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Sample records for keratinocytes identifies similarities

  1. Matrix Metalloproteinase 10 Degradomics in Keratinocytes and Epidermal Tissue Identifies Bioactive Substrates With Pleiotropic Functions.

    PubMed

    Schlage, Pascal; Kockmann, Tobias; Sabino, Fabio; Kizhakkedathu, Jayachandran N; Auf dem Keller, Ulrich

    2015-12-01

    Matrix metalloproteinases (MMPs) are important players in skin homeostasis, wound repair, and in the pathogenesis of skin cancer. It is now well established that most of their functions are related to processing of bioactive proteins rather than components of the extracellular matrix (ECM). MMP10 is highly expressed in keratinocytes at the wound edge and at the invasive front of tumors, but hardly any non-ECM substrates have been identified and its function in tissue repair and carcinogenesis is unclear. To better understand the role of MMP10 in the epidermis, we employed multiplexed iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS) and monitored MMP10-dependent proteolysis over time in secretomes from keratinocytes. Time-resolved abundance clustering of neo-N termini classified MMP10-dependent cleavage events by efficiency and refined the MMP10 cleavage site specificity by revealing a so far unknown preference for glutamate in the P1 position. Moreover, we identified and validated the integrin alpha 6 subunit, cysteine-rich angiogenic inducer 61 and dermokine as novel direct MMP10 substrates and provide evidence for MMP10-dependent but indirect processing of phosphatidylethanolamine-binding protein 1. Finally, we sampled the epidermal proteome and degradome in unprecedented depth and confirmed MMP10-dependent processing of dermokine in vivo by TAILS analysis of epidermis from transgenic mice that overexpress a constitutively active mutant of MMP10 in basal keratinocytes. The newly identified substrates are involved in cell adhesion, migration, proliferation, and/or differentiation, indicating a contribution of MMP10 to local modulation of these processes during wound healing and cancer development. Data are available via ProteomeXchange with identifier PXD002474. PMID:26475864

  2. Using Scientific Publications to Identify People with Similar Interests

    NASA Astrophysics Data System (ADS)

    Loh, Stanley; Lorenzi, Fabiana; Granada, Roger; Lichtnow, Daniel; Wives, Leandro Krug; Palazzo Moreira de Oliveira, José

    In many situations, related to some types of systems or organizations' tasks, it is necessary to identify people with similar profiles. In the case of a collaborative recommender system, items to be recommended are those associated to similar users. Another example, in the academic environment, is to identify new members to be part of a research group (people with similar profiles). This task of identifying people with similar profiles can be time-consuming. In this sense, this work considers that scientific papers written by people can be used to identify users with similar profiles. Considering this assumption, we have done some experiments to identify which parts of papers, which type of indexes (terms or concepts) and which type of similarity functions (Jaccard or a Fuzzy function) are more suitable to identify similar people. The paper presents the results of some experiments and some application scenarios considering academic environments.

  3. Novel in vivo targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach

    PubMed Central

    Birkaya, Barbara; Ortt, Kori; Sinha, Satrajit

    2007-01-01

    Background p63 is a transcription factor that plays an important role in skin epidermal development and differentiation. The p63 gene encodes for two major protein isoforms, those containing an amino-terminal trans-activation domain (TAp63) and those lacking this domain (ΔNp63). Both the TA and ΔN transcripts are also alternatively spliced at the 3' end producing proteins with unique C-termini that are designated as α, β and γ isoforms. Recent research has suggested that ΔNp63 is the predominant isoform expressed and active in keratinocytes. Results To better elucidate the biological role of p63 in regulating gene expression in keratinocytes we performed chromatin immunoprecipitation (ChIP) experiments with ΔNp63-specific antibodies. We included an additional step in the ChIP procedure to enrich for ΔNp63 targets by screening the library of immunoprecipitated DNA for its ability to bind recombinant GST-ΔNp63. Cloning of ΔNp63-ChIP-derived DNA fragments identified more than 60 potential ΔNp63 target loci that were located close to or embedded within known or predicted genes. Identity of these target genes suggests that they may participate in a myriad of cellular processes including transcriptional regulation, signaling and metabolism. Here we confirm the binding of ΔNp63 to several of these genomic loci both by EMSA and replicate ChIP assays. Finally we show that the expression of many of these target genes is altered when ΔNp63 levels in keratinocytes are reduced by siRNA, further confirming that these are bona fide targets. Conclusion This unbiased genomic approach has allowed us to uncover functional targets of ΔNp63 and serves as the initial step in further analysis of the transcriptional regulatory mechanisms that are governed by p63 in keratinocytes. PMID:17521434

  4. Identifying a Superfluid Reynolds Number via Dynamical Similarity.

    PubMed

    Reeves, M T; Billam, T P; Anderson, B P; Bradley, A S

    2015-04-17

    The Reynolds number provides a characterization of the transition to turbulent flow, with wide application in classical fluid dynamics. Identifying such a parameter in superfluid systems is challenging due to their fundamentally inviscid nature. Performing a systematic study of superfluid cylinder wakes in two dimensions, we observe dynamical similarity of the frequency of vortex shedding by a cylindrical obstacle. The universality of the turbulent wake dynamics is revealed by expressing shedding frequencies in terms of an appropriately defined superfluid Reynolds number, Re(s), that accounts for the breakdown of superfluid flow through quantum vortex shedding. For large obstacles, the dimensionless shedding frequency exhibits a universal form that is well-fitted by a classical empirical relation. In this regime the transition to turbulence occurs at Re(s)≈0.7, irrespective of obstacle width. PMID:25933320

  5. Proteome profiling of keratinocytes transforming to malignancy.

    PubMed

    Paulitschke, Verena; Gerner, Christopher; Hofstätter, Elisabeth; Mohr, Thomas; Mayer, Rupert Laurenz; Pehamberger, Hubert; Kunstfeld, Rainer

    2015-02-01

    To shed light on the multistep process of squamous cell carcinoma development and the underlying pathologic mechanisms, we performed comparative proteome analysis of keratinocytes, keratinocytes stimulated with Il-1beta, and A431 epidermoid carcinoma cells. Fractionation of the cells into supernatant, nucleus, and cytoplasm was followed by protein separation, proteolytic digest, and nano-LC separation, and fragmentation using an ion trap mass spectrometer. Specific bioinformatics tools were used to generate a list of keratinocyte-specific proteins. Ninety percent of these proteins were found to be upregulated in keratinocytes versus the A431 cells. Classification of the identified proteins by biologic function and gene set enrichment analysis revealed that keratinocytes produced more proteins involved in cell differentiation, cell adhesion, cell junction, calcium ion, calmodulin binding, cytoskeleton organization, and cytokinesis, whereas A431 produced more proteins involved in cell cycle checkpoint, cell cycle process, RNA processing and transport, DNA damage and repair, RNA and DNA binding, and chromatin remodeling. The protein signatures of A431 and normal keratinocytes treated with IL-1beta showed marked similarity, confirming that inflammation is an important step in malignant transformation in nonmelanoma skin cancer. Thus, proteome profiling and bioinformatic processing may support the understanding of the underlying mechanisms, with the potential to facilitate development of early biomarkers and patient-tailored therapy. PMID:25395074

  6. Identifiable Orthographically Similar Word Primes Interfere in Visual Word Identification

    ERIC Educational Resources Information Center

    Burt, Jennifer S.

    2009-01-01

    University students participated in five experiments concerning the effects of unmasked, orthographically similar, primes on visual word recognition in the lexical decision task (LDT) and naming tasks. The modal prime-target stimulus onset asynchrony (SOA) was 350 ms. When primes were words that were orthographic neighbors of the targets, and…

  7. Beta Adrenergic Receptors in Keratinocytes

    PubMed Central

    Sivamani, Raja K.; Lam, Susanne T.; Isseroff, R. Rivkah

    2007-01-01

    Synopsis Beta2 adrenergic receptors were identified in keratinocytes more than 30 years ago, but their function in the epidermis continues to be elucidated. Abnormalities in their expression, signaling pathway, or in the generation of endogenous catecholamine agonists by keratinocytes have been implicated in the pathogenesis of cutaneous diseases such as atopic dermatitis, vitiligo and psoriasis. New studies also indicate that the beta2AR also modulates keratinocyte migration, and thus can function to regulate wound re-epithelialization. This review focuses on the function of these receptors in keratinocytes and their contribution to cutaneous physiology and disease. PMID:17903623

  8. Distinctive molecular responses to ultraviolet radiation between keratinocytes and melanocytes.

    PubMed

    Sun, Xiaoyun; Kim, Arianna; Nakatani, Masashi; Shen, Yao; Liu, Liang

    2016-09-01

    Solar ultraviolet radiation (UVR) is the major risk factor for skin carcinogenesis. To gain new insights into the molecular pathways mediating UVR effects in the skin, we performed comprehensive transcriptomic analyses to identify shared and distinctive molecular responses to UVR between human keratinocytes and melanocytes. Keratinocytes and melanocytes were irradiated with varying doses of UVB (10, 20 and 30 mJ/cm(2) ) then analysed by RNA-Seq at different time points post-UVB radiation (4, 24 and 72 h). Under basal conditions, keratinocytes and melanocytes expressed similar number of genes, although they each expressed a distinctive subset of genes pertaining to their specific cellular identity. Upon UVB radiation, keratinocytes displayed a clear pattern of time- and dose-dependent changes in gene expression that was different from melanocytes. The early UVB-responsive gene set (4 h post-UVR) differed significantly from delayed UVB-responsive gene sets (24 and 72 h). We also identified multiple novel UVB signature genes including PRSS23, SERPINH1, LCE3D and CNFN, which were conserved between melanocyte and keratinocyte lines from different individuals. Taken together, our findings elucidated both common and distinctive molecular features between melanocytes and keratinocytes and uncovered novel UVB signature genes that might be utilized to predict UVB photobiological effects on the skin. PMID:27119462

  9. Primary structure of keratinocyte transglutaminase

    SciTech Connect

    Phillips, M.A.; Stewart, B.E.; Qin, Q.; Rice, R.H. ); Chakravarty, R. ); Floyd, E.E.; Jetten, A.M. )

    1990-12-01

    The nucleotide and deduced amino acid sequences of the coding regions of human and rat keratinocyte transglutaminases (protein-glutamine: amine {gamma}-glutamyltransferase; EC 2.3.2.13) have been determined. These yield proteins of {approximately}90 kDa that are 92% identical, indicative of the conservation of important structural features. Alignments of amino acid sequences show substantial similarity among the keratinocyte transglutaminase, human clotting factor XIII catalytic subunit, guinea pig liver tissue transglutaminase, and the human erythrocyte band-4.2 protein. The keratinocyte enzyme is most similar to factor XIII, whereas the band-4.2 protein is most similar to the tissue transglutaminase. A salient feature of the keratinocyte transglutaminase is its 105-residue extension beyond the N terminus of the tissue transglutaminase. This extension and the unreltaed activation peptide of factor XIII (a 37-residue extension) appear to be added for specialized functions after divergence of the tissue transglutaminase from their common lineage.

  10. Similarity

    NASA Technical Reports Server (NTRS)

    Apostol, Tom M. (Editor)

    1990-01-01

    In this 'Project Mathematics! series, sponsored by the California Institute for Technology (CalTech), the mathematical concept of similarity is presented. he history of and real life applications are discussed using actual film footage and computer animation. Terms used and various concepts of size, shape, ratio, area, and volume are demonstrated. The similarity of polygons, solids, congruent triangles, internal ratios, perimeters, and line segments using the previous mentioned concepts are shown.

  11. Expression of membrane glycoproteins in normal keratinocytes and squamous carcinoma cell lines

    SciTech Connect

    Rayter, Z. ); McIlhinney, R. ); Gusterson, B. )

    1989-08-01

    Con A acceptor glycoproteins were analyzed by 2D-PAGE and {sup 125}I-Con A overlay in three squamous carcinoma cell lines and compared with those in the simian virus (SV40)-transformed keratinocyte cell line SVK-14 and in normal keratinocytes. The majority of the glycoproteins identified by this technique were expressed at similar levels in all of the cells examined, independent of the culture conditions used. A cell surface glycoprotein gp34 was increased in the tumor cells compared with normal keratinocytes and expression varied with the culture density. Another glycoprotein, gp21, was found to be increased in expression in normal keratinocytes and stratified hyperconfluent cultures of squamous carcinoma cell lines. This paper describes the potential of this technique to identify membrane glycoproteins which may be expressed as a function of proliferation or differentiation.

  12. eTBLAST: a web server to identify expert reviewers, appropriate journals and similar publications.

    PubMed

    Errami, Mounir; Wren, Jonathan D; Hicks, Justin M; Garner, Harold R

    2007-07-01

    Authors, editors and reviewers alike use the biomedical literature to identify appropriate journals in which to publish, potential reviewers for papers or grants, and collaborators (or competitors) with similar interests. Traditionally, this process has either relied upon personal expertise and knowledge or upon a somewhat unsystematic and laborious process of manually searching through the literature for trends. To help with these tasks, we report three utilities that parse and summarize the results of an abstract similarity search to find appropriate journals for publication, authors with expertise in a given field, and documents similar to a submitted query. The utilities are based upon a program, eTBLAST, designed to identify similar documents within literature databases such as (but not limited to) MEDLINE. These services are freely accessible through the Internet at http://invention.swmed.edu/etblast/etblast.shtml, where users can upload a file or paste text such as an abstract into the browser interface. PMID:17452348

  13. Methylation-specific digital karyotyping of HPV16E6E7-expressing human keratinocytes identifies novel methylation events in cervical carcinogenesis.

    PubMed

    Steenbergen, Renske D M; Ongenaert, Maté; Snellenberg, Suzanne; Trooskens, Geert; van der Meide, Wendy F; Pandey, Deeksha; Bloushtain-Qimron, Noga; Polyak, Kornelia; Meijer, Chris J L M; Snijders, Peter J F; Van Criekinge, Wim

    2013-09-01

    Transformation of epithelial cells by high-risk human papillomavirus (hrHPV) types can lead to anogenital carcinomas, particularly cervical cancer, and oropharyngeal cancers. This process is associated with DNA methylation alterations, often affecting tumour suppressor gene expression. This study aimed to comprehensively unravel genome-wide DNA methylation events linked to a transforming hrHPV-infection, which is driven by deregulated expression of the viral oncogenes E6 and E7 in dividing cells. Primary human keratinocytes transduced with HPV16E6E7 and their untransduced counterparts were subjected to methylation-specific digital karyotyping (MSDK) to screen for genome-wide DNA-methylation changes at different stages of HPV-induced transformation. Integration of the obtained methylation profiles with genome-wide gene expression patterns of cervical carcinomas identified 34 genes with increased methylation in HPV-transformed cells and reduced expression in cervical carcinomas. For 12 genes (CLIC3, CREB3L1, FAM19A4, LFNG, LHX1, MRC2, NKX2-8, NPTX-1, PHACTR3, PRDM14, SOST and TNFSF13) specific methylation in HPV-containing cell lines was confirmed by semi-quantitative methylation-specific PCR. Subsequent analysis of FAM19A4, LHX1, NKX2-8, NPTX-1, PHACTR3 and PRDM14 in cervical tissue specimens showed increasing methylation levels for all genes with disease progression. All six genes were frequently methylated in cervical carcinomas, with highest frequencies (up to 100%) seen for FAM19A4, PHACTR3 and PRDM14. Analysis of hrHPV-positive cervical scrapes revealed significantly increased methylation levels of the latter three genes in women with high-grade cervical disease compared to controls. In conclusion, MSDK analysis of HPV16-transduced keratinocytes at different stages of HPV-induced transformation resulted in the identification of novel DNA methylation events, involving FAM19A4, LHX1, NKX2-8, PHACTR3 and PRDM14 genes in cervical carcinogenesis. These genes may

  14. Identifying Similar Patterns of Structural Flexibility in Proteins by Disorder Prediction and Dynamic Programming

    PubMed Central

    Petrovich, Aidan; Borne, Adam; Uversky, Vladimir N.; Xue, Bin

    2015-01-01

    Computational methods are prevailing in identifying protein intrinsic disorder. The results from predictors are often given as per-residue disorder scores. The scores describe the disorder propensity of amino acids of a protein and can be further represented as a disorder curve. Many proteins share similar patterns in their disorder curves. The similar patterns are often associated with similar functions and evolutionary origins. Therefore, finding and characterizing specific patterns of disorder curves provides a unique and attractive perspective of studying the function of intrinsically disordered proteins. In this study, we developed a new computational tool named IDalign using dynamic programming. This tool is able to identify similar patterns among disorder curves, as well as to present the distribution of intrinsic disorder in query proteins. The disorder-based information generated by IDalign is significantly different from the information retrieved from classical sequence alignments. This tool can also be used to infer functions of disordered regions and disordered proteins. The web server of IDalign is available at (http://labs.cas.usf.edu/bioinfo/service.html). PMID:26086829

  15. Application of a Profile Similarity Methodology for Identifying Terrorist Groups That Use or Pursue CBRN Weapons

    NASA Astrophysics Data System (ADS)

    Breiger, Ronald L.; Ackerman, Gary A.; Asal, Victor; Melamed, David; Milward, H. Brinton; Rethemeyer, R. Karl; Schoon, Eric

    No single profile fits all CBRN-active groups, and therefore it is important to identify multiple profiles. In the analysis of terrorist organizations, linear and generalized regression modeling provide a set of tools to apply to data that is in the form of cases (named groups) by variables (traits and behaviors of the groups). We turn the conventional regression modeling "inside out" to reveal a network of relations among the cases on the basis of their attribute and behavioral similarity. We show that a network of profile similarity among the cases is built in to standard regression modeling, and that the exploitation of this aspect leads to new insights helpful in the identification of multiple profiles for actors. Our application builds on a study of 108 Islamic jihadist organizations that predicts use or pursuit of CBRN weapons.

  16. Genetic similarity between cancers and comorbid Mendelian diseases identifies candidate driver genes

    PubMed Central

    Melamed, Rachel D.; Emmett, Kevin J.; Madubata, Chioma; Rzhetsky, Andrey; Rabadan, Raul

    2015-01-01

    Despite large-scale cancer genomics studies, key somatic mutations driving cancer, and their functional roles, remain elusive. Here we propose that analysis of comorbidities of Mendelian diseases with cancers provides a novel, systematic way to discover new cancer genes. If germline genetic variation in Mendelian loci predisposes bearers to common cancers, the same loci may harbor cancer-associated somatic variation. Compilations of clinical records spanning over 100 million patients provide an unprecedented opportunity to assess clinical associations between Mendelian diseases and cancers. We systematically compare these comorbidities against recurrent somatic mutations from more than five thousand patients across many cancers. Using multiple measures of genetic similarity, we show that a Mendelian disease and comorbid cancer indeed have genetic alterations of significant functional similarity. This result provides a basis to identify candidate drivers in cancers including melanoma and glioblastoma. Some Mendelian diseases demonstrate “pan-cancer” comorbidity and shared genetics across cancers. PMID:25926297

  17. Identifying Similar Cases in Document Networks Using Cross-Reference Structures.

    PubMed

    Botsis, Taxiarchis; Scott, John; Woo, Emily Jane; Ball, Robert

    2015-11-01

    Our objective was to explore the creation of document networks based on different thresholds of shared information and different clustering algorithms on those networks to identify document clusters describing similar clinical cases. We created networks from vaccine adverse event report sets using seven approaches for linking reports. We then applied three clustering algorithms [visualization of similarities (VOS), Louvain, k-means] to these networks and evaluated their ability to identify known clusters. The report sets included one simulated set and three sets from the Vaccine Adverse Event Reporting System; each was split into training and testing subsets. Training subsets were used to estimate parameter values for the clustering algorithms and testing subsets to evaluate clusters. We created the networks by linking reports based on shared information in the form either of individual Medical Dictionary for Regulatory Activities Preferred Terms (PTs) or of dyads, triplets, quadruplets, quintuplets, and sextuplets of PTs; we created another network by weighting the single PT network connections by Lin's information theoretic approach to similarity. We then repeated this entire process using networks based on text mining output rather than structured data. We evaluated report clustering using recall, precision, and f-measure. The VOS algorithm outperformed Louvain and k-means in general. The best weighting scheme appeared to be related to the complexity of the known cluster. For example, singleton weighting performed best for an intussusception cluster driven by a single PT. We observed marginal differences between the code- and textual-based clustering. In conclusion, our approach supported identification of similar nodes in a document network. PMID:25122604

  18. A similarity based approach to identify homogeneous regions for seasonal forecasting

    NASA Astrophysics Data System (ADS)

    Schick, Simon; Rössler, Ole; Weingartner, Rolf

    2015-04-01

    Seasonal runoff forecasting using statistical models is challenged by a large number of candidate predictors and a general weak predictor-predictand relationship. As the area of the target basin increases, often also the available data sets do, thus reinforcing the predictor selection challenge. We propose an approach which follows the idea of 'divide and conquer' as developed in computational sciences and machine learning: First, the macroscale target basin is partitioned into homogeneous regions using all its gauged mesoscale subbasins. Second, one representative subbasin per homogeneous region is identified, for which models are fitted and applied. Third, the resulting forecasts are combined at the scale of the macroscale target basin. This approach requires a suitable method to identify homogeneous regions and representative subbasins. We suggest a way based on hydrological similarity, as catchment similarity estimated with respect to physiographic-climatic descriptors does not necessarily imply similar runoff response. Each descriptor is derived from daily runoff series and aimed to reflect a specific catchment characteristic: autocorrelation coefficient, parameters of fitted Gamma distribution and low/high flow indices (based on daily runoff values) fluctuation of the standard deviation within the yearly cycle (based on weekly runoff values) dominant harmonics obtained from the discrete Fourier transform (based on monthly runoff values) long term trend (based on yearly runoff values) Where necessary, the runoff series first need to be standardized, aggregated, detrended or deseasonalized. As a preliminary study we present the results of a cluster analysis for the Swiss Rhine River as macroscale target basin, which leads to about 40 mesoscale subbasins with runoff series for the period 1991-2010. Problems we have to address include the choice of a clustering algorithm, the identification of an appropriate number of regions and the selection of representative

  19. Lineage mapping identifies molecular and architectural similarities between the larval and adult Drosophila central nervous system

    PubMed Central

    Lacin, Haluk; Truman, James W

    2016-01-01

    Neurogenesis in Drosophila occurs in two phases, embryonic and post-embryonic, in which the same set of neuroblasts give rise to the distinct larval and adult nervous systems, respectively. Here, we identified the embryonic neuroblast origin of the adult neuronal lineages in the ventral nervous system via lineage-specific GAL4 lines and molecular markers. Our lineage mapping revealed that neurons born late in the embryonic phase show axonal morphology and transcription factor profiles that are similar to the neurons born post-embryonically from the same neuroblast. Moreover, we identified three thorax-specific neuroblasts not previously characterized and show that HOX genes confine them to the thoracic segments. Two of these, NB2-3 and NB3-4, generate leg motor neurons. The other neuroblast is novel and appears to have arisen recently during insect evolution. Our findings provide a comprehensive view of neurogenesis and show how proliferation of individual neuroblasts is dictated by temporal and spatial cues. DOI: http://dx.doi.org/10.7554/eLife.13399.001 PMID:26975248

  20. A non-target approach to identify disinfection byproducts of structurally similar sulfonamide antibiotics.

    PubMed

    Wang, Mian; Helbling, Damian E

    2016-10-01

    There is growing concern over the formation of new types of disinfection byproducts (DBPs) from pharmaceuticals and other emerging contaminants during drinking water production. Free chlorine is a widely used disinfectant that reacts non-selectively with organic molecules to form a variety of byproducts. In this research, we aimed to investigate the DBPs formed from three structurally similar sulfonamide antibiotics (sulfamethoxazole, sulfathiazole, and sulfadimethoxine) to determine how chemical structure influences the types of chlorination reactions observed. We conducted free chlorination experiments and developed a non-target approach to extract masses from the experimental dataset that represent the masses of candidate DBPs. Structures were assigned to the candidate DBPs based on analytical data and knowledge of chlorine chemistry. Confidence levels were assigned to each proposed structure according to conventions in the field. In total, 11, 12, and 15 DBP structures were proposed for sulfamethoxazole, sulfathiazole, and sulfadimethoxine, respectively. The structures of the products suggest a variety of reaction types including chlorine substitution, SC cleavage, SN hydrolysis, desulfonation, oxidation/hydroxylation, and conjugation reactions. Some reaction types were common to all of the sulfonamide antibiotics, but unique reaction types were also observed for each sulfonamide antibiotic suggesting that selective prediction of DBP structures of other sulfonamide antibiotics based on chemical structure is unlikely to be possible based on these data alone. This research offers an approach to comprehensively identify DBPs of organic molecules and fills in much needed data on the formation of specific DBPs from three environmentally relevant sulfonamide antibiotics. PMID:27348196

  1. Automated identification of epidermal keratinocytes in reflectance confocal microscopy

    NASA Astrophysics Data System (ADS)

    Gareau, Dan

    2011-03-01

    Keratinocytes in skin epidermis, which have bright cytoplasmic contrast and dark nuclear contrast in reflectance confocal microscopy (RCM), were modeled with a simple error function reflectance profile: erf( ). Forty-two example keratinocytes were identified as a training set which characterized the nuclear size a = 8.6+/-2.8 μm and reflectance gradient b = 3.6+/-2.1 μm at the nuclear/cytoplasmic boundary. These mean a and b parameters were used to create a rotationally symmetric erf( ) mask that approximated the mean keratinocyte image. A computer vision algorithm used an erf( ) mask to scan RCM images, identifying the coordinates of keratinocytes. Applying the mask to the confocal data identified the positions of keratinocytes in the epidermis. This simple model may be used to noninvasively evaluate keratinocyte populations as a quantitative morphometric diagnostic in skin cancer detection and evaluation of dermatological cosmetics.

  2. A Hybrid Knowledge-Based and Data-Driven Approach to Identifying Semantically Similar Concepts

    PubMed Central

    Pivovarov, Rimma; Elhadad, Noémie

    2012-01-01

    An open research question when leveraging ontological knowledge is when to treat different concepts separately from each other and when to aggregate them. For instance, concepts for the terms "paroxysmal cough" and "nocturnal cough" might be aggregated in a kidney disease study, but should be left separate in a pneumonia study. Determining whether two concepts are similar enough to be aggregated can help build better datasets for data mining purposes and avoid signal dilution. Quantifying the similarity among concepts is a difficult task, however, in part because such similarity is context-dependent. We propose a comprehensive method, which computes a similarity score for a concept pair by combining data-driven and ontology-driven knowledge. We demonstrate our method on concepts from SNOMED-CT and on a corpus of clinical notes of patients with chronic kidney disease. By combining information from usage patterns in clinical notes and from ontological structure, the method can prune out concepts that are simply related from those which are semantically similar. When evaluated against a list of concept pairs annotated for similarity, our method reaches an AUC (area under the curve) of 92%. PMID:22289420

  3. Content-Based Search on a Database of Geometric Models: Identifying Objects of Similar Shape

    SciTech Connect

    XAVIER, PATRICK G.; HENRY, TYSON R.; LAFARGE, ROBERT A.; MEIRANS, LILITA; RAY, LAWRENCE P.

    2001-11-01

    The Geometric Search Engine is a software system for storing and searching a database of geometric models. The database maybe searched for modeled objects similar in shape to a target model supplied by the user. The database models are generally from CAD models while the target model may be either a CAD model or a model generated from range data collected from a physical object. This document describes key generation, database layout, and search of the database.

  4. 'Gene shaving' as a method for identifying distinct sets of genes with similar expression patterns

    PubMed Central

    Hastie, Trevor; Tibshirani, Robert; Eisen, Michael B; Alizadeh, Ash; Levy, Ronald; Staudt, Louis; Chan, Wing C; Botstein, David; Brown, Patrick

    2000-01-01

    Background: Large gene expression studies, such as those conducted using DNA arrays, often provide millions of different pieces of data. To address the problem of analyzing such data, we describe a statistical method, which we have called 'gene shaving'. The method identifies subsets of genes with coherent expression patterns and large variation across conditions. Gene shaving differs from hierarchical clustering and other widely used methods for analyzing gene expression studies in that genes may belong to more than one cluster, and the clustering may be supervised by an outcome measure. The technique can be 'unsupervised', that is, the genes and samples are treated as unlabeled, or partially or fully supervised by using known properties of the genes or samples to assist in finding meaningful groupings. Results: We illustrate the use of the gene shaving method to analyze gene expression measurements made on samples from patients with diffuse large B-cell lymphoma. The method identifies a small cluster of genes whose expression is highly predictive of survival. Conclusions: The gene shaving method is a potentially useful tool for exploration of gene expression data and identification of interesting clusters of genes worth further investigation. PMID:11178228

  5. Phenotype Similarity Regression for Identifying the Genetic Determinants of Rare Diseases

    PubMed Central

    Greene, Daniel; Richardson, Sylvia; Turro, Ernest

    2016-01-01

    Rare genetic disorders, which can now be studied systematically with affordable genome sequencing, are often caused by high-penetrance rare variants. Such disorders are often heterogeneous and characterized by abnormalities spanning multiple organ systems ascertained with variable clinical precision. Existing methods for identifying genes with variants responsible for rare diseases summarize phenotypes with unstructured binary or quantitative variables. The Human Phenotype Ontology (HPO) allows composite phenotypes to be represented systematically but association methods accounting for the ontological relationship between HPO terms do not exist. We present a Bayesian method to model the association between an HPO-coded patient phenotype and genotype. Our method estimates the probability of an association together with an HPO-coded phenotype characteristic of the disease. We thus formalize a clinical approach to phenotyping that is lacking in standard regression techniques for rare disease research. We demonstrate the power of our method by uncovering a number of true associations in a large collection of genome-sequenced and HPO-coded cases with rare diseases. PMID:26924528

  6. Phenotype Similarity Regression for Identifying the Genetic Determinants of Rare Diseases.

    PubMed

    Greene, Daniel; Richardson, Sylvia; Turro, Ernest

    2016-03-01

    Rare genetic disorders, which can now be studied systematically with affordable genome sequencing, are often caused by high-penetrance rare variants. Such disorders are often heterogeneous and characterized by abnormalities spanning multiple organ systems ascertained with variable clinical precision. Existing methods for identifying genes with variants responsible for rare diseases summarize phenotypes with unstructured binary or quantitative variables. The Human Phenotype Ontology (HPO) allows composite phenotypes to be represented systematically but association methods accounting for the ontological relationship between HPO terms do not exist. We present a Bayesian method to model the association between an HPO-coded patient phenotype and genotype. Our method estimates the probability of an association together with an HPO-coded phenotype characteristic of the disease. We thus formalize a clinical approach to phenotyping that is lacking in standard regression techniques for rare disease research. We demonstrate the power of our method by uncovering a number of true associations in a large collection of genome-sequenced and HPO-coded cases with rare diseases. PMID:26924528

  7. A Model to Predict the Risk of Keratinocyte Carcinomas.

    PubMed

    Whiteman, David C; Thompson, Bridie S; Thrift, Aaron P; Hughes, Maria-Celia; Muranushi, Chiho; Neale, Rachel E; Green, Adele C; Olsen, Catherine M

    2016-06-01

    Basal cell and squamous cell carcinomas of the skin are the commonest cancers in humans, yet no validated tools exist to estimate future risks of developing keratinocyte carcinomas. To develop a prediction tool, we used baseline data from a prospective cohort study (n = 38,726) in Queensland, Australia, and used data linkage to capture all surgically excised keratinocyte carcinomas arising within the cohort. Predictive factors were identified through stepwise logistic regression models. In secondary analyses, we derived separate models within strata of prior skin cancer history, age, and sex. The primary model included terms for 10 items. Factors with the strongest effects were >20 prior skin cancers excised (odds ratio 8.57, 95% confidence interval [95% CI] 6.73-10.91), >50 skin lesions destroyed (odds ratio 3.37, 95% CI 2.85-3.99), age ≥ 70 years (odds ratio 3.47, 95% CI 2.53-4.77), and fair skin color (odds ratio 1.75, 95% CI 1.42-2.15). Discrimination in the validation dataset was high (area under the receiver operator characteristic curve 0.80, 95% CI 0.79-0.81) and the model appeared well calibrated. Among those reporting no prior history of skin cancer, a similar model with 10 factors predicted keratinocyte carcinoma events with reasonable discrimination (area under the receiver operator characteristic curve 0.72, 95% CI 0.70-0.75). Algorithms using self-reported patient data have high accuracy for predicting risks of keratinocyte carcinomas. PMID:26908057

  8. Sphingolipid metabolism in organotypic mouse keratinocyte cultures

    SciTech Connect

    Madison, K.C.; Swartzendruber, D.C.; Wertz, P.W.; Downing, D.T. )

    1990-12-01

    Ceramides are the dominant component of the stratum corneum intercellular lipid lamellae, which constitute the epidermal permeability barrier. Only pig and human epidermal ceramides have been extensively characterized and the structures of the ceramides of cultured keratinocytes have not been previously investigated. In the present studies, we have characterized the ceramides synthesized by organotypic lifted mouse keratinocyte cultures for the first time and compared them to the ceramides of intact mouse epidermis. Both mouse epidermis and cultures contained five ceramides, ceramide 1 being the least polar and ceramide 5 the most polar. Ceramide 1 was a group of acylceramides, i.e., very-long-chain omega-hydroxyceramides with an ester-linked nonhydroxy fatty acid. Ceramide 2 contained medium-length saturated nonhydroxy fatty acids. (In culture, the ceramide 2 band was split into two parts with the slightly more polar ceramide 2' containing short-chain saturated nonhydroxy fatty acids.) Ceramide 5 contained short-chain alpha-hydroxy fatty acids. The structures of ceramides 1, 2, and 5 were analagous to those of pig and human epidermis. Mouse epidermal ceramide 3 was quite unusual, containing beta-hydroxy fatty acids, a structure not previously identified among mammalian ceramides. In contrast, culture ceramide 3 was composed of omega-hydroxy fatty acids with a chain-length distribution similar to that of ceramide 1. Mouse ceramide 4 was composed of fatty acids with chromatographic mobility similar to hydroxy fatty acids but with different chemical reactivity; it remains only partially characterized. Culture ceramide 4 was present in quantities too small for analysis. All ceramides in mouse epidermis and cultures contained only sphingosine bases, whereas pig and human ceramides also contain phytosphingosine.

  9. Growth-regulated synthesis and secretion of biologically active nerve growth factor by human keratinocytes.

    PubMed

    Di Marco, E; Marchisio, P C; Bondanza, S; Franzi, A T; Cancedda, R; De Luca, M

    1991-11-15

    Nerve growth factor (NGF) transcripts were identified in normal human keratinocytes in primary and secondary culture. The expression of the NGF mRNA was strongly down-regulated by corticosteroids and was maximal when keratinocytes were in the exponential phase of growth. Immunofluorescence studies on growing keratinocytes colonies and on elutriated keratinocytes obtained from growing colonies and mature stratified epithelium showed specific staining of the Golgi apparatus only in basal keratinocytes in the exponential phase of growth. The keratinocyte-derived NGF was secreted in a biologically active form as assessed by neurite induction in sensory neurons obtained from chick embryo dorsal root ganglia. Based on these data we suggest that the basal keratinocyte is the cell synthesizing and secreting NGF in the human adult epidermis. The paracrine secretion of NGF by keratinocytes might have a major role in regulating innervation, lymphocyte function, and melanocyte growth and differentiation in epidermal morphogenesis as well as during wound healing. PMID:1718982

  10. Antimycotics suppress the Malassezia extract-induced production of CXC chemokine ligand 10 in human keratinocytes.

    PubMed

    Hau, Carren S; Kanda, Naoko; Makimura, Koichi; Watanabe, Shinichi

    2014-02-01

    Malassezia, a lipophilic yeast, exacerbates atopic dermatitis. Malassezia products can penetrate the disintegrated stratum corneum and encounter subcorneal keratinocytes in the skin of atopic dermatitis patients. Type 1 helper T (Th1) cells infiltrate chronic lesions with atopic dermatitis, and antimycotic agents improve its symptoms. We aimed to identify Malassezia-induced chemokines in keratinocytes and examine whether antimycotics suppressed this induction. Normal human keratinocytes were incubated with a Malassezia restricta extract and antimycotics. Chemokine expression was analyzed by enzyme-linked immunosorbent assays and real-time polymerase chain reaction. Signal transducer and activator of transcription (STAT)1 activity was examined by luciferase assays. The tyrosine-phosphorylation of STAT1 was analyzed by western blotting. The M. restricta extract increased the mRNA and protein expression of Th1-attracting CXC chemokine ligand (CXCL)10 and STAT1 activity and phosphorylation in keratinocytes, which was suppressed by a Janus kinase inhibitor. The antimycotics itraconazole, ketoconazole, luliconazole, terbinafine, butenafine and amorolfine suppressed M. restricta extract-induced CXCL10 mRNA and protein expression and STAT1 activity and phosphorylation. These effects were similarly induced by 15-deoxy-Δ-(12,14) -prostaglandin J2 (15d-PGJ2 ), a prostaglandin D2 metabolite. Antimycotics increased the release of 15d-PGJ2 from keratinocytes. The antimycotic-induced suppression of CXCL10 production and STAT1 activity was counteracted by a lipocalin-type prostaglandin D synthase inhibitor. The antimycotics itraconazole, ketoconazole, luliconazole, terbinafine, butenafine and amorolfine may suppress the M. restricta-induced production of CXCL10 by inhibiting STAT1 through an increase in 15d-PGJ2 production in keratinocytes. These antimycotics may block the Th1-mediated inflammation triggered by Malassezia in the chronic phase of atopic dermatitis. PMID

  11. Combining molecular fingerprints with multidimensional scaling analyses to identify the source of spilled oil from highly similar suspected oils.

    PubMed

    Zhou, Peiyu; Chen, Changshu; Ye, Jianjun; Shen, Wenjie; Xiong, Xiaofei; Hu, Ping; Fang, Hongda; Huang, Chuguang; Sun, Yongge

    2015-04-15

    Oil fingerprints have been a powerful tool widely used for determining the source of spilled oil. In most cases, this tool works well. However, it is usually difficult to identify the source if the oil spill accident occurs during offshore petroleum exploration due to the highly similar physiochemical characteristics of suspected oils from the same drilling platform. In this report, a case study from the waters of the South China Sea is presented, and multidimensional scaling analysis (MDS) is introduced to demonstrate how oil fingerprints can be combined with mathematical methods to identify the source of spilled oil from highly similar suspected sources. The results suggest that the MDS calculation based on oil fingerprints and subsequently integrated with specific biomarkers in spilled oils is the most effective method with a great potential for determining the source in terms of highly similar suspected oils. PMID:25765488

  12. Joint GWAS Analysis: Comparing similar GWAS at different genomic resolutions identifies novel pathway associations with six complex diseases

    PubMed Central

    McGeachie, Michael J.; Clemmer, George L.; Lasky-Su, Jessica; Dahlin, Amber; Raby, Benjamin A.; Weiss, Scott T.

    2014-01-01

    We show here that combining two existing genome wide association studies (GWAS) yields additional biologically relevant information, beyond that obtained by either GWAS separately. We propose Joint GWAS Analysis, a method that compares a pair of GWAS for similarity among the top SNP associations, top genes identified, gene functional clusters, and top biological pathways. We show that Joint GWAS Analysis identifies additional enriched biological pathways that would be missed by traditional Single-GWAS analysis. Furthermore, we examine the similarities of six complex genetic disorders at the SNP-level, gene-level, gene-cluster-level, and pathway-level. We make concrete hypotheses regarding novel pathway associations for several complex disorders considered, based on the results of Joint GWAS Analysis. Together, these results demonstrate that common complex disorders share substantially more genomic architecture than has been previously realized and that the meta-analysis of GWAS needs not be limited to GWAS of the same phenotype to be informative. PMID:25838990

  13. 1,4-dihydroxy-2-naphthoic Acid Induces Apoptosis in Human Keratinocyte: Potential Application for Psoriasis Treatment

    PubMed Central

    Mok, Chong-Fai; Xie, Chuan-Ming; Sham, Kathy Wai-Yan; Lin, Zhi-Xiu; Cheng, Christopher Hon-Ki

    2013-01-01

    Psoriasis, which affects approximately 1–3% of the population worldwide, is a chronic inflammatory skin disorder characterized by epidermal keratinocytes hyperproliferation, abnormal differentiation, and inflammatory infiltration. Decrease in keratinocyte apoptosis is a specific pathogenic phenomenon in psoriasis. Chinese herbs have been used for the treatment of psoriasis in China showing promising effect in clinical trials. A traditional Chinese medicine has relatively fewer side effects with longer remission time and lower recurrence rate. The extract of Rubia cordifolia L. (EA) was previously found by us to induce HaCaT keratinocytes apoptosis. In this study we identified one of the components in Rubia cordifolia L., the anthraquinone precursor 1,4-dihydroxy-2-naphthoic acid (DHNA), induces HaCaT keratinocytes apoptosis through G0/G1 cell cycle arrest. We have also demonstrated that DHNA acts through both caspase-dependent and caspase-independent pathways. Besides, cytotoxicity and IL-1α release assays indicate that DHNA causes less irritation problems than dithranol, which is commonly employed to treat psoriasis in many countries. Since DHNA possesses similar apoptotic effects on keratinocytes as dithranol but causes less irritation, DHNA therefore constitutes a promising alternative agent for treating psoriasis. Our studies also provide an insight on the potential of using EA and DHNA, alternatively, as a safe and effective treatment modality for psoriasis. PMID:23690852

  14. Large-Scale Chemical Similarity Networks for Target Profiling of Compounds Identified in Cell-Based Chemical Screens

    PubMed Central

    Lo, Yu-Chen; Senese, Silvia; Li, Chien-Ming; Hu, Qiyang; Huang, Yong; Damoiseaux, Robert; Torres, Jorge Z.

    2015-01-01

    Target identification is one of the most critical steps following cell-based phenotypic chemical screens aimed at identifying compounds with potential uses in cell biology and for developing novel disease therapies. Current in silico target identification methods, including chemical similarity database searches, are limited to single or sequential ligand analysis that have limited capabilities for accurate deconvolution of a large number of compounds with diverse chemical structures. Here, we present CSNAP (Chemical Similarity Network Analysis Pulldown), a new computational target identification method that utilizes chemical similarity networks for large-scale chemotype (consensus chemical pattern) recognition and drug target profiling. Our benchmark study showed that CSNAP can achieve an overall higher accuracy (>80%) of target prediction with respect to representative chemotypes in large (>200) compound sets, in comparison to the SEA approach (60–70%). Additionally, CSNAP is capable of integrating with biological knowledge-based databases (Uniprot, GO) and high-throughput biology platforms (proteomic, genetic, etc) for system-wise drug target validation. To demonstrate the utility of the CSNAP approach, we combined CSNAP's target prediction with experimental ligand evaluation to identify the major mitotic targets of hit compounds from a cell-based chemical screen and we highlight novel compounds targeting microtubules, an important cancer therapeutic target. The CSNAP method is freely available and can be accessed from the CSNAP web server (http://services.mbi.ucla.edu/CSNAP/). PMID:25826798

  15. RXRα ablation in epidermal keratinocytes enhances UV radiation induced DNA damage, apoptosis, and proliferation of keratinocytes and melanocytes

    PubMed Central

    Wang, Zhixing; Coleman, Daniel J.; Bajaj, Gaurav; Liang, Xiaobo; Ganguli-Indra, Gitali; Indra, Arup Kumar

    2011-01-01

    We show here that keratinocytic nuclear receptor Retinoid X Receptor α (RXRα) regulates mouse keratinocyte and melanocyte homeostasis following acute ultraviolet radiation (UVR). Keratinocytic RXRα has a protective role on UVR-induced keratinocyte and melanocyte proliferation/differentiation, oxidative stress mediated DNA damage and cellular apoptosis. We discovered that keratinocytic RXRα in a cell autonomous manner regulate mitogenic growth responses in skin epidermis via secretion of hbEGF, GMCSF, IL1-α and COX2, and activation of MAPK pathways. We identified altered expression of several keratinocyte-derived mitogenic paracrine growth factors such as ET-1, HGF, α–MSH, SCF and FGF2 in skin of mice lacking RXRα in epidermal keratinocytes (RXRαep−/− mice), which in a non-cell autonomous manner modulated melanocyte proliferation and activation after UVR. RXRαep−/− mouse represents a unique animal model where UVR induces melanocyte proliferation/activation in both epidermis and dermis. Considered together, our results suggest that RXR antagonists, together with inhibitors of cell proliferation can be effective to prevent solar UV radiation induced photo-carcinogenesis. PMID:20944655

  16. Functional analysis of ZFP36 proteins in keratinocytes.

    PubMed

    Prenzler, Frauke; Fragasso, Annunziata; Schmitt, Angelika; Munz, Barbara

    2016-08-01

    The ZFP36 family of zinc finger proteins, including ZFP36, ZFP36L1, and ZFP36L2, regulates the production of growth factors and cytokines via destabilization of the respective mRNAs. We could recently demonstrate that in cultured keratinocytes, expression of the ZFP36, ZFP36L1, and ZFP36L2 genes is induced by growth factors and cytokines and that ZFP36L1 is a potent regulator of keratinocyte VEGF production. We now further analyzed the localization and function of ZFP36 proteins in the skin, specifically in epidermal keratinocytes. We found that in human epidermis, the ZFP36 protein could be detected in basal and suprabasal keratinocytes, whereas ZFP36L1 and ZFP36L2 were expressed mainly in the basal layer, indicating different and non-redundant functions of the three proteins in the epidermis. Consistently, upon inhibition of ZFP36 or ZFP36L1 expression using specific siRNAs, there was no major effect on expression of the respective other gene. In addition, we demonstrate that both ZFP36 and ZFP36L1 influence keratinocyte cell cycle, differentiation, and apoptosis in a distinct manner. Finally, we show that similarly as ZFP36L1, ZFP36 is a potent regulator of keratinocyte VEGF production. Thus, it is likely that both proteins regulate angiogenesis via paracrine mechanisms. Taken together, our results suggest that ZFP36 proteins might control reepithelialization and angiogenesis in the skin in a multimodal manner. PMID:27182009

  17. Hyaluronan-phosphatidylethanolamine polymers form pericellular coats on keratinocytes and promote basal keratinocyte proliferation.

    PubMed

    Symonette, Caitlin J; Kaur Mann, Aman; Tan, Xiao Cherie; Tolg, Cornelia; Ma, Jenny; Perera, Francisco; Yazdani, Arjang; Turley, Eva A

    2014-01-01

    Aged keratinocytes have diminished proliferative capacity and hyaluronan (HA) cell coats, which are losses that contribute to atrophic skin characterized by reduced barrier and repair functions. We formulated HA-phospholipid (phosphatidylethanolamine, HA-PE) polymers that form pericellular coats around cultured dermal fibroblasts independently of CD44 or RHAMM display. We investigated the ability of these HA-PE polymers to penetrate into aged mouse skin and restore epidermal function in vivo. Topically applied Alexa(647)-HA-PE penetrated into the epidermis and dermis, where it associated with both keratinocytes and fibroblasts. In contrast, Alexa(647)-HA was largely retained in the outer cornified layer of the epidermis and quantification of fluorescence confirmed that significantly more Alexa(647)-HA-PE penetrated into and was retained within the epidermis than Alexa(647)-HA. Multiple topical applications of HA-PE to shaved mouse skin significantly stimulated basal keratinocyte proliferation and epidermal thickness compared to HA or vehicle cream alone. HA-PE had no detectable effect on keratinocyte differentiation and did not promote local or systemic inflammation. These effects of HA-PE polymers are similar to those reported for endogenous epidermal HA in youthful skin and show that topical application of HA-PE polymers can restore some of the impaired functions of aged epidermis. PMID:25276814

  18. Hyaluronan-Phosphatidylethanolamine Polymers Form Pericellular Coats on Keratinocytes and Promote Basal Keratinocyte Proliferation

    PubMed Central

    Symonette, Caitlin J.; Tan, Xiao Cherie; Tolg, Cornelia; Ma, Jenny; Perera, Francisco; Turley, Eva A.

    2014-01-01

    Aged keratinocytes have diminished proliferative capacity and hyaluronan (HA) cell coats, which are losses that contribute to atrophic skin characterized by reduced barrier and repair functions. We formulated HA-phospholipid (phosphatidylethanolamine, HA-PE) polymers that form pericellular coats around cultured dermal fibroblasts independently of CD44 or RHAMM display. We investigated the ability of these HA-PE polymers to penetrate into aged mouse skin and restore epidermal function in vivo. Topically applied Alexa647-HA-PE penetrated into the epidermis and dermis, where it associated with both keratinocytes and fibroblasts. In contrast, Alexa647-HA was largely retained in the outer cornified layer of the epidermis and quantification of fluorescence confirmed that significantly more Alexa647-HA-PE penetrated into and was retained within the epidermis than Alexa647-HA. Multiple topical applications of HA-PE to shaved mouse skin significantly stimulated basal keratinocyte proliferation and epidermal thickness compared to HA or vehicle cream alone. HA-PE had no detectable effect on keratinocyte differentiation and did not promote local or systemic inflammation. These effects of HA-PE polymers are similar to those reported for endogenous epidermal HA in youthful skin and show that topical application of HA-PE polymers can restore some of the impaired functions of aged epidermis. PMID:25276814

  19. Expression and modulation of IL-1 alpha in murine keratinocytes

    SciTech Connect

    Ansel, J.C.; Luger, T.A.; Lowry, D.; Perry, P.; Roop, D.R.; Mountz, J.D.

    1988-04-01

    Murine and human keratinocytes produce an IL-1-like factor that appears to be similar if not identical to monocyte-derived IL-1. IL-1 may be an important mediator in cutaneous inflammatory responses, however, little is currently known concerning factors that may modulate IL-1 expression in keratinocytes. To address this issue we examined the effect of LPS, UV, and the cell differentiation state on murine keratinocyte IL-1 mRNA expression. Our results indicated that as with the murine P388D1 monocyte cell line, PAM 212 keratinocytes constitutively express abundant amounts of IL-1 alpha mRNA. On exposure to LPS (100 micrograms/ml) for 8 h there was more than 10 times the increase in PAM 212 IL-1 alpha mRNA which was accompanied by a sixfold increase in supernatant IL-1 activity. Similarly UV irradiation had a significant effect on keratinocyte IL-1 alpha expression. High dose UV (300 mJ/cm2) inhibited PAM 212 IL-1 alpha expression at 4, 8, 24, 48 h post-UV whereas a lower dose of UV (100 mJ/cm2) inhibited UV at 4 and 8 h post-UV, but induced IL-1 expression at 24 and 48 h post-UV. The expression of IL-1 alpha varied with the differentiation state of the keratinocytes. Freshly removed newborn murine keratinocytes were found to constitutively express IL-1 alpha mRNA. Keratinocytes grown in low (Ca2+) tissue culture media (0.05 mM) for 6 days, functionally and phenotypically become undifferentiated and express increased quantities of IL-1 alpha mRNA, whereas cells grown in high (Ca2+) media (1.2 mM) for 6 days become terminally differentiated and IL-1 expression ceased. Keratinocytes cultured for 3 days in low (Ca2+) conditions expressed an intermediate level of IL-1 alpha. In contrast, little or no IL-1 beta mRNA was detected in either the PAM 212 cells or newborn murine keratinocytes.

  20. Automatic Online Educational Game Content Creation by Identifying Similar Chinese Characters with Radical Extraction and Graph Matching Algorithms

    ERIC Educational Resources Information Center

    Lai, Jason Kwong-Hung; Leung, Howard; Hu, Zhi-Hui; Tang, Jeff K. T.; Xu, Yun

    2010-01-01

    One of the difficulties in learning Chinese characters is distinguishing similar characters. This can cause misunderstanding and miscommunication in daily life. Thus, it is important for students learning the Chinese language to be able to distinguish similar characters and understand their proper usage. In this paper, the authors propose a game…

  1. Micronucleus formation in human keratinocytes is dependent on radiation quality and tissue architecture.

    PubMed

    Snijders, Antoine M; Mannion, Brandon J; Leung, Stanley G; Moon, Sol C; Kronenberg, Amy; Wiese, Claudia

    2015-01-01

    The cytokinesis-block micronucleus (MN) assay was used to assess the genotoxicity of low doses of different types of space radiation. Normal human primary keratinocytes and immortalized keratinocytes grown in 2D monolayers each were exposed to graded doses of 0.3 or 1.0 GeV/n silicon ions or similar energies of iron ions. The frequencies of induced MN were determined and compared to γ-ray data. RBE(max) values ranged from 1.6 to 3.9 for primary keratinocytes and from 2.4 to 6.3 for immortalized keratinocytes. At low radiation doses ≤ 0.4 Gy, 0.3 GeV/n iron ions were the most effective at inducing MN in normal keratinocytes. An "over-kill effect" was observed for 0.3 GeV/n iron ions at higher doses, wherein 1.0 GeV/n iron ions were most efficient in inducing MN. In immortalized keratinocytes, 0.3 GeV/n iron ions produced MN with greater frequency than 1.0 GeV/n iron ions, except at the highest dose tested. MN formation was higher in immortalized keratinocytes than in normal keratinocytes for all doses and radiation qualities investigated. MN induction was also assessed in human keratinocytes cultured in 3D to simulate the complex architecture of human skin. RBE values for MN formation in 3D were reduced for normal keratinocytes exposed to iron ions, but were elevated for immortalized keratinocytes. Overall, MN induction was significantly lower in keratinocytes cultured in 3D than in 2D. Together, the results suggest that tissue architecture and immortalization status modulate the genotoxic response to space radiation, perhaps via alterations in DNA repair fidelity. PMID:25041929

  2. Amarogentin Displays Immunomodulatory Effects in Human Mast Cells and Keratinocytes

    PubMed Central

    Wölfle, Ute; Haarhaus, Birgit; Schempp, Christoph M.

    2015-01-01

    Keratinocytes express the bitter taste receptors TAS2R1 and TAS2R38. Amarogentin as an agonist for TAS2R1 and other TAS2Rs promotes keratinocyte differentiation. Similarly, mast cells are known to express bitter taste receptors. The aim of this study was to assess whether bitter compounds display immunomodulatory effects on these immunocompetent cells in the skin, so that they might be a target in chronic inflammatory diseases such as atopic dermatitis and psoriasis. Here, we investigated the impact of amarogentin on substance P-induced release of histamine and TNF-α from the human mast cell line LAD-2. Furthermore, the effect of amarogentin on HaCaT keratinocytes costimulated with TNF-α and histamine was investigated. Amarogentin inhibited in LAD-2 cells substance P-induced production of newly synthesized TNF-α, but the degranulation and release of stored histamine were not affected. In HaCaT keratinocytes histamine and TNF-α induced IL-8 and MMP-1 expression was reduced by amarogentin to a similar extent as with azelastine. In conclusion amarogentin displays immunomodulatory effects in the skin by interacting with mast cells and keratinocytes. PMID:26600671

  3. AMPK regulation of the growth of cultured human keratinocytes

    SciTech Connect

    Saha, Asish K. . E-mail: aksaha@bu.edu; Persons, Kelly; Safer, Joshua D.; Luo Zhijun; Holick, Michael F.; Ruderman, Neil B.

    2006-10-20

    AMP kinase (AMPK) is a fuel sensing enzyme that responds to cellular energy depletion by increasing processes that generate ATP and inhibiting others that require ATP but are not acutely necessary for survival. In the present study, we examined the relationship between AMPK activation and the growth (proliferation) of cultured human keratinocytes and assessed whether the inhibition of keratinocyte growth by vitamin D involves AMPK activation. In addition, we explored whether the inhibition of keratinocyte proliferation as they approach confluence could be AMPK-related. Keratinocytes were incubated for 12 h with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside (AICAR). At concentrations of 10{sup -4} and 10{sup -3} M, AICAR inhibited keratinocyte growth by 50% and 95%, respectively, based on measurements of thymidine incorporation into DNA. It also increased AMPK and acetyl CoA carboxylase phosphorylation (P-AMPK and P-ACC) and decreased the concentration of malonyl CoA confirming that AMPK activation had occurred. Incubation with the thiazolidinedione, troglitazone (10{sup -6} M) caused similar alterations in P-AMPK, P-ACC, and cell growth. In contrast, the well known inhibition of keratinocyte growth by 1,25-dihydroxyvitamin D{sub 3} (10{sup -7} and 10{sup -6} M) was not associated with changes in P-AMPK or P-ACC. Like most cells, the growth of keratinocytes diminished as they approached confluence. Thus, it was of note that we found a progressive increase in P-AMPK (1.5- to 2-fold, p < 0.05) as keratinocytes grown in control medium went from 25% to 100% confluence. In conclusion, the data are consistent with the hypothesis that activation of AMPK acts as a signal to diminish the proliferation of cultured keratinocytes as they approach confluence. They also suggest that AMPK activators, such as AICAR and troglitazone, inhibit keratinocyte growth and that the inhibition of cell growth by 1,25-dihydroxyvitamin D{sub 3} is AMPK-independent.

  4. A Systems Biology Approach for Identifying Hepatotoxicant Groups Based on Similarity in Mechanisms of Action and Chemical Structure.

    PubMed

    Hebels, Dennie G A J; Rasche, Axel; Herwig, Ralf; van Westen, Gerard J P; Jennen, Danyel G J; Kleinjans, Jos C S

    2016-01-01

    When evaluating compound similarity, addressing multiple sources of information to reach conclusions about common pharmaceutical and/or toxicological mechanisms of action is a crucial strategy. In this chapter, we describe a systems biology approach that incorporates analyses of hepatotoxicant data for 33 compounds from three different sources: a chemical structure similarity analysis based on the 3D Tanimoto coefficient, a chemical structure-based protein target prediction analysis, and a cross-study/cross-platform meta-analysis of in vitro and in vivo human and rat transcriptomics data derived from public resources (i.e., the diXa data warehouse). Hierarchical clustering of the outcome scores of the separate analyses did not result in a satisfactory grouping of compounds considering their known toxic mechanism as described in literature. However, a combined analysis of multiple data types may hypothetically compensate for missing or unreliable information in any of the single data types. We therefore performed an integrated clustering analysis of all three data sets using the R-based tool iClusterPlus. This indeed improved the grouping results. The compound clusters that were formed by means of iClusterPlus represent groups that show similar gene expression while simultaneously integrating a similarity in structure and protein targets, which corresponds much better with the known mechanism of action of these toxicants. Using an integrative systems biology approach may thus overcome the limitations of the separate analyses when grouping liver toxicants sharing a similar mechanism of toxicity. PMID:27311473

  5. Human Keratinocytes Are Vanilloid Resistant

    PubMed Central

    Pecze, László; Szabó, Kornélia; Széll, Márta; Jósvay, Katalin; Kaszás, Krisztián; Kúsz, Erzsébet; Letoha, Tamás; Prorok, János; Koncz, István; Tóth, András; Kemény, Lajos; Vizler, Csaba; Oláh, Zoltán

    2008-01-01

    Background Use of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca2+-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. Methods To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. Results Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca2+-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1–50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca2+-cytotoxity ([RTX]>15 µM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca2+-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. Conclusion TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials. PMID:18852901

  6. Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells

    PubMed Central

    Hahn, Hyung Jin; Youn, Hae Jeong; Cha, Hwa Jun; Kim, Karam; An, Sungkwan

    2016-01-01

    Background We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model. Objective We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation. Methods To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation. Results Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation. Conclusion These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation. PMID:27489424

  7. Hi-C analysis in Arabidopsis identifies the KNOT, a structure with similarities to the flamenco locus of Drosophila.

    PubMed

    Grob, Stefan; Schmid, Marc W; Grossniklaus, Ueli

    2014-09-01

    Chromosomes are folded, spatially organized, and regulated by epigenetic marks. How chromosomal architecture is connected to the epigenome is not well understood. We show that chromosomal architecture of Arabidopsis is tightly linked to the epigenetic state. Furthermore, we show how physical constraints, such as nuclear size, correlate with the folding principles of chromatin. We also describe a nuclear structure, termed KNOT, in which genomic regions of all five Arabidopsis chromosomes interact. These KNOT ENGAGED ELEMENT (KEE) regions represent heterochromatic islands within euchromatin. Similar to PIWI-interacting RNA clusters, such as flamenco in Drosophila, KEEs represent preferred landing sites for transposable elements, which may be part of a transposon defense mechanism in the Arabidopsis nucleus. PMID:25132176

  8. Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake*

    PubMed Central

    Chung, Heesung; Jung, Hyejung; Lee, Jung-hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

    2014-01-01

    Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake. PMID:24951591

  9. H(+)/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport.

    PubMed

    Kudo, Michiko; Katayoshi, Takeshi; Kobayashi-Nakamura, Kumiko; Akagawa, Mitsugu; Tsuji-Naito, Kentaro

    2016-07-01

    Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H(+) gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake. PMID:27216463

  10. Dynamic Transcriptional and Epigenetic Regulation of Human Epidermal Keratinocyte Differentiation.

    PubMed

    Cavazza, Alessia; Miccio, Annarita; Romano, Oriana; Petiti, Luca; Malagoli Tagliazucchi, Guidantonio; Peano, Clelia; Severgnini, Marco; Rizzi, Ermanno; De Bellis, Gianluca; Bicciato, Silvio; Mavilio, Fulvio

    2016-04-12

    Human skin is maintained by the differentiation and maturation of interfollicular stem and progenitors cells. We used DeepCAGE, genome-wide profiling of histone modifications and retroviral integration analysis, to map transcripts, promoters, enhancers, and super-enhancers (SEs) in prospectively isolated keratinocytes and transit-amplifying progenitors, and retrospectively defined keratinocyte stem cells. We show that >95% of the active promoters are in common and differentially regulated in progenitors and differentiated keratinocytes, while approximately half of the enhancers and SEs are stage specific and account for most of the epigenetic changes occurring during differentiation. Transcription factor (TF) motif identification and correlation with TF binding site maps allowed the identification of TF circuitries acting on enhancers and SEs during differentiation. Overall, our study provides a broad, genome-wide description of chromatin dynamics and differential enhancer and promoter usage during epithelial differentiation, and describes a novel approach to identify active regulatory elements in rare stem cell populations. PMID:27050947

  11. Dynamic Transcriptional and Epigenetic Regulation of Human Epidermal Keratinocyte Differentiation

    PubMed Central

    Cavazza, Alessia; Miccio, Annarita; Romano, Oriana; Petiti, Luca; Malagoli Tagliazucchi, Guidantonio; Peano, Clelia; Severgnini, Marco; Rizzi, Ermanno; De Bellis, Gianluca; Bicciato, Silvio; Mavilio, Fulvio

    2016-01-01

    Summary Human skin is maintained by the differentiation and maturation of interfollicular stem and progenitors cells. We used DeepCAGE, genome-wide profiling of histone modifications and retroviral integration analysis, to map transcripts, promoters, enhancers, and super-enhancers (SEs) in prospectively isolated keratinocytes and transit-amplifying progenitors, and retrospectively defined keratinocyte stem cells. We show that >95% of the active promoters are in common and differentially regulated in progenitors and differentiated keratinocytes, while approximately half of the enhancers and SEs are stage specific and account for most of the epigenetic changes occurring during differentiation. Transcription factor (TF) motif identification and correlation with TF binding site maps allowed the identification of TF circuitries acting on enhancers and SEs during differentiation. Overall, our study provides a broad, genome-wide description of chromatin dynamics and differential enhancer and promoter usage during epithelial differentiation, and describes a novel approach to identify active regulatory elements in rare stem cell populations. PMID:27050947

  12. Reconstruction of Rabbit Urethral Epithelium with Skin Keratinocytes

    PubMed Central

    Rogovaya, O. S.; Fayzulin, A. K.; Vasiliev, A. V.; Kononov, A. V.; Terskikh, V. V.

    2015-01-01

    We have investigated the living skin equivalent (LSE) as an alternative source of plastic material for closing full-thickness epithelial-stromal urethral injuries. The possibility of transdifferentiation of epidermal keratinocytes, a component of 3D tissue constructs, was investigated in vivo in a model of the recovery of urethral injuries in laboratory rabbits. Autologous grafting of LSE in de-epithelialized urethra showed that skin keratinocytes placed in a specific in vivo microenvironment can be incorporated into the damaged area and function as urothelium. The use of EGFP transfected keratinocytes allowed us to identify transplanted cells. The reconstructed urethral tubes did not develop strictures or fistulas at the site of the grafted LSE. Immunohistochemical studies of neo-urothelium revealed EGFP-positive cells expressing the urothelial markers K7 and UP3. PMID:25927003

  13. Reconstruction of rabbit urethral epithelium with skin keratinocytes.

    PubMed

    Rogovaya, O S; Fayzulin, A K; Vasiliev, A V; Kononov, A V; Terskikh, V V

    2015-01-01

    We have investigated the living skin equivalent (LSE) as an alternative source of plastic material for closing full-thickness epithelial-stromal urethral injuries. The possibility of transdifferentiation of epidermal keratinocytes, a component of 3D tissue constructs, was investigated in vivo in a model of the recovery of urethral injuries in laboratory rabbits. Autologous grafting of LSE in de-epithelialized urethra showed that skin keratinocytes placed in a specific in vivo microenvironment can be incorporated into the damaged area and function as urothelium. The use of EGFP transfected keratinocytes allowed us to identify transplanted cells. The reconstructed urethral tubes did not develop strictures or fistulas at the site of the grafted LSE. Immunohistochemical studies of neo-urothelium revealed EGFP-positive cells expressing the urothelial markers K7 and UP3. PMID:25927003

  14. A comparative molecular similarity index analysis (CoMSIA) study identifies an HLA-A2 binding supermotif.

    PubMed

    Doytchinova, Irini A; Flower, Darren R

    2002-01-01

    The 3D-QSAR CoMSIA technique was applied to a set of 458 peptides binding to the five most widespread HLA-A2-like alleles: A*0201, A*0202, A*0203, A*0206 and A*6802. Models comprising the main physicochemical properties (steric bulk, electron density, hydrophobicity and hydrogen-bond formation abilities) were obtained with acceptable predictivity (q2 ranged from 0.385 to 0.683). The use of coefficient contour maps allowed an A2-supermotif to be identified based on common favoured and disfavoured areas. The CoMSIA definition for the best HLA-A2 binder is as follows: hydrophobic aromatic amino acid at position 1; hydrophobic bulky side chains at positions 2, 6 and 9; non-hydrogen-bond-forming amino acids at position 3; small aliphatic hydrogen-bond donors at position 4; aliphatic amino acids at position 5; small aliphatic side chains at position 7; and small aliphatic hydrophilic and hydrogen-bond forming amino acids at position 8. PMID:12602948

  15. Human melanocytes mitigate keratinocyte-dependent contraction in an in vitro collagen contraction assay.

    PubMed

    Rakar, Jonathan; Krammer, Markus P; Kratz, Gunnar

    2015-08-01

    Scarring is an extensive problem in burn care, and treatment can be especially complicated in cases of hypertrophic scarring. Contraction is an important factor in scarring but the contribution of different cell types remains unclear. We have investigated the contractile behavior of keratinocytes, melanocytes and fibroblasts by using an in vitro collagen gel assay aimed at identifying a modulating role of melanocytes in keratinocyte-mediated contraction. Cells were seeded on a collagen type I gel substrate and the change in gel dimensions were measured over time. Hematoxylin & Eosin-staining and immunohistochemistry against pan-cytokeratin and microphthalmia-associated transcription factor showed that melanocytes integrated between keratinocytes and remained there throughout the experiments. Keratinocyte- and fibroblast-seeded gels contracted significantly over time, whereas melanocyte-seeded gels did not. Co-culture assays showed that melanocytes mitigate the keratinocyte-dependent contraction (significantly slower and 18-32% less). Fibroblasts augmented the contraction in most assays (approximately 6% more). Non-contact co-cultures showed some influence on the keratinocyte-dependent contraction. Results show that mechanisms attributable to melanocytes, but not fibroblasts, can mitigate keratinocyte contractile behavior. Contact-dependent mechanisms are stronger modulators than non-contact dependent mechanisms, but both modes carry significance to the contraction modulation of keratinocytes. Further investigations are required to determine the mechanisms involved and to determine the utility of melanocytes beyond hypopigmentation in improved clinical regimes of burn wounds and wound healing. PMID:25466959

  16. Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation

    PubMed Central

    Nakamura, Takashi; Yoshitomi, Yasuo; Sakai, Kiyoshi; Patel, Vyomesh; Fukumoto, Satoshi; Yamada, Yoshihiko

    2014-01-01

    ABSTRACT The basal layer of the epidermis contains stem cells and transit amplifying cells that rapidly proliferate and differentiate further into the upper layers of the epidermis. A number of molecules have been identified as regulators of this process, including p63 (also known as tumor protein 63) and Notch1. However, little is known about the mechanisms that regulate the transitions from stem cell to proliferating or differentiating transit amplifying cell. Here, we demonstrate that epiprofin (Epfn, also known as Sp6) plays crucial distinct roles in these transition stages as a cell cycle regulator and a transcription factor. Epfn knockout mice have a thickened epidermis, in which p63-expressing basal cells form multiple layers owing to the accumulation of premature transit amplifying cells with reduced proliferation and a reduction in the number of differentiating keratinocytes expressing Notch1. We found that low levels of Epfn expression increased the proliferation of human immortalized keratinocyte (HaCaT) cells by increasing EGF responsiveness and superphosphorylation of Rb. By contrast, high levels of Epfn expression promoted cell cycle exit and differentiation, by reducing E2F transactivation and inducing Notch1 expression. Our findings identify multiple novel functions of Epfn in epidermal development. PMID:25344255

  17. Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation.

    PubMed

    Nakamura, Takashi; Yoshitomi, Yasuo; Sakai, Kiyoshi; Patel, Vyomesh; Fukumoto, Satoshi; Yamada, Yoshihiko

    2014-12-15

    The basal layer of the epidermis contains stem cells and transit amplifying cells that rapidly proliferate and differentiate further into the upper layers of the epidermis. A number of molecules have been identified as regulators of this process, including p63 (also known as tumor protein 63) and Notch1. However, little is known about the mechanisms that regulate the transitions from stem cell to proliferating or differentiating transit amplifying cell. Here, we demonstrate that epiprofin (Epfn, also known as Sp6) plays crucial distinct roles in these transition stages as a cell cycle regulator and a transcription factor. Epfn knockout mice have a thickened epidermis, in which p63-expressing basal cells form multiple layers owing to the accumulation of premature transit amplifying cells with reduced proliferation and a reduction in the number of differentiating keratinocytes expressing Notch1. We found that low levels of Epfn expression increased the proliferation of human immortalized keratinocyte (HaCaT) cells by increasing EGF responsiveness and superphosphorylation of Rb. By contrast, high levels of Epfn expression promoted cell cycle exit and differentiation, by reducing E2F transactivation and inducing Notch1 expression. Our findings identify multiple novel functions of Epfn in epidermal development. PMID:25344255

  18. Dynamic and Physical Clustering of Gene Expression during Epidermal Barrier Formation in Differentiating Keratinocytes

    PubMed Central

    Copley, Richard; Taylor, Martin S.; Hayden, Patrick J.; Stolper, Gina; Mott, Richard; Hein, Jotun; Moffatt, Miriam F.; Cookson, William O. C. M.

    2009-01-01

    The mammalian epidermis is a continually renewing structure that provides the interface between the organism and an innately hostile environment. The keratinocyte is its principal cell. Keratinocyte proteins form a physical epithelial barrier, protect against microbial damage, and prepare immune responses to danger. Epithelial immunity is disordered in many common diseases and disordered epithelial differentiation underlies many cancers. In order to identify the genes that mediate epithelial development we used a tissue model of the skin derived from primary human keratinocytes. We measured global gene expression in triplicate at five times over the ten days that the keratinocytes took to fully differentiate. We identified 1282 gene transcripts that significantly changed during differentiation (false discovery rate <0.01%). We robustly grouped these transcripts by K-means clustering into modules with distinct temporal expression patterns, shared regulatory motifs, and biological functions. We found a striking cluster of late expressed genes that form the structural and innate immune defences of the epithelial barrier. Gene Ontology analyses showed that undifferentiated keratinocytes were characterised by genes for motility and the adaptive immune response. We systematically identified calcium-binding genes, which may operate with the epidermal calcium gradient to control keratinocyte division during skin repair. The results provide multiple novel insights into keratinocyte biology, in particular providing a comprehensive list of known and previously unrecognised major components of the epidermal barrier. The findings provide a reference for subsequent understanding of how the barrier functions in health and disease. PMID:19888454

  19. DNA repair in cultured keratinocytes

    SciTech Connect

    Liu, S.C.; Parsons, S.; Hanawalt, P.C.

    1983-07-01

    Most of our understanding of DNA repair mechanisms in human cells has come from the study of these processes in cultured fibroblasts. The unique properties of keratinocytes and their pattern of terminal differentiation led us to a comparative examination of their DNA repair properties. The relative repair capabilities of the basal cells and the differentiated epidermal keratinocytes as well as possible correlations of DNA repair capacity with respect to age of the donor have been examined. In addition, since portions of human skin are chronically exposed to sunlight, the repair response to ultraviolet (UV) irradiation (254 nm) when the cells are conditioned by chronic low-level UV irradiation has been assessed. The comparative studies of DNA repair in keratinocytes from infant and aged donors have revealed no significant age-related differences for repair of UV-induced damage to DNA. Sublethal UV conditioning of cells from infant skin had no appreciable effect on either the repair or normal replication response to higher, challenge doses of UVL. However, such conditioning resulted in attenuated repair in keratinocytes from adult skin after UV doses above 25 J/m2. In addition, a surprising enhancement in replication was seen in conditioned cells from adult following challenge UV doses.

  20. A novel emerging virus with indistinguishable symptoms and genome structure similar to citrus leprosis virus C identified by small RNA deep sequencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus leprosis disease (CiLD) in Colombia was previously shown to be caused by cytoplasmic Citrus leprosis virus (CiLV-C). In 2011, ELISA and RT-PCR based diagnostic methods failed to identify CiLV-C from CiLD samples, but virions similar to CiLV-C were observed in cytoplasm of the symptomatic leav...

  1. Grape Cells (Multinucleated Keratinocytes) in Noninfectious Dermatoses: Case Series and Review of the Literature

    PubMed Central

    Sulit, Daryl J.; Adams, Erin G.; Shvartsman, Katerina R.; Rapini, Ronald P.

    2015-01-01

    Abstract: Multinucleated keratinocytes (also known as multinucleated epidermal giant cells) are a frequently overlooked histological finding in noninfectious inflammatory dermatoses. They are sometimes found in conditions characterized by chronic rubbing and pruritus, such as lichen simplex chronicus or prurigo nodularis, and may be a helpful clue in making the clinical diagnosis. This finding must be differentiated from other conditions characterized by multinucleated keratinocytes on histopathology, specifically herpes simplex, varicella zoster, or measles viral infections. The authors present a case series of 2 patients with unique clinical noninfectious diagnoses but similar histopathologic findings on biopsy. The histopathologic findings on both cases demonstrated multinucleated keratinocytes, which were related to manipulation of the epidermis. PMID:26588345

  2. Vitiligo patient-derived keratinocytes exhibit characteristics of normal wound healing via epithelial to mesenchymal transition.

    PubMed

    Banerjee, Poulomi; Venkatachalam, Sandhyaa; Mamidi, Murali Krishna; Bhonde, Ramesh; Shankar, Krupa; Pal, Rajarshi

    2015-05-01

    Vitiligo is an autoimmune disorder that leads to depigmentation of skin via melanocyte dysfunction. Keratinocyte-induced toxicity is one among the several etiological factors implicated for vitiligo, and hence, autologous keratinocyte grafting is projected as one of the primary mode of treatment for vitiligo. However, reports indicate that perilesional keratinocytes not only display signatures of apoptosis but also could secrete cytokines and mediators which have antagonistic effect on proliferation or survival. Therefore, we investigated how vitiligo patients' derived keratinocytes respond to surplus amounts of inflammatory cytokines and whether they recapitulate events that take place during conventional wound healing. The primary objective of our study was to determine whether keratinocytes isolated from a vitiligo patient would undergo epithelial-mesenchymal transition similar to their normal counterparts upon induction with inflammatory cytokines such as TGF-b1 and EGF. We found that these keratinocytes undergo EMT during wound repair accompanied with increase in the levels of mesenchymal markers and ECM proteins; decrease in the levels of epithelial markers and enhanced migratory ability. Besides, we also demonstrated that EMT induction leads to activation of SMAD and MAPK pathways via Ras, Raf, PAI 1, Snail, Slug and ZO1. To our knowledge, this is the first report on the characterization of primary keratinocytes isolated from vitiligo patients with respect to their wound healing capacity. PMID:25690925

  3. Protective effects of myricetin against ultraviolet-B-induced damage in human keratinocytes.

    PubMed

    Huang, Jheng-Hua; Huang, Chieh-Chen; Fang, Jia-You; Yang, Cheng; Chan, Chi-Ming; Wu, Nan-Lin; Kang, Shung-Wen; Hung, Chi-Feng

    2010-02-01

    Myricetin is a flavonoid similar to quercetin, which is commonly found in natural foods such as berries, vegetables, teas, wine, and herbs. It is considered to be an antioxidant which is capable of quenching photoaging-causing free radicals within the skin. In this study, we investigated the mechanisms underlying protective effect of myricetin on ultraviolet-B (UVB)-induced damage to keratinocytes. We found that myricetin concentration-dependently attenuated UVB-induced keratinocyte death as determined by a cell viability assay. Pretreatment with myricetin also reduced the UVB-induced malondialdehyde level. Moreover, UVB-induced H(2)O(2) generation in keratinocytes was inhibited by myricetin according to flow cytometry, suggesting that myricetin can act as a free radical scavenger when keratinocytes experience photodamage. Furthermore, UVB-induced activation of c-jun-NH(2) terminal kinase (JNK) in keratinocytes was inhibited by myricetin. UVB-induced pre-G(1) phase arrest leading to apoptotic changes in keratinocytes was blocked by myricetin. Taken together, the protective mechanisms of keratinocyte by myricetin against UVB-induced photodamage occur by the inhibition of UVB-induced intracellular hydrogen peroxide production, lipid peroxidation and JNK activation. Therefore, myricetin is suitable for further development as an anti-aging agent for skin care. PMID:19778600

  4. Systemic suppression of delayed-type hypersensitivity by supernatants from UV-irradiated keratinocytes

    SciTech Connect

    Rivas, J.M.; Ullrich, S.E. )

    1992-12-15

    Exposing murine keratinocyte cultures to UV radiation causes the release of a suppressive cytokine that mimics the immunosuppressive effects of total-body UV exposure. Injecting supernatants from UV-irradiated keratinocyte cultures into mice inhibits their ability to generate a delayed-type hypersensitivity reaction against allogeneic histocompatibility Ag, and spleen cells from mice injected with supernatant do not respond to alloantigen in the in vitro MLR. A unique feature of the immunosuppression induced by either total-body UV-exposure or injecting the suppressive cytokine from UV-irradiated keratinocytes is the selectivity of suppression. Although cellular immune reactions such as delayed-type hypersensitivity are suppressed antibody production is unaffected. Because the selective nature to the UV-induced immunosuppression is similar to the biologic activity of IL-10, the authors examined the hypothesis that UV exposure of keratinocytes causes the release of IL-10. Keratinocyte monolayers were exposed to UV radiation and at specific times after exposure mRNA was isolated or the culture supernatant from the cells was collected. These data indicate that activated keratinocytes are capable of secreting IL-10 and suggest that the release of IL-10 by UV-irradiated keratinocytes plays an essential role in the induction of systemic immunosuppression after total-body UV exposure. 44 refs., 3 figs., 2 tabs.

  5. The protective effects of piceatannol from passion fruit (Passiflora edulis) seeds in UVB-irradiated keratinocytes.

    PubMed

    Maruki-Uchida, Hiroko; Kurita, Ikuko; Sugiyama, Kenkichi; Sai, Masahiko; Maeda, Kazuhisa; Ito, Tatsuhiko

    2013-01-01

    The use of naturally occurring botanicals with substantial antioxidant activity to prevent photoageing is receiving increasing attention. We have previously identified piceatannol and scirpusin B, which is a dimer of piceatannol, as strong antioxidants that are present in passion fruit (Passiflora edulis) seeds. In the present study, the effects of passion fruit seed extract, piceatannol, and scirpusin B on human keratinocytes were investigated. The passion fruit seed extract and piceatannol upregulated the glutathione (GSH) levels in keratinocytes in a dose-dependent manner, indicating that piceatannol is an active component of the passion fruit seed extract in keratinocytes. The pretreatment with piceatannol also suppressed the UVB-induced generation of reactive oxygen species (ROS) in the keratinocytes. In addition, the transfer of the medium from the UVB-irradiated keratinocytes to non-irradiated fibroblasts enhanced matrix-metalloproteinase (MMP)-1 activity, and this MMP-1 induction was reduced when the keratinocytes were pretreated with piceatannol. These results suggest that piceatannol attenuates the UVB-induced activity of MMP-1 along with a reduction of ROS generation in keratinocytes. Thus, piceatannol and passion fruit seed extract containing high amounts of piceatannol are potential anti-photoageing cosmetic ingredients. PMID:23649341

  6. The peanut lectin-binding glycoproteins of human epidermal keratinocytes

    SciTech Connect

    Morrison, A.I. ); Keeble, S.; Watt, F.M. )

    1988-08-01

    The peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes. In order to investigate the significance of this change in cell-surface carbohydrate authors have identified the PNA-binding glycoproteins of cultured human keratinocytes and antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of ({sup 14}C)galactose- or ({sup 14}C)mannose- and ({sup 14}C)glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification.

  7. High glucose inhibits ClC-2 chloride channels and attenuates cell migration of rat keratinocytes

    PubMed Central

    Pan, Fuqiang; Guo, Rui; Cheng, Wenguang; Chai, Linlin; Wang, Wenping; Cao, Chuan; Li, Shirong

    2015-01-01

    Background Accumulating evidence has demonstrated that migration of keratinocytes is critical to wound epithelialization, and defects of this function result in chronic delayed-healing wounds in diabetes mellitus patients, and the migration has been proved to be associated with volume-activated chloride channels. The aim of the study is to investigate the effects of high glucose (HG, 25 mM) on ClC-2 chloride channels and cell migration of keratinocytes. Methods Newborn Sprague Dawley rats were used to isolate and culture the keratinocyte in this study. Immunofluorescence assay, real-time polymerase chain reaction, and Western blot assay were used to examine the expression of ClC-2 protein or mRNA. Scratch wound assay was used to measure the migratory ability of keratinocytes. Transwell cell migration assay was used to measure the invasion and migration of keratinocytes. Recombinant lentivirus vectors were established and transducted to keratinocytes. Whole-cell patch clamp was used to perform the electrophysiological studies. Results We found that the expression of ClC-2 was significantly inhibited when keratinocytes were exposed to a HG (25 mM) medium, accompanied by the decline of volume-activated Cl− current (ICl,vol), migration potential, and phosphorylated PI3K as compared to control group. When knockdown of ClC-2 by RNAi or pretreatment with wortmannin, similar results were observed, including ICl,vol and migration keratinocytes were inhibited. Conclusion Our study proved that HG inhibited ClC-2 chloride channels and attenuated cell migration of rat keratinocytes via inhibiting PI3K signaling. PMID:26355894

  8. Lactobacillus reuteri protects epidermal keratinocytes from Staphylococcus aureus-induced cell death by competitive exclusion.

    PubMed

    Prince, Tessa; McBain, Andrew J; O'Neill, Catherine A

    2012-08-01

    Recent studies have suggested that the topical application of probiotic bacteria can improve skin health or combat disease. We have utilized a primary human keratinocyte culture model to investigate whether probiotic bacteria can inhibit Staphylococcus aureus infection. Evaluation of the candidate probiotics Lactobacillus reuteri ATCC 55730, Lactobacillus rhamnosus AC413, and Lactobacillus salivarius UCC118 demonstrated that both L. reuteri and L. rhamnosus, but not L. salivarius, reduced S. aureus-induced keratinocyte cell death in both undifferentiated and differentiated keratinocytes. Keratinocyte survival was significantly higher if the probiotic was applied prior to (P < 0.01) or simultaneously with (P < 0.01) infection with S. aureus but not when added after infection had commenced (P > 0.05). The protective effect of L. reuteri was not dependent on the elaboration of inhibitory substances such as lactic acid. L. reuteri inhibited adherence of S. aureus to keratinocytes by competitive exclusion (P = 0.026). L. salivarius UCC118, however, did not inhibit S. aureus from adhering to keratinocytes (P > 0.05) and did not protect keratinocyte viability. S. aureus utilizes the α5β1 integrin to adhere to keratinocytes, and blocking of this integrin resulted in a protective effect similar to that observed with probiotics (P = 0.03). This suggests that the protective mechanism for L. reuteri-mediated protection of keratinocytes was by competitive exclusion of the pathogen from its binding sites on the cells. Our results suggest that use of a topical probiotic prophylactically could inhibit the colonization of skin by S. aureus and thus aid in the prevention of infection. PMID:22582077

  9. Lactobacillus reuteri Protects Epidermal Keratinocytes from Staphylococcus aureus-Induced Cell Death by Competitive Exclusion

    PubMed Central

    Prince, Tessa; McBain, Andrew J.

    2012-01-01

    Recent studies have suggested that the topical application of probiotic bacteria can improve skin health or combat disease. We have utilized a primary human keratinocyte culture model to investigate whether probiotic bacteria can inhibit Staphylococcus aureus infection. Evaluation of the candidate probiotics Lactobacillus reuteri ATCC 55730, Lactobacillus rhamnosus AC413, and Lactobacillus salivarius UCC118 demonstrated that both L. reuteri and L. rhamnosus, but not L. salivarius, reduced S. aureus-induced keratinocyte cell death in both undifferentiated and differentiated keratinocytes. Keratinocyte survival was significantly higher if the probiotic was applied prior to (P < 0.01) or simultaneously with (P < 0.01) infection with S. aureus but not when added after infection had commenced (P > 0.05). The protective effect of L. reuteri was not dependent on the elaboration of inhibitory substances such as lactic acid. L. reuteri inhibited adherence of S. aureus to keratinocytes by competitive exclusion (P = 0.026). L. salivarius UCC118, however, did not inhibit S. aureus from adhering to keratinocytes (P > 0.05) and did not protect keratinocyte viability. S. aureus utilizes the α5β1 integrin to adhere to keratinocytes, and blocking of this integrin resulted in a protective effect similar to that observed with probiotics (P = 0.03). This suggests that the protective mechanism for L. reuteri-mediated protection of keratinocytes was by competitive exclusion of the pathogen from its binding sites on the cells. Our results suggest that use of a topical probiotic prophylactically could inhibit the colonization of skin by S. aureus and thus aid in the prevention of infection. PMID:22582077

  10. Systemic suppression of delayed-type hypersensitivity by supernatants from UV-irradiated keratinocytes. An essential role for keratinocyte-derived IL-10.

    PubMed

    Rivas, J M; Ullrich, S E

    1992-12-15

    Exposing murine keratinocyte cultures to UV radiation causes the release of a suppressive cytokine that mimics the immunosuppressive effects of total-body UV exposure. Injecting supernatants from UV-irradiated keratinocyte cultures into mice inhibits their ability to generate a delayed-type hypersensitivity reaction against allogeneic histocompatibility Ag, and spleen cells from mice injected with supernatant do not respond to alloantigen in the in vitro MLR. A unique feature of the immunosuppression induced by either total-body UV-exposure or injecting the suppressive cytokine from UV-irradiated keratinocytes is the selectivity of suppression. Although cellular immune reactions such as delayed-type hypersensitivity are suppressed antibody production is unaffected. Because the selective nature to the UV-induced immunosuppression is similar to the biologic activity of IL-10, we examined the hypothesis that UV exposure of keratinocytes causes the release of IL-10. Keratinocyte monolayers were exposed to UV radiation and at specific times after exposure mRNA was isolated or the culture supernatant from the cells was collected. IL-10 mRNA expression was enhanced in UV-irradiated keratinocytes. The secretion of IL-10 by the irradiated keratinocytes was determined by Western blot analysis. A band reactive with anti-IL-10 mAb was found in supernatants from the UV-irradiated but not the mock-irradiated cells. IL-10 biologic activity was determined by the ability of the supernatants from the UV-irradiated keratinocytes to suppress IFN-gamma production by Ag-activated Th 1 cell clones. Anti-IL-10 mAb neutralized the ability of supernatants from UV-irradiated keratinocytes to suppress the induction of delayed-type hypersensitivity in vivo. Furthermore, injecting UV-irradiated mice with antibodies against IL-10 partially inhibited in vivo immunosuppression. These data indicate that activated keratinocytes are capable of secreting IL-10 and suggest that the release of IL-10 by

  11. Substrate Stiffness Affects Human Keratinocyte Colony Formation

    PubMed Central

    Zarkoob, Hoda; Bodduluri, Sandeep; Ponnaluri, Sailahari V.; Selby, John C.; Sander, Edward A.

    2015-01-01

    Restoration of epidermal organization and function in response to a variety of pathophysiological insults is critically dependent on coordinated keratinocyte migration, proliferation, and stratification during the process of wound healing. These processes are mediated by the reconfiguration of both cell-cell (desmosomes, adherens junctions) and cell-matrix (focal adhesions, hemidesmosomes) junctions and the cytoskeletal filament networks that they serve to interconnect. In this study, we investigated the role of substrate elasticity (stiffness) on keratinocyte colony formation in vitro during the process of nascent epithelial sheet formation as triggered by the calcium switch model of keratinocyte culture. Keratinocytes cultured on pepsin digested type I collagen coated soft (nominal E = 1.2 kPa) polyacrylamide gels embedded with fluorescent microspheres exhibited (i) smaller spread contact areas, (ii) increased migration velocities, and (iii) increased rates of colony formation with more cells per colony than did keratinocytes cultured on stiff (nominal E = 24 kPa) polyacrylamide gels. As assessed by tracking of embedded microsphere displacements, keratinocytes cultured on soft substrates generated large local substrate deformations that appeared to recruit adjacent keratinocytes into joining an evolving colony. Together with the observed differences in keratinocyte kinematics and substrate deformations, we developed two ad hoc analyses, termed distance rank (DR) and radius of cooperativity (RC), that help to objectively ascribe what we perceive as increasingly cooperative behavior of keratinocytes cultured on soft versus stiff gels during the process of colony formation. We hypothesize that the differences in keratinocyte colony formation observed in our experiments could be due to cell-cell mechanical signaling generated via local substrate deformations that appear to be correlated with the increased expression of β4 integrin within keratinocytes positioned

  12. Characterization of prenyl protein transferase enzymes in a human keratinocyte cell line.

    PubMed

    MacNulty, E E; Ryder, N S

    1996-02-01

    Prenylation is a post-translational modification of proteins that involves the attachment of an isoprenoid group derived from mevalonic acid, either 15-carbon farnesyl or 20-carbon geranylgeranyl, to a specific carboxy-terminal domain of acceptor proteins. Three prenyl transferase enzymes have been identified so far. In this paper we report the presence of two prenyl transferases in the HaCaT human keratinocyte cell line. Chromatography of a cytosolic extract from these cells resolved a farnesyl protein transferase (FPT) and geranylgeranyl protein transferase-I (GGPT-I) whose activities were measured using a novel peptide-based assay. Both enzymes were inhibited dose dependently by zaragozic acids A and C. Zaragozic acid C was more active towards the FPT than GGPT-I while zaragozic acid A inhibited both enzymes with similar potency. Incubation of HaCaT cell homogenates with [3H] prenyl precursors resulted in the labelling of a number of proteins which was increased when the cells were pretreated with an inhibitor of hydroxymethylglutaryl CoA reductase. Given the role of prenylated proteins in proliferative and inflammatory processes, our finding that prenyl transferases capable of prenylating endogenous substrates are also present in keratinocytes suggests that these enzymes might provide novel therapeutic targets of dermatological importance. PMID:8605230

  13. Lithium Regulates Keratinocyte Proliferation Via Glycogen Synthase Kinase 3 and NFAT2 (Nuclear Factor of Activated T Cells 2)

    PubMed Central

    Hampton, Philip J; Jans, Ralph; Flockhart, Ross J; Parker, Graeme; Reynolds, Nick J

    2012-01-01

    Certain environmental factors including drugs exacerbate or precipitate psoriasis. Lithium is the commonest cause of drug-induced psoriasis but underlying mechanisms are currently unknown. Lithium inhibits glycogen synthase kinase 3 (GSK-3). As lithium does not exacerbate other T-cell-mediated chronic inflammatory diseases, we investigated whether lithium may be acting directly on epidermal keratinocytes by inhibiting GSK-3. We report that lithium-induced keratinocyte proliferation at therapeutically relevant doses (1–2 mM) and increased the proportion of cells in S phase of the cell cycle. Inhibition of GSK-3 in keratinocytes by retroviral transduction of GSK-binding protein (an endogenous inhibitory protein) or through a highly selective pharmacological inhibitor also resulted in increased keratinocyte proliferation. Nuclear factor of activated T cells (NFAT) is an important substrate for GSK-3 and for cyclosporin, an effective treatment for psoriasis that inhibits NFAT activation in keratinocytes as well as in lymphocytes. Both lithium and genetic/pharmacological inhibition of GSK-3 resulted in increased nuclear localization of NFAT2 (NFATc1) and increased NFAT transcriptional activation. Finally, retroviral transduction of NFAT2 increased keratinocyte proliferation whereas siRNA-mediated knockdown of NFAT2 reduced keratinocyte proliferation and decreased epidermal thickness in an organotypic skin equivalent model. Taken together, these data identify GSK-3 and NFAT2 as key regulators of keratinocyte proliferation and as potential molecular targets relevant to lithium-provoked psoriasis. J. Cell. Physiol. 227: 1529–1537, 2012. © 2011 Wiley Periodicals, Inc. PMID:21678407

  14. Role of taurine accumulation in keratinocyte hydration.

    PubMed

    Janeke, Guido; Siefken, Wilfried; Carstensen, Stefanie; Springmann, Gunja; Bleck, Oliver; Steinhart, Hans; Höger, Peter; Wittern, Klaus-Peter; Wenck, Horst; Stäb, Franz; Sauermann, Gerhard; Schreiner, Volker; Doering, Thomas

    2003-08-01

    Epidermal keratinocytes are exposed to a low water concentration at the stratum corneum-stratum granulosum interface. When epithelial tissues are osmotically perturbed, cellular protection and cell volume regulation is mediated by accumulation of organic osmolytes such as taurine. Previous studies reported the presence of taurine in the epidermis of several animal species. Therefore, we analyzed human skin for the presence of the taurine transporter (TAUT) and studied the accumulation of taurine as one potential mechanism protecting epidermal keratinocytes from dehydration. According to our results, TAUT is expressed as a 69 kDa protein in human epidermis but not in the dermis. For the epidermis a gradient was evident with maximal levels of TAUT in the outermost granular keratinocyte layer and lower levels in the stratum spinosum. No TAUT was found in the basal layer or in the stratum corneum. Keratinocyte accumulation of taurine was induced by experimental induction of skin dryness via application of silica gel to human skin. Cultured human keratinocytes accumulated taurine in a concentration- and osmolarity-dependent manner. TAUT mRNA levels were increased after exposure of human keratinocytes to hyperosmotic culture medium, indicating osmosensitive TAUT mRNA expression as part of the adaptation of keratinocytes to hyperosmotic stress. Keratinocyte uptake of taurine was inhibited by beta-alanine but not by other osmolytes such as betaine, inositol, or sorbitol. Accumulation of taurine protected cultured human keratinocytes from both osmotically induced and ultraviolet-induced apoptosis. Our data indicate that taurine is an important epidermal osmolyte required to maintain keratinocyte hydration in a dry environment. PMID:12880428

  15. High-glucose environment increased thrombospondin-1 expression in keratinocytes via DNA hypomethylation.

    PubMed

    Lan, Cheng-Che E; Huang, Shu-Mei; Wu, Ching-Shuang; Wu, Chin-Han; Chen, Gwo-Shing

    2016-03-01

    Diabetes is an important health issue because of its increasing prevalence and association with impaired wound healing. Epidermal keratinocytes with overexpressed antiangiogenic molecule thrombospondin-1 (TSP1) have been shown to impair proper wound healing. This study examined the potential involvement of keratinocyte-derived TSP1 on diabetic wound healing. Cultured human keratinocytes and diabetic rat model were used to evaluate the effect of high-glucose environment on TSP1 expression in epidermal keratinocytes, and the molecular mechanisms involved in the process were also studied. We demonstrated that high-glucose environment increased TSP1 expression in keratinocytes. In addition, increased oxidative stress induced DNA hypomethylation at the TSP1 promoter region in keratinocytes exposed to high-glucose environment. Similar findings were found in our diabetic rat model. Early antioxidant administration normalized TSP1 expression and global DNA methylation status in diabetic rat skin and improved wound healing in vivo. Because oxidative stress contributed to TSP1 DNA hypomethylation, early recognition of diabetic condition and timely administration of antioxidant are logical approaches to reduce complications associated with diabetes as alterations in epigenome may not be reversible by controlling glucose levels during the later stages of disease course. PMID:26678678

  16. Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo

    SciTech Connect

    Kioka, Noriyuki; Ito, Takuya; Yamashita, Hiroshi; Uekawa, Natsuko; Umemoto, Tsutomu; Motoyoshi, Soh; Imai, Hiroshi; Takahashi, Kenzo; Watanabe, Hideto; Yamada, Masayasu; Ueda, Kazumitsu

    2010-06-10

    In the process of tissue injury and repair, epithelial cells rapidly migrate and form epithelial sheets. Vinexin is a cytoplasmic molecule of the integrin-containing cell adhesion complex localized at focal contacts in vitro. Here, we investigated the roles of vinexin in keratinocyte migration in vitro and wound healing in vivo. Vinexin knockdown using siRNA delayed migration of both HaCaT human keratinocytes and A431 epidermoid carcinoma cells in scratch assay but did not affect cell proliferation. Induction of cell migration by scratching the confluent monolayer culture of these cells activated both EGFR and ERK, and their inhibitors AG1478 and U0126 substantially suppressed scratch-induced keratinocyte migration. Vinexin knockdown in these cells inhibited the scratch-induced activation of EGFR, but not that of ERK, suggesting that vinexin promotes cell migration via activation of EGFR. We further generated vinexin (-/-) mice and isolated their keratinocytes. They similarly showed slow migration in scratch assay. Furthermore, vinexin (-/-) mice exhibited a delay in cutaneous wound healing in both the back skin and tail without affecting the proliferation of keratinocytes. Together, these results strongly suggest a crucial role of vinexin in keratinocyte migration in vitro and cutaneous wound healing in vivo.

  17. Development of an inducible gene expression system for primary murine keratinocytes

    PubMed Central

    Nagarajan, Priyadharsini

    2008-01-01

    Background The tetracycline (Tet) responsive system is a valuable tool that is routinely used in a wide variety of mammalian cells for regulatable expression of gene products. However, technical difficulties such as harsh selection conditions and extensive screening processes to identify suitably responsive clones limit the generation of stable cell lines. Hence, application of this system in mammalian cells with relatively slow growth rates and / or the capacity to undergo terminal differentiation such as primary mouse keratinocytes is particularly challenging. Objective To our knowledge, no Tet-responsive stable cell lines have been generated from mouse keratinocytes, presumably due to their sensitivity to selection conditions. Our goal was to utilize a modified and robust Tet-expression system to generate a stable primary mouse keratinocyte cell line. These cells could be then utilized for conditional expression of potentially toxic proteins in an inducible fashion. Methods We utilized a eukaryotic promoter instead of a viral promoter to express a modified reverse tetracycline transactivator in mouse keratinocytes and optimized the selection process for generating stable cell lines. Results Here, we report the generation of a stable mouse keratinocyte cell line for Tet-regulated gene expression with minimal leakiness and high degree of Tet responsivity. This mouse keratinocyte cell line was further engineered for generation of a double stable cell line, which expresses the transcription factor AP-2α in an inducible manner. Importantly, the selected cells retain their inherent keratinocyte morphology, respond to differentiation signals and exhibit a persistent and highly tunable Tet inducibility upon continuous culturing. Conclusion We have generated a tetracycline inducible gene expression model system in mouse epidermal keratinocytes. Such inducible cell lines will serve as valuable in vitro models for future gain-of-function and loss-of-function studies. PMID

  18. FOXM1 regulates proliferation, senescence and oxidative stress in keratinocytes and cancer cells

    PubMed Central

    Smirnov, Artem; Panatta, Emanuele; Lena, AnnaMaria; Castiglia, Daniele; Di Daniele, Nicola; Melino, Gerry; Candi, Eleonora

    2016-01-01

    Several transcription factors, including the master regulator of the epidermis, p63, are involved in controlling human keratinocyte proliferation and differentiation. Here, we report that in normal keratinocytes, the expression of FOXM1, a member of the Forkhead superfamily of transcription factors, is controlled by p63. We observe that, together with p63, FOXM1 strongly contributes to the maintenance of high proliferative potential in keratinocytes, whereas its expression decreases during differentiation, as well as during replicative-induced senescence. Depletion of FOXM1 is sufficient to induce keratinocyte senescence, paralleled by an increased ROS production and an inhibition of ROS-scavenger genes (SOD2, CAT, GPX2, PRDX). Interestingly, FOXM1 expression is strongly reduced in keratinocytes isolated from old human subjects compared with young subjects. FOXM1 depletion sensitizes both normal keratinocytes and squamous carcinoma cells to apoptosis and ROS-induced apoptosis. Together, these data identify FOXM1 as a key regulator of ROS in normal dividing epithelial cells and suggest that squamous carcinoma cells may also use FOXM1 to control oxidative stress to escape premature senescence and apoptosis. PMID:27385468

  19. Transcriptional regulation of genes involved in keratinocyte differentiation by human papillomavirus 16 oncoproteins.

    PubMed

    Gyöngyösi, Eszter; Szalmás, Anita; Ferenczi, Annamária; Póliska, Szilárd; Kónya, József; Veress, György

    2015-02-01

    The life cycle of human papillomaviruses (HPVs) is strictly linked to the differentiation of their natural host cells. The HPV E6 and E7 oncoproteins can delay the normal differentiation program of keratinocytes; however, the exact mechanisms responsible for this have not yet been identified. The goal of this study was to investigate the effects of HPV16 oncoproteins on the expression of genes involved in keratinocyte differentiation. Primary human keratinocytes transduced by LXSN (control) retroviruses or virus vectors expressing HPV16 E6, E7 or E6/E7 genes were subjected to gene expression profiling. The results of microarray analysis showed that HPV 16 E6 and E7 have the capacity to downregulate the expression of several genes involved in keratinocyte differentiation. Quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed to confirm the microarray data. To investigate the effects of the HPV oncoproteins on the promoters of selected keratinocyte differentiation genes, luciferase reporter assays were performed. Our results suggest that the HPV 16 E6 and/or E7 oncogenes are able to downregulate the expression of several genes involved in keratinocyte differentiation (such as desmocollin 1, keratin 4, S100 calcium-binding protein A8 and small proline-rich protein 1A), at least partially by downregulating their promoter activity. This activity of the HPV oncoproteins may have a role in the productive virus life cycle, and also in virus-induced carcinogenesis. PMID:25488293

  20. Single cell mechanics of keratinocyte cells.

    PubMed

    Lulevich, Valentin; Yang, Hsin-ya; Isseroff, R Rivkah; Liu, Gang-yu

    2010-11-01

    Keratinocytes represent the major cell type of the uppermost layer of human skin, the epidermis. Using AFM-based single cell compression, the ability of individual keratinocytes to resist external pressure and global rupturing forces is investigated and compared with various cell types. Keratinocytes are found to be 6-70 times stiffer than other cell types, such as white blood, breast epithelial, fibroblast, or neuronal cells, and in contrast to other cell types they retain high mechanic strength even after the cell's death. The absence of membrane rupturing peaks in the force-deformation profiles of keratinocytes and their high stiffness during a second load cycle suggests that their unique mechanical resistance is dictated by the cytoskeleton. A simple analytical model enables the quantification of Young's modulus of keratinocyte cytoskeleton, as high as 120-340 Pa. Selective disruption of the two major cytoskeletal networks, actin filaments and microtubules, does not significantly affect keratinocyte mechanics. F-actin is found to impact cell deformation under pressure. During keratinocyte compression, the plasma membrane stretches to form peripheral blebs. Instead of blebbing, cells with depolymerized F-actin respond to pressure by detaching the plasma membrane from the cytoskeleton underneath. On the other hand, the compression force of keratinocytes expressing a mutated keratin (cell line, KEB-7) is 1.6-2.2 times less than that for the control cell line that has normal keratin networks. Therefore, we infer that the keratin intermediate filament network is responsible for the extremely high keratinocyte stiffness and resilience. This could manifest into the rugged protective nature of the human epidermis. PMID:20728993

  1. Powerful Sequence Similarity Search Methods and In-Depth Manual Analyses Can Identify Remote Homologs in Many Apparently “Orphan” Viral Proteins

    PubMed Central

    Kuchibhatla, Durga B.; Chung, Betty Y. W.; Cook, Shelley; Schneider, Georg; Eisenhaber, Birgit

    2014-01-01

    The genome sequences of new viruses often contain many “orphan” or “taxon-specific” proteins apparently lacking homologs. However, because viral proteins evolve very fast, commonly used sequence similarity detection methods such as BLAST may overlook homologs. We analyzed a data set of proteins from RNA viruses characterized as “genus specific” by BLAST. More powerful methods developed recently, such as HHblits or HHpred (available through web-based, user-friendly interfaces), could detect distant homologs of a quarter of these proteins, suggesting that these methods should be used to annotate viral genomes. In-depth manual analyses of a subset of the remaining sequences, guided by contextual information such as taxonomy, gene order, or domain cooccurrence, identified distant homologs of another third. Thus, a combination of powerful automated methods and manual analyses can uncover distant homologs of many proteins thought to be orphans. We expect these methodological results to be also applicable to cellular organisms, since they generally evolve much more slowly than RNA viruses. As an application, we reanalyzed the genome of a bee pathogen, Chronic bee paralysis virus (CBPV). We could identify homologs of most of its proteins thought to be orphans; in each case, identifying homologs provided functional clues. We discovered that CBPV encodes a domain homologous to the Alphavirus methyltransferase-guanylyltransferase; a putative membrane protein, SP24, with homologs in unrelated insect viruses and insect-transmitted plant viruses having different morphologies (cileviruses, higreviruses, blunerviruses, negeviruses); and a putative virion glycoprotein, ORF2, also found in negeviruses. SP24 and ORF2 are probably major structural components of the virions. PMID:24155369

  2. Exosomes released by keratinocytes modulate melanocyte pigmentation

    PubMed Central

    Cicero, Alessandra Lo; Delevoye, Cédric; Gilles-Marsens, Floriane; Loew, Damarys; Dingli, Florent; Guéré, Christelle; André, Nathalie; Vié, Katell; van Niel, Guillaume; Raposo, Graça

    2015-01-01

    Cells secrete extracellular vesicles (EVs), exosomes and microvesicles, which transfer proteins, lipids and RNAs to regulate recipient cell functions. Skin pigmentation relies on a tight dialogue between keratinocytes and melanocytes in the epidermis. Here we report that exosomes secreted by keratinocytes enhance melanin synthesis by increasing both the expression and activity of melanosomal proteins. Furthermore, we show that the function of keratinocyte-derived exosomes is phototype-dependent and is modulated by ultraviolet B. In sum, this study uncovers an important physiological function for exosomes in human pigmentation and opens new avenues in our understanding of how pigmentation is regulated by intercellular communication in both healthy and diseased states. PMID:26103923

  3. Exosomes released by keratinocytes modulate melanocyte pigmentation.

    PubMed

    Lo Cicero, Alessandra; Delevoye, Cédric; Gilles-Marsens, Floriane; Loew, Damarys; Dingli, Florent; Guéré, Christelle; André, Nathalie; Vié, Katell; van Niel, Guillaume; Raposo, Graça

    2015-01-01

    Cells secrete extracellular vesicles (EVs), exosomes and microvesicles, which transfer proteins, lipids and RNAs to regulate recipient cell functions. Skin pigmentation relies on a tight dialogue between keratinocytes and melanocytes in the epidermis. Here we report that exosomes secreted by keratinocytes enhance melanin synthesis by increasing both the expression and activity of melanosomal proteins. Furthermore, we show that the function of keratinocyte-derived exosomes is phototype-dependent and is modulated by ultraviolet B. In sum, this study uncovers an important physiological function for exosomes in human pigmentation and opens new avenues in our understanding of how pigmentation is regulated by intercellular communication in both healthy and diseased states. PMID:26103923

  4. Canine Distemper Virus Infects Canine Keratinocytes and Immune Cells by Using Overlapping and Distinct Regions Located on One Side of the Attachment Protein▿

    PubMed Central

    Langedijk, Johannes P. M.; Janda, Jozef; Origgi, Francesco C.; Örvell, Claes; Vandevelde, Marc; Zurbriggen, Andreas; Plattet, Philippe

    2011-01-01

    The morbilliviruses measles virus (MeV) and canine distemper virus (CDV) both rely on two surface glycoproteins, the attachment (H) and fusion proteins, to promote fusion activity for viral cell entry. Growing evidence suggests that morbilliviruses infect multiple cell types by binding to distinct host cell surface receptors. Currently, the only known in vivo receptor used by morbilliviruses is CD150/SLAM, a molecule expressed in certain immune cells. Here we investigated the usage of multiple receptors by the highly virulent and demyelinating CDV strain A75/17. We based our study on the assumption that CDV-H may interact with receptors similar to those for MeV, and we conducted systematic alanine-scanning mutagenesis on CDV-H throughout one side of the β-propeller documented in MeV-H to contain multiple receptor-binding sites. Functional and biochemical assays performed with SLAM-expressing cells and primary canine epithelial keratinocytes identified 11 residues mutation of which selectively abrogated fusion in keratinocytes. Among these, four were identical to amino acids identified in MeV-H as residues contacting a putative receptor expressed in polarized epithelial cells. Strikingly, when mapped on a CDV-H structural model, all residues clustered in or around a recessed groove located on one side of CDV-H. In contrast, reported CDV-H mutants with SLAM-dependent fusion deficiencies were characterized by additional impairments to the promotion of fusion in keratinocytes. Furthermore, upon transfer of residues that selectively impaired fusion induction in keratinocytes into the CDV-H of the vaccine strain, fusion remained largely unaltered. Taken together, our results suggest that a restricted region on one side of CDV-H contains distinct and overlapping sites that control functional interaction with multiple receptors. PMID:21849439

  5. Evaluation of the skin sensitization potential of chemicals in THP-1/keratinocyte co-cultures.

    PubMed

    Cao, Yu-Ping; Ma, Peng-Cheng; Liu, Wei-Da; Zhou, Wu-Qing; Tao, Yue; Zhang, Meng-Li; Li, Ling-Jun; Chen, Zi-Yi

    2012-04-01

    Many attempts have been made to develop in vitro sensitization tests that employ dendritic cells (DCs), DC-like cell lines or keratinocytes. The aim of the present investigation was to establish a co-culture of THP-1 cells and keratinocytes for evaluation of skin sensitization potential of chemicals. Co-cultures were constructed by THP-1 cells cultured in lower compartments and keratinocytes cultured in upper compartments of cell culture inserts. After 24 h exposure to sensitizers (2, 4-dinitrochlorobenzene, p-phenylenediamine, formaldehyde, nickel sulfate, isoeugenol and eugenol) and non-sensitizers (sodium lauryl sulfate, benzalkonium chloride and lactic acid), the expression of CD86 and CD54 on THP-1 cells were evaluated by flow cytometry, and cell viabilities were determined. The sensitizers induced the augmentation of CD86 and CD54 expression, but the non-sensitizers had no significant effect. Compared with mono-cultures of THP-1 cells, the augmentation of CD86 and CD54 could be detected even at a non-toxic concentration of sensitizers in THP-1 cell/keratinocyte co-cultures. Moreover, isoeugenol was distinguished as a sensitizer in co-cultures, but failed to be identified in mono-cultures. These results revealed that the co-cultures of THP-1 cells and keratinocytes were successfully established and suitable for identifying sensitizers using CD86 and CD54 expression as identification markers. PMID:21721923

  6. MiR-146a negatively regulates TLR2-induced inflammatory responses in keratinocytes.

    PubMed

    Meisgen, Florian; Xu Landén, Ning; Wang, Aoxue; Réthi, Bence; Bouez, Charbel; Zuccolo, Michela; Gueniche, Audrey; Ståhle, Mona; Sonkoly, Enikö; Breton, Lionel; Pivarcsi, Andor

    2014-07-01

    Keratinocytes represent the first line of defense against pathogens in the skin and have important roles in initiating and regulating inflammation during infection and autoimmunity. Here we investigated the role of miR-146a in the regulation of the innate immune response of keratinocytes. Toll-like receptor 2 (TLR2) stimulation of primary human keratinocytes resulted in an NF-κB- and mitogen-activated protein kinase-dependent upregulation of miR-146a expression, which was surprisingly long lasting, contrasting with the rapid and transient induction of inflammatory mediators. Overexpression of miR-146a significantly suppressed the production of IL-8, CCL20, and tumor necrosis factor-α, which functionally suppressed the chemotactic attraction of neutrophils by keratinocytes. Inhibition of endogenous miR-146a induced the production of inflammatory mediators even in nonstimulated keratinocytes, and potentiated the effect of TLR2 stimulation. Transcriptomic profiling revealed that miR-146a suppresses the expression of a large number of immune-related genes in keratinocytes. MiR-146a downregulated interleukin-1 receptor-associated kinase 1 and TNF receptor-associated factor 6, two key adapter molecules downstream of TLR signaling, and suppressed NF-κB promoter-binding activity as shown by promoter luciferase experiments. Together, these data identify miR-146a as a regulatory element in keratinocyte innate immunity, which prevents the production of inflammatory mediators under homeostatic conditions and serves as a potent negative feedback regulator after TLR2 stimulation. PMID:24670381

  7. A Heat-Sensitive TRP Channel Expressed in Keratinocytes

    NASA Astrophysics Data System (ADS)

    Peier, Andrea M.; Reeve, Alison J.; Andersson, David A.; Moqrich, Aziz; Earley, Taryn J.; Hergarden, Anne C.; Story, Gina M.; Colley, Sian; Hogenesch, John B.; McIntyre, Peter; Bevan, Stuart; Patapoutian, Ardem

    2002-06-01

    Mechanical and thermal cues stimulate a specialized group of sensory neurons that terminate in the skin. Three members of the transient receptor potential (TRP) family of channels are expressed in subsets of these neurons and are activated at distinct physiological temperatures. Here, we describe the cloning and characterization of a novel thermosensitive TRP channel. TRPV3 has a unique threshold: It is activated at innocuous (warm) temperatures and shows an increased response at noxious temperatures. TRPV3 is specifically expressed in keratinocytes; hence, skin cells are capable of detecting heat via molecules similar to those in heat-sensing neurons.

  8. Human keratinocyte growth and differentiation on acellular porcine dermal matrix in relation to wound healing potential.

    PubMed

    Zajicek, Robert; Mandys, Vaclav; Mestak, Ondrej; Sevcik, Jan; Königova, Radana; Matouskova, Eva

    2012-01-01

    A number of implantable biomaterials derived from animal tissues are now used in modern surgery. Xe-Derma is a dry, sterile, acellular porcine dermis. It has a remarkable healing effect on burns and other wounds. Our hypothesis was that the natural biological structure of Xe-Derma plays an important role in keratinocyte proliferation and formation of epidermal architecture in vitro as well as in vivo. The bioactivity of Xe-Derma was studied by a cell culture assay. We analyzed growth and differentiation of human keratinocytes cultured in vitro on Xe-Derma, and we compared the results with formation of neoepidermis in the deep dermal wounds treated with Xe-Derma. Keratinocytes cultured on Xe-Derma submerged in the culture medium achieved confluence in 7-10 days. After lifting the cultures to the air-liquid interface, the keratinocytes were stratified and differentiated within one week, forming an epidermis with basal, spinous, granular, and stratum corneum layers. Immunohistochemical detection of high-molecular weight cytokeratins (HMW CKs), CD29, p63, and involucrin confirmed the similarity of organization and differentiation of the cultured epidermal cells to the normal epidermis. The results suggest that the firm natural structure of Xe-Derma stimulates proliferation and differentiation of human primary keratinocytes and by this way improves wound healing. PMID:22629190

  9. Differentiation of human embryonic stem cells into clinically amenable keratinocytes in an autogenic environment.

    PubMed

    Kidwai, Fahad K; Liu, Hua; Toh, Wei Seong; Fu, Xin; Jokhun, Doorgesh S; Movahednia, Mohammad M; Li, Mingming; Zou, Yu; Squier, Christopher A; Phan, Toan T; Cao, Tong

    2013-03-01

    Human embryonic stem cells (hESCs)-derived keratinocytes hold great clinical and research potential. However, the current techniques are hampered by the use of xenogenic components that limits their clinical application. Here we demonstrated an efficient differentiation of H9 hESCs (H9-hESCs) into keratinocytes (H9-Kert) with the minimum use of animal-derived materials. For differentiation, we established two microenvironment systems originated from H9-hESCs (autogenic microenvironment). These autogenic microenvironment systems consist of an autogenic coculture system (ACC) and an autogenic feeder-free system (AFF). In addition, we showed a stage-specific effect of Activin in promoting keratinocyte differentiation from H9-hESCs while repressing the expression of early neural markers in the ACC system. Furthermore, we also explained the effect of Activin in construction of the AFF system made up of extracellular matrix similar to basement membrane extracted from H9-hESC-derived fibroblasts. H9-Kert differentiated in both systems expressed keratinocyte markers at mRNA and protein levels. H9-Kert were also able to undergo terminal differentiation in high Ca(2+) medium. These findings support the transition toward the establishment of an animal-free microenvironment for successful differentiation of hESCs into keratinocytes for potential clinical application. PMID:23235526

  10. Identified lhb-expressing cells from medaka (Oryzias latipes) show similar Ca(2+)-response to all endogenous Gnrh forms, and reveal expression of a novel fourth Gnrh receptor.

    PubMed

    Strandabø, Rønnaug A U; Grønlien, Heidi K; Ager-Wick, Eirill; Nourizadeh-Lillabadi, Rasoul; Hildahl, Jon P; Weltzien, Finn-Arne; Haug, Trude M

    2016-04-01

    We have previously characterized the response to gonadotropin-releasing hormone (Gnrh) 2 in luteinizing hormone (lhb)-expressing cells from green fluorescent protein (Gfp)-transgenic medaka (Oryzias latipes), with regard to changes in the cytosolic Ca(2+) concentration. In the current study we present the corresponding responses to Gnrh1 and Gnrh3. Ca(2+) imaging revealed three response patterns to Gnrh1 and Gnrh3, one monophasic and two types of biphasic patterns. There were few significant differences in the shape of the response patterns between the three Gnrh forms, although the amplitude of the Ca(2+) signal was considerably lower for Gnrh1 and Gnrh3 than for Gnrh2, and the distribution between the two different biphasic patterns differed. The different putative Ca(2+) sources were examined by depleting intracellular Ca(2+) stores with thapsigargin, or preventing influx of extracellular Ca(2+) by either extracellular Ca(2+) depletion or the L-type Ca(2+)-channel blocker verapamil. Both Gnrh1 and 3 relied on Ca(2+) from both intracellular and extracellular sources, with some unexpected differences in the relative contribution. Furthermore, gene expression of Gnrh-receptors (gnrhr) in whole pituitaries was studied during development from juvenile to adult. Only two of the four identified medaka receptors were expressed in the pituitary, gnrhr1b and gnrhr2a, with the newly discovered gnrhr2a showing the highest expression level at all stages as analyzed by quantitative PCR. While both receptors differed in expression level according to developmental stage, only the expression of gnrhr2a showed a clear-cut increase with gonadal maturation. RNA sequencing analysis of FACS-sorted Gfp-positive lhb-cells revealed that both gnrhr1b and gnrhr2a were expressed in lhb-expressing cells, and confirmed the higher expression of gnrhr2a compared to gnrhr1b. These results show that although lhb-expressing gonadotropes in medaka show similar Ca(2+) response patterns to all three

  11. Melatonin protects skin keratinocyte from hydrogen peroxide-mediated cell death via the SIRT1 pathway.

    PubMed

    Lee, Ju-Hee; Moon, Ji-Hong; Nazim, Uddin Md; Lee, You-Jin; Seol, Jae-Won; Eo, Seong-Kug; Lee, John-Hwa; Park, Sang-Youel

    2016-03-15

    Melatonin (N-acetyl-5-methoxytryptamine), which is primarily synthesized in and secreted from the pineal gland, plays a pivotal role in cell proliferation as well as in the regulation of cell metastasis and cell survival in a diverse range of cells. The aim of this study is to investigate protection effect of melatonin on H2O2-induced cell damage and the mechanisms of melatonin in human keratinocytes. Hydrogen peroxide dose-dependently induced cell damages in human keratinocytes and co-treatment of melatonin protected the keratinocytes against H2O2-induced cell damage. Melatonin treatment activated the autophagy flux signals, which were identified by the decreased levels of p62 protein. Inhibition of autophagy flux via an autophagy inhibitor and ATG5 siRNA technique blocked the protective effects of melatonin against H2O2-induced cell death in human keratinocytes. And we found the inhibition of sirt1 using sirtinol and sirt1 siRNA reversed the protective effects of melatonin and induces the autophagy process in H2O2-treated cells. This is the first report demonstrating that autophagy flux activated by melatonin protects human keratinocytes through sirt1 pathway against hydrogen peroxide-induced damages. And this study also suggest that melatonin could potentially be utilized as a therapeutic agent in skin disease. PMID:26918354

  12. Melatonin protects skin keratinocyte from hydrogen peroxide-mediated cell death via the SIRT1 pathway

    PubMed Central

    Lee, Ju-Hee; Moon, Ji-Hong; Nazim, Uddin MD.; Lee, You-Jin; Seol, Jae-Won; Eo, Seong-Kug; Lee, John-Hwa; Park, Sang-Youel

    2016-01-01

    Melatonin (N-acetyl-5-methoxytryptamine), which is primarily synthesized in and secreted from the pineal gland, plays a pivotal role in cell proliferation as well as in the regulation of cell metastasis and cell survival in a diverse range of cells. The aim of this study is to investigate protection effect of melatonin on H2O2-induced cell damage and the mechanisms of melatonin in human keratinocytes. Hydrogen peroxide dose-dependently induced cell damages in human keratinocytes and co-treatment of melatonin protected the keratinocytes against H2O2-induced cell damage. Melatonin treatment activated the autophagy flux signals, which were identified by the decreased levels of p62 protein. Inhibition of autophagy flux via an autophagy inhibitor and ATG5 siRNA technique blocked the protective effects of melatonin against H2O2-induced cell death in human keratinocytes. And we found the inhibition of sirt1 using sirtinol and sirt1 siRNA reversed the protective effects of melatonin and induces the autophagy process in H2O2-treated cells. This is the first report demonstrating that autophagy flux activated by melatonin protects human keratinocytes through sirt1 pathway against hydrogen peroxide-induced damages. And this study also suggest that melatonin could potentially be utilized as a therapeutic agent in skin disease. PMID:26918354

  13. Staphylococcus aureus keratinocyte invasion is mediated by integrin-linked kinase and Rac1.

    PubMed

    Sayedyahossein, Samar; Xu, Stacey X; Rudkouskaya, Alena; McGavin, Martin J; McCormick, John K; Dagnino, Lina

    2015-02-01

    Staphylococcus aureus is a major component of the skin microbiota and causes a large number of serious infections. S. aureus first interacts with epidermal keratinocytes to breach the epidermal barrier through mechanisms not fully understood. By use of primary keratinocytes from mice with epidermis-restricted Ilk gene inactivation and control integrin-linked kinase (ILK)-expressing littermates, we investigated the role of ILK in epidermal S. aureus invasion. Heat-killed, but not live, bacteria were internalized to Rab5- and Rab7-positive phagosomes, and incubation with keratinocyte growth factor increased their uptake 2.5-fold. ILK-deficient mouse keratinocytes internalized bacteria 2- to 4-fold less efficiently than normal cells. The reduced invasion by live S. aureus of ILK-deficient cells was restored in the presence of exogenous, constitutively active Rac1. Thus, Rac1 functions downstream from ILK during invasion. Further, invasion by S. aureus of Rac1-deficient cells was 2.5-fold lower than in normal cells. Paradoxically, staphylococcal cutaneous penetration of mouse skin explants with ILK-deficient epidermis was 35-fold higher than that of normal skin, indicating defects in epidermal barrier function in the absence of ILK. Thus, we identified an ILK-Rac1 pathway essential for bacterial invasion of keratinocytes, and established ILK as a key contributor to prevent invasive staphylococcal cutaneous infection. PMID:25416549

  14. Consistency of the Proteome in Primary Human Keratinocytes With Respect to Gender, Age, and Skin Localization*

    PubMed Central

    Sprenger, Adrian; Weber, Sebastian; Zarai, Mostafa; Engelke, Rudolf; Nascimento, Juliana M.; Gretzmeier, Christine; Hilpert, Martin; Boerries, Melanie; Has, Cristina; Busch, Hauke; Bruckner-Tuderman, Leena; Dengjel, Jörn

    2013-01-01

    Keratinocytes account for 95% of all cells of the epidermis, the stratified squamous epithelium forming the outer layer of the skin, in which a significant number of skin diseases takes root. Immortalized keratinocyte cell lines are often used as research model systems providing standardized, reproducible, and homogenous biological material. Apart from that, primary human keratinocytes are frequently used for medical studies because the skin provides an important route for drug administration and is readily accessible for biopsies. However, comparability of these cell systems is not known. Cell lines may undergo phenotypic shifts and may differ from the in vivo situation in important aspects. Primary cells, on the other hand, may vary in biological functions depending on gender and age of the donor and localization of the biopsy specimen. Here we employed metabolic labeling in combination with quantitative mass spectrometry-based proteomics to assess A431 and HaCaT cell lines for their suitability as model systems. Compared with cell lines, comprehensive profiling of the primary human keratinocyte proteome with respect to gender, age, and skin localization identified an unexpected high proteomic consistency. The data were analyzed by an improved ontology enrichment analysis workflow designed for the study of global proteomics experiments. It enables a quick, comprehensive and unbiased overview of altered biological phenomena and links experimental data to literature. We guide through our workflow, point out its advantages compared with other methods and apply it to visualize differences of cell lines compared with primary human keratinocytes. PMID:23722187

  15. Chitin Modulates Innate Immune Responses of Keratinocytes

    PubMed Central

    Koller, Barbara; Müller-Wiefel, Alisa Sophie; Rupec, Rudolph; Korting, Hans Christian; Ruzicka, Thomas

    2011-01-01

    Background Chitin, after cellulose the second most abundant polysaccharide in nature, is an essential component of exoskeletons of crabs, shrimps and insects and protects these organisms from harsh conditions in their environment. Unexpectedly, chitin has been found to activate innate immune cells and to elicit murine airway inflammation. The skin represents the outer barrier of the human host defense and is in frequent contact with chitin-bearing organisms, such as house-dust mites or flies. The effects of chitin on keratinocytes, however, are poorly understood. Methodology/Principal Findings We hypothesized that chitin stimulates keratinocytes and thereby modulates the innate immune response of the skin. Here we show that chitin is bioactive on primary and immortalized keratinocytes by triggering production of pro-inflammatory cytokines and chemokines. Chitin stimulation further induced the expression of the Toll-like receptor (TLR) TLR4 on keratinocytes at mRNA and protein level. Chitin-induced effects were mainly abrogated when TLR2 was blocked, suggesting that TLR2 senses chitin on keratinocytes. Conclusions/Significance We speculate that chitin-bearing organisms modulate the innate immune response towards pathogens by upregulating secretion of cytokines and chemokines and expression of MyD88-associated TLRs, two major components of innate immunity. The clinical relevance of this mechanism remains to be defined. PMID:21383982

  16. Cataloging of the genes expressed in human keratinocytes: analysis of 607 randomly isolated cDNA sequences.

    PubMed

    Konishi, K; Morishima, Y; Ueda, E; Kibe, Y; Nonomura, K; Yamanishi, K; Yasuno, H

    1994-07-29

    The partial nucleotide sequences of 607 cDNAs randomly isolated from a cDNA library of cultured human epidermal keratinocytes were determined by single pass sequencing. Homology search of the sequences to the non-redundant nucleotide databases revealed that 27% of the cDNAs matched registered human-or non-human genes encoding not only keratinocyte specific genes, but also a variety of functional proteins, the expression of which had not been identified in keratinocytes. Non-matching cDNAs covering 49% of the cDNAs were not homologous even to ESTs from other organs, suggesting that these cDNAs include novel genes expressed in the cells. The large scale sequencing of keratinocyte cDNAs provides a useful molecular source for research into biology and diseases of the skin. PMID:8048971

  17. Expression profiling of human epidermal keratinocyte response following 1-minute JP-8 exposure.

    PubMed

    Chou, Chi-Chung; Yang, Jen-Hung; Chen, San-Duo; Monteiro-Riviere, Nancy A; Li, Han-Ni; Chen, Jeremy J W

    2006-01-01

    The cDNA microarray analysis of 9600 expressed sequence tags was performed to examine the gene expression changes in human epidermal keratinocytes after 1-minute JP-8 exposure; 151 genes were identified as JP-8 responsive and classified into 8 clusters by self organization map. Genes involved in basal transcription and translations were up-regulated, whereas genes related to DNA repair, metabolism, and keratin were mostly down-regulated. Genes encoded for growth factors, apoptosis, signal transduction, and adhesion were also altered. These results indicated that human keratinocyte responds to a single dose of JP-8 insult and revealed several cellular processes previously not associated with jet fuel exposure. PMID:16835149

  18. Skin graft storage and keratinocyte viability.

    PubMed

    Fahmy, F S; Navsaria, H A; Frame, J D; Jones, C R; Leigh, I M

    1993-06-01

    The viability of human split skin grafts stored in four solutions has been assessed by monitoring the percentage of viable keratinocytes in the stored grafts. Skin grafts stored in RM+ (Ready Mix) tissue culture medium remained more viable than those stored in Hartmann's, Marshall's or saline solutions. By day 10 (postoperative), the percentage of viable keratinocytes of those grafts stored in RM+ was around 85%, compared to a value of around 10% for the other media. By day 30, RM+ achieved a value of around 60% keratinocyte viability compared to a value approaching 1% in the other storage media under investigation. RM+ provides mitogens, nutrients, growth factors and physiological pH, all of which are important factors for successful skin graft storage. PMID:8330085

  19. A saw is first identified as an object used on wood: ERP evidence for temporal differences between Thematic and Functional similarity relations.

    PubMed

    Wamain, Yannick; Pluciennicka, Ewa; Kalénine, Solène

    2015-05-01

    The role of functional and motor information in manipulable artifact object semantic organization is still poorly understood. In particular, several types of semantic relations involving object functional knowledge may be distinguished. Functional similarity relations group objects with similar functions at relatively specific (e.g. saw-axe, both used to cut wood) or general (saw-knife, both used to cut) levels. Thematic relations group objects based on their complementarity in events (saw used upon/with wood). Recent eye-tracking data showed distinct temporal time courses for the different semantic relations, with fastest processing of thematic relations and slowest processing of general function similarity relations. Behavioral data suggest the involvement of distinct cognitive mechanisms in manipulable artifact object semantic processing. The aim of the present study was to assess the neural correlates of thematic, and specific and general function similarity relation processing. Specifically, we investigated whether time course differences between semantic relations could be highlighted at the neurophysiological level. We used a protocol combining semantic priming with electroencephalography, and manipulated the type of semantic relation and the duration of the interval between prime and target objects. Two consistent and complementary results were shown. On N1 and P3 components, semantic priming was observed for thematic relations only. On N400 component, the type of semantic relation interacted with interval duration, and semantic priming was visible for all 3 relations after the longest interval only. Results revealed graded processing time courses for thematic, specific function similarity, and general function similarity relations at the neural level, and further indicate that thematic relations impact object processing during the early stages of object recognition. Findings suggest a hierarchical organization of three types of semantic relations based on

  20. Aberrantly Expressed Genes in HaCaT Keratinocytes Chronically Exposed to Arsenic Trioxide

    PubMed Central

    Udensi, Udensi K.; Cohly, Hari H.P.; Graham-Evans, Barbara E.; Ndebele, Kenneth; Garcia-Reyero, Natàlia; Nanduri, Bindu; Tchounwou, Paul B.; Isokpehi, Raphael D.

    2011-01-01

    Inorganic arsenic is a known environmental toxicant and carcinogen of global public health concern. Arsenic is genotoxic and cytotoxic to human keratinocytes. However, the biological pathways perturbed in keratinocytes by low chronic dose inorganic arsenic are not completely understood. The objective of the investigation was to discover the mechanism of arsenic carcinogenicity in human epidermal keratinocytes. We hypothesize that a combined strategy of DNA microarray, qRT-PCR and gene function annotation will identify aberrantly expressed genes in HaCaT keratinocyte cell line after chronic treatment with arsenic trioxide. Microarray data analysis identified 14 up-regulated genes and 21 down-regulated genes in response to arsenic trioxide. The expression of 4 up-regulated genes and 1 down-regulated gene were confirmed by qRT-PCR. The up-regulated genes were AKR1C3 (Aldo-Keto Reductase family 1, member C3), IGFL1 (Insulin Growth Factor-Like family member 1), IL1R2 (Interleukin 1 Receptor, type 2), and TNFSF18 (Tumor Necrosis Factor [ligand] SuperFamily, member 18) and down-regulated gene was RGS2 (Regulator of G-protein Signaling 2). The observed over expression of TNFSF18 (167 fold) coupled with moderate expression of IGFL1 (3.1 fold), IL1R2 (5.9 fold) and AKR1C3 (9.2 fold) with a decreased RGS2 (2.0 fold) suggests that chronic arsenic exposure could produce sustained levels of TNF with modulation by an IL-1 analogue resulting in chronic immunologic insult. A concomitant decrease in growth inhibiting gene (RGS2) and increase in AKR1C3 may contribute to chronic inflammation leading to metaplasia, which may eventually lead to carcinogenicity in the skin keratinocytes. Also, increased expression of IGFL1 may trigger cancer development and progression in HaCaT keratinocytes. PMID:21461292

  1. Epidermal expression of the truncated prelamin A causing Hutchinson–Gilford progeria syndrome: effects on keratinocytes, hair and skin

    PubMed Central

    Wang, Yuexia; Panteleyev, Andrey A.; Owens, David M.; Djabali, Karima; Stewart, Colin L.; Worman, Howard J.

    2008-01-01

    Hutchinson–Gilford progeria syndrome (HGPS) is an accelerated aging disorder caused by point mutation in LMNA encoding A-type nuclear lamins. The mutations in LMNA activate a cryptic splice donor site, resulting in expression of a truncated, prenylated prelamin A called progerin. Expression of progerin leads to alterations in nuclear morphology, which may underlie pathology in HGPS. We generated transgenic mice expressing progerin in epidermis under control of a keratin 14 promoter. The mice had severe abnormalities in morphology of skin keratinocyte nuclei, including nuclear envelope lobulation and decreased nuclear circularity not present in transgenic mice expressing wild-type human lamin A. Primary keratinocytes isolated from these mice had a higher frequency of nuclei with abnormal shape compared to those from transgenic mice expressing wild-type human lamin A. Treatment with a farnesyltransferase inhibitor significantly improved nuclear shape abnormalities and induced the formation of intranuclear foci in the primary keratinocytes expressing progerin. Similarly, spontaneous immortalization of progerin-expressing cultured keratinocytes selected for cells with normal nuclear morphology. Despite morphological alterations in keratinocyte nuclei, mice expressing progerin in epidermis had normal hair grown and wound healing. Hair and skin thickness were normal even after crossing to Lmna null mice to reduce or eliminate expression of normal A-type lamins. Although progerin induces significant alterations in keratinocyte nuclear morphology that are reversed by inhibition of farnesyltransferasae, epidermal expression does not lead to alopecia or other skin abnormalities typically seen in human subjects with HGPS. PMID:18442998

  2. Epidermal expression of the truncated prelamin A causing Hutchinson-Gilford progeria syndrome: effects on keratinocytes, hair and skin.

    PubMed

    Wang, Yuexia; Panteleyev, Andrey A; Owens, David M; Djabali, Karima; Stewart, Colin L; Worman, Howard J

    2008-08-01

    Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated aging disorder caused by point mutation in LMNA encoding A-type nuclear lamins. The mutations in LMNA activate a cryptic splice donor site, resulting in expression of a truncated, prenylated prelamin A called progerin. Expression of progerin leads to alterations in nuclear morphology, which may underlie pathology in HGPS. We generated transgenic mice expressing progerin in epidermis under control of a keratin 14 promoter. The mice had severe abnormalities in morphology of skin keratinocyte nuclei, including nuclear envelope lobulation and decreased nuclear circularity not present in transgenic mice expressing wild-type human lamin A. Primary keratinocytes isolated from these mice had a higher frequency of nuclei with abnormal shape compared to those from transgenic mice expressing wild-type human lamin A. Treatment with a farnesyltransferase inhibitor significantly improved nuclear shape abnormalities and induced the formation of intranuclear foci in the primary keratinocytes expressing progerin. Similarly, spontaneous immortalization of progerin-expressing cultured keratinocytes selected for cells with normal nuclear morphology. Despite morphological alterations in keratinocyte nuclei, mice expressing progerin in epidermis had normal hair grown and wound healing. Hair and skin thickness were normal even after crossing to Lmna null mice to reduce or eliminate expression of normal A-type lamins. Although progerin induces significant alterations in keratinocyte nuclear morphology that are reversed by inhibition of farnesyltransferasae, epidermal expression does not lead to alopecia or other skin abnormalities typically seen in human subjects with HGPS. PMID:18442998

  3. Identification of copper/zinc superoxide dismutase as a nitric oxide-regulated gene in human (HaCaT) keratinocytes: implications for keratinocyte proliferation.

    PubMed

    Frank, S; Kämpfer, H; Podda, M; Kaufmann, R; Pfeilschifter, J

    2000-03-15

    Recent studies have demonstrated an induction of expression of inducible nitric oxide synthase that is associated with several inflammatory diseases of the skin. To define the mechanisms of action of nitric oxide (NO) in the skin, we attempted to identify genes that are regulated by NO in keratinocytes. Using the human keratinocyte cell line HaCaT as a model system, we identified a Cu/Zn superoxide dismutase (SOD) that was strongly induced by high concentrations (500 microM) of NO-donating agents ¿S-nitrosoglutathione, sodium nitroprusside and (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1,2 -diolate (DETA-NO)¿, but not by serum or by single recombinant growth factors and inflammatory cytokines or by treatment with superoxide anions. Furthermore, endogenously produced NO increased the expression of Cu/Zn SOD mRNA in keratinocytes. Moreover, treatment of HaCaT cells with NO was associated with a biphasic effect on cell proliferation, because low doses (100 microM) of different NO donors (S-nitrosoglutathione and DETA-NO) mediated a proliferative signal to the cells, whereas high concentrations (500 microM) were cytostatic. To determine a possible correlation between the close regulation of Cu/Zn SOD expression and proliferation by NO in keratinocytes, we established a cell line (psp1CZ1N) carrying a human Cu/Zn SOD cDNA under the control of a ponasterone-inducible promoter construct. Ponasterone-induced overexpression of Cu/Zn SOD caused a cytostatic effect in proliferating psp1CZ1N cells. We therefore suggest that the up-regulation of Cu/Zn SOD expression by NO establishes an inhibitory mechanism on keratinocyte proliferation. PMID:10698699

  4. Epidermal-like architecture obtained from equine keratinocytes in three-dimensional cultures.

    PubMed

    Sharma, Ruchi; Barakzai, Safia Z; Taylor, Sarah E; Donadeu, F Xavier

    2016-08-01

    Despite the high prevalence of skin conditions in the horse, there is a dearth of literature on the culture and biology of equine skin cells, and this is partially attributable to the lack of suitable in vitro skin models. The objective of this study was to develop a three-dimensional (3D) culture system that would support the proliferation and differentiation of equine keratinocytes, similar to that observed in natural epidermis. Cell monolayers were obtained from explants of equine skin and serially passaged as highly pure keratinocyte populations (> 95% of cells), based on their expression of cytokeratins, including CK-5 and CK-14, which are associated in vivo with proliferating keratinocyte populations. Explant-derived keratinocytes were seeded into Alvetex™ 3D tissue scaffolds for 30 days under conditions that promote cell differentiation. Ultrastructural, immunohistochemical and biochemical analyses revealed that keratinocytes within scaffolds were able to proliferate and attain tissue polarity, including differentiation into basal and suprabasal layers. The basal layer contained distinct cuboidal cells with large nuclei and stained for proliferative markers such as CK-5 and CK-14. In contrast, the suprabasal layers consisted of cells with distinct polyhedral morphology, abundant cytoplasmic processes and desmosomes indicative of stratum spinosum and distinct flattened cornified cells that expressed involucrin, a marker of terminal differentiation. Thus, keratinocytes derived from primary equine skin explants were able to attain epidermal-like architecture in culture. This novel system could provide a very useful tool for modelling skin diseases, drug testing/toxicity studies and, potentially, equine regenerative medicine. Copyright © 2016 John Wiley & Sons, Ltd. PMID:23897780

  5. Mouse keratinocytes express c98, a novel gene homologous to bcl-2, that is stimulated by insulin-like growth factor 1 and prevents dexamethasone-induced apoptosis.

    PubMed

    Su, Hung-Yi; Cheng, Winston T K; Chen, Shih-Chu; Lin, Chen-Tse; Lien, Yi-Yang; Liu, Hung-Jen; Gilmour, R Stewart

    2004-01-20

    Many studies have been undertaken to investigate the mechanisms of skin differentiation. In particular, growth factors and hormones are believed to play important roles in skin proliferation, differentiation and survival. Insulin-like growth factor-1 (IGF-1) has been identified as a survival factor in many tissues including the skin, but the molecular mechanism of IGF-1 in epidermal differentiation is not completely understood. Neonatal mouse skin is useful for studying changes in gene expression, as the mitotic activity of skin cells changes shortly after birth. Using RNA differential display (DD), a 357-nt message that is specifically expressed in the epidermal keratinocytes of IGF-1-injected newborn mice but not in controls, has been identified. Confirmation of expression of this gene by ribonuclease protection assay (RPA) showed that its mRNA expression in the epidermal keratinocytes is induced by IGF-1. Using RNA ligase-mediated rapid amplification of 5' cDNA ends (RLM-5'-RACE), we have successfully isolated a 3473-bp full-length gene, c98, that has 97% sequence homology to a bcl-2-like gene, bcl-w. The latter has been identified as a proto-oncogene in several murine myeloid cell lines. Amino acid sequence analysis of the c98 showed that it has 97% sequence identity to the bcl-w protein and possesses bcl-2 homology domains (BH) 1, 2 and 3. Immunoblotting data revealed similar increases of c98 protein expression to its mRNA expression in the keratinocytes of IGF-1-injected animals. Weak expression of other bcl-2 family member proteins, bax, bcl-2 and bcl-xL, were also found in the immunoblots. Additionally, IGF-1 was found to be able to protect epidermal keratinocytes from dexamethasone (DEX)-induced apoptosis, based on the findings that after the cells were treated with DEX, DNA laddering was present in the control mice but not in those injected with IGF-1. Further, using a photometric enzyme-linked immunoassay to quantitate keratinocyte death, we found that

  6. Differential response of human adipose tissue-derived mesenchymal stem cells, dermal fibroblasts, and keratinocytes to burn wound exudates: potential role of skin-specific chemokine CCL27.

    PubMed

    van den Broek, Lenie J; Kroeze, Kim L; Waaijman, Taco; Breetveld, Melanie; Sampat-Sardjoepersad, Shakun C; Niessen, Frank B; Middelkoop, Esther; Scheper, Rik J; Gibbs, Susan

    2014-01-01

    Many cell-based regenerative medicine strategies toward tissue-engineered constructs are currently being explored. Cell-cell interactions and interactions with different biomaterials are extensively investigated, whereas very few studies address how cultured cells will interact with soluble wound-healing mediators that are present within the wound bed after transplantation. The aim of this study was to determine how adipose tissue-derived mesenchymal stem cells (ASC), dermal fibroblasts, and keratinocytes will react when they come in contact with the deep cutaneous burn wound bed. Burn wound exudates isolated from deep burn wounds were found to contain many cytokines, including chemokines and growth factors related to inflammation and wound healing. Seventeen mediators were identified by ELISA (concentration range 0.0006-9 ng/mg total protein), including the skin-specific chemokine CCL27. Burn wound exudates activated both ASC and dermal fibroblasts, but not keratinocytes, to increase secretion of CXCL1, CXCL8, CCL2, and CCL20. Notably, ASC but not fibroblasts or keratinocytes showed significant increased secretion of vascular endothelial growth factor (5-fold) and interleukin-6 (253-fold), although when the cells were incorporated in bi-layered skin substitute (SS) these differences were less pronounced. A similar discrepancy between ASC and dermal fibroblast mono-cultures was observed when recombinant human-CCL27 was used instead of burn wound exudates. Although CCL27 did not stimulate the secretion of any of the wound-healing mediators by keratinocytes, these cells, in contrast to ASC or dermal fibroblasts, showed increased proliferation and migration. Taken together, these results indicate that on transplantation, keratinocytes are primarily activated to promote wound closure. In contrast, dermal fibroblasts and, in particular, ASC respond vigorously to factors present in the wound bed, leading to increased secretion of angiogenesis/granulation tissue formation

  7. A Method for the Immortalization of Newborn Mouse Skin Keratinocytes

    PubMed Central

    Hammiller, Brianna O.; El-Abaseri, Taghrid Bahig; Dlugosz, Andrzej A.; Hansen, Laura A.

    2015-01-01

    Isolation and culture of mouse primary epidermal keratinocytes is a common technique that allows for easy genetic and environmental manipulation. However, due to their limited lifespan in culture, experiments utilizing primary keratinocytes require large numbers of animals, and are time consuming and expensive. To avoid these issues, we developed a method for the immortalization of primary mouse epidermal keratinocytes. Upon isolation of newborn epidermal keratinocytes according to established methods, the cells were cultured long-term in keratinocyte growth factor-containing medium. The cells senesced within a few weeks and eventually, small, slowly growing colonies emerged. After they regained confluency, the cells were passaged and slowly refilled the dish. With several rounds of subculture, the cells adapted to culture conditions, were easily subcultured, maintained normal morphology, and were apparently immortal. The immortalized cells retained the ability to differentiate with increased calcium concentrations, and were maintained to high passage numbers while maintaining a relatively stable karyotype. Analysis of multiple immortalized cell lines as well as primary keratinocyte cultures revealed increased numbers of chromosomes, especially in the primary keratinocytes, and chromosomal aberrations in most of the immortalized cultures and in the primary keratinocytes. Orthotopic grafting of immortalized keratinocytes together with fibroblasts onto nude mouse hosts produced skin while v-rasHa infection of the immortalized keratinocytes prior to grafting produced squamous cell carcinoma. In summary, this method of cell line generation allows for decreased use of animals, reduces the expense and time involved in research, and provides a useful model for cutaneous keratinocyte experimentation. PMID:26284198

  8. Keratinocyte Microvesicles Regulate the Expression of Multiple Genes in Dermal Fibroblasts.

    PubMed

    Huang, Ping; Bi, Jiarui; Owen, Gethin R; Chen, Weimin; Rokka, Anne; Koivisto, Leeni; Heino, Jyrki; Häkkinen, Lari; Larjava, Hannu

    2015-12-01

    Extracellular vesicles released from cells regulate many normal and pathological conditions. Little is known about the role of epidermal keratinocyte microvesicles (KC-MVs) in epithelial-stromal interaction that is essential for wound healing. We investigated, therefore, whether MV-like structures are present in human wounds and whether they affect wound healing-associated gene expression in dermal fibroblasts. In human wounds, MV-like vesicles were observed during active epithelial migration and early granulation tissue formation. When KC-MVs derived from keratinocyte-like cells (HaCaT) were added to fibroblast cultures, expression of 21 genes was significantly regulated (P<0.05) out of 80 genes investigated, including matrix metalloproteinase-1 and -3, interleukin-6 and -8, and genes associated with transforming growth factor-β signaling. Similar changes were observed at the protein level. MVs from normal epidermal keratinocytes showed similar response to HaCaT cells. KC-MVs activated ERK1/2, JNK, Smad, and p38 signaling pathways in fibroblasts with ERK1/2 signaling having the most prominent role in the MV-induced gene expression changes. KC-MVs stimulated fibroblast migration and induced fibroblast-mediated endothelial tube formation but did not affect collagen gel contraction by fibroblasts. The results demonstrate that keratinocyte microvesicles have a strong and a specific regulatory effect on fibroblasts that may modulate several aspects of wound healing. PMID:26288358

  9. Whole-exome sequencing identified a homozygous FNBP4 mutation in a family with a condition similar to microphthalmia with limb anomalies.

    PubMed

    Kondo, Yukiko; Koshimizu, Eriko; Megarbane, Andre; Hamanoue, Haruka; Okada, Ippei; Nishiyama, Kiyomi; Kodera, Hirofumi; Miyatake, Satoko; Tsurusaki, Yoshinori; Nakashima, Mitsuko; Doi, Hiroshi; Miyake, Noriko; Saitsu, Hirotomo; Matsumoto, Naomichi

    2013-07-01

    Microphthalmia with limb anomalies (MLA), also known as Waardenburg anophthalmia syndrome or ophthalmoacromelic syndrome, is a rare autosomal recessive disorder. Recently, we and others successfully identified SMOC1 as the causative gene for MLA. However, there are several MLA families without SMOC1 abnormality, suggesting locus heterogeneity in MLA. We aimed to identify a pathogenic mutation in one Lebanese family having an MLA-like condition without SMOC1 mutation by whole-exome sequencing (WES) combined with homozygosity mapping. A c.683C>T (p.Thr228Met) in FNBP4 was found as a primary candidate, drawing the attention that FNBP4 and SMOC1 may potentially modulate BMP signaling. PMID:23703728

  10. MicroRNA-191 triggers keratinocytes senescence by SATB1 and CDK6 downregulation

    SciTech Connect

    Lena, A.M.; Mancini, M.; Rivetti di Val Cervo, P. [University of 'Tor Vergata', Department of Experimental Medicine and Biochemical Sciences, Via Montpellier 1, Rome 00133; Istituto Dermopatico dell'Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico , Laboratory of Biochemistry c Saintigny, G.; Mahe, C. [CHANEL Parfums Beaute, 135 av. Charles de Gaulle, F 92521, Neuilly Melino, G. [University of 'Tor Vergata', Department of Experimental Medicine and Biochemical Sciences, Via Montpellier 1, Rome 00133; Istituto Dermopatico dell'Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico , Laboratory of Biochemistry c Association Cell Death and Differentiation c and others

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer miR-191 expression is upregulated in senescencent human epidermal keratinocytes. Black-Right-Pointing-Pointer miR-191 overexpression is sufficient per se to induce senescence in keratinocytes. Black-Right-Pointing-Pointer SATB1 and CDK6 are downregulated in senescence and are direct miR-191 targets. Black-Right-Pointing-Pointer SATB1 and CDK6 silencing by siRNA triggers senescence in HEKn cells. -- Abstract: Keratinocyte replicative senescence has an important role in time-dependent changes of the epidermis, a tissue with high turnover. Senescence encompasses growth arrest during which cells remain metabolically active but acquire a typical enlarged, vacuolar and flattened morphology. It is also accompanied by the expression of endogenous senescence-associated-{beta}-galactosidase and specific gene expression profiles. MicroRNAs levels have been shown to be modulated during keratinocytes senescence, playing key roles in inhibiting proliferation and in the acquisition of senescent markers. Here, we identify miR-191 as an anti-proliferative and replicative senescence-associated miRNA in primary human keratinocytes. Its overexpression is sufficient per se to induce senescence, as evaluated by induction of several senescence-associated markers. We show that SATB1 and CDK6 3 Prime UTRs are two miR-191 direct targets involved in this pathway. Cdk6 and Satb1 protein levels decrease during keratinocytes replicative senescence and their silencing by siRNA is able to induce a G1 block in cell cycle, accompanied by an increase in senescence-associated markers.

  11. Identifying learning disabilities through a cognitive deficit framework: can verbal memory deficits explain similarities between learning disabled and low achieving students?

    PubMed

    Callinan, Sarah; Theiler, Stephen; Cunningham, Everarda

    2015-01-01

    Traditionally, students with learning disabilities (LD) have been identified using an aptitude-achievement discrepancy or response to intervention approach. As profiles of the cognitive deficits of discrepancy-defined students with LD have already been developed using these approaches, these deficits can in turn be used to identify LD using the discrepancy approach as a benchmark for convergent validity. Australian Grade 3 (N = 172) students were administered cognitive processing tests to ascertain whether scores in these tests could accurately allocate students into discrepancy-defined groups using discriminant function analysis. Results showed that 77% to 82% of students could be correctly allocated into LD, low achievement, and regular achievement groups using only measures of phonological processing, rapid naming, and verbal memory. Furthermore, verbal memory deficits were found, along with phonological processing and rapid naming deficits, in students that would be designated as low achieving by the discrepancy method. Because a significant discrepancy or lack of response to intervention is a result of cognitive deficits rather than the other way around, it is argued that LD should be identified via cognitive deficits. PMID:23886581

  12. DNA barcodes effectively identify the morphologically similar Common Opossum (Didelphis marsupialis) and Virginia Opossum (Didelphis virginiana) from areas of sympatry in Mexico.

    PubMed

    Cervantes, Fernando A; Arcangeli, Jésica; Hortelano-Moncada, Yolanda; Borisenko, Alex V

    2010-12-01

    Two morphologically similar species of opossum from the genus Didelphis-Didelphis virginiana and Didelphis marsupialis-cooccur sympatrically in Mexico. High intraspecific variation complicates their morphological discrimination, under both field and museum conditions. This study aims to evaluate the utility and reliability of using DNA barcodes (short standardized genome fragments used for DNA-based identification) to distinguish these two species. Sequences of the cytochrome c oxidase subunit I (Cox1) mitochondrial gene were obtained from 12 D. marsupialis and 29 D. virginiana individuals and were compared using the neighbor-joining (NJ) algorithm with Kimura's two-parameter (K2P) model of nucleotide substitution. Average K2P distances were 1.56% within D. virginiana and 1.65% in D. marsupialis. Interspecific distances between D. virginiana and D. marsupialis varied from 7.8 to 9.3% and their barcode sequences formed distinct non-overlapping clusters on NJ trees. All sympatric specimens of both species were effectively discriminated, confirming the utility of Cox1 barcoding as a tool for taxonomic identification of these morphologically similar taxa. PMID:21271858

  13. Sulfation of estradiol in human epidermal keratinocyte.

    PubMed

    Kushida, Akira; Hattori, Kenji; Yamaguchi, Nozomi; Kobayashi, Tetsuyuki; Date, Akira; Tamura, Hiroomi

    2011-01-01

    Epidermis is one of the well-known estrogen target tissues. Information regarding estrogen metabolism in epidermis is still very limited compared to that of estrogen action. In the breast cancer tissue, 17β-estradiol (E(2)) is inactivated by sulfation and the expression level of estrogen sulfotransferase (SULT1E1) is inversely correlated with its malignancy. However, there is little datum about inactivation of estradiol in skin. In order to detect and measure E(2) and its metabolites simultaneously, we established an assay method with radio HPLC. A majority of [(3)H] labeled E(2) was converted to E(2) sulfate in normal human epidermal keratinocyte (NHEK) cells. The estimated activity of sulfotransferase toward E(2) at 20 nM was 0.11±0.01 (pmol/min/mg protein). Significant induction of estrogen sulfotransferase activity was observed in calcium-differentiated NHEK cells (0.58±0.07 (pmol/min/mg protein)). The gene expression of SULT1E1 was fifteen-fold higher in differentiated keratinocyte than in proliferating keratinocyte, whereas that of steroid sulfatase was reduced. These results suggest that E(2) inactivation is primarily mediated by SULT1E1 in keratinocyte and E(2) action is likely suppressed in epidermal differentiation. PMID:21720030

  14. Adherence of human oral keratinocytes and gingival fibroblasts to nano-structured titanium surfaces

    PubMed Central

    2014-01-01

    Background A key element for long-term success of dental implants is integration of the implant surface with the surrounding host tissues. Modification of titanium implant surfaces can enhance osteoblast activity but their effects on soft-tissue cells are unclear. Adherence of human keratinocytes and gingival fibroblasts to control commercially pure titanium (CpTi) and two surfaces prepared by anodic oxidation was therefore investigated. Since implant abutments are exposed to a bacteria-rich environment in vivo, the effect of oral bacteria on keratinocyte adhesion was also evaluated. Methods The surfaces were characterized using scanning electron microscopy (SEM). The number of adhered cells and binding strength, as well as vitality of fibroblasts and keratinocytes were evaluated using confocal scanning laser microscopy after staining with Live/Dead Baclight. To evaluate the effect of bacteria on adherence and vitality, keratinocytes were co-cultured with a four-species streptococcal consortium. Results SEM analysis showed the two anodically oxidized surfaces to be nano-structured with differing degrees of pore-density. Over 24 hours, both fibroblasts and keratinocytes adhered well to the nano-structured surfaces, although to a somewhat lesser degree than to CpTi (range 42-89% of the levels on CpTi). The strength of keratinocyte adhesion was greater than that of the fibroblasts but no differences in adhesion strength could be observed between the two nano-structured surfaces and the CpTi. The consortium of commensal streptococci markedly reduced keratinocyte adherence on all the surfaces as well as compromising membrane integrity of the adhered cells. Conclusion Both the vitality and level of adherence of soft-tissue cells to the nano-structured surfaces was similar to that on CpTi. Co-culture with streptococci reduced the number of keratinocytes on all the surfaces to approximately the same level and caused cell damage, suggesting that commensal bacteria could

  15. Normalization of keratinocyte-type integrins during the establishment of the oral mucosa phenotype in vitro.

    PubMed

    Tomakidi, P; Breitkreutz, D; Kohl, A; Komposch, G

    1999-01-01

    In stratified epithelia, integrins play a fundamental role in mediating basal cell attachment to a variety of extracellular matrix molecules. To assess whether keratinocyte-specific integrins are expressed in a similar way as in the normal situation also under in vivo related conditions, we processed oral mucosa equivalents consisting of keratinocytes and fibroblasts from non-cornified gingiva. In this model histomorphology, the expression of differentiation-specific keratins and keratinocyte-type integrins exhibited similarity to the tissue of origin. The stages of tissue normalization were assessed on frozen sections by indirect immunofluorescence. The initial activated stage (1 week) was characterized by (i) incomplete epithelial organization and a weak presence of the suprabasal mucosa type keratin K4, (ii) diffuse expression of the integrin chains beta 1 and alpha 6 and (iii) abundance of the wound healing-associated integrin alpha v throughout the whole epithelium. After 2 weeks, the increase in epithelial organization was characterized by (i) the presence of a basal and suprabasal cell compartment, (ii) extension of K4 in the suprabasal compartment, (iii) extended expression of the keratinocyte integrins beta 1 and alpha 6 and (iv) concentration of alpha v integrin underneath basal cells. Further normalization of tissue architecture was indicated by (i) a slight increase in K4 extension, (ii) appearance of keratinocyte integrins beta 1 and alpha 6 in basal and parabasal cells and (iii) interruption of the band-like alpha v integrin immunolocalization at the subepithelial site. The findings in the in vitro model system indicate that these oral mucosa equivalents exhibit similarities to the in vivo situation of non-cornified gingiva, thus rendering them a suitable model for the assessment of stages during epithelial reconstruction or in vivo relevant studies on material effects. PMID:10081576

  16. Novel sphingolipid derivatives promote keratinocyte differentiation.

    PubMed

    Paragh, György; Schling, Petra; Ugocsai, Peter; Kel, Alexander E; Liebisch, Gerhard; Heimerl, Susanne; Moehle, Christoph; Schiemann, Yvonne; Wegmann, Michael; Farwick, Mike; Wikonkál, Norbert M; Mandl, József; Langmann, Thomas; Schmitz, Gerd

    2008-12-01

    Sphingolipids are important components of the water permeability barrier of the skin. Moreover, ceramides were also shown to influence keratinocyte differentiation and regulate cellular signalling. A confluence-induced differentiation model of normal human keratinocytes was established to allow evaluation of pro- and anti-differentiation effects of exogenous compounds. The effects of phytosphingosine (PS), sphingosine (SO), sphinganine (SA) and their hexanoyl (-C6), stearoyl (-C18) and salicyl (-SLC) derivatives, C12-alkylamine-salicylate (C12-SLC), salicylate (SLC) along with vitamin D3 (VD3) and retinol as control substances were tested in this system. Cytotoxicity assays were carried out to optimize the incubation conditions of compounds and whole genome expression changes were monitored by DNA-microarray on days 0, 1 and 4. Geometric means of gene expression levels of a subset of known keratinocyte differentiation-related genes were calculated from the microarray data to compare effects of the sphingolipid derivatives. Compound treatment-induced transcriptional changes were analysed by the ExPlain software (BIOBASE GmbH). Five of the assayed substances (SA, SO-C6, PS-C6, SO-SLC, PS-SLC) were found to be potent promoters of keratinocyte differentiation compared with VD3, and C12-SLC revealed potential anti-differentiation properties. ExPlain analysis found a different regulatory profile in the computed transcriptional networks of the sphingoid bases versus their -C6 and especially -SLC derivatives suggesting that the change in their keratinocyte differentiation modifying potential is due to a unique effect of the covalent attachment of the salicylic acid. Taken together, these results demonstrate the gene regulatory potential of sphingolipid species that could be valuable for dermatological or cosmetic applications. PMID:18631249

  17. Role of solar conditioning in DNA repair response and survival of human epidermal keratinocytes following UV irradiation

    SciTech Connect

    Liu, S.C.; Meagher, K.; Hanawalt, P.C.

    1985-08-01

    The authors have investigated the cumulative effects of sunlight exposure upon the excision-repair of UV radiation damage to DNA in epidermal keratinocytes from human donors of different ages as well as the possible effect on DNA repair of periodic conditioning of the cultured keratinocytes with sublethal UV radiation exposures. The authors have also compared the growth properties of UV-irradiated keratinocytes derived from habitually sun-exposed and nonexposed areas from the bodies of young and aged donors. DNA repair replication in keratinocytes from habitually sun-exposed facial skin and the less sun-exposed abdominal skin of middle-aged adults was found to be similar, with respect to both the UV dose response and the time course of repair after 20 J/m2, 254 nm. Growth and survival (after exposure up to 50 J/m2, 254 nm) were greater for keratinocytes from protected areas of the upper arm of young donors (under 18 years) than for cells from their own sun-exposed areas. Growth and survival were markedly reduced for all keratinocyte cultures from aged donors, especially those cultures developed from sun-exposed areas. Nevertheless, the DNA repair response to UV radiation was similar in all cases. The evident uncoupling of UV sensitivity from DNA repair capacity remains to be understood. These studies confirm that the cumulative effect of sunlight exposure indeed contributes to some skin aging processes. However, the authors have found no indication that an overall reduction in capacity for excision-repair of UV photoproducts in keratinocyte DNA accompanies senescence in human skin.

  18. Donating blood and organs: using an extended theory of planned behavior perspective to identify similarities and differences in individual motivations to donate.

    PubMed

    Hyde, Melissa K; Knowles, Simon R; White, Katherine M

    2013-12-01

    Due to the critical shortage and continued need of blood and organ donations (ODs), research exploring similarities and differences in the motivational determinants of these behaviors is needed. In a sample of 258 university students, we used a cross-sectional design to test the utility of an extended theory of planned behavior (TPB) including moral norm, self-identity and in-group altruism (family/close friends and ethnic group), to predict people's blood and OD intentions. Overall, the extended TPB explained 77.0% and 74.6% of variance in blood and OD intentions, respectively. In regression analyses, common contributors to intentions across donation contexts were attitude, self-efficacy and self-identity. Normative influences varied with subjective norm as a significant predictor related to OD intentions but not blood donation intentions at the final step of regression analyses. Moral norm did not contribute significantly to blood or OD intentions. In-group altruism (family/close friends) was significantly related to OD intentions only in regressions. Future donation strategies should increase confidence to donate, foster a perception of self as the type of person who donates blood and/or organs, and address preferences to donate organs to in-group members only. PMID:23943782

  19. (-)-Epicatechin-3-gallate, a green tea polyphenol is a potent agent against UVB-induced damage in HaCaT keratinocytes.

    PubMed

    Huang, Chieh-Chen; Wu, Wen-Bin; Fang, Jia-You; Chiang, Han-Sun; Chen, Shao-Kuan; Chen, Bing-Huei; Chen, Ying-Ting; Hung, Chi-Feng

    2007-01-01

    (-)-Epicatechin-3-gallate (ECG) is a polyphenolic compound similar to (-)-epigallocatechin-3-gallate (EGCG) which is abundant in green tea. Numerous workers have proposed that EGCG protects epidermal cells against UVB-induced damage. However, little has been known about whether ECG protects keratinocytes against UVB-induced damage. We decided to investigate the protective effects and underlying mechanisms of ECG on UVB-induced damage. Cell viability was determined by the MTT assay. Activation of ERK1/2, p38 and JNK was analyzed by Western blotting. Intracellular H2O2 production and DNA content was analyzed by flow cytometry. Lipid peroxidation was assayed by colorimetry. In our study, we found that ECG dose-dependently attenuated UVB-induced keratinocyte death. Moreover, ECG markedly inhibited UVB-induced cell membrane lipid peroxidation and H2O2 generation in keratinocytes, suggesting that ECG can act as a free radical scavenger when keratinocytes were photodamaged. In parallel, H2O2-induced the activation of ERK1/2, p38 and JNK in keratinocytes could be inhibited by ECG. UVB-induced pre-G1 arrest leading to apoptotic changes of keratinocytes were blocked by ECG. Taken together, we provide here evidence that ECG protects keratinocytes from UVB-induced photodamage and H2O2-induced oxidative stress, possibly through inhibition of the activation of ERK1/2, p38 and JNK and/or scavenging of free radicals. PMID:17960092

  20. Improvement of hind-limb paralysis following traumatic spinal cord injury in rats by grafting normal human keratinocytes: new cell-therapy strategy for nerve regeneration.

    PubMed

    Inoue, Hajime; Takenaga, Mitsuko; Ohta, Yuki; Tomioka, Miyuki; Watabe, Yu-Ichi; Aihara, Masaki; Kumagai, Norio

    2011-12-01

    Somatic (adult) stem cells are thought to have pluripotency, just as do embryotic stem (ES) cells. We investigated the possibility that grafted epithelial keratinocytes could induce spinal cord regeneration in an animal model of spinal cord injury (SCI). Normal human keratinocytes were cultured by the routine technique, and normal human dermal fibroblasts were cultured by a similar method as a control group. SCI model was prepared by dropping a 10-g weight onto the exposed spinal cord of rats from a height of 25 mm, and 8 days later, the cultured cells were grafted into the injury site. Motor function was significantly improved in the cultured-keratinocyte-grafted group compared with that in the fibroblast-grafted group. After functional observation, human nestin- and nuclei-positive cells were found at the grafted spinal cord. Grafted cultured keratinocytes induced in vitro morphological changes in the neural induction medium. These results indicated one possibility that some of the grafted cultured keratinocytes survived and could have contributed to neural regeneration. On the other hand, it should be noted that the grafted cultured keratinocytes secreted a large amount of enzymes and/or growth factors. Therefore, another possibility is that the grafted-keratinocyte-derived factors could induce survived cell growth and endogenous neural differentiation of spinal-nerve-derived stem cells surrounding the injured spinal cord, leading to functional recovery. Epithelial stem cell therapy may be applied clinically in the near future to treat SCI. PMID:21842261

  1. Different effects of pemphigus antibody and plasmin on the distribution of keratin intermediate filaments and desmoplakins between cultured oral and epidermal keratinocytes.

    PubMed

    Jinbu, Y; Kitajima, Y; Koto, S; Akasaka, Y; Yaoita, H

    1992-01-01

    In order to clarify the molecular mechanism of blister formation in oral mucosa in pemphigus vulgaris (PV) comparing with that in epidermis, we analyzed the effects of PV serum on the distribution of keratin intermediate filaments (KIFs) and desmoplakins in oral as well as epidermal cultured keratinocytes by immunofluorescence microscopy using anti-keratin and anti-desmoplakin I/II monoclonal antibodies. After incubation with PV serum for 96 h at 37 degrees C, clusters of anti-keratin positive dots were formed around the nucleus in some of the keratinocytes from normal gingiva and soft palate but not in keratinocytes from tongue and skin, and desmoplakins also changed their distribution from linear arrangement at cell-cell contacts to clusters of dots around the nucleus in gingiva but not in epidermal keratinocytes. The dotted structures similar to those induced by pemphigus serum were formed also by incubation with human plasmin in gingival keratinocytes. However, no dot-formation of keratins was induced in these cells after incubation with trypsin. Furthermore, in epidermal keratinocytes, no keratin-dot formation was observed even after incubation with plasmin or trypsin. These results suggest that the dotted structures of KIFs caused by PV serum and plasmin might be a feature characteristic for the response of oral keratinocytes to PV serum and that there are some distinct differences in susceptibility to, and mode of, bulla formation between oral epithelium and epidermis. PMID:1375506

  2. Knockdown of filaggrin in a three-dimensional reconstructed human epidermis impairs keratinocyte differentiation.

    PubMed

    Pendaries, Valérie; Malaisse, Jeremy; Pellerin, Laurence; Le Lamer, Marina; Nachat, Rachida; Kezic, Sanja; Schmitt, Anne-Marie; Paul, Carle; Poumay, Yves; Serre, Guy; Simon, Michel

    2014-12-01

    Atopic dermatitis is a chronic inflammatory skin disorder characterized by defects in the epidermal barrier and keratinocyte differentiation. The expression of filaggrin, a protein thought to have a major role in the function of the epidermis, is downregulated. However, the impact of this deficiency on keratinocytes is not really known. This was investigated using lentivirus-mediated small-hairpin RNA interference in a three-dimensional reconstructed human epidermis (RHE) model, in the absence of other cell types than keratinocytes. Similar to what is known for atopic skin, the experimental filaggrin downregulation resulted in hypogranulosis, a disturbed corneocyte intracellular matrix, reduced amounts of natural moisturizing factor components, increased permeability and UV-B sensitivity of the RHE, and impaired keratinocyte differentiation at the messenger RNA and protein levels. In particular, the amounts of two filaggrin-related proteins and one protease involved in the degradation of filaggrin, bleomycin hydrolase, were lower. In addition, caspase-14 activation was reduced. These results demonstrate the importance of filaggrin for the stratum corneum properties/functions. They indicate that filaggrin downregulation in the epidermis of atopic patients, either acquired or innate, may be directly responsible for some of the disease-related alterations in the epidermal differentiation program and epidermal barrier function. PMID:24940654

  3. IFN-γ Primes Keratinocytes for HSV-1-Induced Inflammasome Activation.

    PubMed

    Strittmatter, Gerhard E; Sand, Jennifer; Sauter, Marlies; Seyffert, Michael; Steigerwald, Robin; Fraefel, Cornel; Smola, Sigrun; French, Lars E; Beer, Hans-Dietmar

    2016-03-01

    Inflammasomes are immune complexes that induce an inflammatory response upon sensing of different stress signals. This effect is mainly mediated by activation and secretion of the proinflammatory cytokines proIL-1β and -18. Here we report that infection of human primary keratinocytes with the double-stranded DNA viruses modified vaccinia virus Ankara (MVA) or herpes simplex virus type 1 (HSV-1)-induced secretion of mature IL-1β and -18. This secretion was dependent on several inflammasome complexes; however, the absent in melanoma 2 (AIM2) inflammasome, which is activated by binding of double-stranded DNA, played the most important role. Whereas prestimulation of keratinocytes with IFN-γ moderately increased MVA-induced IL-1β and IL-18 secretion, it was essential for substantial secretion of these cytokines in response to herpes simplex virus type 1 infection. IFN-γ partially restored HSV-1 suppressed proIL-1β expression and was also required for inflammasome activation. Most importantly, IFN-γ strongly suppressed virus replication in keratinocytes in vitro and ex vivo, which was independent of inflammasome activation. Our results suggest that, similar to Herpesviridae infection in mice, HSV-1 replication in human skin is controlled by a positive feedback loop of keratinocyte-derived IL-1/IL-18 and IFN-γ expressed by immune cells. PMID:26739094

  4. Cultured mucosal cell sheet with a double layer of keratinocytes and fibroblasts on a collagen membrane.

    PubMed

    Imaizumi, Fumiko; Asahina, Izumi; Moriyama, Takashi; Ishii, Masatoshi; Omura, Ken

    2004-01-01

    The aim of this study was to develop a novel cultured mucosal membrane that was facile to prepare and easy to handle, and that could be applied to mucosal defects in the oral cavity. Human oral keratinocytes and fibroblasts were prepared from the oral mucosa. We made the following two types of cultured mucosal cell sheets: a monolayer sheet of keratinocytes cultured on a collagen membrane (K-S) and a double-layered sheet of keratinocytes and fibroblasts on a collagen membrane (KF-S). A collagen membrane was used as a control. Each type of sheet was transplanted onto dorsal skin defects of nude mice. The wound area was measured for the assessment of wound contraction and a specimen was harvested for histologic evaluation 1 week and 4 weeks after grafting. Wound contraction was minimal with KF-S grafts. Although histologic examination showed normal differentiation of the epithelium in all graft types, the involucrin expression pattern of KFS was most similar to that of normal epithelium. These results indicate that a double-layered sheet of keratinocytes and fibroblasts cultured on a collagen membrane may facilitate epithelial healing and prevent wound contraction. PMID:15265283

  5. Vimentin coordinates fibroblast proliferation and keratinocyte differentiation in wound healing via TGF-β-Slug signaling.

    PubMed

    Cheng, Fang; Shen, Yue; Mohanasundaram, Ponnuswamy; Lindström, Michelle; Ivaska, Johanna; Ny, Tor; Eriksson, John E

    2016-07-26

    Vimentin has been shown to be involved in wound healing, but its functional contribution to this process is poorly understood. Here we describe a previously unrecognized function of vimentin in coordinating fibroblast proliferation and keratinocyte differentiation during wound healing. Loss of vimentin led to a severe deficiency in fibroblast growth, which in turn inhibited the activation of two major initiators of epithelial-mesenchymal transition (EMT), TGF-β1 signaling and the Zinc finger transcriptional repressor protein Slug, in vimentin-deficient (VIM(-/-)) wounds. Correspondingly, VIM(-/-) wounds exhibited loss of EMT-like keratinocyte activation, limited keratinization, and slow reepithelialization. Furthermore, the fibroblast deficiency abolished collagen accumulation in the VIM(-/-) wounds. Vimentin reconstitution in VIM(-/-) fibroblasts restored both their proliferation and TGF-β1 production. Similarly, restoring paracrine TGF-β-Slug-EMT signaling reactivated the transdifferentiation of keratinocytes, reviving their migratory properties, a critical feature for efficient healing. Our results demonstrate that vimentin orchestrates the healing by controlling fibroblast proliferation, TGF-β1-Slug signaling, collagen accumulation, and EMT processing, all of which in turn govern the required keratinocyte activation. PMID:27466403

  6. Analysis of epithelial–mesenchymal transition markers in psoriatic epidermal keratinocytes

    PubMed Central

    Man, Xiao-Yong; Chen, Xi-Bei; Li, Wei; Landeck, Lilla; Dou, Ting-Ting; Chen, Jia-qi; Zhou, Jiong; Cai, Sui-Qing; Zheng, Min

    2015-01-01

    Psoriasis is similar to endpoints of epithelial–mesenchymal transition (EMT), a process of epithelial cells transformed into fibroblast-like cells. The molecular epithelial and mesenchymal markers were analysed in psoriatic keratinocytes. No obvious alteration of epithelial markers E-cadherin (E-cad), keratin 10 (K10), K14 and K16 was detected in psoriatic keratinocytes. However, significantly increased expression of Vim, FN, plasminogen activator inhibitor 1 (PAI-1) and Slug was seen. IL-17A and IL-13 at 50 ng ml−1 strongly decreased expression of K10, Vim and FN. TGF-β1 at 50 ng ml−1 promoted the production of N-cad, Vim, FN and PAI-1. Slug was decreased by dexamethasone (Dex), but E-cad was upregulated by Dex. Silencing of ERK partially increased E-cad and K16, but remarkably inhibited K14, FN, Vim, β-catenin, Slug and α5 integrin. Moreover, inhibition of Rho and GSK3 by their inhibitors Y27632 and SB216763, respectively, strongly raised E-cad, β-catenin and Slug. Dex decreased Y27632-mediated increase of β-catenin. Dex at 2.0 µM inhibited SB216763-regulated E-cad, β-catenin and slug. In conclusion, EMT in psoriatic keratinocytes may be defined as an intermediate phenotype of type 2 EMT. ERK, Rho and GSK3 play active roles in the process of EMT in psoriatic keratinocytes. PMID:26269426

  7. Melanocytes and keratinocytes have distinct and shared responses to ultraviolet radiation and arsenic.

    PubMed

    Cooper, K L; Yager, J W; Hudson, L G

    2014-01-30

    The rise of melanoma incidence in the United States is a growing public health concern. A limited number of epidemiology studies suggest an association between arsenic levels and melanoma risk. Arsenic acts as a co-carcinogen with ultraviolet radiation (UVR) for the development of squamous cell carcinoma and proposed mechanisms include generation of oxidative stress by arsenic and UVR and inhibition of UVR-induced DNA repair by arsenic. In this study, we investigate similarities and differences in response to arsenic and UVR in keratinocytes and melanocytes. Normal melanocytes are markedly more resistant to UVR-induced cytotoxicity than normal keratinocytes, but both cell types are equally sensitive to arsenite. Melanocytes were more resistant to arsenite and UVR stimulation of superoxide production than keratinocytes, but the concentration of arsenite necessary to inhibit the activity of the DNA repair protein poly(ADP-ribose)polymerase and enhance retention of UVR-induced DNA damage was essentially equivalent in both cell types. These findings suggest that although melanocytes are less sensitive than keratinocytes to initial UVR-mediated DNA damage, both of these important target cells in the skin share a mechanism related to arsenic inhibition of DNA repair. These findings suggest that concurrent chronic arsenic exposure could promote retention of unrepaired DNA damage in melanocytes and act as a co-carcinogen in melanoma. PMID:24270004

  8. Local Glucocorticoid Activation by 11β-Hydroxysteroid Dehydrogenase 1 in Keratinocytes: The Role in Hapten-Induced Dermatitis.

    PubMed

    Terao, Mika; Itoi, Saori; Matsumura, Sayaka; Yang, Lingli; Murota, Hiroyuki; Katayama, Ichiro

    2016-06-01

    Over the past decade, extra-adrenal cortisol production was reported in various tissues. The enzyme that catalyzes the conversion of hormonally inactive cortisone into active cortisol in cells is 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1). We recently reported that 11β-HSD1 is also expressed in keratinocytes and regulates inflammation and keratinocyte proliferation. To investigate the function of 11β-HSD1 in keratinocytes during inflammation in vivo, we created keratinocyte-specific 11β-HSD1 knockout (K5-Hsd11b1-KO) mice and analyzed the inflammatory response in models of hapten-induced contact irritant dermatitis. K5-Hsd11b1-KO mice showed enhanced ear swelling in low-dose oxazolone-, 2,4,6-trinitro-1-chlorobenzene (TNCB)-, and 2,4-dinitrofluorobenzene-induced irritant dermatitis associated with increased inflammatory cell infiltration. Topical application of corticosterone dose dependently suppressed TNCB-induced ear swelling and cytokine expression. Similarly in mouse keratinocytes in vitro, corticosterone dose dependently suppressed 2,4,6-trinitrobenzenesulfonic acid-induced IL-1α and IL-1β expression. The effect of 11-dehydrocorticosterone was attenuated in TNCB-induced irritant dermatitis in K5-Hsd11b1-KO mice compared with wild-type mice. In human samples, 11β-HSD1 expression was decreased in epidermis of psoriasis vulgaris compared with healthy skin. Taken together, these data suggest that corticosterone activation by 11β-HSD1 in keratinocytes suppresses hapten-induced irritant dermatitis through suppression of expression of cytokines, such as IL-1α and IL-1β, in keratinocytes. PMID:27070821

  9. p53 and TAp63 Promote Keratinocyte Proliferation and Differentiation in Breeding Tubercles of the Zebrafish

    PubMed Central

    Fischer, Boris; Metzger, Manuel; Richardson, Rebecca; Knyphausen, Philipp; Ramezani, Thomas; Franzen, Rainer; Schmelzer, Elmon; Bloch, Wilhelm; Carney, Thomas J.; Hammerschmidt, Matthias

    2014-01-01

    p63 is a multi-isoform member of the p53 family of transcription factors. There is compelling genetic evidence that ΔNp63 isoforms are needed for keratinocyte proliferation and stemness in the developing vertebrate epidermis. However, the role of TAp63 isoforms is not fully understood, and TAp63 knockout mice display normal epidermal development. Here, we show that zebrafish mutants specifically lacking TAp63 isoforms, or p53, display compromised development of breeding tubercles, epidermal appendages which according to our analyses display more advanced stratification and keratinization than regular epidermis, including continuous desquamation and renewal of superficial cells by derivatives of basal keratinocytes. Defects are further enhanced in TAp63/p53 double mutants, pointing to partially redundant roles of the two related factors. Molecular analyses, treatments with chemical inhibitors and epistasis studies further reveal the existence of a linear TAp63/p53->Notch->caspase 3 pathway required both for enhanced proliferation of keratinocytes at the base of the tubercles and their subsequent differentiation in upper layers. Together, these studies identify the zebrafish breeding tubercles as specific epidermal structures sharing crucial features with the cornified mammalian epidermis. In addition, they unravel essential roles of TAp63 and p53 to promote both keratinocyte proliferation and their terminal differentiation by promoting Notch signalling and caspase 3 activity, ensuring formation and proper homeostasis of this self-renewing stratified epithelium. PMID:24415949

  10. Deregulation of keratinocyte differentiation and activation: a hallmark of venous ulcers

    PubMed Central

    Stojadinovic, Olivera; Pastar, Irena; Vukelic, Sasa; Mahoney, Mỹ G; Brennan, Donna; Krzyzanowska, Agata; Golinko, Michael; Brem, Harold; Tomic-Canic, Marjana

    2008-01-01

    Epidermal morphology of chronic wounds differs from that of normal epidermis. Biopsies of non-healing edges obtained from patients with venous ulcers show thick and hyperproliferative epidermis with mitosis present in suprabasal layers. This epidermis is also hyper-keratotic and parakeratotic. This suggests incomplete activation and differentiation of keratinocytes. To identify molecular changes that lead to pathogenic alterations in keratinocyte activation and differentiation pathways we isolated mRNA from non-healing edges deriving from venous ulcers patients and determined transcriptional profiles using Affymetrix chips. Obtained transcriptional profiles were compared to those from healthy, unwounded skin. As previously indicated by histology, we found deregulation of differentiation and activation markers. We also found differential regulation of signalling molecules that regulate these two processes. Early differentiation markers, keratins K1/K10 and a subset of small proline-rich proteins, along with the late differentiation marker filaggrin were suppressed, whereas late differentiation markers involucrin, transgultaminase 1 and another subset of small proline-rich proteins were induced in ulcers when compared to healthy skin. Surprisingly, desomosomal and tight junction components were also deregulated. Keratinocyte activation markers keratins K6/K16/K17 were induced. We conclude that keratinocytes at the non-healing edges of venous ulcers do not execute either activation or differentiation pathway, resulting in thick callus-like formation at the edge of a venous ulcers. PMID:18373736

  11. IL-24 is Expressed During Wound Repair and Inhibits TGFα induced Migration and Proliferation of Keratinocytes

    PubMed Central

    Poindexter, Nancy J.; Williams, Ryan R.; Powis, Garth; Jen, Emily; Caudle, Abigail S.; Chada, Sunil; Grimm, Elizabeth A.

    2011-01-01

    Interleukin (IL)-24 is the protein product of melanoma differentiation-associated gene 7 (MDA-7). Originally identified as a tumor suppressor molecule, MDA-7 was renamed IL-24 and classified as a cytokine because of its chromosomal location in the IL-10 locus, its mRNA expression in leukocytes, and its secretory sequence elements. We previously reported that IL-24 is expressed by cytokine-activated monocytes and T lymphocytes. Here, we show that IL-24 is expressed in keratinocytes during wound repair. Paraffin-embedded tissues prepared from human skin sampled at days 2, 6, and 10 after wounding were examined by immunohistochemistry for expression of IL-24. Protein expression was detected in the keratinocyte population with maximum expression at days 2 and 6; and no expression by day 10 (4 of 4 subjects). In vitro studies showed that cytokines involved in wound repair, most notably TGFα, TGFβ, IFNγ and IFNβ, upregulated IL-24 protein expression in normal human epidermal keratinocytes (NHEK). Examination of the function of IL-24 in both in vitro wound repair and migration assays demonstrated that IL-24 inhibits TGFα induced proliferation and migration of NHEKs. These data support the hypothesis that IL-24 functions during an inflammatory response in the skin by inhibiting the proliferation and migration of keratinocytes. PMID:20545760

  12. Hypotonic stress influence the membrane potential and alter the proliferation of keratinocytes in vitro.

    PubMed

    Gönczi, Mónika; Szentandrássy, Norbert; Fülöp, László; Telek, Andrea; Szigeti, Gyula P; Magyar, János; Bíró, Tamás; Nánási, Péter P; Csernoch, László

    2007-04-01

    Keratinocyte proliferation and differentiation is strongly influenced by mechanical forces. We investigated the effect of osmotic changes in the development of HaCaT cells in culture using intracellular calcium measurements, electrophysiological recordings and molecular biology techniques. The application of hypotonic stress (174 mOsmol/l) caused a sustained hyperpolarization of HaCaT cells from a resting potential of -27 +/- 4 to -51 +/- 9 mV. This change was partially reversible. The surface membrane channels involved in the hyperpolarization were identified as chloride channels due to the lack of response in the absence of the anion. Cells responded with an elevation of intracellular calcium concentration to hypotonic stress, which critically depended on external calcium. The presence of phorbol-12-myristate-13-acetate in the culture medium for 12 h augmented the subsequent response to hypotonic stress. A sudden switch from iso- to hypotonic solution increased cell proliferation and suppressed the production of involucrin, filaggrin and transglutaminase, markers of keratinocyte differentiation. It is concluded that sudden mechanical forces increase the proliferation of keratinocytes through alterations in their membrane potential and intracellular calcium concentration. These changes together with additional modifications in channel expression and intracellular signalling mechanisms could underlie the increased proliferation of keratinocytes in hyperproliferative skin diseases. PMID:17359336

  13. Human keratinocytes have two interconvertible modes of proliferation.

    PubMed

    Roshan, Amit; Murai, Kasumi; Fowler, Joanna; Simons, Benjamin D; Nikolaidou-Neokosmidou, Varvara; Jones, Philip H

    2016-02-01

    Single stem cells, including those in human epidermis, have a remarkable ability to reconstitute tissues in vitro, but the cellular mechanisms that enable this are ill-defined. Here we used live imaging to track the outcome of thousands of divisions in clonal cultures of primary human epidermal keratinocytes. Two modes of proliferation were seen. In 'balanced' mode, similar proportions of proliferating and differentiating cells were generated, achieving the 'population asymmetry' that sustains epidermal homeostasis in vivo. In 'expanding' mode, an excess of cycling cells was produced, generating large expanding colonies. Cells in expanding mode switched their behaviour to balanced mode once local confluence was attained. However, when a confluent area was wounded in a scratch assay, cells near the scratch switched back to expanding mode until the defect was closed. We conclude that the ability of a single epidermal stem cell to reconstitute an epithelium is explained by two interconvertible modes of proliferation regulated by confluence. PMID:26641719

  14. Histamine induces proliferation in keratinocytes from atopic dermatitis patients

    PubMed Central

    Glatzer, Franziska; Gschwandtner, Maria; Ehling, Sarah; Rossbach, Kristine; Janik, Katrin; Klos, Andreas; Bäumer, Wolfgang; Kietzmann, Manfred; Werfel, Thomas; Gutzmer, Ralf

    2015-01-01

    Background Epidermal hyperproliferation resulting in acanthosis is an important clinical observation in atopic dermatitis and its underlying mechanisms are not completely understood by now. Objective Since elevated levels of histamine are present in lesional skin, we investigated the effect of histamine, especially with regard to H4R activation, on the proliferation of human and murine keratinocytes. Methods The expression of H4R on human and murine keratinocytes was detected by real-time PCR. Keratinocyte proliferation was evaluated by different in vitro cell proliferation assays, scratch assays and measurement of epidermal thickness of murine skin. Results We detected H4R mRNA on foreskin keratinocytes and on outer root sheath keratinocytes; H4R mRNA was more abundant in keratinocytes from patients with atopic dermatitis as compared to non-atopic donors. Stimulation of foreskin keratinocytes, atopic dermatitis outer root sheath keratinocytes and H4R transfected HaCaT cells with histamine and H4R agonist resulted in an increase of proliferation, which was blocked with the H4R-specific antagonist JNJ7777120. Abdominal epidermis of H4R-deficient mice was significantly thinner and the in vitro proliferation of keratinocytes derived from H4R-deficient mice was lower compared to control mice. Interestingly, we only detected H4R expression on murine keratinocytes after stimulation with lipopolysaccharide and peptidoglycane. Conclusion The H4R is highly expressed on keratinocytes from atopic dermatitis patients and its stimulation induces keratinocyte proliferation. This might represent a mechanism that contributes to the epidermal hyperplasia observed in atopic dermatitis. PMID:23932072

  15. Human keratinocyte culture from the peritonsillar mucosa.

    PubMed

    Neugebauer, P; Bonnekoh, B; Wevers, A; Michel, O; Mahrle, G; Krieg, T; Stennert, E

    1996-01-01

    Tonsillectomy tissue can be used as a routine source for cultures of oropharyngeal keratinocytes. In so doing, a peritonsillar strip of unaltered mucosa was dissected in the upper submucosa. Subsequent trypsinization yielded 7.0 +/- 3.4 x 10(6) keratinocytes per bilateral tonsillectomy. Keratinocyte attachment and growth in primary culture were promoted by sublethally irradiated 3T3 murine fibroblasts. Three subcultures could be performed without a feeder layer and were characterized by a population doubling time of 4.5 days during log growth phase. Electrophoretic and immunoblot analysis of the third subculture revealed a strong expression of keratin pairs 5/14 and 6/16 as well as keratins 7 and 19, whereas keratins 8/18 were expressed less intensely. The lowest intensity, was found for keratin 13, which is known to be indicative of the differentiated mucosa. The culture technique thus provides an easily available in vitro model for morphological and functional studies on the epithelial compartment of human oropharyngeal mucosa. PMID:8737778

  16. Methicillin-Resistant Staphylococcus aureus Adaptation to Human Keratinocytes

    PubMed Central

    Soong, Grace; Paulino, Franklin; Wachtel, Sarah; Parker, Dane; Wickersham, Matthew; Zhang, Dongni; Brown, Armand; Lauren, Christine; Dowd, Margaret; West, Emily; Horst, Basil; Planet, Paul

    2015-01-01

    ABSTRACT Skin is the most common site of Staphylococcus aureus infection. While most of these infections are self-limited, recurrent infections are common. Keratinocytes and recruited immune cells participate in skin defense against infection. We postulated that S. aureus is able to adapt to the milieu within human keratinocytes to avoid keratinocyte-mediated clearance. From a collection of S. aureus isolated from chronically infected patients with atopic dermatitis, we noted 22% had an agr mutant-like phenotype. Using several models of human skin infection, we demonstrate that toxin-deficient, agr mutants of methicillin-resistant S. aureus (MRSA) USA300 are able to persist within keratinocytes by stimulating autophagy and evading caspase-1 and inflammasome activation. MRSA infection induced keratinocyte autophagy, as evidenced by galectin-8 and LC3 accumulation. Autophagy promoted the degradation of inflammasome components and facilitated staphylococcal survival. The recovery of more than 58% agr or RNAIII mutants (P < 0.0001) of an inoculum of wild-type (WT) MRSA from within wortmannin-treated keratinocytes compared to control keratinocytes reflected the survival advantage for mutants no longer expressing agr-dependent toxins. Our results illustrate the dynamic interplay between S. aureus and keratinocytes that can result in the selection of mutants that have adapted specifically to evade keratinocyte-mediated clearance mechanisms. PMID:25900653

  17. NOVEL NON-CALCEMIC SECOSTEROIDS THAT ARE PRODUCED BY HUMAN EPIDERMAL KERATINOCYTES PROTECT AGAINST SOLAR RADIATION

    PubMed Central

    Slominski, Andrzej T.; Janjetovic, Zorica; Kim, Tae-Kang; Wasilewski, Piotr; Rosas, Sofia; Hanna, Sherie; Sayre, Robert M.; Dowdy, John C.; Li, Wei; Tuckey, Robert C.

    2015-01-01

    CYP11A1 hydroxylates the side chain of vitamin D3 (D3) in a sequential fashion [D3→20S(OH)D3→20,23(OH)2D3→ 17,20,23(OH)3D3], in an alternative to the classical pathway of activation [D3→25(OH)D3→1,25(OH)2D3]. The products/intermediates of the pathway can be further modified by the action of CYP27B1. The CYP11A1-derived products are biologically active with functions determined by the lineage of the target cells. This pathway can operate in epidermal keratinocytes. To further define the role of these novel secosteroids we tested them for protective effects against UVB-induced damage in human epidermal keratinocytes, melanocytes and HaCaT keratinocytes, cultured in vitro. The secosteroids attenuated ROS, H2O2 and NO production by UVB-irradiated keratinocytes and melanocytes, with an efficacy similar to 1,25(OH)2D3, while 25(OH)D3 had lower efficacy. These attenuations were also seen to some extent for the 20(OH)D3 precursor, 20S-hydroxy-7-dehydrocholesterol. These effects were accompanied by upregulation of genes encoding enzymes responsible for defence against oxidative stress. Using immunofluorescent staining we observed that the secosteroids reduced the generation cyclobutane pyrimidine dimers in response to UVB and enhanced expression of p53 phosphorylated at Ser-15, but not at Ser-46. Additional evidence for protection against DNA damage in cells exposed to UVB and treated with secosteroids was provided by the Comet assay where DNA fragmentation was markedly reduced by 20(OH)D3 and 20,23(OH)2D3. In conclusion, novel secosteroids that can be produced by the action of CYP11A1 in epidermal keratinocytes have protective effects against UVB radiation. PMID:25617667

  18. Procedures for Identifying Rocks with Similar Features.

    ERIC Educational Resources Information Center

    Powell, William E.

    1984-01-01

    The purpose of this article is to provide college level physical geography and geology teachers with practical and simple techniques to help students classify and understand igneous, sedimentary, and metamorphic rocks. Essential equipment is also discussed, and recommended readings are listed. (RM)

  19. Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes.

    PubMed

    Grøn, Birgitte; Stoltze, Kaj; Andersson, Anders; Dabelsteen, Erik

    2002-12-01

    The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co-culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations that could explain regional variation in epithelial growth and wound healing. Normal human fibroblasts were cultured on polystyrene or maintained in collagen matrix and stimulated with keratinocytes cultured on membranes. The amount of HGF and KGF protein in the culture medium was determined every 24 h for 5 days by ELISA. When cultured on polystyrene, the constitutive level of KGF and HGF in periodontal fibroblasts was higher than the level in buccal and skin fibroblasts. In the presence of keratinocytes, all three types of fibroblasts in general increased their HGF and KGF production 2-3 times. When cells were maintained in collagen, the level of HGF and KGF was decreased mainly in skin cultures. However, in oral fibroblasts, induction after stimulation was at a similar level in collagen compared to on polystyrene. Skin fibroblasts maintained in collagen produced almost no HGF whether with or without stimulation. The results demonstrate that the secretion of KGF and HGF in both unstimulated fibroblasts and in fibroblasts co-cultured with keratinocytes is dependent on the type of fibroblasts. In general, the periodontal fibroblasts had the highest level of cytokine production. This high level of growth factor production may influence the proliferation and the migration of junctional epithelium and thereby influence the development of periodontal disease. PMID:12645668

  20. The effect of pantothenic acid deficiency on keratinocyte proliferation and the synthesis of keratinocyte growth factor and collagen in fibroblasts.

    PubMed

    Kobayashi, Daisaku; Kusama, Miho; Onda, Masaaki; Nakahata, Norimichi

    2011-01-01

    It has been reported that pantothenic acid (vitamin B5) and panthenol, an alcohol derivative of pantothenic acid, have beneficial moisturizing effects on the skin. However, few studies have investigated the mechanism of action of pantothenic acid on skin tissues. We tried to clarify the role of pantothenic acid on skin function by using keratinocytes and fibroblasts. The depletion of pantothenic acid from the culture medium suppressed keratinocyte proliferation and promoted differentiation. Moreover, pantothenic acid depletion decreased the synthesis of keratinocyte growth factor and procollagen 4a2 in fibroblasts. These results suggest that pantothenic acid is essential for maintaining keratinocyte proliferation and differentiation. PMID:21258175

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

    PubMed

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

    2015-02-01

    Peripheral intracrine sex steroid synthesis from adrenal precursors dehydroepiandrosterone (DHEA) and DHEA-sulfate has evolved in humans. We sought to establish if there are differences in intracrine, paracrine, and endocrine regulation of sex steroids by primary cultures of human skin epidermal keratinocytes and dermal fibroblasts. Microarray analysis identified multifunctional genes modulated by steroids, quantitative RT-PCR (qRT-PCR) mRNA expression, enzymatic assay aromatase activity, scratch assay cell migration, immunocytochemistry α-smooth muscle actin (α-SMA), and collagen gel fibroblast contraction. All steroidogenic components were present, although only keratinocytes expressed the organic anion organic anion transporter protein (OATP) 2B1 transporter. Both expressed the G-protein-coupled estrogen receptor (GPER1). Steroids modulated multifunctional genes, up-regulating genes important in repair and aging [angiopoietin-like 4 (ANGPTL4), chemokine (C-X-C motif) ligand 1 (CXCL1), lamin B1 (LMNB1), and thioredoxin interacting protein (TXNIP)]. DHEA-sulfate (DHEA-S), DHEA, and 17β-estradiol stimulated keratinocyte and fibroblast migration at early (4 h) and late (24-48 h) time points, suggesting involvement of genomic and nongenomic signaling. Migration was blocked by aromatase and steroid sulfatase (STS) inhibitors confirming intracrine synthesis to estrogen. Testosterone had little effect, implying it is not an intermediate. Steroids stimulated fibroblast contraction but not α-SMA expression. Mechanical wounding reduced fibroblast aromatase activity but increased keratinocyte activity, amplifying the bioavailability of intracellular estrogen. Cultured fibroblasts and keratinocytes provide a biologically relevant model system to investigate the complex pathways of sex steroid intracrinology in human skin. PMID:25392269

  2. α6 Integrin and CD44 Enrich for a Primary Keratinocyte Population That Displays Resistance to UV-Induced Apoptosis

    PubMed Central

    Wray, Helen; Mackenzie, Ian C.; Storey, Alan; Navsaria, Harshad

    2012-01-01

    Epidermal human keratinocytes are exposed to a wide range of environmental genotoxic insults, including the UV component of solar radiation. Epidermal homeostasis in response to cellular or tissue damage is maintained by a population of keratinocyte stem cells (KSC) that reside in the basal layer of the epithelium. Using cell sorting based on cell-surface markers, we have identified a novel α6 integrinhigh+/CD44+ sub-population of basal keratinocytes. These α6 integrinhigh+/CD44+ keratinocytes have both high proliferative potential, form colonies in culture that have characteristics of holoclones and have a unique pattern of resistance to apoptosis induced by UVB radiation or by agents that induce single- or double strand DNA breaks. Resistance to UVB induced apoptosis in the α6 integrinhigh+/CD44+ cells involved increased expression of TAp63 and was overcome by PI-3 kinase inhibition. In marked contrast, the α6 integrinhigh+/CD44+ cells were sensitive to apoptosis induced by the cross-linking agent cisplatin, and imatinib inhibition of c-Abl blocked the ability of cisplatin to kill α6 integrinhigh+/CD44+ cells. Our findings reveal a population of basal keratinocytes with long-term proliferative properties that display specific patterns of apoptotic resistance that is dependent upon the genotoxic stimulus, and provide insights into how these cells can be targeted with chemotherapeutic agents. PMID:23071680

  3. Oligonucleotide uptake in cultured keratinocytes: influence of confluence, cationic liposomes, and keratinocyte cell type.

    PubMed

    White, P J; Fogarty, R D; McKean, S C; Venables, D J; Werther, G A; Wraight, C J

    1999-05-01

    The success of anti-sense strategies has been limited, at least in part, by the poor uptake of these agents into the target cells. In keratinocytes, there is conflicting evidence as to the amount and location of oligonucleotide uptake into these cells, with variable proportions of cells reported to take up oligodeoxynucleotide, and also cytoplasmic and nuclear localization reported. In this study, the uptake of oligodeoxynucleotides in cultured normal human keratinocytes and the HaCaT cell line was quantitated in the presence of various lipids designed to enhance uptake and in varying culture conditions. About 12% of cells in a confluent normal human keratinocyte culture showed nuclear uptake, with a small and variable proportion showing cytoplasmic localization after 24 h incubation with 1 microM oligodeoxynucleotide. Uptake of oligodeoxynucleotide was found to be increased by liposome encapsulation (to a maximum of 28.1% +/- 2.1% of cells), low confluence (39.5% +/- 2.5%), and further increased by a combination of the two conditions (55.4% +/- 4.3%). HaCaT cell populations showed sparse but consistent uptake of oligodeoxynucleotide, with about 1% of cells showing nuclear localization in the presence of 1 microM oligodeoxynucleotide, increasing to 13.5% +/- 4.9% in the presence of cationic lipid (Tfx-50) in low confluence HaCaT monolayers. We conclude that normal keratinocytes exhibit reliable, substantial uptake of oligonucleotides in conditions controlled for confluence and aided by liposome encapsulation. PMID:10233759

  4. Reorganization of the interchromosomal network during keratinocyte differentiation.

    PubMed

    Sehgal, Nitasha; Seifert, Brandon; Ding, Hu; Chen, Zihe; Stojkovic, Branislav; Bhattacharya, Sambit; Xu, Jinhui; Berezney, Ronald

    2016-06-01

    The well-established human epidermal keratinocyte (HEK) differentiation model was investigated to determine possible alterations in chromosome territory (CT) association during differentiation. The seven human chromosomes (1, 4, 11, 12, 16, 17, and 18) selected for this analysis are representative of the chromosome size and gene density range of the overall human genome as well as including a majority of genes involved in epidermal development and differentiation (CT1, 12, and 17). Induction with calcium chloride (Ca(2+)) resulted in morphological changes characteristic of keratinocyte differentiation. Combined multi-fluorescence in situ hybridization (FISH) and computational image analysis on the undifferentiated (0 h) and differentiated (24 h after Ca(2+) treatment) HEK revealed that (a) increases in CT volumes correspond to overall nuclear volume increases, (b) radial positioning is gene density-dependent at 0 h but neither gene density- nor size-dependent at 24 h, (c) the average number of interchromosomal associations for each CT is gene density-dependent and similar at both time points, and (d) there are striking differences in the single and multiple pairwise interchromosomal association profiles. Probabilistic network models of the overall interchromosomal associations demonstrate major reorganization of the network during differentiation. Only ~40 % of the CT pairwise connections in the networks are common to both 0 and 24 h HEK. We propose that there is a probabilistic chromosome positional code which can be significantly altered during cell differentiation in coordination with reprogramming of gene expression. PMID:26490167

  5. Interleukin-36 potently stimulates human M2 macrophages, Langerhans cells and keratinocytes to produce pro-inflammatory cytokines.

    PubMed

    Dietrich, Damien; Martin, Praxedis; Flacher, Vincent; Sun, Yu; Jarrossay, David; Brembilla, Nicolo; Mueller, Christopher; Arnett, Heather A; Palmer, Gaby; Towne, Jennifer; Gabay, Cem

    2016-08-01

    Interleukin (IL)-36 cytokines belong to the IL-1 family and include three agonists, IL-36 α, β and γ and one inhibitor, IL-36 receptor antagonist (IL-36Ra). IL-36 and IL-1 (α and β) activate similar intracellular pathways via their related heterodimeric receptors, IL-36R/IL-1RAcP and IL-1R1/IL-1RAcP, respectively. However, excessive IL-36 versus IL-1 signaling induces different phenotypes in humans, which may be related to differential expression of their respective receptors. We examined the expression of IL-36R, IL-1R1 and IL-1RAcP mRNA in human peripheral blood, tonsil and skin immune cells by RT-qPCR. Monocyte-derived dendritic cells (MDDC), M0, M1 or M2-polarized macrophages, primary keratinocytes, dermal macrophages and Langerhans cells (LC) were stimulated with IL-1β or IL-36β. Cytokine production was assessed by RT-qPCR and immunoassays. The highest levels of IL-36R mRNA were found in skin-derived keratinocytes, LC, dermal macrophages and dermal CD1a(+) DC. In the blood and in tonsils, IL-36R mRNA was predominantly found in myeloid cells. By contrast, IL-1R1 mRNA was detected in almost all cell types with higher levels in tonsil and skin compared to peripheral blood immune cells. IL-36β was as potent as IL-1β in stimulating M2 macrophages, keratinocytes and LC, less potent than IL-1β in stimulating M0 macrophages and MDDC, and exerted no effects in M1 and dermal macrophages. Levels of IL-1Ra diminished the ability of M2 macrophages to respond to IL-1. Taken together, these data are consistent with the association of excessive IL-36 signaling with an inflammatory skin phenotype and identify human LC and M2 macrophages as new IL-36 target cells. PMID:27259168

  6. Phlorizin, an Active Ingredient of Eleutherococcus senticosus, Increases Proliferative Potential of Keratinocytes with Inhibition of MiR135b and Increased Expression of Type IV Collagen

    PubMed Central

    Choi, Hye-Ryung; Nam, Kyung-Mi; Lee, Hyun-Sun; Yang, Seung-Hye; Kim, Young-Soo; Lee, Jongsung; Date, Akira; Toyama, Kazumi; Park, Kyoung-Chan

    2016-01-01

    E. senticosus extract (ESE), known as antioxidant, has diverse pharmacologic effects. It is also used as an antiaging agent for the skin and phlorizin (PZ) is identified as a main ingredient. In this study, the effects of PZ on epidermal stem cells were investigated. Cultured normal human keratinocytes and skin equivalents are used to test whether PZ affects proliferative potential of keratinocytes and how it regulates these effects. Skin equivalents (SEs) were treated with ESE and the results showed that the epidermis became slightly thickened on addition of 0.002% ESE. The staining intensity of p63 as well as proliferating cell nuclear antigen (PCNA) is increased, and integrin α6 was upregulated. Analysis of ESE confirmed that PZ is the main ingredient. When SEs were treated with PZ, similar findings were observed. In particular, the expression of integrin α6, integrin β1, and type IV collagen was increased. Levels of mRNA for type IV collagen were increased and levels of miR135b were downregulated. All these findings suggested that PZ can affect the proliferative potential of epidermal cells in part by microenvironment changes via miR135b downregulation and following increased expression of type IV collagen. PMID:27042261

  7. Phlorizin, an Active Ingredient of Eleutherococcus senticosus, Increases Proliferative Potential of Keratinocytes with Inhibition of MiR135b and Increased Expression of Type IV Collagen.

    PubMed

    Choi, Hye-Ryung; Nam, Kyung-Mi; Lee, Hyun-Sun; Yang, Seung-Hye; Kim, Young-Soo; Lee, Jongsung; Date, Akira; Toyama, Kazumi; Park, Kyoung-Chan

    2016-01-01

    E. senticosus extract (ESE), known as antioxidant, has diverse pharmacologic effects. It is also used as an antiaging agent for the skin and phlorizin (PZ) is identified as a main ingredient. In this study, the effects of PZ on epidermal stem cells were investigated. Cultured normal human keratinocytes and skin equivalents are used to test whether PZ affects proliferative potential of keratinocytes and how it regulates these effects. Skin equivalents (SEs) were treated with ESE and the results showed that the epidermis became slightly thickened on addition of 0.002% ESE. The staining intensity of p63 as well as proliferating cell nuclear antigen (PCNA) is increased, and integrin α6 was upregulated. Analysis of ESE confirmed that PZ is the main ingredient. When SEs were treated with PZ, similar findings were observed. In particular, the expression of integrin α6, integrin β1, and type IV collagen was increased. Levels of mRNA for type IV collagen were increased and levels of miR135b were downregulated. All these findings suggested that PZ can affect the proliferative potential of epidermal cells in part by microenvironment changes via miR135b downregulation and following increased expression of type IV collagen. PMID:27042261

  8. Vitamin D enhances mitogenesis mediated by keratinocyte growth factor receptor in keratinocytes.

    PubMed

    Gamady, Anat; Koren, Ruth; Ron, Dina; Liberman, Uri A; Ravid, Amiram

    2003-06-01

    The hormonally active vitamin D metabolite, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and keratinocyte growth factor (KGF) belong to the network of autocrine and paracrine mediators in the skin. Both were shown to modulate keratinocyte proliferation, to reverse epidermal atrophy, to increase wound healing, and to reduce chemotherapy-induced alopecia. The overlap between their activities may suggest that vitamin D exerts some of its actions by modulation of KGF activities in the skin. This notion was examined by using HaCaT keratinocytes cultured in serum-free medium in the absence of exogenous growth factors and in the presence of the EGF receptor tyrosine kinase inhibitor AG 1478 that blocks their autonomous proliferation. These cells could be stimulated to proliferate by different fibroblast growth factors (FGFs). The relative mitogenic efficacy of basic FGF, acidic FGF, or KGF was in correlation with their affinities for the KGF receptor (KGFR). Forty-eight hour co-treatment with 1,25(OH)(2)D(3) enhanced KGFR-mediated cell proliferation in a dose dependent manner. Both ERK1/2 and c-Jun N-terminal kinase (JNK) were activated by the FGFs. Treatment with 1,25(OH)(2)D(3) increased the activation of ERK but reduced the activation of JNK. Treatment with 1,25(OH)(2)D(3) increased the levels of KGFR in the presence but not in the absence of KGF, probably due to inhibition of ligand-induced receptor degradation. Inhibition of protein kinase C with bisindolylmaleimide did not interfere with the effect of 1,25(OH)(2)D(3) on KGFR-mediated ERK activation. Our results support the notion that the paracrine KGF-KGFR system in the skin can act in concert with the autocrine vitamin D system in keratinocytes to promote keratinocyte proliferation and survival under situations of stress and injury. PMID:12761878

  9. Decorin gene expression and its regulation in human keratinocytes

    SciTech Connect

    Velez-DelValle, Cristina; Marsch-Moreno, Meytha; Castro-Munozledo, Federico; Kuri-Harcuch, Walid

    2011-07-22

    Highlights: {yields} We showed that cultured human diploid epidermal keratinocytes express and synthesize decorin. {yields} Decorin is found intracytoplasmic in suprabasal cells of cultures and in human epidermis. {yields} Decorin mRNA expression in cHEK is regulated by pro-inflammatory and proliferative cytokines. {yields} Decorin immunostaining of psoriatic lesions showed a lower intensity and altered intracytoplasmic arrangements. -- Abstract: In various cell types, including cancer cells, decorin is involved in regulation of cell attachment, migration and proliferation. In skin, decorin is seen in dermis, but not in keratinocytes. We show that decorin gene (DCN) is expressed in the cultured keratinocytes, and the protein is found in the cytoplasm of differentiating keratinocytes and in suprabasal layers of human epidermis. RT-PCR experiments showed that DCN expression is regulated by pro-inflammatory and proliferative cytokines. Our data suggest that decorin should play a significant role in keratinocyte terminal differentiation, cutaneous homeostasis and dermatological diseases.

  10. Mathematical Modeling of Calcium Waves Induced by Mechanical Stimulation in Keratinocytes

    PubMed Central

    Kobayashi, Yasuaki; Sanno, Yumi; Sakai, Akihiko; Sawabu, Yusuke; Tsutsumi, Moe; Goto, Makiko; Kitahata, Hiroyuki; Nakata, Satoshi; Kumamoto, Junichi; Denda, Mitsuhiro; Nagayama, Masaharu

    2014-01-01

    Recent studies have shown that the behavior of calcium in the epidermis is closely related to the conditions of the skin, especially the differentiation of the epidermal keratinocytes and the permeability barrier function, and therefore a correct understanding of the calcium dynamics is important in explaining epidermal homeostasis. Here we report on experimental observations of in vitro calcium waves in keratinocytes induced by mechanical stimulation, and present a mathematical model that can describe the experimentally observed wave behavior that includes finite-range wave propagation and a ring-shaped pattern. A mechanism of the ring formation hypothesized by our model may be related to similar calcium propagation patterns observed during the wound healing process in the epidermis. We discuss a possible extension of our model that may serve as a tool for investigating the mechanisms of various skin diseases. PMID:24663805

  11. Spatial Distribution of Stem Cell-Like Keratinocytes in Dissected Compound Hair Follicles of the Dog

    PubMed Central

    Wiener, Dominique J.; Doherr, Marcus G.; Müller, Eliane J.; Welle, Monika M.

    2016-01-01

    Hair cycle disturbances are common in dogs and comparable to some alopecic disorders in humans. A normal hair cycle is maintained by follicular stem cells which are predominately found in an area known as the bulge. Due to similar morphological characteristics of the bulge area in humans and dogs, the shared particularity of compound hair follicles as well as similarities in follicular biomarker expression, the dog is a promising model to study human hair cycle and stem cell disorders. To gain insight into the spatial distribution of follicular keratinocytes with stem cell potential in canine compound follicles, we microdissected hair follicles in anagen and telogen from skin samples of freshly euthanized dogs. The keratinocytes isolated from different locations were investigated for their colony forming efficiency, growth and differentiation potential as well as clonal growth. Our results indicate that i) compound and single hair follicles exhibit a comparable spatial distribution pattern with respect to cells with high growth potential and stem cell-like characteristics, ii) the lower isthmus (comprising the bulge) harbors most cells with high growth potential in both, the anagen and the telogen hair cycle stage, iii) unlike in other species, colonies with highest growth potential are rather small with an irregular perimeter and iv) the keratinocytes derived from the bulbar region exhibit characteristics of actively dividing transit amplifying cells. Our results now provide the basis to conduct comparative studies of normal dogs and those with hair cycle disorders with the possibility to extend relevant findings to human patients. PMID:26788850

  12. Spatial Distribution of Stem Cell-Like Keratinocytes in Dissected Compound Hair Follicles of the Dog.

    PubMed

    Wiener, Dominique J; Doherr, Marcus G; Müller, Eliane J; Welle, Monika M

    2016-01-01

    Hair cycle disturbances are common in dogs and comparable to some alopecic disorders in humans. A normal hair cycle is maintained by follicular stem cells which are predominately found in an area known as the bulge. Due to similar morphological characteristics of the bulge area in humans and dogs, the shared particularity of compound hair follicles as well as similarities in follicular biomarker expression, the dog is a promising model to study human hair cycle and stem cell disorders. To gain insight into the spatial distribution of follicular keratinocytes with stem cell potential in canine compound follicles, we microdissected hair follicles in anagen and telogen from skin samples of freshly euthanized dogs. The keratinocytes isolated from different locations were investigated for their colony forming efficiency, growth and differentiation potential as well as clonal growth. Our results indicate that i) compound and single hair follicles exhibit a comparable spatial distribution pattern with respect to cells with high growth potential and stem cell-like characteristics, ii) the lower isthmus (comprising the bulge) harbors most cells with high growth potential in both, the anagen and the telogen hair cycle stage, iii) unlike in other species, colonies with highest growth potential are rather small with an irregular perimeter and iv) the keratinocytes derived from the bulbar region exhibit characteristics of actively dividing transit amplifying cells. Our results now provide the basis to conduct comparative studies of normal dogs and those with hair cycle disorders with the possibility to extend relevant findings to human patients. PMID:26788850

  13. Ultraviolet B, melanin and mitochondrial DNA: Photo-damage in human epidermal keratinocytes and melanocytes modulated by alpha-melanocyte-stimulating hormone

    PubMed Central

    Böhm, Markus; Hill, Helene Z.

    2016-01-01

    Alpha-melanocyte-stimulating hormone (alpha-MSH) increases melanogenesis and protects from UV-induced DNA damage. However, its effect on mitochondrial DNA (mtDNA) damage is unknown. We have addressed this issue in a pilot study using human epidermal keratinocytes and melanocytes incubated with alpha-MSH and irradiated with UVB. Real-time touchdown PCR was used to quantify total and deleted mtDNA. The deletion detected encompassed the common deletion but was more sensitive to detection. There were 4.4 times more mtDNA copies in keratinocytes than in melanocytes. Irradiation alone did not affect copy numbers. Alpha-MSH slightly increased copy numbers in both cell types in the absence of UVB and caused a similar small decrease in copy number with dose in both cell types. Deleted copies were nearly twice as frequent in keratinocytes as in melanocytes. Alpha-MSH reduced the frequency of deleted copies by half in keratinocytes but not in melanocytes. UVB dose dependently led to an increase in the deleted copy number in alpha-MSH-treated melanocytes. UVB irradiation had little effect on deleted copy number in alpha-MSH-treated keratinocytes. In summary, alpha-MSH enhances mtDNA damage in melanocytes presumably by increased melanogenesis, while α-MSH is protective in keratinocytes, the more so in the absence of irradiation. PMID:27303631

  14. Ultraviolet B, melanin and mitochondrial DNA: Photo-damage in human epidermal keratinocytes and melanocytes modulated by alpha-melanocyte-stimulating hormone.

    PubMed

    Böhm, Markus; Hill, Helene Z

    2016-01-01

    Alpha-melanocyte-stimulating hormone (alpha-MSH) increases melanogenesis and protects from UV-induced DNA damage. However, its effect on mitochondrial DNA (mtDNA) damage is unknown. We have addressed this issue in a pilot study using human epidermal keratinocytes and melanocytes incubated with alpha-MSH and irradiated with UVB. Real-time touchdown PCR was used to quantify total and deleted mtDNA. The deletion detected encompassed the common deletion but was more sensitive to detection. There were 4.4 times more mtDNA copies in keratinocytes than in melanocytes. Irradiation alone did not affect copy numbers. Alpha-MSH slightly increased copy numbers in both cell types in the absence of UVB and caused a similar small decrease in copy number with dose in both cell types. Deleted copies were nearly twice as frequent in keratinocytes as in melanocytes. Alpha-MSH reduced the frequency of deleted copies by half in keratinocytes but not in melanocytes. UVB dose dependently led to an increase in the deleted copy number in alpha-MSH-treated melanocytes. UVB irradiation had little effect on deleted copy number in alpha-MSH-treated keratinocytes. In summary, alpha-MSH enhances mtDNA damage in melanocytes presumably by increased melanogenesis, while α-MSH is protective in keratinocytes, the more so in the absence of irradiation. PMID:27303631

  15. Aldo-keto reductase 1C subfamily genes in skin are UV-inducible: possible role in keratinocytes survival.

    PubMed

    Marín, Yarí E; Seiberg, Miri; Lin, Connie B

    2009-07-01

    Please cite this paper as: Aldo-keto reductase 1C subfamily genes in skin are UV-inducible: possible role in keratinocytes survival. Experimental Dermatology 2009; 18: 611-618.Abstract: Human skin is endowed with the capacity to synthesize and metabolize steroid hormones, a function of importance in skin physiology and pathology. It is the hormone-regulatory enzymes, including the aldo-keto reductase 1C subfamily (AKR1Cs) that are largely responsible for the local levels of active steroid hormones. AKR1C1 and AKR1C2 inactivate progesterone and 5alpha-dihydrotestosterone, respectively, whereas AKR1C3 activates oestradiol and testosterone. Here, we show that AKR1C1-3 are expressed in keratinocytes and fibroblasts, with marginal expression in melanocytes. In human primary keratinocytes, AKR1C1 and -2 were UVB-inducible in a dose-dependent manner, as shown by quantitative PCR and Western blot analyses. The induction of AKR1C1 by UVB was concomitant with the presence of an apoptotic marker, the cleavage product of poly-ADP ribose polymerase. Similarly, the activation of AKR1C1 and -2 upon UVB exposure was demonstrated in swine skin in vivo and in human skin explants. As expected, hydrogen peroxide-derived reactive oxygen species also induced AKR1C1 and -2 mRNA and protein levels in keratinocytes in a dose-dependent manner. Furthermore, down-regulation of AKR1Cs by small interfering ribonucleic acid led to significantly reduced cell viability. Based on the combined evidence of the presence of an apoptotic marker in the UVB-exposed keratinocytes with increased AKR1Cs expression and reduced cell viability in down-regulated AKR1Cs, we suggest that AKR1C subfamily genes are stress-inducible and might function as survival factors in keratinocytes. PMID:19320734

  16. Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes.

    PubMed Central

    Santulli, R J; Derian, C K; Darrow, A L; Tomko, K A; Eckardt, A J; Seiberg, M; Scarborough, R M; Andrade-Gordon, P

    1995-01-01

    Thrombin receptor activation was explored in human epidermal keratinocytes and human dermal fibroblasts, cells that are actively involved in skin tissue repair. The effects of thrombin, trypsin, and the receptor agonist peptides SFLLRN and TFRIFD were assessed in inositolphospholipid hydrolysis and calcium mobilization studies. Thrombin and SFLLRN stimulated fibroblasts in both assays to a similar extent, whereas TFRIFD was less potent. Trypsin demonstrated weak efficacy in these assays in comparison with thrombin. Results in fibroblasts were consistent with human platelet thrombin receptor activation. Keratinocytes, however, exhibited a distinct profile, with trypsin being a far better activator of inositolphospholipid hydrolysis and calcium mobilization than thrombin. Furthermore, SFLLRN was more efficacious than thrombin, whereas no response was observed with TFRIFD. Since our data indicated that keratinocytes possess a trypsin-sensitive receptor, we addressed the possibility that these cells express the human homologue of the newly described murine protease-activated receptor, PAR-2 [Nystedt, S., Emilsson, K., Wahlestedt, C. & Sundelin, J. (1994) Proc. Natl. Acad. Sci. USA 91, 9208-9212]. PAR-2 is activated by nanomolar concentrations of trypsin and possesses the tethered ligand sequence SLIGRL. SLIGRL was found to be equipotent with SFLLRN in activating keratinocyte inositolphospholipid hydrolysis and calcium mobilization. Desensitization studies indicated that SFLLRN, SLIGRL, and trypsin activate a common receptor, PAR-2. Northern blot analyses detected a transcript of PAR-2 in total RNA from keratinocytes but not fibroblasts. Levels of thrombin receptor message were equivalent in the two cell types. Our results indicate that human keratinocytes possess PAR-2, suggesting a potential role for this receptor in tissue repair and/or skin-related disorders. Images Fig. 6 PMID:7568091

  17. Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor-beta

    SciTech Connect

    Matsumoto, K.; Hashimoto, K.; Hashiro, M.; Yoshimasa, H.; Yoshikawa, K. )

    1990-10-01

    The effect of transforming growth factor-type beta 1(TGF-beta) on the growth and differentiation of normal human skin keratinocytes cultured in serum-free medium was investigated. TGF-beta markedly inhibited the growth of keratinocytes at the concentrations greater than 2 ng/ml under low Ca2+ conditions (0.1 mM). Growth inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of (3H)thymidine incorporation. The decrease of (3H)thymidine incorporation was observed as early as 3 hr after addition of TGF-beta. TGF-beta also decreased c-myc messenger RNA (mRNA) expression 30 min after addition of TGF-beta. This rapid reduction of c-myc mRNA expression by TGF-beta treatment is possibly one of the main factors in the process of TGF-beta-induced growth inhibition of human keratinocytes. Since growth inhibition and induction of differentiation are closely related in human keratinocytes, the growth-inhibitory effect of TGF-beta under high Ca2+ conditions was examined. TGF-beta inhibited the growth of keratinocytes under high Ca2+ conditions in the same manner as under low Ca2+ conditions, suggesting that it is a strong growth inhibitor in both low and high Ca2+ environments. The induction of keratinocyte differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF-beta at 20 ng/ml increased involucrin expression from 9.3% to 18.8% under high Ca2+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca2+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF-beta: TGF-beta at 20 ng/ml decreased cornified envelope formation by 53% under low Ca2+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca2+ conditions.

  18. Removal of Reprogramming Transgenes Improves the Tissue Reconstitution Potential of Keratinocytes Generated From Human Induced Pluripotent Stem Cells

    PubMed Central

    Kokubu, Chikara; Yusa, Kosuke; Horie, Kyoji; Yoshimura, Yasuhide; Yamauchi, Kaori; Suemori, Hirofumi; Yokozeki, Hiroo; Toyoda, Masashi; Kiyokawa, Nobutaka; Okita, Hajime; Miyagawa, Yoshitaka; Akutsu, Hidenori; Umezawa, Akihiro; Katayama, Ichiro

    2014-01-01

    Human induced pluripotent stem cell (hiPSC) lines have a great potential for therapeutics because customized cells and organs can be induced from such cells. Assessment of the residual reprogramming factors after the generation of hiPSC lines is required, but an ideal system has been lacking. Here, we generated hiPSC lines from normal human dermal fibroblasts with piggyBac transposon bearing reprogramming transgenes followed by removal of the transposon by the transposase. Under this condition, we compared the phenotypes of transgene-residual and -free hiPSCs of the same genetic background. The transgene-residual hiPSCs, in which the transcription levels of the reprogramming transgenes were eventually suppressed, were quite similar to the transgene-free hiPSCs in a pluripotent state. However, after differentiation into keratinocytes, clear differences were observed. Morphological, functional, and molecular analyses including single-cell gene expression profiling revealed that keratinocytes from transgene-free hiPSC lines were more similar to normal human keratinocytes than those from transgene-residual hiPSC lines, which may be partly explained by reactivation of residual transgenes upon induction of keratinocyte differentiation. These results suggest that transgene-free hiPSC lines should be chosen for therapeutic purposes. PMID:25024429

  19. Transient receptor potential vanilloid 4 (TRPV4)-dependent calcium influx and ATP release in mouse oesophageal keratinocytes

    PubMed Central

    Mihara, Hiroshi; Boudaka, Ammar; Sugiyama, Toshiro; Moriyama, Yoshinori; Tominaga, Makoto

    2011-01-01

    Abstract Gastro-oesophageal reflux disease (GERD) is a multi-factorial disease that may involve oesophageal hypersensitivity to mechanical or heat stimulus as well as acids. Intraganglionic laminar endings (IGLEs) are the most prominent terminal structures of oesophageal vagal mechanosensitive afferents and may modulate mechanotransduction via purinergic receptors. Transient receptor potential channel vanilloid 4 (TRPV4) can detect various stimuli such as warm temperature, stretch and some chemicals, including 4α-phorbol 12,13-didecanoate (4α-PDD) and GSK1016790A. TRPV4 is expressed in many tissues, including renal epithelium, skin keratinocytes and urinary bladder epithelium, but its expression and function in the oesophagus is poorly understood. Here, we show anatomical and functional TRPV4 expression in mouse oesophagus and its involvement in ATP release. TRPV4 mRNA and protein were detected in oesophageal keratinocytes. Several known TRPV4 activators (chemicals, heat and stretch stimulus) increased cytosolic Ca2+ concentrations in cultured WT keratinocytes but not in TRPV4 knockout (KO) cells. Moreover, the TRPV4 agonist GSK1016790A and heat stimulus evoked TRPV4-like current responses in isolated WT keratinocytes, but not in TRPV4KO cells. GSK1016790A and heat stimulus also significantly increased ATP release from WT oesophageal keratinocytes compared to TRPV4KO cells. The vesicle-trafficking inhibitor brefeldin A (BFA) inhibited the ATP release. This ATP release could be mediated by the newly identified vesicle ATP transporter, VNUT, which is expressed by oesophageal keratinocytes at the mRNA and protein levels. In conclusion, in response to heat, chemical and possibly mechanical stimuli, TRPV4 contributes to ATP release in the oesophagus. Thus, TRPV4 could be involved in oesophageal mechano- and heat hypersensitivity. PMID:21540339

  20. A modified method for the culture of naturally HPV-infected high-grade cervical intraepithelial neoplasia keratinocytes from human neoplastic cervical biopsies

    PubMed Central

    LIU, YU-ZHEN; WANG, TIAN-TIAN; ZHANG, YOU-ZHONG

    2016-01-01

    Few studies on cervical intraepithelial neoplasia (CIN) keratinocyte cultures are available due to the numerous technical and methodological problems associated with the in vitro cultivation of these cells. The present study investigated an applicable and effective method for the in vitro cultivation of high-grade CIN keratinocytes from human neoplastic cervical biopsies. Human neoplastic cervical tissue sections were obtained and digested using type I collagen in order to dissociate the cells. The cells were seeded in tissue culture plastic plates that were coated with rat tail collagen type I and contained modified keratinocyte serum-free medium (K-SFM) supplemented with 5% fetal bovine serum. The medium was replaced with K-SFM on days 3, 5 and 7, respectively. The unattached cells were recovered and the cell viability was determined accurately using the Trypan Blue exclusion method. The expression of keratin 14 (K14), keratin 19 (K19), keratin 17 (K17) and P63 was assayed using immunofluorescence in order to identify the presence of CIN keratinocytes. The present results indicated that the attachment rate of CIN keratinocytes significantly increased between 56.75±1.76% on day 3 and 77.09±3.55% on day 5, and became relatively stable between days 5 and 7. The cell viability significantly decreased between 83.00±0.50% on day 5 and 68.17±1.04% on day 7. The passaged CIN keratinocytes maintained the original unequally sized, abnormally shaped morphology and did not undergo differentiation. In addition, the passaged CIN keratinocytes exhibited the same human papilloma virus (HPV) genotype that was detected in the original primary cells. K14 and K19 were expressed in the majority of the normal and CIN keratinocytes, whereas K17 and P63 were expressed only in high-grade CIN keratinocytes. The present study proposes a simple and practical method for rapidly obtaining highly purified naturally HPV-infected high-grade CIN keratinocytes from small neoplastic cervical

  1. Acute and chronic wound fluids influence keratinocyte function differently.

    PubMed

    Thamm, Oliver C; Koenen, Paola; Bader, Nicola; Schneider, Alina; Wutzler, Sebastian; Neugebauer, Edmund A M; Spanholtz, Timo A

    2015-04-01

    Wound healing requires a proper functioning of keratinocytes that migrate, proliferate and lead to a competent wound closure. Impaired wound healing might be due to a disturbed keratinocyte function caused by the wound environment. Basically, chronic wound fluid (CWF) differs from acute wound fluid (AWF). The aim of this study was to analyse the effects of AWF and CWF on keratinocyte function. We therefore investigated keratinocyte migration and proliferation under the influence of AWF and CWF using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test and scratch assay. We further measured the gene expression by qRT-PCR regarding growth factors and matrixmetalloproteinases (MMPs) involved in regeneration processes. AWF had a positive impact on keratinocyte proliferation over time, whereas CWF had an anti-proliferative effect. Keratinocyte migration was significantly impaired by CWF in contrast to an undisturbed wound closure under the influence of AWF. MMP-9 expression was strongly upregulated by CWF compared with AWF. Keratinocyte function was significantly impaired by CWF. An excessive induction of MMP-9 by CWF might lead to a permanent degradation of extracellular matrix and thereby prevent wounds from healing. PMID:23517467

  2. Stathmin Regulates Keratinocyte Proliferation and Migration during Cutaneous Regeneration

    PubMed Central

    Schmitt, Sabrina; Safferling, Kai; Westphal, Kathi; Hrabowski, Manuel; Müller, Ute; Angel, Peter; Wiechert, Lars; Ehemann, Volker; Müller, Benedikt; Holland-Cunz, Stefan; Stichel, Damian; Harder, Nathalie; Rohr, Karl; Germann, Günter; Matthäus, Franziska; Schirmacher, Peter; Grabe, Niels; Breuhahn, Kai

    2013-01-01

    Cutaneous regeneration utilizes paracrine feedback mechanisms to fine-tune the regulation of epidermal keratinocyte proliferation and migration. However, it is unknown how fibroblast-derived hepatocyte growth factor (HGF) affects these mutually exclusive processes in distinct cell populations. We here show that HGF stimulates the expression and phosphorylation of the microtubule-destabilizing factor stathmin in primary human keratinocytes. Quantitative single cell- and cell population-based analyses revealed that basal stathmin levels are important for the migratory ability of keratinocytes in vitro; however, its expression is moderately induced in the migration tongue of mouse skin or organotypic multi-layered keratinocyte 3D cultures after full-thickness wounding. In contrast, clearly elevated stathmin expression is detectable in hyperproliferative epidermal areas. In vitro, stathmin silencing significantly reduced keratinocyte proliferation. Automated quantitative and time-resolved analyses in organotypic cocultures demonstrated a high correlation between Stathmin/phospho-Stathmin and Ki67 positivity in epidermal regions with proliferative activity. Thus, activation of stathmin may stimulate keratinocyte proliferation, while basal stathmin levels are sufficient for keratinocyte migration during cutaneous regeneration. PMID:24066165

  3. Serum-free primary human fibroblast and keratinocyte coculture.

    PubMed

    Mujaj, Sally; Manton, Kerry; Upton, Zee; Richards, Sean

    2010-04-01

    Research has shown that the inclusion of a fibroblast cell support layer is required for the isolation and expansion of primary keratinocytes. Recent advances have provided keratinocyte culture with fibroblast-free alternatives. However, these technologies are often undefined and rely on the incorporation of purified proteins/components. To address this problem we developed a medium that used recombinant proteins to support the serum-free isolation and expansion of human dermal fibroblasts and keratinocytes. The human dermal fibroblasts were able to be isolated serum free by adding recombinant human albumin to a collagenase solution. These fibroblasts were then expanded using a serum-free medium containing recombinant proteins: epidermal growth factor, basic fibroblast growth factor, chimeric vitronectin:insulin-like growth factor-I protein, and recombinant human albumin. These fibroblasts maintained a typical morphology and expressed fibroblast markers during their serum-free isolation, expansion, and freezing. Moreover, these fibroblasts were able to support the serum-free isolation and expansion of primary keratinocytes using these recombinant proteins. Real-time polymerase chain reaction and immunofluorescence analysis confirmed that there were no differences in expression levels of p63 or keratins 1, 6, and 10 when keratinocytes were grown in either serum-supplemented or serum-free medium. Using a three-dimensional human skin equivalent model we demonstrated that these keratinocytes also maintained their ability to reform an epidermal layer. In summary, the techniques described provide a valuable alternative for culturing fibroblasts and keratinocytes using recombinant proteins. PMID:19929322

  4. Distinct Effects of Different Phosphatidylglycerol Species on Mouse Keratinocyte Proliferation

    PubMed Central

    Xie, Ding; Seremwe, Mutsa; Edwards, John G.; Podolsky, Robert; Bollag, Wendy B.

    2014-01-01

    We have previously shown that liposomes composed of egg-derived phosphatidylglycerol (PG), with a mixed fatty acid composition (comprising mainly palmitate and oleate), inhibit the proliferation and promote the differentiation of rapidly dividing keratinocytes, and stimulate the growth of slowly proliferating epidermal cells. To determine the species of PG most effective at modulating keratinocyte proliferation, primary mouse keratinocytes were treated with different PG species, and proliferation was measured. PG species containing polyunsaturated fatty acids were effective at inhibiting rapidly proliferating keratinocytes, whereas PG species with monounsaturated fatty acids were effective at promoting proliferation in slowly dividing cells. Thus, palmitoyl-arachidonyl-PG (16∶0/20∶4), palmitoyl-linoleoyl-PG (16∶0/18∶2), dilinoleoyl-PG (18∶2/18∶2) and soy PG (a PG mixture with a large percentage of polyunsaturated fatty acids) were particularly effective at inhibiting proliferation in rapidly dividing keratinocytes. Conversely, palmitoyl-oleoyl-PG (16∶0/18∶1) and dioleoyl-PG (18∶1/18∶1) were especially effective proproliferative PG species. This result represents the first demonstration of opposite effects of different species of a single class of phospholipid and suggests that these different PG species may signal to diverse effector enzymes to differentially affect keratinocyte proliferation and normalize keratinocyte proliferation. Thus, different PG species may be useful for treating skin diseases characterized by excessive or insufficient proliferation. PMID:25233484

  5. Dyskeratosis Congenita Dermal Fibroblasts are Defective in Supporting the Clonogenic Growth of Epidermal Keratinocytes

    PubMed Central

    Buckingham, Erin M.; Goldman, Frederick D.; Klingelhutz, Aloysius J.

    2012-01-01

    Telomere shortening is associated with cellular senescence and aging. Dyskeratosis congenita (DC) is a premature aging syndrome caused by mutations in genes for telomerase components or telomere proteins. DC patients have very short telomeres and exhibit aging-associated pathologies including epidermal abnormalities and bone marrow failure. Here, we show that DC skin fibroblasts are defective in their ability to support the clonogenic growth of epidermal keratinocytes. Conditioned media transfer experiments demonstrated that this defect was largely due to lack of a factor or factors secreted from the DC fibroblasts. Compared to early passage normal fibroblasts, DC fibroblasts express significantly lower transcript levels of several genes that code for secreted proteins, including Insulin-like Growth Factor 1 (IGF1) and Hepatocyte Growth Factor (HGF). Aged normal fibroblasts with short telomeres also had reduced levels of IGF1 and HGF, similar to early passage DC fibroblasts. Knockdown of IGF1 or HGF in normal fibroblasts caused a reduction in the capacity of conditioned media from these fibroblasts to support keratinocyte clonogenic growth. Surprisingly, reconstitution of telomerase in DC fibroblasts did not significantly increase transcript levels of IGF1 or HGF or substantially increase the ability of the fibroblasts to support keratinocyte growth, indicating that the gene expression defect is not readily reversible. Our results suggest that telomere shortening in dermal fibroblasts leads to reduction in expression of genes such as IGF1 and HGF and that this may cause a defect in supporting normal epidermal proliferation. PMID:23251848

  6. Stereotyped distribution of proliferating keratinocytes in disorders affecting the epidermis

    SciTech Connect

    Pierard-Franchimont, C.; Pierard, G.E.

    1989-06-01

    We used the technique of autoradiography after incorporation of tritiated thymidine (/sup 3/H-TdR) to evaluate keratinocyte proliferation in basal, epibasal, and other epidermal layers in 30 diseases affecting the epidermis. The number and proportion of /sup 3/H-TdR-labeled keratinocytes were counted in the different layers of the epidermis. Significant correlations were found between the proliferative indices of the different epidermal layers. Such links indicate that the epidermis responds in a rather stereotyped way to various pathological conditions. There exists some regulation in the distribution, number, and proportion of /sup 3/H-TdR-labeled keratinocytes in the various layers of the epidermis.

  7. Differential effects of UV irradiation on nuclear retinoid receptor levels in cultured keratinocytes and melanocytes.

    PubMed

    Andersson, Eva; Rosdahl, Inger; Törmä, Hans; Vahlquist, Anders

    2003-10-01

    A major risk factor for skin cancer is UV irradiation, which not only damages DNA and other photosensitive compounds like vitamin A, but may also perturb cellular signaling, e.g. via the retinoid receptor system believed to be important for cancer protection. We used cultured normal human keratinocytes and melanocytes to examine the effects of UV irradiation on the expression of the predominant retinoid receptors in the human skin (RARalpha, RARgamma and RXRalpha) and the AP-1 protein c-Jun; mRNA levels were studied by real-time PCR and protein levels by Western blot. In keratinocytes, a single dose of UVB (50 mJ/cm2) caused a rapid drop in the expression of all three receptors (mRNA levels minus 35-50% after 4 h; protein levels minus 20-45% after 8 h), which was followed over the next 40 h by a variable response, leading to full normalization for RARalpha only. In contrast, the levels of c-Jun did not change significantly after UV exposure. In melanocytes, UVB caused a similar drop of the retinoid receptor levels as in keratinocytes but this was soon followed by an increased expression leading to a complete normalization of all receptor levels within 1-3 days. The c-Jun levels in melanocytes increased 1 day after UV exposure and remained high (plus 50%) thereafter. In both cell types, a approximately 3-fold increase in apoptosis (measured by DNA fragmentation) was observed 8-48 h after UVB irradiation. In conclusion, a depletion of vitamin A and retinoid receptors by UV irradiation, together with unchanged or even increased c-Jun levels, might seriously interfere with retinoid signaling and thus promote future tumor development, especially in keratinocytes. PMID:14705796

  8. Modeling keratinocyte wound healing dynamics: Cell-cell adhesion promotes sustained collective migration.

    PubMed

    Nardini, John T; Chapnick, Douglas A; Liu, Xuedong; Bortz, David M

    2016-07-01

    The in vitro migration of keratinocyte cell sheets displays behavioral and biochemical similarities to the in vivo wound healing response of keratinocytes in animal model systems. In both cases, ligand-dependent Epidermal Growth Factor Receptor (EGFR) activation is sufficient to elicit collective cell migration into the wound. Previous mathematical modeling studies of in vitro wound healing assays assume that physical connections between cells have a hindering effect on cell migration, but biological literature suggests a more complicated story. By combining mathematical modeling and experimental observations of collectively migrating sheets of keratinocytes, we investigate the role of cell-cell adhesion during in vitro keratinocyte wound healing assays. We develop and compare two nonlinear diffusion models of the wound healing process in which cell-cell adhesion either hinders or promotes migration. Both models can accurately fit the leading edge propagation of cell sheets during wound healing when using a time-dependent rate of cell-cell adhesion strength. The model that assumes a positive role of cell-cell adhesion on migration, however, is robust to changes in the leading edge definition and yields a qualitatively accurate density profile. Using RNAi for the critical adherens junction protein, α-catenin, we demonstrate that cell sheets with wild type cell-cell adhesion expression maintain migration into the wound longer than cell sheets with decreased cell-cell adhesion expression, which fails to exhibit collective migration. Our modeling and experimental data thus suggest that cell-cell adhesion promotes sustained migration as cells pull neighboring cells into the wound during wound healing. PMID:27105673

  9. The impact of extracellular syntaxin4 on HaCaT keratinocyte behavior

    SciTech Connect

    Kadono, Nanako; Miyazaki, Takafumi; Okugawa, Yoji; Nakajima, Kiichiro; Hirai, Yohei

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer A subpopulation of syntaxin4 localizes extracellularly in the keratinocytes. Black-Right-Pointing-Pointer Epimorphin and syntaxin4 confer the resistance to the oxidative stress. Black-Right-Pointing-Pointer Epimorphin suppresses and syntaxin4 accelerates the CCE formation. Black-Right-Pointing-Pointer The antagonistic peptide to syntaxin4 blocks the syntaxin4-dependent CCE formation. -- Abstract: Syntaxin4 belongs to t-SNARE protein family and functions as a vesicular fusion mediator in the plasma membrane in a wide variety of cell types. This protein resembles another family member, epimorphin, a subpopulation of which has been shown to be secreted extracellularly in order to exert signaling functions. Here, we demonstrate the secretion of syntaxin4 via a non-classical pathway and its extracellular functions by using the functionally normal keratinocyte HaCaT. Extracellularly presented syntaxin4 appeared to elicit many cell responses similar to epimorphin with an important exception: it clearly facilitated keratinocyte cornification. The circularized peptide ST4n1 was synthesized from the putative functional core of syntaxin4 (a.a. 103-108), which is equivalent to the previously generated antagonist of epimorphin, and neutralized this contradictory effect. Intriguingly, an epimorphin mutant (EP4M) in which the functional core was replaced by that of syntaxin4 behaved like epimorphin, which was again antagonized by ST4n1. Electrophoresis-based analyses demonstrated the distinct structure of syntaxin4 compared to epimorphin or EP4M. These results revealed, for the first time, the extracellular role of syntaxin4 and shed light on the division of the extracellular effects exerted by epimorphin and syntaxin4 on keratinocyte cornification.

  10. Similar names for similar biologics.

    PubMed

    Casadevall, Nicole; Felix, Thomas; Strober, Bruce E; Warnock, David G

    2014-10-01

    Approval of the first biosimilar in the USA may occur by the end of 2014, yet a naming approach for biosimilars has not been determined. Biosimilars are highly similar to their biologic reference product but are not identical to it, because of their structural complexity and variations in manufacturing processes among companies. There is a need for a naming approach that can distinguish a biosimilar from its reference product and other biosimilars and ensure accurate tracing of adverse events (AEs) to the administered product. In contrast, generic small-molecule drugs are identical to their reference product and, therefore, share the same nonproprietary name. Clinical trials required to demonstrate biosimilarity for approval may not detect rare AEs or those occurring after prolonged use, and the incidence of such events may differ between a biosimilar and its reference product. The need for precise biologic identification is further underscored by the possibility of biosimilar interchangeability, a US designation that will allow substitution without prescriber intervention. For several biologics, the US Food and Drug Administration (FDA) has used a naming approach that adds a prefix to a common root nonproprietary name, enabling healthcare providers to distinguish between products, avoid medication errors, and facilitate pharmacovigilance. We recommend that the FDA implement a biosimilars naming policy that likewise would add a distinguishable prefix or suffix to the root nonproprietary name of the reference product. This approach would ensure that a biosimilar could be distinguished from its reference product and other biosimilars in patient records and pharmacovigilance databases/reports, facilitating accurate attribution of AEs. PMID:25001080

  11. Scanning Ion Conductance Microscopy of Live Keratinocytes

    NASA Astrophysics Data System (ADS)

    Hegde, V.; Mason, A.; Saliev, T.; Smith, F. J. D.; McLean, W. H. I.; Campbell, P. A.

    2012-07-01

    Scanning ion conductance microscopy (SICM) is perhaps the least well known technique from the scanning probe microscopy (SPM) family of instruments. As with its more familiar counterpart, atomic force microscopy (AFM), the technique provides high-resolution topographic imaging, with the caveat that target structures must be immersed in a conducting solution so that a controllable ion current may be utilised as the basis for feedback. In operation, this non-contact characteristic of SICM makes it ideal for the study of delicate structures, such as live cells. Moreover, the intrinsic architecture of the instrument, incorporating as it does, a scanned micropipette, lends itself to combination approaches with complementary techniques such as patch-clamp electrophysiology: SICM therefore boasts the capability for both structural and functional imaging. For the present observations, an ICnano S system (Ionscope Ltd., Melbourn, UK) operating in 'hopping mode' was used, with the objective of assessing the instrument's utility for imaging live keratinocytes under physiological buffers. In scans employing cultured HaCaT cells (spontaneously immortalised, human keratinocytes), we compared the qualitative differences of live cells imaged with SICM and AFM, and also with their respective counterparts after chemical fixation in 4% paraformaldehyde. Characteristic surface microvilli were particularly prominent in live cell imaging by SICM. Moreover, time lapse SICM imaging on live cells revealed that changes in the pattern of microvilli could be tracked over time. By comparison, AFM imaging on live cells, even at very low contact forces (

  12. A synthetic isoflavone, DCMF, promotes human keratinocyte migration by activating Src/FAK signaling pathway.

    PubMed

    Sophors, Phorl; Kim, Young Mee; Seo, Ga Young; Huh, Jung-Sik; Lim, Yoongho; Koh, Dong Soo; Cho, Moonjae

    2016-04-01

    Flavonoids are plant secondary compounds with various pharmacological properties. We previously showed that one flavonoid, trimethoxyisoflavone (TMF), could promote wound healing by inducing keratinocyte migration. Here, we screened TMF derivatives for enhanced activity and identified one compound, 2',6 Dichloro-7-methoxyisoflavone (DCMF), as most effective at promoting migration in a scratch wound assay. Using the HaCaT keratinocyte cell line, we found DCMF treatment induced phosphorylation of both FAK and Src, and increased keratinocyte migration. DCMF-induced Src kinase could promote activation of ERK, AKT, and p38 signaling pathways, and DCMF-induced secretion of matrix metalloproteinase (MMP)-2 and MMP-9 and partial epithelial-mesenchymal transition (EMT), whereas Src inhibition abolished DCMF-induced EMT. Using an in vivo excisional wound model, we observed improved wound closure and re-epithelialization in DCMF-treated mice, as compared to controls. Collectively, our data demonstrate that DCMF induces cell migration and promotes wound healing through activation of Src/FAK, ERK, AKT, and p38 MAPK signaling. PMID:26923073

  13. Loss of tumorigenic potential upon transdifferentiation from keratinocytic into melanocytic lineage.

    PubMed

    Fehrenbach, Sabrina; Novak, Daniel; Bernhardt, Mathias; Larribere, Lionel; Boukamp, Petra; Umansky, Viktor; Utikal, Jochen

    2016-01-01

    Lineage-specific transcription factors determine the cell fate during development. Direct conversion of several cell types into other lineages has been achieved by the overexpression of specific transcription factors. Even cancer cells have been demonstrated to be amenable to transdifferentiation. Here, we identified a distinct set of transcription factors, which are sufficient to transform cells of the keratinocytic lineage to melanocyte-like cells. Melanocyte marker expression was induced and melanosome formation was observed in non-tumorigenic keratinocytes (HaCaT) and tumorigenic squamous cell carcinoma (MET-4) cells. Moreover, reduced proliferation, cell metabolism, invasion and migration were measured in vitro in transdifferentiated MT-MET-4 cells. A loss of tumorigenic potential of squamous cell carcinoma cells could be due to the upregulation of the melanocyte differentiation associated gene IL-24. Our data show that cells from the keratinocytic lineage can be transdifferented into the melanocytic lineage and provide a proof of principle for a potential new therapeutic strategy. PMID:27387763

  14. Extracellular group A Streptococcus induces keratinocyte apoptosis by dysregulating calcium signalling.

    PubMed

    Cywes Bentley, Colette; Hakansson, Anders; Christianson, Jennifer; Wessels, Michael R

    2005-07-01

    Group A Streptococcus (GAS) colonizes the oropharynx and damaged skin. To cause local infection or severe invasive syndromes the bacteria must gain access into deeper tissues. Host cell death may facilitate this process. GAS internalization has been identified to induce apoptosis. We now report an alternate mechanism of GAS-mediated apoptosis of primary human keratinocytes, initiated by extracellular GAS and involving dysregulation of intracellular calcium to produce endoplasmic reticulum stress. Two bacterial virulence factors are required for effective induction of apoptosis by extracellular GAS: (i) hyaluronic acid capsule that inhibits bacterial internalization and (ii) secreted cytolysin, streptolysin O (SLO), that forms transmembrane pores that permit extracellular calcium influx into the cytosol. Induction of keratinocyte apoptosis by wild-type GAS was accompanied by cell detachment and loss of epithelial integrity, a phenomenon not observed with GAS deficient in capsule or SLO. We propose that cell signalling initiated by extracellular GAS compromises the epithelial barrier by inducing premature keratinocyte differentiation and apoptosis, thereby facilitating GAS invasion of deeper tissues. PMID:15953027

  15. Candidate EDA targets revealed by expression profiling of primary keratinocytes from Tabby mutant mice

    PubMed Central

    Esibizione, Diana; Cui, Chang-Yi; Schlessinger, David

    2009-01-01

    EDA, the gene mutated in anhidrotic ectodermal dysplasia, encodes ectodysplasin, a TNF superfamily member that activates NF-kB mediated transcription. To identify EDA target genes, we have earlier used expression profiling to infer genes differentially expressed at various developmental time points in Tabby (Eda-deficient) compared to wild-type mouse skin. To increase the resolution to find genes whose expression may be restricted to epidermal cells, we have now extended studies to primary keratinocyte cultures established from E19 wild-type and Tabby skin. Using microarrays bearing 44,000 gene probes, we found 385 preliminary candidate genes whose expression was significantly affected by Eda loss. By comparing expression profiles to those from Eda-A1 transgenic skin, we restricted the list to 38 “candidate EDA targets”, 14 of which were already known to be expressed in hair follicles or epidermis. We confirmed expression changes for 3 selected genes, Tbx1, Bmp7, and Jag1, both in keratinocytes and in whole skin, by Q-PCR and Western blotting analyses. Thus, by the analysis of keratinocytes, novel candidate pathways downstream of EDA were detected. PMID:18848976

  16. Focal Contact and Hemidesmosomal Proteins in Keratinocyte Migration and Wound Repair

    PubMed Central

    Hopkinson, Susan B.; Hamill, Kevin J.; Wu, Yvonne; Eisenberg, Jessica L.; Hiroyasu, Sho; Jones, Jonathan C.R.

    2014-01-01

    Significance: During wound healing of the skin, keratinocytes should move over while still adhering to their underlying matrix. Thus, mechanistic insights into the wound-healing process require an understanding of the forms and functions of keratinocyte matrix adhesions, specifically focal contacts and hemidesmosomes, and their components. Recent Advances: Although the structure and composition of focal contacts and hemidesmosomes are relatively well defined, the functions of their components are only now being delineated using mouse genetic models and knockdown approaches in cell culture systems. Remarkably, both focal contact and hemidesmosomal proteins appear involved in determining the speed and directional migration of epidermal cells by modulating several signal transduction pathways. Critical Issues: Although many publications are centered on focal contacts, their existence in tissues such as the skin is controversial. Nonetheless, focal contact proteins are central to mechanisms that regulate skin cell motility. Conversely, hemidesmosomes have been identified in intact skin but whether hemidesmosomal components play a positive regulatory function in keratinocyte motility remains debated in the field. Future Directions: Defective wound healing is a developing problem in the aged, hospitalized and diabetic populations. Hence, deriving new insights into the molecular roles of matrix adhesion proteins in wound healing is a prerequisite to the development of novel therapeutics to enhance tissue repair and regeneration. PMID:24669360

  17. Loss of tumorigenic potential upon transdifferentiation from keratinocytic into melanocytic lineage

    PubMed Central

    Fehrenbach, Sabrina; Novak, Daniel; Bernhardt, Mathias; Larribere, Lionel; Boukamp, Petra; Umansky, Viktor; Utikal, Jochen

    2016-01-01

    Lineage-specific transcription factors determine the cell fate during development. Direct conversion of several cell types into other lineages has been achieved by the overexpression of specific transcription factors. Even cancer cells have been demonstrated to be amenable to transdifferentiation. Here, we identified a distinct set of transcription factors, which are sufficient to transform cells of the keratinocytic lineage to melanocyte-like cells. Melanocyte marker expression was induced and melanosome formation was observed in non-tumorigenic keratinocytes (HaCaT) and tumorigenic squamous cell carcinoma (MET-4) cells. Moreover, reduced proliferation, cell metabolism, invasion and migration were measured in vitro in transdifferentiated MT-MET-4 cells. A loss of tumorigenic potential of squamous cell carcinoma cells could be due to the upregulation of the melanocyte differentiation associated gene IL-24. Our data show that cells from the keratinocytic lineage can be transdifferented into the melanocytic lineage and provide a proof of principle for a potential new therapeutic strategy. PMID:27387763

  18. Microenvironment-induced myofibroblast-like conversion of engrafted keratinocytes.

    PubMed

    Li, MeiRong; Ti, DongDong; Han, WeiDong; Fu, XiaoBing

    2014-02-01

    Myofibroblasts, recognized classically by α-smooth muscle actin (α-SMA) expression, play a key role in the wound-healing process, promoting wound closure and matrix deposition. Although a body of evidence shows that keratinocytes explanted onto a wound bed promote closure of a skin injury, the underlying mechanisms are not well understood. The basal layer of epidermis is rich in undifferentiated keratinocytes (UKs). We showed that UKs injected into granulation tissue could switch into α-SMA positive cells, and accelerate the rate of skin wound healing. In addition, when the epidermis sheets isolated from foreskin cover up the wound bed or are induced in vitro, keratinocytes located at the basal layers or adjacent sites were observed to convert into myofibroblast-like cells. Thus, UKs have a potential for myofibroblastic transition, which provides a novel mechanism by which keratinocyte explants accelerate skin wound healing. PMID:24443179

  19. A review on quality of life in keratinocyte carcinoma patients.

    PubMed

    Waalboer-Spuij, R; Nijsten, T E C

    2013-06-01

    Health-related quality of life issues in patients with cutaneous malignancies is being re-explored. This is motivated by the heavy burden they put on dermatological care, it is more and more considered a chronic disease and new non-invasive therapies are being introduced. The purpose of this review is to identify the relevant quality of life (QOL) issues and to summarize the instruments used for investigating QOL in keratinocyte carcinoma patients. With a systematic literature search in Embase, MEDLINE OvidSP, PubMed publisher and Cochrane Central, 10 questionnaires and 4 studies reporting on quality of life issues were identified. Generic (UK Sickness Impact profile [UKSIP], Short Form 36-item Health Survey [SF-36], Functional Assessment of Cancer Therapy-General [FACT-G]) and dermatology specific (Dermatology Life Quality Index [DLQI] and Skindex-29, -16, -17) instruments demonstrated little to no QOL impairment. This may be explained by failing to capture the relevant domains such as "emotions", "appearance" and "anxiety". Skin cancer specific questionnaires (Skin Cancer Index [SCI], Skin Cancer Quality of Life Impact Tool [SCQOLIT] and Actinic Keratosis Quality of Life [AKQoL]) demonstrated good validity and responsiveness and represent the effect on QOL properly. However, there are some points of critique to these questionnaires. Optimal management of patients with actinic neoplasia syndrome and the selection and evaluation of therapies may benefit from the use of PROs in this ever increasing population. PMID:23670061

  20. Cytotoxicity of HBD3 for dendritic cells, normal human epidermal keratinocytes, hTERT keratinocytes, and primary oral gingival epithelial keratinocytes in cell culture conditions.

    PubMed

    Leelakanok, Nattawut; Fischer, Carol L; Bates, Amber M; Guthmiller, Janet M; Johnson, Georgia K; Salem, Aliasger K; Brogden, Kim A; Brogden, Nicole K

    2015-12-01

    Human β-defensin 3 (HBD3) is a prominent host defense peptide. In our recent work, we observed that HBD3 modulates pro-inflammatory agonist-induced chemokine and cytokine responses in human myeloid dendritic cells (DCs), often at 20.0 μM concentrations. Since HBD3 can be cytotoxic in some circumstances, it is necessary to assess its cytotoxicity for DCs, normal human epidermal keratinocytes (NHEKs), human telomerase reverse transcriptase (hTERT) keratinocytes, and primary oral gingival epithelial (GE) keratinocytes in different cell culture conditions. Cells, in serum free media with resazurin and in complete media with 10% fetal bovine serum and resazurin, were incubated with 5, 10, 20, and 40 μM HBD3. Cytotoxicity was determined by measuring metabolic conversion of resazurin to resorufin. The lethal dose 50 (LD50, mean μM±Std Err) values were determined from the median fluorescent intensities of test concentrations compared to live and killed cell controls. The LD50 value range of HBD3 was 18.2-35.9 μM in serum-free media for DCs, NHEKs, hTERT keratinocytes, and GE keratinocytes, and >40.0 μM in complete media. Thus, HBD3 was cytotoxic at higher concentrations, which must be considered in future studies of HBD3-modulated chemokine and cytokine responses in vitro. PMID:26367466

  1. Vimentin coordinates fibroblast proliferation and keratinocyte differentiation in wound healing via TGF-β–Slug signaling

    PubMed Central

    Cheng, Fang; Shen, Yue; Mohanasundaram, Ponnuswamy; Lindström, Michelle; Ivaska, Johanna; Ny, Tor; Eriksson, John E.

    2016-01-01

    Vimentin has been shown to be involved in wound healing, but its functional contribution to this process is poorly understood. Here we describe a previously unrecognized function of vimentin in coordinating fibroblast proliferation and keratinocyte differentiation during wound healing. Loss of vimentin led to a severe deficiency in fibroblast growth, which in turn inhibited the activation of two major initiators of epithelial–mesenchymal transition (EMT), TGF-β1 signaling and the Zinc finger transcriptional repressor protein Slug, in vimentin-deficient (VIM−/−) wounds. Correspondingly, VIM−/− wounds exhibited loss of EMT-like keratinocyte activation, limited keratinization, and slow reepithelialization. Furthermore, the fibroblast deficiency abolished collagen accumulation in the VIM−/− wounds. Vimentin reconstitution in VIM−/− fibroblasts restored both their proliferation and TGF-β1 production. Similarly, restoring paracrine TGF-β–Slug–EMT signaling reactivated the transdifferentiation of keratinocytes, reviving their migratory properties, a critical feature for efficient healing. Our results demonstrate that vimentin orchestrates the healing by controlling fibroblast proliferation, TGF-β1–Slug signaling, collagen accumulation, and EMT processing, all of which in turn govern the required keratinocyte activation. PMID:27466403

  2. Alteration in the arrangement of the keratin-type intermediate filaments during mitosis in cultured human keratinocytes.

    PubMed

    Kitajima, Y; Inoue, S; Yoneda, K; Mori, S; Yaoita, H

    1985-09-01

    The behavior of the keratin-type intermediate filaments (KIFs) during mitosis was characterized in cultured human keratinocytes by immunofluorescence microscopy using polyclonal antibodies to keratin. The structural relationship of KIFs with microtubules (MTs) was also studied at the same time using a monoclonal antibody to alpha-tubulin. The KIFs and MTs showed similar but different cytoskeletal networks and underwent structural rearrangements independently during the cell cycle. KIFs in keratinocytes formed two different arrangements during meta- and anaphase: a global aggregation of filaments around the spindle and a fibrous array radiating from the central, global aggregation of filaments to the cell periphery where they were connected with those of the adjacent cells at desmosomal sites. These radiating fibrous portions of KIFs appeared to play a role in retaining the cell in its correct relationship to the surrounding cells during mitosis. This behavior of KIFs in normal keratinocytes was different from the KIF-alterations which had been previously described in SV40-transformed keratinocytes and other cells which expressed two different IFs (keratin and vimentin). PMID:2412816

  3. Transient receptor potential vanilloid 4 (TRPV4) is downregulated in keratinocytes in human non-melanoma skin cancer.

    PubMed

    Fusi, Camilla; Materazzi, Serena; Minocci, Daiana; Maio, Vincenza; Oranges, Teresa; Massi, Daniela; Nassini, Romina

    2014-09-01

    A subgroup of the transient receptor potential (TRP) channels, including vanilloid 1 (TRPV1), TRPV2, TRPV3, TRPV4, and TRP ankyrin 1 (TRPA1), is expressed in cutaneous peptidergic somatosensory neurons, and has been found in skin non-neuronal cells, such as keratinocytes. Different cancer cells express TRPs, where they may exert either pro- or antitumorigenic roles. Expression and function of TRPs in skin cancers have been, however, poorly investigated. Here, we have studied the distribution and expression of TRPs by immunohistochemistry and messenger RNA (mRNA) in human healthy skin and human keratinocytic tumors, including intraepidermal proliferative disorders (solar keratosis (SK) and Bowen's disease), and non-melanoma skin cancer (NMSC; basal cell and squamous cell carcinomas). Similar TRPV1, TRPV2, and TRPV3 staining was found in keratinocytes from healthy and tumor tissues. TRPA1 staining was increased solely in SK samples. However, the marked TRPV4 staining and TRPV4 mRNA expression, observed in healthy or inflamed skin, was abrogated both in premalignant lesions and NMSC. In a human keratinocyte cell line (HaCaT), TRPV4 stimulation released IL-8, which in turn downregulated TRPV4 expression. Selective reduction in TRPV4 expression could represent an early biomarker of skin carcinogenesis. Whether the cytokine-dependent, autocrine pathway that results in TRPV4 downregulation contributes to NMSC mechanism remains to be determined. PMID:24643128

  4. Cellular and molecular facets of keratinocyte reepithelization during wound healing

    SciTech Connect

    Santoro, Massimo M. . E-mail: msantoro@unipmn.it; Gaudino, Giovanni

    2005-03-10

    Cutaneous wound healing is a highly coordinated physiological process that rapidly and efficiently restores skin integrity. Reepithelization is a crucial step during wound healing, which involves migration and proliferation of keratinocytes to cover the denuded dermal surface. Recent advances in wound biology clarified the molecular pathways governing keratinocyte reepithelization at wound sites. These new findings point towards novel therapeutic targets and provide suitable methods to promote faster tissue regeneration in vivo.

  5. Polarized Integrin Mediates Human Keratinocyte Adhesion to Basal Lamina

    NASA Astrophysics Data System (ADS)

    de Luca, Michele; Tamura, Richard N.; Kajiji, Shama; Bondanza, Sergio; Rossino, Paola; Cancedda, Ranieri; Carlo Marchisio, Pier; Quaranta, Vito

    1990-09-01

    Epithelial cell interactions with matrices are critical to tissue organization. Indirect immunofluorescence and immunoprecipitations of cell lysates prepared from stratified cultures of human epidermal cells showed that the major integrins expressed by keratinocytes are α_Eβ_4 (also called α_6β_4) and α_2β_1/α_3β_1. The α_Eβ_4 integrin is localized at the surface of basal cells in contact with the basement membrane, whereas α_2β_1/ α_3β_1 integrins are absent from the basal surface and are localized only on the lateral surface of basal and spinous keratinocytes. Anti-β_4 antibodies potently inhibited keratinocyte adhesion to matrigel or purified laminin, whereas anti-β_1 antibodies were ineffective. Only anti-β_4 antibodies were able to detach established keratinocyte colonies. These data suggest that α_Eβ_4 mediates keratinocyte adhesion to basal lamina, whereas the β_1 subfamily is involved in cell-cell adhesion of keratinocytes.

  6. Incorporation of linoleic acid by cultured human keratinocytes.

    PubMed

    Vicanová, J; Weerheim, A M; Kempenaar, J A; Ponec, M

    1999-01-01

    Linoleic acid is required for the formation and maintenance of the epidermal barrier, but most of the current in vitro keratinocyte culture systems are linoleic acid-deficient. The aim of the present study was to examine the efficiency of linoleic acid uptake in human keratinocyte cultures grown under submerged and air-exposed conditions in serum-free medium. The water-insoluble linoleic acid was bound to carrier molecules (cyclodextrin or bovine serum albumin). Comparable results were obtained with home-made and commercially available linoleic acid complexes. In the submerged cultures, the increase of the linoleic acid medium concentration (ranging from 0 to 20 microg/ml) resulted in a gradual increase in the linoleic acid cellular content, which exceeded 1.4 times the value found in native epidermis when the highest concentration of linoleic acid was used. The addition of linoleic acid did not alter the profile of the other epidermal fatty acids, with the exception of oleic acid, which decreased in parallel with the increasing linoleic acid content. While the content of linoleic acid found in phospholipids was similar to that in native epidermis, a large excess of linoleic acid was detected in triglycerides, the synthesis of which was markedly increased in cultures grown submerged in medium containing higher concentrations of linoleic acid. Under air-exposed conditions, the dermal substrate used seemed to be the most limiting factor for efficient linoleic acid supplementation. A low linoleic acid cellular content was detected when an inert filter was used. De-epidermized dermis was found to be the most permeable substrate for linoleic acid complexes. The cellular linoleic acid content increased in a parallel with the increasing linoleic acid concentration (ranging from 4 to 30 microg/ml), but the overall amount incorporated was lower than that in submerged cultures. The content of linoleic acid in the phospholipid and ceramide fractions isolated from

  7. Microgravity influences circadian clock oscillation in human keratinocytes

    PubMed Central

    Ranieri, Danilo; Cucina, Alessandra; Bizzarri, Mariano; Alimandi, Maurizio; Torrisi, Maria Rosaria

    2015-01-01

    Microgravity and sudden changes of gravitational forces exert numerous effects on tissues, organs and apparatus. Responses to these forces variably applied to cells indicate the existence of mechanotransduction pathways able to modulate transcription. Oscillation of circadian clocks similarly influences many cellular and metabolic processes. Here we hypothesized that signals derived from changes of gravitational forces applied to epidermal cells might influence their physiology in harmony with the oscillation of the molecular clock. In this study, we describe amplified oscillations of Bmal1 circadian clock gene in human keratinocytes exposed to short simulated microgravity and to rapid variation of gravitational forces. We found that exposure to microgravity enhances the amplitude of the Bmal1 feedback loop sustained by an apparently lower variability of Rev-erbα transcription, while recovery from microgravity is characterized by increased amplitude of Bmal1 expression and elongation of the oscillatory periods of Bmal1 and Rev-erbα. These data highlight the existence of integrated signaling network connecting mechanosensitive pathways to circadian gene regulation. PMID:26448904

  8. Increase developmental plasticity of human keratinocytes with gene suppression.

    PubMed

    Li, Shengwen Calvin; Jin, Yangsun; Loudon, William G; Song, Yahui; Ma, Zhiwei; Weiner, Leslie P; Zhong, Jiang F

    2011-08-01

    Recent evidence indicates that p53 suppression increased the efficiency of induced pluripotent stem cell (iPSC) generation. This occurred even with the enforced expression of as few as two canonical transcription factors, Oct4 and Sox2. In this study, primary human keratinocytes were successfully induced into a stage of plasticity by transient inactivation of p53, without enforced expression of any of the transcription factors previously used in iPSC generation. These cells were later redifferentiated into neural lineages. The gene suppression plastic cells were morphologically indistinguishable from human ES cells. Gene suppression plastic cells were alkaline phosphatase-positive, had normal karyotypes, and expressed p53. Together with the accumulating evidence of similarities and overlapping mechanisms between iPSC generation and cancer formation, this finding sheds light on the emerging picture of p53 sitting at the crossroads between two intricate cellular potentials: stem cell vs. cancer cell generation. This finding further supports the crucial role played by p53 in cellular reprogramming and suggests an alternative method to switch the lineage identity of human cells. This reported method offers the potential for directed lineage switching with the goal of generating autologous cell populations for novel clinical applications for neurodegenerative diseases. PMID:21768375

  9. Human Keratinocytes have two interconvertible modes of proliferation

    PubMed Central

    Roshan, Amit; Murai, Kasumi; Fowler, Joanna; Simons, Benjamin D; Nikolaidou-Neokosmidou, Varvara; Jones, Philip H

    2016-01-01

    Summary Single stem cells, including those in human epidermis, have a remarkable ability to reconstitute tissues in vitro, but the cellular mechanisms that enable this are ill defined. We used live imaging to track the outcome of thousands of divisions in clonal cultures of primary human epidermal keratinocytes. Two modes of proliferation were seen. In ‘balanced’ mode, similar proportions of proliferating and differentiating cells were generated, achieving the ‘population asymmetry’ that sustains epidermal homeostasis in vivo. In ‘expanding’ mode, an excess of cycling cells was produced, generating large expanding colonies. Cells in expanding mode switched their behaviour to balanced mode once local confluence was attained. However when a confluent area is wounded in a scratch assay, cells near the scratch switch back to expanding mode until the defect is closed. We conclude that the ability of a single epidermal stem cell to reconstitute an epithelium is explained by two interconvertible modes of proliferation regulated by confluence. PMID:26641719

  10. Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

    SciTech Connect

    Walter, M.N.M.; Wright, K.T.; Fuller, H.R.; MacNeil, S.; Johnson, W.E.B.

    2010-04-15

    We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-{beta}1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

  11. Novel non-calcemic secosteroids that are produced by human epidermal keratinocytes protect against solar radiation.

    PubMed

    Slominski, Andrzej T; Janjetovic, Zorica; Kim, Tae-Kang; Wasilewski, Piotr; Rosas, Sofia; Hanna, Sherie; Sayre, Robert M; Dowdy, John C; Li, Wei; Tuckey, Robert C

    2015-04-01

    CYP11A1 hydroxylates the side chain of vitamin D3 (D3) in a sequential fashion [D3→20S(OH)D3→20,23(OH)2D3→17,20,23(OH)3D3], in an alternative to the classical pathway of activation [D3→25(OH)D3→1,25(OH)2D3]. The products/intermediates of the pathway can be further modified by the action of CYP27B1. The CYP11A1-derived products are biologically active with functions determined by the lineage of the target cells. This pathway can operate in epidermal keratinocytes. To further define the role of these novel secosteroids we tested them for protective effects against UVB-induced damage in human epidermal keratinocytes, melanocytes and HaCaT keratinocytes, cultured in vitro. The secosteroids attenuated ROS, H2O2 and NO production by UVB-irradiated keratinocytes and melanocytes, with an efficacy similar to 1,25(OH)2D3, while 25(OH)D3 had lower efficacy. These attenuations were also seen to some extent for the 20(OH)D3 precursor, 20S-hydroxy-7-dehydrocholesterol. These effects were accompanied by upregulation of genes encoding enzymes responsible for defense against oxidative stress. Using immunofluorescent staining we observed that the secosteroids reduced the generation cyclobutane pyrimidine dimers in response to UVB and enhanced expression of p53 phosphorylated at Ser-15, but not at Ser-46. Additional evidence for protection against DNA damage in cells exposed to UVB and treated with secosteroids was provided by the Comet assay where DNA fragmentation was markedly reduced by 20(OH)D3 and 20,23(OH)2D3. In conclusion, novel secosteroids that can be produced by the action of CYP11A1 in epidermal keratinocytes have protective effects against UVB radiation. This article is part of a special issue entitled '17th Vitamin D Workshop'. PMID:25617667

  12. Lithospermum erythrorhizon extract protects keratinocytes and fibroblasts against oxidative stress.

    PubMed

    Yoo, Hee Geun; Lee, Bong Han; Kim, Wooki; Lee, Jong Suk; Kim, Gun Hee; Chun, Ock K; Koo, Sung I; Kim, Dae-Ok

    2014-11-01

    Oxidative stress damages dermal and epidermal cells and degrades extracellular matrix proteins, such as collagen, ultimately leading to skin aging. The present study evaluated the potential protective effect of the aqueous methanolic extract obtained from Lithospermum erythrorhizon (LE) against oxidative stress, induced by H2O2 and ultraviolet (UV) irradiation, on human keratinocyte (HaCaT) and human dermal fibroblast-neonatal (HDF-n) cells. Exposure of cells to H2O2 or UVB irradiation markedly increased oxidative stress and reduced cell viability. However, pretreatment of cells with the LE extract not only increased cell viability (up to 84.5%), but also significantly decreased oxidative stress. Further, the LE extract downregulated the expression of matrix metalloproteinase-1, an endopeptidase that degrades extracellular matrix collagen. In contrast, treatment with the LE extract did not affect the expression of procollagen type 1 in HDF-n cells exposed to UVA irradiation. Thirteen phenolic compounds, including derivatives of shikonin and caffeic acid, were identified by ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. These results suggest that LE-derived extracts may protect oxidative-stress-induced skin aging by inhibiting degradation of skin collagen, and that this protection may derive at least in part from the antioxidant phenolics present in these extracts. Further studies are warranted to determine the potential utility of LE-derived extracts in both therapeutic and cosmetic applications. PMID:25136892

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

    PubMed Central

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

    2015-01-01

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

  14. Japanese Cedar (Cryptomeria japonica) pollen allergen induces elevation of intracellular calcium in human keratinocytes and impairs epidermal barrier function of human skin ex vivo.

    PubMed

    Kumamoto, Junichi; Tsutsumi, Moe; Goto, Makiko; Nagayama, Masaharu; Denda, Mitsuhiro

    2016-01-01

    Cry j1 is the major peptide allergen of Japanese cedar (Sugi), Cryptomeria japonica. Since some allergens disrupt epidermal permeability barrier homeostasis, we hypothesized that Cry j1 might have a similar effect. Intracellular calcium level in cultured human keratinocytes was measured with a ratiometric fluorescent probe, Fura-2 AM. Application of Cry j1 significantly increased the intracellular calcium level of keratinocytes, and this increase was inhibited by trypsin inhibitor or a protease-activated receptor 2 (PAR-2) antagonist. We found that Cry j1 itself did not show protease activity, but application of Cry j1 to cultured keratinocytes induced a rapid (within 30 s) and transient increase of protease activity in the medium. This transient increase was blocked by trypsin inhibitor or PAR-2 antagonist. The effect of Cry j1 on transepidermal water loss (TEWL) of cultured human skin was measured in the presence and absence of a trypsin inhibitor and PAR-2 antagonist. Cry j1 significantly impaired the barrier function of human skin ex vivo, and this action was blocked by co-application of trypsin inhibitor or PAR-2 antagonist. Our results suggested that interaction of Cry j1 with epidermal keratinocytes leads to the activation of PAR-2, which induces elevation of intracellular calcium and disruption of barrier function. Blocking the interaction of Cry j1 with epidermal keratinocytes might ameliorate allergic reaction and prevent disruption of epidermal permeability barrier homeostasis. PMID:26498292

  15. Keratinocyte cytoskeletal roles in cell sheet engineering

    PubMed Central

    2013-01-01

    Background There is an increasing need to understand cell-cell interactions for cell and tissue engineering purposes, such as optimizing cell sheet constructs, as well as for examining adhesion defect diseases. For cell-sheet engineering, one major obstacle to sheet function is that cell sheets in suspension are fragile and, over time, will contract. While the role of the cytoskeleton in maintaining the structure and adhesion of cells cultured on a rigid substrate is well-characterized, a systematic examination of the role played by different components of the cytoskeleton in regulating cell sheet contraction and cohesion in the absence of a substrate has been lacking. Results In this study, keratinocytes were cultured until confluent and cell sheets were generated using dispase to remove the influence of the substrate. The effects of disrupting actin, microtubules or intermediate filaments on cell-cell interactions were assessed by measuring cell sheet cohesion and contraction. Keratin intermediate filament disruption caused comparable effects on cell sheet cohesion and contraction, when compared to actin or microtubule disruption. Interfering with actomyosin contraction demonstrated that interfering with cell contraction can also diminish cell cohesion. Conclusions All components of the cytoskeleton are involved in maintaining cell sheet cohesion and contraction, although not to the same extent. These findings demonstrate that substrate-free cell sheet biomechanical properties are dependent on the integrity of the cytoskeleton network. PMID:23442760

  16. Characterization of microfluidic human epidermal keratinocyte culture

    PubMed Central

    O’Neill, Adrian T.; Monteiro-Riviere, Nancy A.

    2008-01-01

    Human epidermal keratinocytes (HEK) are skin cells of primary importance in maintaining the body’s defensive barrier and are used in vitro to assess the irritation potential and toxicity of chemical compounds. Microfluidic systems hold promise for high throughput irritant and toxicity assays, but HEK growth kinetics have yet to be characterized within microscale culture chambers. This research demonstrates HEK patterning on microscale patches of Type I collagen within microfluidic channels and maintenance of these cells under constant medium perfusion for 72 h. HEK were shown to maintain 93.0%–99.6% viability at 72 h under medium perfusion ranging from 0.025–0.4 μl min−1. HEK maintained this viability while ∼100% confluent—a level not possible in 96 well plates. Microscale HEK cultures offer the ability to precisely examine the morphology, behavior and viability of individual cells which may open the door to new discoveries in toxicological screening methods and wound healing techniques. PMID:19002858

  17. Human Keratinocytes Radioprotection with Mentha Longifolia

    NASA Astrophysics Data System (ADS)

    Rizzo, Angela Maria; Berselli, P.; Zava, S.; Negroni, M.; Corsetto, P.; Montorfano, G.; Bertolotti, A.; Ranza, E.; Ottolenghi, A.; Berra, B.

    Antioxidants are suggested to act as radioprotectors, and dietary supplements based on antiox-idants have been proposed for astronauts involved in long-term space missions. Plant extracts with antioxidant properties may be used in dietetic supplements for astronauts; in fact recent nutritional guidelines suggest that "fruits and vegetables may become as important on space-going vessels as limes were on the sea-going vessels of old". Mint presents a large variety of biological properties, such as antiallergenic, antibacterial, anti-inflammatory, antitumor, an-tiviral, gastrointestinal protective, hepatoprotective, chemopreventive activities, most of which are attributable to its antioxidant activity. The aim of the present study is to evaluate the antioxidant properties and protective bio-efficacy of a phenol enriched Mentha longifolia ex-tract on gamma rays stressed human keratinocytes (NCTC2544). We assessed first the in vitro antioxidant activity (ABTS and DPPH), and then evaluated different stress markers in order to investigate various oxidative stress targets: cell viability (MTT); retained proliferating ca-pability (CA); DNA damage (histone H2AX) and protein damage (HSP70 induction). Results indicate that this Mint extract has a higher antioxidant activity respect to fresh extracts, that could be responsible of its really interesting radio-protective effects.

  18. miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes

    SciTech Connect

    McKenna, Declan J.; Patel, Daksha; McCance, Dennis J.

    2014-01-05

    A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in the cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes. - Highlights: • miR-24 and miR-205 are induced during keratinocyte differentiation. • This induction is lost in keratinocytes expressing HPV onco-proteins E6 and E7. • miR-205 is dependent upon pRb expression. • miR-24 targets p27 in cycling keratinocytes.

  19. Quantitative analysis of laminin 5 gene expression in human keratinocytes.

    PubMed

    Akutsu, Nobuko; Amano, Satoshi; Nishiyama, Toshio

    2005-05-01

    To examine the expression of laminin 5 genes (LAMA3, LAMB3, and LAMC2) encoding the three polypeptide chains alpha3, beta3, and gamma2, respectively, in human keratinocytes, we developed novel quantitative polymerase chain reaction (PCR) methods utilizing Thermus aquaticus DNA polymerase, specific primers, and fluorescein-labeled probes with the ABI PRISM 7700 sequence detector system. Gene expression levels of LAMA3, LAMB3, and LAMC2 and glyceraldehyde-3-phosphate dehydrogenase were quantitated reproducibly and sensitively in the range from 1 x 10(2) to 1 x 10(8) gene copies. Basal gene expression level of LAMB3 was about one-tenth of that of LAMA3 or LAMC2 in human keratinocytes, although there was no clear difference among immunoprecipitated protein levels of alpha3, beta3, and gamma2 synthesized in radio-labeled keratinocytes. Human serum augmented gene expressions of LAMA3, LAMB3, and LAMC2 in human keratinocytes to almost the same extent, and this was associated with an increase of the laminin 5 protein content measured by a specific sandwich enzyme-linked immunosorbent assay. These results demonstrate that the absolute mRNA levels generated from the laminin 5 genes do not determine the translated protein levels of the laminin 5 chains in keratinocytes, and indicate that the expression of the laminin 5 genes may be controlled by common regulation mechanisms. PMID:15854126

  20. Transcription Factor MafB Coordinates Epidermal Keratinocyte Differentiation.

    PubMed

    Miyai, Masashi; Hamada, Michito; Moriguchi, Takashi; Hiruma, Junichiro; Kamitani-Kawamoto, Akiyo; Watanabe, Hajime; Hara-Chikuma, Mariko; Takahashi, Kenzo; Takahashi, Satoru; Kataoka, Kohsuke

    2016-09-01

    Mammalian epidermis is a stratified epithelium composed of distinct layers of keratinocytes. The outermost cornified layer is a primary barrier that consists of a cornified envelope, an insoluble structure assembled by cross-linked scaffold proteins, and a surrounding mixture of lipids. Skin keratinocytes undergo a multistep differentiation process, but the mechanism underlying this process is not fully understood. We demonstrate that the transcription factor MafB is expressed in differentiating keratinocytes in mice and is transcriptionally upregulated upon human keratinocyte differentiation in vitro. In MafB-deficient mice, epidermal differentiation was partially impaired and the cornified layer was thinner than in wild-type mice. On the basis of transcriptional profiling, we detected reduced expression levels of a subset of cornified envelope genes, for example, filaggrin and repetin, in the MafB(-/-) epidermis. By contrast, the expression levels of lipid metabolism-related genes, such as Alox12e and Smpd3, increased. The upregulated genes in the MafB(-/-) epidermis were enriched for putative target genes of the transcription factors Gata3, Grhl3, and Klf4. Immunohistochemical analysis of skin biopsy samples revealed that the expression levels of filaggrin and MafB were significantly reduced in patients with human atopic dermatitis and psoriasis vulgaris. Our results indicate that MafB is a component of the gene expression program that regulates epidermal keratinocyte differentiation. PMID:27208706

  1. Induction of differentiation in psoriatic keratinocytes by propylthiouracil and fructose.

    PubMed

    Arul, Santhosh; Dayalan, Haripriya; Jegadeesan, Muhilan; Damodharan, Prabhavathy

    2016-12-01

    Psoriasis is characterized by uncontrolled proliferation and poor differentiation. Sirtuin1 (SIRT1) a class III deacetylase, crucial for differentiation in normal keratinocytes, is reduced in psoriasis. Down regulated SIRT1 levels may contribute to poor differentiation in psoriasis. In addition, the levels of early differentiation factors Keratin1 (K1) and Keratin10 (K10) are depleted in psoriasis. We attempted to study a possible effect of fructose, a SIRT1 upregulator and Propylthiouracil (PTU) to augment differentiation in psoriatic keratinocytes. Keratinocytes were cultured from lesional biopsies obtained from psoriatic patients and control cells were obtained from patients undergoing abdominoplasty. Cells were treated with fructose and PTU individually. K1 and K10 transcript levels were measured to evaluate early differentiation; SIRT1 protein expression was also studied to decipher its role in the mechanism of differentiation. The K1, K10 transcript levels, SIRT1 protein and transcript levels in fructose treated psoriatic keratinocytes were improved. This suggests keratinocyte differentiation was induced by fructose through SIRT1 upregulation. Whereas PTU induced differentiation, as confirmed by improved K1, K10 transcript levels followed a non-SIRT1 mechanism. We conclude that the use of fructose and PTU may be an adjunct to the existing therapies for psoriasis. PMID:27453822

  2. Polymeric membranes modulate human keratinocyte differentiation in specific epidermal layers.

    PubMed

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

    2016-10-01

    In vitro models of human bioengineered skin substitutes are an alternative to animal experimentation for testing the effects and toxicity of drugs, cosmetics and pollutants. For the first time specific and distinct human epidermal strata were engineered by using membranes and keratinocytes. To this purpose, biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT-PCL were prepared by phase-inversion technique and characterized in order to evaluate their morphological, physico-chemical and mechanical properties. The capability of membranes to modulate keratinocyte differentiation inducing specific interactions in epidermal membrane systems was investigated. The overall results demonstrated that the membrane properties strongly influence the cell morpho-functional behaviour of human keratinocytes, modulating their terminal differentiation, with the creation of specific epidermal strata or a fully proliferative epidermal multilayer system. In particular, human keratinocytes adhered on CHT and CHT-PCL membranes, forming the structure of the epidermal top layers, such as the corneum and granulosum strata, characterized by withdrawal or reduction from the cell cycle and cell proliferation. On the PCL membrane, keratinocytes developed an epidermal basal lamina, with high proliferating cells that stratified and migrated over time to form a complete differentiating epidermal multilayer system. PMID:27371895

  3. Kinetics of growth and differentiation of cultured human epidermal keratinocytes

    SciTech Connect

    Albers, K.M.

    1985-01-01

    A study was made of the interrelationship between replication and differentiation in cultures of human epidermal keratinocytes. Measures of both parameters were made using newly developed methods to quantify the rate at which keratinocytes replicate and the rate at which they withdraw from the cell cycle. Keratinocyte replication was measured by determining the cell doubling time, labeling index, and cell cycle duration. Cell cycle length was measured using a double label assay that determines the length of time between two successive phases of DNA synthesis. The first DNA synthesis phase was marked by labeling keratinocytes with /sup 14/C-thymidine. At the next round of DNA synthesis, cells were labeled with bromodeoxyuridine, a heavy analog of thymidine. The cell cycle length is given by the time required for the /sup 14/C-labeled DNA to become double labeled. To measure keratinocyte differentiation, the rate at which cells withdraw from the cell cycle was determined. To measure withdrawal, the percentage of cells labeled by a pulse of /sup 14/C-thymidine that failed to undergo a second cycle of DNA synthesis, as measured by bromodeoxyuridine incorporation, was determined. Cells which failed to undergo a second cycle of synthesis were considered to have differentiated and withdrawn from the cell cycle.

  4. Chemosensory Information Processing between Keratinocytes and Trigeminal Neurons

    PubMed Central

    Sondersorg, Anna Christina; Busse, Daniela; Kyereme, Jessica; Rothermel, Markus; Neufang, Gitta; Gisselmann, Günter; Hatt, Hanns; Conrad, Heike

    2014-01-01

    Trigeminal fibers terminate within the facial mucosa and skin and transmit tactile, proprioceptive, chemical, and nociceptive sensations. Trigeminal sensations can arise from the direct stimulation of intraepithelial free nerve endings or indirectly through information transmission from adjacent cells at the peripheral innervation area. For mechanical and thermal cues, communication processes between skin cells and somatosensory neurons have already been suggested. High concentrations of most odors typically provoke trigeminal sensations in vivo but surprisingly fail to activate trigeminal neuron monocultures. This fact favors the hypothesis that epithelial cells may participate in chemodetection and subsequently transmit signals to neighboring trigeminal fibers. Keratinocytes, the major cell type of the epidermis, express various receptors that enable reactions to multiple environmental stimuli. Here, using a co-culture approach, we show for the first time that exposure to the odorant chemicals induces a chemical communication between human HaCaT keratinocytes and mouse trigeminal neurons. Moreover, a supernatant analysis of stimulated keratinocytes and subsequent blocking experiments with pyrodoxalphosphate-6-azophenyl-2′,4′-disulfonate revealed that ATP serves as the mediating transmitter molecule released from skin cells after odor stimulation. We show that the ATP release resulting from Javanol® stimulation of keratinocytes was mediated by pannexins. Consequently, keratinocytes act as chemosensors linking the environment and the trigeminal system via ATP signaling. PMID:24790106

  5. Concentration of Fibrin and Presence of Plasminogen Affect Proliferation, Fibrinolytic Activity, and Morphology of Human Fibroblasts and Keratinocytes in 3D Fibrin Constructs

    PubMed Central

    Reinertsen, Erik; Skinner, Michael; Wu, Benjamin

    2014-01-01

    Fibrin is a hemostatic protein found in the clotting cascade. It is used in the operating room to stop bleeding and deliver cells and growth factors to heal wounds. However, formulations of clinically approved fibrin are optimized for hemostasis, and the extent to which biochemical and physical cues in fibrin mediate skin cell behavior is not fully understood nor utilized in the design of biomaterials. To determine if the concentration of fibrinogen and the presence of plasminogen affect cell behavior relevant to wound healing, we fabricated three-dimensional fibrin constructs made from 5, 10, or 20 mg/mL of clinical fibrin or plasminogen-depleted (PD) fibrin. We cultured dermal fibroblasts or epidermal keratinocytes in these constructs. Fibroblasts proliferated similarly in both types of fibrin, but keratinocytes proliferated more in low concentrations of clinical fibrin and less in PD fibrin. Clinical fibrin constructs with fibroblasts were less stiff and degraded faster than PD fibrin constructs with fibroblasts. Similarly, keratinocytes degraded clinical fibrin, but not PD fibrin. Fibroblast spreading varied with fibrin concentration in both types of fibrin. In conclusion, the concentration of fibrinogen and the presence of plasminogen affect fibroblast and keratinocyte proliferation, morphology, and fibrin degradation. Creating materials with heterogeneous regions of fibrin formulations and concentrations could be a novel strategy for controlling the phenotype of encapsulated fibroblasts and keratinocytes, and the subsequent biomechanical properties of the construct. However, other well-investigated aspects of wound healing remain to be utilized in the design of fibrin biomaterials, such as autocrine and paracrine signaling between fibroblasts, keratinocytes, and immune cells. PMID:24738616

  6. A confocal three-dimensional micro X-ray scattering technology based on Rayleigh to Compton ratio for identifying materials with similar density and different weight percentages of low-Z elements

    NASA Astrophysics Data System (ADS)

    Li, Fangzuo; Liu, Zhiguo; Sun, Tianxi; Yang, Chaolin; Sun, Xuepeng; Sun, Weiyuan; He, Jialin; Ding, Xunliang

    2015-07-01

    A point-by-point Rayleigh to Compton scattering ratio (R/C) imaging for two polymer materials with similar density and different weight percentages of low-Z elements was carried out by using the confocal three-dimensional (3D) micro X-ray scatter tomographic technology based on polycapillary X-ray optics. This confocal 3D micro X-ray scatter tomographic technique was based on the confocal configuration of a polycapillary focusing X-ray lens (PFXRL) in the excitation channel and a polycapillary parallel X-ray lens (PPXRL) in the detection channel, which let only the X-rays scattered from the confocal micro-volume overlapped by the input focal spot of the PPXRL and the output focal spot of the PFXRL be detected by the detector. The main scope of this study was using the confocal 3D micro X-ray scattering tomography based on the R/C ratio to characterize and identify materials with nearly equal low density and different weight percentages of low-Z elements, as other radiological techniques are difficult to discriminate them for their very close attenuation coefficients μ . A mapping of R/C ratios for two thermoplastic polymer materials was obtained, which provided the spatially resolved distribution of their effective atom numbers, and their differences were accordingly presented. This confocal 3D micro X-ray scatter tomographic technique has potential applications in fields such as material identification, dosimetry, medical imaging, carbonation cancer, and so on.

  7. Angiopoietin-related growth factor (AGF) supports adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells through interaction with RGD-binding integrins

    SciTech Connect

    Zhang Yueqing; Hu Xiaobo; Tian Ruiyang; Wei Wangui; Hu Wei; Chen Xia; Han Wei; Chen Huayou; Gong Yi . E-mail: ygong@sibs.ac.cn

    2006-08-18

    Angiopoietin-related growth factor (AGF) is a newly identified member of angiopoietin-related proteins (ARPs)/angiopoietin-like proteins (Angptls). AGF has been considered as a novel growth factor in accelerating cutaneous wound healing, as it is capable of stimulating keratinocytes proliferation as well as angiogenesis. But in our paper, we demonstrate that AGF stimulates keratinocytes proliferation only at high protein concentration, however, it can potently promote adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells. Furthermore, we confirm that the adhesion and migration cellular events are mediated by RGD-binding integrins, most possibly the {alpha}{sub v}-containing integrins, by in vitro inhibition assays using synthetic competitive peptides. Our results strongly suggest that AGF is an integrin ligand as well as a mitogenic growth factor and theoretically participates in cutaneous wound healing in a more complex mechanism.

  8. The Effect of Secretory Factors of Adipose-Derived Stem Cells on Human Keratinocytes

    PubMed Central

    Moon, Kyoung Mi; Park, Ye-Hyoung; Lee, Jae Seol; Chae, Yong-Byung; Kim, Moon-Moo; Kim, Dong-Soo; Kim, Byung-Woo; Nam, Soo-Wan; Lee, Jong-Hwan

    2012-01-01

    The beneficial effects of adipose-derived stem cell conditioned medium (ADSC-CM) on skin regeneration have been reported. Although the mechanism of how ADSC-CM promotes skin regeneration is unclear, ADSC-CM contained various growth factors and it is an excellent raw material for skin treatment. ADSC-CM produced in a hypoxia condition of ADSC—in other words, Advanced Adipose-Derived Stem cell Protein Extract (AAPE)—has great merits for skin regeneration. In this study, human primary keratinocytes (HKs), which play fundamental roles in skin tissue, was used to examine how AAPE affects HK. HK proliferation was significantly higher in the experimental group (1.22 μg/mL) than in the control group. DNA gene chip demonstrated that AAPE in keratinocytes (p < 0.05) notably affected expression of 290 identified transcripts, which were associated with cell proliferation, cycle and migration. More keratinocyte wound healing and migration was shown in the experimental group (1.22 μg/mL). AAPE treatment significantly stimulated stress fiber formation, which was linked to the RhoA-ROCK pathway. We identified 48 protein spots in 2-D gel analysis and selected proteins were divided into 64% collagen components and 30% non-collagen components as shown by the MALDI-TOF analysis. Antibody array results contained growth factor/cytokine such as HGF, FGF-1, G-CSF, GM-CSF, IL-6, VEGF, and TGF-β3 differing from that shown by 2-D analysis. Conclusion: AAPE activates HK proliferation and migration. These results highlight the potential of the topical application of AAPE in the treatment of skin regeneration. PMID:22312315

  9. Simarouba amara extract increases human skin keratinocyte differentiation.

    PubMed

    Bonté, F; Barré, P; Pinguet, P; Dusser, I; Dumas, M; Meybeck, A

    1996-08-01

    An aqueous extract of Simarouba amara was studied for its activity on the differentiation of human skin keratinocytes. Submerged and air-exposed treated keratinocyte cultures displayed a more highly differentiated histoarchitecture, with presence of ultrastructural differentiated elements, than untreated controls. Immunohistochemistry of involucrin and activation of transglutaminase activity provided further evidence for the increase in corneocyte envelope formation observed ultrastructurally. Lipid analysis of air-exposed cultures revealed an increase in the cholesterol sulphate, cholesterol and ceramide contents. After 4 weeks of treatment on the hemiface of volunteers, the capacitance and transepidermal water loss evaluation revealed the potential interest of this extract for improvement of skin hydration. Electron microscopic examination of the corneocyte envelope on tape strips confirmed its actions. Taken together these data demonstrated that an aqueous extract of S. amara increases human keratinocyte differentiation. PMID:8844461

  10. A review of keratinocyte delivery to the wound bed.

    PubMed

    Chester, D L; Balderson, D S; Papini, R P G

    2004-01-01

    Over the last 20 years, confluent sheets of cultured epithelial autograft have been used for patients with major burns. Problems with the lack of "take" and long-term durability, as well as the time delay to produce such grafts, have led to the development of delivery systems to transfer keratinocytes to the wound bed. This review article describes the problems of using cultured epithelial autograft and the advantages of using preconfluent keratinocytes. Despite the numerous delivery systems that have been reported, most studies are limited to animal wound bed models. There are a few small clinical studies that have demonstrated enhanced healing using mainly subjective methods. There is a need for controlled, randomized clinical trials to prove the efficacy of keratinocyte delivery systems. Proposals for the use of this technology are made. PMID:15273468

  11. Activation of Nrf2 in keratinocytes causes chloracne (MADISH)-like skin disease in mice.

    PubMed

    Schäfer, Matthias; Willrodt, Ann-Helen; Kurinna, Svitlana; Link, Andrea S; Farwanah, Hany; Geusau, Alexandra; Gruber, Florian; Sorg, Olivier; Huebner, Aaron J; Roop, Dennis R; Sandhoff, Konrad; Saurat, Jean-Hilaire; Tschachler, Erwin; Schneider, Marlon R; Langbein, Lutz; Bloch, Wilhelm; Beer, Hans-Dietmar; Werner, Sabine

    2014-04-01

    The transcription factor Nrf2 is a key regulator of the cellular stress response, and pharmacological Nrf2 activation is a promising strategy for skin protection and cancer prevention. We show here that prolonged Nrf2 activation in keratinocytes causes sebaceous gland enlargement and seborrhea in mice due to upregulation of the growth factor epigen, which we identified as a novel Nrf2 target. This was accompanied by thickening and hyperkeratosis of hair follicle infundibula. These abnormalities caused dilatation of infundibula, hair loss, and cyst development upon aging. Upregulation of epigen, secretory leukocyte peptidase inhibitor (Slpi), and small proline-rich protein 2d (Sprr2d) in hair follicles was identified as the likely cause of infundibular acanthosis, hyperkeratosis, and cyst formation. These alterations were highly reminiscent to the phenotype of chloracne/"metabolizing acquired dioxin-induced skin hamartomas" (MADISH) patients. Indeed, SLPI, SPRR2, and epigen were strongly expressed in cysts of MADISH patients and upregulated by dioxin in human keratinocytes in an NRF2-dependent manner. These results identify novel Nrf2 activities in the pilosebaceous unit and point to a role of NRF2 in MADISH pathogenesis. PMID:24503019

  12. Optimal Differentiation of In Vitro Keratinocytes Requires Multifactorial External Control

    PubMed Central

    Borowiec, Anne-Sophie; Delcourt, Philippe; Dewailly, Etienne; Bidaux, Gabriel

    2013-01-01

    For almost 30 years, keratinocyte differentiation has been studied in numerous cell models including keratinocyte primary culture with various supplemented culture media. In this respect, it has become quite difficult to draw comparisons between studies using such a variety of culture conditions. Serum-free condition with low calcium has been used to culture basal proliferating cells, though differentiation is induced by various procedures. These latter include the addition of calcium at mM concentration and a concomitant addition of serum and calcium. Lowering the incubation temperature of cells has also been reported to induce a premature differentiation of keratinocytes in organotypic skin culture. This effect of temperature on keratinocyte differentiation has been poorly depicted, although average human skin temperature has been shown to be about 32°C. However, studying differentiation and quantifying shifts in the differentiation rate of a cell population implies to precisely know i) the proportion of differentiated cells in the whole population, and ii) to which extent and to which level of expression, the induction of a gene or a protein might be considered as a marker of differentiation. This lack has rarely been taken into consideration and has surely led to over-interpretations of single protein induction and to consequent extrapolations to real differentiation processes. By means of paralleled analyses with immunocytofluorescence, flow cytometry, and with multiple differentiation markers quantify by qPCR and western-blot, we studied the paradoxical connection between calcium, serum, multilayer culture and incubation temperature on the differentiation of in vitro keratinocytes. Conversely to previous reports, we have shown that calcium switch is indeed a potent model for inducing calcium-dependent genes, but is not an efficient procedure when one wishes to assess the keratinocyte differentiation rate. Moreover, we have demonstrated that a synergic

  13. Characterization of primary human keratinocytes transformed by human papillomavirus type 18

    SciTech Connect

    Kaur, P.; McDougall, J.K. )

    1988-06-01

    Primary human epithelial cells were cotransfected with pHPV-18 and pSV2neo, and cell strains were generated by selecting in G418. Southern blot analysis revealed the presence of at least one intact, integrated viral genome in these cells. FE-A cells showed altered growth properties, characterized by a change in morphology, and clonal density. Differentiation markers analyzed by Western blotting (immunoblotting), such as cytokeratins and involucrin, indicated that the cells resembled a partially differentiated epithelial population. Increased expression of the 40-kilodalton cytokeratin was observed in FE-A cells, similar to that observed in simian virus 40-immortalized human keratinocytes. Calcium and 12-O-tetradecanoyl-phorbol-13-acetate treatment induced normal epithelial cells to differentiate, whereas the human papillomavirus 18 (HPV-18)-containing keratinocytes were resistant to these signals, indicating their partially transformed nature. These cells were not able to induce tumors in nude mice over a period of up to 8 months. A second cell strain, FE-H18L, also generated by transfecting HPV-18, also exhibited an extended life span and similar alterations in morphology. Viral RNA transcribed from the early region of HPV-18 was detected in both cell strains by Northern (RNA) blot analysis. These cell strains should provide a useful model for determining the role of HPV in carcinogenesis.

  14. Proliferative Defects in Dyskeratosis Congenita Skin Keratinocytes are Corrected by Expression of the Telomerase Reverse Transcriptase, TERT, or by Activation of Endogenous Telomerase through Expression of Papillomavirus E6/E7 or the Telomerase RNA Component, TERC

    PubMed Central

    Gourronc, Francoise A.; Robertson, Mckaylee M.; Herrig, Annie K.; Lansdorp, Peter M.; Goldman, Frederick D.; Klingelhutz, Aloysius J.

    2010-01-01

    Dyskeratosis congenita (DC) is characterized by the triad of reticulate skin pigmentation, nail dystrophy, and leukoplakia. Epidermal atrophy, hair growth defects, bone marrow failure, and increased risk of cancer are also common in DC patients. DC is caused by mutations in genes encoding for telomerase complex factors. Although there is an association of epidermal abnormalities with DC, epidermal cells from DC donors have not been previously characterized. We have isolated skin keratinocytes from affected members of a family with an autosomal dominant form of DC that is due to a mutation in the RNA component of telomerase, TERC. Here we demonstrate that, similar to DC fibroblasts from these donors, DC keratinocytes have short telomeres and a short lifespan. DC keratinocytes also exhibited impaired colony forming efficiency and migration capacity. Exogenous expression of the reverse transcriptase component of telomerase, TERT, activated telomerase levels to half that of TERT expressing normal cells and maintained telomeres at a short length with concomitant extension of lifespan. Unlike fibroblasts, transduction of human papillomavirus type 16 E6/E7 genes into DC keratinocytes activated telomerase to half that of E6/E7 expressing normal cells, and robust proliferation was observed. While expression of TERC has no measurable effect on telomerase in fibroblasts, expression of TERC in keratinocytes upregulated telomerase activity and, rarely, allowed rescue of proliferative defects. Our results point to important differences between DC fibroblasts and keratinocytes and show, for the first time, that expression of TERC can increase the lifespan of primary human epithelial cells. PMID:19558498

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

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

  16. Mechanisms of Mitochondrial Damage in Keratinocytes by Pemphigus Vulgaris Antibodies*

    PubMed Central

    Kalantari-Dehaghi, Mina; Chen, Yumay; Deng, Wu; Chernyavsky, Alex; Marchenko, Steve; Wang, Ping H.; Grando, Sergei A.

    2013-01-01

    The development of nonhormonal treatment of pemphigus vulgaris (PV) has been hampered by a lack of clear understanding of the mechanisms leading to keratinocyte (KC) detachment and death in pemphigus. In this study, we sought to identify changes in the vital mitochondrial functions in KCs treated with the sera from PV patients and healthy donors. PV sera significantly increased proton leakage from KCs, suggesting that PV IgGs increase production of reactive oxygen species. Indeed, measurement of intracellular reactive oxygen species production showed a drastic increase of cell staining in response to treatment by PV sera, which was confirmed by FACS analysis. Exposure of KCs to PV sera also caused dramatic changes in the mitochondrial membrane potential detected with the JC-1 dye. These changes can trigger the mitochondria-mediated intrinsic apoptosis. Although sera from different PV patients elicited unique patterns of mitochondrial damage, the mitochondria-protecting drugs nicotinamide (also called niacinamide), minocycline, and cyclosporine A exhibited a uniform protective effect. Their therapeutic activity was validated in the passive transfer model of PV in neonatal BALB/c mice. The highest efficacy of mitochondrial protection of the combination of these drugs found in mitochondrial assay was consistent with the ability of the same drug combination to abolish acantholysis in mouse skin. These findings provide a theoretical background for clinical reports of the efficacy of mitochondria-protecting drugs in PV patients. Pharmacological protection of mitochondria and/or compensation of an altered mitochondrial function may therefore become a novel approach to development of personalized nonhormonal therapies of patients with this potentially lethal autoimmune blistering disease. PMID:23599429

  17. Multivariate Hypergeometric Similarity Measure

    PubMed Central

    Kaddi, Chanchala D.; Parry, R. Mitchell; Wang, May D.

    2016-01-01

    We propose a similarity measure based on the multivariate hypergeometric distribution for the pairwise comparison of images and data vectors. The formulation and performance of the proposed measure are compared with other similarity measures using synthetic data. A method of piecewise approximation is also implemented to facilitate application of the proposed measure to large samples. Example applications of the proposed similarity measure are presented using mass spectrometry imaging data and gene expression microarray data. Results from synthetic and biological data indicate that the proposed measure is capable of providing meaningful discrimination between samples, and that it can be a useful tool for identifying potentially related samples in large-scale biological data sets. PMID:24407308

  18. Knowledge Building Insights on Biomarkers of Arsenic Toxicity to Keratinocytes and Melanocytes

    PubMed Central

    Isokpehi, Raphael D.; Udensi, Udensi K.; Anyanwu, Matthew N.; Mbah, Andreas N.; Johnson, Matilda O.; Edusei, Kafui; Bauer, Michael A.; Hall, Roger A.; Awofolu, Omotayo R.

    2012-01-01

    Exposure to inorganic arsenic induces skin cancer and abnormal pigmentation in susceptible humans. High-throughput gene transcription assays such as DNA microarrays allow for the identification of biological pathways affected by arsenic that lead to initiation and progression of skin cancer and abnormal pigmentation. The overall purpose of the reported research was to determine knowledge building insights on biomarker genes for arsenic toxicity to human epidermal cells by integrating a collection of gene lists annotated with biological information. The information sets included toxicogenomics gene-chemical interaction; enzymes encoded in the human genome; enriched biological information associated with genes; environmentally relevant gene sequence variation; and effects of non-synonymous single nucleotide polymorphisms (SNPs) on protein function. Molecular network construction for arsenic upregulated genes TNFSF18 (tumor necrosis factor [ligand] superfamily member 18) and IL1R2 (interleukin 1 Receptor, type 2) revealed subnetwork interconnections to E2F4, an oncogenic transcription factor, predominantly expressed at the onset of keratinocyte differentiation. Visual analytics integration of gene information sources helped identify RAC1, a GTP binding protein, and TFRC, an iron uptake protein as prioritized arsenic-perturbed protein targets for biological processes leading to skin hyperpigmentation. RAC1 regulates the formation of dendrites that transfer melanin from melanocytes to neighboring keratinocytes. Increased melanocyte dendricity is correlated with hyperpigmentation. TFRC is a key determinant of the amount and location of iron in the epidermis. Aberrant TFRC expression could impair cutaneous iron metabolism leading to abnormal pigmentation seen in some humans exposed to arsenicals. The reported findings contribute to insights on how arsenic could impair the function of genes and biological pathways in epidermal cells. Finally, we developed visual analytics

  19. Effects of the Novel Compound DK223 ([1E,2E-1,2-Bis(6-methoxy-2H-chromen-3-yl)methylene]hydrazine) on Migration and Proliferation of Human Keratinocytes and Primary Dermal Fibroblasts

    PubMed Central

    Ho, Manh Tin; Kang, Hyun Sik; Huh, Jung Sik; Kim, Young Mee; Lim, Yoongho; Cho, Moonjae

    2014-01-01

    Wound healing plays an important role in protecting the human body from external infection. Cell migration and proliferation of keratinocytes and dermal fibroblasts are essential for proper wound healing. Recently, several studies have demonstrated that secondary compounds produced in plants could affect skin cells migration and proliferation. In this study, we identified a novel compound DK223 ([1E,2E-1,2-bis(6-methoxy-2H-chromen-3-yl)methylene]hydrazine) that concomitantly induced human keratinocyte migration and dermal fibroblast proliferation. We evaluated the regulation of epithelial and mesenchymal protein markers, such as E-cadherin and Vimentin, in human keratinocytes, as well as extracellular matrix (ECM) secretion and metalloproteinase families in dermal fibroblasts. DK223 upregulated keratinocyte migration and significantly increased the epithelial marker E-cadherin in a time-dependent manner. We also found that reactive oxygen species (ROS) increased significantly in keratinocytes after 2 h of DK223 exposure, returning to normal levels after 24 h, which indicated that DK223 had an early shock effect on ROS production. DK223 also stimulated fibroblast proliferation, and induced significant secretion of ECM proteins, such as collagen I, III, and fibronectin. In dermal fibroblasts, DK223 treatment induced TGF-β1, which is involved in a signaling pathway that mediates proliferation. In conclusion, DK223 simultaneously induced both keratinocyte migration via ROS production and fibroblast proliferation via TGF-β1 induction. PMID:25056546

  20. TLR3 drives IRF6-dependent IL-23p19 expression and p19/EBI3 heterodimer formation in keratinocytes.

    PubMed

    Ramnath, Divya; Tunny, Kathryn; Hohenhaus, Daniel M; Pitts, Claire M; Bergot, Anne-Sophie; Hogarth, P Mark; Hamilton, John A; Kapetanovic, Ronan; Sturm, Richard A; Scholz, Glen M; Sweet, Matthew J

    2015-10-01

    Interferon regulatory factor (IRF) family members impart cell-type specificity to toll-like receptor (TLR) signalling, and we recently identified a role for IRF6 in TLR2 signalling in epithelial cells. TLR3 has a well-characterized role in wound healing in the skin, and here, we examined TLR3-dependent IRF6 functions in human keratinocytes. Primary keratinocytes responded robustly to the TLR3 agonist poly(IC) with upregulation of mRNAs for interferon-β (IFN-β), the interleukin-12 (IL-12) family member IL-23p19 and the chemokines IL-8 and chemokine (C-C motif) ligand 5 (CCL5). Silencing of IRF6 expression enhanced poly(IC)-inducible IFN-β mRNA levels and inhibited poly(IC)-inducible IL-23p19 mRNA expression in primary keratinocytes. Consistent with these data, co-transfection of IRF6 increased poly(IC)-inducible IL-23p19 promoter activity, but inhibited poly(IC)-inducible IFN-β promoter activity in reporter assays. Surprisingly, poly(IC) did not regulate IL-12p40 expression in keratinocytes, suggesting that TLR3-inducible IL-23p19 may have an IL-23-independent function in these cells. The only other IL-12 family member that was strongly poly(IC) inducible was EBI3, which has not been shown to heterodimerize with IL-23p19. Both co-immunoprecipitation and proximity ligation assays revealed that IL-23p19 and EBI3 interact in cells. Co-expression of IL-23p19 and EBI3, as compared with IL-23p19 alone, resulted in increased levels of secreted IL-23p19, implying a functional role for this heterodimer. In summary, we report that IRF6 regulates a subset of TLR3 responses in human keratinocytes, including the production of a novel IL-12 family heterodimer (p19/EBI3). We propose that the TLR3-IRF6-p19/EBI3 axis may regulate keratinocyte and/or immune cell functions in the context of cell damage and wound healing in the skin. PMID:26303210

  1. Absence of premature senescence in Werner's syndrome keratinocytes.

    PubMed

    Ibrahim, Badr; Sheerin, Angela N; Jennert-Burston, Katrin; Bird, Joe L E; Massala, M V; Illsley, Matthew; James, S Elizabeth; Faragher, Richard G A

    2016-10-01

    Werner's syndrome (WS) is an autosomal recessive genetic disorder caused by loss of function mutation in wrn and is a useful model of premature in vivo ageing. Cellular senescence is a plausible causal mechanism of mammalian ageing and, at the cellular level, WS fibroblasts show premature senescence resulting from a combination of telomeric attrition and replication fork stalling. Over 90% of WS fibroblast cultures achieve <20 population doublings (PD) in vitro compared to wild type human fibroblast cultures. It has been proposed that some cell types, capable of proliferation, will fail to show a premature senescence phenotype in response to wrn mutations. To test this hypothesis, human dermal keratinocytes (derived from both WS and wild type patients) were cultured long term. WS Keratinocytes showed a replicative lifespan in excess of 100 population doublings but maintained functional growth arrest mechanisms based on p16 and p53. The karyotype of the cells was superficially normal and the cultures retained markers characteristic of keratinocyte holoclones (stem cells) including p63 expression and telomerase activity. Accordingly we conclude that, in contrast to WS fibroblasts, WS keratinocytes do not demonstrate slow growth rates or features of premature senescence. These findings suggest that the epidermis is among the tissue types that do not display symptoms of premature ageing caused by loss of function of wrn. This is in support that Werner's syndrome is a segmental progeroid syndrome. PMID:27492502

  2. Keratinocytes can modulate and directly initiate nociceptive responses

    PubMed Central

    Baumbauer, Kyle M; DeBerry, Jennifer J; Adelman, Peter C; Miller, Richard H; Hachisuka, Junichi; Lee, Kuan Hsien; Ross, Sarah E; Koerber, H Richard; Davis, Brian M; Albers, Kathryn M

    2015-01-01

    How thermal, mechanical and chemical stimuli applied to the skin are transduced into signals transmitted by peripheral neurons to the CNS is an area of intense study. Several studies indicate that transduction mechanisms are intrinsic to cutaneous neurons and that epidermal keratinocytes only modulate this transduction. Using mice expressing channelrhodopsin (ChR2) in keratinocytes we show that blue light activation of the epidermis alone can produce action potentials (APs) in multiple types of cutaneous sensory neurons including SA1, A-HTMR, CM, CH, CMC, CMH and CMHC fiber types. In loss of function studies, yellow light stimulation of keratinocytes that express halorhodopsin reduced AP generation in response to naturalistic stimuli. These findings support the idea that intrinsic sensory transduction mechanisms in epidermal keratinocytes can directly elicit AP firing in nociceptive as well as tactile sensory afferents and suggest a significantly expanded role for the epidermis in sensory processing. DOI: http://dx.doi.org/10.7554/eLife.09674.001 PMID:26329459

  3. Dental metal-induced innate reactivity in keratinocytes.

    PubMed

    Rachmawati, Dessy; Buskermolen, Jeroen K; Scheper, Rik J; Gibbs, Susan; von Blomberg, B Mary E; van Hoogstraten, Ingrid M W

    2015-12-25

    Gold, nickel, copper and mercury, i.e. four metals frequently used in dental applications, were explored for their capacity to induce innate immune activation in keratinocytes (KC). Due to their anatomical location the latter epithelial cells are key in primary local irritative responses of skin and mucosa. Fresh foreskin-derived keratinocytes and skin and gingiva KC cell lines were studied for IL-8 release as a most sensitive parameter for NF-kB activation. First, we verified that viral-defense mediating TLR3 is a key innate immune receptor in both skin- and mucosa derived keratinocytes. Second, we found that, in line with our earlier finding that ionized gold can mimic viral dsRNA in triggering TLR3, gold is very effective in KC activation. It would appear that epithelial TLR3 can play a key role in both skin- and mucosa localized irritation reactivities to gold. Subsequently we found that not only gold, but also nickel, copper and mercury salts can activate innate immune reactivity in keratinocytes, although the pathways involved remain unclear. Although current alloys have been optimized for minimal leakage of metal ions, secondary factors such as mechanical friction and acidity may still facilitate such leakage. Subsequently, these metal ions may create local irritation, itching and swelling by triggering innate immune reactions, potentially also facilitating the development of metal specific adaptive immunity. PMID:26456670

  4. The Galvanotactic Migration of Keratinocytes is Enhanced by Hypoxic Preconditioning

    PubMed Central

    Guo, Xiaowei; Jiang, Xupin; Ren, Xi; Sun, Huanbo; Zhang, Dongxia; Zhang, Qiong; Zhang, Jiaping; Huang, Yuesheng

    2015-01-01

    The endogenous electric field (EF)-directed migration of keratinocytes (galvanotaxis) into wounds is an essential step in wound re-epithelialization. Hypoxia, which occurs immediately after injury, acts as an early stimulus to initiate the healing process; however, the mechanisms for this effect, remain elusive. We show here that the galvanotactic migration of keratinocytes was enhanced by hypoxia preconditioning as a result of the increased directionality rather than the increased motility of keratinocytes. This enhancement was both oxygen tension- and preconditioning time-dependent, with the maximum effects achieved using 2% O2 preconditioning for 6 hours. Hypoxic preconditioning (2% O2, 6 hours) decreased the threshold voltage of galvanotaxis to < 25 mV/mm, whereas this value was between 25 and 50 mV/mm in the normal culture control. In a scratch-wound monolayer assay in which the applied EF was in the default healing direction, hypoxic preconditioning accelerated healing by 1.38-fold compared with the control conditions. Scavenging of the induced ROS by N-acetylcysteine (NAC) abolished the enhanced galvanotaxis and the accelerated healing by hypoxic preconditioning. Our data demonstrate a novel and unsuspected role of hypoxia in supporting keratinocyte galvanotaxis. Enhancing the galvanotactic response of cells might therefore be a clinically attractive approach to induce improved wound healing. PMID:25988491

  5. Desmoglein 3-Dependent Signaling Regulates Keratinocyte Migration and Wound Healing.

    PubMed

    Rötzer, Vera; Hartlieb, Eva; Winkler, Julia; Walter, Elias; Schlipp, Angela; Sardy, Miklós; Spindler, Volker; Waschke, Jens

    2016-01-01

    The desmosomal transmembrane adhesion molecules desmoglein 3 (Dsg3) and desmocollin 3 (Dsc3) are required for strong keratinocyte cohesion. Recently, we have shown that Dsg3 associates with p38 mitogen-activated protein kinase (p38MAPK) and suppresses its activity. Here, we further investigated the role of Dsg3-dependent control of p38MAPK function. Dsg3-deficient mice display recurrent spontaneously healing skin erosions. In lesional and perilesional biopsies, p38MAPK activation was detectable compared with control animals. This led us to speculate that Dsg3 regulates wound repair in a p38MAPK-dependent manner. Indeed, scratch-wounded keratinocyte monolayers exhibited p38MAPK activation and loss of Dsg3 in cells lining the wound edge. Human keratinocytes after silencing of Dsg3 as well as primary cells isolated from Dsg3 knockout animals exhibited accelerated migration, which was further corroborated in an ex vivo skin outgrowth assay. Importantly, migration was efficiently blocked by inhibition of p38MAPK, indicating that p38MAPK mediates the effects observed upon loss of Dsg3. In line with this, we show that levels of active p38MAPK associated with Dsc3 are increased in Dsg3-deficient cells. These data indicate that Dsg3 controls a switch from an adhesive to a migratory keratinocyte phenotype via p38MAPK inhibition. Thus, loss of Dsg3 adhesion may foster wound closure by allowing p38MAPK-dependent migration. PMID:26763450

  6. Basal and stress-inducible expression of HSPA6 in human keratinocytes is regulated by negative and positive promoter regions.

    PubMed

    Ramirez, Vincent P; Stamatis, Michael; Shmukler, Anastasia; Aneskievich, Brian J

    2015-01-01

    Epidermal keratinocytes serve as the primary barrier between the body and environmental stressors. They are subjected to numerous stress events and are likely to respond with a repertoire of heat shock proteins (HSPs). HSPA6 (HSP70B') is described in other cell types with characteristically low to undetectable basal expression, but is highly stress induced. Despite this response in other cells, little is known about its control in keratinocytes. We examined endogenous human keratinocyte HSPA6 expression and localized some responsible transcription factor sites in a cloned HSPA6 3 kb promoter. Using promoter 5' truncations and deletions, negative and positive regulatory regions were found throughout the 3 kb promoter. A region between -346 and -217 bp was found to be crucial to HSPA6 basal expression and stress inducibility. Site-specific mutations and DNA-binding studies show that a previously uncharacterized AP1 site contributes to the basal expression and maximal stress induction of HSPA6. Additionally, a new heat shock element (HSE) within this region was defined. While this element mediates increased transcriptional response in thermally stressed HaCaT keratinocytes, it preferentially binds a stress-inducible factor other than heat shock factor (HSF)1 or HSF2. Intriguingly, this newly characterized HSPA6 HSE competes HSF1 binding a consensus HSE and binds both HSF1 and HSF2 from other epithelial cells. Taken together, our results demonstrate that the HSPA6 promoter contains essential negative and positive promoter regions and newly identified transcription factor targets, which are key to the basal and stress-inducible expression of HSPA6. Furthermore, these results suggest that an HSF-like factor may preferentially bind this newly identified HSPA6 HSE in HaCaT cells. PMID:25073946

  7. Differential miRNA expression profiles in proliferating or differentiated keratinocytes in response to gamma irradiation

    PubMed Central

    2013-01-01

    Background MicroRNAs (miRNAs), a group of short non-coding RNAs that negatively regulate gene expression, have recently emerged as potential modulators of cellular response to ionizing radiations both in vitro and in vivo in various cell types and tissues. However, in epidermal cells, the involvement of the miRNA machinery in the cellular response to ionizing radiations remains to be clarified. Indeed, understanding the mechanisms of cutaneous radiosensitivity is an important issue since skin is the most exposed organ to ionizing radiations and among the most sensitive. Results We settled up an expression study of miRNAs in primary human skin keratinocytes using a microfluidic system of qPCR assay, which permits to assess the expression of almost 700 annotated miRNAs. The keratinocytes were cultured to a proliferative or a differentiated state mimicking basal or suprabasal layers of human epidermis. These cells were irradiated at 10 mGy or 6 Gy and RNA was extracted 3 hours after irradiation. We found that proliferative cells irradiated at 6 Gy display a global fall of miRNA expression whereas differentiated cells exposed to the same dose display a global increase of miRNAs expression. We identified twenty miRNAs weakly but significantly modulated after 6 Gy irradiation, whereas only 2 miRNAs were modulated after low-dose irradiation in proliferating cells. To go further into the biological meaning of this miRNA response, we over-expressed some of the responding miRNA in proliferating cells: we observed a significant decrease of cell viability 72 hours after irradiation. Functional annotation of their predicted targets revealed that G-protein related pathways might be regulated by these responding miRNAs. Conclusions Our results reveal that human primary keratinocytes exposed to ionizing irradiation expressed a miRNA pattern strongly related to the differentiation status of irradiated cells. We also demonstrate that some miRNAs play a role in the radiation

  8. cdk4 Deficiency Inhibits Skin Tumor Development but Does Not Affect Normal Keratinocyte Proliferation

    PubMed Central

    Rodriguez-Puebla, Marcelo L.; Miliani de Marval, Paula L.; LaCava, Margaret; Moons, David S.; Kiyokawa, Hiroaki; Conti, Claudio J.

    2002-01-01

    Most human tumors have mutations that result in deregulation of the cdk4/cyclin-Ink4-Rb pathway. Overexpression of D-type cyclins or cdk4 and inactivation of Ink4 inhibitors are common in human tumors. Conversely, lack of cyclin D1 expression results in significant reduction in mouse skin and mammary tumor development. However, complete elimination of tumor development was not observed in these models, suggesting that other cyclin/cdk complexes play an important role in tumorigenesis. Here we described the effects of cdk4 deficiency on mouse skin proliferation and tumor development. Cdk4 deficiency resulted in a 98% reduction in the number of tumors generated through the two-stage carcinogenesis model. The absence of cdk4 did not affect normal keratinocyte proliferation and both wild-type and cdk4 knockout epidermis are equally affected after topical treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), resulting in epidermal hyperplasia. In similar fashion, cdk4 knockout keratinocytes proliferated well in an in vivo model of wound-induced proliferation. Biochemical studies in mouse epidermis showed that cdk6 activity increased twofold in cdk4-deficient mice compared to wild-type siblings. These results suggest that therapeutic approaches to inhibit cdk4 activity could provide a target to inhibit tumor development with minimal or no effect in normal tissue. PMID:12163365

  9. The Tumor Necrosis Factor Superfamily Molecule LIGHT Promotes Keratinocyte Activity and Skin Fibrosis

    PubMed Central

    Herro, Rana; Da Silva Antunes, Ricardo; Aguilera, Amelia Roman; Tamada, Koji; Croft, Michael

    2015-01-01

    Several inflammatory diseases including scleroderma and atopic dermatitis display dermal thickening, epidermal hypertrophy, or excessive accumulation of collagen. Factors that might promote these features are of interest for clinical therapy. We previously reported that LIGHT, a TNF superfamily molecule, mediated collagen deposition in the lungs in response to allergen. We therefore tested whether LIGHT might similarly promote collagen accumulation and features of skin fibrosis. Strikingly, injection of recombinant soluble LIGHT into naïve mice, either subcutaneously or systemically, promoted collagen deposition in the skin, and dermal and epidermal thickening. This replicated the activity of bleomycin, an antibiotic that has been previously used in models of scleroderma in mice. Moreover skin fibrosis induced by bleomycin was dependent on endogenous LIGHT activity. The action of LIGHT in vivo was mediated via both of its receptors, HVEM and LTβR, and was dependent on the innate cytokine TSLP and TGF-β. Furthermore, we found that HVEM and LTβR were expressed on human epidermal keratinocytes, and that LIGHT could directly promote TSLP expression in these cells. We reveal an unappreciated activity of LIGHT on keratinocytes and suggest that LIGHT may be an important mediator of skin inflammation and fibrosis in diseases such as scleroderma or atopic dermatitis. PMID:25789702

  10. Induction of micronuclei in mouse keratinocytes by polycyclic aromatic hydrocarbons and n-heterocyclic aromatics

    SciTech Connect

    Krishnan, S.P.; Warshawsky, D.

    1994-12-31

    This research is concerned with the ability of environmental carcinogens, benzo[a]pyrene (BaP), 7,12-dimethylbenz[a]anthracene (DMBA) and 7H-dibenzo(c,g)carbazole (DBC) to induce micronuclei (MN) in mouse keratinocytes. Mouse (HSD:ICR[Br]) keratinocytes (primary cultures) are grown on collagen coated microscope slides/dishes and dosed with the carcinogen (dissolved in 0.5% DMSO) for 12/24 hours. After carcinogen treatment, the medium is replaced with the medium containing cytochalasin-B and the cells are cultures for 72 hours. The binucleate fraction is used as a general indicator of cytotoxicity. MN are scored in 1000 acridine organs stained binucleated cells. Adriamycin, a direct acting compound, is used as a positive control. DBC induces a dose-dependent (0.01-1 {mu}g/ml) increase in MN (13-40.3 MN/1000 BN cells ) over controls (9.7{+-}1.8); N=3). In addition, 1 nmole/ml (256 ng/ml of DMBA induces twice the number of MN over controls (21.5{+-}1.1). In contrast, skin painting of higher non-cytotoxic doses of BaP (12 {mu}g/ml; 24 {mu}g/ml) showed BaP to be a weak inducer of MN over controls. Similar results were obtained in vitro. The lack of a significant response with BaP indicates that negative results in the mouse kerotinocyte MN assay should be interpreted with caution.

  11. Similarity of molecular shape.

    PubMed

    Meyer, A Y; Richards, W G

    1991-10-01

    The similarity of one molecule to another has usually been defined in terms of electron densities or electrostatic potentials or fields. Here it is expressed as a function of the molecular shape. Formulations of similarity (S) reduce to very simple forms, thus rendering the computerised calculation straightforward and fast. 'Elements of similarity' are identified, in the same spirit as 'elements of chirality', except that the former are understood to be variable rather than present-or-absent. Methods are presented which bypass the time-consuming mathematical optimisation of the relative orientation of the molecules. Numerical results are presented and examined, with emphasis on the similarity of isomers. At the extreme, enantiomeric pairs are considered, where it is the dissimilarity (D = 1 - S) that is of consequence. We argue that chiral molecules can be graded by dissimilarity, and show that D is the shape-analog of the 'chirality coefficient', with the simple form of the former opening up numerical access to the latter. PMID:1770379

  12. Sulfur mustard induces the formation of keratin aggregates in human epidermal keratinocytes.

    PubMed

    Dillman, James F; McGary, Kriston L; Schlager, John J

    2003-12-01

    The vesicant sulfur mustard is an alkylating agent that has the capacity to cross-link biological molecules. We are interested in identifying specific proteins that are altered upon sulfur mustard exposure. Keratins are particularly important for the structural integrity of skin, and several genetically inherited blistering diseases have been linked to mutations in keratin 5 and keratin 14. We examined whether sulfur mustard exposure alters keratin biochemistry in cultured human epidermal keratinocytes. Western blotting with specific monoclonal antibodies revealed the formation of stable high-molecular-weight "aggregates" containing keratin 14 and/or keratin 5. These aggregates begin to form within 15 min after sulfur mustard exposure. These aggregates display a complex gel electrophoresis pattern between approximately 100 and approximately 200 kDa. Purification and analysis of these aggregates by one- and two-dimensional gel electrophoresis and mass spectrometry confirmed the presence of keratin 14 and keratin 5 and indicate that at least some of the aggregates are composed of keratin 14-keratin 14, keratin 14-keratin 5, or keratin 5-keratin 5 dimers. These studies demonstrate that sulfur mustard induces keratin aggregation in keratinocytes and support further investigation into the role of keratin aggregation in sulfur mustard-induced vesication. PMID:14644625

  13. High Throughput, High Content Screening for Novel Pigmentation Regulators Using a Keratinocyte/Melanocyte Co-culture System

    PubMed Central

    Lee, Ju Hee; Chen, Hongxiang; Kolev, Vihren; Aull, Katherine H.; Jung, Inhee; Wang, Jun; Miyamoto, Shoko; Hosoi, Junichi; Mandinova, Anna; Fisher, David E.

    2014-01-01

    Skin pigmentation is a complex process including melanogenesis within melanocytes and melanin transfer to the keratinocytes. To develop a comprehensive screening method for novel pigmentation regulators, we used immortalized melanocytes and keratinocytes in co-culture to screen large numbers of compounds. High-throughput screening plates were subjected to digital automated microscopy to quantify the pigmentation via brightfield microscopy. Compounds with pigment suppression were secondarily tested for their effects on expression of MITF and several pigment regulatory genes, and further validated in terms of non-toxicity to keratinocytes/melanocytes and dose dependent activity. The results demonstrate a high-throughput, high-content screening approach, which is applicable to the analysis of large chemical libraries using a co-culture system. We identified candidate pigmentation inhibitors from 4,000 screened compounds including zoxazolamine, 3-methoxycatechol, and alpha-mangostin, which were also shown to modulate expression of MITF and several key pigmentation factors, and are worthy of further evaluation for potential translation to clinical use. PMID:24438532

  14. Identification of avarol derivatives as potential antipsoriatic drugs using an in vitro model for keratinocyte growth and differentiation.

    PubMed

    Amigó, María; Schalkwijk, Joost; Olthuis, Diana; De Rosa, Salvatore; Payá, Miguel; Terencio, María Carmen; Lamme, Evert

    2006-11-17

    Avarol, a marine sesquiterpenoid hydroquinone, and 14 avarol derivatives have shown interesting anti-inflammatory properties in previous studies. In this study, avarol and derivatives were evaluated in high-throughput keratinocyte culture models using cytokeratin 10 and SKALP/Elafin expression as markers for respectively normal and psoriatic differentiation. Avarol and five of its derivatives (5, 10, 13, 14 and 15) were selected for further study. Only 10, 13, 14 and 15 were able to inhibit keratinocyte cell growth. Changes in expression levels of 22 genes were assessed by quantitative real time PCR (qPCR). From these genes, TNFalpha mRNA levels showed the strongest changes. For compound 13, 15 and dithranol (used as a model antipsoriatic drug), a dose-dependent downregulation of TNFalpha mRNA was found. The changes in TNFalpha mRNA were confirmed at the protein level for compound 13. Additionally, this compound was able to reduce also IL-8 and COX-2 mRNA levels and this effect was correlated with a reduction in COX-2 protein expression. The mechanism of action of this compound involves at least the inhibition of NF-kappaB-DNA binding activity. In conclusion, our high-throughput screening models in combination with quantitative assessment of changes in gene expression profiles identified the avarol derivative 13, a benzylamine derivative of avarol at the 4' position of benzoquinone ring, as an interesting anti-psoriatic drug candidate that inhibits keratinocyte cell growth and TNFalpha and COX-2 expression. PMID:16973179

  15. XPC silencing in normal human keratinocytes triggers metabolic alterations that drive the formation of squamous cell carcinomas

    PubMed Central

    Rezvani, Hamid Reza; Kim, Arianna L.; Rossignol, Rodrigue; Ali, Nsrein; Daly, Meaghan; Mahfouf, Walid; Bellance, Nadège; Taïeb, Alain; de Verneuil, Hubert; Mazurier, Frédéric; Bickers, David R.

    2010-01-01

    DNA damage is a well-known initiator of tumorigenesis. Studies have shown that most cancer cells rely on aerobic glycolysis for their bioenergetics. We sought to identify a molecular link between genomic mutations and metabolic alterations in neoplastic transformation. We took advantage of the intrinsic genomic instability arising in xeroderma pigmentosum C (XPC). The XPC protein plays a key role in recognizing DNA damage in nucleotide excision repair, and patients with XPC deficiency have increased incidence of skin cancer and other malignancies. In cultured human keratinocytes, we showed that lentivirus-mediated knockdown of XPC reduced mitochondrial oxidative phosphorylation and increased glycolysis, recapitulating cancer cell metabolism. Accumulation of unrepaired DNA following XPC silencing increased DNA-dependent protein kinase activity, which subsequently activated AKT1 and NADPH oxidase-1 (NOX1), resulting in ROS production and accumulation of specific deletions in mitochondrial DNA (mtDNA) over time. Subcutaneous injection of XPC-deficient keratinocytes into immunodeficient mice led to squamous cell carcinoma formation, demonstrating the tumorigenic potential of transduced cells. Conversely, simultaneous knockdown of either NOX1 or AKT1 blocked the neoplastic transformation induced by XPC silencing. Our results demonstrate that genomic instability resulting from XPC silencing results in activation of AKT1 and subsequently NOX1 to induce ROS generation, mtDNA deletions, and neoplastic transformation in human keratinocytes. PMID:21123941

  16. Genome-Wide DNA Methylation as an Epigenetic Consequence of Epstein-Barr Virus Infection of Immortalized Keratinocytes

    PubMed Central

    Birdwell, Christine E.; Queen, Krista J.; Kilgore, Phillip C. S. R.; Rollyson, Phoebe; Trutschl, Marjan; Cvek, Urska

    2014-01-01

    ABSTRACT The oral cavity is a persistent reservoir for Epstein-Barr virus (EBV) with lifelong infection of resident epithelial and B cells. Infection of these cell types results in distinct EBV gene expression patterns regulated by epigenetic modifications involving DNA methylation and chromatin structure. Regulation of EBV gene expression relies on viral manipulation of the host epigenetic machinery that may result in long-lasting host epigenetic reprogramming. To identify epigenetic events following EBV infection, a transient infection model was established to map epigenetic changes in telomerase-immortalized oral keratinocytes. EBV-infected oral keratinocytes exhibited a predominantly latent viral gene expression program with some lytic or abortive replication. Calcium and methylcellulose-induced differentiation was delayed in EBV-positive clones and in clones that lost EBV compared to uninfected controls, indicating a functional consequence of EBV epigenetic modifications. Analysis of global cellular DNA methylation identified over 13,000 differentially methylated CpG residues in cells exposed to EBV compared to uninfected controls, with CpG island hypermethylation observed at several cellular genes. Although the vast majority of the DNA methylation changes were silent, 65 cellular genes that acquired CpG methylation showed altered transcript levels. Genes with increased transcript levels frequently acquired DNA methylation within the gene body while those with decreased transcript levels acquired DNA methylation near the transcription start site. Treatment with the DNA methyltransferase inhibitor, decitabine, restored expression of some hypermethylated genes in EBV-infected and EBV-negative transiently infected clones. Overall, these observations suggested that EBV infection of keratinocytes leaves a lasting epigenetic imprint that can enhance the tumorigenic phenotype of infected cells. IMPORTANCE Here, we show that EBV infection of oral keratinocytes led to

  17. Interleukin-1β-induced Reduction of CD44 Ser-325 Phosphorylation in Human Epidermal Keratinocytes Promotes CD44 Homomeric Complexes, Binding to Ezrin, and Extended, Monocyte-adhesive Hyaluronan Coats*

    PubMed Central

    Jokela, Tiina; Oikari, Sanna; Takabe, Piia; Rilla, Kirsi; Kärnä, Riikka; Tammi, Markku; Tammi, Raija

    2015-01-01

    The proinflammatory cytokine interleukin-1β (IL-1β) attracts leukocytes to sites of inflammation. One of the recruitment mechanisms involves the formation of extended, hyaluronan-rich pericellular coats on local fibroblasts, endothelial cells, and epithelial cells. In the present work, we studied how IL-1β turns on the monocyte adhesion of the hyaluronan coat on human keratinocytes. IL-1β did not influence hyaluronan synthesis or increase the amount of pericellular hyaluronan in these cells. Instead, we found that the increase in the hyaluronan-dependent monocyte binding was associated with the CD44 of the keratinocytes. Although IL-1β caused a small increase in the total amount of CD44, a more marked impact was the decrease of CD44 phosphorylation at serine 325. At the same time, IL-1β increased the association of CD44 with ezrin and complex formation of CD44 with itself. Treatment of keratinocyte cultures with KN93, an inhibitor of calmodulin kinase 2, known to phosphorylate Ser-325 in CD44, caused similar effects as IL-1β (i.e. homomerization of CD44 and its association with ezrin) and resulted in increased monocyte binding to keratinocytes in a hyaluronan-dependent way. Overexpression of wild type CD44 standard form, but not a corresponding CD44 mutant mimicking the Ser-325-phosphorylated form, was able to induce monocyte binding to keratinocytes. In conclusion, treatment of human keratinocytes with IL-1β changes the structure of their hyaluronan coat by influencing the amount, post-translational modification, and cytoskeletal association of CD44, thus enhancing monocyte retention on keratinocytes. PMID:25809479

  18. Binding of antibodies to the extractable nuclear antigens SS-A/Ro and SS-B/La is induced on the surface of human keratinocytes by ultraviolet light (UVL): Implications for the pathogenesis of photosensitive cutaneous lupus

    SciTech Connect

    Furukawa, F.; Kashihara-Sawami, M.; Lyons, M.B.; Norris, D.A. )

    1990-01-01

    Autoantibodies to the non-histone nucleoprotein antigens SS-A/Ro, SS-B/La, and RNP are highly associated with photosensitive cutaneous lupus erythematosus (LE). In order to better understand the potential mechanisms of ultraviolet (UV) light on photosensitivity in patients with cutaneous LE, we designed immunopathologic in vitro and in vivo experiments to evaluate the effects of UV on the binding of such autoantibodies to the surface of human keratinocytes, one major target of immunologic damage in photosensitive LE. Short-term 2% paraformaldehyde fixation of suspensions of cultured human keratinocytes previously incubated with monospecific antiserum probes enabled the detection of ENA expression on the cell surface by flow-cytometry analysis. UVB light (280-320 nm) induced the binding of monospecific antibody probes for SS-A/Ro and SS-B/La on keratinocytes in a dose-dependent pattern with maximal induction observed at the dose of 200 mJ/cm2 UVB. Binding of SS-A/Ro, SS-B/La, and RNP antibody was augmented strongly, but binding of anti-Sm was very weak. In contrast, UVA (320-400 nm) light had no effect on the induction of binding of these antibody probes. Identical results were seen by standard immunofluorescence techniques. Hydroxyurea-treated keratinocytes showed similar induction of those antigens by UVB irradiation, which suggested that ENA expression on cultured keratinocytes by UVB were cell-cycle independent. Tunicamycin, an inhibitor of glycosylation of proteins, reduced UVB light effect on the SS-A/Ro and SS-B/La antigen's expression. These in vitro FACS analyses revealed that ENA augmentation on the keratinocyte cell surface was dose dependent, UVB dependent, glycosylation dependent, and cell-cycle independent. In vivo ENA augmentation on the keratinocyte surface was examined in suction blister epidermal roofs.

  19. Impaired keratinocyte function on matrix metalloproteinase-1 (MMP-1) damaged collagen

    PubMed Central

    Perone, Patricia; Deming, Monica O’Brien; Warner, Roscoe L.; Aslam, Muhammad N.; Bhagavathula, Narasimharao; Dame, Michael K.; Voorhees, John J.

    2010-01-01

    Healing of superficial skin wounds depends on the proliferation and migration of keratinocytes at the wound margin. When human epidermal keratinocytes were incubated on polymerized type I collagen, they rapidly attached and spread. The cells underwent a proliferative response and, over the subsequent 6-day period, covered the collagen surface with a monolayer of cells. When keratinocytes were plated on collagen that had been fragmented by exposure to matrix metalloproteinase-1 (MMP-1, collagenase-1), the cells attached as readily as to intact collagen but spread more slowly and less completely. Growth was reduced by approximately 50%. Instead of covering the collagen surface, the keratinocytes remained localized to the site of attachment. Keratinocytes on fragmented collagen expressed a more differentiated phenotype as indicated by a higher level of surface E-cadherin. Based on these findings, we suggest that damage to the underlying collagenous matrix may impede efficient keratinocyte function and retard wound closure. PMID:19352688

  20. Subcellular localisation of BAG-1 and its regulation of vitamin D receptor-mediated transactivation and involucrin expression in oral keratinocytes: Implications for oral carcinogenesis

    SciTech Connect

    Lee, San San; Crabb, Simon J.; Janghra, Nari; Carlberg, Carsten; Williams, Ann C.; Cutress, Ramsey I.; Packham, Graham; Hague, Angela

    2007-09-10

    In oral cancers, cytoplasmic BAG-1 overexpression is a marker of poor prognosis. BAG-1 regulates cellular growth, differentiation and survival through interactions with diverse proteins, including the vitamin D receptor (VDR), a key regulator of keratinocyte growth and differentiation. BAG-1 is expressed ubiquitously in human cells as three major isoforms of 50 kDa (BAG-1L), 46 kDa (BAG-1M) and 36 kDa (BAG-1S) from a single mRNA. In oral keratinocytes BAG-1L, but not BAG-1M and BAG-1S, enhanced VDR transactivation in response to 1{alpha},25-dihydroxyvitamin D{sub 3.} BAG-1L was nucleoplasmic and nucleolar, whereas BAG-1S and BAG-1M were cytoplasmic and nucleoplasmic in localisation. Having identified the nucleolar localisation sequence in BAG-1L, we showed that mutation of this sequence did not prevent BAG-1L from potentiating VDR activity. BAG-1L also potentiated transactivation of known vitamin-D-responsive gene promoters, osteocalcin and 24-hydroxylase, and enhanced VDR-dependent transcription and protein expression of the keratinocyte differentiation marker, involucrin. These results demonstrate endogenous gene regulation by BAG-1L by potentiating nuclear hormone receptor function and suggest a role for BAG-1L in 24-hydroxylase regulation of vitamin D metabolism and the cellular response of oral keratinocytes to 1{alpha},25-dihydroxyvitamin D{sub 3}. By contrast to the cytoplasmic BAG-1 isoforms, BAG-1L may act to suppress tumorigenesis.

  1. Neoplastic transformation of immortalized human epidermal keratinocytes by ionizing radiation

    SciTech Connect

    Thraves, P.; Salehi, Z.; Dritschilo, A.; Rhim, J.S. )

    1990-02-01

    Efforts to investigate the progression of events that cause human cells to become neoplastic in response to ionizing radiation have been aided by the development of tissue culture systems of epithelial cells. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus type 12 and simian virus 40 have been transformed by exposure to x-ray irradiation. Such transformants showed morphological alterations, formed colonies in soft agar, and induced carcinomas when transplanted into nude mice, whereas primary human epidermal keratinocytes exposed to radiation in this manner failed to show any evidence of transformation. These findings demonstrate the malignant transformation of human primary epithelial cells in culture by the combined action of a DNA tumor virus and radiation, indicating a multistep process for radiation-induced neoplastic conversion. This in vitro system may be useful as a tool for dissecting the process of radiation-induced neoplastic transformation of human epithelial cells and for detecting previously unreported human oncogenes.

  2. Transcriptional Network of p63 in Human Keratinocytes

    PubMed Central

    Pozzi, Silvia; Zambelli, Federico; Merico, Daniele; Pavesi, Giulio; Robert, Amélie; Maltère, Peggy; Gidrol, Xavier; Mantovani, Roberto; Vigano, M. Alessandra

    2009-01-01

    p63 is a transcription factor required for the development and maintenance of ectodermal tissues in general, and skin keratinocytes in particular. The identification of its target genes is fundamental for understanding the complex network of gene regulation governing the development of epithelia. We report a list of almost 1000 targets derived from ChIP on chip analysis on two platforms; all genes analyzed changed in expression during differentiation of human keratinocytes. Functional annotation highlighted unexpected GO terms enrichments and confirmed that genes involved in transcriptional regulation are the most significant. A detailed analysis of these transcriptional regulators in condition of perturbed p63 levels confirmed the role of p63 in the regulatory network. Rather than a rigid master-slave hierarchical model, our data indicate that p63 connects different hubs involved in the multiple specific functions of the skin. PMID:19390658

  3. [Cytokinetics and keratins of keratinocytes from skin of the elderly].

    PubMed

    Wevers, A; Schulze, H J; Mahrle, G; Steigleder, G K

    1989-11-15

    Regarding the keratin pattern of non-exposed skin, we found no significant qualitative or quantitative differences between 6 old persons (mean age 85 years) and 4 young adults (mean age 20 years). There was, however, a slight increase of proliferation keratins (K6, K16) in aged skin. In non-exposed skin taken from 6 old (mean age 70 years) and 5 young persons (mean age 37 years), longterm primary submersion cultures of keratinocytes did not show any significant differences as far as the classical parameters of growth behavior were concerned (i.e. plating efficiency, cell count, and labeled thymidine incorporation). In accordance with these findings, daily measurements of the thymidine kinase activity in the supernatants revealed discrete but not significant differences between keratinocytes in aged people and those in young persons. PMID:2481907

  4. Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes

    PubMed Central

    Varma, Sandeep R.; Sivaprakasam, Thiyagarajan O.; Mishra, Abheepsa; Kumar, L. M. Sharath; Prakash, N. S.; Prabhu, Sunil; Ramakrishnan, Shyam

    2016-01-01

    Human skin is body’s vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC50 values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H2O2) induced RBC haemolysis (IC50 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H2O2-induced damage; inhibited H2O2 induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations. PMID:26731545

  5. Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes.

    PubMed

    Varma, Sandeep R; Sivaprakasam, Thiyagarajan O; Mishra, Abheepsa; Kumar, L M Sharath; Prakash, N S; Prabhu, Sunil; Ramakrishnan, Shyam

    2016-01-01

    Human skin is body's vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC50 values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H2O2) induced RBC haemolysis (IC50 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H2O2-induced damage; inhibited H2O2 induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations. PMID:26731545

  6. Autophagy in human keratinocytes: an early step of the differentiation?

    PubMed

    Aymard, Elodie; Barruche, Vincent; Naves, Thomas; Bordes, Sylvie; Closs, Brigitte; Verdier, Mireille; Ratinaud, Marie-Hélène

    2011-03-01

    Studies have established that autophagy constitutes an efficient process to recycle cellular components and certain proteins. The phenomenon was demonstrated primarily in response to nutrient starvation, and there are increasing evidences that it is implied in differentiation. Keratinocyte differentiation was going along an activation of lysosomal enzymes and organelle clearance, and terminal steps are sometimes described as a specialized form of cell death leading to corneocytes. We examined whether initiation of the process in human keratinocyte HaCaT involves autophagy. The KSFM™ culture medium was substituted by M199, which contains a low glucose concentration but a high calcium level (known to induce differentiation). Metabolic stress reduced enhanced cell number in G(1) phase, without apoptotic features (ΔΨmt and membrane integrity are unchanged). Morphological changes were associated with a lower integrin ß1 expression and modifications of protein levels involved in keratinocyte differentiation (involucrin, keratin K10 and ΔNp63α). Whereas autophagic signalling was supported by SIRT1 and pAMPK (T172) increase according to time kinetic, which led to the disappearance of mTOR phosphorylated on S2448 residue. The significant Bcl-X(L) level reduction with stress promoted autophagy, by the release of Beclin-1, whereas ATG5-ATG12 and LC3-II that are involved in autophagosome formation were enhanced significantly. Then, the level of lysosomal protein cathepsin B rose to execute autophagy. Kinetic studies established that autophagy would constitute an early signalling process required for keratinocyte commitment in differentiation pathway. PMID:21166723

  7. Epidermal nevi with aberrant epidermal structure in keratinocytes and melanocytes.

    PubMed

    Oiso, Naoki; Sugawara, Koji; Yonamine, Ayano; Tsuruta, Daisuke; Kawada, Akira

    2015-04-01

    Epidermal nevi are congenital cutaneous hamartomas caused by embryonic somatic mutations. Ultrastructural features of adult epidermal nevi have rarely been investigated. Herein, we report a case involving a Japanese adult who had epidermal nevi with right congenital blindness and a right accessory nipple. The histopathologic and ultrastructural studies showed divergent abnormal epidermal structures in both melanocytes and keratinocytes. Our case indicates the need to further investigate histopathologic, ultrastructural, and genetic associations in adult epidermal nevi. PMID:25657059

  8. Exposure to Carbon Nanotube Material: Assessment of Nanotube Cytotoxicity Using Human Keratinocyte Cells

    NASA Technical Reports Server (NTRS)

    Shvedova, Anna A.; Castranova, Vincent; Kisin, Elena R.; Schwegler-Berry, Diane; Murray, Ashley R.; Gandelsman, Vadim Z.; Maynard, Andrew; Baron, Paul

    2003-01-01

    Carbon nanotubes are new members of carbon allotropes similar to fullerenes and graphite. Because of their unique electrical, mechanical, and thermal properties, carbon nanotubes are important for novel applications in the electronics, aerospace, and computer industries. Exposure to graphite and carbon materials has been associated with increased incidence of skin diseases, such as carbon fiber dermatitis, hyperkeratosis, and naevi. We investigated adverse effects of single-wall carbon nanotubes (SWCNT) using a cell culture of immortalized human epidermal keratinocytes (HaCaT). After 18 h of exposure of HaCaT to SWCNT, oxidative stress and cellular toxicity were indicated by formation of free radicals, accumulation of peroxidative products, antioxidant depletion, and loss of cell viability. Exposure to SWCNT also resulted in ultrastructural and morphological changes in cultured skin cells. These data indicate that dermal exposure to unrefined SWCNT may lead to dermal toxicity due to accelerated oxidative stress in the skin of exposed workers.

  9. Enhanced Keratinocyte Proliferation and Migration in Co-culture with Fibroblasts

    PubMed Central

    Wang, Zhenxiang; Wang, Ying; Farhangfar, Farhang; Zimmer, Monica; Zhang, Yongxin

    2012-01-01

    Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days) of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11th to 15th day), keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF), IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage. PMID:22911722

  10. Ultraviolet B suppresses vitamin D receptor gene expression in keratinocytes.

    PubMed

    Courtois, S J; Segaert, S; Degreef, H; Bouillon, R; Garmyn, M

    1998-05-01

    Keratinocytes not only produce vitamin D3 in response to ultraviolet B light (UVB) and convert 25-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D) but also possess the vitamin D receptor (VDR) and respond to 1,25(OH)2D. We characterized the regulation of the expression of the VDR gene in primary human keratinocytes following UVB irradiation. We report a marked dose-dependent down-regulation of the VDR mRNA and protein within a few hours after irradiation. This occurs independently of de novo protein synthesis and is not due to a change in the half-life of the VDR mRNA. Interestingly, treatment of the cells with sodium salicylate, caffeic acid phenethyl ester and tosylphenylchloromethylketone inhibited this down-regulation. Our results strongly suggest the existence of a feedback mechanism in that UVB initiates vitamin D synthesis in keratinocytes and at the same time limits VDR abundance. They also provide a rational explanation for the reported lack of any additive effect between 1,25(OH)2D and UVB phototherapy in the treatment of psoriasis. PMID:9600069