered immune responses such as the oxidative burst or activation of mitogen-activated protein kinases. RACB also supports rather than restricts expression of defence-related genes in barley. Instead, silencing of RACB expression by RNAi leads to defects in cell polarity. In particular, initiation and maintenance of root hair growth and development of stomatal subsidiary cells by asymmetric cell division is affected by silencing expression of RACB. Nucleus migration is a common factor of developmental cell polarity and cell-autonomous interaction with Bgh. RACB is required for positioning of the nucleus near the site of attack from Bgh. We therefore suggest that Bgh profits from RACB’s function in cell polarity rather than from immunity-regulating functions of RACB. PMID:27056842

  1. Barley disease susceptibility factor RACB acts in epidermal cell polarity and positioning of the nucleus.

    PubMed

    Scheler, Björn; Schnepf, Vera; Galgenmüller, Carolina; Ranf, Stefanie; Hückelhoven, Ralph

    2016-05-01

    RHO GTPases are regulators of cell polarity and immunity in eukaryotes. In plants, RHO-like RAC/ROP GTPases are regulators of cell shaping, hormone responses, and responses to microbial pathogens. The barley (Hordeum vulgare L.) RAC/ROP protein RACB is required for full susceptibility to penetration by Blumeria graminis f.sp. hordei (Bgh), the barley powdery mildew fungus. Disease susceptibility factors often control host immune responses. Here we show that RACB does not interfere with early microbe-associated molecular pattern-triggered immune responses such as the oxidative burst or activation of mitogen-activated protein kinases. RACB also supports rather than restricts expression of defence-related genes in barley. Instead, silencing of RACB expression by RNAi leads to defects in cell polarity. In particular, initiation and maintenance of root hair growth and development of stomatal subsidiary cells by asymmetric cell division is affected by silencing expression of RACB. Nucleus migration is a common factor of developmental cell polarity and cell-autonomous interaction with Bgh RACB is required for positioning of the nucleus near the site of attack from Bgh We therefore suggest that Bgh profits from RACB's function in cell polarity rather than from immunity-regulating functions of RACB.

  2. Effects of electromagnetic radiation from a cellular telephone on epidermal Merkel cells.

    PubMed

    Irmak, M Kemal; Oztas, Emin; Yagmurca, Murat; Fadillioglu, Ersin; Bakir, Bilal

    2003-02-01

    The number of reports on the effects induced by electromagnetic radiation (EMR) from cellular telephones in various cellular systems is still increasing. Until now, no satisfactory mechanism has been proposed to explain the biological effects of this radiation except a role suggested for mast cells. Merkel cells may also play a role in the mechanisms of biological effects of EMR. This study was undertaken to investigate the influence of EMR from a cellular telephone (900 MHz) on Merkel cells in rats. A group of rats was exposed to a cellular telephone in speech position for 30 min. Another group of rats was sham-exposed under the same environmental conditions for 30 min. Exposure led to significantly higher exocytotic activity in Merkel cells compared with the sham exposure group. This finding may indicate the possible role of Merkel cells in the pathophysiology of the effects of EMR.

  3. Autocrine epidermal growth factor signaling stimulates directionally persistent mammary epithelial cell migration

    SciTech Connect

    Maheshwari, Gargi; Wiley, H Steven ); Lauffenburger, Douglas A.

    2001-12-24

    Autocrine receptor/ligand signaling loops were first identified in tumor cells, where it was found that transformation of cells resulted in overexpression of certain growth factors leading to unregulated proliferation of the tumor cells (Sporn and Todaro, 1980). However, in the ensuing decades autocrine signaling has been found to operate in numerous physiological situations (Sporn and Roberts, 1992), including wound healing (Tokumaru et al., 2000), angiogenesis (Seghezzi et al., 1998), and tissue organization during development (Wasserman and Freeman, 1998) and reproductive cycles (Xie et al., 1997). Although it is becoming evident that autocrine loops play crucial roles in regulation of cell function within tissue contexts, it is unclear whether their effects on cell responses are different from the effects of the same ligand presented in exogenous or paracrine manner.

  4. Amlexanox Blocks the Interaction between S100A4 and Epidermal Growth Factor and Inhibits Cell Proliferation.

    PubMed

    Cho, Ching Chang; Chou, Ruey-Hwang; Yu, Chin

    2016-01-01

    The human S100A4 protein binds calcium, resulting in a change in its conformation to promote the interaction with its target protein. Human epidermal growth factor (EGF) is the target protein of S100A4 and a critical ligand of the receptor EGFR. The EGF/EGFR system promotes cell survival, differentiation, and growth by activating several signaling pathways. Amlexanox is an anti-inflammatory and anti-allergic drug that is used to treat recurrent aphthous ulcers. In the present study, we determined that amlexanox interacts with S100A4 using heteronuclear single quantum correlation titration. We elucidated the interactions of S100A4 with EGF and amlexanox using fluorescence and nuclear magnetic resonance spectroscopy. We generated two binary models (for the S100A4-EGF and S100A4-amlexanox complexes) and observed that amlexanox and EGF share a similar binding region in mS100A4. We also used a WST-1 assay to investigate the bioactivity of S100A4, EGF, and amlexanox, and found that amlexanox blocks the binding between S100A4 and EGF, and is therefore useful for the development of new anti-proliferation drugs. PMID:27559743

  5. Epidermal growth factor receptor tyrosine kinase inhibitors with conventional chemotherapy for the treatment of non-small cell lung cancer

    PubMed Central

    Gao, Yuan; Song, PingPing; Li, Hui; Guo, HongBo; Jia, Hui; Zhang, BaiJiang

    2016-01-01

    We report a Chinese male patient with advanced stage lung squamous cell carcinoma who developed brain metastases after responding to treatment comprising six cycles of conventional chemotherapy with docetaxel and cisplatin. The patient was then treated with oral erlotinib (150 mg/day) and whole-brain radiation therapy followed by four cycles of docetaxel and carboplatin chemotherapy. The patient then received gefitinib (250 mg/day) as a maintenance therapy until the end of the follow-up period. In this patient, progression-free survival, defined as the interval from the initiation of first-line chemotherapy to the cessation of erlotinib due to progressive disease or death from any cause, was 3 months. Overall survival, defined as the interval from the initiation of first-line chemotherapy to death from any cause, was 75 months. Erlotinib was well tolerated in combination with whole-brain radiation therapy and a favorable objective response rate was observed. Furthermore, targeted drug treatment warrants consideration in patients with a negative epidermal growth factor receptor mutation status and male patients with a history of smoking. PMID:26719713

  6. Golgi- and trans-Golgi network-mediated vesicle trafficking is required for wax secretion from epidermal cells.

    PubMed

    McFarlane, Heather E; Watanabe, Yoichiro; Yang, Weili; Huang, Yan; Ohlrogge, John; Samuels, A Lacey

    2014-03-01

    Lipid secretion from epidermal cells to the plant surface is essential to create the protective plant cuticle. Cuticular waxes are unusual secretory products, consisting of a variety of highly hydrophobic compounds including saturated very-long-chain alkanes, ketones, and alcohols. These compounds are synthesized in the endoplasmic reticulum (ER) but must be trafficked to the plasma membrane for export by ATP-binding cassette transporters. To test the hypothesis that wax components are trafficked via the endomembrane system and packaged in Golgi-derived secretory vesicles, Arabidopsis (Arabidopsis thaliana) stem wax secretion was assayed in a series of vesicle-trafficking mutants, including gnom like1-1 (gnl1-1), transport particle protein subunit120-4, and echidna (ech). Wax secretion was dependent upon GNL1 and ECH. Independent of secretion phenotypes, mutants with altered ER morphology also had decreased wax biosynthesis phenotypes, implying that the biosynthetic capacity of the ER is closely related to its structure. These results provide genetic evidence that wax export requires GNL1- and ECH-dependent endomembrane vesicle trafficking to deliver cargo to plasma membrane-localized ATP-binding cassette transporters. PMID:24468625

  7. Amlexanox Blocks the Interaction between S100A4 and Epidermal Growth Factor and Inhibits Cell Proliferation

    PubMed Central

    Cho, Ching Chang; Chou, Ruey-Hwang; Yu, Chin

    2016-01-01

    The human S100A4 protein binds calcium, resulting in a change in its conformation to promote the interaction with its target protein. Human epidermal growth factor (EGF) is the target protein of S100A4 and a critical ligand of the receptor EGFR. The EGF/EGFR system promotes cell survival, differentiation, and growth by activating several signaling pathways. Amlexanox is an anti-inflammatory and anti-allergic drug that is used to treat recurrent aphthous ulcers. In the present study, we determined that amlexanox interacts with S100A4 using heteronuclear single quantum correlation titration. We elucidated the interactions of S100A4 with EGF and amlexanox using fluorescence and nuclear magnetic resonance spectroscopy. We generated two binary models (for the S100A4-EGF and S100A4-amlexanox complexes) and observed that amlexanox and EGF share a similar binding region in mS100A4. We also used a WST-1 assay to investigate the bioactivity of S100A4, EGF, and amlexanox, and found that amlexanox blocks the binding between S100A4 and EGF, and is therefore useful for the development of new anti-proliferation drugs. PMID:27559743

  8. Fermented milk containing Lactobacillus GG alleviated DSS-induced colitis in mice and activated epidermal growth factor receptor and Akt signaling in intestinal epithelial cells.

    PubMed

    Yoda, Kazutoyo; He, Fang; Miyazawa, Kenji; Hiramatsu, Masaru; Yan, Fang

    2012-01-01

    Lactobacillus rhamnosus GG was assessed for its ability to alleviate DSS-induced colitis in mice and activate epidermal growth factor receptor and Akt signaling in intestinal epithelial cells. In this study mice were treated with DSS to induce colitis and they were given Lactobacillus GG fermented milk to assess the effect of probiotic on colitis. Lactobacillus GG fermented milk significantly reduced the colitis associated changes suggesting a protective effect against DSS induced colitis.

  9. Mn-doped Zinc Sulphide nanocrystals for immunofluorescent labeling of epidermal growth factor receptors on cells and clinical tumor tissues

    NASA Astrophysics Data System (ADS)

    J, Aswathy; V, Seethalekshmy N.; R, Hiran K.; R, Bindhu M.; K, Manzoor; Nair, Shantikumar V.; Menon, Deepthy

    2014-11-01

    The field of molecular detection and targeted imaging has evolved considerably with the introduction of fluorescent semiconductor nanocrystals. Manganese-doped zinc sulphide nanocrystals (ZnS:Mn NCs), which are widely used in electroluminescent displays, have been explored for the first time for direct immunofluorescent (IF) labeling of clinical tumor tissues. ZnS:Mn NCs developed through a facile wet chemistry route were capped using amino acid cysteine, conjugated to streptavidin and thereafter coupled to biotinylated epidermal growth factor receptor (EGFR) antibody utilizing the streptavidin-biotin linkage. The overall conjugation yielded stable EGFR antibody conjugated ZnS:Mn NCs (EGFR ZnS:Mn NCs) with a hydrodynamic diameter of 65 ± 15 nm, and having an intense orange-red fluorescence emission at 598 nm. Specific labeling of EGF receptors on EGFR+ve A431 cells in a co-culture with EGFR-ve NIH3T3 cells was demonstrated using these nanoprobes. The primary antibody conjugated fluorescent NCs could also clearly delineate EGFR over-expressing cells on clinical tumor tissues processed by formalin fixation as well as cryopreservation with a specificity of 86% and accuracy of 88%, in comparison to immunohistochemistry. Tumor tissues labeled with EGFR ZnS:Mn NCs showed good fluorescence emission when imaged after storage even at 15 months. Thus, ZnS nanobioconjugates with dopant-dependent and stable fluorescence emission show promise as an efficient, target-specific fluorophore that would enable long term IF labeling of any antigen of interest on clinical tissues.

  10. Strain differences in the expression of an H-2K/sup k/ gene product by epidermal and spleen cells

    SciTech Connect

    Hadley, G.A.; Steinmuller, D.

    1986-03-01

    Cytotoxic T lymphocytes (CTL) directed against Epa-1, a non-H-2 alloantigen expressed by epidermal cells (EC) but no lymphoid cells, lyse EC of different H-2/sup k/, Epa-1/sup +/ strains at different levels. For example, the mean percent lysis values for EC of strains CBA, AKR, C58, and RF are 60, 46, 41, and 35 respectively. Since the CTL used to obtain these values recognize Epa-1 only in the context of H-2K/sup k/, the different levels of lysis could reflect differences in either Epa-1 or K/sup k/ antigens. The goal of this investigation was to test the second alternative. For this purpose, the authors obtained hybridoma 16-1-11N that secretes a K/sup k/-specific MoAb. They first demonstrated the capacity of MoAb 16-1-11N to block the lysis of CBA EC by Epa-1-specific CTL. They then utilized it as the probe in a cellar RIA, with /sup 125/I-protein A as the second reagent, to quantitate the expression of K/sup k/ antigens on EC of strains CBA, AKR, C58, and RF. They found that C58 and RF EC bound significantly less of the K/sup k/ MoAb than CBA EC. Although AKR EC also bound less of the MoAb than CBA EC, the difference was not significant. Nonetheless, these data support the hypothesis that the differential susceptibility of the strains to lysis by Epa-1-specific CTL is due to differences in the expression of the H-2 restricting element. The authors also tested spleen cells (SC) of the four strains in the RIA described above and found that SC of RF, but not of C58 or AKR, express reduced levels of K/sup k/ antigens compared to CBA SC.

  11. Platelet-activating factor induces ovine fetal pulmonary venous smooth muscle cell proliferation: role of epidermal growth factor receptor transactivation.

    PubMed

    Zhou, Weilin; Ibe, Basil O; Raj, J Usha

    2007-06-01

    We have previously reported that platelet-activating factor (PAF) is present in very high levels in the ovine fetal lung and circulation and that PAF serves as an important physiological vasoconstrictor of the pulmonary circulation in utero. However, it is not known whether PAF stimulates pulmonary vascular smooth muscle cell (SMC) proliferation. In this study, we used ovine fetal pulmonary venous SMCs as our model system to study the effects and mechanisms of action of PAF on SMC proliferation. We found that PAF induced SMC proliferation in a dose-dependent manner. PAF also stimulated activation of both ERK and p38 but not c-Jun NH(2) terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. PAF (10 nM) induced phosphorylation of epidermal growth factor receptor (EGFR). Specific inhibition of EGFR by AG-1478 and by the expression of a dominant-negative EGFR mutant in SMCs attenuated PAF-stimulated cell proliferation. Inhibition of heparin-binding EGF-like growth factor (HB-EGF) release by CRM-197 and inhibition of matrix metalloproteinases (MMP) by GM-6001 abolished PAF-induced MAP kinase activation and cell proliferation. Increased alkaline phosphatase (AP) activity after PAF treatment in AP-HB-EGF fusion construct-transfected SMCs indicated that PAF induced the release of HB-EGF within 1 min. Gelatin zymography data showed that PAF stimulated MMP-2 activity and MMP-9 activity within 1 min. These results suggest that PAF promotes pulmonary vascular SMC proliferation via transactivation of EGFR through MMP activation and HB-EGF, resulting in p38 and ERK activation and that EGFR transactivation is essential for the mitogenic effect of PAF in pulmonary venous SMC. PMID:17322418

  12. Antiestrogen fulvestrant enhances the antiproliferative effects of epidermal growth factor receptor inhibitors in human non-small cell lung cancer

    PubMed Central

    Garon, Edward B.; Pietras, Richard J.; Finn, Richard S.; Kamranpour, Naeimeh; Pitts, Sharon; Márquez-Garbán, Diana C.; Desai, Amrita J.; Dering, Judy; Hosmer, Wylie; von Euw, Erika M.; Dubinett, Steven M.; Slamon, Dennis J.

    2012-01-01

    Introduction Estrogen receptor (ER) signaling and its interaction with epidermal growth factor receptor (EGFR) is a potential therapeutic target in non-small cell lung cancer (NSCLC). To explore cross-communication between ER and EGFR, we have correlated ER pathway gene and protein expression profiles and examined effects of antiestrogens with or without EGFR inhibitors in preclinical models of human NSCLC. Methods We evaluated 54 NSCLC cell lines for growth inhibition with EGFR inhibitors, antiestrogen treatment or the combination. Each line was evaluated for baseline ER pathway protein expression. The majority were also evaluated for baseline ER pathway gene expression. Human NSCLC xenografts were evaluated for effects of inhibition of each pathway either individually or in combination. Results The specific antiestrogen fulvestrant has modest single agent activity in vitro, but in many lines fulvestrant adds to effects of EGFR inhibitors, including synergy in the EGFR mutant, erlotinib-resistant H1975 line. ERα, ERβ, progesterone receptor (PR)-A, PR-B and aromatase proteins are expressed in all lines to varying degrees, with trends towards lower aromatase in more sensitive cell lines. Sensitivity to fulvestrant correlates with greater baseline ERα gene expression. Tumor stability is achieved in human tumor xenografts with either fulvestrant or EGFR inhibitors, but tumors regress significantly when both pathways are inhibited. Conclusions These data provide a rationale for further investigation of the antitumor activity of combined therapy with antiestrogen and anti-EGFR agents in the clinic. Future work should also evaluate dual ER and EGFR inhibition in the setting of secondary resistance to EGFR inhibition. PMID:23399957

  13. Human epidermal keratinocyte cell response on integrin-specific artificial extracellular matrix proteins.

    PubMed

    Tjin, Monica Suryana; Chua, Alvin Wen Choong; Ma, Dong Rui; Lee, Seng Teik; Fong, Eileen

    2014-08-01

    Cell-matrix interactions play critical roles in regulating cellular behavior in wound repair and regeneration of the human skin. In particular, human skin keratinocytes express several key integrins such as alpha5beta1, alpha3beta1, and alpha2beta1 for binding to the extracellular matrix (ECM) present in the basement membrane in uninjured skin. To mimic these key integrin-ECM interactions, artificial ECM (aECM) proteins containing functional domains derived from laminin 5, type IV collagen, fibronectin, and elastin are prepared. Human skin keratinocyte cell responses on the aECM proteins are specific to the cell-binding domain present in each construct. Keratinocyte attachment to the aECM protein substrates is also mediated by specific integrin-material interactions. In addition, the aECM proteins are able to support the proliferation of keratinocyte stem cells, demonstrating their promise for use in skin tissue engineering.

  14. Proton Irradiation Sensitizes Radioresistant Non-small Cell Lung Cancer Cells by Modulating Epidermal Growth Factor Receptor-mediated DNA Repair.

    PubMed

    Park, Hyung Ju; Oh, Jeong Su; Chang, Jong Wook; Hwang, Sang-Gu; Kim, Jae-Sung

    2016-01-01

    Although proton radiotherapy is effective in treating various types of cancer, little is known on the biological responses triggered by proton irradiation. In the present study, we investigated protein profiles following proton irradiation of non-small cell lung cancer (NSCLC) cells and defined the role of proton-induced epidermal growth factor receptor (EGFR) expression in NSCLC cells. We found that proton irradiation more effectively sensitized NSCLC cells than gamma irradiation did. The expression profiles of radiosensitive and radioresistant NSCLC cells following proton and gamma irradiation were examined using antibody arrays. With regard to proteins, expression of EGFR was the most highly induced by proton irradiation. In addition, we found that EGFR inhibition with gefinitib significantly increased the radiosensitivity of NSCLC cells, and that increased radiosensitivity due to gefinitib was mediated by the suppression of DNA repair in radioresistant NSCLC cells. Thus, our data provide the first evidence that proton irradiation sensitizes radioresistant NSCLC cancer cells by modulating EGFR-mediated DNA repair.

  15. Blockade of Hedgehog Signaling Synergistically Increases Sensitivity to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Cancer Cell Lines.

    PubMed

    Bai, Xiao-Yan; Zhang, Xu-Chao; Yang, Su-Qing; An, She-Juan; Chen, Zhi-Hong; Su, Jian; Xie, Zhi; Gou, Lan-Ying; Wu, Yi-Long

    2016-01-01

    Aberrant activation of the hedgehog (Hh) signaling pathway has been implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human cancer. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells, while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells, accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression, resulting in gefitinib tolerance (P < 0.001) in EGFR-TKI-sensitive cells. Blockade of the Hh signaling pathway using the SMO antagonist SANT-1 restored E-cadherin expression and downregulate Snail and ABCG2 in EGFR-TKI-resistant cells. A combination of SANT-1 and gefitinib markedly inhibited tumorigenesis and proliferation in EGFR-TKI-resistant cells (P < 0.001). These findings indicate that hyperactivity of Hh signaling resulted in EGFR-TKI resistance, by EMT introduction and ABCG2 upregulation, and blockade of Hh signaling synergistically increased sensitivity to EGFR-TKIs in primary and secondary resistant NSCLC cells. E-cadherin expression may be a potential biomarker of the suitability of the combined application of an Hh inhibitor and EGFR-TKIs in EGFR-TKI-resistant NSCLCs. PMID:26943330

  16. Epidermal stem cells and skin tissue engineering in hair follicle regeneration.

    PubMed

    Balañá, María Eugenia; Charreau, Hernán Eduardo; Leirós, Gustavo José

    2015-05-26

    The reconstitution of a fully organized and functional hair follicle from dissociated cells propagated under defined tissue culture conditions is a challenge still pending in tissue engineering. The loss of hair follicles caused by injuries or pathologies such as alopecia not only affects the patients' psychological well-being, but also endangers certain inherent functions of the skin. It is then of great interest to find different strategies aiming to regenerate or neogenerate the hair follicle under conditions proper of an adult individual. Based upon current knowledge on the epithelial and dermal cells and their interactions during the embryonic hair generation and adult hair cycling, many researchers have tried to obtain mature hair follicles using different strategies and approaches depending on the causes of hair loss. This review summarizes current advances in the different experimental strategies to regenerate or neogenerate hair follicles, with emphasis on those involving neogenesis of hair follicles in adult individuals using isolated cells and tissue engineering. Most of these experiments were performed using rodent cells, particularly from embryonic or newborn origin. However, no successful strategy to generate human hair follicles from adult cells has yet been reported. This review identifies several issues that should be considered to achieve this objective. Perhaps the most important challenge is to provide three-dimensional culture conditions mimicking the structure of living tissue. Improving culture conditions that allow the expansion of specific cells while protecting their inductive properties, as well as methods for selecting populations of epithelial stem cells, should give us the necessary tools to overcome the difficulties that constrain human hair follicle neogenesis. An analysis of patent trends shows that the number of patent applications aimed at hair follicle regeneration and neogenesis has been increasing during the last decade. This

  17. Epidermal stem cells and skin tissue engineering in hair follicle regeneration

    PubMed Central

    Balañá, María Eugenia; Charreau, Hernán Eduardo; Leirós, Gustavo José

    2015-01-01

    The reconstitution of a fully organized and functional hair follicle from dissociated cells propagated under defined tissue culture conditions is a challenge still pending in tissue engineering. The loss of hair follicles caused by injuries or pathologies such as alopecia not only affects the patients’ psychological well-being, but also endangers certain inherent functions of the skin. It is then of great interest to find different strategies aiming to regenerate or neogenerate the hair follicle under conditions proper of an adult individual. Based upon current knowledge on the epithelial and dermal cells and their interactions during the embryonic hair generation and adult hair cycling, many researchers have tried to obtain mature hair follicles using different strategies and approaches depending on the causes of hair loss. This review summarizes current advances in the different experimental strategies to regenerate or neogenerate hair follicles, with emphasis on those involving neogenesis of hair follicles in adult individuals using isolated cells and tissue engineering. Most of these experiments were performed using rodent cells, particularly from embryonic or newborn origin. However, no successful strategy to generate human hair follicles from adult cells has yet been reported. This review identifies several issues that should be considered to achieve this objective. Perhaps the most important challenge is to provide three-dimensional culture conditions mimicking the structure of living tissue. Improving culture conditions that allow the expansion of specific cells while protecting their inductive properties, as well as methods for selecting populations of epithelial stem cells, should give us the necessary tools to overcome the difficulties that constrain human hair follicle neogenesis. An analysis of patent trends shows that the number of patent applications aimed at hair follicle regeneration and neogenesis has been increasing during the last decade. This

  18. Epidermal stem cells and skin tissue engineering in hair follicle regeneration.

    PubMed

    Balañá, María Eugenia; Charreau, Hernán Eduardo; Leirós, Gustavo José

    2015-05-26

    The reconstitution of a fully organized and functional hair follicle from dissociated cells propagated under defined tissue culture conditions is a challenge still pending in tissue engineering. The loss of hair follicles caused by injuries or pathologies such as alopecia not only affects the patients' psychological well-being, but also endangers certain inherent functions of the skin. It is then of great interest to find different strategies aiming to regenerate or neogenerate the hair follicle under conditions proper of an adult individual. Based upon current knowledge on the epithelial and dermal cells and their interactions during the embryonic hair generation and adult hair cycling, many researchers have tried to obtain mature hair follicles using different strategies and approaches depending on the causes of hair loss. This review summarizes current advances in the different experimental strategies to regenerate or neogenerate hair follicles, with emphasis on those involving neogenesis of hair follicles in adult individuals using isolated cells and tissue engineering. Most of these experiments were performed using rodent cells, particularly from embryonic or newborn origin. However, no successful strategy to generate human hair follicles from adult cells has yet been reported. This review identifies several issues that should be considered to achieve this objective. Perhaps the most important challenge is to provide three-dimensional culture conditions mimicking the structure of living tissue. Improving culture conditions that allow the expansion of specific cells while protecting their inductive properties, as well as methods for selecting populations of epithelial stem cells, should give us the necessary tools to overcome the difficulties that constrain human hair follicle neogenesis. An analysis of patent trends shows that the number of patent applications aimed at hair follicle regeneration and neogenesis has been increasing during the last decade. This

  19. Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition

    PubMed Central

    Hata, Aaron N; Niederst, Matthew J; Archibald, Hannah L; Gomez-Caraballo, Maria; Siddiqui, Faria M; Mulvey, Hillary E; Maruvka, Yosef E; Ji, Fei; Bhang, Hyo-eun C; Radhakrishna, Viveksagar Krishnamurthy; Siravegna, Giulia; Hu, Haichuan; Raoof, Sana; Lockerman, Elizabeth; Kalsy, Anuj; Lee, Dana; Keating, Celina L; Ruddy, David A; Damon, Leah J; Crystal, Adam S; Costa, Carlotta; Piotrowska, Zofia; Bardelli, Alberto; Iafrate, Anthony J; Sadreyev, Ruslan I; Stegmeier, Frank; Getz, Gad; Sequist, Lecia V; Faber, Anthony C; Engelman, Jeffrey A

    2016-01-01

    Although mechanisms of acquired resistance of EGFR mutant non-small cell lung cancers to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. Here, we observe that acquired resistance caused by the T790M gatekeeper mutation can occur either by selection of pre-existing T790M clones or via genetic evolution of initially T790M-negative drug tolerant cells. The path to resistance impacts the biology of the resistant clone, as those that evolved from drug tolerant cells had a diminished apoptotic response to third generation EGFR inhibitors that target T790M EGFR; treatment with navitoclax, an inhibitor of BCL-XL and BCL-2 restored sensitivity. We corroborated these findings using cultures derived directly from EGFR inhibitor-resistant patient tumors. These findings provide evidence that clinically relevant drug resistant cancer cells can both pre-exist and evolve from drug tolerant cells, and point to therapeutic opportunities to prevent or overcome resistance in the clinic. PMID:26828195

  20. Non-small-cell lung cancer cells combat epidermal growth factor receptor tyrosine kinase inhibition through immediate adhesion-related responses

    PubMed Central

    Wang, Hsian-Yu; Hsu, Min-Kung; Wang, Kai-Hsuan; Tseng, Ching-Ping; Chen, Feng-Chi; Hsu, John T-A

    2016-01-01

    Background Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib, and afatinib, have greatly improved treatment efficacy in non-small cell lung cancer (NSCLC) patients with drug-sensitive EGFR mutations. However, in some TKI responders, the benefits of such targeted therapies are limited by the rapid development of resistance, and strategies to overcome this resistance are urgently needed. Studies of drug resistance in cancer cells typically involve long term in vitro induction to obtain stably acquired drug-resistant cells followed by elucidation of resistance mechanisms, but the immediate responses of cancer cells upon drug treatment have been ignored. The aim of this study was to investigate the immediate responses of NSCLC cells upon treatment with EGFR TKIs. Results Both NSCLC cells, ie, PC9 and H1975, showed immediate enhanced adhesion-related responses as an apoptosis-countering mechanism upon first-time TKI treatment. By gene expression and pathway analysis, adhesion-related pathways were enriched in gefitinib-treated PC9 cells. Pathway inhibition by small-hairpin RNAs or small-molecule drugs revealed that within hours of EGFR TKI treatment, NSCLC cells used adhesion-related responses to combat the drugs. Importantly, we show here that the Src family inhibitor, dasatinib, dramatically inhibits cell adhesion-related response and greatly enhances the cell-killing effects of EGFR TKI (gefitinib for the PC9 cells; afatinib for the H1975 cells) in NSCLC cells, which would otherwise escape the TKI-induced apoptosis. Conclusion Results from this study indicate that NSCLC cells can employ the adhesion response as a survival pathway to survive under EGFR-targeted therapy. Simultaneous targeting of EGFR signaling and adhesion pathways would further boost the efficacy of EGFR-targeted therapy in NSCLC. PMID:27284246

  1. Targeting Cancer Stem Cell Plasticity Through Modulation of Epidermal Growth Factor and Insulin-Like Growth Factor Receptor Signaling in Head and Neck Squamous Cell Cancer

    PubMed Central

    Leong, Hui Sun; Chong, Fui Teen; Sew, Pui Hoon; Lau, Dawn P.; Wong, Bernice H.; Teh, Bin-Tean

    2014-01-01

    Emerging data suggest that cancer stem cells (CSCs) exist in equilibrium with differentiated cells and that stochastic transitions between these states can account for tumor heterogeneity and drug resistance. The aim of this study was to establish an in vitro system that recapitulates stem cell plasticity in head and neck squamous cell cancers (HNSCCs) and identify the factors that play a role in the maintenance and repopulation of CSCs. Tumor spheres were established using patient-derived cell lines via anchorage-independent cell culture techniques. These tumor spheres were found to have higher aldehyde dehydrogenase (ALD) cell fractions and increased expression of Kruppel-like factor 4, SRY (sex determining region Y)-box 2, and Nanog and were resistant to γ-radiation, 5-fluorouracil, cisplatin, and etoposide treatment compared with monolayer culture cells. Monolayer cultures were subject to single cell cloning to generate clones with high and low ALD fractions. ALDHigh clones showed higher expression of stem cell and epithelial-mesenchymal transition markers compared with ALDLow clones. ALD fractions, representing stem cell fractions, fluctuated with serial passaging, equilibrating at a level specific to each cell line, and could be augmented by the addition of epidermal growth factor (EGF) and/or insulin. ALDHigh clones showed increased EGF receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation, with increased activation of downstream pathways compared with ALDLow clones. Importantly, blocking these pathways using specific inhibitors against EGFR and IGF-1R reduced stem cell fractions drastically. Taken together, these results show that HNSCC CSCs exhibit plasticity, with the maintenance of the stem cell fraction dependent on the EGFR and IGF-1R pathways and potentially amenable to targeted therapeutics. PMID:25024430

  2. Action of staphylococcal exfoliative toxins on epidermal cell cultures and organotypic skin.

    PubMed

    Gentilhomme, E; Faure, M; Piemont, Y; Binder, P; Thivolet, J

    1990-09-01

    In the staphylococcal scalded skin syndrome, spontaneous intraepithelial cleavages are due to the exfoliative toxins A or B (ETA or ETB). Until now, these toxins have been studied either on epidermis or on organotypic skin cultures. In the present study, we compare the effects of these toxins on human keratinocyte cell cultures to those on human and mouse organotypic skin cultures. With concentrations of ETA or ETB of 1 mg/ml for 3 hours, spontaneous intraepithelial cleavages were noted in both cell and organotypic cultures. Keratinocyte cell cultures were as sensitive as organotypic skin cultures to these toxins. Since keratohyaline granules may represent a possible binding site for ETA or ETB, we tried to correlate the expression of keratohyaline granules with the appearance of intraepithelial clefts due to the toxins. However, when cultured in liquid medium, epithelia were not differentiated enough to allow the detection of the binding site of ETA-ETB. PMID:1703553

  3. Epidermal growth factor: Porcine uterine luminal epithelial cell migratory signal during the peri-implantation period of pregnancy.

    PubMed

    Jeong, Wooyoung; Jung, Seoungo; Bazer, Fuller W; Song, Gwonhwa; Kim, Jinyoung

    2016-01-15

    The majority of early conceptus mortality in pregnancy occurs during the peri-implantation period, suggesting that this period is important for conceptus viability and the establishment of pregnancy. Successful establishment of pregnancy in all mammalian species depends on the orchestrated molecular events that transpire at the conceptus-uterine interface during the peri-implantation period of pregnancy. This maternal-conceptus interaction is especially crucial in pigs because they have a non-invasive epitheliochorial placentation during a protracted peri-implantation period. During the pre-implantation period of pregnancy, conceptus survival and the establishment of pregnancy depend on the developing conceptus receiving an adequate supply of histotroph which contains a wide range of nutrients and growth factors. Evidence links epidermal growth factor (EGF) to embryogenesis or implantation in various mammalian species. EGF exhibits potential growth-promoting activities on the conceptus and endometrium; however, in the case of pigs, little is known its functions, especially their regulatory mechanisms at the maternal-conceptus interface. EGF receptor (EGFR) mRNA and protein are abundant in endometrial luminal (LE) and glandular (GE) epithelia and conceptus trophectoderm on Days 13-14 of pregnancy, suggesting that EGF provides an autocrine signal to uterine LE and GE just prior to implantation. Therefore, the objectives of this study were to determine: 1) the potential intracellular signaling pathways responsible for the activities of EGF in porcine uterine LE (pLE) cells; and 2) the changes in cellular activities induced by EGF. EGF treatment of pLE cells increased the abundance of phosphorylated (p)-ERK1/2, p-P70RSK and p-RPS6 compared to that for control cells. Furthermore, EGF-stimulated phosphorylation of ERK1/2 MAPK was inhibited in pLE cells transfected with an EGFR siRNA compared with control siRNA-transfected pLE cells. Moreover, EGF stimulated migration of

  4. 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

    SciTech Connect

    Kasten-Pisula, Ulla; Saker, Jarob; Eicheler, Wolfgang; Krause, Mechthild; Yaromina, Ala; Meyer-Staeckling, Soenke; Scherkl, Benjamin; Kriegs, Malte; Brandt, Burkhard; Grenman, Reidar; Petersen, Cordula; Baumann, Michael; Dikomey, Ekkehard

    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 blot 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.

  5. Blockade of Ets-1 attenuates epidermal growth factor-dependent collagen loss in human carotid plaque smooth muscle cells.

    PubMed

    Rao, Velidi H; Rai, Vikrant; Stoupa, Samantha; Agrawal, Devendra K

    2015-09-15

    Although degradation of extracellular matrix by matrix metalloproteinases (MMPs) is thought to be involved in symptomatic (S) carotid plaques in atherosclerosis, the mechanisms of MMP expression are poorly understood. Here, we demonstrate that collagen loss in vascular smooth vessel cells (VSMCs) isolated from S plaques was induced by epidermal growth factor (EGF) through the activation of p38-MAPK and JNK-MAPK pathways. Inhibitors of p38-MAPK and JNK-MAPK signaling pathways downregulated the expression of MMP-1 and MMP-9. In addition, we examined whether v-ets erythroblastosis virus E26 oncogene homologue 1 (Ets-1), an important regulator of different genes, is involved in destabilizing S plaques in patients with carotid stenosis. We demonstrate that EGF induces Ets-1 expression and decreases interstitial and basement membrane collagen in vascular smooth muscle cells (VSMCs) from patients with carotid stenosis. Increased expression of MMP-1 and -9 and decreased collagen mRNA transcripts were also found in Ets-1-overexpressed VSMCs. Transfection with both dominant-negative form of Ets-1 and small interfering RNA blocked EGF-induced MMP-1 and -9 expressions and increased the mRNA transcripts for collagen I (α1) and collagen III (α1) in S compared with asymptomatic (AS) carotid plaques. Inhibitors of p38-MAPK (SB202190) and JNK-MAPK (SP600125) signaling pathways decreased the expression of Ets-1, MMP-1, and MMP-9 and increased collagen type I and III expression in EGF-treated VSMCs. This study provides a mechanistic insight into the role of Ets-1 in the plaque destabilization in patients with carotid stenosis involving p38-MAPK and JNK signaling pathways.

  6. Arabidopsis Actin Depolymerizing Factor4 Modulates the Stochastic Dynamic Behavior of Actin Filaments in the Cortical Array of Epidermal Cells[C][W

    PubMed Central

    Henty, Jessica L.; Bledsoe, Samuel W.; Khurana, Parul; Meagher, Richard B.; Day, Brad; Blanchoin, Laurent; Staiger, Christopher J.

    2011-01-01

    Actin filament arrays are constantly remodeled as the needs of cells change as well as during responses to biotic and abiotic stimuli. Previous studies demonstrate that many single actin filaments in the cortical array of living Arabidopsis thaliana epidermal cells undergo stochastic dynamics, a combination of rapid growth balanced by disassembly from prolific severing activity. Filament turnover and dynamics are well understood from in vitro biochemical analyses and simple reconstituted systems. However, the identification in living cells of the molecular players involved in controlling actin dynamics awaits the use of model systems, especially ones where the power of genetics can be combined with imaging of individual actin filaments at high spatial and temporal resolution. Here, we test the hypothesis that actin depolymerizing factor (ADF)/cofilin contributes to stochastic filament severing and facilitates actin turnover. A knockout mutant for Arabidopsis ADF4 has longer hypocotyls and epidermal cells when compared with wild-type seedlings. This correlates with a change in actin filament architecture; cytoskeletal arrays in adf4 cells are significantly more bundled and less dense than in wild-type cells. Several parameters of single actin filament turnover are also altered. Notably, adf4 mutant cells have a 2.5-fold reduced severing frequency as well as significantly increased actin filament lengths and lifetimes. Thus, we provide evidence that ADF4 contributes to the stochastic dynamic turnover of actin filaments in plant cells. PMID:22010035

  7. In Vivo Quantification of Peroxisome Tethering to Chloroplasts in Tobacco Epidermal Cells Using Optical Tweezers.

    PubMed

    Gao, Hongbo; Metz, Jeremy; Teanby, Nick A; Ward, Andy D; Botchway, Stanley W; Coles, Benjamin; Pollard, Mark R; Sparkes, Imogen

    2016-01-01

    Peroxisomes are highly motile organelles that display a range of motions within a short time frame. In static snapshots, they can be juxtaposed to chloroplasts, which has led to the hypothesis that they are physically interacting. Here, using optical tweezers, we tested the dynamic physical interaction in vivo. Using near-infrared optical tweezers combined with TIRF microscopy, we were able to trap peroxisomes and approximate the forces involved in chloroplast association in vivo in tobacco (Nicotiana tabacum) and observed weaker tethering to additional unknown structures within the cell. We show that chloroplasts and peroxisomes are physically tethered through peroxules, a poorly described structure in plant cells. We suggest that peroxules have a novel role in maintaining peroxisome-organelle interactions in the dynamic environment. This could be important for fatty acid mobilization and photorespiration through the interaction with oil bodies and chloroplasts, highlighting a fundamentally important role for organelle interactions for essential biochemistry and physiological processes.

  8. In Vivo Quantification of Peroxisome Tethering to Chloroplasts in Tobacco Epidermal Cells Using Optical Tweezers.

    PubMed

    Gao, Hongbo; Metz, Jeremy; Teanby, Nick A; Ward, Andy D; Botchway, Stanley W; Coles, Benjamin; Pollard, Mark R; Sparkes, Imogen

    2016-01-01

    Peroxisomes are highly motile organelles that display a range of motions within a short time frame. In static snapshots, they can be juxtaposed to chloroplasts, which has led to the hypothesis that they are physically interacting. Here, using optical tweezers, we tested the dynamic physical interaction in vivo. Using near-infrared optical tweezers combined with TIRF microscopy, we were able to trap peroxisomes and approximate the forces involved in chloroplast association in vivo in tobacco (Nicotiana tabacum) and observed weaker tethering to additional unknown structures within the cell. We show that chloroplasts and peroxisomes are physically tethered through peroxules, a poorly described structure in plant cells. We suggest that peroxules have a novel role in maintaining peroxisome-organelle interactions in the dynamic environment. This could be important for fatty acid mobilization and photorespiration through the interaction with oil bodies and chloroplasts, highlighting a fundamentally important role for organelle interactions for essential biochemistry and physiological processes. PMID:26518344

  9. Photothermal optical coherence tomography of epidermal growth factor receptor in live cells using immunotargeted gold nanospheres

    NASA Astrophysics Data System (ADS)

    Skala, Melissa C.; Crow, Matthew J.; Wax, Adam; Izatt, Joseph A.

    2009-02-01

    Molecular imaging is a powerful tool for investigating disease processes and potential therapies in both in vivo and in vitro systems. However, high resolution molecular imaging has been limited to relatively shallow penetration depths that can be accessed with microscopy. Optical coherence tomography (OCT) is an optical analogue to ultrasound with relatively good penetration depth (1-2 mm) and resolution (~1-10 μm). We have developed and characterized photothermal OCT as a molecular contrast mechanism that allows for high resolution molecular imaging at deeper penetration depths than microscopy. Our photothermal system consists of an amplitude-modulated heating beam that spatially overlaps with the focused spot of the sample arm of a spectral-domain OCT microscope. Validation experiments in tissue-like phantoms containing gold nanospheres that absorb at 532 nm revealed a sensitivity of 14 parts per million nanospheres (weight/weight) in a tissue-like environment. The nanospheres were then conjugated to anti-EGFR, and molecular targeting was confirmed in cells that over-express EGFR (MDA-MB-468) and cells that express low levels of EGFR (MDA-MB-435). Molecular imaging in three-dimensional tissue constructs was confirmed with a significantly lower photothermal signal (p<0.0001) from the constructs composed of cells that express low levels of EGFR compared to the over-expressing cell constructs (300% signal increase). This technique could potentially augment confocal and multiphoton microscopy as a method for deep-tissue, depth-resolved molecular imaging with relatively high resolution and target sensitivity, without photobleaching or cytotoxicity.

  10. Matriptase promotes inflammatory cell accumulation and progression of established epidermal tumors

    PubMed Central

    Sales, Katiuchia Uzzun; Friis, Stine; Abusleme, Loreto; Moutsopoulos, Niki M.; Bugge, Thomas H.

    2014-01-01

    Deregulation of matriptase is a consistent feature of human epithelial cancers and correlates with poor disease outcome. We have previously shown that matriptase promotes multi-stage squamous cell carcinogenesis in transgenic mice through dual activation of pro-hepatocyte growth factor-cMet-Akt-mTor proliferation/survival signaling and PAR-2-Gαi-NFκB inflammatory signaling. Matriptase was congenitally and constitutively deregulated in our prior studies, and therefore it was unclear if aberrant matriptase signaling supports only initiation of tumor formation or if it is also critical for the progression of established tumors. To determine this, we here have generated triple-transgenic mice with constitutive deregulation of matriptase and simultaneous inducible expression of the cognate matriptase inhibitor, hepatocyte growth factor inhibitor (HAI)-2. As expected, constitutive expression of HAI-2 suppressed the formation of matriptase-dependent tumors in 7,12-Dimethylbenz(a)anthracene (DMBA)-treated mouse skin. Interestingly, however, the induction of HAI-2 expression in already established tumors markedly impaired malignant progression and caused regression of individual tumors. Tumor regression correlated with reduced accumulation of tumor-associated inflammatory cells, likely caused by diminished expression of pro-tumorigenic inflammatory cytokines. The data suggest that matriptase-dependent signaling may be a therapeutic target for both squamous cell carcinoma chemoprevention and for the treatment of established tumors. PMID:25486433

  11. HDAC6 regulates epidermal growth factor receptor (EGFR) endocytic trafficking and degradation in renal epithelial cells.

    PubMed

    Liu, Wei; Fan, Lucy X; Zhou, Xia; Sweeney, William E; Avner, Ellis D; Li, Xiaogang

    2012-01-01

    We present for the first time that histone deacetylase 6 (HDAC6) regulates EGFR degradation and trafficking along microtubules in Pkd1 mutant renal epithelial cells. HDAC6, the microtubule-associated α-tubulin deacetylase, demonstrates increased expression and activity in Pkd1 mutant mouse embryonic kidney cells. Targeting HDAC6 with a general HDAC inhibitor, trichostatin (TSA), or a specific HDAC6 inhibitor, tubacin, increased the acetylation of α-tubulin and downregulated the expression of EGFR in Pkd1 mutant renal epithelial cells. HDAC6 was co-localized with EGF induced endocytic EGFR and endosomes, respectively. Inhibition of the activity of HDAC6 accelerated the trafficking of EGFR from early endosomes to late endosomes along the microtubules. Without EGF stimulation EGFR was randomly distributed while after stimulation with EGF for 30 min, EGFR was accumulated around α-tubulin labeled microtubule bundles. These data suggested that the Pkd1 mutation induced upregulation of HDAC6 might act to slow the trafficking of EGFR from early endosomes to late endosomes along the microtubules for degradation through deacetylating α-tubulin. In addition, inhibition of HDAC activity decreased the phosphorylation of ERK1/2, the downstream target of EGFR axis, and normalized EGFR localization from apical to basolateral in Pkd1 knockout mouse kidneys. Thus, targeting HDAC6 to downregulate EGFR activity may provide a potential therapeutic approach to treat polycystic kidney disease.

  12. Modulation of estrogen and epidermal growth factor receptors by rosemary extract in breast cancer cells.

    PubMed

    González-Vallinas, Margarita; Molina, Susana; Vicente, Gonzalo; Sánchez-Martínez, Ruth; Vargas, Teodoro; García-Risco, Mónica R; Fornari, Tiziana; Reglero, Guillermo; Ramírez de Molina, Ana

    2014-06-01

    Breast cancer is the leading cause of cancer-related mortality among females worldwide, and therefore the development of new therapeutic approaches is still needed. Rosemary (Rosmarinus officinalis L.) extract possesses antitumor properties against tumor cells from several organs, including breast. However, in order to apply it as a complementary therapeutic agent in breast cancer, more information is needed regarding the sensitivity of the different breast tumor subtypes and its effect in combination with the currently used chemotherapy. Here, we analyzed the antitumor activities of a supercritical fluid rosemary extract (SFRE) in different breast cancer cells, and used a genomic approach to explore its effect on the modulation of ER-α and HER2 signaling pathways, the most important mitogen pathways related to breast cancer progression. We found that SFRE exerts antitumor activity against breast cancer cells from different tumor subtypes and the downregulation of ER-α and HER2 receptors by SFRE might be involved in its antitumor effect against estrogen-dependent (ER+) and HER2 overexpressing (HER2+) breast cancer subtypes. Moreover, SFRE significantly enhanced the effect of breast cancer chemotherapy (tamoxifen, trastuzumab, and paclitaxel). Overall, our results support the potential utility of SFRE as a complementary approach in breast cancer therapy.

  13. Selective Killing Effects of Cold Atmospheric Pressure Plasma with NO Induced Dysfunction of Epidermal Growth Factor Receptor in Oral Squamous Cell Carcinoma

    PubMed Central

    Lee, Jung-Hwan; Om, Ji-Yeon; Kim, Yong-Hee; Kim, Kwang-Mahn; Choi, Eun-Ha; Kim, Kyoung-Nam

    2016-01-01

    The aim of this study is to investigate the effects of cold atmospheric pressure plasma (CAP)-induced radicals on the epidermal growth factor receptor (EGFR), which is overexpressed by oral squamous cell carcinoma, to determine the underlying mechanism of selective killing. CAP-induced highly reactive radicals were observed in both plasma plume and cell culture media. The selective killing effect was observed in oral squamous cell carcinoma compared with normal human gingival fibroblast. Degradation and dysfunction of EGFRs were observed only in the EGFR-overexpressing oral squamous cell carcinoma and not in the normal cell. Nitric oxide scavenger pretreatment in cell culture media before CAP treatment rescued above degradation and dysfunction of the EGFR as well as the killing effect in oral squamous cell carcinoma. CAP may be a promising cancer treatment method by inducing EGFR dysfunction in EGFR-overexpressing oral squamous cell carcinoma via nitric oxide radicals. PMID:26919318

  14. Selective Killing Effects of Cold Atmospheric Pressure Plasma with NO Induced Dysfunction of Epidermal Growth Factor Receptor in Oral Squamous Cell Carcinoma.

    PubMed

    Lee, Jung-Hwan; Om, Ji-Yeon; Kim, Yong-Hee; Kim, Kwang-Mahn; Choi, Eun-Ha; Kim, Kyoung-Nam

    2016-01-01

    The aim of this study is to investigate the effects of cold atmospheric pressure plasma (CAP)-induced radicals on the epidermal growth factor receptor (EGFR), which is overexpressed by oral squamous cell carcinoma, to determine the underlying mechanism of selective killing. CAP-induced highly reactive radicals were observed in both plasma plume and cell culture media. The selective killing effect was observed in oral squamous cell carcinoma compared with normal human gingival fibroblast. Degradation and dysfunction of EGFRs were observed only in the EGFR-overexpressing oral squamous cell carcinoma and not in the normal cell. Nitric oxide scavenger pretreatment in cell culture media before CAP treatment rescued above degradation and dysfunction of the EGFR as well as the killing effect in oral squamous cell carcinoma. CAP may be a promising cancer treatment method by inducing EGFR dysfunction in EGFR-overexpressing oral squamous cell carcinoma via nitric oxide radicals.

  15. Characterization of A Three-Dimensional Organotypic Co-Culture Skin Model for Epidermal Differentiation of Rat Adipose-Derived Stem Cells

    PubMed Central

    Ghanavati, Zeinab; Orazizadeh, Mahmoud; Bayati, Vahid; Abbaspour, Mohammad Reza; Khorsandi, Layasadat; Mansouri, Esrafil; Neisi, Niloofar

    2016-01-01

    Objective The organotypic co-culture is a well-known technique to examine cellular interactions and their roles in stem cell proliferation and differentiation. This study aims to evaluate the effects of dermal fibroblasts (DFs) on epidermal differentiation of adipose-derived stem cells (ASCs) using a three-dimensional (3D) organotypic co- culture technique. Materials and Methods In this experimental research study, rat DFs and ASCs were isolated and cultured separately on electrospun polycaprolactone (PCL) matrices. The PCL matrices seeded by ASCs were superimposed on to the matrices seeded by DFs in order to create a 3D organotypic co-culture. In the control groups, PCL matrices seeded by ASCs were placed on matrices devoid of DFs. After 10 days, we assessed the expressions of keratinocyte-related genes by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and expression of pan-cytokeratin protein by immunofluorescence in the differentiated keratinocyte-like cells from co- culture and control groups. Keratinocyte-like cell morphologies were also observed by scanning electron microscopy (SEM). Results The early, intermediate, and terminal differentiation keratinocyte markers-Cytokeratin14, Filaggrin, and Involucrin significantly expressed in the co-culture groups com- pared to the control ones (P<0.05). We observed pan-cytokeratin in keratinocyte-like cells of both groups by immunofluorescence. SEM observation of the co-culture groups showed that the differentiated keratinocyte-like cells developed a polygonal cobblestone shape, considered characteristic of keratinocytes. Conclusion The 3D organotypic co-culture bilayered construct that consisted of DFs and ASCs was an effective technique for epidermal differentiation of ASCs. This co-culture might be useful for epidermal differentiation of stem cells for future applications in skin regeneration.

  16. Characterization of A Three-Dimensional Organotypic Co-Culture Skin Model for Epidermal Differentiation of Rat Adipose-Derived Stem Cells

    PubMed Central

    Ghanavati, Zeinab; Orazizadeh, Mahmoud; Bayati, Vahid; Abbaspour, Mohammad Reza; Khorsandi, Layasadat; Mansouri, Esrafil; Neisi, Niloofar

    2016-01-01

    Objective The organotypic co-culture is a well-known technique to examine cellular interactions and their roles in stem cell proliferation and differentiation. This study aims to evaluate the effects of dermal fibroblasts (DFs) on epidermal differentiation of adipose-derived stem cells (ASCs) using a three-dimensional (3D) organotypic co- culture technique. Materials and Methods In this experimental research study, rat DFs and ASCs were isolated and cultured separately on electrospun polycaprolactone (PCL) matrices. The PCL matrices seeded by ASCs were superimposed on to the matrices seeded by DFs in order to create a 3D organotypic co-culture. In the control groups, PCL matrices seeded by ASCs were placed on matrices devoid of DFs. After 10 days, we assessed the expressions of keratinocyte-related genes by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and expression of pan-cytokeratin protein by immunofluorescence in the differentiated keratinocyte-like cells from co- culture and control groups. Keratinocyte-like cell morphologies were also observed by scanning electron microscopy (SEM). Results The early, intermediate, and terminal differentiation keratinocyte markers-Cytokeratin14, Filaggrin, and Involucrin significantly expressed in the co-culture groups com- pared to the control ones (P<0.05). We observed pan-cytokeratin in keratinocyte-like cells of both groups by immunofluorescence. SEM observation of the co-culture groups showed that the differentiated keratinocyte-like cells developed a polygonal cobblestone shape, considered characteristic of keratinocytes. Conclusion The 3D organotypic co-culture bilayered construct that consisted of DFs and ASCs was an effective technique for epidermal differentiation of ASCs. This co-culture might be useful for epidermal differentiation of stem cells for future applications in skin regeneration. PMID:27602310

  17. Lipopolysaccharide (LPS)-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR).

    PubMed

    Trussoni, Christy E; Tabibian, James H; Splinter, Patrick L; O'Hara, Steven P

    2015-01-01

    Cholangiocytes (biliary epithelial cells) actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells), or low passage normal human cholangiocytes (NHC), were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE) inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (p<0.05) and proliferation (p<0.01). Additionally, cholangiocytes from LPS-treated mouse livers and human primary sclerosing cholangitis (PSC) livers exhibited increased phospho-EGFR (p<0.01). Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes.

  18. Tyrosine kinase inhibitors for epidermal growth factor receptor gene mutation-positive non-small cell lung cancers: an update for recent advances in therapeutics.

    PubMed

    Chung, Clement

    2016-06-01

    The presence of activating gene mutations in the epidermal growth factor receptor of non-small cell lung cancer patients is predictive (improved progression-free survival and improved response rate) when treated with small molecule tyrosine kinase inhibitors such as gefitinib, erlotinib and afatinib. The two most common mutations that account for greater than 85% of all EGFR gene mutations are in-frame deletions in exon 19 (LREA deletions) and substitution in exon 21 (L858R). Exon 18 mutations occur much less frequently at about 4% of all EGFR gene mutations. Together, exon 19 deletion and exon 21 L858R gene substitution are present in about 10% of Caucasian patients and 20-40% of Asian patients with non-small cell lung cancer. T790M gene mutation at exon 20 is associated with acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors. Early studies showed that activating EGFR gene mutations are most common in patients with adenocarcinoma histology, women, never smokers and those of Asian ethnicity. A recent multi-center phase III trial suggested that frontline epidermal growth factor receptor tyrosine kinase inhibitor therapy with afatinib is associated with improved progression-free survival compared to chemotherapy regardless of race. Moreover, guidelines now suggest EGFR gene mutation testing should be conducted in all patients with lung adenocarcinoma or mixed lung cancers with an adenocarcinoma component, regardless of characteristics such as smoking status, gender or race. The success of targeted therapies in non-small cell lung cancer patients has changed the treatment paradigm in metastatic non-small cell lung cancer. However, despite a durable response of greater than a year, resistance to epidermal growth factor receptor tyrosine kinase inhibitors inevitably occurs. This mini-review describes the clinically relevant EGFR gene mutations and the efficacy/toxicity of small molecule epidermal growth factor receptor tyrosine kinase

  19. Multiple Mechanisms are Responsible for Transactivation of the Epidermal Growth Factor Receptor in Mammary Epithelial Cells

    SciTech Connect

    Rodland, Karin D.; Bollinger, Nikki; Ippolito, Danielle L.; Opresko, Lee; Coffey, Robert J.; Zangar, Richard C.; Wiley, H. S.

    2008-11-14

    REVIEW ENTIRE DOCUMENT AT: https://pnlweb.pnl.gov/projects/bsd/ERICA%20Manuscripts%20for%20Review/KD%20Rodland%20D7E80/HMEC_transactivation_ms01_15+Figs.pdf ABSTRACT: Using a single nontransformed strain of human mammary epithelial cells, we found that the ability of multiple growth factors and cytokines to induce ERK phosphorylation was dependent on EGFR activity. These included lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factoralpha. In contrast, hepatocyte growth factor could stimulate ERK phosphorylation independent of EGFR activity...

  20. Muscarinic Type 3 Receptor Induces Cytoprotective Signaling in Salivary Gland Cells through Epidermal Growth Factor Receptor Transactivation

    PubMed Central

    Kajiya, Mikihito; Ichimonji, Isao; Min, Christine; Zhu, Tongbo; Jin, Jun-O; Yu, Qing; Almazrooa, Soulafa A.; Cha, Seunghee

    2012-01-01

    Muscarinic type 3 receptor (M3R) plays a pivotal role in the induction of glandular fluid secretions. Although M3R is often the target of autoantibodies in Sjögren's syndrome (SjS), chemical agonists for M3R are clinically used to stimulate saliva secretion in patients with SjS. Aside from its activity in promoting glandular fluid secretion, however, it is unclear whether activation of M3R is related to other biological events in SjS. This study aimed to investigate the cytoprotective effect of chemical agonist-mediated M3R activation on apoptosis induced in human salivary gland (HSG) cells. Carbachol (CCh), a muscarinic receptor-specific agonist, abrogated tumor necrosis factor α/interferon γ-induced apoptosis through pathways involving caspase 3/7, but its cytoprotective effect was decreased by a M3R antagonist, a mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) inhibitor, a phosphatidylinositol 3-kinase/Akt inhibitor, or an epidermal growth factor receptor (EGFR) inhibitor. Ligation of M3R with CCh transactivated EGFR and phosphorylated ERK and Akt, the downstream targets of EGFR. Inhibition of intracellular calcium release or protein kinase C δ, both of which are involved in the cell signaling of M3R-mediated fluid secretion, did not affect CCh-induced ERK or Akt phosphorylation. CCh stimulated Src phosphorylation and binding to EGFR. A Src inhibitor attenuated the CCh/M3R-induced cytoprotective effect and EGFR transactivation cascades. Overall, these results indicated that CCh/M3R induced transactivation of EGFR through Src activation leading to ERK and Akt phosphorylation, which in turn suppressed caspase 3/7-mediated apoptotic signals in HSG cells. This study, for the first time, proposes that CCh-mediated M3R activation can promote not only fluid secretion but also survival of salivary gland cells in the inflammatory context of SjS. PMID:22511543

  1. Rapid and simple isolation of vascular, epidermal and mesophyll cells from plant leaf tissue.

    PubMed

    Endo, Motomu; Shimizu, Hanako; Araki, Takashi

    2016-08-01

    To understand physiological phenomena at the tissue level, elucidation of tissue-specific molecular functions in vivo is required. As an example of the current state of affairs, many genes in plants have been reported to have discordant levels of expression between bulk tissues and the specific tissues in which the respective gene product is principally functional. The principal challenge in deciphering such tissue-specific functions lies in separating tissues with high spatiotemporal resolution to evaluate accurate gene expression profiles. Here, we provide a simple and rapid tissue isolation protocol to isolate all three major leaf tissues (mesophyll, vasculature and epidermis) from Arabidopsis within 30 min with high purity. On the basis of the different cell-to-cell connectivities of tissues, the mesophyll isolation is achieved by making protoplasts, and the vasculature and epidermis isolation is achieved through sonication and enzymatic digestion of leaves. We have successfully tested the protocol on several other plant species, including crop plants such as soybean, tomato and wheat. Furthermore, isolated tissues can be used not only for tissue-specific transcriptome assays but also potentially for tissue-specific proteome and methylome assays. PMID:27388555

  2. Rapid and simple isolation of vascular, epidermal and mesophyll cells from plant leaf tissue.

    PubMed

    Endo, Motomu; Shimizu, Hanako; Araki, Takashi

    2016-08-01

    To understand physiological phenomena at the tissue level, elucidation of tissue-specific molecular functions in vivo is required. As an example of the current state of affairs, many genes in plants have been reported to have discordant levels of expression between bulk tissues and the specific tissues in which the respective gene product is principally functional. The principal challenge in deciphering such tissue-specific functions lies in separating tissues with high spatiotemporal resolution to evaluate accurate gene expression profiles. Here, we provide a simple and rapid tissue isolation protocol to isolate all three major leaf tissues (mesophyll, vasculature and epidermis) from Arabidopsis within 30 min with high purity. On the basis of the different cell-to-cell connectivities of tissues, the mesophyll isolation is achieved by making protoplasts, and the vasculature and epidermis isolation is achieved through sonication and enzymatic digestion of leaves. We have successfully tested the protocol on several other plant species, including crop plants such as soybean, tomato and wheat. Furthermore, isolated tissues can be used not only for tissue-specific transcriptome assays but also potentially for tissue-specific proteome and methylome assays.

  3. Epigenetic modifications of triterpenoid ursolic acid in activating Nrf2 and blocking cellular transformation of mouse epidermal cells.

    PubMed

    Kim, Hyuck; Ramirez, Christina N; Su, Zheng-Yuan; Kong, Ah-Ng Tony

    2016-07-01

    Ursolic acid (UA), a well-known natural triterpenoid found in abundance in blueberries, cranberries and apple peels, has been reported to possess many beneficial health effects. These effects include anticancer activity in various cancers, such as skin cancer. Skin cancer is the most common cancer in the world. Nuclear factor E2-related factor 2 (Nrf2) is a master regulator of antioxidative stress response with anticarcinogenic activity against UV- and chemical-induced tumor formation in the skin. Recent studies show that epigenetic modifications of Nrf2 play an important role in cancer prevention. However, the epigenetic impact of UA on Nrf2 signaling remains poorly understood in skin cancer. In this study, we investigated the epigenetic effects of UA on mouse epidermal JB6 P+ cells. UA inhibited cellular transformation by 12-O-tetradecanoylphorbol-13-acetate at a concentration at which the cytotoxicity was no more than 25%. Under this condition, UA induced the expression of the Nrf2-mediated detoxifying/antioxidant enzymes heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferase 1A1. DNA methylation analysis revealed that UA demethylated the first 15 CpG sites of the Nrf2 promoter region, which correlated with the reexpression of Nrf2. Furthermore, UA reduced the expression of epigenetic modifying enzymes, including the DNA methyltransferases DNMT1 and DNMT3a and the histone deacetylases (HDACs) HDAC1, HDAC2, HDAC3 and HDAC8 (Class I) and HDAC6 and HDAC7 (Class II), and HDAC activity. Taken together, these results suggest that the epigenetic effects of the triterpenoid UA could potentially contribute to its beneficial effects, including the prevention of skin cancer. PMID:27260468

  4. Epidermal growth factor/TNF-α transactivation modulates epithelial cell proliferation and apoptosis in a mouse model of parenteral nutrition

    PubMed Central

    Feng, Yongjia

    2012-01-01

    Epidermal growth factor (EGF) and tumor necrosis factor-α (TNF-α) signaling are critical for effective proliferative and apoptotic actions; however, little is known about the codependency of these signaling pathways in the intestinal epithelium. Because total parenteral nutrition (TPN) is associated with loss of intestinal epithelial cell (IEC) proliferation and increased apoptosis, we utilized a mouse model to explore these transactivation pathways in small bowel epithelium. Mice underwent intravenous cannulation and were given enteral nutrition or TPN for 7 days. Outcomes included IEC proliferation, apoptosis, and survival. To address transactivation or dependence of EGF and TNF on IEC physiology, TNF-α receptor knockout (KO) mice, TNFR1-KO, R2-KO, or R1R2-double KO, were used. Exogenous EGF and pharmacological blockade of ErbB1 were performed in other groups to examine the relevance of the ErB1 pathway. TPN increased IEC TNFR1 and decreased EGF and ErbB1 abundance. Loss of IEC proliferation was prevented by exogenous EGF or blockade of TNFR1. However, EGF action was prevented without effective TNFR2 signaling. Also, blockade of TNFR1 could not prevent loss of IEC proliferation without effective ErbB1 signaling. TPN increased IEC apoptosis and was due to increased TNFR1 signaling. Exogenous EGF or blockade of TNFR1 could prevent increased apoptosis, and both pathways were dependent on effective ErbB1 signaling. Exogenous EGF prevented increased apoptosis in mice lacking TNFR2 signaling. TPN mice had significantly decreased survival vs. controls, and this was associated with the TNFR1 signaling pathway. We concluded that these findings identify critical mechanisms that contribute to TPN-associated mucosal atrophy via altered TNF-α/EGF signaling. It emphasizes the importance of both TNFR1 and TNFR2 pathways, as well as the strong interdependence on an intact EGF/ErbB1 pathway. PMID:22075779

  5. UV-induced mutations in epidermal cells of mice defective in DNA polymerase η and/or ι.

    PubMed

    Kanao, Rie; Yokoi, Masayuki; Ohkumo, Tsuyoshi; Sakurai, Yasutaka; Dotsu, Kantaro; Kura, Shinobu; Nakatsu, Yoshimichi; Tsuzuki, Teruhisa; Masutani, Chikahide; Hanaoka, Fumio

    2015-05-01

    Xeroderma pigmentosum variant (XP-V) is a human rare inherited recessive disease, predisposed to sunlight-induced skin cancer, which is caused by deficiency in DNA polymerase η (Polη). Polη catalyzes accurate translesion synthesis (TLS) past pyrimidine dimers, the most prominent UV-induced lesions. DNA polymerase ι (Polι) is a paralog of Polη that has been suggested to participate in TLS past UV-induced lesions, but its function in vivo remains uncertain. We have previously reported that Polη-deficient and Polη/Polι double-deficient mice showed increased susceptibility to UV-induced carcinogenesis. Here, we investigated UV-induced mutation frequencies and spectra in the epidermal cells of Polη- and/or Polι-deficient mice. While Polη-deficient mice showed significantly higher UV-induced mutation frequencies than wild-type mice, Polι deficiency did not influence the frequencies in the presence of Polη. Interestingly, the frequencies in Polη/Polι double-deficient mice were statistically lower than those in Polη-deficient mice, although they were still higher than those of wild-type mice. Sequence analysis revealed that most of the UV-induced mutations in Polη-deficient and Polη/Polι double-deficient mice were base substitutions at dipyrimidine sites. An increase in UV-induced mutations at both G:C and A:T pairs associated with Polη deficiency suggests that Polη contributes to accurate TLS past both thymine- and cytosine-containing dimers in vivo. A significant decrease in G:C to A:T transition in Polη/Polι double-deficient mice when compared with Polη-deficient mice suggests that Polι is involved in error-prone TLS past cytosine-containing dimers when Polη is inactivated.

  6. Intercalated chemotherapy and erlotinib for non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutations

    PubMed Central

    Zwitter, Matjaz; Rajer, Mirjana; Stanic, Karmen; Vrankar, Martina; Doma, Andrej; Cuderman, Anka; Grmek, Marko; Kern, Izidor; Kovac, Viljem

    2016-01-01

    ABSTRACT Among attempts to delay development of resistance to tyrosine kinase inhibitors (TKIs) in patients with advanced non-small cell lung cancer (NSCLC) with activating mutations of epidermal growth factor receptor (EGFR), intercalated therapy has not been properly evaluated. In a phase II trial, 38 patients with EGFR mutated NSCLC in advanced stage were treated with 4 to 6 3-weekly cycles of intercalated schedule with gemcitabine (1250 mg/m2, days 1 and 4), cisplatin (75 mg/m2, day 2) and erlotinib (150 mg, days 5 – 15), followed by continuous erlotinib as maintenance. In addition to standard radiologic evaluation according to RECIST, PET/CT was done prior to treatment and at 6 months, using PERCIST as a method for assessment of response. The primary endpoint was progression-free survival (PFS). In general, tolerance to treatment was good, even among 8 patients with performance status 2–3 and 13 patients with brain metastases; grade 4 toxicity included 2 cases of neutropenia and 4 thrombo-embolic events. Complete response (CR) or partial response (PR) were seen in 15 (39.5%) and 17 (44.7%) cases, respectively. All cases of CR were confirmed also by PET/CT. Median PFS was 23.4 months and median overall survival (OS) was 38.3  months. After a median follow-up of 35 months, 8 patients are still in CR and on maintenance erlotinib. In conclusion, intercalated treatment for treatment-naive patients with EGFR activating mutations leads to excellent response rate and prolonged PFS and survival. Comparison of the intercalated schedule to monotherapy with TKIs in a randomized trial is warranted. PMID:27261103

  7. Epigenetic modifications of triterpenoid ursolic acid in activating Nrf2 and blocking cellular transformation of mouse epidermal cells.

    PubMed

    Kim, Hyuck; Ramirez, Christina N; Su, Zheng-Yuan; Kong, Ah-Ng Tony

    2016-07-01

    Ursolic acid (UA), a well-known natural triterpenoid found in abundance in blueberries, cranberries and apple peels, has been reported to possess many beneficial health effects. These effects include anticancer activity in various cancers, such as skin cancer. Skin cancer is the most common cancer in the world. Nuclear factor E2-related factor 2 (Nrf2) is a master regulator of antioxidative stress response with anticarcinogenic activity against UV- and chemical-induced tumor formation in the skin. Recent studies show that epigenetic modifications of Nrf2 play an important role in cancer prevention. However, the epigenetic impact of UA on Nrf2 signaling remains poorly understood in skin cancer. In this study, we investigated the epigenetic effects of UA on mouse epidermal JB6 P+ cells. UA inhibited cellular transformation by 12-O-tetradecanoylphorbol-13-acetate at a concentration at which the cytotoxicity was no more than 25%. Under this condition, UA induced the expression of the Nrf2-mediated detoxifying/antioxidant enzymes heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferase 1A1. DNA methylation analysis revealed that UA demethylated the first 15 CpG sites of the Nrf2 promoter region, which correlated with the reexpression of Nrf2. Furthermore, UA reduced the expression of epigenetic modifying enzymes, including the DNA methyltransferases DNMT1 and DNMT3a and the histone deacetylases (HDACs) HDAC1, HDAC2, HDAC3 and HDAC8 (Class I) and HDAC6 and HDAC7 (Class II), and HDAC activity. Taken together, these results suggest that the epigenetic effects of the triterpenoid UA could potentially contribute to its beneficial effects, including the prevention of skin cancer.

  8. MMP-1 and MMP-9 regulate epidermal growth factor-dependent collagen loss in human carotid plaque smooth muscle cells.

    PubMed

    Rao, Velidi H; Kansal, Vikash; Stoupa, Samantha; Agrawal, Devendra K

    2014-02-01

    Mechanisms underlying the rupture of atherosclerotic plaque, a crucial factor in the development of myocardial infarction and stroke, are not well defined. Here, we examined the role of epidermal growth factor (EGF)-mediated matrix metalloproteinases (MMP) on the stability of interstitial collagens in vascular smooth muscle cells (VSMCs) isolated from carotid endarterectomy tissues of symptomatic and asymptomatic patients with carotid stenosis. VSMCs isolated from the carotid plaques of both asymptomatic and symptomatic patients were treated with EGF. The MMP-9 activity was quantified by gelatin zymography and the analysis of mRNA transcripts and protein for MMP-9, MMP-1, EGFR and collagen types I, Col I(α1) and collagen type III, Col III(α1) were analyzed by qPCR and immunofluorescence, respectively. The effect of EGF treatment to increase MMP-9 activity and mRNA transcripts for MMP-9, MMP-1, and EGFR and to decrease mRNA transcripts for Col I(α1) and Col III(α1) was threefold to fourfold greater in VSMCs isolated from the carotid plaques of symptomatic than asymptomatic patients. Inhibitors of EGFR (AG1478) and a small molecule inhibitor of MMP-9 decreased the MMP9 expression and upregulated Col I(α1) and Col III(α1) in EGF-treated VSMCs of both groups. Additionally, the magnitude in decreased MMP-9 mRNA and increased Col I(α1) and Col III(α1) due to knockdown of MMP-9 gene with siRNA in EGF-treated VSMCs was significantly greater in the symptomatic group than the asymptomatic group. Thus, a selective blockade of both EGFR and MMP-9 may be a novel strategy and a promising target for stabilizing vulnerable plaques in patients with carotid stenosis.

  9. Expressed miRNAs target feather related mRNAs involved in cell signaling, cell adhesion and structure during chicken epidermal development.

    PubMed

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

    2016-10-15

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Previous studies have shown that miRNA regulation contributes to a diverse set of processes including cellular differentiation and morphogenesis which leads to the creation of different cell types in multicellular organisms and is thus key to animal development. Feathers are one of the most distinctive features of extant birds and are important for multiple functions including flight, thermal regulation, and sexual selection. However, the role of miRNAs in feather development has been woefully understudied despite the identification of cell signaling pathways, cell adhesion molecules and structural genes involved in feather development. In this study, we performed a microarray experiment comparing the expression of miRNAs and mRNAs among three embryonic stages of development and two tissues (scutate scale and feather) of the chicken. We combined this expression data with miRNA target prediction tools and a curated list of feather related genes to produce a set of 19 miRNA-mRNA duplexes. These targeted mRNAs have been previously identified as important cell signaling and cell adhesion genes as well as structural genes involved in feather and scale morphogenesis. Interestingly, the miRNA target site of the cell signaling pathway gene, Aldehyde Dehydrogenase 1 Family, Member A3 (ALDH1A3), is unique to birds indicating a novel role in Aves. The identified miRNA target site of the cell adhesion gene, Tenascin C (TNC), is only found in specific chicken TNC splice variants that are differentially expressed in developing scutate scale and feather tissue indicating an important role of miRNA regulation in epidermal differentiation. Additionally, we found that β-keratins, a major structural component of avian and reptilian epidermal appendages, are targeted by multiple miRNA genes. In conclusion, our work provides quantitative expression data on miRNAs and m

  10. Expressed miRNAs target feather related mRNAs involved in cell signaling, cell adhesion and structure during chicken epidermal development.

    PubMed

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

    2016-10-15

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Previous studies have shown that miRNA regulation contributes to a diverse set of processes including cellular differentiation and morphogenesis which leads to the creation of different cell types in multicellular organisms and is thus key to animal development. Feathers are one of the most distinctive features of extant birds and are important for multiple functions including flight, thermal regulation, and sexual selection. However, the role of miRNAs in feather development has been woefully understudied despite the identification of cell signaling pathways, cell adhesion molecules and structural genes involved in feather development. In this study, we performed a microarray experiment comparing the expression of miRNAs and mRNAs among three embryonic stages of development and two tissues (scutate scale and feather) of the chicken. We combined this expression data with miRNA target prediction tools and a curated list of feather related genes to produce a set of 19 miRNA-mRNA duplexes. These targeted mRNAs have been previously identified as important cell signaling and cell adhesion genes as well as structural genes involved in feather and scale morphogenesis. Interestingly, the miRNA target site of the cell signaling pathway gene, Aldehyde Dehydrogenase 1 Family, Member A3 (ALDH1A3), is unique to birds indicating a novel role in Aves. The identified miRNA target site of the cell adhesion gene, Tenascin C (TNC), is only found in specific chicken TNC splice variants that are differentially expressed in developing scutate scale and feather tissue indicating an important role of miRNA regulation in epidermal differentiation. Additionally, we found that β-keratins, a major structural component of avian and reptilian epidermal appendages, are targeted by multiple miRNA genes. In conclusion, our work provides quantitative expression data on miRNAs and m

  11. Quantitative differences in host cell reactivation of ultraviolet-damaged virus in human skin fibroblasts and epidermal keratinocytes cultured from the same foreskin biopsy

    SciTech Connect

    Tyrrell, R.M.; Pidoux, M.

    1986-06-01

    Repair efficiency of cultured cells may be estimated by measuring the ability of a particular cell type to support virus damaged by an appropriate agent. In this study we have compared the inactivation of ultraviolet (254 nm)-damaged herpes simplex virus in human fibroblast and epidermal keratinocyte cell lines derived from the same foreskin biopsy and found the epithelial cells to be a factor of 3 times less efficient in supporting the damaged virus. The two different cell types show comparable ultraviolet inactivation of clone-forming ability, indicating that the difference is specific to viral host cell reactivation. This study required the development of a quantitative infectious centers assay for the measurement of viral titer in human epithelial cells, a system which may be of more general application in studies of potential human carcinogens.

  12. Transcriptional and Secretomic Profiling of Epidermal Cells Exposed to Alpha Particle Radiation

    PubMed Central

    Chauhan, Vinita; Howland, Matthew; Greene, Hillary Boulay; Wilkins, Ruth C

    2012-01-01

    Alpha (α)-particle emitters are probable isotopes to be used in a terrorist attack. The development of biological assessment tools to identify those who have handled these difficult to detect materials would be an asset to our current forensic capacity. In this study, for the purposes of biomarker discovery, human keratinocytes were exposed to α-particle and X-radiation (0.98 Gy/h at 0, 0.5, 1.0, 1.5 Gy) and assessed for differential gene and protein expression using microarray and Bio-Plex technology, respectively. Secretomic analysis of supernatants showed expression of two pro-inflammatory cytokines (IL-13 and PDGF-bb) to be exclusively affected in α-particle exposed cells. The highest dose of α-particle radiation modulated a total of 67 transcripts (fold change>|1.5|, (False discovery rate) FDR<0.05) in exposed cells. Several genes which responded with high expression levels (>2 fold) included KIF20A, NEFM, C7orf10, HIST1H2BD, BMP6, and HIST1H2AC. Among the high expressing genes, five (CCNB2, BUB1, NEK2, CDC20, AURKA) were also differentially expressed at the medium (1.0 Gy) dose however, these genes were unmodulated following exposure to X-irradiation. Networks of these genes clustered around tumor protein-53 and transforming growth factor-beta signaling. This study has identified some potential gene /protein responses and networks that may be validated further to confirm their specificity and potential to be signature biomarkers of α-particle exposure. PMID:23002402

  13. Differential regulation of cell proliferation and protease secretion by epidermal growth factor and amphiregulin in tumoral versus normal breast epithelial cells

    PubMed Central

    Silvy, M; Giusti, C; Martin, P-M; Berthois, Y

    2001-01-01

    Amphiregulin (AR) is a heparin-binding epidermal growth factor (EGF)-related peptide that seems to play an important role in mammary epithelial cell growth regulation. We have investigated the regulation of AR-gene expression and -protein secretion by EGF in normal breast epithelial cells (HMECs), as well as in the tumoral breast epithelial cell lines MCF-7 and MDA-MB231. EGF induced a dose-dependent increase of AR mRNA level in both normal and tumoral cells. Thus, 10−8M EGF stimulated AR expression in HMECs to 140–300% of control. A similar EGF concentration increased AR mRNA level to 550% and 980% of control in MCF-7 and MDA-MB231 cells, respectively. This was accompanied by an accumulation of AR into conditioned culture media. However, HMECs secreted in response to EGF, 5–10 fold more AR than tumour cells. Furthermore, the potential participation of AR in the regulation of the plasminogen activator (PA)/plasmin system was investigated. Whereas HMEC-proliferation was stimulated by AR, the levels of secreted urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAi-1) remained unaffected. Conversely, AR failed to regulate the proliferation of tumoral cell lines but induced an accumulation of uPA and PAi-1 into culture media. This was accompanied by an increase of the number of tumoral cells that invaded matrigel in vitro. Moreover, the presence of a neutralizing anti-uPA receptor antibody reversed the increased invasiveness of MDA-MB231 cells induced by AR. These data reveal differential behaviour of normal versus tumoral breast epithelial cells in regard to the action of AR and demonstrate that, in a number of cases, AR might play a significant role in tumour progression through the regulation of the PA/plasmin system. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11286474

  14. Crosstalk with cancer-associated fibroblasts induces resistance of non-small cell lung cancer cells to epidermal growth factor receptor tyrosine kinase inhibition

    PubMed Central

    Choe, Chungyoul; Shin, Yong-Sung; Kim, Changhoon; Choi, So-Jung; Lee, Jinseon; Kim, So Young; Cho, Yong Beom; Kim, Jhingook

    2015-01-01

    Although lung cancers with activating mutations in the epidermal growth factor receptor (EGFR) are highly sensitive to selective EGFR tyrosine kinase inhibitors (TKIs), these tumors invariably develop acquired drug resistance. Host stromal cells have been found to have a considerable effect on the sensitivity of cancer cells to EGFR TKIs. Little is known, however, about the signaling mechanisms through which stromal cells contribute to the response to EGFR TKI in non-small cell lung cancer. This work examined the role of hedgehog signaling in cancer-associated fibroblast (CAF)-mediated resistance of lung cancer cells to the EGFR TKI erlotinib. PC9 cells, non-small cell lung cancer cells with EGFR-activating mutations, became resistant to the EGFR TKI erlotinib when cocultured in vitro with CAFs. Polymerase chain reaction and immunocytochemical assays showed that CAFs induced epithelial to mesenchymal transition phenotype in PC9 cells, with an associated change in the expression of epithelial to mesenchymal transition marker proteins including vimentin. Importantly, CAFs induce upregulation of the 7-transmembrane protein smoothened, the central signal transducer of hedgehog, suggesting that the hedgehog signaling pathway is active in CAF-mediated drug resistance. Indeed, downregulation of smoothened activity with the smoothened antagonist cyclopamine induces remodeling of the actin cytoskeleton independently of Gli-mediated transcriptional activity in PC9 cells. These findings indicate that crosstalk with CAFs plays a critical role in resistance of lung cancer to EGFR TKIs through induction of the epithelial to mesenchymal transition and may be an ideal therapeutic target in lung cancer. PMID:26676152

  15. The combination of epidermal growth factor and transforming growth factor-beta induces novel phenotypic changes in mouse liver stem cell lines.

    PubMed

    Isfort, R J; Cody, D B; Stuard, S B; Randall, C J; Miller, C; Ridder, G M; Doersen, C J; Richards, W G; Yoder, B K; Wilkinson, J E; Woychik, R P

    1997-12-01

    Mouse liver stem cell (oval cell) lines were investigated in order to determine the role which two families of growth and differentiation factors (GDFs), epidermal growth factor (EGF) family and transforming growth factor beta (TGF-beta) family, play in liver regeneration. EGF family members, including EGF, amphiregulin, betacellulin, heparin-binding epidermal growth factor, and TGF-alpha, were mitogenic for oval cell lines while TGF-beta family members, including TGF-beta1, TGF-beta2 and TGF-beta3, inhibited mitogenesis and induced apoptosis in oval cell lines. Surprisingly, the combination of EGF family members and TGF-ss family members resulted in neither proliferation nor apoptosis but instead in a novel cellular response, cellular scattering in tissue culture and morphological differentiation in Matrigel. Analysis of the signal transduction pathways activated by exposure of oval cell lines to either EGF, EGF+TGF-beta, or TGF-beta indicated that novel combinations of intracellular signals result following stimulation of the cells with the combination of EGF+TGF-beta. These data reveal that the dynamics of synergistic GDF action following tissue injury and regeneration results in a new level of complexity not obvious from the study of individual GDFs.

  16. Role of oxygen intermediates in UV-induced epidermal cell injury

    SciTech Connect

    Danno, K.; Horio, T.; Takigawa, M.; Imamura, S.

    1984-09-01

    To investigate the role of oxygen intermediates (OIs) in sunburn cell (SC) formation and development of UV-inflammation in vivo, groups of mice were injected intravenously with OI scavengers, including bovine blood superoxide dismutase (SOD), bovine liver catalase, L-histidine, D-mannitol, and saline (controls) before and/or after UV irradiation with sunlamp tubes (mainly 280-320 nm; 300 mJ/cm2; UVR). Ear thickness was measured before and 6 and 24 h after UVR. Ears were removed 24 h after UVR and the number of SCs per unit length of ear epidermis was counted using hematoxylineosin stained sections. The number of SCs was significantly decreased (p less than 0.02) by a single injection of SOD (10-30 units/g body weight) given either just before or immediately after (less than 15 min) UVR, while SC formation was no longer suppressed by injections given more than 2 h before or after UVR. Four repeated injections of SOD (10 units/g) also reduced SC counts but did not significantly alter ear-swelling responses (ESR). Neither SC counts nor ESR were remarkably suppressed by 4 injections of any of the other active OI scavengers, inactivated SOD, or bovine serum albumin. A single injection of diethyldithiocarbamate, an SOD inactivator, significantly augmented SC formation (p less than 0.05), but did not change ESR. These findings suggest that OIs generated by UVR participate in SC formation but are not apparently involved in UV-edema.

  17. Desmosomal molecules in and out of adhering junctions: normal and diseased States of epidermal, cardiac and mesenchymally derived cells.

    PubMed

    Pieperhoff, Sebastian; Barth, Mareike; Rickelt, Steffen; Franke, Werner W

    2010-01-01

    Current cell biology textbooks mention only two kinds of cell-to-cell adhering junctions coated with the cytoplasmic plaques: the desmosomes (maculae adhaerentes), anchoring intermediate-sized filaments (IFs), and the actin microfilament-anchoring adherens junctions (AJs), including both punctate (puncta adhaerentia) and elongate (fasciae adhaerentes) structures. In addition, however, a series of other junction types has been identified and characterized which contain desmosomal molecules but do not fit the definition of desmosomes. Of these special cell-cell junctions containing desmosomal glycoproteins or proteins we review the composite junctions (areae compositae) connecting the cardiomyocytes of mature mammalian hearts and their importance in relation to human arrhythmogenic cardiomyopathies. We also emphasize the various plakophilin-2-positive plaques in AJs (coniunctiones adhaerentes) connecting proliferatively active mesenchymally-derived cells, including interstitial cells of the heart and several soft tissue tumor cell types. Moreover, desmoplakin has also been recognized as a constituent of the plaques of the complexus adhaerentes connecting certain lymphatic endothelial cells. Finally, we emphasize the occurrence of the desmosomal transmembrane glycoprotein, desmoglein Dsg2, out of the context of any junction as dispersed cell surface molecules in certain types of melanoma cells and melanocytes. This broadening of our knowledge on the diversity of AJ structures indicates that it may still be too premature to close the textbook chapters on cell-cell junctions. PMID:20671973

  18. Progressive stages of "transdifferentiation" from epidermal to mesenchymal phenotype induced by MyoD1 transfection, 5-aza-2'- deoxycytidine treatment, and selection for reduced cell attachment in the human keratinocyte line HaCaT

    PubMed Central

    1992-01-01

    The ability of the myogenic determination gene (MyoD1) to convert differentiating human keratinocytes (HaCaT cell-line) to the myogenic pathway and the effect of MyoD1 on the epidermal phenotype was studied in culture and in surface transplants on nude mice. MyoD1 transfection induced the synthesis of myosin, desmin, and vimentin without substantially altering the epidermal differentiation properties (morphology, keratin profile) in vitro nor epidermal morphogenesis (formation of a complex stratified squamous epithelium) in surface transplants, demonstrating the stability of the keratinocyte phenotype. 5-Aza-CdR treatment of these MyoD1-transfected cells had little effect on the cultured cells but a morphologically unstructured epithelium was formed with no indications of typical cell layers including cornification. Since prevention of epidermal strata in transplants was not accompanied by blocked epidermal differentiation markers (keratins K1 and K10, involucrin, and filaggrin), the dissociation of morphogenesis and expression of these markers argues for independently controlled processes. A subpopulation of less adhesive cells, isolated from the 5-aza-CdR treated MyoD1-transfectants, had lost most epithelial characteristics in culture (epidermal keratins, desmosomal proteins, and surface-glycoprotein Gp90) and had shifted to a mesenchymal/myogenic phenotype (fibroblastic morphology, transactivation of Myf3 and myogenin, expression of myosin, desmin, vimentin, and Gp130). Moreover, the cells had lost the ability to stratify and remained as a monolayer of flat elongated cells in transplants. These subsequent changes from a fully differentiated keratinocyte to a mesenchymal/myogenic phenotype strongly argue for a complex "transdifferentiation" process which occurred in the original monoclonal human epidermal HaCaT cells. PMID:1371288

  19. Altered growth, differentiation, and responsiveness to epidermal growth factor of human embryonic mesenchymal cells of palate by persistent rubella virus infection

    SciTech Connect

    Yoneda, T.; Urade, M.; Sakuda, M.; Miyazaki, T.

    1986-05-01

    We previously demonstrated that human embryonic mesenchymal cells derived from the palate (HEMP cells) retain alkaline phosphatase (ALP) content and capacity for collagen synthesis after long-term culture, and their growth is markedly stimulated by epidermal growth factor (EGF). There was a dramatic decrease in ALP content and capacity to synthesize collagen in HEMP cells (HEMP-RV cells) persistently infected with rubella virus (RV). EGF increased ALP activity and decreased collagen synthesis in HEMP cells, whereas EGF showed no effect on these activities in HEMP-RV cells. Growth of HEMP-RV cells was slightly reduced compared with that of HEMP cells. EGF stimulated growth of HEMP cells and to a lesser extent of HEMP-RV cells. Binding of /sup 125/I-EGF to cell-surface receptors in HEMP-RV cells was, to our surprise, twice as much as that in HEMP cells. However, internalization of bound /sup 125/I-EGF in HEMP-RV cells was profoundly diminished. Thus, persistent RV infection causes not only changes in HEMP cell growth and differentiation but a decrease in or loss of HEMP cell responsiveness to EGF. The effects of persistent RV infection on palatal cell differentiation as well as growth may be responsible for the pathogenesis of congenital rubella. Furthermore, since HEMP cells appear to be closely related to osteoblasts, these results suggest a mechanism for RV-induced osseous abnormalities manifested in congenital rubella patients.

  20. Enhanced gefitinib-induced repression of the epidermal growth factor receptor pathway by ataxia telangiectasia-mutated kinase inhibition in non-small-cell lung cancer cells.

    PubMed

    Misumi, Keizo; Sun, Jiying; Kinomura, Aiko; Miyata, Yoshihiro; Okada, Morihito; Tashiro, Satoshi

    2016-04-01

    The epidermal growth factor receptor (EGFR) tyrosine kinase signaling pathways regulate cellular activities. The EGFR tyrosine kinase inhibitors (EGFR-TKIs) repress the EGFR pathway constitutively activated by somatic EGFR gene mutations and have drastically improved the prognosis of non-small-cell lung cancer (NSCLC) patients. However, some problems, including resistance, remain to be solved. Recently, combination therapy with EGFR-TKIs and cytotoxic agents has been shown to improve the prognosis of NSCLC patients. To enhance the anticancer effects of EGFR-TKIs, we examined the cross-talk of the EGFR pathways with ataxia telangiectasia-mutated (ATM) signaling pathways. ATM is a key protein kinase in the DNA damage response and is known to phosphorylate Akt, an EGFR downstream factor. We found that the combination of an ATM inhibitor, KU55933, and an EGFR-TKI, gefitinib, resulted in synergistic cell growth inhibition and induction of apoptosis in NSCLC cell lines carrying the sensitive EGFR mutation. We also found that KU55933 enhanced the gefitinib-dependent repression of the phosphorylation of EGFR and/or its downstream factors. ATM inhibition may facilitate the gefitinib-dependent repression of the phosphorylation of EGFR and/or its downstream factors, to exert anticancer effects against NSCLC cells with the sensitive EGFR mutation.

  1. Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor, epidermal growth factor, and thrombin.

    PubMed

    Gospodarowicz, D; Brown, K D; Birdwell, C R; Zetter, B R

    1978-06-01

    Because the response of human endothelial cells to growth factors and conditioning agents has broad implications for our understanding of wound healing angiogenesis, and human atherogenesis, we have investigated the responses of these cells to the fibroblast (FGF) and epidermal growth factors (EGF), as well as to the protease thrombin, which has been previously shown to potentiate the growth response of other cell types of FGF and EGF. Because the vascular endothelial cells that form the inner lining of blood vessels may be expected to be exposed to high thrombin concentrations after trauma or in pathological states associated with thrombosis, they are of particular interest with respect to the physiological role of this protease in potentiating cell proliferation. Our results indicate that human vascular endothelial cells respond poorly to either FGF or thrombin alone. In contrast, when cells are maintained in the presence of thrombin, their proliferative response to FGF is greatly increased even in cultures seeded at a density as low as 3 cells/mm2. Human vascular endothelial cells also respond to EGF and thrombin, although their rate of proliferation is much slower than when maintained with FGF and thrombin. In contrast, bovine vascular endothelial cells derived from vascular territories as diverse as the bovine heart, aortic arch, and umbilical vein respond maximally to FGF alone and neither respond to nor bind EGF. Furthermore, the response of bovine vascular endothelial cells to FGF was not potentiated by thrombin, indicating that the set of factors controlling the proliferation of vascular endothelial cells could be species-dependent. The requirement of cultured human vascular endothelial cells for thrombin could explain why the human cells, in contrast to bovine endothelial cells, are so difficult to maintain in tissue culture. Our results demonstrate that by using FGF and thrombin one can develop cultures of human vascular endothelial cells capable of

  2. Responses of epidermal cell turgor pressure and photosynthetic activity of leaves of the atmospheric epiphyte Tillandsia usneoides (Bromeliaceae) after exposure to high humidity.

    PubMed

    Martin, Craig E; Rux, Guido; Herppich, Werner B

    2013-01-01

    It has been well-established that many epiphytic bromeliads of the atmospheric-type morphology, i.e., with leaf surfaces completely covered by large, overlapping, multicellular trichomes, are capable of absorbing water vapor from the atmosphere when air humidity increases. It is much less clear, however, whether this absorption of water vapor can hydrate the living cells of the leaves and, as a consequence, enhance physiological processes in such cells. The goal of this research was to determine if the absorption of atmospheric water vapor by the atmospheric epiphyte Tillandsia usneoides results in an increase in turgor pressure in leaf epidermal cells that subtend the large trichomes, and, by using chlorophyll fluorescence techniques, to determine if the absorption of atmospheric water vapor by leaves of this epiphyte results in increased photosynthetic activity. Results of measurements on living cells of attached leaves of this epiphytic bromeliad, using a pressure probe and of whole-shoot fluorescence imaging analyses clearly illustrated that the turgor pressure of leaf epidermal cells did not increase, and the photosynthetic activity of leaves did not increase, following exposure of the leaves to high humidity air. These results experimentally demonstrate, for the first time, that the absorption of water vapor following increases in atmospheric humidity in atmospheric epiphytic bromeliads is mostly likely a physical phenomenon resulting from hydration of non-living leaf structures, e.g., trichomes, and has no physiological significance for the plant's living tissues.

  3. Responses of epidermal cell turgor pressure and photosynthetic activity of leaves of the atmospheric epiphyte Tillandsia usneoides (Bromeliaceae) after exposure to high humidity.

    PubMed

    Martin, Craig E; Rux, Guido; Herppich, Werner B

    2013-01-01

    It has been well-established that many epiphytic bromeliads of the atmospheric-type morphology, i.e., with leaf surfaces completely covered by large, overlapping, multicellular trichomes, are capable of absorbing water vapor from the atmosphere when air humidity increases. It is much less clear, however, whether this absorption of water vapor can hydrate the living cells of the leaves and, as a consequence, enhance physiological processes in such cells. The goal of this research was to determine if the absorption of atmospheric water vapor by the atmospheric epiphyte Tillandsia usneoides results in an increase in turgor pressure in leaf epidermal cells that subtend the large trichomes, and, by using chlorophyll fluorescence techniques, to determine if the absorption of atmospheric water vapor by leaves of this epiphyte results in increased photosynthetic activity. Results of measurements on living cells of attached leaves of this epiphytic bromeliad, using a pressure probe and of whole-shoot fluorescence imaging analyses clearly illustrated that the turgor pressure of leaf epidermal cells did not increase, and the photosynthetic activity of leaves did not increase, following exposure of the leaves to high humidity air. These results experimentally demonstrate, for the first time, that the absorption of water vapor following increases in atmospheric humidity in atmospheric epiphytic bromeliads is mostly likely a physical phenomenon resulting from hydration of non-living leaf structures, e.g., trichomes, and has no physiological significance for the plant's living tissues. PMID:23000465

  4. ADP-ribosylation factor 1 controls the activation of the phosphatidylinositol 3-kinase pathway to regulate epidermal growth factor-dependent growth and migration of breast cancer cells.

    PubMed

    Boulay, Pierre-Luc; Cotton, Mathieu; Melançon, Paul; Claing, Audrey

    2008-12-26

    Activation of intracellular signaling pathways by growth factors is one of the major causes of cancer development and progression. Recent studies have demonstrated that monomeric G proteins of the Ras family are key regulators of cell proliferation, migration, and invasion. Using an invasive breast cancer cell lines, we demonstrate that the ADP-ribosylation factor 1 (ARF1), a small GTPase classically associated with the Golgi, is an important regulator of the biological effects induced by epidermal growth factor. Here, we show that this ARF isoform is activated following epidermal growth factor stimulation and that, in MDA-MB-231 cells, ARF1 is found in dynamic plasma membrane ruffles. Inhibition of endogenous ARF1 expression results in the inhibition of breast cancer cell migration and proliferation. The underlying mechanism involves the activation of the phosphatidylinositol 3-kinase pathway. Our data demonstrate that depletion of ARF1 markedly impairs the recruitment of the phosphatidylinositol 3-kinase catalytic subunit (p110alpha) to the plasma membrane, and the association of the regulatory subunit (p85alpha) to the activated receptor. These results uncover a novel molecular mechanism by which ARF1 regulates breast cancer cell growth and invasion during cancer progression.

  5. Monoclonal antibody-conjugated superparamagnetic iron oxide nanoparticles for imaging of epidermal growth factor receptor-targeted cells and gliomas.

    PubMed

    Mu, Ketao; Zhang, Shun; Ai, Tao; Jiang, Jingjing; Yao, Yihao; Jiang, Lingyu; Zhou, Qing; Xiang, Hongbing; Zhu, Yanhong; Yang, Xiangliang; Zhu, Wenzhen

    2015-01-01

    The objective of this study was to successfully synthesize epidermal growth factor receptor monoclonal antibody-conjugated superparamagnetic iron oxide nanoparticles (EGFRmAb-SPIONs) and explore their biocompatibility and potential applications as a targeted magnetic resonance imaging (MRI) contrast agent for the EGFR-specific detection of brain glioma in vivo. After conjugation of EGFRmAb with SPIONs, the magnetic characteristics of EGFRmAb-SPIONs were investigated. Thereafter, the targeting abilities of EGFRmAb-SPIONs with MRI were qualitatively and quantitatively assessed in EGFR-positive C6 glioma cells in vitro and in a Wistar rat model bearing C6 glioma in vivo. Furthermore, the preliminary biocompatibility and toxicity of EGFRmAb-SPIONs were evaluated in normal rats through hematology assays and histopathologic analyses. Statistical analysis was performed using one-way analysis of variance and Student t-test, with a significance level of p < .05. From the results of EGFRmAb-SPION characterizations, the average particle size was 10.21 nm and the hydrodynamic diameter was 161.5 ± 2.12 nm. The saturation magnetization was 55 emu/g·Fe, and T2 relaxivity was 92.73 s-1mM-1 in distilled water. The preferential accumulation of the EGFRmAb-SPIONs within glioma and subsequent MRI contrast enhancement were demonstrated both in vitro in C6 cells and in vivo in rats bearing C6 glioma. After intravenous administration of EGFRmAb-SPIONs, T2-weighted MRI of the rat model with brain glioma exhibited an apparent hypointense region within glioma from 2 to 48 hours. The maximal image contrast was reached at 24 hours, where the signal intensity decreased and the R2 value increased by 30% compared to baseline. However, T2-weighted imaging of the rat model administered with SPIONs showed no visible signal changes within the tumor over the same time period. Moreover, no evident toxicities in vitro and in vivo with EGFRmAb-SPIONs were clearly identified based on the laboratory

  6. The tricornered gene, which is required for the integrity of epidermal cell extensions, encodes the Drosophila nuclear DBF2-related kinase.

    PubMed

    Geng, W; He, B; Wang, M; Adler, P N

    2000-12-01

    During their differentiation epidermal cells of Drosophila form a rich variety of polarized structures. These include the epidermal hairs that decorate much of the adult cuticular surface, the shafts of the bristle sense organs, the lateral extensions of the arista, and the larval denticles. These cuticular structures are produced by cytoskeletal-mediated outgrowths of epidermal cells. Mutations in the tricornered gene result in the splitting or branching of all of these structures. Thus, tricornered function appears to be important for maintaining the integrity of the outgrowths. tricornered mutations however do not have major effects on the growth or shape of these cellular extensions. Inhibiting actin polymerization in differentiating cells by cytochalasin D or latrunculin A treatment also induces the splitting of hairs and bristles, suggesting that the actin cytoskeleton might be a target of tricornered. However, the drugs also result in short, fat, and occasionally malformed hairs and bristles. The data suggest that the function of the actin cytoskeleton is important for maintaining the integrity of cellular extensions as well as their growth and shape. Thus, if tricornered causes the splitting of cellular extensions by interacting with the actin cytoskeleton it likely does so in a subtle way. Consistent with this possibility we found that a weak tricornered mutant is hypersensitive to cytochalasin D. We have cloned the tricornered gene and found that it encodes the Drosophila NDR kinase. This is a conserved ser/thr protein kinase found in Caenorhabditis elegans and humans that is related to a number of kinases that have been found to be important in controlling cell structure and proliferation.

  7. The tricornered gene, which is required for the integrity of epidermal cell extensions, encodes the Drosophila nuclear DBF2-related kinase.

    PubMed Central

    Geng, W; He, B; Wang, M; Adler, P N

    2000-01-01

    During their differentiation epidermal cells of Drosophila form a rich variety of polarized structures. These include the epidermal hairs that decorate much of the adult cuticular surface, the shafts of the bristle sense organs, the lateral extensions of the arista, and the larval denticles. These cuticular structures are produced by cytoskeletal-mediated outgrowths of epidermal cells. Mutations in the tricornered gene result in the splitting or branching of all of these structures. Thus, tricornered function appears to be important for maintaining the integrity of the outgrowths. tricornered mutations however do not have major effects on the growth or shape of these cellular extensions. Inhibiting actin polymerization in differentiating cells by cytochalasin D or latrunculin A treatment also induces the splitting of hairs and bristles, suggesting that the actin cytoskeleton might be a target of tricornered. However, the drugs also result in short, fat, and occasionally malformed hairs and bristles. The data suggest that the function of the actin cytoskeleton is important for maintaining the integrity of cellular extensions as well as their growth and shape. Thus, if tricornered causes the splitting of cellular extensions by interacting with the actin cytoskeleton it likely does so in a subtle way. Consistent with this possibility we found that a weak tricornered mutant is hypersensitive to cytochalasin D. We have cloned the tricornered gene and found that it encodes the Drosophila NDR kinase. This is a conserved ser/thr protein kinase found in Caenorhabditis elegans and humans that is related to a number of kinases that have been found to be important in controlling cell structure and proliferation. PMID:11102376

  8. Epidermal growth factor and Ras regulate gene expression in GH4 pituitary cells by separate, antagonistic signal transduction pathways.

    PubMed Central

    Pickett, C A; Gutierrez-Hartmann, A

    1995-01-01

    We have previously demonstrated that epidermal growth factor (EGF) produces activation of the rat prolactin (rPRL) promoter in GH4 neuroendocrine cells via a Ras-independent mechanism. This Ras independence of the EGF response appears to be cell rather than promoter specific. Oncogenic Ras also produces activation of the rPRL promoter when transfected into GH4 cells and requires the sequential activation of Raf kinase, mitogen-activated protein (MAP) kinase, and c-Ets-1/GHF-1 to mediate this response. In these studies, we have investigated the interaction between EGF and Ras in stimulating rPRL promoter activity and the role of Raf and MAP kinases in mediating the EGF response. We have also examined the role of several transcription factors and used various promoter mutants of the rPRL gene in order to better define the trans- and cis-acting components of the EGF response. EGF treatment of GH4 cells inhibits activation of the rPRL promoter produced by transfection of V12Ras from 24- to 4-fold in an EGF dose-dependent manner. This antagonistic effect of EGF and Ras is mutual in that transfection of V12Ras also blocks EGF-induced activation of the rPRL promoter in a Ras dose-dependent manner, from 5.5- to 1.6-fold. Transfection of a plasmid encoding the dominant-negative Raf C4 blocks Ras-induced activation by 66% but fails to inhibit EGF-mediated activation of the rPRL promoter. Similarly, transfection of a construct encoding an inhibitory form of MAP kinase decreases the Ras response by 50% but does not inhibit the EGF response. Previous studies have demonstrated that c-Ets-1 is necessary and that GHF-1 acts synergistically with c-Ets-1 in the Ras response of the rPRL promoter. In contrast, overexpression of neither c-Ets-1 nor GHF-1 enhanced EGF-mediated activation of the rPRL promoter, and dominant-negative forms of these transcription factors failed to inhibit the EGF response. Using 5' deletion and site-specific mutations, we have mapped the EGF response to two

  9. Epidermal Electronics

    NASA Astrophysics Data System (ADS)

    Kim, Dae-Hyeong; Lu, Nanshu; Ma, Rui; Kim, Yun-Soung; Kim, Rak-Hwan; Wang, Shuodao; Wu, Jian; Won, Sang Min; Tao, Hu; Islam, Ahmad; Yu, Ki Jun; Kim, Tae-il; Chowdhury, Raeed; Ying, Ming; Xu, Lizhi; Li, Ming; Chung, Hyun-Joong; Keum, Hohyun; McCormick, Martin; Liu, Ping; Zhang, Yong-Wei; Omenetto, Fiorenzo G.; Huang, Yonggang; Coleman, Todd; Rogers, John A.

    2011-08-01

    We report classes of electronic systems that achieve thicknesses, effective elastic moduli, bending stiffnesses, and areal mass densities matched to the epidermis. Unlike traditional wafer-based technologies, laminating such devices onto the skin leads to conformal contact and adequate adhesion based on van der Waals interactions alone, in a manner that is mechanically invisible to the user. We describe systems incorporating electrophysiological, temperature, and strain sensors, as well as transistors, light-emitting diodes, photodetectors, radio frequency inductors, capacitors, oscillators, and rectifying diodes. Solar cells and wireless coils provide options for power supply. We used this type of technology to measure electrical activity produced by the heart, brain, and skeletal muscles and show that the resulting data contain sufficient information for an unusual type of computer game controller.

  10. Effects of transforming growth factor beta and epidermal growth factor on cell proliferation and the formation of bone nodules in isolated fetal rat calvaria cells.

    PubMed

    Antosz, M E; Bellows, C G; Aubin, J E

    1989-08-01

    When cells enzymatically isolated from fetal rat calvaria (RC cells) are cultured in vitro in the presence of ascorbic acid and Na beta-glycerophosphate, discrete three-dimensional nodules form with the histologic, immunohistochemical, and ultrastructural characteristics of bone (Bellows et al; Calcified Tissue International 38:143-154, 1986; Bhargava et al., Bone, 9:155-163, 1988). Quantitation of the number of bone nodules that forms provides a colony assay for osteoprogenitor cells present in the RC population (Bellows and Aubin, Develop. Biol., 133:8-13, 1989). Continuous culture with either epidermal growth factor (EGF) or transforming growth factor beta (TGF-beta) results in dose-dependent inhibition of bone nodule formation; however, the former causes increased proliferation and saturation density, while the latter reduces both parameters. Addition of EGF (48 h pulse, 2-200 ng/ml) to RC cells at day 1 after plating results in increased proliferation and population saturation density and an increased number of bone nodules formed. Similar pulses at confluence and in postconfluent multilayered cultures when nodules first begin forming (approx. day 11) inhibited bone nodule formation and resulted in a smaller stimulation of cell proliferation. Forty-eight hour pulses of TGF-beta (0.01-1 ng/ml) reduced bone nodule formation and proliferation at all times examined, with pulses on day 1 causing maximum inhibition. The effects of pulses with TGF-beta and EGF on inhibition of nodule formation are independent of the presence of serum in the culture medium during the pulse. The data suggest that whereas EGF can either stimulate or inhibit the formation of bone nodules depending upon the time and duration of exposure, TGF-B inhibits bone nodule formation under all conditions tested. Moreover, these effects on osteoprogenitor cell differentiation do not always correlate with the effects of the growth factors on RC cell proliferation. PMID:2787326

  11. Microsurgical removal of epidermal and cortical cells: evidence that the gravitropic signal moves through the outer cell layers in primary roots of maize

    NASA Technical Reports Server (NTRS)

    Yang, R. L.; Evans, M. L.; Moore, R.

    1990-01-01

    There is general agreement that during root gravitropism some sort of growth-modifying signal moves from the cap to the elongation zone and that this signal ultimately induces the curvature that leads to reorientation of the root. However, there is disagreement regarding both the nature of the signal and the pathway of its movement from the root cap to the elongation zone. We examined the pathway of movement by testing gravitropism in primary roots of maize (Zea mays L.) from which narrow (0.5 mm) rings of epidermal and cortical tissue were surgically removed from various positions within the elongation zone. When roots were girdled in the apical part of the elongation zone gravitropic curvature occurred apical to the girdle but not basal to the girdle. Filling the girdle with agar allowed curvature basal to the girdle to occur. Shallow girdles, in which only two or three cell layers (epidermis plus one or two cortical cell layers) were removed, prevented or greatly delayed gravitropic curvature basal to the girdle. The results indicate that the gravitropic signal moves basipetally through the outermost cell layers, perhaps through the epidermis itself.

  12. In vitro primary sensitization and restimulation of hapten-specific T cells by fresh and cultured human epidermal Langerhans' cells.

    PubMed Central

    Moulon, C; Péguet-Navarro, J; Courtellemont, P; Redziniak, G; Schmitt, D

    1993-01-01

    We examined the capacity of human Langerhans's cells (LC) to sensitize autologous T cells to the trinitrophenyl hapten (TNP) in vitro. Two-day cultured Langerhans' cells, but not freshly prepared Langerhans' cells, can induce in vitro primary proliferative reactions to the TNP hapten. Using a CD45RA+ naive T-cell subset, similar results were found, therefore making the possibility of a previous in vivo T-cell contact with the hapten unlikely. The primary in vitro response was strongly inhibited by monoclonal antibodies to major histocompatibility complex (MHC) class I and II, CD4 antigens and ICAM-1 and LFA-3 adhesion molecules. Furthermore, we found that fresh LC can prime T cells to TNP, as revealed by a significant secondary T-cell proliferation after restimulation of the recovered T lymphocytes by fresh hapten-modified autologous LC. Nevertheless, the ability of these fresh LC to stimulate in vitro secondary hapten-specific T-cell proliferation was very limited in comparison with that of 2-day incubated Langerhans' cells. After secondary stimulation with TNP-cultured LC, sensitized T cells could be non-specifically expanded without losing hapten specificity. The TNP-specific T-cell lines were mostly of the CD4+ phenotype. The present findings extend previous studies in the mouse, showing that culture LC are potent antigen-presenting cells (APC) in primary hapten-dependent proliferation assays. Furthermore, this in vitro priming assay, using cultured human Langerhans' cells as APC, might be useful to analyse the early steps of T-cell sensitization and subsequently to develop in vitro predictive tests allowing detection of sensitizing compounds. PMID:7507088

  13. The role of fetal epithelial tissues in the maturation/differentiation of bone marrow-derived precursors into dendritic epidermal T cells (DETC) of the mouse.

    PubMed

    Stingl, G; Elbe, A; Paer, E; Kilgus, O; Strohal, R; Schreiber, S

    1991-01-01

    Our attempts to clarify the contribution of the thymic vs. the cutaneous microenvironment in the maturation of dendritic epidermal T cell (DETC) precursors into DETC gave diverse results. In one series of experiments, we found that i.v. injection of fetal thymocytes (containing a TCR V gamma 3-expressing subpopulation), but not of adult thymocytes (containing no TCR V gamma 3+ cells) results in the appearance of CD3/TCR V gamma 3+ dendritic epidermal cells (=DETC). In other experiments, we have obtained evidence that transplantation of day 16 fetal skin onto a Thy-1-disparate recipient results in the appearance of donor-type DETC. Our further observation that the transplanted skin contains CD45+/Thy-1+/CD3- lymphocytes, but no mature T cells, therefore implies that fetal skin can provide stimuli promoting the expression of CD3/TCR genes in immature (CD3-) DETC precursors. It remains to be seen whether both or only one of these maturational pathways are (is) followed under physiological conditions.

  14. A Barley ROP GTPase ACTIVATING PROTEIN Associates with Microtubules and Regulates Entry of the Barley Powdery Mildew Fungus into Leaf Epidermal Cells[C][W

    PubMed Central

    Hoefle, Caroline; Huesmann, Christina; Schultheiss, Holger; Börnke, Frederik; Hensel, Götz; Kumlehn, Jochen; Hückelhoven, Ralph

    2011-01-01

    Little is known about the function of host factors involved in disease susceptibility. The barley (Hordeum vulgare) ROP (RHO of plants) G-protein RACB is required for full susceptibility of the leaf epidermis to invasion by the biotrophic fungus Blumeria graminis f. sp hordei. Stable transgenic knockdown of RACB reduced the ability of barley to accommodate haustoria of B. graminis in intact epidermal leaf cells and to form hairs on the root epidermis, suggesting that RACB is a common element of root hair outgrowth and ingrowth of haustoria in leaf epidermal cells. We further identified a barley MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN (MAGAP1) interacting with RACB in yeast and in planta. Fluorescent MAGAP1 decorated cortical microtubules and was recruited by activated RACB to the cell periphery. Under fungal attack, MAGAP1-labeled microtubules built a polarized network at sites of successful defense. By contrast, microtubules loosened where the fungus succeeded in penetration. Genetic evidence suggests a function of MAGAP1 in limiting susceptibility to penetration by B. graminis. Additionally, MAGAP1 influenced the polar organization of cortical microtubules. These results add to our understanding of how intact plant cells accommodate fungal infection structures and suggest that RACB and MAGAP1 might be antagonistic players in cytoskeleton organization for fungal entry. PMID:21685259

  15. Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells

    PubMed Central

    Zhang, Hui-ming; Talbot, Mark J.; McCurdy, David W.; Patrick, John W.; Offler, Christina E.

    2015-01-01

    Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca2+ levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane–microtubule inter-relationship is discussed. PMID:26136268

  16. Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells.

    PubMed

    Zhang, Hui-ming; Talbot, Mark J; McCurdy, David W; Patrick, John W; Offler, Christina E

    2015-09-01

    Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca(2+) levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane-microtubule inter-relationship is discussed.

  17. Epidermal growth factor promotes a mesenchymal over an amoeboid motility of MDA-MB-231 cells embedded within a 3D collagen matrix

    NASA Astrophysics Data System (ADS)

    Geum, Dongil T.; Kim, Beum Jun; Chang, Audrey E.; Hall, Matthew S.; Wu, Mingming

    2016-01-01

    The receptor of epidermal growth factor (EGFR) critically regulates tumor cell invasion and is a potent therapeutic target for treatment of many types of cancers, including carcinomas and glioblastomas. It is known that EGF regulates cell motility when tumor cells are embedded within a 3D biomatrix. However, roles of EGF in modulating tumor cell motility phenotype are largely unknown. In this article, we report that EGF promotes a mesenchymal over an amoeboid motility phenotype using a malignant breast tumor cell line, MDA-MB-231, embedded within a 3D collagen matrix. Amoeboid cells are rounded in shape, while mesenchymal cells are elongated, and their migrations are governed by a distinctly different set of biomolecules. Using single cell tracking analysis, we also show that EGF promotes cell dissemination through a significant increase in cell persistence along with a moderate increase of speed. The increase of persistence is correlated with the increase of the percentage of the mesenchymal cells within the population. Our work reveals a novel role of microenvironmental cue, EGF, in modulating heterogeneity and plasticity of tumor cell motility phenotype. In addition, it suggests a potential visual cue for diagnosing invasive states of breast cancer cells. This work can be easily extended beyond breast cancer cells.

  18. Baseline, Trend, and Normalization of Carcinoembryonic Antigen as Prognostic Factors in Epidermal Growth Factor Receptor-Mutant Nonsmall Cell Lung Cancer Patients Treated With First-Line Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors

    PubMed Central

    Chen, Yu-Mu; Lai, Chien-Hao; Chang, Huang-Chih; Chao, Tung-Ying; Tseng, Chia-Cheng; Fang, Wen-Feng; Wang, Chin-Chou; Chung, Yu-Hsiu; Huang, Kuo-Tung; Chen, Hung-Cheng; Chang, Ya-Chun; Lin, Meng-Chih

    2015-01-01

    Abstract Among epidermal growth factor receptor (EGFR) mutation status unknown nonsmall cell lung cancer (NSCLC) patients, those with higher carcinoembryonic antigen (CEA) level are more likely to response to EGFR-tyrosine kinase inhibitors (TKIs) because they tend to have mutant epidermal growth factor receptor (EGFR). However, patients with higher CEA also have more tumor burden. With the above paradoxical evidence, it is prudent to understand the prognostic significance of baseline CEA in patients with EGFR-mutant NSCLC treated with first-line EGFR-TKIs. The clinical significance of the trend in CEA after treatment and the impact of CEA normalization during EGFR-TKI therapy are also unknown and potentially important. A total of 241 patients who received first-line EGFR-TKIs were included. As to baseline CEA, patients were divided into normal, low, and high baseline CEA by cut point determined by receiver operating characteristic curves. As to CEA responses, patients were divided into 3 groups accordingly to their amount of CEA change after taking TKIs. In group A, 1-month follow-up CEA level decreased more than 35% with nadir CEA normalization; in group B, 1-month follow-up CEA level decreased more than 35% without nadir CEA normalization; and in group C, 1-month follow-up CEA level decreased less than 35% or increased. Patients with higher baseline CEA levels had shorter progression-free survival (PFS) and overall survival (OS) (CEA > 32 vs 5–32 vs <5 ng/mL, PFS = 8.8 vs 11.3 vs 14.4 months, respectively, P < 0.001; OS = 17.8 vs 22.0 vs 27.9 months, respectively, P = 0.01). For trend and CEA normalization in groups A, B, and C, PFS was 14.3, 10.6, and 7.1 months, respectively (P < 0.001); OS was 29.7, 20.0, and 16.2 months, respectively (P < 0.001). Baseline, trend, and normalization of CEA levels are potential prognostic markers for patients with EGFR-mutant advanced NSCLC treated with first line EGFR-TKIs. PMID:26683939

  19. Epidermal chalone and cyclic AMP: an in vivo study.

    PubMed

    Elgjo, K

    1975-01-01

    Water extracts of skin contain two factors that inhibit epidermal cell proliferation: one substance inhibits epidermal cells in the G2 phase (the epidermal G2 inhibitor), and another inhibits the transit of cells from the G1 phase into the S phase (the epidermal G1 inhibitor). Pretreatment of mice with a beta-receptor antagonist (propranolol) abolished the activity of the G2 inhibitor but not that of the G1 inhibitor. After pretreatment with both propranolol and a phosphodiesterase inhibitor (caffine)the G2 inhibitor had full effect. Cafine alone had a moderately inhibitory effect on epidermal G2 cells and enhanced the depressing effect of the G1 inhibitor on epidermal DNA synthesis. AMP level in epidermis to be active. Cyclic AMP is probably also involved in the regulation of the rate of transit of epidermal G1 cells into the S phase but the epidermal cyclic AMP level seems not to be so critical for the efficacy of the epidermal G2 inhibitor in epidermal cell differentiation. PMID:162919

  20. Epidermal growth factor receptor tyrosine kinase inhibitors as initial therapy for non-small cell lung cancer: focus on epidermal growth factor receptor mutation testing and mutation-positive patients.

    PubMed

    Roengvoraphoj, Monic; Tsongalis, Gregory J; Dragnev, Konstantin H; Rigas, James R

    2013-12-01

    Activation of the epidermal growth factor receptor (EGFR) pathway has been implicated in tumorigenesis in non-small cell lung cancer (NSCLC), the most common type of lung cancer. As a result, EGFR has become a key focus for the development of personalized therapy, with several molecular biomarkers having been investigated as potential predictors of response with EGFR tyrosine kinase inhibitors (TKIs) in NSCLC (e.g., EGFR expression, EGFR gene copy gain, and EGFR mutations). Of these, activating mutations in EGFR have thus far given the most consistent results based on the available evidence from preclinical studies and clinical trials. In an attempt to identify patients who are most likely to benefit from treatment with EGFR TKIs, EGFR mutation testing is being increasingly utilized in clinical practice. Currently in the United States, no EGFR TKI or accompanying mutational test is approved for the identification and first-line treatment of patients with advanced NSCLC. However, the first-generation EGFR TKIs, erlotinib and gefitinib, as well as investigational ErbB family TKIs and EGFR mutation testing methods are being evaluated in this setting. This review will discuss EGFR mutation testing as a biomarker of response to EGFR TKIs and the evolution of EGFR mutational analysis in NSCLC. Completed and ongoing clinical trials evaluating currently available or investigational EGFR TKIs as first-line therapy in molecularly and clinically selected patients with NSCLC, with a focus on trials in patients whose tumors have EGFR mutations, will also be reviewed.

  1. Release of infectious cells from epidermal ulcers in Ichthyophonus sp.–infected Pacific herring (Clupea pallasii): Evidence for multiple mechanisms of transmission

    USGS Publications Warehouse

    Hershberger, Paul K.; Gregg, Jacob L.; Kocan, R.M.

    2010-01-01

    A common clinical sign of ichthyophoniasis in herring and trout is “sandpaper” skin, a roughening of the epidermis characterized by the appearance of small papules, followed by ulceration and sloughing of the epithelium; early investigators hypothesized that these ulcers might be a means of transmitting the parasite, Ichthyophonus sp., without the necessity of ingesting an infected host. We examined the cells associated with the epidermal lesions and confirmed that they were viable Ichthyophonus sp. cells that were readily released from the skin into the mucous layer and ultimately into the aquatic environment. The released cells were infectious when injected into the body cavity of specific-pathogen-free herring. Our hypothesis is that different mechanisms of transmission occur in carnivorous and planktivorous hosts: Planktonic feeders become infected by ingestion of ulcer-derived cells, while carnivores become infected by ingestion of whole infected fish.

  2. Construction of Epidermal Growth Factor Receptor Peptide Magnetic Nanovesicles with Lipid Bilayers for Enhanced Capture of Liver Cancer Circulating Tumor Cells.

    PubMed

    Ding, Jian; Wang, Kai; Tang, Wen-Jie; Li, Dan; Wei, You-Zhen; Lu, Ying; Li, Zong-Hai; Liang, Xiao-Fei

    2016-09-20

    Highly effective targeted tumor recognition via vectors is crucial for cancer detection. In contrast to antibodies and proteins, peptides are direct targeting ligands with a low molecular weight. In the present study, a peptide magnetic nanovector platform containing a lipid bilayer was designed using a peptide amphiphile (PA) as a skeleton material in a controlled manner without surface modification. Fluorescein isothiocyanate-labeled epidermal growth factor receptor (EGFR) peptide nanoparticles (NPs) could specifically bind to EGFR-positive liver tumor cells. EGFR peptide magnetic vesicles (EPMVs) could efficiently recognize and separate hepatoma carcinoma cells from cell solutions and treated blood samples (ratio of magnetic EPMVs versus anti-EpCAM NPs: 3.5 ± 0.29). Analysis of the circulating tumor cell (CTC) count in blood samples from 32 patients with liver cancer showed that EPMVs could be effectively applied for CTC capture. Thus, this nanoscale, targeted cargo-packaging technology may be useful for designing cancer diagnostic systems.

  3. Synergistic effect of targeting the epidermal growth factor receptor and hyaluronan synthesis in oesophageal squamous cell carcinoma cells

    PubMed Central

    Kretschmer, I; Freudenberger, T; Twarock, S; Fischer, J W

    2015-01-01

    Background and Purpose Worldwide, oesophageal cancer is the eighth most common cancer and has a very poor survival rate. In order to identify new tolerable treatment options for oesophageal squamous cell carcinoma (ESCC), erlotinib was tested with moderate efficacy in phase I and II studies. As 4-methylumbelliferone (4-MU), an hyaluronan (HA) synthesis inhibitor showed anti-cancer effects in vitro, and in ESCC xenograft tumours, we investigated whether the anti-cancer effects of erlotinib could be augmented by combining it with 4-MU. Experimental Approach ESCC cell lines were treated with erlotinib or gefitinib (1 μmol·L−1) and 4-MU (300 μmol·L−1), and the cell count, cell cycle progression and migration were determined as compared to the single agents and the solvent-control. Key Results The combination of erlotinib and 4-MU synergistically inhibited the proliferation of ESCC cell lines. Furthermore, the migration speed of ESCC cell line KYSE-410 in gap closure assays was significantly reduced by the combination of erlotinib and 4-MU. Decreased ERK phosphorylation could explain the anti-proliferative and anti-migratory effects in the combined treatment group. Finally, the combination was additionally able to decrease the growth of multicellular tumour spheroids, a three-dimensional cell culture model that was associated with sustained inhibition of ERK1/2 phosphorylation. Conclusions and Implications The combination of 4-MU and erlotinib showed promising anti-cancer efficacies in the ESCC cell lines. PMID:26140525

  4. Size-dependent effects of tungsten carbide-cobalt particles on oxygen radical production and activation of cell signaling pathways in murine epidermal cells

    SciTech Connect

    Ding, M.; Kisin, E.R.; Zhao, J.; Bowman, L.; Lu, Y.; Jiang, B.; Leonard, S.; Vallyathan, V.; Castranova, V.; Murray, A.R.; Fadeel, B.; Shvedova, A.A.

    2009-12-15

    Hard metal or cemented carbide consists of a mixture of tungsten carbide (WC) (85%) and metallic cobalt (Co) (5-15%). WC-Co is considered to be potentially carcinogenic to humans. However, no comparison of the adverse effects of nano-sized WC-Co particles is available to date. In the present study, we compared the ability of nano- and fine-sized WC-Co particles to form free radicals and propensity to activate the transcription factors, AP-1 and NF-kappaB, along with stimulation of mitogen-activated protein kinase (MAPK) signaling pathways in a mouse epidermal cell line (JB6 P{sup +}). Our results demonstrated that nano-WC-Co generated a higher level of hydroxyl radicals, induced greater oxidative stress, as evidenced by a decrease of GSH levels, and caused faster JB6 P{sup +} cell growth/proliferation than observed after exposure of cells to fine WC-Co. In addition, nano-WC-Co activated AP-1 and NF-kappaB more efficiently in JB6{sup +/+} cells as compared to fine WC-Co. Experiments using AP-1-luciferase reporter transgenic mice confirmed the activation of AP-1 by nano-WC-Co. Nano- and fine-sized WC-Co particles also stimulated MAPKs, including ERKs, p38, and JNKs with significantly higher potency of nano-WC-Co. Finally, co-incubation of the JB6{sup +/+} cells with N-acetyl-cysteine decreased AP-1 activation and phosphorylation of ERKs, p38 kinase, and JNKs, thus suggesting that oxidative stress is involved in WC-Co-induced toxicity and AP-1 activation.

  5. Faster DNA Repair of Ultraviolet-Induced Cyclobutane Pyrimidine Dimers and Lower Sensitivity to Apoptosis in Human Corneal Epithelial Cells than in Epidermal Keratinocytes.

    PubMed

    Mallet, Justin D; Dorr, Marie M; Drigeard Desgarnier, Marie-Catherine; Bastien, Nathalie; Gendron, Sébastien P; Rochette, Patrick J

    2016-01-01

    Absorption of UV rays by DNA generates the formation of mutagenic cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP). These damages are the major cause of skin cancer because in turn, they can lead to signature UV mutations. The eye is exposed to UV light, but the cornea is orders of magnitude less prone to UV-induced cancer. In an attempt to shed light on this paradox, we compared cells of the corneal epithelium and the epidermis for UVB-induced DNA damage frequency, repair and cell death sensitivity. We found similar CPD levels but a 4-time faster UVB-induced CPD, but not 6-4PP, repair and lower UV-induced apoptosis sensitivity in corneal epithelial cells than epidermal. We then investigated levels of DDB2, a UV-induced DNA damage recognition protein mostly impacting CPD repair, XPC, essential for the repair of both CPD and 6-4PP and p53 a protein upstream of the genotoxic stress response. We found more DDB2, XPC and p53 in corneal epithelial cells than in epidermal cells. According to our results analyzing the protein stability of DDB2 and XPC, the higher level of DDB2 and XPC in corneal epithelial cells is most likely due to an increased stability of the protein. Taken together, our results show that corneal epithelial cells have a better efficiency to repair UV-induced mutagenic CPD. On the other hand, they are less prone to UV-induced apoptosis, which could be related to the fact that since the repair is more efficient in the HCEC, the need to eliminate highly damaged cells by apoptosis is reduced. PMID:27611318

  6. Faster DNA Repair of Ultraviolet-Induced Cyclobutane Pyrimidine Dimers and Lower Sensitivity to Apoptosis in Human Corneal Epithelial Cells than in Epidermal Keratinocytes

    PubMed Central

    Mallet, Justin D.; Bastien, Nathalie; Gendron, Sébastien P.; Rochette, Patrick J.

    2016-01-01

    Absorption of UV rays by DNA generates the formation of mutagenic cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts (6-4PP). These damages are the major cause of skin cancer because in turn, they can lead to signature UV mutations. The eye is exposed to UV light, but the cornea is orders of magnitude less prone to UV-induced cancer. In an attempt to shed light on this paradox, we compared cells of the corneal epithelium and the epidermis for UVB-induced DNA damage frequency, repair and cell death sensitivity. We found similar CPD levels but a 4-time faster UVB-induced CPD, but not 6-4PP, repair and lower UV-induced apoptosis sensitivity in corneal epithelial cells than epidermal. We then investigated levels of DDB2, a UV-induced DNA damage recognition protein mostly impacting CPD repair, XPC, essential for the repair of both CPD and 6-4PP and p53 a protein upstream of the genotoxic stress response. We found more DDB2, XPC and p53 in corneal epithelial cells than in epidermal cells. According to our results analyzing the protein stability of DDB2 and XPC, the higher level of DDB2 and XPC in corneal epithelial cells is most likely due to an increased stability of the protein. Taken together, our results show that corneal epithelial cells have a better efficiency to repair UV-induced mutagenic CPD. On the other hand, they are less prone to UV-induced apoptosis, which could be related to the fact that since the repair is more efficient in the HCEC, the need to eliminate highly damaged cells by apoptosis is reduced. PMID:27611318

  7. The role of peroxisome proliferator-activated receptor-{beta}/{delta} in epidermal growth factor-induced HaCaT cell proliferation

    SciTech Connect

    Liang Pengfei; Jiang Bimei; Yang Xinghua; Xiao Xianzhong Huang Xu; Long Jianhong; Zhang Pihong; Zhang Minghua; Xiao Muzhang; Xie Tinghong; Huang Xiaoyuan

    2008-10-15

    Epidermal growth factor (EGF) has been shown to be a potent mitogen for epidermal cells both in vitro and in vivo, thus contributing to the development of an organism. It has recently become clear that peroxisome proliferator-activated receptor-{beta}/{delta} (PPAR{beta}/{delta}) expression and activation is involved in the cell proliferation. However, little is known about the role of PPAR{beta}/{delta} in EGF-induced proliferation of HaCaT keratinocytes. In this study, HaCaT cells were cultured in the presence and absence of EGF and we identified that EGF induced an increase of PPAR{beta}/{delta} mRNA and protein level expression in time-dependent and dose-dependent manner, and AG1487, an EGF receptor (EGFR) special inhibitor, caused attenuation of PPAR{beta}/{delta} protein expression. Electrophoretic mobility shift assay (EMSA) revealed that EGF significantly increased PPAR{beta}/{delta} binding activity in HaCaT keratinocytes. Antisense phosphorothioate oligonucleotides (asODNs) against PPAR{beta}/{delta} caused selectively inhibition of PPAR{beta}/{delta} protein content induced by EGF and significantly attenuated EGF-mediated cell proliferation. Treatment of the cells with L165041, a specific synthetic ligand for PPAR{beta}/{delta}, significantly enhanced EGF-mediated cell proliferation. Finally, c-Jun ablation inhibited PPAR{beta}/{delta} up-regulation induced by EGF, and chromatin immunoprecipitation (ChIP) showed that c-Jun bound to the PPAR{beta}/{delta} promoter and the binding increased in EGF-stimulated cells. These results demonstrate that EGF induces PPAR{beta}/{delta} expression in a c-Jun-dependent manner and PPAR{beta}/{delta} plays a vital role in EGF-stimulated proliferation of HaCaT cells.

  8. Epidermal Fatty Acid Binding Protein (E-FABP) Is Not Required for the Generation or Maintenance of Effector and Memory T Cells following Infection with Listeria monocytogenes.

    PubMed

    Li, Bing; Schmidt, Nathan W

    2016-01-01

    Following activation of naïve T cells there are dynamic changes in the metabolic pathways used by T cells to support both the energetic needs of the cell and the macromolecules required for growth and proliferation. Among other changes, lipid metabolism undergoes dynamic transitions between fatty acid oxidation and fatty acid synthesis as cells progress from naïve to effector and effector to memory T cells. The hydrophobic nature of lipids requires that they be bound to protein chaperones within a cell. Fatty acid binding proteins (FABPs) represent a large class of lipid chaperones, with epidermal FABP (E-FABP) expressed in T cells. The objective of this study was to determine the contribution of E-FABP in antigen-specific T cell responses. Following infection with Listeria monocytogenes, we observed similar clonal expansion, contraction and formation of memory CD8 T cells in WT and E-FABP-/- mice, which also exhibited similar phenotypic and functional characteristics. Analysis of Listeria-specific CD4 T cells also revealed no defect in the expansion, contraction, and formation of memory CD4 T cells in E-FABP-/- mice. These data demonstrate that E-FABP is dispensable for antigen-specific T cell responses following a bacterial infection. PMID:27588422

  9. Epidermal Fatty Acid Binding Protein (E-FABP) Is Not Required for the Generation or Maintenance of Effector and Memory T Cells following Infection with Listeria monocytogenes

    PubMed Central

    Li, Bing; Schmidt, Nathan W.

    2016-01-01

    Following activation of naïve T cells there are dynamic changes in the metabolic pathways used by T cells to support both the energetic needs of the cell and the macromolecules required for growth and proliferation. Among other changes, lipid metabolism undergoes dynamic transitions between fatty acid oxidation and fatty acid synthesis as cells progress from naïve to effector and effector to memory T cells. The hydrophobic nature of lipids requires that they be bound to protein chaperones within a cell. Fatty acid binding proteins (FABPs) represent a large class of lipid chaperones, with epidermal FABP (E-FABP) expressed in T cells. The objective of this study was to determine the contribution of E-FABP in antigen-specific T cell responses. Following infection with Listeria monocytogenes, we observed similar clonal expansion, contraction and formation of memory CD8 T cells in WT and E-FABP-/- mice, which also exhibited similar phenotypic and functional characteristics. Analysis of Listeria-specific CD4 T cells also revealed no defect in the expansion, contraction, and formation of memory CD4 T cells in E-FABP-/- mice. These data demonstrate that E-FABP is dispensable for antigen-specific T cell responses following a bacterial infection. PMID:27588422

  10. Epidermal growth factor receptors in the oesophagus.

    PubMed Central

    Jankowski, J; Murphy, S; Coghill, G; Grant, A; Wormsley, K G; Sanders, D S; Kerr, M; Hopwood, D

    1992-01-01

    The quantity and distribution of epidermal growth factor receptors (EGF-R) in oesophageal mucosa was studied in the oesophagus in order to determine its role in oesophageal disease. Fifty five biopsies were taken from different levels of the oesophagus in 25 consecutive patients undergoing endoscopy. Another group of eight patients with histologically proven Barrett's oesophagitis had a biopsy taken from the area of columnar lined oesophagus. A peripheral, membranous pattern was seen predominantly confined to the basal and immediately suprabasal cells in all of the first group of patients. In the superficial cells a few granular cytoplasmic structures were positive. All patients with Barrett's oesophagitis showed EGF-R staining of the surface epithelium. A computerised planimeter was used to determine the proportion of stained areas of squamous cells which were expressed as a percentage of the total area of squamous cells. The difference in the area of cells stained for EGF-R between normal and inflamed oesophageal mucosa (29.5% and 43.1% respectively) was significant (p less than 0.001). Images Figure 1 PMID:1582583

  11. The epidermal cell structure of the secondary pollen presenter in Vangueria infausta (Rubiaceae: Vanguerieae) suggests a functional association with protruding onci in pollen grains.

    PubMed

    Tilney, Patricia M; van Wyk, Abraham E; van der Merwe, Chris F

    2014-01-01

    Secondary pollen presentation is a well-known phenomenon in the Rubiaceae with particularly conspicuous pollen presenters occurring in the tribe Vanguerieae. These knob-like structures are formed by a modification of the upper portion of the style and stigma, together known as the stylar head complex. In the flower bud and shortly before anthesis, the anthers surrounding the stylar head complex dehisce and release pollen grains which adhere to the pollen presenter. The epidermal cells of the pollen presenter facing the anthers are radially elongated with a characteristic wall thickening encircling the anticlinal walls of each cell towards the distal end. These cells were studied in the pollen presenter of Vangueria infausta using electron and light microscopy in conjunction with histochemical tests and immunohistochemical methods. Other prominent thickenings of the cell wall were also observed on the distal and proximal walls. All these thickenings were found to be rich in pectin and possibly xyloglucan. The terms "thickenings of Igersheim" and "bands of Igersheim" are proposed to refer, respectively, to these wall structures in general and those encircling the anticlinal walls of each cell near the distal end. The epidermal cells have an intricate ultrastructure with an abundance of organelles, including smooth and rough endoplasmic reticulum, Golgi apparatus, mitochondria and secretory vesicles. This indicates that these cells are likely to have an active physiological role. The pollen grains possess prominent protruding onci and observations were made on their structure and development. Walls of the protruding onci are also rich in pectin. Pectins are hydrophilic and known to be involved in the dehydration and rehydration of pollen grains. We hypothesise that the thickenings of Igersheim, as well as the protruding onci of the pollen grains, are functionally associated and part of the adaptive syndrome of secondary pollen presentation, at least in the Vanguerieae.

  12. Novel Antioxidant Tripeptide "ACQ" Can Prevent UV-Induced Cell Death and Preserve the Number of Epidermal Stem Cells.

    PubMed

    Choi, Hye-Ryung; Shin, Jung-Won; Na, Jung-Im; Nam, Kyung-Mi; Lee, Hyun-Sun; Park, Kyoung-Chan

    2015-01-01

    We found that tripeptide "ACQ: alanine-cysteine-glutamine" has significant DPPH scavenging activity compared to that of glutathione. Antioxidant effects of ACQ were tested in in vitro and in vivo models. When treated with H2O2, mock treated fibroblasts and keratinocytes showed strong staining by H2DCFA. But, ACQ showed good protective effects against hydrogen peroxide treatment. When mice were fed for 2 or 4 weeks, similar protective effects were observed. In the control group, epidermis was severely damaged by UV irradiation and apoptotic keratinocytes were observed. There were also numerous TUNEL positive cells. But in the ACQ group, epidermis became thicker and there was no sign of severe damage. Interestingly, the number of p63 cells was also higher in ACQ fed mice. To confirm the stem cell rescuing effects of ACQ, three-dimensional skin samples were constructed. Results showed that ACQ increased the expression of integrin α6 and the number of p63 positive cells. These findings showed that ACQ has good antioxidant activity and may increase stem cell activities by the regulation of integrin α6.

  13. Ganoderma tsugae extract inhibits expression of epidermal growth factor receptor and angiogenesis in human epidermoid carcinoma cells: In vitro and in vivo.

    PubMed

    Hsu, Shih-Chung; Ou, Chien-Chih; Chuang, Tzu-Chao; Li, Jhy-Wei; Lee, Yi-Jen; Wang, Vinchi; Liu, Jah-Yao; Chen, Chin-Shiang; Lin, Song-Chow; Kao, Ming-Ching

    2009-08-18

    We examined the anti-angiogenic effects of Ganoderma tsugae methanol extract (GTME) on human epidermoid carcinoma A-431 cells. Our data indicate that GTME inhibits the expression of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) in vitro and in vivo, and also inhibits the capillary tube formation of human umbilical vein endothelial cells (HUVECs). We also show that the suppression of VEGF expression by GTME can be restored by treatment with EGF. These results suggest that GTME inhibits VEGF expression via the suppression of EGFR expression, resulting in the downregulation of VEGF secretion from epidermoid carcinoma A-431 cells. These findings reveal a novel role for G. tsugae in inhibiting EGFR and VEGF expression, which are important for tumor angiogenesis and growth. Thus, GTME may provide a potential therapeutic approach for anti-tumor treatment. PMID:19332363

  14. Rapid auxin-mediated changes in the proteome of the epidermal cells in rye coleoptiles: Implications for the initiation of growth

    PubMed Central

    Deng, Z.; Xu, S.; Chalkley, R. J.; Oses-Prieto, J. A.; Burlingame, A. L.; Wang, Z.-Y.; Kutschera, U.

    2011-01-01

    In axial organs of juvenile plants, the phytohormone auxin (indole-3-acetic acid, IAA) rapidly allows cell wall loosening and hence promotes turgor-driven elongation. In this study, we used rye (Secale cereale) coleoptile sections to investigate possible effects of IAA on the proteome of cells. In a first set of experiments, we document that IAA causes organ elongation via promotion of expansion of the rigid outer wall of the outer epidermis. A quantitative comparison of the proteome (membrane-associated proteins), using two-dimensional difference gel electrophoresis (2-D DIGE), revealed that, within 2 h of auxin treatment, at least 16 protein spots were up- or down-regulated by IAA. These proteins were identified using reverse-phase liquid chromatography electrospray tandem mass spectrometry. Four of these proteins were detected in the growth-controlling outer epidermis and were further analysed. One epidermal polypeptide, a small Ras-related GTP-binding protein, was rapidly down-regulated by IAA (after 0.5 h of incubation) by −35% compared to the control. Concomitantly, a subunit of the 26S proteasome was up-regulated by IAA (+30% within 1 h). In addition, this protein displayed IAA-mediated post-translational modification. The implications of these rapid auxin effects with respect to signal transduction and IAA-mediated secretion of glycoproteins (osmiophilic nano-particles) into the growth-controlling outer epidermal wall are discussed. PMID:22117532

  15. Templated green synthesis of plasmonic silver nanoparticles in onion epidermal cells suitable for surface-enhanced Raman and hyper-Raman scattering.

    PubMed

    Espina Palanco, Marta; Bo Mogensen, Klaus; Gühlke, Marina; Heiner, Zsuzsanna; Kneipp, Janina; Kneipp, Katrin

    2016-01-01

    We report fast and simple green synthesis of plasmonic silver nanoparticles in the epidermal cells of onions after incubation with AgNO3 solution. The biological environment supports the generation of silver nanostructures in two ways. The plant tissue delivers reducing chemicals for the initial formation of small silver clusters and their following conversion to plasmonic particles. Additionally, the natural morphological structures of the onion layers, in particular the extracellular matrix provides a biological template for the growth of plasmonic nanostructures. This is indicated by red glowing images of extracellular spaces in dark field microscopy of onion layers a few hours after AgNO3 exposure due to the formation of silver nanoparticles. Silver nanostructures generated in the extracellular space of onion layers and within the epidermal cell walls can serve as enhancing plasmonic structures for one- and two-photon-excited spectroscopy such as surface enhanced Raman scattering (SERS) and surface enhanced hyper-Raman scattering (SEHRS). Our studies demonstrate a templated green preparation of enhancing plasmonic nanoparticles and suggest a new route to deliver silver nanoparticles as basic building blocks of plasmonic nanosensors to plants by the uptake of solutions of metal salts.

  16. Templated green synthesis of plasmonic silver nanoparticles in onion epidermal cells suitable for surface-enhanced Raman and hyper-Raman scattering

    PubMed Central

    Espina Palanco, Marta; Bo Mogensen, Klaus; Gühlke, Marina; Heiner, Zsuzsanna; Kneipp, Janina

    2016-01-01

    Summary We report fast and simple green synthesis of plasmonic silver nanoparticles in the epidermal cells of onions after incubation with AgNO3 solution. The biological environment supports the generation of silver nanostructures in two ways. The plant tissue delivers reducing chemicals for the initial formation of small silver clusters and their following conversion to plasmonic particles. Additionally, the natural morphological structures of the onion layers, in particular the extracellular matrix provides a biological template for the growth of plasmonic nanostructures. This is indicated by red glowing images of extracellular spaces in dark field microscopy of onion layers a few hours after AgNO3 exposure due to the formation of silver nanoparticles. Silver nanostructures generated in the extracellular space of onion layers and within the epidermal cell walls can serve as enhancing plasmonic structures for one- and two-photon-excited spectroscopy such as surface enhanced Raman scattering (SERS) and surface enhanced hyper-Raman scattering (SEHRS). Our studies demonstrate a templated green preparation of enhancing plasmonic nanoparticles and suggest a new route to deliver silver nanoparticles as basic building blocks of plasmonic nanosensors to plants by the uptake of solutions of metal salts. PMID:27547600

  17. Templated green synthesis of plasmonic silver nanoparticles in onion epidermal cells suitable for surface-enhanced Raman and hyper-Raman scattering.

    PubMed

    Espina Palanco, Marta; Bo Mogensen, Klaus; Gühlke, Marina; Heiner, Zsuzsanna; Kneipp, Janina; Kneipp, Katrin

    2016-01-01

    We report fast and simple green synthesis of plasmonic silver nanoparticles in the epidermal cells of onions after incubation with AgNO3 solution. The biological environment supports the generation of silver nanostructures in two ways. The plant tissue delivers reducing chemicals for the initial formation of small silver clusters and their following conversion to plasmonic particles. Additionally, the natural morphological structures of the onion layers, in particular the extracellular matrix provides a biological template for the growth of plasmonic nanostructures. This is indicated by red glowing images of extracellular spaces in dark field microscopy of onion layers a few hours after AgNO3 exposure due to the formation of silver nanoparticles. Silver nanostructures generated in the extracellular space of onion layers and within the epidermal cell walls can serve as enhancing plasmonic structures for one- and two-photon-excited spectroscopy such as surface enhanced Raman scattering (SERS) and surface enhanced hyper-Raman scattering (SEHRS). Our studies demonstrate a templated green preparation of enhancing plasmonic nanoparticles and suggest a new route to deliver silver nanoparticles as basic building blocks of plasmonic nanosensors to plants by the uptake of solutions of metal salts. PMID:27547600

  18. Requirement for neurogenesis to proceed through the division of neuronal progenitors following differentiation of epidermal growth factor and fibroblast growth factor-2-responsive human neural stem cells.

    PubMed

    Ostenfeld, Thor; Svendsen, Clive N

    2004-01-01

    Epidermal growth factor (EGF)- and fibroblast growth factor-2 (FGF-2)-responsive human neural stem cells may provide insight into mechanisms of neural development and have applications in cell-based therapeutics for neurological disease. However, their biology after expansion in vitro is currently poorly understood. Cells grown in either EGF or FGF-2 or a combination of both mitogens displayed characteristically similar levels of transcriptional activation and comparable proliferative profiles with linear cell-cycle kinetics and possessed similar neuronal differentiation capabilities. These data support the view that human neurospheres at later stages of expansion (>10 weeks) are comprised overwhelmingly of a single type of stem cell responsive to both EGF and FGF-2. After mitogen withdrawal and neurosphere plating, bromodeoxyuridine pulse-chase experiments revealed that the stem cells did not undergo differentiation directly into neurons. Instead, most immature neurons arose via the division of emerging progenitor cells in the absence of exogenous EGF or FGF-2. Neurogenesis was abolished by application of high concentrations of either EGF/FGF-2 or the mitotic inhibitor cytosine-b-arabinofuranoside, suggesting that there is an obligatory requirement for at least one round of cell division in the absence of mitogens as a prelude to terminal neuronal differentiation. The differentiation of human neurospheres provides a useful model of human neurogenesis, and the data presented indicate that it proceeds through the division of committed neuronal progenitor cells rather than directly from the neural stem cell. PMID:15342944

  19. The response to epidermal growth factor of human maxillary tumor cells in terms of tumor growth, invasion and expression of proteinase inhibitors.

    PubMed

    Mizoguchi, H; Komiyama, S; Matsui, K; Hamanaka, R; Ono, M; Kiue, A; Kobayashi, M; Shimizu, N; Welgus, H G; Kuwano, M

    1991-11-11

    Three cancer cell lines, IMC-2, IMC-3 and IMC-4, were established from a single tumor of a patient with maxillary cancer. We examined responses to epidermal growth factor (EGF) of these 3 cell lines with regard to cell growth and tumor invasion. The growth rate of IMC-2 in nude mice was markedly faster than that of the IMC-3 and IMC-4 cell lines. Assay for invasion through fibrin gels showed significantly enhanced invasive capacity of IMC-2 cells in response to EGF, but no change for IMC-3 and IMC-4 cells. We examined response to EGF of IMC-2 cells with regard to expression of a growth-related oncogene (c-fos), proteinases and their inhibitors. Expression of c-fos was transiently increased in IMC-2 cells at rates comparable to those seen in the 2 other lines in the presence of EGF. There was no apparent effect of EGF on the expression of urokinase-type plasminogen activator and 72-kDa type-IV collagenase in IMC-2 cells. In contrast, EGF specifically enhanced the expression of plasminogen activator inhibitor-I (PAI-I) and tissue inhibitor of metalloproteinases-I (TIMP-I) in IMC-2 cells. Our data suggest that proteinase inhibitors or other related factors may play an important role in tumor growth and invasion in response to EGF.

  20. Large area space solar cell assemblies

    NASA Technical Reports Server (NTRS)

    Spitzer, M. B.; Nowlan, M. J.

    1982-01-01

    Development of a large area space solar cell assembly is presented. The assembly consists of an ion implanted silicon cell and glass cover. The important attributes of fabrication are (1) use of a back surface field which is compatible with a back surface reflector, and (2) integration of coverglass application and call fabrication.

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

    SciTech Connect

    Zhang Dongyun; Li Jingxia; Gao Jimin; Huang Chuanshu

    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 cell 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.

  2. Combinatorial-Designed Epidermal Growth Factor Receptor-Targeted Chitosan Nanoparticles for Encapsulation and Delivery of Lipid-Modified Platinum Derivatives in Wild-Type and Resistant Non-Small-Cell Lung Cancer Cells.

    PubMed

    Nascimento, Ana Vanessa; Singh, Amit; Bousbaa, Hassan; Ferreira, Domingos; Sarmento, Bruno; Amiji, Mansoor M

    2015-12-01

    Development of efficient and versatile drug delivery platforms to overcome the physical and biological challenges in cancer therapeutics is an area of great interest, and novel materials are actively sought for such applications. Recent strides in polymer science have led to a combinatorial approach for generating a library of materials with different functional identities that can be "mixed and matched" to attain desired characteristics of a delivery vector. We have applied the combinatorial design to chitosan (CS), where the polymer backbone has been modified with polyethylene glycol, epidermal growth factor receptor-binding peptide, and lipid derivatives of varying chain length to encapsulate hydrophobic drugs. Cisplatin, cis-([PtCl2(NH3)2]), is one of the most potent chemotherapy drugs broadly administered for cancer treatment. Cisplatin is a hydrophilic drug, and in order for it to be encapsulated in the developed nanosystems, it was modified with lipids of varying chain length. The library of four CS derivatives and six platinum derivatives was self-assembled in aqueous medium and evaluated for physicochemical characteristics and cytotoxic effects in platinum-sensitive and -resistant lung cancer cells. The results show that the lipid-modified platinate encapsulation into CS nanoparticles significantly improved cellular cytotoxicity of the drug. In this work, we have also reinforced the idea that CS is a multifaceted system that can be as successful in delivering small molecules as it has been as a nucleic acids carrier.

  3. A role for G-proteins in the epidermal growth factor stimulation of phospholipase A2 in rat kidney mesangial cells.

    PubMed

    Hack, N; Clayman, P; Skorecki, K

    1990-08-01

    We have previously demonstrated phospholipase C (PLC) independent activation of phospholipase A2(PLA2) by epidermal growth factor (EGF) in glomerular mesangial cells in culture. In the current study using glass beads to permeabilize [3H]- or [14C]-arachidonate labelled mesangial cells we demonstrate that guanine nucleotides modulate the EGF-mediated stimulation of arachidonic acid release (75% inhibition with 100 microM GDP beta S and 108% augmentation with 100 microM GTP gamma S). GTP gamma S alone stimulated both the release of free arachidonic acid and production of diacylglycerol (DAG)