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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 Central

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

    2015-01-01

    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. 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.

  3. Sonicated and stirred copper oxide nanoparticles induce similar toxicity and pro-inflammatory response in N-hTERT keratinocytes and SZ95 sebocytes

    NASA Astrophysics Data System (ADS)

    Piret, Jean-Pascal; Mejia, Jorge; Lucas, Stéphane; Zouboulis, Christos C.; Saout, Christelle; Toussaint, Olivier

    2014-04-01

    The potential toxic and pro-inflammatory effects of rod-shaped copper oxide (CuO) nanoparticles (NPs; 10 ± 3 nm in thickness and 74 ± 17 nm in length) were studied on N-hTERT keratinocytes and SZ95 sebocytes and on reconstructed human epidermis. Non-sonicated and sonicated CuO NPs induced similar cellular toxicity. The toxic effect of CuO NPs (non-sonicated and sonicated) was more pronounced in keratinocytes than in sebocytes. Pro-oxidant effects of CuO NPs were demonstrated by showing increase in the production of reactive oxygen species and decrease of cellular glutathione. In addition, DNA-binding activities suggested that redox-sensitive transcription factors Nrf2 and NF-κB were implicated in the response of keratinocytes to CuO NPs. Transcriptomic analysis showed an increase in the abundance of transcript species coding for pro-inflammatory interleukins (e.g. IL-8 and IL-1α) and chemokines. In reconstituted human epidermis exposed topically to raw CuO NPs, no effect on the integrity, viability and inflammatory response was noticed.

  4. [Peptide-agonist of protease-activated receptor (PAR 1), similar to activated protein C, promotes proliferation in keratinocytes and wound healing of epithelial layer].

    PubMed

    Kiseleva, E V; Sidorova, M V; Gorbacheva, L R; Strukova, S M

    2014-01-01

    Activated protein C (APC) is serine protease hemostasis, independent of its anticoagulant activity, exhibits anti-inflammatory and anti-apoptotic properties that determine the possibility of the protective effects of APC in different diseases, including sepsis and chronic wound healing. APC, binding of endothelial protein C receptor (EPCR) and specifically cleaving PAR1 receptor and releasing peptide agonist PAR1 stabilizes not only endothelial cells, but also many others, including epidermal keratinocytes of the skin. We develop the hypothesis that the cytoprotective effect of APC on the cells, involved in wound healing, seem to imitate peptide - analogous of PAR1 "tethered ligand" that activate PAR1. In our work, we synthesized a peptide (AP9) - analogue of PAR1 tethered ligand, released by APC, and firstly showed that peptide AP9 (0.1-10 мM), like to APC (0.01-100 nM), stimulates the proliferative activity of human primary keratinocytes. Using a model of the formation of epithelial wounds in vitro we found that peptide AP9, as well as protease APC, accelerates wound healing. Using specific antibodies to the receptor PAR1 and EPCR was studied the receptor mechanism of AP9 action in wound healing compared with the action of APС. The necessity of both receptors - PAR1 and EPСR, for proliferative activity of agonists was revealed. Identified in our work imitation by peptide AP9 - PAR1 ligand, APC acts on keratinocytes suggests the possibility of using a peptide AP9 to stimulate tissue repair.

  5. Identifying Similarities in Cognitive Subtest Functional Requirements: An Empirical Approach

    ERIC Educational Resources Information Center

    Frisby, Craig L.; Parkin, Jason R.

    2007-01-01

    In the cognitive test interpretation literature, a Rational/Intuitive, Indirect Empirical, or Combined approach is typically used to construct conceptual taxonomies of the functional (behavioral) similarities between subtests. To address shortcomings of these approaches, the functional requirements for 49 subtests from six individually…

  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. Gene expression profiling to identify markers associated with deregulated hTERT in HPV-transformed keratinocytes and cervical cancer.

    PubMed

    de Wilde, Jillian; Wilting, Saskia M; Meijer, Chris J L M; van de Wiel, Mark A; Ylstra, Bauke; Snijders, Peter J F; Steenbergen, Renske D M

    2008-02-15

    Although high-risk human papillomavirus (HPV) infection plays a major role in the development of cervical cancer, additive oncogenic events are involved as well. One key event involves increased activity of telomerase resulting from a deregulated expression of its catalytic subunit hTERT. Our previous microcell-mediated chromosome transfer studies revealed that introduction of human chromosome 6 in the HPV16-immortalized keratinocyte cell line FK16A and in the HPV16-containing cervical cancer cell line SiHa induced growth arrest, resulting from a repression of hTERT mRNA expression and telomerase activity. Here, this model was used to analyze expression profiles associated with hTERT deregulation in HPV-transformed cells. Microarray expression analysis of 12 FK16A/chromosome 6 hybrids, 4 of which were negative for endogenous hTERT and 8 of which were positive for endogenous hTERT, resulted in the identification of 164 differentially expressed genes. Differential expression of a selection of 5 genes was verified by real-time RT-PCR. Of these 164 genes, 32 were also differentially expressed in other HPV transformed cells with deregulated hTERT. For 2 of these genes, encoding AQP3 and MGP, altered expression in hTERT positive cervical carcinomas was confirmed by real-time RT-PCR and immunohistochemistry, respectively. Moreover, increased MGP protein expression was significantly more frequent in high-grade cervical premalignant lesions with elevated hTERT mRNA expression compared to those without. In summary, we identified 32 candidate biomarkers for deregulated hTERT mRNA expression, which may enable the identification of cervical premalignant lesions that are at highest risk to progress to invasive cancer.

  8. Quantitative keratinocyte assay detects two biological activities of human papillomavirus DNA and identifies viral types associated with cervical carcinoma.

    PubMed Central

    Schlegel, R; Phelps, W C; Zhang, Y L; Barbosa, M

    1988-01-01

    Keratinocytes electroporated with human papillomavirus (HPV) DNA (HPV-6, 11, 16 and 18) exhibited an increased cellular proliferation which was quantitated as microcolony and macrocolony formation. However, only macrocolonies induced by HPV-16 or HPV-18 DNA (the two viral types most commonly found in human cervical carcinomas) gave rise to proliferating, poorly-stratified colonies when grown in the presence of serum and calcium. Hydrocortisone increased the frequency of these differentiation-resistant colonies, and studies showed that they were immortalized, contained one copy of viral DNA per cell, expressed three discrete species of viral RNA and synthesized the viral E7 protein. HPV-induced cellular proliferation and altered differentiation are therefore separable events and may represent the activity of different viral genes. Images PMID:2460337

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

    PubMed Central

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

    2017-01-01

    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/cm2) 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

  10. 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.

  11. Primary structure of keratinocyte transglutaminase.

    PubMed Central

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

    1990-01-01

    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 unrelated 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. Images PMID:1979171

  12. Human papillomavirus type 16 (HPV-16) genomes integrated in head and neck cancers and in HPV-16-immortalized human keratinocyte clones express chimeric virus-cell mRNAs similar to those found in cervical cancers.

    PubMed

    Lace, Michael J; Anson, James R; Klussmann, Jens P; Wang, Dong Hong; Smith, Elaine M; Haugen, Thomas H; Turek, Lubomir P

    2011-02-01

    Many human papillomavirus (HPV)-positive high-grade lesions and cancers of the uterine cervix harbor integrated HPV genomes expressing the E6 and E7 oncogenes from chimeric virus-cell mRNAs, but less is known about HPV integration in head and neck cancer (HNC). Here we compared viral DNA status and E6-E7 mRNA sequences in HPV-16-positive HNC tumors to those in independent human keratinocyte cell clones derived from primary tonsillar or foreskin epithelia immortalized with HPV-16 genomes. Three of nine HNC tumors and epithelial clones containing unintegrated HPV-16 genomes expressed mRNAs spliced from HPV-16 SD880 to SA3358 and terminating at the viral early gene p(A) signal. In contrast, most integrated HPV genomes in six HNCs and a set of 31 keratinocyte clones expressed HPV-16 major early promoter (MEP)-initiated mRNAs spliced from viral SD880 directly to diverse cellular sequences, with a minority spliced to SA3358 followed by a cellular DNA junction. Sequence analysis of chimeric virus-cell mRNAs from HNC tumors and keratinocyte clones identified viral integration sites in a variety of chromosomes, with some located in or near growth control genes, including the c-myc protooncogene and the gene encoding FAP-1 phosphatase. Taken together, these findings support the hypothesis that HPV integration in cancers is a stochastic process resulting in clonal selection of aggressively expanding cells with altered gene expression of integrated HPV genomes and potential perturbations of cellular genes at or near viral integration sites. Furthermore, our results demonstrate that this selection also takes place and can be studied in primary human keratinocytes in culture.

  13. 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.

  14. 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.

  15. Epidermal keratinocytes initiate wound healing and pro-inflammatory immune responses following percutaneous schistosome infection.

    PubMed

    Bourke, Claire D; Prendergast, Catriona T; Sanin, David E; Oulton, Tate E; Hall, Rebecca J; Mountford, Adrian P

    2015-03-01

    Keratinocytes constitute the majority of cells in the skin's epidermis, the first line of defence against percutaneous pathogens. Schistosome larvae (cercariae) actively penetrate the epidermis to establish infection, however the response of keratinocytes to invading cercariae has not been investigated. Here we address the hypothesis that cercariae activate epidermal keratinocytes to promote the development of a pro-inflammatory immune response in the skin. C57BL/6 mice were exposed to Schistosoma mansoni cercariae via each pinna and non-haematopoietic cells isolated from epidermal tissue were characterised for the presence of different keratinocyte sub-sets at 6, 24 and 96 h p.i. We identified an expansion of epidermal keratinocyte precursors (CD45(-), CD326(-), CD34(+)) within 24 h of infection relative to naïve animals. Following infection, cells within the precursor population displayed a more differentiated phenotype (α6integrin(-)) than in uninfected skin. Parallel immunohistochemical analysis of pinnae cryosections showed that this expansion corresponded to an increase in the intensity of CD34 staining, specifically in the basal bulge region of hair follicles of infected mice, and a higher frequency of keratinocyte Ki67(+) nuclei in both the hair follicle and interfollicular epidermis. Expression of pro-inflammatory cytokine and stress-associated keratin 6b genes was also transiently upregulated in the epidermal tissue of infected mice. In vitro exposure of keratinocyte precursors isolated from neonatal mouse skin to excretory/secretory antigens released by penetrating cercariae elicited IL-1α and IL-1β production, supporting a role for keratinocyte precursors in initiating cutaneous inflammatory immune responses. Together, these observations indicate that S.mansoni cercariae and their excretory/secretory products act directly upon epidermal keratinocytes, which respond by initiating barrier repair and pro-inflammatory mechanisms similar to those

  16. Hyaluronan Does Not Regulate Human Epidermal Keratinocyte Proliferation and Differentiation.

    PubMed

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

    2016-03-18

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

  17. Pathway-specific profiling identifies the NF-kappa B-dependent tumor necrosis factor alpha-regulated genes in epidermal keratinocytes.

    PubMed

    Banno, Tomohiro; Gazel, Alix; Blumenberg, Miroslav

    2005-05-13

    Identification of tumor necrosis factor alpha (TNF alpha) as the key agent in inflammatory disorders led to new therapies specifically targeting TNF alpha and avoiding many side effects of earlier anti-inflammatory drugs. However, because of the wide spectrum of systems affected by TNF alpha, drugs targeting TNF alpha have a potential risk of delaying wound healing, secondary infections, and cancer. Indeed, increased risks of tuberculosis and carcinogenesis have been reported as side effects after anti-TNF alpha therapy. TNF alpha regulates many processes (e.g. immune response, cell cycle, and apoptosis) through several signal transduction pathways that convey the TNF alpha signals to the nucleus. Hypothesizing that specific TNF alpha-dependent pathways control specific processes and that inhibition of a specific pathway may yield even more precisely targeted therapies, we used oligonucleotide microarrays and parthenolide, an NF-kappa B-specific inhibitor, to identify the NF-kappa B-dependent set of the TNF alpha-regulated genes in human epidermal keratinocytes. Expression of approximately 40% of all TNF alpha-regulated genes depends on NF-kappa B; 17% are regulated early (1-4 h post-treatment), and 23% are regulated late (24-48 h). Cytokines and apoptosis-related and cornification proteins belong to the "early" NF-kappa B-dependent group, and antigen presentation proteins belong to the "late" group, whereas most cell cycle, RNA-processing, and metabolic enzymes are not NF-kappa B-dependent. Therefore, inflammation, immunomodulation, apoptosis, and differentiation are on the NF-kappa B pathway, and cell cycle, metabolism, and RNA processing are not. Most early genes contain consensus NF-kappaB binding sites in their promoter DNA and are, presumably, directly regulated by NF-kappa B, except, curiously, the cornification markers. Using siRNA silencing, we identified cFLIP/CFLAR as an essential NF-kappa B-dependent antiapoptotic gene. The results confirm our

  18. A combined assay of hTERT and E6 oncoprotein to identify virus-infected keratinocytes with higher telomerase activity in human papillomaviruses 16 and 18-related bowenoid papulosis.

    PubMed

    Li, Dongsheng; Dong, Bilin; Hu, Zhimin; Chen, Liuqing; Zeng, Xianyu; Chen, Jinbo; Duan, Yiqun

    2012-12-01

    In the present study, we aim to evaluate the application potential of a combined assay of human telomerase reverse transcriptase (hTERT) and E6 oncoprotein in screening the virus-infected keratinocytes with higher telomerase activity in human papillomaviruses (HPV) 16- and 18-related bowenoid papulosis (BP). HPV16/18 DNA in BP (n = 123) was identified by in situ hybridization, the expression of hTERT and E6 in HPV16/18-related BP (n = 68) was determined by immunohistochemistry. We demonstrated that the expression of hTERT correlated well with that of E6 oncoprotein in HPV16/18-related BP lesions (Spearman rho = 0.868, P < 0.01). Furthermore, the majority of keratinocytes with positive nuclear staining for hTERT or E6 in the consecutive sections of each HPV16/18-related BP lesion showed nuclear paleomorphism or nuclear mitosis. In conclusion, we suggested that a combined assay of hTERT and E6 oncoprotein can be used to screen the HPV-infected keratinocytes with higher telomerase activity in HPV16-related and HPV18-related BP lesions.

  19. The waveform similarity approach to identify dependent events in instrumental seismic catalogues

    NASA Astrophysics Data System (ADS)

    Barani, S.; Ferretti, G.; Massa, M.; Spallarossa, D.

    2007-01-01

    In this paper, waveform similarity analysis is adapted and implemented in a declustering procedure to identify foreshocks and aftershocks, to obtain instrumental catalogues that are cleaned of dependent events and to perform an independent check of the results of traditional declustering techniques. Unlike other traditional declustering methods (i.e. windowing techniques), the application of cross-correlation analysis allows definition of groups of dependent events (multiplets) characterized by similar location, fault mechanism and propagation pattern. In this way the chain of intervening related events is led by the seismogenetic features of earthquakes. Furthermore, a time-selection criterion is used to define time-independent seismic episodes eventually joined (on the basis of waveform similarity) into a single multiplet. The results, obtained applying our procedure to a test data set, show that the declustered catalogue is drawn by the Poisson distribution with a degree of confidence higher than using the Gardner and Knopoff method. The declustered catalogues, applying these two approaches, are similar with respect to the frequency-magnitude distribution and the number of earthquakes. Nevertheless, the application of our approach leads to declustered catalogues properly related to the seismotectonic background and the reology of the investigated area and the success of the procedure is ensured by the independence of the results on estimated location errors of the events collected in the raw catalogue.

  20. Pollution trees: identifying similarities among complex pollutant mixtures in water and correlating them to mutagenicity.

    PubMed

    Zheng, Weiwei; Wang, Xia; Tian, Dajun; Zhang, Hao; Tian, Weidong; Andersen, Melvin E; Zheng, Yuxin; Sun, Xin; Jiang, Songhui; Cao, Zhaojin; He, Gengsheng; Qu, Weidong

    2012-07-03

    There are relatively few tools available for computing and visualizing similarities among complex mixtures and in correlating the chemical composition clusters with toxicological clusters of mixtures. Using the "intersection and union ratio (IUR)" and other traditional distance matrices on contaminant profiles of 33 specific water samples, we used "pollution trees" to compare these mixtures. The "pollution trees" constructed by neighbor-joining (NJ), maximum parsimony (MP), and maximum likelihood (ML) methods allowed comparison of similarities among these samples. The mutagenicity of each sample was then mapped to the "pollution tree". The IUR-distance-based measure proved effective in comparing chemical composition and compound level differences between mixtures. We found a robust "pollution tree" containing seven major lineages with certain broad characteristics: treated municipal water samples were different from raw water samples and untreated rural drinking water samples were similar with local water sources. The IUR-distance-based tree was more highly correlated to mutagenicity than were other distance matrices, i.e., MP/ML methods, sampling group, region, or water type. IUR-distance-based "pollution trees" may become important tools for identifying similarities among real mixtures and examining chemical composition clusters in a toxicological context.

  1. 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

  2. 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.

  3. 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

  4. Interferon regulatory factor 6 regulates keratinocyte migration

    PubMed Central

    Biggs, Leah C.; Naridze, Rachelle L.; DeMali, Kris A.; Lusche, Daniel F.; Kuhl, Spencer; Soll, David R.; Schutte, Brian C.; Dunnwald, Martine

    2014-01-01

    ABSTRACT Interferon regulatory factor 6 (Irf6) regulates keratinocyte proliferation and differentiation. In this study, we tested the hypothesis that Irf6 regulates cellular migration and adhesion. Irf6-deficient embryos at 10.5 days post-conception failed to close their wound compared with wild-type embryos. In vitro, Irf6-deficient murine embryonic keratinocytes were delayed in closing a scratch wound. Live imaging of the scratch showed deficient directional migration and reduced speed in cells lacking Irf6. To understand the underlying molecular mechanisms, cell–cell and cell–matrix adhesions were investigated. We show that wild-type and Irf6-deficient keratinocytes adhere similarly to all matrices after 60 min. However, Irf6-deficient keratinocytes were consistently larger and more spread, a phenotype that persisted during the scratch-healing process. Interestingly, Irf6-deficient keratinocytes exhibited an increased network of stress fibers and active RhoA compared with that observed in wild-type keratinocytes. Blocking ROCK, a downstream effector of RhoA, rescued the delay in closing scratch wounds. The expression of Arhgap29, a Rho GTPase-activating protein, was reduced in Irf6-deficient keratinocytes. Taken together, these data suggest that Irf6 functions through the RhoA pathway to regulate cellular migration. PMID:24777480

  5. 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

  6. 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

  7. Fragment Finder: a web-based software to identify similar three-dimensional structural motif

    PubMed Central

    Ananthalakshmi, P.; Kumar, Ch. Kiran; Jeyasimhan, M.; Sumathi, K.; Sekar, K.

    2005-01-01

    FF (Fragment Finder) is a web-based interactive search engine developed to retrieve the user-desired similar 3D structural fragments from the selected subset of 25 or 90% non-homologous protein chains. The search is based on the comparison of the main chain backbone conformational angles (φ and ϕ). Additionally, the queried motifs can be superimposed to find out how similar the structural fragments are, so that the information can be effectively used in molecular modeling. The engine has facilities to view the resultant superposed or individual 3D structure(s) on the client machine. The proposed web server is made freely accessible at the following URL: or . PMID:15980587

  8. 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.

  9. Similarities and Differences between Children with and without Disabilities on Identified Clinical Findings

    ERIC Educational Resources Information Center

    Reinke, Diane C.

    2005-01-01

    This study was conducted to examine the types and proportions of identified clinical findings among children with and without disabilities. Using data from the Canadian Incidence Study of Reported Child Abuse and Neglect (CIS), this study compared 7672 children aged 0 to 15 years (n=1067 with disabilities and n=6605 without disabilities) who were…

  10. DNAc: A clustering method for identifying kinship relations between DNA profiles using a novel similarity measure.

    PubMed

    Ntwari, Aimé; Kelil, Abdellali; Drouin, Régen; Monga, Ernest; Wang, Shengrui; Brzezinski, Ryszard; Bronsard, Marc; Yan, Ju

    2011-01-01

    After decades of refinement, DNA testing methods have become essential tools in forensic sciences. They are essentially based on likelihood ratio test principle, which is utilized specifically, by using as prior knowledge the allele frequencies in the population, to confirm or refute a given kinship hypothesis made on two genotypes. This makes these methods ill suited when allele frequencies or kinship hypotheses are unavailable. In this paper, we introduce DNAc, a new clustering methodology for DNA testing based on a new similarity measure that allows an accurate retrieval of the degree of relatedness among two or more genotypes, without relying on kinship hypotheses or allele frequencies in the population. We used DNAc in analyzing microsatellite DNA sequences distributed among 12 genotypes from normal individuals from two distinct families. The results show that DNAc accurately determines kinship among genotypes and further gathers them in the appropriate kinship groups.

  11. 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.

  12. Autoantibody profiles in autoimmune hepatitis and chronic hepatitis C identifies similarities in patients with severe disease

    PubMed Central

    Amin, Kawa; Rasool, Aram H; Hattem, Ali; Al-Karboly, Taha AM; Taher, Taher E; Bystrom, Jonas

    2017-01-01

    AIM To determine how the auto-antibodies (Abs) profiles overlap in chronic hepatitis C infection (CHC) and autoimmune hepatitis (AIH) and correlate to liver disease. METHODS Levels of antinuclear Ab, smooth muscle antibody (SMA) and liver/kidney microsomal-1 (LKM-1) Ab and markers of liver damage were determined in the sera of 50 patients with CHC infection, 20 AIH patients and 20 healthy controls using enzyme linked immunosorbent assay and other immune assays. RESULTS We found that AIH patients had more severe liver disease as determined by elevation of total IgG, alkaline phosphatase, total serum bilirubin and serum transaminases and significantly higher prevalence of the three non-organ-specific autoantibodies (auto-Abs) than CHC patients. Antinuclear Ab, SMA and LKM-1 Ab were also present in 36% of CHC patients and related to disease severity. CHC cases positive for auto-Abs were directly comparable to AIH in respect of most markers of liver damage and total IgG. These cases had longer disease duration compared with auto-Ab negative cases, but there was no difference in gender, age or viral load. KLM-1+ Ab CHC cases showed best overlap with AIH. CONCLUSION Auto-Ab levels in CHC may be important markers of disease severity and positive cases have a disease similar to AIH. Auto-Abs might have a pathogenic role as indicated by elevated markers of liver damage. Future studies will unravel any novel associations between these two diseases, whether genetic or other. PMID:28293081

  13. 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

  14. 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.

  15. 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

  16. Large-scale chemical similarity networks for target profiling of compounds identified in cell-based chemical screens.

    PubMed

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

    2015-03-01

    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/).

  17. A mammary stem cell population identified and characterized in late embryogenesis reveals similarities to human breast cancer.

    PubMed

    Spike, Benjamin T; Engle, Dannielle D; Lin, Jennifer C; Cheung, Samantha K; La, Justin; Wahl, Geoffrey M

    2012-02-03

    Gene expression signatures relating mammary stem cell populations to breast cancers have focused on adult tissue. Here, we identify, isolate, and characterize the fetal mammary stem cell (fMaSC) state since the invasive and proliferative processes of mammogenesis resemble phases of cancer progression. fMaSC frequency peaks late in embryogenesis, enabling more extensive stem cell purification than achieved with adult tissue. fMaSCs are self-renewing, multipotent, and coexpress multiple mammary lineage markers. Gene expression, transplantation, and in vitro analyses reveal putative autocrine and paracrine regulatory mechanisms, including ErbB and FGF signaling pathways impinging on fMaSC growth. Expression profiles from fMaSCs and associated stroma exhibit significant similarities to basal-like and Her2+ intrinsic breast cancer subtypes. Our results reveal links between development and cancer and provide resources to identify new candidates for diagnosis, prognosis, and therapy.

  18. NCR1 Expression Identifies Canine Natural Killer Cell Subsets with Phenotypic Similarity to Human Natural Killer Cells

    PubMed Central

    Foltz, Jennifer A.; Somanchi, Srinivas S.; Yang, Yanwen; Aquino-Lopez, Arianexys; Bishop, Erin E.; Lee, Dean A.

    2016-01-01

    Canines spontaneously develop many cancers similar to humans – including osteosarcoma, leukemia, and lymphoma – offering the opportunity to study immune therapies in a genetically heterogeneous and immunocompetent environment. However, a lack of antibodies recognizing canine NK cell markers has resulted in suboptimal characterization and unknown purity of NK cell products, hindering the development of canine models of NK cell adoptive immunotherapy. To this end, we generated a novel antibody to canine NCR1 (NKp46), the putative species-wide marker of NK cells, enabling purification of NK cells for further characterization. We demonstrate that CD3−/NKp46+ cells in healthy and osteosarcoma-bearing canines have phenotypic similarity to human CD3−/NKp46+ NK cells, expressing mRNA for CD16 and the natural cytotoxicity receptors NKp30, NKp44, and NKp80. Functionally, we demonstrate with the calcein release assay that canine CD3−/NKp46+ cells kill canine tumor cell lines without prior sensitization and secrete IFN-γ, TNF-α, IL-8, IL-10, and granulocyte-macrophage colony-stimulating factor as measured by Luminex. Similar to human NK cells, CD3−/NKp46+ cells expand rapidly on feeder cells expressing 4-1BBL and membrane-bound IL-21 (median = 20,283-fold in 21 days). Furthermore, we identify a minor Null population (CD3−/CD21−/CD14−/NKp46−) with reduced cytotoxicity against osteosarcoma cells, but similar cytokine secretion as CD3−/NKp46+ cells. Null cells in canines and humans have reduced expression of NKG2D, NKp44, and CD16 compared to NKp46+ NK cells and can be induced to express NKp46 with further expansion on feeder cells. In conclusion, we have identified and characterized canine NK cells, including an NKp46− subset of canine and human NK cells, using a novel anti-canine NKp46 antibody, and report robust ex vivo expansion of canine NK cells sufficient for adoptive immunotherapy. PMID:27933061

  19. A Systematic In Silico Search for Target Similarity Identifies Several Approved Drugs with Potential Activity against the Plasmodium falciparum Apicoplast

    PubMed Central

    Bispo, Nadlla Alves; Culleton, Richard; Silva, Lourival Almeida; Cravo, Pedro

    2013-01-01

    Most of the drugs in use against Plasmodium falciparum share similar modes of action and, consequently, there is a need to identify alternative potential drug targets. Here, we focus on the apicoplast, a malarial plastid-like organelle of algal source which evolved through secondary endosymbiosis. We undertake a systematic in silico target-based identification approach for detecting drugs already approved for clinical use in humans that may be able to interfere with the P. falciparum apicoplast. The P. falciparum genome database GeneDB was used to compile a list of ≈600 proteins containing apicoplast signal peptides. Each of these proteins was treated as a potential drug target and its predicted sequence was used to interrogate three different freely available databases (Therapeutic Target Database, DrugBank and STITCH3.1) that provide synoptic data on drugs and their primary or putative drug targets. We were able to identify several drugs that are expected to interact with forty-seven (47) peptides predicted to be involved in the biology of the P. falciparum apicoplast. Fifteen (15) of these putative targets are predicted to have affinity to drugs that are already approved for clinical use but have never been evaluated against malaria parasites. We suggest that some of these drugs should be experimentally tested and/or serve as leads for engineering new antimalarials. PMID:23555651

  20. 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.

  1. 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

  2. Transcriptional profiling of epidermal keratinocytes: comparison of genes expressed in skin, cultured keratinocytes, and reconstituted epidermis, using large DNA microarrays.

    PubMed

    Gazel, Alix; Ramphal, Patricia; Rosdy, Martin; De Wever, Bart; Tornier, Carine; Hosein, Nadia; Lee, Brian; Tomic-Canic, Marjana; Blumenberg, Miroslav

    2003-12-01

    Epidermal keratinocytes are complex cells that create a unique three-dimensional (3-D) structure, differentiate through a multistage process, and respond to extracellular stimuli from nearby cells. Consequently, keratinocytes express many genes, i.e., have a relatively large "transcriptome." To determine which of the expressed genes are innate to keratinocytes, which are specific for the differentiation and 3-D architecture, and which are induced by other cell types, we compared the transcriptomes of skin from human subjects, differentiating 3-D reconstituted epidermis, cultured keratinocytes, and nonkeratinocyte cell types. Using large oligonucleotide microarrays, we analyzed five or more replicates of each, which yielded statistically consistent data and allowed identification of the differentially expressed genes. Epidermal keratinocytes, unlike other cells, express many proteases and protease inhibitors and genes that protect from UV light. Skin specifically expresses a higher number of receptors, secreted proteins, and transcription factors, perhaps influenced by the presence of nonkeratinocyte cell types. Surprisingly, mitochondrial proteins were significantly suppressed in skin, suggesting a low metabolic rate. Three-dimensional samples, skin and reconstituted epidermis, are similar to each other, expressing epidermal differentiation markers. Cultured keratinocytes express many cell-cycle and DNA replication genes, as well as integrins and extracellular matrix proteins. These results define innate, architecture-specific, and cell-type-regulated genes in epidermis.

  3. 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…

  4. 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.

  5. Identifying Potential Protein Targets for Toluene Using a Molecular Similarity Search, in Silico Docking and in Vitro Validation

    DTIC Science & Technology

    2015-01-01

    the applicability of an exploratory in silico toxicity tool, based on a molecular similarity search and protein-ligand docking for identification of...toluene-induced aggregation. These results demonstrate the applicability of an exploratory in silico toxicity tool, based on a molecular simi- larity... Molecular Similarity Search, in Silico Docking and in Vitro Validation 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6

  6. Basal keratinocytes contribute to all strata of the adult zebrafish epidermis.

    PubMed

    Lee, Raymond T H; Asharani, P V; Carney, Thomas J

    2014-01-01

    The epidermis of terrestrial vertebrates is a stratified epithelium and forms an essential protective barrier. It is continually renewed, with dead corneocytes shed from the surface and replaced from a basal keratinocyte stem cell population. Whilst mouse is the prime model system used for epidermal studies, there is increasing employment of the zebrafish to analyse epidermis development and homeostasis, however the architecture and ontogeny of the epidermis in this system are incompletely described. In particular, it is unclear if adult zebrafish epidermis is derived entirely from the basal epidermal stem cell layer, as in the mouse, or if the most superficial keratinocyte layer is a remnant of the embryonic periderm. Furthermore, a relative paucity of cellular markers and genetic reagents to label and manipulate the basal epidermal stem cell compartment has hampered research. Here we show that the type I keratin, krtt1c19e, is a suitable marker of the basal epidermal layer and identify a krtt1c19e promoter fragment able to drive strong and specific expression in this cell type. Use of this promoter to express an inducible Cre recombinase allowed permanent labelling of basal cells during embryogenesis, and demonstrated that these cells do indeed generate keratinocytes of all strata in the adult epidermis. Further deployment of the Cre-Lox system highlighted the transient nature of the embryonic periderm. We thus show that the epidermis of adult zebrafish, as in the mouse, derives from basal stem cells, further expanding the similarities of epidermal ontogeny across vertebrates. Future use of this promoter will assist genetic analysis of basal keratinocyte biology in zebrafish.

  7. Basal Keratinocytes Contribute to All Strata of the Adult Zebrafish Epidermis

    PubMed Central

    Carney, Thomas J.

    2014-01-01

    The epidermis of terrestrial vertebrates is a stratified epithelium and forms an essential protective barrier. It is continually renewed, with dead corneocytes shed from the surface and replaced from a basal keratinocyte stem cell population. Whilst mouse is the prime model system used for epidermal studies, there is increasing employment of the zebrafish to analyse epidermis development and homeostasis, however the architecture and ontogeny of the epidermis in this system are incompletely described. In particular, it is unclear if adult zebrafish epidermis is derived entirely from the basal epidermal stem cell layer, as in the mouse, or if the most superficial keratinocyte layer is a remnant of the embryonic periderm. Furthermore, a relative paucity of cellular markers and genetic reagents to label and manipulate the basal epidermal stem cell compartment has hampered research. Here we show that the type I keratin, krtt1c19e, is a suitable marker of the basal epidermal layer and identify a krtt1c19e promoter fragment able to drive strong and specific expression in this cell type. Use of this promoter to express an inducible Cre recombinase allowed permanent labelling of basal cells during embryogenesis, and demonstrated that these cells do indeed generate keratinocytes of all strata in the adult epidermis. Further deployment of the Cre-Lox system highlighted the transient nature of the embryonic periderm. We thus show that the epidermis of adult zebrafish, as in the mouse, derives from basal stem cells, further expanding the similarities of epidermal ontogeny across vertebrates. Future use of this promoter will assist genetic analysis of basal keratinocyte biology in zebrafish. PMID:24400120

  8. 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

  9. 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

  10. Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.

    PubMed

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

    2014-08-01

    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.

  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. miR-21 promotes keratinocyte migration and re-epithelialization during wound healing.

    PubMed

    Yang, Xue; Wang, Jun; Guo, Shui-Long; Fan, Kai-Ji; Li, Jun; Wang, You-Liang; Teng, Yan; Yang, Xiao

    2011-01-01

    MicroRNAs involved in keratinocyte migration and wound healing are largely unknown. Here, we revealed the indispensable role of miR-21 in keratinocyte migration and in re-epithelialization during wound healing in mice. In HaCaT cell, miR-21 could be upregulated by TGF-β1. Similar to the effect of TGF-β1, miR-21 overexpression promoted keratinocyte migration. Conversely, miR-21 knockdown attenuated TGF-β1-induced keratinocyte migration, suggesting that miR-21 was essential for TGF-β-driven keratinocyte migration. Furthermore, we found that miR-21 was upregulated during wound healing, coincident with the temporal expression pattern of TGF-β1. Consistently, knockdown of endogenous miR-21 using a specific antagomir dramatically delayed re-epithelialization possibly due to the reduced keratinocyte migration. TIMP3 and TIAM1, direct targets of miR-21, were verified to be regulated by miR-21 in vitro and in vivo, indicating that these two molecules might contribute to miR-21-induced keratinocyte migration. Taken together, our results demonstrate that miR-21 promotes keratinocyte migration and boosts re-epithelialization during skin wound healing.

  13. The signaling involved in autophagy machinery in keratinocytes and therapeutic approaches for skin diseases

    PubMed Central

    Li, Li

    2016-01-01

    Autophagy is responsible for the lysosomal degradation of proteins, organelles, microorganisms and exogenous particles. Epidermis primarily consists of keratinocytes which functions as an extremely important barrier. Investigation on autophagy in keratinocytes has been continuously renewing, but is not so systematic due to the complexity of the autophagy machinery. Here we reviewed recent studies on the autophagy in keratinocyte with a focus on interplay between autophagy machinery and keratinocytes biology, and novel autophagy regulators identified in keratinocytes. In this review, we discussed the roles of autophagy in apoptosis, differentiation, immune response, survival and melanin metabolism, trying to reveal the possible involvement of autophagy in skin aging, skin disorders and skin color formation. Since autophagy routinely plays a double-edged sword role in various conditions, its functions in skin homeostasis and potential application as a therapeutic target for skin diseases remains to be clarified. Furthermore, more investigations are needed on optimizing designed strategies to inhibit or enhance autophagy for clinical efficacy. PMID:27191982

  14. CKD-EPI and cockcroft-gault equations identify similar candidates for neoadjuvant chemotherapy in muscle-invasive bladder cancer.

    PubMed

    Pal, Sumanta K; Ruel, Nora; Villegas, Sergio; Chang, Mark; DeWalt, Kara; Wilson, Timothy G; Vogelzang, Nicholas J; Yuh, Bertram E

    2014-01-01

    Clinical guidelines suggest neoadjuvant cisplatin-based chemotherapy prior to cystectomy in the setting of muscle-invasive bladder cancer (MIBC). A creatinine clearance (CrCl) >60 mL/min is frequently used to characterize cisplatin-eligible patients, and use of the CKD-EPI equation to estimate CrCl has been advocated. From a prospectively maintained institutional database, patients with MIBC who received cystectomy were identified and clinicopathologic information was ascertained. CrCl prior to surgery was computed using three equations: (1) Cockcroft-Gault (CG), (2) CKD-EPI, and (3) MDRD. The primary objective was to determine if the CG and CKD-EPI equations identified a different proportion of patients who were cisplatin-eligible, based on an estimated CrCl of >60 mL/min. Cisplatin-eligibility was also assessed in subsets based on age, CCI score and race. Actuarial rates of neoadjuvant cisplatin-based chemotherapy use were also reported. Of 126 patients, 70% and 71% of patients were found to be cisplatin-eligible by the CKD-EPI and CG equations, respectively (P = 0.9). The MDRD did not result in significantly different characterization of cisplatin-eligibility as compared to the CKD-EPI and CG equations. In the subset of patients age >80, the CKD-EPI equation identified a much smaller proportion of cisplatin-eligible patients (25%) as compared to the CG equation (50%) or the MDRD equation (63%). Only 34 patients (27%) received neoadjuvant cisplatin-based chemotherapy. Of the 92 patients who did not receive neoadjuvant chemotherapy, 64% had a CrCl >60 mL/min by CG. In contrast to previous reports, the CKD-EPI equation does not appear to characterize a broader span of patients as cisplatin-eligible. Older patients (age >80) may less frequently be characterized as cisplatin-eligible by CKD-EPI. The discordance between actual rates of neoadjuvant chemotherapy use and rates of cisplatin eligibility suggest that other factors (e.g., patient and physician preference

  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.

  16. 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.

  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.

  18. Differentiation-dependent p53 regulation of nucleotide excision repair in keratinocytes.

    PubMed Central

    Li, G.; Ho, V. C.; Mitchell, D. L.; Trotter, M. J.; Tron, V. A.

    1997-01-01

    The role of the tumor suppressor p53 in repair of ultraviolet light (UV)-induced DNA damage was evaluated using a host-cell reactivation (HCR) assay. HCR determines a cell's ability to repair UV-damaged DNA through reactivation of a transfected CAT reported plasmid. Most UV damage is removed through nucleotide excision repair (NER). Primary murine keratinocytes isolated from p53-deficient and wild-type p53 mice were used in the HCR assay. The NER was reduced in p53-/- keratinocytes as compared with p53+/+ keratinocytes. The reduced DNA repair in p53-/- mice was confirmed with a radioimmunoassay comparing cyclobutane dimers (CPDs) and (6-4) photoproducts in p53+/+ and p53-/- keratinocytes after the cells were exposed to UV irradiation. Our results demonstrate that wildtype p53 plays a significant role in regulating NER. Furthermore, as there is evidence that p53 protein levels decrease after keratinocytes become differentiated, we sought to determine whether p53 plays a role in NER in differentiated keratinocytes. Differentiation of the keratinocytes by increasing the Ca2+ concentration in the culture media resulted in a marked reduction in NER equally in both p53+/+ and p53-/- groups. This finding suggests that reduced DNA repair after differentiation is p53 independent. A similar reduction in HCR was confirmed in differentiated human keratinocytes. These data, taken together, indicate that p53 or p53-regulated proteins enhance NER in basal undifferentiated keratinocytes but not in differentiated cells. As nonmelanoma skin cancers originate from the basal keratinocytes, our findings suggest that loss of p53 may contribute to the pathogenesis of this common skin cancer. PMID:9095000

  19. Human keratinocytes synthesize and secrete the extracellular matrix protein, thrombospondin.

    PubMed

    Wikner, N E; Dixit, V M; Frazier, W A; Clark, R A

    1987-02-01

    Thrombospondin (TSP) a glycoprotein originally identified as the endogenous lectin of platelets, is also synthesized by fibroblasts, endothelial cells, pneumocytes, smooth muscle cells, and macrophages. Thrombospondin is subdivided into functional domains which bind specifically to heparin, fibronectin, collagen, and to specific cellular receptors. It is found within the basement membranes of kidney, lung, smooth muscle, and skin. Thus TSP may serve as an important link between cells and matrices. Thrombospondin also has been reported at the epidermal-dermal junction. We wished to determine whether human keratinocytes synthesize and secrete TSP. Pure human keratinocytes were grown in defined medium without fibroblast feeder layers. Immunofluorescent staining with either rabbit polyclonal or mouse monoclonal antibodies to human platelet TSP yielded specific granular staining within the cytoplasm of keratinocytes. Culture media and cellular lysates were harvested from cultures metabolically labeled with [35S]methionine. Trichloroacetic acid precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography revealed a major labeled band comigrating with purified platelet TSP in both the media and the cellular lysates. Immunoprecipitation with either the polyclonal or the monoclonal anti-TSP antibodies followed by SDS-PAGE and autoradiography identified this band as TSP. Thus keratinocytes in culture synthesize and secrete TSP. Thrombospondin may play an important role in epidermal interactions with extracellular matrix.

  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. Role of keratin 24 in human epidermal keratinocytes

    PubMed Central

    Min, Min; Chen, Xi-Bei; Wang, Ping; Landeck, Lilla; Chen, Jia-Qi; Li, Wei; Cai, Sui-Qing; Zheng, Min; Man, Xiao-Yong

    2017-01-01

    Keratin 24 (K24) is a new kind of keratin genes, which encodes a novel keratin protein, K24 that bears high similarity to the type I keratins and displays a unique expression profile. However, the role of K24 is incompletely understood. In our study, we investigated the localization of K24 within the epidermis and possible functions. Keratin 24 was found to be modestly overexpressed in senescent keratinocytes and was mainly restricted to the upper stratum spinosum of epidermis. The protein was required for terminal differentiation upon CaCl2-induced differentiation. In vitro results showed that increased K24 in keratinocytes dramatically changed the differentiation of primary keratinocytes. It also inhibited cell survival by G1/S phase cell cycle arrest and induced senescence, autophagy and apoptosis of keratinocytes. In addition, K24 activated PKCδ signal pathway involving in cellular survival. In summary, K24 may be suggested as a potential differentiation marker and anti-proliferative factor in the epidermis. PMID:28362807

  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.

  3. Prediction of drug-induced liver injury using keratinocytes.

    PubMed

    Hirashima, Rika; Itoh, Tomoo; Tukey, Robert H; Fujiwara, Ryoichi

    2017-01-31

    Drug-induced liver injury (DILI) is one of the most common adverse drug reactions. DILI is often accompanied by skin reactions, including rash and pruritus. However, it is still unknown whether DILI-associated genes such as S100 calcium-binding protein A and interleukin (IL)-1β are involved in drug-induced skin toxicity. In the present study, most of the tested hepatotoxic drugs such as pioglitazone and diclofenac induced DILI-associated genes in human and mouse keratinocytes. Keratinocytes of mice at higher risk for DILI exhibited an increased IL-1β basal expression. They also showed a higher inducibility of IL-1β when treated by pioglitazone. Mice at higher risk for DILI showed even higher sums of DILI-associated gene basal expression levels and induction rates in keratinocytes. Our data suggest that DILI-associated genes might be involved in the onset and progression of drug-induced skin toxicity. Furthermore, we might be able to identify individuals at higher risk of developing DILI less invasively by examining gene expression patterns in keratinocytes. Copyright © 2017 John Wiley & Sons, Ltd.

  4. 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.

  5. Canine distemper virus infection: proliferation of canine footpad keratinocytes.

    PubMed

    Gröne, A; Engelhardt, P; Zurbriggen, A

    2003-09-01

    The proliferation of footpad keratinocytes of canine distemper virus (CDV)-infected dogs was investigated. Footpads of 19 dogs inoculated experimentally with a virulent distemper strain (A75/17) and of two noninoculated control dogs were collected at necropsy. Dogs were divided into four groups according to results of the postmortem examination: dogs with severe distemper (group 1), dogs with mild distemper (group 2), inoculated dogs without distemper (group 3) and noninoculated dogs (group 4). There was no distinct difference of epidermal thickness among the four groups. Infection of the footpad epidermis with CDV was demonstrated using immunohistochemistry for viral nucleoprotein and in situ hybridization for nucleoprotein messenger ribonucleic acid (mRNA). Only group 1 dogs had viral antigen and mRNA in the footpad epidermis with the same distribution. Footpad epidermis of group 1 dogs had more mitotic figures in the basal layer, and significantly more basal keratinocytes were positive for the proliferation markers Ki-67 and proliferating cell nuclear antigen. Double-staining for Ki-67 and viral nucleoprotein identified rare double-labeled basal keratinocytes. These findings suggest that the presence of CDV particles in the footpad epidermis is associated with keratinocyte proliferation.

  6. Connexin43 reduces melanoma growth within a keratinocyte microenvironment and during tumorigenesis in vivo.

    PubMed

    Ableser, Mark J; Penuela, Silvia; Lee, Jack; Shao, Qing; Laird, Dale W

    2014-01-17

    Connexins (Cx) have been identified as tumor suppressors or enhancers, a distinction that appears to be dependent on the type and stage of disease. However, the role of connexins in melanoma tumorigenesis and their status during cancer onset and progression remain controversial and unclear. Here, we show that the aggressive B16-BL6 mouse melanoma cell line expresses low basal levels of Cx26 and Cx43, rendering them gap junctional intercellular communication-deficient as elucidated by immunofluorescence, Western blotting, and dye transfer studies. Following ectopic expression of green fluorescent protein-tagged Cx26 and Cx43 in these connexin-deficient melanomas, punctate gap junction-like plaques were evident at sites of cell-cell apposition, and the incidence of dye transfer was significantly increased similar to connexin-rich keratinocytes. We found that the expression of Cx43, but not Cx26, significantly reduced cellular proliferation and anchorage-independent growth from control melanomas, whereas migration was unaffected. Additionally, melanomas expressing Cx43 displayed significantly reduced growth within the in situ-like microenvironment of keratinocytes, despite a lack of heterocellular gap junctional intercellular communication between the two cell types. Furthermore, when grown in vivo in the chicken chorioallantoic membrane, primary tumors derived from Cx43-expressing melanomas were significantly smaller than controls, whereas Cx26-expressing melanomas produced tumors similar to controls. Collectively, these results suggest that Cx43, and not Cx26, can act as a tumor suppressor during melanoma tumorigenesis.

  7. Altered expression of keratinocyte growth factor and its receptor in psoriasis.

    PubMed

    Finch, P W; Murphy, F; Cardinale, I; Krueger, J G

    1997-12-01

    One of the biological characteristics of psoriasis is excessive flaking of the skin. This is directly related to the marked hyperplasia of epidermal keratinocytes and to incomplete epidermal differentiation. Keratinocyte growth factor (KGF), a potent mitogen for human keratinocytes, is expressed by stromal cells. Alterations in the KGF signaling pathway might account for the epidermal hyperplasia associated with psoriasis. To test this hypothesis, we investigated the expression of KGF and its receptor (KGFR) in psoriasis tissue. KGF and KGFR mRNA levels were found to be frequently elevated in psoriatic skin specimens as compared with normal skin. Increased KGF transcript expression was localized to the dermal layer of the involved skin specimen using in situ hybridization. In contrast, KGFR transcript and protein expression was localized to the basal layer of keratinocytes in normal skin and to the basal and suprabasal layers of the psoriatic epidermis, coincident with the expanded proliferative keratinocyte pool. To identify molecules that might regulate KGFR expression we investigated the effects of various pharmacological agents and cytokines on KGFR synthesis by keratinocytes. Phorbol ester, interleukin-6, interferon-gamma, and ultraviolet B (UVB) treatment all led to substantial down-regulation of KGFR expression. The down-regulation of KGFR synthesis by UVB suggests a possible mechanism for the antiproliferative action of this agent in the treatment of psoriasis. Taken together, these results suggest that increased KGFR-mediated signaling in keratinocytes in the lesional epidermis might account in part for the epidermal hyperplasia in psoriasis.

  8. 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.

  9. Transdifferentiation of Adipose-Derived Stem Cells into Keratinocyte-Like Cells: Engineering a Stratified Epidermis

    PubMed Central

    Chavez-Munoz, Claudia; Nguyen, Khang T.; Xu, Wei; Hong, Seok-Jong; Mustoe, Thomas A.; Galiano, Robert D.

    2013-01-01

    Skin regeneration is an important area of research in the field of tissue-engineering, especially for cases involving loss of massive areas of skin, where current treatments are not capable of inducing permanent satisfying replacements. Human adipose-derived stem cells (ASC) have been shown to differentiate in-vitro into both mesenchymal lineages and non-mesenchymal lineages, confirming their transdifferentiation ability. This versatile differentiation potential, coupled with their ease of harvest, places ASC at the advancing front of stem cell-based therapies. In this study, we hypothesized that ASC also have the capacity to transdifferentiate into keratinocyte-like cells and furthermore are able to engineer a stratified epidermis. ASC were successfully isolated from lipoaspirates and cell sorted (FACS). After sorting, ASC were either co-cultured with human keratinocytes or with keratinocyte conditioned media. After a 14-day incubation period, ASC developed a polygonal cobblestone shape characteristic of human keratinocytes. Western blot and q-PCR analysis showed the presence of specific keratinocyte markers including cytokeratin-5, involucrin, filaggrin and stratifin in these keratinocyte-like cells (KLC); these markers were absent in ASC. To further evaluate if KLC were capable of stratification akin to human keratinocytes, ASC were seeded on top of human decellularized dermis and cultured in the presence or absence of EGF and high Ca2+ concentrations. Histological analysis demonstrated a stratified structure similar to that observed in normal skin when cultured in the presence of EGF and high Ca2+. Furthermore, immunohistochemical analysis revealed the presence of keratinocyte markers such as involucrin, cytokeratin-5 and cytokeratin-10. In conclusion this study demonstrates for the first time that ASC have the capacity to transdifferentiate into KLC and engineer a stratified epidermis. This study suggests that adipose tissue is potentially a readily available

  10. Vimentin is necessary for colony growth of human diploid keratinocytes.

    PubMed

    Castro-Muñozledo, Federico; Velez-DelValle, Cristina; Marsch-Moreno, Meytha; Hernández-Quintero, Miriam; Kuri-Harcuch, Walid

    2015-01-01

    The role of vimentin (Vim) in diploid epithelial cells is not well known. To understand its biological function, we cultured human epidermal keratinocytes under conditions that support migration, proliferation, stratification and terminal differentiation. We identified a keratinocyte subpopulation that shows a p63(+)/α5β1(bright) phenotype and displays Vim intermediate filaments (IFs) besides their keratin IF network. These cells were mainly located at the proliferative/migratory rim of the growing colonies; but also, they were scarce and scattered or formed small groups of basal cells in confluent stratified epithelia. Stimulation of cells with EGF and wounding experiments in confluent arrested epithelia increased the number of Vim(+) keratinocytes in an extent higher to the expected for a cell population doubling. BrdU labeling demonstrated that most of the proliferative cells located at the migratory border of the colony have Vim, in contrast with proliferative cells located at the basal layer at the center of big colonies which lacked of Vim IFs, suggesting that Vim expression was not solely linked to proliferation. Therefore, we silenced Vim mRNA in the cultured keratinocytes and observed an inhibition of colony growth. Such results, together with long-term cultivation assays which showed that Vim might be associated to pattern formation in cultured epithelia, suggest that Vim expression is essential for a highly motile phenotype, which is necessary for keratinocyte colony growth and possibly for development and wound healing. Vim(+)/p63(+)/α5β1(bright) epithelial cells may play a significant physiological role in embryonic morphogenetic movements; wound healing and other pathologies such as carcinomas and hyperproliferative diseases.

  11. In Vitro Toxicity of Aluminum Nanoparticles in Human Keratinocytes

    DTIC Science & Technology

    2008-03-01

    absorption of aluminum has been reported after application of underarm deodorant (Becaria, 2002:311). Two adult volunteers were dermally exposed to one...NPs can be classified as a sensitizer agent. Since sensitization has been identified in persons exposed to aluminum in deodorant , vaccines, and...IN VITRO TOXICITY OF ALUMINUM NANOPARTICLES IN HUMAN KERATINOCYTES THESIS Stephanie McCormack-Brown, Major, USAF, BSC AFIT/GIH/ENV

  12. 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.

  13. Control of keratinocyte proliferation and differentiation by p63.

    PubMed

    Truong, Amy B; Khavari, Paul A

    2007-02-01

    The p53 family member p63 has been implicated in both the development and maintenance of stratified epithelial tissues, including the epidermis. Increasing data support p63 function in the regenerative capacity of basal keratinocytes by maintaining cell proliferation. Recent studies further suggest this regulation relies on inhibition of p53 activity. In addition, p63 appears to exert separate control over epidermal differentiation, which may involve control of such key signaling molecules as IKKalpha and Notch. While studies over the past decade have greatly expanded our knowledge of p63 function, much remains to be understood regarding how p63 regulates epidermal homeostasis. Future efforts to identify and validate direct p63 target genes as well as to understand the expression and function of individual p63 isoforms will be important to further define how p63 functions in the control of keratinocyte proliferation and differentiation.

  14. 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.

  15. Cytokine release and cytotoxicity in human keratinocytes and fibroblasts induced by phenols and sodium dodecyl sulfate.

    PubMed

    Newby, C S; Barr, R M; Greaves, M W; Mallet, A I

    2000-08-01

    Phenolic compounds used in pharmaceutical and industrial products can cause irritant contact dermatitis. We studied the effects of resorcinol, phenol, 3,5-xylenol, chloroxylenol, and 4-hexyl-resorcinol on normal human epidermal keratinocytes and dermal fibroblasts for cytotoxicity and cytokine release, determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide methodology and enzyme-linked immunosorbent assay, respectively. An inverse correlation between phenol concentrations causing a 50% reduction in keratinocyte and fibroblast viability at 24 h and their octanol water-partition coefficients (i.e., hydrophobicity) was observed. 3,5-xylenol, chloroxylenol, hexyl-resorcinol, and sodium dodecyl sulfate, but not resorcinol or phenol, induced release of interleukin-1alpha from keratinocytes at cytotoxic concentrations. Variable release of tumor necrosis factor-alpha and interleukin-8 from keratinocytes occurred only at toxic threshold concentrations of the phenols or sodium dodecyl sulfate. Subtoxic concentrations of phenols or sodium dodecyl sulfate did not induce cytokine release from keratinocytes. Neither the phenols nor sodium dodecyl sulfate induced release of the chemokines interleukin-8, growth-related oncogene-alpha or monocyte chemotactic protein-1 from fibroblasts. Conditioned media from keratinocytes treated with cytotoxic concentrations of 3,5-xylenol, chloroxylenol, hexyl-resorcinol, or sodium dodecyl sulfate stimulated further release of the chemokines from fibroblasts above that obtained with control media. Rabbit anti-interleukin-1alpha serum inhibited keratinocyte-conditioned media induction of chemokine release. We have shown a structure-cytotoxicity relationship for a series of phenols as well as an association of interleukin-1alpha release with a cytotoxic effect. We demonstrated a cytokine cascade amplification step by the actions of stimulated keratinocyte media on cultured dermal fibroblasts, identifying interleukin-1alpha as

  16. 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

  17. 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

    2015-06-24

    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.

  18. C/EBPalpha and beta couple interfollicular keratinocyte proliferation arrest to commitment and terminal differentiation.

    PubMed

    Lopez, Rodolphe G; Garcia-Silva, Susana; Moore, Susan J; Bereshchenko, Oksana; Martinez-Cruz, Ana B; Ermakova, Olga; Kurz, Elke; Paramio, Jesus M; Nerlov, Claus

    2009-10-01

    The transcriptional regulators that couple interfollicular basal keratinocyte proliferation arrest to commitment and differentiation are yet to be identified. Here we report that the basic region leucine zipper transcription factors C/EBPalpha and C/EBPbeta are co-expressed in basal keratinocytes, and are coordinately upregulated as keratinocytes exit the basal layer and undergo terminal differentiation. Mice lacking both C/EBPalpha and beta in the epidermis showed increased proliferation of basal keratinocytes and impaired commitment to differentiation. This led to ectopic expression of keratin 14 (K14) and DeltaNp63 in suprabasal cells, decreased expression of spinous and granular layer proteins, parakeratosis and defective epidermal water barrier function. Knock-in mutagenesis revealed that C/EBP-E2F interaction was required for control of interfollicular epidermis (IFE) keratinocyte proliferation, but not for induction of spinous and granular layer markers, whereas C/EBP DNA binding was required for DeltaNp63 downregulation and K1/K10 induction. Finally, loss of C/EBPalpha/beta induced stem cell gene expression signatures in the epidermis. C/EBPs, therefore, couple basal keratinocyte cell cycle exit to commitment to differentiation through E2F repression and DNA binding, respectively, and may act to restrict the epidermal stem cell compartment.

  19. Reprogramming Postnatal Human Epidermal Keratinocytes toward Functional Neural Crest Fates.

    PubMed

    Bajpai, Vivek K; Kerosuo, Laura; Tseropoulos, Georgios; Cummings, Kirstie A; Wang, Xiaoyan; Lei, Pedro; Liu, Biao; Liu, Song; Popescu, Gabriela; Bronner, Marianne E; Andreadis, Stelios T

    2017-01-31

    During development, neural crest cells are induced by signaling events at the neural plate border of all vertebrate embryos. Initially arising within the central nervous system, neural crest cells subsequently undergo an epithelial to mesenchymal transition to migrate into the periphery, where they differentiate into diverse cell types. Here we provide evidence that postnatal human epidermal keratinocytes, in response to FGF2 and IGF1 signals, can be reprogrammed toward a neural crest fate. Genome-wide transcriptome analyses show that keratinocyte-derived neural crest cells are similar to those derived from human embryonic stem cells. Moreover, they give rise in vitro and in vivo to neural crest derivatives such as peripheral neurons, melanocytes, Schwann cells and mesenchymal cells (osteocytes, chondrocytes, adipocytes and smooth muscle). By demonstrating that human KRT14+ keratinocytes can form neural crest cells, even from clones of single cells, our results have important implications in stem cell biology and regenerative medicine. This article is protected by copyright. All rights reserved.

  20. Cytosolic DNA triggers inflammasome activation in keratinocytes in psoriatic lesions.

    PubMed

    Dombrowski, Yvonne; Peric, Mark; Koglin, Sarah; Kammerbauer, Claudia; Göss, Christine; Anz, David; Simanski, Maren; Gläser, Regine; Harder, Jürgen; Hornung, Veit; Gallo, Richard L; Ruzicka, Thomas; Besch, Robert; Schauber, Jürgen

    2011-05-11

    The proinflammatory cytokine interleukin-1β (IL-1β) plays a central role in the pathogenesis and the course of inflammatory skin diseases, including psoriasis. Posttranscriptional activation of IL-1β is mediated by inflammasomes; however, the mechanisms triggering IL-1β processing remain unknown. Recently, cytosolic DNA has been identified as a danger signal that activates inflammasomes containing the DNA sensor AIM2. In this study, we detected abundant cytosolic DNA and increased AIM2 expression in keratinocytes in psoriatic lesions but not in healthy skin. In cultured keratinocytes, interferon-γ induced AIM2, and cytosolic DNA triggered the release of IL-1β via the AIM2 inflammasome. Moreover, the antimicrobial cathelicidin peptide LL-37, which can interact with DNA in psoriatic skin, neutralized cytosolic DNA in keratinocytes and blocked AIM2 inflammasome activation. Together, these data suggest that cytosolic DNA is an important disease-associated molecular pattern that can trigger AIM2 inflammasome and IL-1β activation in psoriasis. Furthermore, cathelicidin LL-37 interfered with DNA-sensing inflammasomes, which thereby suggests an anti-inflammatory function for this peptide. Thus, our data reveal a link between the AIM2 inflammasome, cathelicidin LL-37, and autoinflammation in psoriasis, providing new potential targets for the treatment of this chronic skin disease.

  1. Inhibition of JNK promotes differentiation of epidermal keratinocytes.

    PubMed

    Gazel, Alix; Banno, Tomohiro; Walsh, Rebecca; Blumenberg, Miroslav

    2006-07-21

    In inflamed tissue, normal signal transduction pathways are altered by extracellular signals. For example, the JNK pathway is activated in psoriatic skin, which makes it an attractive target for treatment. To define comprehensively the JNK-regulated genes in human epidermal keratinocytes, we compared the transcriptional profiles of control and JNK inhibitor-treated keratinocytes, using DNA microarrays. We identified the differentially expressed genes 1, 4, 24, and 48 h after the treatment with SP600125. Surprisingly, the inhibition of JNK in keratinocyte cultures in vitro induces virtually all aspects of epidermal differentiation in vivo: transcription of cornification markers, inhibition of motility, withdrawal from the cell cycle, stratification, and even production of cornified envelopes. The inhibition of JNK also induces the production of enzymes of lipid and steroid metabolism, proteins of the diacylglycerol and inositol phosphate pathways, mitochondrial proteins, histones, and DNA repair enzymes, which have not been associated with differentiation previously. Simultaneously, basal cell markers, including integrins, hemidesmosome and extracellular matrix components, are suppressed. Promoter analysis of regulated genes finds that the binding sites for the forkhead family of transcription factors are over-represented in the SP600125-induced genes and c-Fos sites in the suppressed genes. The JNK-induced proliferation appears to be secondary to inhibition of differentiation. The results indicate that the inhibition of JNK in epidermal keratinocytes is sufficient to initiate their differentiation program and suggest that augmenting JNK activity could be used to delay cornification and enhance wound healing, whereas attenuating it could be a differentiation therapy-based approach for treating psoriasis.

  2. Canine distemper virus infects canine keratinocytes and immune cells by using overlapping and distinct regions located on one side of the attachment protein.

    PubMed

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

    2011-11-01

    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.

  3. 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

  4. Arachidonic acid metabolism in cultured mouse keratinocytes

    SciTech Connect

    Kondoh, H.; Sato, Y.; Kanoh, H.

    1985-07-01

    The authors attempted to characterize the general features of arachidonate metabolism in cultured mouse keratinocytes. The cells labeled with (/sup 3/H)arachidonate were stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), ionophore A23187, and fetal bovine serum (FBS). Common to the three substances, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine almost equally served as sources of arachidonate liberated by the action of phospholipase A2. The stimulation of phospholipase A2 action was observed in the order of A23187 greater than FBS greater than TPA. When stimulated by TPA or A23187, the radioactivity released into the extracellular medium was mostly found in prostaglandin (PG) E2. Formation of other PGs and hydroxyeicosatetraenoate (HETE) was extremely limited. In the case of stimulation by FBS, however, the released radioactivity was mainly associated with non-converted arachidonate. FBS also inhibited the TPA- and A23187-induced conversion of arachidonate to PGE2. Phospholipid degradation induced by the three stimulators was similarly dependent on extracellular Ca/sup 2 +/. The stimulation by FBS and A23187 was suppressed by calmodulin antagonists, though the effect of A23187 was much more sensitive to the antagonists when compared to that of FBS. The authors observed more than additive effects of the three stimulators when tested together.

  5. Enhanced constitutive invasion activity in human nontumorigenic keratinocytes exposed to a low level of barium for a long time.

    PubMed

    Thang, Nguyen D; Yajima, Ichiro; Ohnuma, Shoko; Ohgami, Nobutaka; Kumasaka, Mayuko Y; Ichihara, Gaku; Kato, Masashi

    2015-02-01

    We have recently demonstrated that exposure to barium for a short time (≤4 days) and at a low level (5 µM = 687 µg/L) promotes invasion of human nontumorigenic HaCaT cells, which have characteristics similar to those of normal keratinocytes, suggesting that exposure to barium for a short time enhances malignant characteristics. Here we examined the effect of exposure to low level of barium for a long time, a condition mimicking the exposure to barium through well water, on malignant characteristics of HaCaT keratinocytes. Constitutive invasion activity, focal adhesion kinase (FAK) protein expression and activity, and matrix metalloproteinase 14 (MMP14) protein expression in primary cultured normal human epidermal keratinocytes, HaCaT keratinocytes, and HSC5 and A431 human squamous cell carcinoma cells were augmented following an increase in malignancy grade of the cells. Constitutive invasion activity, FAK phosphorylation, and MMP14 expression levels of HaCaT keratinocytes after treatment with 5 µM barium for 4 months were significantly higher than those of control untreated HaCaT keratinocytes. Taken together, our results suggest that exposure to a low level of barium for a long time enhances constitutive malignant characteristics of HaCaT keratinocytes via regulatory molecules (FAK and MMP14) for invasion.

  6. A heat-sensitive TRP channel expressed in keratinocytes.

    PubMed

    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

    2002-06-14

    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.

  7. Effect of Absolute From Hibiscus syriacus L. Flower on Wound Healing in Keratinocytes

    PubMed Central

    Yoon, Seok Won; Lee, Kang Pa; Kim, Do-Yoon; Hwang, Dae Il; Won, Kyung-Jong; Lee, Dae Won; Lee, Hwan Myung

    2017-01-01

    Background: Proliferation and migration of keratinocytes are essential for the repair of cutaneous wounds. Hibiscus syriacus L. has been used in Asian medicine; however, research on keratinocytes is inadequate. Objective: To establish the dermatological properties of absolute from Hibiscus syriacus L. flower (HSF) and to provide fundamental research for alternative medicine. Materials and Methods: We identified the composition of HSF absolute using gas chromatography-mass spectrometry analysis. We also examined the effect of HSF absolute in HaCaT cells using the XTT assay, Boyden chamber assay, sprout-out growth assay, and western blotting. We conducted an in-vivo wound healing assay in rat tail-skin. Results: Ten major active compounds were identified from HSF absolute. As determined by the XTT assay, Boyden chamber assay, and sprout-out growth assay results, HSF absolute exhibited similar effects as that of epidermal growth factor on the proliferation and migration patterns of keratinocytes (HaCaT cells), which were significantly increased after HSF absolute treatment. The expression levels of the phosphorylated signaling proteins relevant to proliferation, including extracellular signal-regulated kinase 1/2 (Erk 1/2) and Akt, were also determined by western blot analysis. Conclusion: These results of our in-vitro and ex-vivo studies indicate that HSF absolute induced cell growth and migration of HaCaT cells by phosphorylating both Erk 1/2 and Akt. Moreover, we confirmed the wound-healing effect of HSF on injury of the rat tail-skin. Therefore, our results suggest that HSF absolute is promising for use in cosmetics and alternative medicine. SUMMARY Hisbiscus syriacus L. flower absolute increases HaCaT cell migration and proliferation.Hisbiscus syriacus L. flower absolute regulates phosphorylation of ERK 1/2 and Akt in HaCaT cell.Treatment with Hisbiscus syriacus L. flower induced sprout outgrowth.The wound in the tail-skin of rat was reduced by Hisbiscus syriacus

  8. Expression and functional role of Sox9 in human epidermal keratinocytes.

    PubMed

    Shi, Ge; Sohn, Kyung-Cheol; Li, Zhengjun; Choi, Dae-Kyoung; Park, Young Min; Kim, Jin-Hwa; Fan, Yi-Ming; Nam, Yong Hee; Kim, Sooyeon; Im, Myung; Lee, Young; Seo, Young-Joon; Kim, Chang Deok; Lee, Jeung-Hoon

    2013-01-01

    In this study, we investigated the expression and putative role of Sox9 in epidermal keratinocyte. Immunohistochemical staining showed that Sox9 is predominantly expressed in the basal layer of normal human skin epidermis, and highly expressed in several skin diseases including psoriasis, basal cell carcinoma, keratoacanthoma and squamous cell carcinoma. In calcium-induced keratinocyte differentiation model, the expression of Sox9 was decreased in a time dependent manner. When Sox9 was overexpressed using a recombinant adenovirus, cell growth was enhanced, while the expression of differentiation-related genes such as loricrin and involucrin was markedly decreased. Similarly, when rat skin was intradermally injected with the adenovirus expressing Sox9, the epidermis was thickened with increase of PCNA positive cells, while the epidermal differentiation was decreased. Finally, UVB irradiation induced Sox9 expression in cultured human epidermal keratinocytes, and keratinocytes are protected from UVB-induced apoptosis by Sox9 overexpression. Together, these results suggest that Sox9 is an important regulator of epidermal keratinocytes with putative pro-proliferation and/or pro-survival functions, and may be related to several cutaneous diseases that are characterized by abnormal differentiation and hyperproliferation.

  9. 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.

  10. The influence of microtextured basal lamina analog topography on keratinocyte function and epidermal organization.

    PubMed

    Downing, Brett R; Cornwell, Kevin; Toner, Mehmet; Pins, George D

    2005-01-01

    The rational design of future bioengineered skin substitutes requires an understanding of the mechanisms by which the three-dimensional microarchitecture of tissue scaffolds modulates keratinocyte function. Microtextured basal lamina analogs were developed to investigate the relationship between the characteristic topography at the dermal-epidermal interface of native skin and keratinocyte function. Microfabrication techniques were used to create master patterns, negative replicates, and collagen membranes with ridges and channels of length scales (e.g., grooves of 50-200 microm in depth and width) similar to the invaginations found in basal lamina at the dermal-epidermal junction of native skin. Keratinocytes were seeded on the surfaces of basal lamina analogs, and histological analyses were performed after 7 days of tissue culture at the air-liquid interface. The keratinocytes formed a differentiated and stratified epidermis that conformed to the features of the microtextured membranes. Morphometric analyses of immunostained skin equivalents suggest that keratinocyte stratification and differentiation increases as channel depth increases and channel width decreases. This trend was most pronounced in channels with the highest depth-to-width ratios (i.e., 200 microm deep, 50 microm wide). It is anticipated that the findings from these studies will elucidate design parameters to enhance the performance of future bioengineered skin substitutes.

  11. RIP4 is an ankyrin repeat-containing kinase essential for keratinocyte differentiation.

    PubMed

    Holland, Pamela; Willis, Cynthia; Kanaly, Suzanne; Glaccum, Moira; Warren, Annjanette; Charrier, Keith; Murison, J; Derry, Jonathan; Virca, G; Bird, Timothy; Peschon, Jacques

    2002-08-20

    The epidermis is a stratified, continually renewing epithelium dependent on a balance among cell proliferation, differentiation, and death for homeostasis. In normal epidermis, a mitotically active basal layer gives rise to terminally differentiating keratinocytes that migrate outward and are ultimately sloughed from the skin surface as enucleated squames. Although many proteins are known to function in maintaining epidermal homeostasis, the molecular coordination of these events is poorly understood. RIP4 is a novel RIP (receptor-interacting protein) family kinase with ankyrin repeats cloned from a keratinocyte cDNA library. RIP4 deficiency in mice results in perinatal lethality associated with abnormal epidermal differentiation. The phenotype of RIP4(-/-) mice in part resembles that of mice lacking IKKalpha, a component of a complex that regulates NF-kappaB. Despite the similar keratinocyte defects in RIP4- and IKKalpha-deficient mice, these kinases function in distinct pathways. RIP4 functions cell autonomously within the keratinocyte lineage. Unlike IKKalpha, RIP4-deficient skin fails to fully differentiate when grafted onto a normal host. Instead, abnormal hair follicle development and epidermal dysplasia, indicative of progression into a more pathologic state, are observed. Thus, RIP4 is a critical component of a novel pathway that controls keratinocyte differentiation.

  12. Effective inhibition of melanosome transfer to keratinocytes by lectins and niacinamide is reversible.

    PubMed

    Greatens, Amanda; Hakozaki, Tomohiro; Koshoffer, Amy; Epstein, Howard; Schwemberger, Sandy; Babcock, George; Bissett, Donald; Takiwaki, Hirotsugu; Arase, Seiji; Wickett, R Randall; Boissy, Raymond E

    2005-07-01

    Skin pigmentation results in part from the transfer of melanized melanosomes synthesized by melanocytes to neighboring keratinocytes. Plasma membrane lectins and their glycoconjugates expressed by these epidermal cells are critical molecules involved in this transfer process. In addition, the derivative of vitamin B(3), niacinamide, can inhibit melanosome transfer and induce skin lightening. We investigated the effects of these molecules on the viability of melanocytes and keratinocytes and on the reversibility of melanosome-transfer inhibition induced by these agents using an in vitro melanocyte-keratinocyte coculture model system. While lectins and neoglycoproteins could induce apoptosis in a dose-dependent manner to melanocytes or keratinocytes in monoculture, similar dosages of the lectins, as opposed to neoglycoproteins, did not induce apoptosis to either cell type when treated in coculture. The dosages of lectins and niacinamide not affecting cell viability produced an inhibitory effect on melanosome transfer, when used either alone or together in cocultures of melanocytes-keratinocytes. Cocultures treated with lectins or niacinamide resumed normal melanosome transfer in 3 days after removal of the inhibitor, while cocultures treated with a combination of lectins and niacinamide demonstrated a lag in this recovery. Subsequently, we assessed the effect of niacinamide on facial hyperpigmented spots using a vehicle-controlled, split-faced design human clinical trial. Topical application of niacinamide resulted in a dose-dependent and reversible reduction in hyperpigmented lesions. These results suggest that lectins and niacinamide at concentrations that do not affect cell viability are reversible inhibitors of melanosome transfer.

  13. 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.

  14. RoCaSCA: A contour tracing grid-based algorithm to identify similarity regions and clusters in spatial geographical data

    NASA Astrophysics Data System (ADS)

    Hazenberg, P.; Torfs, P. J. J. F.; Leijnse, H.; Uijlenhoet, R.

    2012-04-01

    Over the last decades the amount of spatial geographic data obtained from satellite and radar remote sensing, geographical and other types of spatial information has increased tremendously, making it impossible for a user to examine all in detail. Therefore, a considerable amount of research has focused on smart and efficient solutions to segment a spatial image into its dominant regions, extracting most essential information. The current research presents a new spatial image cluster identification method. The delineation of clusters is performed in two separate steps. First, we identify a regions outer contour using the properties of a rotating carpenter square. Secondly, we define all inner pixels belonging to a cluster based on the same principle, excluding inner contour regions if necessary. As such, a cluster identification method will be presented which has considerable similarity to some of the tracing type and connected component image segmentation algorithms developed in the literature during the last decade. However, since the characteristic shape of a carpenter square can easily be extended, the algorithm presented here does not strictly label neighboring pixels to the same component only. On the contrary, our algorithm is able to connect non-neighboring pixels for varying pixel distances as well. In addition, since our algorithm takes a continuous grid as input, it is possible to define transition pixels, that connect pixels that belong to a given cluster. Therefore, this newly developed algorithm presents a link between the traditional image segmentation methods implemented on binary grids and the partitional density and grid-based cluster identification methods that use continuous datasets. We will demonstrate the impact of this new cluster identification method for a number of typical geophysical cases ranging from global drought identification to weather radar based precipitation cell delineation.

  15. Proteome and metabolome profiling of cytokinin action in Arabidopsis identifying both distinct and similar responses to cytokinin down- and up-regulation

    PubMed Central

    Hoehenwarter, Wolfgang; Brzobohatý, Břetislav

    2013-01-01

    In plants, numerous developmental processes are controlled by cytokinin (CK) levels and their ratios to levels of other hormones. While molecular mechanisms underlying the regulatory roles of CKs have been intensely researched, proteomic and metabolomic responses to CK deficiency are unknown. Transgenic Arabidopsis seedlings carrying inducible barley cytokinin oxidase/dehydrogenase (CaMV35S>GR>HvCKX2) and agrobacterial isopentenyl transferase (CaMV35S>GR>ipt) constructs were profiled to elucidate proteome- and metabolome-wide responses to down- and up-regulation of CK levels, respectively. Proteome profiling identified >1100 proteins, 155 of which responded to HvCKX2 and/or ipt activation, mostly involved in growth, development, and/or hormone and light signalling. The metabolome profiling covered 79 metabolites, 33 of which responded to HvCKX2 and/or ipt activation, mostly amino acids, carbohydrates, and organic acids. Comparison of the data sets obtained from activated CaMV35S>GR>HvCKX2 and CaMV35S>GR>ipt plants revealed unexpectedly extensive overlaps. Integration of the proteomic and metabolomic data sets revealed: (i) novel components of molecular circuits involved in CK action (e.g. ribosomal proteins); (ii) previously unrecognized links to redox regulation and stress hormone signalling networks; and (iii) CK content markers. The striking overlaps in profiles observed in CK-deficient and CK-overproducing seedlings might explain surprising previously reported similarities between plants with down- and up-regulated CK levels. PMID:24064926

  16. 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

  17. Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes.

    PubMed

    Herz, Corinna; Aumailley, Monique; Schulte, Carsten; Schlötzer-Schrehardt, Ursula; Bruckner-Tuderman, Leena; Has, Cristina

    2006-11-24

    A novel family of focal adhesion proteins, the kindlins, is involved in attachment of the actin cytoskeleton to the plasma membrane and in integrin-mediated cellular processes. Deficiency of kindlin-1, as a result of loss-of-function mutations in the KIND1 gene, causes Kindler syndrome, an autosomal recessive genodermatosis characterized by skin blistering, progressive skin atrophy, photosensitivity and, occasionally, carcinogenesis. Here we characterized authentic and recombinantly expressed kindlin-1 and show that it is localized in basal epidermal keratinocytes in a polar fashion, close to the cell surface facing the basement membrane, in the areas between the hemidesmosomes. We identified two forms of kindlin-1 in keratinocytes, with apparent molecular masses of 78 and 74 kDa, corresponding to phosphorylated and desphosphorylated forms of the protein. In kindlin-1-deficient skin, basal keratinocytes show multiple abnormalities: cell polarity is lost, proliferation is strongly reduced, and several cells undergo apoptosis. In vitro, deficiency of kindlin-1 in keratinocytes leads to strongly reduced cell proliferation, decreased adhesion, undirected motility, and intense protrusion activity of the plasma membrane. Taken together, these results show that kindlin-1 plays a role in keratinocyte adhesion, polarization, proliferation, and migration. It is involved in organization and anchorage of the actin cytoskeleton to integrin-associated signaling platforms.

  18. Immunomodulatory effects of a set of amygdalin analogues on human keratinocyte cells.

    PubMed

    Baroni, A; Paoletti, I; Greco, R; Satriano, R A; Ruocco, E; Tufano, M A; Perez, J J

    2005-11-01

    Peptide T (PT) is an octapeptide shown to resolve psoriatic lesions. Our previous investigations suggest that keratinocytes play an important role in conditioning the therapeutic effects of the PT in psoriasis. However, peptides are not good therapeutic agents, because they exhibit poor absorption, are easily metabolized and are immunogenic. Using computational methods, the natural product amygdalin was identified as peptidomimetic of PT. However, amygdalin exhibits a toxic profile due to its cyanide group. To overcome this deleterious effect, we synthesized analogues lacking the cyanide group. Human keratinocytes were treated with PT or with three different peptidomimetics of PT. To study its effects on the expression of HSP-70, TGF-beta, alpha-v integrin, ICAM-1 and cytokines, we analysed the protein levels by Western blot and ELISA. Our results show that the different peptidomimetics of PT tested exhibit a similar biological behaviour in regard to the overexpression of HSP-70, TGF-beta and alpha-v integrin than the native peptide. TNF-alpha is overexpressed by PT and SVT-03018; between the other two analogs, SVT-03016 do not produce any significant change in regard to the control, while SVT-03017 shows only a moderate increase in regard to control. SVT-03018 provokes a remarkable upregulation of IL-10, stronger than SVT-03016, SVT-03017 and PT. All the other three analogues reduce comparably to the PT, the expression of ICAM-1 and do not increase the release of proinflammatory cytokines. The results highlighted that the three analogues of amygdalin with the cyanide group removed exhibit the same biological effects of PT. Therefore, they can be considered peptidomimetics, suggesting their possible use in the treatment of psoriasis.

  19. Streptococcus pyogenes M49 plasminogen/plasmin binding facilitates keratinocyte invasion via integrin-integrin-linked kinase (ILK) pathways and protects from macrophage killing.

    PubMed

    Siemens, Nikolai; Patenge, Nadja; Otto, Juliane; Fiedler, Tomas; Kreikemeyer, Bernd

    2011-06-17

    The entry into epithelial cells and the prevention of primary immune responses are a prerequisite for a successful colonization and subsequent infection of the human host by Streptococcus pyogenes (group A streptococci, GAS). Here, we demonstrate that interaction of GAS with plasminogen promotes an integrin-mediated internalization of the bacteria into keratinocytes, which is independent from the serine protease activity of potentially generated plasmin. α(1)β(1)- and α(5)β(1)-integrins were identified as the major keratinocyte receptors involved in this process. Inhibition of integrin-linked kinase (ILK) expression by siRNA silencing or blocking of PI3K and Akt with specific inhibitors, reduced the GAS M49-plasminogen/plasmin-mediated invasion of keratinocytes. In addition, blocking of actin polymerization significantly reduced GAS internalization into keratinocytes. Altogether, these results provide a first model of plasminogen-mediated GAS invasion into keratinocytes. Furthermore, we demonstrate that plasminogen binding protects the bacteria against macrophage killing.

  20. Activation of TLR3 in keratinocytes increases expression of genes involved in formation of the epidermis, lipid accumulation and epidermal organelles

    PubMed Central

    Borkowski, Andrew W.; Park, Kyungho; Uchida, Yoshikazu; Gallo, Richard L.

    2013-01-01

    Injury to the skin, and the subsequent release of non-coding double-stranded RNA from necrotic keratinocytes, has been identified as an endogenous activator of Toll-like receptor 3 (TLR3). Since changes in keratinocyte growth and differentiation follow injury, we hypothesized that TLR3 might trigger some elements of the barrier repair program in keratinocytes. Double-stranded RNA was observed to induce TLR3-dependent increases in human keratinocyte mRNA abundance for ABCA12 (ATP-binding cassette, sub-family A, member 12), glucocerebrosidase, acid sphingomyelinase, and transglutaminase 1. Additionally, treatment with double-stranded RNA resulted in increases in sphingomyelin and morphologic changes including increased epidermal lipid staining by oil-red O and TLR3-dependent increases in lamellar bodies and keratohyalin granules. These observations show that double-stranded RNA can stimulate some events in keratinocytes that are important for skin barrier repair and maintenance. PMID:23353987

  1. The PSORS1 locus gene CCHCR1 affects keratinocyte proliferation in transgenic mice.

    PubMed

    Tiala, Inkeri; Wakkinen, Janica; Suomela, Sari; Puolakkainen, Pauli; Tammi, Raija; Forsberg, Sofi; Rollman, Ola; Kainu, Kati; Rozell, Björn; Kere, Juha; Saarialho-Kere, Ulpu; Elomaa, Outi

    2008-04-01

    The CCHCR1 gene (Coiled-Coil alpha-Helical Rod protein 1) within the major psoriasis susceptibility locus PSORS1 is a plausible candidate gene for the risk effect. We have previously generated transgenic mice overexpressing either the psoriasis-associated risk allele CCHCR1*WWCC or the normal allele of CCHCR1. All transgenic CCHCR1 mice appeared phenotypically normal, but exhibited altered expression of genes relevant to the pathogenesis of psoriasis, including upregulation of hyperproliferation markers keratins 6, 16 and 17. Here, we challenged the skin of CCHCR1 transgenic mice with wounding or 12-O-tetradecanoyl-13-acetate (TPA), treatments able to induce epidermal hyperplasia and proliferation that both are hallmarks of psoriasis. These experiments revealed that CCHCR1 regulates keratinocyte proliferation. Early wound healing on days 1 and 4 was delayed, and TPA-induced epidermal hyperproliferation was less pronounced in mice with the CCHCR1*WWCC risk allele than in mice with the normal allele or in wild-type animals. Finally, we demonstrated that overexpression of CCHCR1 affects basal keratinocyte proliferation in mice; CCHCR1*WWCC mice had less proliferating keratinocytes than the non-risk allele mice. Similarly, keratinocytes isolated from risk allele mice proliferated more slowly in culture than wild-type cells when measured by BrdU labeling and ELISA. Our data show that CCHCR1 may function as a negative regulator of keratinocyte proliferation. Thus, aberrant function of CCHCR1 may lead to abnormal keratinocyte proliferation which is a key feature of psoriatic epidermis.

  2. Keratinocyte detachment-differentiation connection revisited, or anoikis-pityriasi nexus redux.

    PubMed

    Banno, Tomohiro; Blumenberg, Miroslav

    2014-01-01

    Epidermis, a continuously self-renewing and differentiating organ, produces a protective stratum corneum that shields us from external chemical, physical and microbial threats. Epidermal differentiation is a multi-step process regulated by influences, some unknown, others insufficiently explored. Detachment of keratinocytes from the basement membrane is one such pro-differentiation stimulus. Here, we define the transcriptional changes during differentiation, especially those caused by detachment from the substratum. Using comprehensive transcriptional profiling, we revisited the effects of detachment as a differentiation signal to keratinocytes. We identified the genes regulated by detachment, the corresponding ontological categories and, using metaanalysis, compared the genes and categories to those regulated by other pro-differentiating stimuli. We identified 762 genes overexpressed in suspended keratinocyte, including known and novel differentiation markers, and 1427 in attached cells, including basal layer markers. Detachment induced epidermis development, cornification and desmosomal genes, but also innate immunity, proliferation inhibitors, transcription regulators and MAPKs; conversely the attached cells overexpressed cell cycle, anchoring, motility, splicing and mitochondrial genes, and both positive and negative regulators of apoptosis. Metaanalysis identified which detachment-regulated categories overlap with those induced by suprabasal location in vivo, by reaching confluency in vitro, and by inhibition of JUN kinases. Attached and in vivo basal cells shared overexpression of mitochondrial components. Interestingly, melanosome trafficking components were also overexpressed in the attached and in vivo basal keratinocytes. These results suggest that specific pro-differentiation signals induce specific features of the keratinization process, which are in vivo orchestrated into harmonious epidermal homeostasis.

  3. 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.

  4. Sphingosine 1-phosphate protects primary human keratinocytes from apoptosis via nitric oxide formation through the receptor subtype S1P₃.

    PubMed

    Schmitz, Elisabeth I; Potteck, Henrik; Schüppel, Melanie; Manggau, Marianti; Wahydin, Elly; Kleuser, Burkhard

    2012-12-01

    Although the lipid mediator sphingosine 1-phosphate (S1P) has been identified to induce cell growth arrest of human keratinocytes, the sphingolipid effectively protects these epidermal cells from apoptosis. The molecular mechanism of the anti-apoptotic action induced by S1P is less characterized. Apart from S1P, endogenously produced nitric oxide (NO•) has been recognized as a potent modulator of apoptosis in keratinocytes. Therefore, it was of great interest to elucidate whether S1P protects human keratinocytes via a NO•-dependent signalling pathway. Indeed, S1P induced an activation of endothelial nitric oxide synthase (eNOS) in human keratinocytes leading to an enhanced formation of NO•. Most interestingly, the cell protective effect of S1P was almost completely abolished in the presence of the eNOS inhibitor L-NAME as well as in eNOS-deficient keratinocytes indicating that the sphingolipid metabolite S1P protects human keratinocytes from apoptosis via eNOS activation and subsequent production of protective amounts of NO•. It is well established that most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. Therefore, the involvement of S1P-receptor subtypes in S1P-mediated eNOS activation has been examined. Indeed, this study clearly shows that the S1P(3) is the exclusive receptor subtype in human keratinocytes which mediates eNOS activation and NO• formation in response to S1P. In congruence, when the S1P(3) receptor subtype is abrogated, S1P almost completely lost its ability to protect human keratinocytes from apoptosis.

  5. 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

  6. Keratinocyte Antiviral Response to Poly(dA:dT) Stimulation and Papillomavirus Infection in a Canine Model of X-Linked Severe Combined Immunodeficiency

    PubMed Central

    Luff, Jennifer A.; Yuan, Hang; Kennedy, Douglas; Schlegel, Richard; Felsburg, Peter; Moore, Peter F.

    2014-01-01

    X-linked severe combined immunodeficiency (XSCID) is caused by a genetic mutation within the common gamma chain (γc), an essential component of the cytokine receptors for interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21. XSCID patients are most commonly treated with bone marrow transplants (BMT) to restore systemic immune function. However, BMT-XSCID humans and dogs remain at an increased risk for development of cutaneous papillomavirus (PV) infections and their associated neoplasms, most typically cutaneous papillomas. Since basal keratinocytes are the target cell for the initial PV infection, we wanted to determine if canine XSCID keratinocytes have a diminished antiviral cytokine response to poly(dA:dT) and canine papillomavirus-2 (CPV-2) upon initial infection. We performed quantitative RT-PCR for antiviral cytokines and downstream interferon stimulated genes (ISG) on poly(dA:dT) stimulated and CPV-2 infected monolayer keratinocyte cultures derived from XSCID and normal control dogs. We found that XSCID keratinocytes responded similarly to poly(dA:dT) as normal keratinocytes by upregulating antiviral cytokines and ISGs. CPV-2 infection of both XSCID and normal keratinocytes did not result in upregulation of antiviral cytokines or ISGs at 2, 4, or 6 days post infection. These data suggest that the antiviral response to initial PV infection of basal keratinocytes is similar between XSCID and normal patients, and is not the likely source for the remaining immunodeficiency in XSCID patients. PMID:25025687

  7. Differential susceptibility to hydrogen sulfide-induced apoptosis between PHLDA1-overexpressing oral cancer cell lines and oral keratinocytes: role of PHLDA1 as an apoptosis suppressor.

    PubMed

    Murata, Takatoshi; Sato, Tsutomu; Kamoda, Takeshi; Moriyama, Hiromitsu; Kumazawa, Yasuo; Hanada, Nobuhiro

    2014-01-15

    Hydrogen sulfide (H2S) is a novel gasotransmitter that plays multiple biological roles in various body systems. In addition to its endogenous production, H2S is produced by bacteria colonizing digestive organs, including the oral cavity. H2S was previously shown to enhance pro-apoptotic effects in cancer cell lines, although the mechanisms involved remain unclear. To properly assess the anti-cancer effects of H2S, however, investigations of apoptotic effects in normal cells are also necessary. The aims of this study were (1) to compare the susceptibility to H2S-induced apoptosis between the oral cancer cell line Ca9-22 and oral keratinocytes that were derived from healthy gingiva, and (2) to identify candidate genes involved in the induction of apoptosis by H2S. The susceptibility to H2S-induced apoptosis in Ca9-22 cells was significantly higher than that in keratinocytes. H2S exposure in Ca9-22 cells, but not keratinocytes, enhanced the expression of pleckstrin homology-like domain, family A, member 1 (PHLDA1), which was identified through a differential display method. In addition, PHLDA1 expression increased during actinomycin D-induced apoptosis in Ca9-22 cells. Knockdown of PHLDA1 expression by small interfering RNA in Ca9-22 cells led to expression of active caspase 3, thus indicating apoptosis induction. The tongue cancer cell line SCC-25, which expresses PHLDA1 at a high level, showed similar effects. Our data indicate that H2S is an anti-cancer compound that may contribute to the low incidence of oral cancer. Furthermore, we demonstrated the role of PHLDA1 as an apoptosis suppressor.

  8. ADA3: a gene, identified by resistance to GAL4-VP16, with properties similar to and different from those of ADA2.

    PubMed Central

    Piña, B; Berger, S; Marcus, G A; Silverman, N; Agapite, J; Guarente, L

    1993-01-01

    We describe the isolation of a yeast gene, ADA3, mutations in which prevent the toxicity of GAL4-VP16 in vivo. Toxicity was previously proposed to be due to the trapping of general transcription factors required at RNA polymerase II promoters (S. L. Berger, B. Piña, N. Silverman, G. A. Marcus, J. Agapite, J. L. Regier, S. J. Triezenberg, and L. Guarente, Cell 70:251-265, 1992). trans activation by VP16 as well as the acidic activation domain of GCN4 is reduced in the mutant. Other activation domains, such as those of GAL4 and HAP4, are only slightly affected in the mutant. This spectrum is similar to that observed for mutants with lesions in ADA2, a gene proposed to encode a transcriptional adaptor. The ADA3 gene is not absolutely essential for cell growth, but gene disruption mutants grow slowly and are temperature sensitive. Strains doubly disrupted for ada2 and ada3 grow no more slowly than single mutants, providing further evidence that these genes function in the same pathway. Selection of initiation sites by the general transcriptional machinery in vitro is altered in the ada3 mutant, providing a clue that ADA3 could be a novel general transcription factor involved in the response to acidic activators. Images PMID:8413201

  9. 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.

  10. STAT3-dependent effects of IL-22 in human keratinocytes are counterregulated by sirtuin 1 through a direct inhibition of STAT3 acetylation.

    PubMed

    Sestito, Rosanna; Madonna, Stefania; Scarponi, Claudia; Cianfarani, Francesca; Failla, Cristina M; Cavani, Andrea; Girolomoni, Giampiero; Albanesi, Cristina

    2011-03-01

    IL-22 has a pathogenetic role in psoriasis, where it is responsible for the altered proliferation and differentiation of keratinocytes and induces inflammatory molecules. The IL-22-induced effects are mediated by STAT3, whose activity is proportional to acetylation in lysine (Lys)685 and phosphorylation in tyrosine (Tyr)705. Lys 685 acetylation of STAT3 is inhibited by sirtuin (SIRT)1, a class III deacetylase promoting keratinocyte differentiation. Due to the opposite effects of IL-22 and SIRT1, we investigated whether IL-22-induced effects in keratinocytes could be regulated by SIRT1 through control of STAT3. We found that SIRT1 opposes the IL-22-induced STAT3 activity by deacetylating STAT3 and reducing STAT3 Tyr705 phosphorylation. By controlling STAT3, SIRT1 also influences the IL-22-induced expression of molecules involved in proliferation and inflammation as well as proliferation and migration processes in cultured keratinocytes. Although SIRT1 levels were similar in keratinocytes of healthy individuals and patients with psoriasis, they were reduced in psoriatic skin lesions, with the lymphokine IFN-γ inhibiting SIRT1 expression. Concomitantly, IFN-γ enhanced basal acetylation of STAT3 and its phosphorylation induced by IL-22. In conclusion, STAT3-dependent IL-22 signaling and effects in keratinocytes are negatively regulated by SIRT1. In skin affected by psoriasis, SIRT1 is down-regulated by IFN-γ, which thus renders psoriatic keratinocytes more prone to respond to IL-22.

  11. Differentiation of cultured keratinocytes promotes the adherence of Streptococcus pyogenes.

    PubMed Central

    Darmstadt, G L; Fleckman, P; Jonas, M; Chi, E; Rubens, C E

    1998-01-01

    Based on a consideration of the histopathology of nonbullous impetigo that shows localization of Streptococcus pyogenes to highly differentiated, subcorneal keratinocytes, we hypothesized that adherence of an impetigo strain of S. pyogenes would be promoted by terminal differentiation of keratinocytes. An assay was developed in which S. pyogenes adhered via pilus-like projections from the cell wall to the surface of cultured human keratinocytes in a time- and inoculum-dependent manner suggestive of a receptor-mediated process. Terminal differentiation of keratinocytes was induced by increasing the calcium concentration in the growth medium, and was confirmed by morphologic analysis using electron microscopy. Adherence of S. pyogenes was three and fourfold greater to keratinocytes differentiated in 1.0 and 1.5 mM calcium, respectively, compared with undifferentiated keratinocytes in 0.15 mM calcium. The presence of calcium during the adherence assay further enhanced adherence nearly twofold. Adherence occurred preferentially to sites of contact between adjacent keratinocytes, suggesting that the keratinocyte receptor may be a molecule involved in cell-to-cell adhesion. In contrast, nonpathogenic Streptococcus gordonii adhered poorly to keratinocytes regardless of their state of terminal differentiation, and adherence of a pharyngeal strain of S. pyogenes was twofold greater to undifferentiated than differentiated keratinocytes. This is the first report of in vitro adherence of S. pyogenes to keratinocytes in a manner that emulates human impetigo. Adherence of only the impetigo strain, and not the pharyngeal strain of S. pyogenes or the nonpathogenic S. gorgonii isolate, was promoted by keratinocyte differentiation. This result provides a model system for investigating the molecular pathogenesis of streptococcal skin infections. PMID:9421474

  12. p53 and TAp63 promote keratinocyte proliferation and differentiation in breeding tubercles of the zebrafish.

    PubMed

    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.

  13. Ultraviolet A radiation transiently disrupts gap junctional communication in human keratinocytes.

    PubMed

    Provost, Nicolas; Moreau, Marielle; Leturque, Armelle; Nizard, Carine

    2003-01-01

    Ultraviolet A (UVA) (320-400 nm) radiation is known to cause cutaneous aging and skin cancer. We studied the effect of UVA (365 nm) radiation on the human epidermis by focusing on keratinocyte gap junction-mediated intercellular communication (GJIC). We observed a dose-dependent 10-fold decrease in GJIC induced by UVA in normal human keratinocytes. This decrease in GJIC was associated with time-dependent internalization of connexin43 (Cx43). UVA radiation also damaged the actin cytoskeleton, as shown by microfilament disappearance. Importantly, the decrease in GJIC was transient when keratinocytes were irradiated with 10 J/cm(2) UVA, with a return to baseline values after 8 h. Concomitantly, Cx43 was relocalized and the actin cytoskeleton was restored. UVA irradiation and 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment activated protein kinase C and reduced GJIC. However, Cx43 localization and phosphorylation were differently regulated by the two treatments. This suggests that at least two different pathways may mediate the observed fall in GJIC. These findings identify keratinocyte GJIC as a new UVA target that might sensitize human skin to photoaging and cancer formation.

  14. 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

  15. Test system for evaluating the influence of polymer properties on primary human keratinocytes and fibroblasts in mono- and coculture.

    PubMed

    Trescher, Karoline; Roch, Toralf; Cui, Jing; Kratz, Karl; Lendlein, Andreas; Jung, Friedrich

    2013-06-20

    Interactions of cells with polymer-based biomaterials are influenced by properties of the substrate. Polymers, which are able to induce cell specific effects, gain increasing importance for biotechnology and regenerative therapies. A test system was developed, which allows studying primary human keratinocytes and fibroblasts in mono- and cocultures to analyze and operate the effect of polymer properties. This system offers to identify polymers for keratinocyte cultivation or wound dressings, since adherence, viability and functionality can be analyzed. Especially the coculture system enables the characterization of potential cell specific effects of polymer-based biomaterials. To establish a coculture test system, it is challenging to find a suitable culture medium, to identify initial seeding densities for comparable cell growth and to develop methods to distinguish and characterize both cell types. Poly(n-butyl acrylate) networks (cPnBAs) as model biomaterials were used to demonstrate the applicability of our newly developed coculture screening system for differential cell growth. The apparent Young's modulus of the cPnBAs differentially regulated fibroblasts and keratinocytes. Particularly, cPnBA73 with an apparent Young's modulus of 930±140 kPa measured in phosphate buffered saline (PBS) solution at ambient temperature seemed to have favoring properties for keratinocyte adhesion, while fibroblast adhesion was not affected. For keratinocytes the concentration of some pro-inflammatory cytokines was lower on cPnBA73 and a decreased deposition of collagen, elastin and fibronectin was observed in the coculture.

  16. Distinct epidermal keratinocytes respond to extremely low-frequency electromagnetic fields differently.

    PubMed

    Huang, Chao-Ying; Chuang, Chun-Yu; Shu, Wun-Yi; Chang, Cheng-Wei; Chen, Chaang-Ray; Fan, Tai-Ching; Hsu, Ian C

    2014-01-01

    Following an increase in the use of electric appliances that can generate 50 or 60 Hz electromagnetic fields, concerns have intensified regarding the biological effects of extremely low-frequency electromagnetic fields (ELF-EMFs) on human health. Previous epidemiological studies have suggested the carcinogenic potential of environmental exposure to ELF-EMFs, specifically at 50 or 60 Hz. However, the biological mechanism facilitating the effects of ELF-EMFs remains unclear. Cellular studies have yielded inconsistent results regarding the biological effects of ELF-EMFs. The inconsistent results might have been due to diverse cell types. In our previous study, we indicated that 1.5 mT, 60 Hz ELF-EMFs will cause G1 arrest through the activation of the ATM-Chk2-p21 pathway in human keratinocyte HaCaT cells. The aim of the current study was to investigate whether ELF-EMFs cause similar effects in a distinct epidermal keratinocyte, primary normal human epidermal keratinocytes (NHEK), by using the same ELF-EMF exposure system and experimental design. We observed that ELF-EMFs exerted no effects on cell growth, cell proliferation, cell cycle distribution, and the activation of ATM signaling pathway in NHEK cells. We demonstrated that the 2 epidermal keratinocytes responded to ELF-EMFs differently. To further validate this finding, we simultaneously exposed the NHEK and HaCaT cells to ELF-EMFs in the same incubator for 168 h and observed the cell growths. The simultaneous exposure of the two cell types results showed that the NHEK and HaCaT cells exhibited distinct responses to ELF-EMFs. Thus, we confirmed that the biological effects of ELF-EMFs in epidermal keratinocytes are cell type specific. Our findings may partially explain the inconsistent results of previous studies when comparing results across various experimental models.

  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. Molecular similarity and property similarity.

    PubMed

    Barbosa, Frédérique; Horvath, Dragos

    2004-01-01

    This paper reviews the main efforts undertaken up to date in order to understand, rationalize and apply the similarity principle (similar compounds=>similar properties) as a computational tool in modern drug discovery. The best suited mathematical expression of this classical working hypothesis of medicinal chemistry needs to be carefully chosen (out of the virtually infinite possible implementations in terms of molecular descriptors and molecular similarity metrics), in order to achieve an optimal validation of the hypothesis that molecules that are neighbors in the Structural Space will also display similar properties. This overview will show why no single "absolute" measure of molecular similarity can be conceived, and why molecular similarity scores should be considered tunable tools that need to be adapted to each problem to solve.

  19. UVB Radiation-Induced β-catenin Signaling is Enhanced by COX-2 Expression in Keratinocytes

    PubMed Central

    Smith, Kimberly A.; Tong, Xin; Abu-Yousif, Adnan O.; Mikulec, Carol C.; Gottardi, Cara J.; Fischer, Susan M.; Pelling, Jill C.

    2014-01-01

    UVB radiation is the major carcinogen responsible for skin carcinogenesis, thus elucidation of the molecular pathways altered in skin in response to UVB would reveal novel targets for therapeutic intervention. It is well established that UVB leads to upregulation of cyclooxygenase 2 (COX-2) in the skin which contributes to skin carcinogenesis. Overexpression of COX-2 has been shown to promote colon cancer cell growth through β-catenin signaling, however, little is known about the connection between UVB, COX-2 and β-catenin in the skin. In the present study, we have identified a novel pathway in which UVB induces β-catenin signaling in keratinocytes, which is modulated by COX-2 expression. Exposure of the mouse 308 keratinocyte cell line (308 cells) and primary normal human epidermal keratinocytes (NHEKs) to UVB resulted in increased protein levels of both N-terminally unphosphorylated and total β-catenin. In addition, we found that UVB enhanced β-catenin-dependent TOPflash reporter activity and expression of a downstream β-catenin target gene. We demonstrated that UVB-induced β-catenin signaling is modulated by COX-2, as treatment of keratinocytes with the specific COX-2 inhibitor NS398 blocked UVB induction of β-catenin. Additionally, β-catenin target gene expression was reduced in UVB-treated COX-2 knockout (KO) MEFs compared to wild-type (WT) MEFs. Furthermore, epidermis from UVB-exposed SKH-1 mice exhibited increased N-terminally unphosphorylated and total β-catenin protein levels and increased staining for total β-catenin, and both responses were reduced in COX-2 heterozygous mice. Taken together, these results suggest a novel pathway in which UVB induces β-catenin signaling in keratinocytes which is enhanced by COX-2 expression. PMID:21853475

  20. Wnt-3a-activated human fibroblasts promote human keratinocyte proliferation and matrix destruction.

    PubMed

    Sobel, Katrin; Tham, Marius; Stark, Hans-Jürgen; Stammer, Hermann; Prätzel-Wunder, Silke; Bickenbach, Jackie R; Boukamp, Petra

    2015-06-15

    Aberrant Wnt regulation, detectable by nuclear translocation of beta-catenin, is a hallmark of many cancers including skin squamous cell carcinomas (SCCs). By analyzing primary human skin SCCs, we demonstrate that nuclear beta-catenin is not restricted to SCC cells but also detected in stromal fibroblasts, suggesting an important role for aberrant Wnt regulation also in the tumor microenvironment. When human keratinocytes and fibroblasts were treated with Wnt-3a, fibroblasts proved to be more responsive. Accordingly, Wnt-3a did not alter HaCaT cell functions in a cell-autonomous manner. However, when organotypic cultures (OTCs) were treated with Wnt-3a, HaCaT keratinocytes responded with increased proliferation. As nuclear beta-catenin was induced only in the fibroblasts, this argued for a Wnt-dependent, paracrine keratinocyte stimulation. Global gene expression analysis of Wnt-3a-stimulated fibroblasts identified genes encoding interleukin-8 (IL-8) and C-C motif chemokine 2 (CCL-2) as well as matrix metalloproteinase-1 (MMP-1) as Wnt-3a targets. In agreement, we show that IL-8 and CCL-2 were secreted in high amounts by Wnt-3a-stimulated fibroblasts also in OTCs. The functional role of IL-8 and CCL-2 as keratinocyte growth regulators was confirmed by directly stimulating HaCaT cell proliferation in conventional cultures. Most important, neutralizing antibodies against IL-8 and CCL-2 abolished the Wnt-dependent HaCaT cell hyperproliferation in OTCs. Additionally, MMP-1 was expressed in high amounts in Wnt-3a-stimulated OTCs and degraded the stromal matrix. Thus, our data show that Wnt-3a stimulates fibroblasts to secrete both keratinocyte proliferation-inducing cytokines and stroma-degrading metalloproteinases, thereby providing evidence for a novel Wnt deregulation in the tumor-stroma directly contributing to skin cancer progression.

  1. 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)

  2. α6 Integrin and CD44 enrich for a primary keratinocyte population that displays resistance to UV-induced apoptosis.

    PubMed

    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 integrin(high+)/CD44(+) sub-population of basal keratinocytes. These α6 integrin(high+)/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 integrin(high+)/CD44(+) cells involved increased expression of TAp63 and was overcome by PI-3 kinase inhibition. In marked contrast, the α6 integrin(high+)/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 integrin(high+)/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.

  3. 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.

  4. Melanocytes expressing MC1R polymorphisms associated with red hair color have altered MSH-ligand activated pigmentary responses in coculture with keratinocytes.

    PubMed

    Roberts, Donald W; Newton, Richard A; Leonard, J Helen; Sturm, Richard A

    2008-05-01

    The occurrence of red hair and pale skin in individuals, which is associated with UV-radiation sensitivity and increased skin cancer risk, is mainly due to polymorphisms in the melanocortin-1 receptor (MC1R) expressed in melanocytes. We have established a serum free human melanocyte-keratinocyte coculture system to study the behavior and functional abilities of melanocytes expressing MC1R red hair color (RHC) variants in order to identify differences from their wild type (WT) counterparts. This model revealed the importance of elevated calcium levels in promoting strong melanocyte interaction with the surrounding keratinocytes and resulted in a dendritic melanocyte morphology similar to that in skin. However, the dendricity response following agonist activation of the MC1R receptor by NDP-MSH peptide, was markedly enhanced in WT melanocytes in comparison to RHC strains. Analysis of mRNA expression and protein levels of the major pigmentation markers following NDP-MSH treatment distinguished the enzyme dopachrome tautomerase as preferentially upregulated in cocultures of WT strains, with negligible or a much reduced response in melanocytes with RHC variant alleles. These results highlight the use of the coculture system in determining fundamental differences in the physiology of melanocytes expressing RHC MC1R receptors and those of WT genotype, which are likely to contribute to the increased skin cancer risk for individuals that carry these variants.

  5. 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.

  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. 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

  8. 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

  9. 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

  10. Sodium arsenite-induced stress-related gene expression in normal human epidermal, HaCaT, and HEL30 keratinocytes.

    PubMed Central

    Trouba, Kevin J; Geisenhoffer, Kristen M; Germolec, Dori R

    2002-01-01

    Arsenic is a carcinogen that poses a significant health risk in humans. Based on evidence that arsenic has differential effects on human, rodent, normal, and transformed cells, these studies addressed the relative merits of using normal human epidermal keratinocytes (NHEK) and immortalized human (HaCaT) and mouse (HEL30) keratinocytes when examining stress-induced gene expression that may contribute to carcinogenesis. We hypothesize that redox-related gene expression is differentially modulated by arsenic in normal versus immortalized keratinocytes. To test the hypothesis, we exposed keratinocytes to sodium arsenite for 4 or 24 hr, at which time serine threonine kinase-25 (stk25) and nicotine adenine dinucleotide phosphate [nad(p)h] quinone oxidoreductase gene expression were measured. The effect of glutathione reduction on arsenite-induced cytotoxicity and gene expression in NHEK also was evaluated by addition of l-buthionine-[S,R]-sulfoximine (BSO) to culture media. Results indicate the term LC(50) for arsenite is approximately 10-15 microM in NHEK and HEL30 keratinocytes and 30 microM in HaCaT keratinocytes. Compared with HaCaT and HEL30 keratinocytes, a nontoxic concentration of arsenite (2.5 microM) increases stk25 and nad(p)h quinone oxidoreductase gene expression in NHEK, an effect partially attenuated by BSO. These data indicate that NHEK and HaCaT/HEL30 keratinocytes have similar sensitivities toward arsenite-induced cytotoxicity but unique gene expression responses. They also suggest that arsenite modulates gene expression in NHEK involved in cellular signaling and other aspects of intermediary metabolism that may contribute to the carcinogenic process. PMID:12426128

  11. Prolonged Integration Site Selection of a Lentiviral Vector in the Genome of Human Keratinocytes

    PubMed Central

    Qian, Wei; Wang, Yong; Li, Rui-fu; Zhou, Xin; Liu, Jing; Peng, Dai-zhi

    2017-01-01

    Background Lentiviral vectors have been successfully used for human skin cell gene transfer studies. Defining the selection of integration sites for retroviral vectors in the host genome is crucial in risk assessment analysis of gene therapy. However, genome-wide analyses of lentiviral integration sites in human keratinocytes, especially after prolonged growth, are poorly understood. Material/Methods In this study, 874 unique lentiviral vector integration sites in human HaCaT keratinocytes after long-term culture were identified and analyzed with the online tool GTSG-QuickMap and SPSS software. Results The data indicated that lentiviral vectors showed integration site preferences for genes and gene-rich regions. Conclusions This study will likely assist in determining the relative risks of the lentiviral vector system and in the design of a safe lentiviral vector system in the gene therapy of skin diseases. PMID:28255155

  12. Heavy metals chromium and neodymium reduced phosphorylation level of heat shock protein 27 in human keratinocytes.

    PubMed

    Zhang, Qihao; Zhang, Lei; Xiao, Xue; Su, Zhijian; Zou, Ping; Hu, Hao; Huang, Yadong; He, Qing-Yu

    2010-06-01

    Heavy metals may exert their acute and chronic effects on the human skin through stress signals. In the present study, 2DE-based proteomics was used to analyze the protein expression in human keratinocytes exposed to heavy metals, chromium and neodymium, and 10 proteins with altered expression were identified. Among these proteins, small heat shock protein 27 (HSP27) was up-regulated significantly and the up-regulation was validated by Western blot and immunofluorescence. In addition, the mRNA expression level of HSP27 markedly increased as detected by quantitative PCR. More interestingly, the ratio of phosphorylated HSP27 and total HSP27 significantly decreased in keratinocytes treated with the heavy metals. These findings suggested that heavy metals reduced the phosphorylation level of HSP27, and that the ratio of p-HSP27 and HSP27 may represent a potential marker or additional endpoint for the hazard assessment of skin irritation caused by chemical products.

  13. Stathmin regulates keratinocyte proliferation and migration during cutaneous regeneration.

    PubMed

    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.

  14. Constitutive and UV-induced metabolism of melatonin in keratinocytes and cell-free systems.

    PubMed

    Fischer, Tobias W; Sweatman, Trevor W; Semak, Igor; Sayre, Robert M; Wortsman, Jacobo; Slominski, Andrzej

    2006-07-01

    Melatonin, which can be produced in the skin, exerts a protective effect against damage induced by UV radiation (UVR). We have investigated the effect of UVB, the most damaging component of UVR, on melatonin metabolism in HaCaT keratinocytes and in a cell-free system. Four metabolites were identified by HPLC and LC-MS: 6-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), 2-hydroxymelatonin (the main intermediate between melatonin and AFMK), and 4-hydroxymelatonin. Concentrations of these photoproducts were directly proportional to UVR-dose and to melatonin substrate content, and their accumulation was time-dependent. The UVR-dependent increase of AFMK and 2-hydroxymelatonin was also detected in keratinocytes, where it was accompanied by simultaneous consumption of intracellular melatonin. Of note, melatonin and its two major metabolites, 2-hydroxymelatonin and AFMK, were also detected in untreated keratinocytes, neither irradiated nor preincubated with melatonin. Thus, intracellular melatonin metabolism is enhanced under exposure to UVR. The additional biological activity of these individual melatonin metabolites increases the spectrum of potential actions of the recently identified cutaneous melatoninergic system.

  15. Oral keratinocyte stem/progenitor cells: specific markers, molecular signaling pathways and potential uses.

    PubMed

    Calenic, Bogdan; Greabu, Maria; Caruntu, Constantin; Tanase, Cristiana; Battino, Maurizio

    2015-10-01

    Oral keratinocyte stem cells reside in the basal layers of the oral epithelium, representing a minor population of cells with a great potential to self-renew and proliferate over the course of their lifetime. As a result of the potential uses of oral keratinocyte stem cells in regenerative medicine and the key roles they play in tissue homeostasis, inflammatory conditions, wound healing and tumor initiation and progression, intense scientific efforts are currently being undertaken to identify, separate and reprogram these cells. Although currently there is no specific marker that can characterize and isolate oral keratinocyte stem cells, several suggestions have been made. Thus, different stem/progenitor-cell subpopulations have been categorized based on combinations of positive and/or negative membrane-surface markers, which include integrins, clusters of differentiation and cytokeratins. Important advances have also been made in understanding the molecular pathways that govern processes such as self-renewal, differentiation, proliferation, wound healing and programmed cell death. A thorough understanding of stem-cell biology and the molecular players that govern cellular fate is paramount in the quest for using stem-cell-derived therapies in the treatment of various oral pathologies. The current review focuses on recent advances in understanding the molecular signaling pathways coordinating the behavior of these cells and in identifying suitable markers used for their isolation and characterization. Special emphasis will also be placed on the roles played by oral keratinocyte stem and progenitor cells in normal and diseased oral tissues and on their potential uses in the fields of general medicine and dentistry.

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

    PubMed

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

    2011-07-15

    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.

  17. 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.

  18. A new mass-spectrometric C-terminal sequencing technique finds a similarity between gamma-interferon and alpha 2-interferon and identifies a proteolytically clipped gamma-interferon that retains full antiviral activity.

    PubMed Central

    Rose, K; Simona, M G; Offord, R E; Prior, C P; Otto, B; Thatcher, D R

    1983-01-01

    A novel mass-spectrometric technique is described that permits the identification of the C-terminal peptide of a protein. The technique involves the incorporation of 18O into all alpha-carboxy groups liberated during enzyme-catalysed partial hydrolysis of the protein, followed by mass spectrometry to identify as the C-terminal peptide the only peptide that did not incorporate any 18O. The technique has been used to identify the true C-terminal tryptic peptide of a bacterially produced gamma-interferon and to distinguish it from a peptide produced by anomalous tryptic cleavage. It was found that a closely similar sequence segment of bacterially produced alpha 2-interferon undergoes an analogous cleavage. The technique was also used to identify the C-terminus of a clipped gamma-interferon that retains full antiviral activity. PMID:6418141

  19. A decorin-deficient matrix affects skin chondroitin/dermatan sulfate levels and keratinocyte function

    PubMed Central

    Nikolovska, Katerina; Renke, Jana K.; Jungmann, Oliver; Grobe, Kay; Iozzo, Renato V.; Zamfir, Alina D.; Seidler, Daniela G.

    2016-01-01

    Decorin is a small leucine-rich proteoglycan harboring a single glycosaminoglycan chain, which, in skin, is mainly composed of dermatan sulfate (DS). Mutant mice with targeted disruption of the decorin gene (Dcn−/−) exhibit an abnormal collagen architecture in the dermis and reduced tensile strength, collectively leading to a skin fragility phenotype. Notably, Ehlers-Danlos patients with mutations in enzymes involved in the biosynthesis of DS display a similar phenotype, and recent studies indicate that DS is involved in growth factor binding and signaling. To determine the impact of the loss of DS-decorin in the dermis, we analyzed the glycosaminoglycan content of Dcn−/− and wild-type mouse skin. The total amount of chondroitin/dermatan sulfate (CS/DS) was increased in the Dcn−/− skin, but was overall less sulfated with a significant reduction in bisulfated ΔDiS2,X (X=4 or 6) disaccharide units, due to the reduced expression of uronyl 2-O sulfotransferase (Ust). With increasing age, sulfation declined; however, Dcn−/− CS/DS was constantly undersulfated vis-à-vis wild-type. Functionally, we found altered fibroblast growth factor (Fgf)-7 and -2 binding due to changes in the micro-heterogeneity of skin Dcn−/− CS/DS. To better delineate the role of decorin, we used a 3D Dcn−/− fibroblast cell culture model. We found that the CS/DS extracts of wild-type and Dcn−/− fibroblasts were similar to the skin sugars, and this correlated with the lack of uronyl 2-O sulfotransferase in the Dcn−/− fibroblasts. Moreover, Ffg7 binding to total CS/DS was attenuated in the Dcn−/− samples. Surprisingly, wild-type CS/DS significantly reduced the binding of Fgf7 to keratinocytes in concentration dependent manner unlike the Dcn−/− CS/DS that only affected the binding at higher concentrations. Although binding to cell-surfaces was quite similar at higher concentrations, keratinocyte proliferation was differentially affected. Higher concentration of

  20. 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 (

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

    PubMed

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

    2016-07-07

    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.

  2. 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.

  3. The M4 muscarinic receptor-selective effects on keratinocyte crawling locomotion.

    PubMed

    Chernyavsky, Alex I; Nguyen, Vu Thuong; Arredondo, Juan; Ndoye, Assane; Zia, Shaheen; Wess, Jürgen; Grando, Sergei A

    2003-03-28

    We have investigated how the cholinergic system of epidermal keratinocytes (KC) controls migratory function of these cells. Several molecular subtypes of muscarinic acetylcholine receptors (mAChRs) have been detected in KC. Early results suggested that M(4) is the predominant mAChR regulating cell motility. To determine muscarinic effects on lateral migration of KC, we used an agarose gel keratinocyte outgrowth system (AGKOS) which provides for measurements of the response of large cell populations (> 10(4) cells). Muscarine produced a dose-dependent stimulatory effect on cell migration (p < 0.05). This activity was abolished by atropine, which decreased migration distance when given alone. To identify the mAChR subtype(s) mediating these muscarinic effects, we substituted atropine with subtype-selective antagonists. Tropicamide (M(4)-selective) was more effective at decreasing the migration distance than pirenzepine and 4-DAMP at nanomolar concentrations. We then compared lateral migration of KC obtained from M(4) mAChR knockout mice with that of wild-type murine KC, using AGKOS. In the absence of M(4) mAChR, the migration distance of KC was significantly (p < 0.05) decreased. These results indicate that the M(4) mAChR plays a central role in mediating cholinergic control of keratinocyte migration by endogenous acetylcholine produced by these cells.

  4. Confluence switch signaling regulates ECM composition and the plasmin proteolytic cascade in keratinocytes.

    PubMed

    Botta, Adrien; Delteil, Frédéric; Mettouchi, Amel; Vieira, Andhira; Estrach, Soline; Négroni, Luc; Stefani, Caroline; Lemichez, E; Meneguzzi, Guerrino; Gagnoux-Palacios, Laurent

    2012-09-15

    In culture, cell confluence generates signals that commit actively growing keratinocytes to exit the cell cycle and differentiate to form a stratified epithelium. Using a comparative proteomic approach, we studied this 'confluence switch' and identified a new pathway triggered by cell confluence that regulates basement membrane (BM) protein composition by suppressing the uPA-uPAR-plasmin pathway. Indeed, confluence triggers adherens junction maturation and enhances TGF-β and activin A activity, resulting in increased deposition of PAI-1 and perlecan in the BM. Extracellular matrix (ECM)-accumulated PAI-1 suppresses the uPA-uPAR-plasmin pathway and further enhances perlecan deposition by inhibiting its plasmin-dependent proteolysis. We show that perlecan deposition in the ECM strengthens cell adhesion, inhibits keratinocyte motility and promotes additional accumulation of PAI-1 in the ECM at confluence. In agreement, during wound-healing, perlecan concentrates at the wound-margin, where BM matures to stabilize keratinocyte adhesion. Our results demonstrate that confluence-dependent signaling orchestrates not only growth inhibition and differentiation, but also controls ECM proteolysis and BM formation. These data suggest that uncontrolled integration of confluence-dependent signaling, might favor skin disorders, including tumorigenesis, not only by promoting cell hyperproliferation, but also by altering protease activity and deposition of ECM components.

  5. An alternatively spliced IL-15 isoform modulates abrasion-induced keratinocyte activation.

    PubMed

    Lee, Tsung-Lin; Chang, Mei-Ling; Lin, Yu-Jei; Tsai, Ming-Hsun; Chang, Yi-Hsuan; Chuang, Che-Ming; Chien, Yun; Sosinowski, Tomasz; Wang, Chih-Hsiu; Chen, Yi-Yuan; Lee, Chien-Kuo; Chen, Jau-Shiuh; Wang, Li-Fang; Kung, John T; Ku, Chia-Chi

    2015-05-01

    In a routine phenotype-driven screen, we identified a point mutation in exon 7 of the IL-15 gene in Pedigree 191 (deficient memory (DM)) of N-ethyl-N-nitrosourea mutagenized mice. The DM epidermis expressed an alternatively spliced IL-15 mRNA isoform, IL-15ΔE7, and a wild-type (WT) IL-15 isoform at comparable levels. Mechanical stimulation of DM skin or DM skin graft transplanted onto the WT host resulted in reduced keratinocyte activation and inhibition of neutrophil infiltration into the dermis, demonstrating that DM keratinocytes produced less inflammatory response to external stimulation. Ectopic expression of IL-15ΔE7 in WT skin prevented abrasion-induced epidermal thickening, blocked the accumulation of nuclear antigen Ki67(+) cells in the basal and the suprabasal cell layers, increased loricrin expression, and also increased keratinocyte CXCL1 and G-CSF production. IL-15ΔE7 also profoundly blocked neutrophil infiltration in SDS- or immiquimod (IMQ)-treated WT skin. Recombinant IL-15ΔE7 failed to activate STAT-5 and its downstream target bcl-2 expression. Our study points to IL-15ΔE7 as a potential therapeutic agent for treating neutrophilia-associated inflammatory skin disorders.

  6. Caspase-1 activity is required for UVB-induced apoptosis of human keratinocytes.

    PubMed

    Sollberger, Gabriel; Strittmatter, Gerhard E; Grossi, Serena; Garstkiewicz, Martha; Auf dem Keller, Ulrich; French, Lars E; Beer, Hans-Dietmar

    2015-05-01

    Caspase-1 has a crucial role in innate immunity as the protease activates the proinflammatory cytokine prointerleukin(IL)-1β. Furthermore, caspase-1 induces pyroptosis, a lytic form of cell death that supports inflammation. Activation of caspase-1 occurs in multi-protein complexes termed inflammasomes, which assemble upon sensing of stress signals. In the skin and in skin-derived keratinocytes, UVB irradiation induces inflammasome-dependent IL-1 secretion and sunburn. Here we present evidence that caspase-1 and caspase-4 are required for UVB-induced apoptosis. In UVB-irradiated human primary keratinocytes, apoptosis occurs significantly later than inflammasome activation but depends on caspase-1 activity. However, it proceeds independently of inflammasome activation. By a proteomics approach, we identified the antiapoptotic Bap31 as a putative caspase-1 substrate. Caspase-1-dependent apoptosis is possibly a recent process in evolution as it was not detected in mice. These results suggest a protective role of caspase-1 in keratinocytes during UVB-induced skin cancer development through the induction of apoptosis.

  7. 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.

  8. Knockdown of PKD1 in normal human epidermal keratinocytes increases mRNA expression of keratin 10 and involucrin: early markers of keratinocyte differentiation.

    PubMed

    Ivanova, Petya; Atanasova, Ganka; Poumay, Yves; Mitev, Vanyo

    2008-03-01

    Subconfluent normal human keratinocytes exhibit autonomous (autocrine growth factor driven) proliferation and express the specific markers for keratinocyte proliferation K5 (keratin 5) and K14 (keratin 14). Utilizing this model the effects of PKD1 (Protein kinase D1) knockdown on activation of differentiation was studied. siRNA approach was applied to achieve specific knockdown of PKD1 and the mRNA levels of different keratinocyte markers -- K14 and PCNA (markers of basal proliferating keratinocytes), involucrin and K10 (early differentiation markers) were analyzed. Treatment of cultured keratinocytes with siRNA for PKD1 resulted in reduction of mRNA levels of PKD1, altered cell phenotype and promotion of keratinocyte differentiation, demonstrated by increased expression of involucrin and K10 mRNAs. No significant changes in K14 mRNA expression levels were detected, but the expression of PCNA mRNA was markedly diminished. This study was the first to show that mRNA expression of PKD1 in subconfluent normal human keratinocytes is very low, the PKD1 mRNA levels were more than 8-fold lower than the same ones in hTert keratinocytes. These findings suggest antidifferentiative role of PKD1 in normal human keratinocytes, contrary to the prodiferentiative role of PKD1 in human hTert keratinocytes. We came to the conclusion that there are differences between transduction pathways involving PKD1 in primary human keratinocyte cultures and these in immortalized hTert keratinocytes.

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

    PubMed

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

    2013-02-01

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

  10. Functional differences between neonatal and adult fibroblasts and keratinocytes: Donor age affects epithelial-mesenchymal crosstalk in vitro

    PubMed Central

    Mateu, Rosana; Živicová, Veronika; Krejčí, Eliška Drobná; Grim, Miloš; Strnad, Hynek; Vlček, Čestmír; Kolář, Michal; Lacina, Lukáš; Gál, Peter; Borský, Jiří; Smetana, Karel; Dvořánková, Barbora

    2016-01-01

    Clinical evidence suggests that healing is faster and almost scarless at an early neonatal age in comparison with that in adults. In this study, the phenotypes of neonatal and adult dermal fibroblasts and keratinocytes (nestin, smooth muscle actin, keratin types 8, 14 and 19, and fibronectin) were compared. Furthermore, functional assays (proliferation, migration, scratch wound closure) including mutual epithelial-mesenchymal interactions were also performed to complete the series of experiments. Positivity for nestin and α smooth muscle actin was higher in neonatal fibroblasts (NFs) when compared with their adult counterparts (adult fibroblasts; AFs). Although the proliferation of NFs and AFs was similar, they significantly differed in their migration potential. The keratinocyte experiments revealed small, poorly differentiated cells (positive for keratins 8, 14 and 19) in primary cultures isolated from neonatal tissues. Moreover, the neonatal keratinocytes exhibited significantly faster rates of healing the experimentally induced in vitro defects in comparison with adult cells. Notably, the epithelial/mesenchymal interaction studies showed that NFs in co-culture with adult keratinocytes significantly stimulated the adult epithelial cells to acquire the phenotype of small, non-confluent cells expressing markers of poor differentiation. These results indicate the important differences between neonatal and adult cells that may be associated with improved wound healing during the early neonatal period. PMID:27513730

  11. 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

  12. Functional differences between neonatal and adult fibroblasts and keratinocytes: Donor age affects epithelial-mesenchymal crosstalk in vitro.

    PubMed

    Mateu, Rosana; Živicová, Veronika; Krejčí, Eliška Drobná; Grim, Miloš; Strnad, Hynek; Vlček, Čestmír; Kolář, Michal; Lacina, Lukáš; Gál, Peter; Borský, Jiří; Smetana, Karel; Dvořánková, Barbora

    2016-10-01

    Clinical evidence suggests that healing is faster and almost scarless at an early neonatal age in comparison with that in adults. In this study, the phenotypes of neonatal and adult dermal fibroblasts and keratinocytes (nestin, smooth muscle actin, keratin types 8, 14 and 19, and fibronectin) were compared. Furthermore, functional assays (proliferation, migration, scratch wound closure) including mutual epithelial‑mesenchymal interactions were also performed to complete the series of experiments. Positivity for nestin and α smooth muscle actin was higher in neonatal fibroblasts (NFs) when compared with their adult counterparts (adult fibroblasts; AFs). Although the proliferation of NFs and AFs was similar, they significantly differed in their migration potential. The keratinocyte experiments revealed small, poorly differentiated cells (positive for keratins 8, 14 and 19) in primary cultures isolated from neonatal tissues. Moreover, the neonatal keratinocytes exhibited significantly faster rates of healing the experimentally induced in vitro defects in comparison with adult cells. Notably, the epithelial/mesenchymal interaction studies showed that NFs in co-culture with adult keratinocytes significantly stimulated the adult epithelial cells to acquire the phenotype of small, non-confluent cells expressing markers of poor differentiation. These results indicate the important differences between neonatal and adult cells that may be associated with improved wound healing during the early neonatal period.

  13. 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

  14. The lysosomal cysteine protease cathepsin L regulates keratinocyte proliferation by control of growth factor recycling.

    PubMed

    Reinheckel, Thomas; Hagemann, Sascha; Dollwet-Mack, Susanne; Martinez, Elke; Lohmüller, Tobias; Zlatkovic, Gordana; Tobin, Desmond J; Maas-Szabowski, Nicole; Peters, Christoph

    2005-08-01

    Mice deficient for cathepsin L (CTSL) show epidermal hyperplasia due to a hyperproliferation of basal keratinocytes. Here we show that the critical function of CTSL in the skin is keratinocyte specific. This is revealed by transgenic re-expression of CTSL in the keratinocytes of ctsl-/- mice, resulting in a rescue of the ctsl-/- skin phenotype. Cultivation of primary mouse keratinocytes with fibroblast- and keratinocyte-conditioned media, as well as heterologous organotypic co-cultures of mouse fibroblasts and human keratinocytes, showed that the altered keratinocyte proliferation is caused primarily by CTSL-deficiency in keratinocytes. In the absence of EGF, wild type and CTSL-knockout keratinocytes proliferate with the same rates, while in presence of EGF, ctsl-/- keratinocytes showed enhanced proliferation compared with controls. Internalization and degradation of radioactively labeled EGF was identical in both ctsl-/- and ctsl+/+ keratinocytes. However, ctsl-/- keratinocytes recycled more EGF to the cell surface, where it is bound to the EGF-receptor, which is also more abundant in ctsl-/- cells. We conclude that the hyperproliferation of keratinocytes in CTSL-knockout mice is caused by an enhanced recycling of growth factors and growth factor receptors from the endosomes to the keratinocyte plasma membrane, which result in sustained growth stimulation.

  15. 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.

  16. The effect of two endogenous retinoids on the mRNA expression profile in human primary keratinocytes, focusing on genes causing autosomal recessive congenital ichthyosis.

    PubMed

    Törmä, H; Bergström, A; Ghiasifarahani, G; Berne, B

    2014-10-01

    Retinoids (natural forms and synthetic derivatives of vitamin A) are used as therapeutic agents for numerous skin diseases such as keratinization disorders (e.g. ichthyoses) and psoriasis. Two endogenous ligands for retinoic acid receptors exist, retinoic acid (atRA) and 3,4-didehydroretinoic acid (ddRA). In primary human epidermal keratinocytes many transcriptional targets for atRA are known, whereas the targets for ddRA are unknown. In an attempt to determine the targets, we compared the effect of atRA and ddRA on transcriptional profiles in undifferentiated and differentiating human primary keratinocytes. First, as expected, many genes were induced or suppressed in response to keratinocyte differentiation. Furthermore, the two retinoids affected substantially more genes in differentiated keratinocytes (>350) than in proliferating keratinocytes (≈20). In differentiating keratinocytes markers of cornification were suppressed suggesting a de-differentiating effect by the two retinoids. When comparing the expression profile of atRA to that of ddRA, no differently regulated genes were found. The array analysis also found that a minor number of miRNAs and a large number of non-coding transcripts were changed during differentiation and in response to the two retinoids. Furthermore, the expression of all, except one, genes known to cause autosomal recessive congenital ichthyosis (ARCI) were found to be induced by differentiation. These results comprehensively document that atRA and ddRA exert similar transcriptional changes in keratinocytes and also add new insights into the molecular mechanism influenced by retinoids in the epidermis. Furthermore, it suggests which ARCI patients could benefit from therapy with retinoids.

  17. Induction of PDGF-B in TCA-treated epidermal keratinocytes.

    PubMed

    Yonei, Nozomi; Kanazawa, Nobuo; Ohtani, Toshio; Furukawa, Fukumi; Yamamoto, Yuki

    2007-11-01

    Trichloroacetic acid (TCA) is one of the most widely used peeling agents, and induces full necrosis of the whole epidermis, followed by reconstitution of the epidermis and the matrix of the papillary dermis. The cytotoxic effects of TCA, such as suppressing proliferation of keratinocytes and fibroblasts and protein synthesis by fibroblasts, have already been reported. However, the entire biological mechanism responsible for TCA peeling has yet to be determined. Hypothetical activation effects of TCA treatment on epidermal cells to induce production of growth factors and cytokines are examined, and are compared with its cytotoxic effects in terms of time course and applied TCA concentrations. After various periods of incubation with TCA, viability of Pam212 murine keratinocytes was investigated with MTT assay and dye exclusion assay, and production of growth factors and cytokines with reverse transcription-polymerase chain reaction (RT-PCR). Changes in platelet-derived growth factor (PDGF)-B mRNA expression and protein production in the human skin specimens after TCA application were then examined by RT-PCR and immunohistochemistry, respectively. Incubation with TCA showed cytotoxicity and induced death of Pam212 cells, depending on the incubation period and the TCA concentration. In addition, expressions of PDGF-B, tumor growth factor (TGF)-alpha, TGF- beta1 and vascular endothelial growth factor, which are the growth factors reportedly secreted from keratinocytes during wound healing, were all detected in Pam212 cells after short-term treatment with TCA. Expressions of inflammatory cytokines such as interleukin (IL)-1 and IL-10 were also induced. In TCA-treated NIH-3T3 fibroblasts, in contrast, observed was upregulation of only keratinocyte growth factor, which is reportedly secreted from fibroblasts, as well as the similar cytotoxic effect. In human skin, PDGF-B mRNA expression became significantly upregulated after TCA application, and then immediately

  18. 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.

  19. 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.

  20. Wound re-epithelialization: modulating keratinocyte migration in wound healing.

    PubMed

    Raja; Sivamani, K; Garcia, Miki Shirakawa; Isseroff, R Rivkah

    2007-05-01

    An essential feature of a healed wound is the restoration of an intact epidermal barrier through wound epithelialization, also known as re-epithelialization. The directed migration of keratinocytes is critical to wound epithelialization and defects in this function are associated with the clinical phenotype of chronic non-healing wounds. A complex balance of signaling factors and surface proteins are expressed and regulated in a temporospatial manner that promote keratinocyte motility and survival to activate wound re-epithelialization. The majority of this review focuses on the mechanisms that regulate keratinocyte migration in the re-epithelialization process. This includes a review of cell attachments via desmosomes, hemidesmosomes, and integrins, the expression of keratins, the role of growth factors, cytokines and chemokines, eicosanoids, oxygen tension, antimicrobial peptides, and matrix metalloproteinases. Also reviewed are recently emerging novel mediators of keratinocyte motility including the role of electric fields, and signaling via the acetylcholine and beta-adrenergic receptors. These multiple regulators impact the ability of keratinocytes to migrate from the wound edge or other epidermal reservoirs to efficiently re-epithelialize a breach in the integrity of the epidermis. New discoveries will continue to uncover the elegant network of events that result in restoration of epidermal integrity and complete the wound repair process.

  1. 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.

  2. 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

  3. Genome-Wide Association Studies of Multiple Keratinocyte Cancers

    PubMed Central

    Verkouteren, Joris A. C.; Hofman, Albert; Uitterlinden, André G.; Kraft, Peter; Turman, Constance; Han, Jiali; Cho, Eunyoung; Murabito, Joanne M.; Levy, Daniel; Qureshi, Abrar A.; Nijsten, Tamar

    2017-01-01

    There is strong evidence for a role of environmental risk factors involved in susceptibility to develop multiple keratinocyte cancers (mKCs), but whether genes are also involved in mKCs susceptibility has not been thoroughly investigated. We investigated whether single nucleotide polymorphisms (SNPs) are associated with susceptibility for mKCs. A genome-wide association study (GWAS) of 1,666 cases with mKCs and 1,950 cases with single KC (sKCs; controls) from Harvard cohorts (the Nurses' Health Study [NHS], NHS II, and the Health Professionals Follow-Up Study) and the Framingham Heart Study was carried-out using over 8 million SNPs (stage-1). We sought to replicate the most significant statistical associations (p-value≤ 5.5x10-6) in an independent cohort of 574 mKCs and 872 sKCs from the Rotterdam Study. In the discovery stage, 40 SNPs with suggestive associations (p-value ≤5.5x10-6) were identified, with eight independent SNPs tagging all 40 SNPs. The most significant SNP was located at chromosome 9 (rs7468390; p-value = 3.92x10-7). In stage-2, none of these SNPs replicated and only two of them were associated with mKCs in the same direction in the combined meta-analysis. We tested the associations for 19 previously reported basal cell carcinoma-related SNPs (candidate gene association analysis), and found that rs1805007 (MC1R locus) was significantly associated with risk of mKCs (p-value = 2.80x10-4). Although the suggestive SNPs with susceptibility for mKCs were not replicated, we found that previously identified BCC variants may also be associated with mKC, which the most significant association (rs1805007) located at the MC1R gene. PMID:28081215

  4. Concentration of fibrin and presence of plasminogen affect proliferation, fibrinolytic activity, and morphology of human fibroblasts and keratinocytes in 3D fibrin constructs.

    PubMed

    Reinertsen, Erik; Skinner, Michael; Wu, Benjamin; Tawil, Bill

    2014-11-01

    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.

  5. Optimal differentiation of in vitro keratinocytes requires multifactorial external control.

    PubMed

    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

  6. Study of HLA-DR synthesis in cultured human keratinocytes.

    PubMed

    Wikner, N E; Huff, J C; Norris, D A; Boyce, S T; Cary, M; Kissinger, M; Weston, W L

    1986-11-01

    Within the normal human epidermis only Langerhans and indeterminate cells express HLA-DR. Human keratinocytes (HK), however, may also express HLA-DR in certain disease states characterized by mononuclear cell infiltrates. Previous studies have shown that HK synthesize HLA-DR in response to stimulation by interferon gamma (INF-gamma). The purposes of this study were to define conditions under which cultured HK might express HLA-DR and to compare the HLA-DR synthesis of HK with that of monocytes. HLA-DR expression by HK as determined by indirect immunofluorescence of HK cultures was absent under standard low calcium conditions and remained absent with the addition of calcium, serum, mitogens, and supernatants from Pam-212 cells containing epidermal thymocyte-activating factor. HLA-DR expression in HK was induced by cocultivation with concanavalin A-stimulated peripheral blood mononuclear cells (PBMC), but not unstimulated PBMC. This effect was time-dependent and directly related to the number of PBMC. HLA-DR expression was also induced in a time- and dose-dependent manner by addition of supernatant from stimulated PBMC (SS) or by addition of recombinant INF-gamma but not by addition of interleukin (IL)-1 or IL-2. Induction by either SS or INF-gamma was blocked by an antiserum to INF-gamma. As determined by a semiquantitative immunoprecipitation technique, HLA-DR synthesis by HK was directly related to INF-gamma concentration. The pattern of HLA-DR peptides produced by HK was similar to that of monocytes, but the relative quantity synthesized was far less than that of monocytes.

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

    PubMed Central

    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

    2014-01-01

    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

  9. 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.

  10. 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.

  11. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line

    PubMed Central

    1988-01-01

    In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation- specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long- term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation. PMID:2450098

  12. Mechanisms of mitochondrial damage in keratinocytes by pemphigus vulgaris antibodies.

    PubMed

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

    2013-06-07

    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.

  13. 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

  14. 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

  15. Cell shape controls terminal differentiation of human epidermal keratinocytes.

    PubMed Central

    Watt, F M; Jordan, P W; O'Neill, C H

    1988-01-01

    Cultures of human epidermal keratinocytes provide a useful experimental model with which to study the factors that regulate cell proliferation and terminal differentiation. One situation that is known to trigger premature terminal differentiation is suspension culture, when keratinocytes are deprived of substratum and intercellular contact. We have now investigated whether area of substratum contact, and hence cell shape, can regulate terminal differentiation. Keratinocytes were grown on circular adhesive islands that prevented cell-cell contact. By varying island area we could vary cell shape from fully spread to almost spherical. We found that when substratum contact was restricted, DNA synthesis was inhibited and expression of involucrin, a marker of terminal differentiation, was stimulated. Inhibition of proliferation was not a sufficient stimulus for involucrin synthesis in fully spread cells. When DNA synthesis and involucrin expression were plotted against contact area, classic dose-response curves were obtained. Thus cell shape acts as a signal for the terminal differentiation of keratinocytes in culture. Images PMID:2456572

  16. 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

  17. Recovery and Cultivation of Keratinocytes From Shipped Mouse Skin.

    PubMed

    Yang, Hsin-Ya; La, Thi Dinh; Gurenko, Zhanna; Steenhuis, Pieter; Liu, Wei; Isseroff, R Rivkah

    2015-02-01

    Murine keratinocyte culture from neonatal skin is an important tool for studying the functional role of specific genes in epithelial biology. However, when the transgenic animal is only available in a geographically distant local, obtaining viable keratinocytes can be problematic. A method for transferring the isolated murine skin from collaborating labs could decrease the cost of shipping live animals, and would allow the efficient use of the tissues from the transgenic animals. Here we optimized shipping conditions and characterized the cells retrieved and cultured from mouse skin shipped for 48 h at 0 °C. The cultured keratinocytes from the control, non-shipped skin and the 2-day shipped skin were 43.6 +/- 7.8% viable, doubled every 2 days, and expressed comparable amounts of heat shock proteins and CD29/integrin beta-1. However, under the same shipping conditions, the 3-day shipped tissue failed to establish colonies in the culture. Therefore, this 2-day shipping technique allows the transfer mouse skin from distant locations with recovery of viable, propagatable keratinocytes, facilitating long-distance collaborations.

  18. 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.

  19. Oxidative damage in keratinocytes exposed to cigarette smoke and aldehydes.

    PubMed

    Avezov, Katia; Reznick, Abraham Z; Aizenbud, Dror

    2014-06-01

    Cigarette smoke (CS) is a significant environmental source of human exposure to chemically active saturated (acetaldehyde) and α,β-unsaturated aldehydes (acrolein) inducing protein carbonylation and dysfunction. The exposure of oral tissues to environmental hazards is immense, especially in smokers. The objectives of the current study were to examine the effect of aldehydes originating from CS on intracellular proteins of oral keratinocytes and to observe the antioxidant response in these cells. Intracellular protein carbonyl modification under CS, acrolein and acetaldehyde exposure in the HaCaT keratinocyte cell line, representing oral keratinocytes was examined by Western blot. Possible intracellular enzymatic dysfunction under the above conditions was examined by lactate dehydrogenase (LDH) activity assay. Oxidative stress response was investigated, by DCF (2,7-dichlorodihydrofluorescein) assay and GSH (glutathione) oxidation. Intracellular protein carbonyls increased 5.2 times after CS exposure and 2.7 times after exposure to 1 μmol of acrolein. DCF assay revealed an increase of fluorescence intensity 3.2 and 3.1 times after CS and acrolein exposure, respectively. CS caused a 72.5% decrease in intracellular GSH levels compared to controls. Activity of intracellular LDH was preserved. α,β-Unsaturated aldehydes from CS are capable of intracellular protein carbonylation and have a role in intracellular oxidative stress elevation in keratinocytes, probably due to the reduction in GSH levels.

  20. Gamma Interferon Reduces the Synthesis of Fibronectin by Human Keratinocytes

    DTIC Science & Technology

    1988-10-06

    mmm mm m m m m mmm• m 13. Wikner NE, Dixit VM, Frazier WA, Clark RAF: Human keratinocytes synthesize and secrete the extracellular matrix protein...keratinoctye-derived lymphocyte inhibitory factor. J Investig Dermatol 90:592A, 1988 18. Wikner NE, Baskin JB, Nielsen LD, McPherson JM, Clark RAF

  1. FOXO1 expression in keratinocytes promotes connective tissue healing

    PubMed Central

    Zhang, Chenying; Lim, Jason; Liu, Jian; Ponugoti, Bhaskar; Alsadun, Sarah; Tian, Chen; Vafa, Rameen; Graves, Dana T.

    2017-01-01

    Wound healing is complex and highly orchestrated. It is well appreciated that leukocytes, particularly macrophages, are essential for inducing the formation of new connective tissue, which requires the generation of signals that stimulate mesenchymal stem cells (MSC), myofibroblasts and fibroblasts. A key role for keratinocytes in this complex process has yet to be established. To this end, we investigated possible involvement of keratinocytes in connective tissue healing. By lineage-specific deletion of the forkhead box-O 1 (FOXO1) transcription factor, we demonstrate for the first time that keratinocytes regulate proliferation of fibroblasts and MSCs, formation of myofibroblasts and production of collagen matrix in wound healing. This stimulation is mediated by a FOXO1 induced TGFβ1/CTGF axis. The results provide direct evidence that epithelial cells play a key role in stimulating connective tissue healing through a FOXO1-dependent mechanism. Thus, FOXO1 and keratinocytes may be an important therapeutic target where healing is deficient or compromised by a fibrotic outcome. PMID:28220813

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

    PubMed Central

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

    2015-01-01

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

  3. Atypical Diabetic Foot Ulcer Keratinocyte Protein Signaling Correlates with Impaired Wound Healing

    PubMed Central

    Hoke, Glenn D.; Ramos, Corrine; Hoke, Nicholas N.; Crossland, Mary C.; Shawler, Lisa G.

    2016-01-01

    Diabetes mellitus is associated with chronic diabetic foot ulcers (DFUs) and wound infections often resulting in lower extremity amputations. The protein signaling architecture of the mechanisms responsible for impaired DFU healing has not been characterized. In this preliminary clinical study, the intracellular levels of proteins involved in signal transduction networks relevant to wound healing were non-biasedly measured using reverse-phase protein arrays (RPPA) in keratinocytes isolated from DFU wound biopsies. RPPA allows for the simultaneous documentation and assessment of the signaling pathways active in each DFU. Thus, RPPA provides for the accurate mapping of wound healing pathways associated with apoptosis, proliferation, senescence, survival, and angiogenesis. From the study data, we have identified potential diagnostic, or predictive, biomarkers for DFU wound healing derived from the ratios of quantified signaling protein expressions within interconnected pathways. These biomarkers may allow physicians to personalize therapeutic strategies for DFU management on an individual basis based upon the signaling architecture present in each wound. Additionally, we have identified altered, interconnected signaling pathways within DFU keratinocytes that may help guide the development of therapeutics to modulate these dysregulated pathways, many of which parallel the therapeutic targets which are the hallmarks of molecular therapies for treating cancer. PMID:27840833

  4. EMILIN1-α4/α9 integrin interaction inhibits dermal fibroblast and keratinocyte proliferation.

    PubMed

    Danussi, Carla; Petrucco, Alessandra; Wassermann, Bruna; Pivetta, Eliana; Modica, Teresa Maria Elisa; Del Bel Belluz, Lisa; Colombatti, Alfonso; Spessotto, Paola

    2011-10-03

    EMILIN1 promotes α4β1 integrin-dependent cell adhesion and migration and reduces pro-transforming growth factor-β processing. A knockout mouse model was used to unravel EMILIN1 functions in skin where the protein was abundantly expressed in the dermal stroma and where EMILIN1-positive fibrils reached the basal keratinocyte layer. Loss of EMILIN1 caused dermal and epidermal hyperproliferation and accelerated wound closure. We identified the direct engagement of EMILIN1 to α4β1 and α9β1 integrins as the mechanism underlying the homeostatic role exerted by EMILIN1. The lack of EMILIN1-α4/α9 integrin interaction was accompanied by activation of PI3K/Akt and Erk1/2 pathways as a result of the reduction of PTEN. The down-regulation of PTEN empowered Erk1/2 phosphorylation that in turn inhibited Smad2 signaling by phosphorylation of residues Ser245/250/255. These results highlight the important regulatory role of an extracellular matrix component in skin proliferation. In addition, EMILIN1 is identified as a novel ligand for keratinocyte α9β1 integrin, suggesting prospective roles for this receptor-ligand pair in skin homeostasis.

  5. Hypoxia enhances the wound-healing potential of adipose-derived stem cells in a novel human primary keratinocyte-based scratch assay.

    PubMed

    Riis, Simone; Newman, Rhonda; Ipek, Hilal; Andersen, Jens I; Kuninger, David; Boucher, Shayne; Vemuri, Mohan C; Pennisi, Cristian P; Zachar, Vladimir; Fink, Trine

    2017-03-01

    Preclinical studies have suggested that paracrine factors from adipose-derived stem cells (ASCs) promote the healing of chronic wounds, and that the exposure of ASCs to hypoxia enhances their wound healing effect. To aid the translation of these findings into clinical use, robust wound models are necessary to explore each aspect of wound healing. The aspect of re-epithelization is often studied in a scratch assay based on transformed keratinocytes. However, there are concerns regarding the validity of this model, since these cell lines differ from normal keratinocytes, both in terms of proliferative capacity and differentiation, and sensitivity to environmental cues. In this study, the main challenge of using primary keratinocytes to examine the effects of ASCs was identified to be their different requirements for calcium in the culture media. We confirmed that a high calcium content led to morphological and cytoskeletal changes in primary keratinocytes, and demonstrated that a low calcium content compromised the growth of ASCs. We found that it is possible to perform the wound healing assay with primary keratinocytes, if the conditioned media from the ASCs is dialyzed to reduce the calcium concentration. Additionally, using this model of re-epithelization, conditioned media from normoxic ASCs was shown to markedly increase the rate of wound closure by primary keratinocytes, and this effect was significantly enhanced with media from the hypoxia-exposed ASCs. These findings, which are in line with the observations from previous in vivo studies, highlight the validity of this modified assay to investigate the wound healing properties of ASCs in vitro.

  6. Hypoxia enhances the wound-healing potential of adipose-derived stem cells in a novel human primary keratinocyte-based scratch assay

    PubMed Central

    Riis, Simone; Newman, Rhonda; Ipek, Hilal; Andersen, Jens I.; Kuninger, David; Boucher, Shayne; Vemuri, Mohan C.; Pennisi, Cristian P.; Zachar, Vladimir; Fink, Trine

    2017-01-01

    Preclinical studies have suggested that paracrine factors from adipose-derived stem cells (ASCs) promote the healing of chronic wounds, and that the exposure of ASCs to hypoxia enhances their wound healing effect. To aid the translation of these findings into clinical use, robust wound models are necessary to explore each aspect of wound healing. The aspect of re-epithelization is often studied in a scratch assay based on transformed keratinocytes. However, there are concerns regarding the validity of this model, since these cell lines differ from normal keratinocytes, both in terms of proliferative capacity and differentiation, and sensitivity to environmental cues. In this study, the main challenge of using primary keratinocytes to examine the effects of ASCs was identified to be their different requirements for calcium in the culture media. We confirmed that a high calcium content led to morphological and cytoskeletal changes in primary keratinocytes, and demonstrated that a low calcium content compromised the growth of ASCs. We found that it is possible to perform the wound healing assay with primary keratinocytes, if the conditioned media from the ASCs is dialyzed to reduce the calcium concentration. Additionally, using this model of re-epithelization, conditioned media from normoxic ASCs was shown to markedly increase the rate of wound closure by primary keratinocytes, and this effect was significantly enhanced with media from the hypoxia-exposed ASCs. These findings, which are in line with the observations from previous in vivo studies, highlight the validity of this modified assay to investigate the wound healing properties of ASCs in vitro. PMID:28204820

  7. 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

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

    2014-07-23

    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.

  8. 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

  9. Growth regulation of primary human keratinocytes by prostaglandin E receptor EP2 and EP3 subtypes.

    PubMed

    Konger, R L; Malaviya, R; Pentland, A P

    1998-02-04

    We examined the contribution of specific EP receptors in regulating cell growth. By RT-PCR and northern hybridization, adult human keratinocytes express mRNA for three PGE2 receptor subtypes associated with cAMP signaling (EP2, EP3, and small amounts of EP4). In actively growing, non-confluent primary keratinocyte cultures, the EP2 and EP4 selective agonists, 11-deoxy PGE1 and 1-OH PGE1, caused complete reversal of indomethacin-induced growth inhibition. The EP3/EP2 agonist (misoprostol), and the EP1/EP2 agonist (17-phenyl trinor PGE2), showed less activity. Similar results were obtained with agonist-induced cAMP formation. The ability of exogenous dibutyryl cAMP to completely reverse indomethacin-induced growth inhibition support the conclusion that growth stimulation occurs via an EP2 and/or EP4 receptor-adenylyl cyclase coupled response. In contrast, activation of EP3 receptors by sulprostone, which is virtually devoid of agonist activity at EP2 or EP4 receptors, inhibited bromodeoxyuridine uptake in indomethacin-treated cells up to 30%. Although human EP3 receptor variants have been shown in other cell types to markedly inhibit cAMP formation via a pertussis toxin sensitive mechanisms, EP3 receptor activation and presumably growth inhibition was independent of adenylyl cyclase, suggesting activation of other signaling pathways.

  10. Transcriptome analysis of airborne PM2.5-induced detrimental effects on human keratinocytes.

    PubMed

    Kim, Hyoung-June; Bae, Il-Hong; Son, Eui Dong; Park, Juyearl; Cha, Nari; Na, Hye-Won; Jung, Changjo; Go, You-Seak; Kim, Dae-Yong; Lee, Tae Ryong; Shin, Dong Wook

    2017-03-21

    Ambient air pollution is becoming more severe worldwide, posing a serious threat to human health. Fine airborne particles of particulate matter (PM2.5) show higher cytotoxicity than other coarse fractions. Indeed, PM2.5 induces cardiovascular or respiratory damage; however, few studies have evaluated the detrimental effect of PM2.5 to normal human skin. We used a next-generation sequencing-based (RNA-Seq) method with transcriptome and Gene Ontology (GO) enrichment analysis to determine the harmful influences of PM2.5 on human normal epidermal keratinocytes. DAVID analysis showed that the most significantly enriched GO terms were associated with epidermis-related biological processes such as "epidermis development (GO: 0008544)" and "keratinocyte differentiation (GO: 0030216)", suggesting that PM2.5 has some deleterious effects to the human epidermis. In addition, Ingenuity Pathway Analysis predicted inflammation-related signaling as one of the major PM2.5-induced signaling pathways, and pro-inflammatory cytokines as upstream regulators with symptoms similar to psoriasis as downstream effects. PM2.5 caused considerable changes in the expression of pro-inflammatory cytokines and psoriatic skin disease-related genes, might lead to epidermal dysfunctions. Our results might help to understand the mechanism of air pollution-induced skin barrier perturbation and contribute to the development of a new strategy for the prevention or recovery of the consequent damage.

  11. Epithelial mesenchymal transition traits in honey-driven keratinocyte wound healing: comparison among different honeys.

    PubMed

    Ranzato, Elia; Martinotti, Simona; Burlando, Bruno

    2012-01-01

    Honey has been used since ancient times for wound repair, but the subjacent mechanisms are almost unknown. We have tried to elucidate the modulatory role of honey in an in vitro model of HaCaT keratinocyte re-epithelialization by using acacia, buckwheat, and manuka honeys. Scratch wound and migration assays showed similar increases of re-epithelialization rates and chemoattractant effects in the presence of different types of honey (0.1%, v/v). However, the use of kinase and calcium inhibitors suggested the occurrence of different mechanisms. All honeys activated cyclin-dependent kinase 2, focal adhesion kinase, and rasGAP SH3 binding protein 1. However, vasodilator-stimulated phosphoprotein, integrin-β3, cdc25C, and p42/44 mitogen activated protein kinase showed variable activation pattern. Re-epithelialization recapitulates traits of epithelial-mesenchymal transition (EMT) and the induction of this process was evaluated by a polymerase chain reaction array, revealing marked differences among honeys. Manuka induced few significant changes in the expression of EMT-regulatory genes, while the other two honeys acted on a wider number of genes and partially showed a common profile of up- and down-regulation. In conclusion, our findings have shown that honey-driven wound repair goes through the activation of keratinocyte re-epithelialization, but the ability of inducing EMT varies sensibly among honeys, according to their botanical origin.

  12. Cdk4 deficiency inhibits skin tumor development but does not affect normal keratinocyte proliferation.

    PubMed

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

    2002-08-01

    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.

  13. Incorporation and distribution of dihomo-gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes

    SciTech Connect

    Punnonen, K.; Puustinen, T.; Jansen, C.T.

    1986-02-01

    Human keratinocytes in culture were labelled with /sup 14/C-dihomo-gamma-linolenic acid, /sup 14/C-arachidonic acid or /sup 14/C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. Most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.

  14. Selective peroxidation and externalization of phosphatidylserine in normal human epidermal keratinocytes during oxidative stress induced by cumene hydroperoxide.

    PubMed

    Shvedova, Anna A; Tyurina, Julia Y; Kawai, Kazuaki; Tyurin, Vladimir A; Kommineni, Choudari; Castranova, Vincent; Fabisiak, James P; Kagan, Valerian E

    2002-06-01

    Reactive oxygen species not only modulate important signal transduction pathways, but also induce DNA damage and cytotoxicity in keratinocytes. Hydrogen peroxide and organic peroxides are particularly important as these chemicals are widely used in dermally applied cosmetics and pharmaceuticals, and also represent endogenous metabolic intermediates. Lipid peroxidation is of fundamental interest in the cellular response to peroxides, as lipids are extremely sensitive to oxidation and lipid-based signaling systems have been implicated in a number of cellular processes, including apoptosis. Oxidation of specific phospholipid classes was measured in normal human epidermal keratinocytes exposed to cumene hydroperoxide after metabolic incorporation of the fluorescent oxidation-sensitive fatty acid, cis-parinaric acid, using a fluorescence high-performance liquid chromatography assay. In addition, lipid oxidation was correlated with changes in membrane phospholipid asymmetry and other markers of apoptosis. Although cumene hydroperoxide produced significant oxidation of cis-parinaric acid in all phospholipid classes, one phospholipid, phosphatidylserine, appeared to be preferentially oxidized above all other species. Using fluorescamine derivatization and annexin V binding it was observed that specific oxidation of phosphatidylserine was accompanied by phosphatidylserine translocation from the inner to the outer plasma membrane surface where it may serve as a recognition signal for interaction with phagocytic macrophages. These effects occurred much earlier than any detectable changes in other apoptotic markers such as caspase-3 activation, DNA fragmentation, or changes in nuclear morphology. Thus, normal human epidermal keratinocytes undergo profound lipid oxidation with preference for phosphatidylserine followed by phosphatidylserine externalization upon exposure to cumene hydroperoxide. It is therefore likely that normal human epidermal keratinocytes exposed to similar

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

    PubMed

    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

    2011-01-01

    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.

  17. Diacerein inhibits the pro-atherogenic & pro-inflammatory effects of IL-1 on human keratinocytes & endothelial cells

    PubMed Central

    Bao, Lei; Many, Benjamin; Chan, Lawrence S.

    2017-01-01

    We investigated IL-1-induced regulation of genes related to inflammation and atherogenesis in human keratinocytes and endothelial cells, and if ‘diacerein’, an oral IL-1 inhibiting drug currently approved for use in osteoarthritis, would reverse IL-1’s effects on these cells. Primary human keratinocytes and coronary artery endothelial cells were treated with either IL-1α or IL-1β, with and without diacerein. Using PCR-array, we assessed differential gene-expression regulated by IL-1 and diacerein. We identified 34 pro-atherogenic genes in endothelial cells and 68 pro-inflammatory genes in keratinocytes significantly (p<0.05) regulated at least 2-fold by IL-1, in comparison to control. Diacerein completely or partially reversed this regulation on almost all genes. Using ELISA, we confirmed diacerein’s ability to reverse IL-1-driven gene-regulation of 11 selected factors, at the protein level. The results support a novel idea that diacerein acts as an inhibitor of the pro-atherogenic and pro-inflammatory effects of IL-1. Diacerein may have therapeutic applications to diminish IL-1-induced skin inflammation in psoriasis and attenuate IL-1-induced development of atherosclerosis. Further investigation into diacerein’s effect on skin inflammation, atherogenesis and cardiovascular risk in animal models or humans is warranted. PMID:28323859

  18. Kaempferol-3-O-rutinoside from Afgekia mahidoliae promotes keratinocyte migration through FAK and Rac1 activation.

    PubMed

    Petpiroon, Nareerat; Suktap, Chalermlat; Pongsamart, Sunanta; Chanvorachote, Pithi; Sukrong, Suchada

    2015-07-01

    The restoration of the epidermal epithelium through re-epithelialization is a critical process in wound healing. Directed keratinocyte migration to the wound is required, and the retardation of this process may result in a chronic, non-healing wound. The present study contributes to research aiming to identify promising compounds that promote wound healing using a human keratinocyte model. The effects of three kaempferol glycosides from an Afgekia mahidoliae leaf extract, kaempferol-3-O-arabinoside, kaempferol-3-O-glucoside, and kaempferol-3-O-rutinoside, on keratinocyte migration were determined. Interestingly, kaempferol-3-O-rutinoside exhibited a pronounced effect on wound closure in comparison to the parental kaempferol and other glycosides. The mechanism by which kaempferol-3-O-rutinoside enhances cell migration involves the induction of filopodia and lamellipodia formation, increased cellular levels of phosphorylated FAK (Tyr 397) and phosphorylated Akt (Ser 473), and up-regulation of active Rac1-GTP. The data obtained in this study may support the development of this compound for use in wound healing therapies.

  19. High-throughput, high-content screening for novel pigmentation regulators using a keratinocyte/melanocyte co-culture system.

    PubMed

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

    2014-02-01

    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 microphthalmia transcription factor (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 4000 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.

  20. Signal transduction and nuclear responses in Staphylococcus aureus-induced expression of human beta-defensin 3 in skin keratinocytes.

    PubMed

    Menzies, Barbara E; Kenoyer, Aimee

    2006-12-01

    The human beta-defensin 3 (hBD-3) is an inducible epithelial peptide antibiotic that has potent antistaphylococcal activity. Infection of skin epithelial cells with viable Staphylococcus aureus, a common skin pathogen, induces increased gene expression of hBD-3 and other antimicrobial peptides. The aim of this study was to identify signaling pathways and nuclear responses that contribute to the gene expression of hBD-3 in primary human keratinocytes upon contact with S. aureus. Increased hBD-3 peptide was observed by immunofluorescence microscopy in keratinocytes exposed to S. aureus and to lipoteichoic acid (LTA). Both are ligands for the cell surface Toll-like receptor 2 (TLR2), and thus the contribution of TLR2 signaling in hBD-3 expression was examined. Functional inhibition of TLR2 prior to S. aureus stimulation significantly decreased hBD-3 mRNA levels by 37%, attesting to the involvement of this surface receptor in the initial recognition and downstream signaling for hBD-3 expression. Treatment of keratinocytes with a p38 mitogen-activated protein kinase (MAPK) inhibitor prior to either S. aureus or LTA stimulation was associated with reduced hBD-3 mRNA transcripts and peptide. We also propose a role for the MAPK-regulated transcriptional activating protein 1 in S. aureus-induced hBD-3 gene expression. Combined, these studies indicate a role for TLR2 signaling and MAPK activation in the upregulation of hBD-3 and demonstrate the innate immune capacity of skin keratinocytes under conditions of S. aureus challenge to enhance the local expression of this antistaphylococcal peptide antibiotic.

  1. Transcriptional responses of human epidermal keratinocytes to Oncostatin-M.

    PubMed

    Finelt, Nika; Gazel, Alix; Gorelick, Steven; Blumenberg, Miroslav

    2005-08-21

    Oncostatin-M (OsM) plays an important role in inflammatory and oncogenic processes in skin, including psoriasis and Kaposi sarcoma. However, the molecular responses to OsM in keratinocytes have not been explored in depth. Here we show the results of transcriptional profiling in OsM-treated primary human epidermal keratinocytes, using high-density DNA microarrays. We find that OsM strongly and specifically affects the expression of many genes, in particular those involved with innate immunity, angiogenesis, adhesion, motility, tissue remodeling, cell cycle and transcription. The timing of the responses to OsM comprises two waves, early at 1h, and late at 48 h, with much fewer genes regulated in the intervening time points. Secreted cytokines and growth factors and their receptors, as well as nuclear transcription factors, are primary targets of OsM regulation, and these, in turn, effect the secondary changes.

  2. Functional interplay between p63 and p53 controls RUNX1 function in the transition from proliferation to differentiation in human keratinocytes.

    PubMed

    Masse, I; Barbollat-Boutrand, L; Molina, M; Berthier-Vergnes, O; Joly-Tonetti, N; Martin, M T; Caron de Fromentel, C; Kanitakis, J; Lamartine, J

    2012-06-07

    The interfollicular epidermis is continuously renewed, thanks to a regulated balance between proliferation and differentiation. The ΔNp63 transcription factor has a key role in the control of this process. It has been shown that ΔNp63 directly regulates Runt-related transcription factor 1 (RUNX1) transcription factor expression in mouse keratinocytes. The present study showed for the first time that RUNX1 is expressed in normal human interfollicular epidermis and that its expression is tightly regulated during the transition from proliferation to differentiation. It demonstrated that ΔNp63 directly binds two different RUNX1 regulatory DNA sequences and modulates RUNX1 expression differentially in proliferative or differentiated human keratinocytes. It also showed that the regulation of RUNX1 expression by ΔNp63 is dependent on p53 and that this coregulation relies on differential binding and activation of RUNX1 regulatory sequences by ΔNp63 and p53. We also found that RUNX1 inhibits keratinocyte proliferation and activates directly the expression of KRT1, a critical actor in early keratinocyte differentiation. Finally, we described that RUNX1 expression, similar to ΔNp63 and p53, was strongly expressed and downregulated in basal cell carcinomas and squamous cell carcinomas respectively. Taken together, these data shed light on the importance of tight control of the functional interplay between ΔNp63 and p53 in regulating RUNX1 transcription factor expression for proper regulation of interfollicular epidermal homeostasis.

  3. 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

  4. Hypoxia induces an undifferentiated phenotype of oral keratinocytes in vitro.

    PubMed

    Kato, Hiroko; Izumi, Kenji; Uenoyama, Atsushi; Shiomi, Aki; Kuo, Shiuhyang; Feinberg, Stephen E

    2014-01-01

    The aim of this study was to determine the effects of hypoxia on the proliferating potential and phenotype of primary human oral keratinocytes cultured at ambient oxygen tension (20%) or at different levels of hypoxia (2 and 0.5% O2). The effects of oxygen tensions on cellular metabolic activity, cell proliferation, clonogenicity and proliferation heterogeneity were measured. Cell cycle profiles were analyzed by a fluorescent-activated cell sorter, and p21(WAF1/CIP1) expression in the G0/G1 phase was also concomitantly quantitated. The expression levels of cell cycle regulatory proteins were examined by immunoblotting, and the cellular senescence was assessed by senescence-associated β-galactosidase staining. Basal and suprabasal keratinocyte phenotypes were determined by the expression levels of 14-3-3σ, p75(NTR) and α6 integrin. Despite having a lower metabolism, the proliferation rate and clonogenic potential were remarkably enhanced in hypoxic cells. The significantly higher percentage of cells in the G0/G1 phase under hypoxia and the expression patterns of cell cycle regulatory proteins in hypoxic cells were indicative of a state of cell cycle arrest in hypoxia. Furthermore, a decrease in the expression of p21(WAF1/CIP1) and p16(INK4A) and fewer β-galactosidase-positive cells suggested a quiescent phenotype rather than a senescent one in hypoxic cells. Compared with normoxic cells, the differential expression patterns of keratinocyte phenotypic markers suggest that hypoxic cells that generate minimal reactive oxygen species, suppress the mammalian target of rapamycin activity and express hypoxia-inducible factor-1α favor a basal cell phenotype. Thus, regardless of the predisposition to the state of cell cycle arrest, hypoxic conditions can maintain oral keratinocytes in vitro in an undifferentiated and quiescent state.

  5. Keratinocytes at the uppermost layer of epidermis might act as sensors of atmospheric pressure change.

    PubMed

    Denda, Mitsuhiro

    2016-01-01

    It has long been suggested that climate, especially atmospheric pressure change, can cause health problems ranging from migraine to myocardial infarction. Here, I hypothesize that the sensory system of epidermal keratinocytes mediates the influence of atmospheric pressure change on the human physiological condition. We previously demonstrated that even subtle changes of atmospheric pressure (5-20 hPa) induce elevation of intracellular calcium level in cultured human keratinocytes (excitation of keratinocytes). It is also established that communication occurs between epidermal keratinocytes and peripheral nerve systems. Moreover, various neurotransmitters and hormones that influence multiple systems (nervous, cardiovascular, endocrine, and immune systems) are generated and released from epidermal keratinocytes in response to various external stimuli. Thus, I suggest that pathophysiological phenomena induced by atmospheric pressure changes might be triggered by epidermal keratinocytes.

  6. Membrane-Tethered Intracellular Domain of Amphiregulin Promotes Keratinocyte Proliferation

    PubMed Central

    Stoll, Stefan W.; Stuart, Philip E.; Lambert, Sylviane; Gandarillas, Alberto; Rittié, Laure; Johnston, Andrew; Elder, James T.

    2016-01-01

    The EGF receptor (EGFR) and its ligands are essential regulators of epithelial biology, which are often amplified in cancer cells. We have previously shown that shRNA-mediated silencing of one of these ligands, amphiregulin (AREG), results in keratinocyte growth arrest that cannot be rescued by soluble extracellular EGFR ligands. To further explore the functional importance of specific AREG domains, we stably transduced keratinocytes expressing tetracycline-inducible AREG-targeted shRNA with lentiviruses expressing silencing-proof, membrane-tethered AREG cytoplasmic and extracellular domains (AREG-CTD and AREG-ECD), as well as full-length AREG precursor (proAREG). Here we show that growth arrest of AREG-silenced keratinocytes occurs in G2/M and is significantly restored by proAREG and AREG-CTD, but not by AREG-ECD. Moreover, the AREG-CTD was sufficient to normalize cell cycle distribution profiles and expression of mitosis-related genes. Our findings uncover an important role of the AREG-CTD in regulating cell division, which may be relevant to tumor resistance to EGFR-directed therapies. PMID:26802239

  7. Candida albicans-induced inflammatory response in human keratinocytes.

    PubMed

    Wollina, U; Künkel, W; Bulling, L; Fünfstück, C; Knöll, B; Vennewald, I; Hipler, U-C

    2004-06-01

    Candida albicans strains 3153a, ATCC 48867, CBS 2730, DSM 70014, and Vir 13 were cultivated and sterile C. albicans filtrates were produced. The interaction of soluble Candida factors of these infiltrates with human HaCaT keratinocytes was assayed in vitro. The following parameters were analyzed: cell proliferation, protein synthesis, nuclear matrix protein (NMP) 41 release, cytokine release (IL-1beta, soluble IL-2 receptor, IL-6, and IL-8), and reactive oxygen species (ROS). Cell counts at 1, 12, and 24 h were significantly lower for C. albicans strains CBS 2730 and VIR 13 (P < 0.05). There was no significant change for the remaining strains. Neither the protein synthesis nor the NMP-41 release was significantly affected. IL-6 and IL-8 were stimulated by C. albicans filtrates to different amounts with higher levels in strains of low virulence. There was no effect on the other cytokines. The production of ROS by HaCaT keratinocytes was suppressed. The induction of an inflammatory keratinocyte response by soluble C. albicans factors may play a role among the host-yeast interactions.

  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. 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.

  10. Newly discovered olfactory receptors in epidermal keratinocytes are associated with proliferation, migration, and re-epithelialization of keratinocytes.

    PubMed

    Denda, Mitsuhiro

    2014-11-01

    Skin contains receptors for various environmental factors. In this issue of the Journal, Busse et al. cloned a new olfactory receptor, OR2AT4, in keratinocytes. They show that the activation of OR2AT4 induces phosphorylation of extracellular signal-regulated kinases and p38 mitogen-activated protein kinases, and that it accelerates wound healing. OR2AT4 may be a promising candidate as a target in clinical drug development.

  11. 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.

  12. Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract.

    PubMed

    Housley, R M; Morris, C F; Boyle, W; Ring, B; Biltz, R; Tarpley, J E; Aukerman, S L; Devine, P L; Whitehead, R H; Pierce, G F

    1994-11-01

    Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, was identified as a specific keratinocyte mitogen after isolation from a lung fibroblast line. Recently, recombinant (r)KGF was found to influence proliferation and differentiation patterns of multiple epithelial cell lineages within skin, lung, and the reproductive tract. In the present study, we designed experiments to identify additional target tissues, and focused on the rat gastrointestinal (GI) system, since a putative receptor, K-sam, was originally identified in a gastric carcinoma. Expression of KGF receptor and KGF mRNA was detected within the entire GI tract, suggesting the gut both synthesized and responded to KGF. Therefore, rKGF was administered to adult rats and was found to induce markedly increased proliferation of epithelial cells from the foregut to the colon, and of hepatocytes, one day after systemic treatment. Daily treatment resulted in the marked selective induction of mucin-producing cell lineages throughout the GI tract in a dose-dependent fashion. Other cell lineages were either unaffected (e.g., Paneth cells), or relatively decreased (e.g., parietal cells, enterocytes) in rKGF-treated rats. The direct effect of rKGF was confirmed by demonstrating markedly increased carcinoembryonic antigen production in a human colon carcinoma cell line, LIM1899. Serum levels of albumin were specifically and significantly elevated after daily treatment. These results demonstrate rKGF can induce epithelial cell activation throughout the GI tract and liver. Further, endogenous KGF may be a normal paracrine mediator of growth within the gut.

  13. Matrix metalloproteinase-19 expression in keratinocytes is repressed by transcription factors Tst-1 and Skn-1a: implications for keratinocyte differentiation.

    PubMed

    Beck, Inken M; Müller, Matthias; Mentlein, Rolf; Sadowski, Thorsten; Mueller, Markus S; Paus, Ralf; Sedlacek, Radislav

    2007-05-01

    Matrix metalloproteinase-19 (MMP-19), unlike other members of the MMP family, is expressed in basal keratinocytes of intact epidermis whereas keratinocytes in suprabasal and higher epidermal layers express this enzyme only during cutaneous disorders. As the activity of MMP-19 effects proliferation, migration, and adhesion of keratinocytes we examined whether transcription factors involved in keratinocyte differentiation repress the expression of MMP-19. Using luciferase reporter assays, POU transcription factors Tst-1 (Oct-6) and Skn-1a (Oct-11) markedly downregulated the activity of MMP-19 promoter in COS-7 cells and HaCaT keratinocytes. Tst-1 alone was able to inhibit 85% of the promoter activity. Skn-1a exhibited a weak inhibitory effect although it synergistically increased effects of Tst-1. HaCaT cells stably transfected with Tst-1 showed a strong decrease of activity of MMP-19 promoter that correlated with suppression of MMP-19, cytokeratin 14 and 5, decreased cell proliferation, and altered expression of involucrin and loricrin. The expression of MMP-9 was also significantly reduced in Tst-1 expressing keratinocytes. MMP-2 was substantially affected during its activation whereas the expression of MMP-28 was unchanged. Our results suggest that Tst-1 and Skn-1a regulate expression of MMPs in keratinocytes and effect both the expression and activation of these proteolytic enzymes.

  14. HSP70 and EndoG modulate cell death by heat in human skin keratinocytes in vitro.

    PubMed

    Chinnathambi, Sathivel; Tomanek-Chalkley, Ann; Bickenbach, Jackie R

    2008-01-01

    We examined how young and old keratinocytes died from heat stress in vitro. We found that keratinocyte cell death was not due to oxidative stress as neither Mn-SOD nor Cu-Zn-SOD was produced in either young or old heated keratinocytes. Instead, analysis of the anti-apoptotic factors, Bcl2 and HSP70, and the pro-apoptotic factors, caspase 3, caspase 8, Apaf-1, cytochrome c, AIF, and EndoG, indicated that keratinocyte cell death occurred via the caspase-independent EndoG apoptotic pathway. We found that both young and old keratinocytes died via the same pathway, and that we could specifically reduce both young and old keratinocyte death by addition of the EndoG inhibitor NEM. Further analysis suggested that the difference between young and old keratinocyte death was due to the synthesis of HSP70 protein, with the increase in response to heat more pronounced in young keratinocytes than in old keratinocytes. When we inhibited HSP70 by adding quercetin, death was increased in both young and old keratinocytes, but more so in old keratinocytes. These data suggest that old keratinocytes may die more readily than young keratinocytes when heated because they synthesize HSP70 at a lower efficiency. Such findings suggest that HSP70 production may be age-dependent.

  15. Biallelic ATM alterations detected at diagnosis identify a subset of treatment-naïve chronic lymphocytic leukemia patients with reduced overall survival similar to patients with p53 deletion.

    PubMed

    Lozano-Santos, Carol; García-Vela, José A; Pérez-Sanz, Nuria; Nova-Gurumeta, Sara; Fernandez-Cuevas, Belen; Gomez-Lozano, Natalia; Sánchez-Beato, Margarita; Sanchez-Godoy, Pedro; Bueno, José Luis; Garcia-Marco, José A

    2017-04-01

    The prognostic impact of biallelic ATM abnormalities (ATM mutation and concurrent 11q deletion) remains unknown. We studied ATM, BIRC3, SF3B1, and NOTCH1 genes in 118 treatment-naïve CLL patients at diagnosis. Patients with biallelic ATM alteration had a similar time to first treatment (TTFT) and shorter overall survival (OS) compared with patients with isolated 11q deletion and shorter TTFT and OS when compared to patients with wild-type ATM. Furthermore, biallelic ATM alteration (HR: 6.4; p ≤ 0.007) was significantly associated with an increased risk of death similar to p53 deletion (HR: 6.1; p ≤ 0.004), superior to 11q deletion alone (HR: 2.8; p ≤ 0.022) and independent of other significant parameters such as age, advanced clinical stage, and complex karyotype. Our results suggest the identification of ATM mutations in CLL patients with 11q deletion at diagnosis is clinically relevant and predicts disease progression, poor response to the treatment, and reduced OS independent of other molecular prognostic factors.

  16. Agent Based Modelling Helps in Understanding the Rules by Which Fibroblasts Support Keratinocyte Colony Formation

    PubMed Central

    Sun, Tao; McMinn, Phil; Holcombe, Mike; Smallwood, Rod; MacNeil, Sheila

    2008-01-01

    Background Autologous keratincoytes are routinely expanded using irradiated mouse fibroblasts and bovine serum for clinical use. With growing concerns about the safety of these xenobiotic materials, it is desirable to culture keratinocytes in media without animal derived products. An improved understanding of epithelial/mesenchymal interactions could assist in this. Methodology/Principal Findings A keratincyte/fibroblast o-culture model was developed by extending an agent-based keratinocyte colony formation model to include the response of keratinocytes to both fibroblasts and serum. The model was validated by comparison of the in virtuo and in vitro multicellular behaviour of keratinocytes and fibroblasts in single and co-culture in Greens medium. To test the robustness of the model, several properties of the fibroblasts were changed to investigate their influence on the multicellular morphogenesis of keratinocyes and fibroblasts. The model was then used to generate hypotheses to explore the interactions of both proliferative and growth arrested fibroblasts with keratinocytes. The key predictions arising from the model which were confirmed by in vitro experiments were that 1) the ratio of fibroblasts to keratinocytes would critically influence keratinocyte colony expansion, 2) this ratio needed to be optimum at the beginning of the co-culture, 3) proliferative fibroblasts would be more effective than irradiated cells in expanding keratinocytes and 4) in the presence of an adequate number of fibroblasts, keratinocyte expansion would be independent of serum. Conclusions A closely associated computational and biological approach is a powerful tool for understanding complex biological systems such as the interactions between keratinocytes and fibroblasts. The key outcome of this study is the finding that the early addition of a critical ratio of proliferative fibroblasts can give rapid keratinocyte expansion without the use of irradiated mouse fibroblasts and bovine

  17. Members of the src and ras oncogene families supplant the epidermal growth factor requirement of BALB/MK-2 keratinocytes and induce distinct alterations in their terminal differentiation program.

    PubMed Central

    Weissman, B; Aaronson, S A

    1985-01-01

    BALB-/MK-2 mouse epidermal keratinocytes required epidermal growth factor for proliferation and terminally differentiated in response to high Ca2+ concentration. Infection with retroviruses containing transforming genes of the src and ras oncogene families led to rapid loss of epidermal growth factor dependence, in some cases, accompanied by alterations in cellular morphology. The virus-altered cells continued to proliferate in the presence of high levels of extracellular calcium but exhibited alterations in normal keratinocyte terminal differentiation that appear to be specific to the particular oncogene. These alterations bore similarities to abnormalities in differentiation observed in naturally occurring squamous epithelial malignancies. Images PMID:2427928

  18. Regulation of interleukin 1 and its receptor in human keratinocytes

    SciTech Connect

    Blanton, R.A.; McDougall, J.K. ); Kupper, T.S. ); Dower, S. )

    1989-02-01

    Keratinocytes in culture synthesize and respond to interleukin 1 (IL-1). The authors have measured surface IL-1 receptor (IL-1R) on keratinocytes in culture using radiolabeled IL-1 binding assays. Surface IL-1R levels are <2,000 receptors per cell in postconfluent cultures but increase 9- to 20-fold 24 hr after treatment with phorbol 12-myristate 13-acetate (PMA) at 10 ng/ml or after raising the extracellular Ca{sup 2+} concentration to 2 mM. This induction of surface IL-1R can be blocked by the addition of retinoic acid and parallels induction of squamous differentiation markers. These results imply that IL-1R levels may be related to the degree of differentiation of these cells. In parallel studies IL-1 protein levels were determined by bioassay and by Western blotting (immunoblots). All detectable IL-1 protein and essentially all IL-1 activity was cell-associated. Although constitutive levels of IL-1 biological activity and protein are significant in these cultures, IL-1 levels increase when either PMA or retinoic acid alone are added to cultures. IL-1 does not increase when PMA and retinoic acid are added simultaneously to cultures; nor is it induced when extracellular Ca{sup 2+} concentrations are raised to 2 mM. Thus, cell-associated IL-1 levels do not necessarily parallel surface IL-1R levels in these cultures. Taken together, these results demonstrate that IL-1 and surface IL-1R levels are differentially and complexly regulated in keratinocyte cultures. Possible implications of these results in terms of normal and abnormal regulation of proliferation and differentiation are discussed.

  19. Arsenite suppression of BMP signaling in human keratinocytes

    SciTech Connect

    Phillips, Marjorie A.; Qin, Qin; Hu, Qin; Zhao, Bin; Rice, Robert H.

    2013-06-15

    Arsenic, a human skin carcinogen, suppresses differentiation of cultured keratinocytes. Exploring the mechanism of this suppression revealed that BMP-6 greatly increased levels of mRNA for keratins 1 and 10, two of the earliest differentiation markers expressed, a process prevented by co-treatment with arsenite. BMP also stimulated, and arsenite suppressed, mRNA for FOXN1, an important transcription factor driving early keratinocyte differentiation. Keratin mRNAs increased slowly after BMP-6 addition, suggesting they are indirect transcriptional targets. Inhibition of Notch1 activation blocked BMP induction of keratins 1 and 10, while FOXN1 induction was largely unaffected. Supporting a requirement for Notch1 signaling in keratin induction, BMP increased levels of activated Notch1, which was blocked by arsenite. BMP also greatly decreased active ERK, while co-treatment with arsenite maintained active ERK. Inhibition of ERK signaling mimicked BMP by inducing keratin and FOXN1 mRNAs and by increasing active Notch1, effects blocked by arsenite. Of 6 dual-specificity phosphatases (DUSPs) targeting ERK, two were induced by BMP unless prevented by simultaneous exposure to arsenite and EGF. Knockdown of DUSP2 or DUSP14 using shRNAs greatly reduced FOXN1 and keratins 1 and 10 mRNA levels and their induction by BMP. Knockdown also decreased activated Notch1, keratin 1 and keratin 10 protein levels, both in the presence and absence of BMP. Thus, one of the earliest effects of BMP is induction of DUSPs, which increases FOXN1 transcription factor and activates Notch1, both required for keratin gene expression. Arsenite prevents this cascade by maintaining ERK signaling, at least in part by suppressing DUSP expression. - Highlights: • BMP induces FOXN1 transcription. • BMP induces DUSP2 and DUSP14, suppressing ERK activation. • Arsenite suppresses levels of phosphorylated Smad1/5 and FOXN1 and DUSP mRNA. • These actions rationalize arsenite suppression of keratinocyte

  20. Stanniocalcin-1 regulates re-epithelialization in human keratinocytes.

    PubMed

    Yeung, Bonnie H Y; Wong, Chris K C

    2011-01-01

    Stanniocalcin-1 (STC1), a glycoprotein hormone, is believed to be involved in various biological processes such as inflammation, oxidative responses and cell migration. Riding on these emerging evidences, we hypothesized that STC1 may participate in the re-epithelialization during wound healing. Re-epithelialization is a critical step that involves keratinocyte lamellipodia (e-lam) formation, followed by cell migration. In this study, staurosporine (STS) treatment induced human keratinocyte (HaCaT) e-lam formation on fibronectin matrix and migration via the activation of focal adhesion kinase (FAK), the surge of intracellular calcium level [Ca²⁺]i and the inactivation of Akt. In accompanied with these migratory features, a time- and dose-dependent increase in STC1 expression was detected. STC1 gene expression was found not the downstream target of FAK-signaling as illustrated by FAK inhibition using PF573228. The reduction of [Ca²⁺]i by BAPTA/AM blocked the STS-mediated keratinocyte migration and STC1 gene expression. Alternatively the increase of [Ca²⁺]i by ionomycin exerted promotional effect on STS-induced STC1 gene expression. The inhibition of Akt by SH6 and GSK3β by lithium chloride (LiCl) could respectively induce and inhibit the STS-mediated e-lam formation, cell migration and STC1 gene expression. The STS-mediated e-lam formation and cell migration were notably hindered or induced respectively by STC1 knockdown or overexpression. This notion was further supported by the scratched wound assay. Collectively the findings provide the first evidence that STC1 promotes re-epithelialization in wound healing.

  1. The p53 status of cultured human premalignant oral keratinocytes.

    PubMed Central

    Burns, J. E.; Clark, L. J.; Yeudall, W. A.; Mitchell, R.; Mackenzie, K.; Chang, S. E.; Parkinson, E. K.

    1994-01-01

    Around 60% of oral squamous cell carcinomas (SCCs) have been shown to harbour p53 mutations, and other studies have demonstrated mutant p53 genes in normal and dysplastic squamous epithelium adjacent to these SCCs. In line with these earlier studies we show here that DOK, a keratinocyte cell line derived from a dysplasia, displays elevated levels of p53 protein and harbours a 12 bp in-frame deletion of the p53 gene spanning codons 188-191. In contrast, the coding region of the p53 gene was normal in a series of six benign recurrent laryngeal papillomas and a series of four premalignant oral erythroplakia biopsies and their cell cultures. All but one of these lesions were free of malignancy at the time of biopsy, in contrast to the premalignant lesions studied by previous investigators, but keratinocytes cultured from these lesions all displayed a partially transformed phenotype that was less pronounced than that of DOK. Since three out of four of the erythroplakia patients developed SCC within 1 year of biopsy, these lesions were by definition premalignant. The availability of strains of partially transformed keratinocytes from premalignant erythroplakias which possess normal p53 genes should enable us to test the role of mutant p53 in the progression of erythroplakia to SCC. The premalignant tissues and cultures were also tested for the presence of human papillomavirus (HPV), which is known to inactivate p53 function in some cases. Only the benign papillomas were shown to contain high levels of either HPV 6 or HPV 11 E6 DNA, but not both, and none of the samples contained detectable levels of HPV 16, HPV 18 or HPV 33 E6 DNA or L1 DNA of several other HPV types. There was therefore no evidence to suggest that p53 was being inactivated by a highly oncogenic HPV in these samples. Images Figure 1 Figure 2 Figure 3 PMID:7917902

  2. The TRAF-interacting protein (TRIP) is a regulator of keratinocyte proliferation.

    PubMed

    Almeida, Stéphanie; Ryser, Stephan; Obarzanek-Fojt, Magdalena; Hohl, Daniel; Huber, Marcel

    2011-02-01

    The TRAF-interacting protein (TRIP/TRAIP) is a RING-type E3 ubiquitin ligase inhibiting tumor necrosis factor-α (TNF-α)-mediated NF-κB activation. TRIP ablation results in early embryonic lethality in mice. To investigate TRIP function in epidermis, we examined its expression and the effect of TRIP knockdown (KD) in keratinocytes. TRIP mRNA expression was strongly downregulated in primary human keratinocytes undergoing differentiation triggered by high cell density or high calcium. Short-term phorbol-12-myristate-13-acetate (TPA) treatment or inhibition of phosphatidylinositol-3 kinase signaling in proliferative keratinocytes suppressed TRIP transcription. Inhibition by TPA was protein kinase C dependent. Keratinocytes undergoing KD of TRIP expression by lentiviral short-hairpin RNA (shRNA; T4 and T5) had strongly reduced proliferation rates compared with control shRNA. Cell cycle analysis demonstrated that TRIP-KD caused growth arrest in the G1/S phase. Keratinocytes with TRIP-KD resembled differentiated cells consistent with the augmented expression of differentiation markers keratin 1 and filaggrin. Luciferase-based reporter assays showed no increase in NF-κB activity in TRIP-KD keratinocytes, indicating that NF-κB activity in keratinocytes is not regulated by TRIP. TRIP expression was increased by ∼2-fold in basal cell carcinomas compared with normal skin. These results underline the important role of TRIP in the regulation of cell cycle progression and the tight linkage of its expression to keratinocyte proliferation.

  3. The suppressor of cytokine signaling (SOCS)-3 determines keratinocyte proliferative and migratory potential during skin repair.

    PubMed

    Linke, Andreas; Goren, Itamar; Bösl, Michael R; Pfeilschifter, Josef; Frank, Stefan

    2010-03-01

    Recently, the suppressor of cytokine signaling (SOCS)-3 has been shown to be expressed in disturbed wound margin epithelia during diabetes-impaired wound healing in mice. To functionally connect a potential contribution of SOCS-3 expression to the control of wound keratinocyte behavior in skin repair, we created a transgenic mouse (tsgn-K5/SOCS3) overexpressing SOCS-3 in keratinocytes using the bovine keratin 5 promoter. Tsgn-K5/SOCS3 mice showed a constitutive expression of SOCS-3 in the basal layer of skin epidermis. Keratinocytes of tsgn-K5/SOCS3 mice showed full inhibition of signal transducer and activator of transcription (STAT)-3 phosphorylation. Tsgn-K5/SOCS3 keratinocytes also showed a strong inhibition of migratory and proliferative potential in vitro. In addition, tsgn-K5/SOCS3 keratinocytes co-expressed the differentiation marker loricrin in the basal layer of nonwounded skin in vivo. Upon wounding, wound tissues of tsgn-K5/SOCS3 mice showed an impairment of wound closure characterized by strongly atrophied wound margin epithelia. Atrophied epithelia of tsgn-K5/SOCS3 mice exhibited a marked reduction in proliferating cells and reduced total keratinocyte numbers. In summary, this study suggests that the presence of SOCS-3 in keratinocytes strongly disturbs epithelial repair of cutaneous wounds by interfering with keratinocyte proliferation and migration.

  4. Foreskin-isolated keratinocytes provide successful extemporaneous autologous paediatric skin grafts.

    PubMed

    Mcheik, Jiad N; Barrault, Christine; Pedretti, Nathalie; Garnier, Julien; Juchaux, Franck; Levard, Guillaume; Morel, Franck; Lecron, Jean-Claude; Bernard, François-Xavier

    2016-03-01

    Severe burns in children are conventionally treated with split-thickness skin autografts or epidermal sheets. However, neither early complete healing nor quality of epithelialization is satisfactory. An alternative approach is to graft isolated keratinocytes. We evaluated paediatric foreskin and auricular skin as donor sources, autologous keratinocyte transplantation, and compared the graft efficiency to the in vitro capacities of isolated keratinocytes to divide and reconstitute epidermal tissue. Keratinocytes were isolated from surgical samples by enzymatic digestion. Living cell recovery, in vitro proliferation and epidermal reconstruction capacities were evaluated. Differentiation status was analysed, using qRT-PCR and immunolabelling. Eleven children were grafted with foreskin-derived (boys) or auricular (girls) keratinocyte suspensions dripped onto deep severe burns. The aesthetic and functional quality of epithelialization was monitored in a standardized way. Foreskin keratinocyte graft in male children provides for the re-epithelialization of partial deep severe burns and accelerates wound healing, thus allowing successful wound closure, and improves the quality of scars. In accordance, in vitro studies have revealed a high yield of living keratinocyte recovery from foreskin and their potential in terms of regeneration and differentiation. We report a successful method for grafting paediatric males presenting large severe burns through direct spreading of autologous foreskin keratinocytes. This alternative method is easy to implement, improves the quality of skin and minimizes associated donor site morbidity. In vitro studies have highlighted the potential of foreskin tissue for graft applications and could help in tissue selection with the prospect of grafting burns for girls.

  5. Keratinocyte cultures from involved skin in vitiligo patients show an impaired in vitro behaviour.

    PubMed

    Bondanza, Sergio; Maurelli, Riccardo; Paterna, Patrizia; Migliore, Eleonora; Giacomo, Fabio Di; Primavera, Giovanni; Paionni, Emanuel; Dellambra, Elena; Guerra, Liliana

    2007-08-01

    Vitiligo depigmentation is considered a consequence of either melanocyte disappearance or loss of functioning melanocytes in the involved areas. However, it has been reported that keratinocytes in involved vitiligo skin are damaged too. Based on this evidence, we evaluated the in vitro behaviour, in life span cultures, of involved and uninvolved vitiligo keratinocytes and their expression of proliferation, differentiation and senescence markers. An additional purpose was to investigate whether vitiligo keratinocytes from depigmented skin are able to sustain survival and growth of normal melanocytes (when added in co-culture experiments), as normal human keratinocytes manage to do. Our results demonstrate that almost all involved vitiligo keratinocytes have a shorter life span in vitro than the uninvolved cells and all of them do not maintain melanocytes in culture in a physiological ratio. Modification of proliferation and senescence marker expression also occurs. Indeed, we detected low initial expression levels of the senescence marker p16 in involved vitiligo keratinocytes, despite their shorter in vitro life span, and increased expression of proliferating cell nuclear antigen and p53. This preliminary analysis of a small number of in vitro cultured vitiligo keratinocytes suggests an impaired senescence process in lesional vitiligo keratinocytes and attempts to regulate it.

  6. Act1: A Psoriasis Susceptibility Gene Playing Its Part in Keratinocytes.

    PubMed

    Hobbs, Ryan P; Smith, Susan H; Getsios, Spiro

    2017-04-05

    Unchecked inflammation, impaired keratinocyte differentiation, and heightened host defense responses typify psoriasis. Lambert et al. make clever use of psoriasis patient genetics and whole transcriptome RNA-Seq analysis to implicate Act1 in these seemingly variegated processes by keeping IL-17 receptor signaling in check while supporting differentiation and limiting innate immune responses in human keratinocytes.

  7. Morphological evidence for the role of suprabasal keratinocytes in wound reepithelialization.

    PubMed

    Usui, Marcia L; Underwood, Robert A; Mansbridge, Jonathan N; Muffley, Lara A; Carter, William G; Olerud, John E

    2005-01-01

    The process by which wounds reepithelialize remains controversial. Two models have been proposed to describe reepithelialization: the "sliding" model and the "rolling" model. In the "sliding" model, basal keratinocytes are the principal cells responsible for migration and wound closure. In this model, basal and suprabasal keratinocytes remain strongly attached to leading edge basal keratinocytes and are then passively dragged along as a sheet. The "rolling" model postulates that basal keratinocytes remain strongly attached to the basement membrane zone while suprabasal keratinocytes at the wound margin are activated to roll into the wound site. The purpose of this study was to determine which populations of keratinocytes are actively involved in reepithelialization. We evaluated expression of keratins K14, K15, K10, K2e, and K16 as well as the proliferation marker Ki67 in the migrating tongue of normal human incisional 1-hour to 28-day wounds and normal human 3 mm diameter excisional 1- to 7-day wounds. Our results show dramatic changes in phenotype and protein expression of keratins K10, K2e, K14, K15, and K16 in suprabasal keratinocytes in response to injury. We conclude that this large population of suprabasal keratinocytes actively participates in wound closure.

  8. Melatonin promotes diabetic wound healing in vitro by regulating keratinocyte activity

    PubMed Central

    Song, Ruipeng; Ren, Lijun; Ma, Haoli; Hu, Ruijing; Gao, Honghong; Wang, Li; Chen, Xuehui; Zhao, Zhigang; Liu, Jialin

    2016-01-01

    Diabetic patients are at high risk of developing delayed cutaneous wound healing. Proper keratinocyte proliferation and migration are crucial steps during re-epithelialization. Melatonin (Mel) accelerates wound repair in full-thickness incisional wounds; however, its role in diabetic wound healing is unknown. This study explored the role of Mel in diabetic wound healing in vitro by using high glucose (HG)-cultured keratinocytes. Mel reduced the HG-induced mRNA expression and release of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-8, in keratinocytes. Mel inhibited oxidative stress, as evidenced by reduced production of reactive oxygen species and malondialdehyde and increased activity of superoxide dismutase in HG-stimulated keratinocytes. Mel also inhibited HG-induced nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasome activation in keratinocytes. HG-induced reduced migration and proliferation and increased apoptosis of keratinocytes were counteracted by Mel treatment. The pro-proliferative, pro-migratory, and anti-apoptotic effects of Mel on HG-treated keratinocytes were mediated by extracellular signal-regulated kinase signaling pathway. Results collectively suggested that Mel is an alternative therapeutic strategy to ameliorate poor condition for diabetic wound healing by regulating keratinocyte activity. PMID:27904671

  9. Steroid synthesis by primary human keratinocytes; implications for skin disease

    SciTech Connect

    Hannen, Rosalind F.; Michael, Anthony E.; Jaulim, Adil; Bhogal, Ranjit; Burrin, Jacky M.; Philpott, Michael P.

    2011-01-07

    Research highlights: {yields} Primary keratinocytes express the steroid enzymes required for cortisol synthesis. {yields} Normal primary human keratinocytes can synthesise cortisol. {yields} Steroidogenic regulators, StAR and MLN64, are expressed in normal epidermis. {yields} StAR expression is down regulated in eczema and psoriatic epidermis. -- Abstract: Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary human keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3{beta}HSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7-{sup 3}H]-pregnenolone through each steroid intermediate to [7-{sup 3}H]-cortisol in cultured PHK. Trilostane (a 3{beta}HSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively these data

  10. Stimulatory effect of Brazilian propolis on hair growth through proliferation of keratinocytes in mice.

    PubMed

    Miyata, Shota; Oda, Yozo; Matsuo, Chika; Kumura, Haruto; Kobayashi, Ken

    2014-12-10

    Propolis is a natural honeybee hive product with the potential for use in the treatment of dermatological conditions, such as cutaneous abrasions, burns, and acne. In this study, we investigated whether propolis stimulates hair growth in mice. Ethanol-extracted propolis, which contains various physiologically active substances such as caffeic acid and kaempferol, stimulated anagen induction in shaved back skin. Anagen induction occurred without any detectable abnormalities in the shape of the hair follicles (HFs), hair stem cells in the bulge, proliferating hair matrix keratinocytes in the hair bulb, or localization of versican in the dermal papilla. Propolis treatment also stimulated migration of hair matrix keratinocytes into the hair shaft in HFs during late anagen in the depilated back skin. Organotypic culture of skin containing anagen stage HFs revealed significant stimulation of hair matrix keratinocyte proliferation by propolis. Furthermore, propolis facilitated the proliferation of epidermal keratinocytes. These results indicate that propolis stimulates hair growth by inducing hair keratinocyte proliferation.

  11. Exposure to Carbon Ions Triggers Proinflammatory Signals and Changes in Homeostasis and Epidermal Tissue Organization to a Similar Extent as Photons

    PubMed Central

    Simoniello, Palma; Wiedemann, Julia; Zink, Joana; Thoennes, Eva; Stange, Maike; Layer, Paul G.; Kovacs, Maximilian; Podda, Maurizio; Durante, Marco; Fournier, Claudia

    2016-01-01

    The increasing application of charged particles in radiotherapy requires a deeper understanding of early and late side effects occurring in skin, which is exposed in all radiation treatments. We measured cellular and molecular changes related to the early inflammatory response of human skin irradiated with carbon ions, in particular cell death induction and changes in differentiation and proliferation of epidermal cells during the first days after exposure. Model systems for human skin from healthy donors of different complexity, i.e., keratinocytes, coculture of skin cells, 3D skin equivalents, and skin explants, were used to investigate the alterations induced by carbon ions (spread-out Bragg peak, dose-averaged LET 100 keV/μm) in comparison to X-ray and UV-B exposure. After exposure to ionizing radiation, in none of the model systems, apoptosis/necrosis was observed. Carbon ions triggered inflammatory signaling and accelerated differentiation of keratinocytes to a similar extent as X-rays at the same doses. High doses of carbon ions were more effective than X-rays in reducing proliferation and inducing abnormal differentiation. In contrast, changes identified following low-dose exposure (≤0.5 Gy) were induced more effectively after X-ray exposure, i.e., enhanced proliferation and change in the polarity of basal cells. PMID:26779439

  12. Transcriptional profiling defines the effects of nickel in human epidermal keratinocytes.

    PubMed

    Gazel, Alix; Rosdy, Martin; Tornier, Carine; De Fraissinette, Anne De Brugerolle; Blumenberg, Miroslav

    2008-12-01

    Nickel is a ubiquitous and virtually unavoidable environmental pollutant and occupational hazard, but its molecular and cellular effects are not well understood. Human epidermal keratinocytes are the sentinel and the primary target for nickel. We treated with nickel salts skin equivalents containing differentiating epidermal keratinocytes grown on air-liquid interface in standard cell culture conditions. We identified the transcriptional profiles affected by nickel in reconstructed human epidermis (RHE) using DNA microarrays. The Ni-regulated genes were determined at two time points, immediate-early, 30 min after treatment, and late, at 6 h. Using in silico data analysis, we determined that 134 genes are regulated by nickel; of these, 97 are induced and 37 suppressed. Functional categories of regulated genes suggest that Ni inhibits apoptosis, promotes cell cycle and induces synthesis of extracellular matrix proteins and extracellular proteases. Importantly, Ni also regulates a set of secreted signaling proteins, inducing VEGF, amphiregulin, PGF, GDF15, and BST2, while suppressing IL-18, galectin-3, and LITAF. These secreted proteins may be important in Ni-caused allergic reactions. Ni induced inhibitors of the NFkappaB signaling pathway, and suppressed its activators. Correspondingly, NFkappaB binding sites were found to be overrepresented in the Ni-suppressed genes, whereas cFOS/AP1 binding sites were common in the Ni-induced genes. Significant parallels were found between the Ni-regulated genes and the genes regulated by TGFbeta, EGF, glucocorticoids, or Oncostatin-M. The comprehensive identification of Ni-regulated genes in human epidermal equivalents significantly advances our understanding of the molecular effects of nickel in skin.

  13. Cortactin involvement in the keratinocyte growth factor and fibroblast growth factor 10 promotion of migration and cortical actin assembly in human keratinocytes

    SciTech Connect

    Ceccarelli, Simona; Cardinali, Giorgia; Aspite, Nicaela; Picardo, Mauro; Marchese, Cinzia; Torrisi, Maria Rosaria; Mancini, Patrizia . E-mail: patrizia.mancini@uniroma1.it

    2007-05-15

    Keratinocyte growth factor (KGF/FGF7) and fibroblast growth factor 10 (FGF10/KGF2) regulate keratinocyte proliferation and differentiation by binding to the tyrosine kinase KGF receptor (KGFR). KGF induces keratinocyte motility and cytoskeletal rearrangement, whereas a direct role of FGF10 on keratinocyte migration is not clearly established. Here we analyzed the motogenic activity of FGF10 and KGF on human keratinocytes. Migration assays and immunofluorescence of actin cytoskeleton revealed that FGF10 is less efficient than KGF in promoting migration and exerts a delayed effect in inducing lamellipodia and ruffles formation. Both growth factors promoted phosphorylation and subsequent membrane translocation of cortactin, an F-actin binding protein involved in cell migration; however, FGF10-induced cortactin phosphorylation was reduced, more transient and delayed with respect to that promoted by KGF. Cortactin phosphorylation induced by both growth factors was Src-dependent, while its membrane translocation and cell migration were blocked by either Src and PI3K inhibitors, suggesting that both pathways are involved in KGF- and FGF10-dependent motility. Furthermore, siRNA-mediated downregulation of cortactin inhibited KGF- and FGF10-induced migration. These results indicate that cortactin is involved in keratinocyte migration promoted by both KGF and FGF10.

  14. Overexpression of CRABPI in suprabasal keratinocytes enhances the proliferation of epidermal basal keratinocytes in mouse skin topically treated with all-trans retinoic acid.

    PubMed

    Tang, Xiao-Han; Vivero, Marina; Gudas, Lorraine J

    2008-01-01

    We investigated whether ectopic expression of CRABPI, a cellular retinoic acid binding protein, influenced the actions of all-trans retinoic acid (ATRA) in transgenic (TG) mice. We targeted CRABPI to the basal vs. suprabasal layers of mouse epidermis by using the keratin 14 (K14) and keratin 10 (K10) promoters, respectively. Greater CRABPI protein levels were detected in the epidermis of adult transgenic(+) mice than in transgenic(-) mice for both transgenes. In adult mouse skin CRABPI overexpression in the basal or suprabasal keratinocytes did not cause morphological abnormalities, but did result in decreased CRABPII mRNA levels. Ectopically overexpressed CRABPI in suprabasal keratinocytes, but not in basal keratinocytes, enhanced the thickening of the epidermis induced by topical ATRA treatments (10 microM, 400 microl for 4 days) by 1.59+/-0.2-fold (p<0.05). ATRA treatment (10 microM) resulted in a 59.9+/-9.8% increase (p<0.05) in the BrdU labeling index in K10/FLAG-CRABPI TG(+) mice vs. TG(-) mice. Retinoid topical treatments reduced p27 and CYP26A1 mRNA levels in TG(+) and TG(-) mouse skin in K14 and K10/FLAG-CRABPI transgenic mice. As epidermal basal keratinocyte proliferation is stimulated by paracrine growth factors secreted by ATRA activated suprabasal keratinocytes, our results indicate that CRABPI overexpression in suprabasal keratinocytes enhances the physiological functions of ATRA.

  15. Overexpression of CRABPI in suprabasal keratinocytes enhances the proliferation of epidermal basal keratinocytes in mouse skin topically treated with all-trans retinoic acid

    SciTech Connect

    Tang, X.-H.; Vivero, Marina; Gudas, Lorraine J.

    2008-01-01

    We investigated whether ectopic expression of CRABPI, a cellular retinoic acid binding protein, influenced the actions of all-trans retinoic acid (ATRA) in transgenic (TG) mice. We targeted CRABPI to the basal vs. suprabasal layers of mouse epidermis by using the keratin 14 (K14) and keratin 10 (K10) promoters, respectively. Greater CRABPI protein levels were detected in the epidermis of adult transgenic(+) mice than in transgenic(-) mice for both transgenes. In adult mouse skin CRABPI overexpression in the basal or suprabasal keratinocytes did not cause morphological abnormalities, but did result in decreased CRABPII mRNA levels. Ectopically overexpressed CRABPI in suprabasal keratinocytes, but not in basal keratinocytes, enhanced the thickening of the epidermis induced by topical ATRA treatments (10 {mu}M, 400 {mu}l for 4 days) by 1.59 {+-} 0.2-fold (p < 0.05). ATRA treatment (10 {mu}M) resulted in a 59.9 {+-} 9.8% increase (p < 0.05) in the BrdU labeling index in K10/FLAG-CRABPI TG(+) mice vs. TG(-) mice. Retinoid topical treatments reduced p27 and CYP26A1 mRNA levels in TG(+) and TG(-) mouse skin in K14 and K10/FLAG-CRABPI transgenic mice. As epidermal basal keratinocyte proliferation is stimulated by paracrine growth factors secreted by ATRA activated suprabasal keratinocytes, our results indicate that CRABPI overexpression in suprabasal keratinocytes enhances the physiological functions of ATRA.

  16. Keratinocyte growth factor causes cystic dilation of the mammary glands of mice. Interactions of keratinocyte growth factor, estrogen, and progesterone in vivo.

    PubMed

    Yi, E S; Bedoya, A A; Lee, H; Kim, S; Housley, R M; Aukerman, S L; Tarpley, J E; Starnes, C; Yin, S; Pierce, G F

    1994-11-01

    Keratinocyte growth factor (KGF) is a paracrine mediator of epithelial cell proliferation that has been reported to induce marked proliferation of mammary epithelium in rats. In this study, systemic administration of KGF into naive and oophorectomized mice causes mammary gland proliferation, as evidenced histologically by the appearance of cysts lined by a single layer of epithelium and by hyperplastic epithelium. Whole mount preparations of the mammary glands reveal that the histologically noted cysts are actually ducts that are dilated along much of their length. The histology of the mammary glands of KGF-treated mice is similar to the histology of fibrocystic disease in the human female breast. The response in mice differs significantly from the appearance of the mammary glands in KGF-treated rats in which ductal epithelial proliferation is most prominent. Estrogen and progesterone when administered in combination but not alone cause the development of numerous endbuds in the mouse mammary gland. KGF in estrogen- and progesterone-pretreated mice causes the growth of dilated ducts, hyperplastic epithelium within ducts and endbuds, and a fibrous metamorphosis of periductal adipose tissue. The mammary epithelial hyperplasia caused by KGF is rapidly reversible in both mice and rats after cessation of KGF treatment. The spectrum of KGF-, estrogen-, and progesterone-induced mammary histopathology in mice provides a model for the study of fibrocystic and hyperplastic breast disease.

  17. TOXICITY OF AMORPHOUS SILICA NANOPARTICLES IN MOUSE KERATINOCYTES

    SciTech Connect

    Yu, Kyung; Wang, Wei; Gu, Baohua; Hussain, Saber

    2009-01-01

    The present study was designed to examine the uptake, localization and the cytotoxic effects of well-dispersed amorphous silica nanoparticles in mouse keratinocytes (HEL-30). Mouse keratinocytes were exposed for 24h to various concentrations of amorphous silica nanoparticles in homogeneous suspensions of average size distribution (30, 48, 118 and 535 nm SiO2) then assessed for uptake and biochemical changes. Results of transmission electron microscopy revealed all sizes of silica were taken up into the cells and localized into the cytoplasm. The lactate dehydrogenase (LDH) assay shows LDH leakage was dose- and size-dependent with exposure to 30 and 48 nm nanoparticles. However, no LDH leakage was observed for either 118 or 535 nm nanoparticles. The mitochondrial viability assay (MTT) showed significant toxicity for 30 and 48 nm at high concentrations (100 g/mL) compare to the 118 and 535 nm particles. Further studies were carried out to investigate if cellular reduced GSH and mitochondria membrane potential are involved in the mechanism of SiO2 toxicity. The redox potential of cells (GSH) was reduced significantly at concentrations of 50, 100 and 200 g/mL at 30 nm nanoparticle exposures. However, silica nanoparticles larger than 30 nm showed no changes in GSH levels. Reactive oxygen species (ROS) formation did not show any significant change between controls and the exposed cells. In summary, amorphous silica nanoparticles below 100 nm induced cytotoxicity suggest size-of the particles is critical to produce biological effects.

  18. Regulation of fos-lacZ fusion gene expression in primary mouse epidermal keratinocytes isolated from transgenic mice.

    PubMed Central

    Bollag, W B; Xiong, Y; Ducote, J; Harmon, C S

    1994-01-01

    The expression of a fos-lacZ fusion gene was studied in primary mouse epidermal keratinocytes obtained from transgenic mice. This gene construct contains the entire upstream regulatory sequence of c-fos, and expression of the endogenous and fusion gene was shown by Northern analysis to correlate upon induction with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Using a chromogenic substrate of beta-galactosidase, we also demonstrated that expression of the fusion gene product, like that of Fos, was localized to the cell nucleus. In addition, we showed that epidermal keratinocytes responded to dialysed fetal bovine serum (FBS), TPA and high-calcium medium with enhanced Fos-lacZ expression and an inhibition of proliferation. The time course of induction of Fos-lacZ expression was similar for dialysed FBS and TPA, with a peak approximately 2 h after exposure. Exposure for approximately 24 h to an elevated extracellular calcium concentration was required to elicit an increase in Fos-lacZ expression. The lack of an immediate effect of raising medium calcium levels on Fos-lacZ expression contrasted with the rapidity of its effect on DNA synthesis, which was significantly inhibited within 6-8 h. In addition, we found that the protein kinase C inhibitor Ro 31-7549 blocked Fos-lacZ expression induced by TPA but had little or no effect on that elicited by high calcium levels. Thus, although our results indicate that the fos gene product may be involved in mediating epidermal keratinocyte growth arrest in response to differentiative agents such as FBS, TPA and high medium calcium levels, the exact role of this gene product remains unclear. Images Figure 1 Figure 2 PMID:8198544

  19. Regulation of fos-lacZ fusion gene expression in primary mouse epidermal keratinocytes isolated from transgenic mice.

    PubMed

    Bollag, W B; Xiong, Y; Ducote, J; Harmon, C S

    1994-05-15

    The expression of a fos-lacZ fusion gene was studied in primary mouse epidermal keratinocytes obtained from transgenic mice. This gene construct contains the entire upstream regulatory sequence of c-fos, and expression of the endogenous and fusion gene was shown by Northern analysis to correlate upon induction with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Using a chromogenic substrate of beta-galactosidase, we also demonstrated that expression of the fusion gene product, like that of Fos, was localized to the cell nucleus. In addition, we showed that epidermal keratinocytes responded to dialysed fetal bovine serum (FBS), TPA and high-calcium medium with enhanced Fos-lacZ expression and an inhibition of proliferation. The time course of induction of Fos-lacZ expression was similar for dialysed FBS and TPA, with a peak approximately 2 h after exposure. Exposure for approximately 24 h to an elevated extracellular calcium concentration was required to elicit an increase in Fos-lacZ expression. The lack of an immediate effect of raising medium calcium levels on Fos-lacZ expression contrasted with the rapidity of its effect on DNA synthesis, which was significantly inhibited within 6-8 h. In addition, we found that the protein kinase C inhibitor Ro 31-7549 blocked Fos-lacZ expression induced by TPA but had little or no effect on that elicited by high calcium levels. Thus, although our results indicate that the fos gene product may be involved in mediating epidermal keratinocyte growth arrest in response to differentiative agents such as FBS, TPA and high medium calcium levels, the exact role of this gene product remains unclear.

  20. Protective Effects of Minor Components of Curcuminoids on Hydrogen Peroxide-Treated Human HaCaT Keratinocytes.

    PubMed

    Liu, Yuh-Hwa; Lin, Yin-Shiou; Huang, Yu-Wei; Fang, Sheng-Uei; Lin, Shyr-Yi; Hou, Wen-Chi

    2016-05-11

    Hydrogen peroxide, one of the reactive oxygen species (ROS), can cause intracellular oxidative stress associated with skin aging and/or photoaging. Curcumin, a polyphenol in turmeric, has been reported to exhibit biological activity. In this study, five naturally occurring curcuminoids [curcumin, demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), monohydroxy-DMC, and monohydroxy-BDMC] were used to investigate their protective roles against hydrogen peroxide-induced oxidative stress in the immortalized human keratinocyte cell lines (HaCaT cells). These five curcuminoids at 10 μM, but not at 5 μM, were shown to exhibit cytotoxicities toward HaCaT keratinocytes. Therefore, a 5 μM concentration of the five curcuminoids was selected for further investigations. Cells were pretreated with or without curcuminoids for 2.5 h before 24-h hydrogen peroxide (150 μM) treatments. Pretreatments with the minor components monohydroxy-DMC or monohydroxy-BDMC, but not curcumin, DMC, and BDMC, showed protective activity, elevating cell viability compared to cells with direct hydrogen peroxide treatments. Pretreatments with monohydroxy-DMC and monohydroxy-BDMC showed the best protective effects, reducing apoptotic cell populations and intracellular ROS, as demonstrated by flow cytometry, as well as reducing the changes of the mitochondrial membrane potential compared to cells with direct hydrogen peroxide treatments. The pretreatments with monohydroxy-DMC and monohydroxy-BDMC reduced c-jun and c-fos mRNA expression and p53 tumor suppressor protein expression and increased HO-1 protein expression and glutathione peroxidase (GPx) activity, respectively, compared to cells with direct hydrogen peroxide treatments. The five curcuminoids exhibited similar hydrogen peroxide-scavenging activity in vitro. It was proposed that monohydroxy-DMC and monohydroxy-BDMC could induce antioxidant defense systems better than curcumin, DMC, or BDMC could against hydrogen peroxide-induced oxidative

  1. The human E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion

    PubMed Central

    1995-01-01

    The E48 antigen, a putative human homologue of the 20-kD protein present in desmosomal preparations of bovine muzzle, and formerly called desmoglein III (dg4), is a promising target antigen for antibody- based therapy of squamous cell carcinoma in man. To anticipate the effect of high antibody dose treatment, and to evaluate the possible biological involvement of the antigen in carcinogenesis, we set out to molecularly characterize the antigen. A cDNA clone encoding the E48 antigen was isolated by expression cloning in COS cells. Sequence analysis revealed that the clone contained an open reading frame of 128 amino acids, encoding a core protein of 13,286 kD. Database searching showed that the E48 antigen has a high level of sequence similarity with the mouse ThB antigen, a member of the Ly-6 antigen family. Phosphatidylinositol-specific (PI-specific) phospholipase-C treatment indicated that the E48 antigen is glycosylphosphatidylinositol-anchored (GPI-anchored) to the plasma membrane. The gene encoding the E48 antigen is a single copy gene, located on human chromosome 8 in the 8q24-qter region. The expression of the gene is confined to keratinocytes and squamous tumor cells. The putative mouse homologue, the ThB antigen, originally identified as an antigen on cells of the lymphocyte lineage, was shown to be highly expressed in squamous mouse epithelia. Moreover, the ThB expression level is in keratinocytes, in contrast to that in lymphocytes, not mouse strain related. Transfection of mouse SV40-polyoma transformed mouse NIH/3T3 cells with the E48 cDNA confirmed that the antigen is likely to be involved in cell-cell adhesion. PMID:7790363

  2. Exogenous stimulation with Eclipta alba promotes hair matrix keratinocyte proliferation and downregulates TGF-β1 expression in nude mice.

    PubMed

    Begum, Shahnaz; Lee, Mi Ra; Gu, Li Juan; Hossain, Jamil; Sung, Chang Keun

    2015-02-01

    Eclipta alba (L.) Hassk (E. alba) is a traditionally acclaimed medicinal herb used for the promotion of hair growth. However, to the best of our knowledge, no report has been issued to date on its effects on genetically distorted hair follicles (HFs). In this study, we aimed to identify an agent (stimuli) that may be beneficial for the restoration of human hair loss and which may be used as an alternative to synthetic drugs. We investigated the effects of petroleum ether extract (PEE) and different solvent fractions of E. alba on HFs of nude mice. Treatment was performed by topical application on the backs of nude mice and the changes in hair growth patterns were evaluated. Histological analysis was carried out to evaluate the HF morphology and the structural differences. Immunohistochemical (IHC) staining was performed to visualize follicular keratinocyte proliferation. The histological assessments revealed that the PEE-treated skin specimens exhibited prominent follicular hypertrophy. Subsequently, IHC staining revealed a significant increase (p<0.001) in the number of follicular keratinocytes in basal epidermal and matrix cells. Our results also demonstrated that PEE significantly (p<0.001) reduced the levels of transforming growth factor-β1 (TGF-β1) expression during early anagen and anagen-catagen transition. Our results suggest that PEE of E. alba acts as an important exogenous mediator that stimulates follicular keratinocyte proliferation and delays terminal differentiation by downregulating TGF-β1 expression. Thus, this study highlights the potential use of PEE of E. alba in the treatment of certain types of alopecia.

  3. Effects of tumor necrosis factor-alpha (TNF alpha) in epidermal keratinocytes revealed using global transcriptional profiling.

    PubMed

    Banno, Tomohiro; Gazel, Alix; Blumenberg, Miroslav

    2004-07-30

    Identification of tumor necrosis factor-alpha (TNF alpha) as the key agent in inflammatory disorders, e.g. rheumatoid arthritis, Crohn's disease, and psoriasis, led to TNF alpha-targeting therapies, which, although avoiding many of the side-effects of previous drugs, nonetheless causes other side-effects, including secondary infections and cancer. By controlling gene expression, TNF alpha orchestrates the cutaneous responses to environmental damage and inflammation. To define TNF alpha action in epidermis, we compared the transcriptional profiles of normal human keratinocytes untreated and treated with TNF alpha for 1, 4, 24, and 48 h by using oligonucleotide microarrays. We found that TNF alpha regulates not only immune and inflammatory responses but also tissue remodeling, cell motility, cell cycle, and apoptosis. Specifically, TNF alpha regulates innate immunity and inflammation by inducing a characteristic large set of chemokines, including newly identified TNF alpha targets, that attract neutrophils, macrophages, and skin-specific memory T-cells. This implicates TNF alpha in the pathogenesis of psoriasis, fixed drug eruption, atopic and allergic contact dermatitis. TNF alpha promotes tissue repair by inducing basement membrane components and collagen-degrading proteases. Unexpectedly, TNF alpha induces actin cytoskeleton regulators and integrins, enhancing keratinocyte motility and attachment, effects not previously associated with TNF alpha. Also unanticipated was the influence of TNF alpha upon keratinocyte cell fate by regulating cell-cycle and apoptosis-associated genes. Therefore, TNF alpha initiates not only the initiation of inflammation and responses to injury, but also the subsequent epidermal repair. The results provide new insights into the harmful and beneficial TNF alpha effects and define the mechanisms and genes that achieve these outcomes, both of which are important for TNF alpha-targeted therapies.

  4. 20-Hydroxyvitamin D3, a Product of Vitamin D3 Hydroxylation by Cytochrome P450scc, Stimulates Keratinocyte Differentiation

    PubMed Central

    Zbytek, Blazej; Janjetovic, Zorica; Tuckey, Robert C.; Zmijewski, Michal A.; Sweatman, Trevor W.; Jones, Emily; Nguyen, Minh N.; Slominski, Andrzej T.

    2008-01-01

    It has been shown that mammalian cytochrome P450scc can metabolize vitamin D3 to 20-hydroxyvitamin D3 (20(OH)D3) and 20,22(OH)2D3. To define the biological significance of this pathway, we tested the effects of 20(OH)D3 on the differentiation program of keratinocytes and on the expression of enzymes engaged in vitamin D3 metabolism. Immortalized HaCaT and adult human epidermal keratinocytes were used as a model and the effects of 20(OH)D3 were compared with those of 25(OH)D3 and 1,25(OH)2D3. 20(OH)D3 inhibited proliferation and caused G2/M arrest. 20(OH)D3 stimulated involucrin and inhibited cytokeratin 14 expression. The potency of 20(OH)D3 was comparable to that of 1,25(OH)2D3. 20(OH)D3 decreased the expression of cytochrome P450 enzyme (CYP)27A1 and CYP27B1, however, having only slight effect on CYP24. The effect of 20(OH)D3 was dependent on the vitamin D receptor (VDR). As shown by electrophoretic mobility shift assay, 20(OH)D3 stimulated the binding of nuclear proteins to the VDRE. Transfection of cells with VDR-specific siRNA decreased 20(OH)D3-stimulated transcriptional activity of the VDRE promoter and the expression of involucrin and CYP24 mRNA. Therefore, the above studies identify 20(OH)D3 as a biologically active secosteroid that induces keratinocyte differentiation. These data imply that the previously unreported pathway of vitamin D3 metabolism by P450scc may have wider biological implications depending, for example, on the extent of adrenal gland or cutaneous metabolism. PMID:18368131

  5. Urethral reconstruction with a 3D porous bacterial cellulose scaffold seeded with lingual keratinocytes in a rabbit model.

    PubMed

    Huang, Jian-Wen; Lv, Xiang-Guo; Li, Zhe; Song, Lu-Jie; Feng, Chao; Xie, Min-Kai; Li, Chao; Li, Hong-Bin; Wang, Ji-Hong; Zhu, Wei-Dong; Chen, Shi-Yan; Wang, Hua-Ping; Xu, Yue-Min

    2015-09-11

    The goal of this study was to evaluate the effects of urethral reconstruction with a three-dimensional (3D) porous bacterial cellulose (BC) scaffold seeded with lingual keratinocytes in a rabbit model. A novel 3D porous BC scaffold was prepared by gelatin sponge interfering in the BC fermentation process. Rabbit lingual keratinocytes were isolated, expanded, and seeded onto 3D porous BC. BC alone (group 1, N  =  10), 3D porous BC alone (group 2, N  =  10), and 3D porous BC seeded with lingual keratinocytes (group 3, N  =  10) were used to repair rabbit ventral urethral defects (2.0   ×   0.8 cm). Scanning electron microscopy revealed that BC consisted of a compact laminate while 3D porous BC was composed of a porous sheet buttressed by a dense outer layer. The average pore diameter and porosity of the 3D porous BC were 4.23   ±   1.14 μm and 67.00   ±   6.80%, respectively. At 3 months postoperatively, macroscopic examinations and retrograde urethrograms of urethras revealed that all urethras maintained wide calibers in group 3. Strictures were found in all rabbits in groups 1 and 2. Histologically, at 1 month postoperatively, intact epithelium occurred in group 3, and discontinued epithelium was found in groups 1 and 2. However, groups 2 and 3 exhibited similar epithelial regeneration, which was superior to that of group 1 at 3 months (p  <  0.05). Comparisons of smooth muscle content and endothelia density among the three groups revealed a significant increase at each time point (p  <  0.05). Our results demonstrated that 3D porous BC seeded with lingual keratinocytes enhanced urethral tissue regeneration. 3D porous BC could potentially be used as an optimized scaffold for urethral reconstruction.

  6. Conjugation of extracellular matrix proteins to basal lamina analogs enhances keratinocyte attachment.

    PubMed

    Bush, Katie A; Downing, Brett R; Walsh, Sarah E; Pins, George D

    2007-02-01

    The dermal-epidermal junction of skin contains extracellular matrix proteins that are involved in initiating and controlling keratinocyte signaling events such as attachment, proliferation, and terminal differentiation. To characterize the relationship between extracellular matrix proteins and keratinocyte attachment, a biomimetic design approach was used to precisely tailor the surface of basal lamina analogs with biochemistries that emulate the native biochemical composition found at the dermal-epidermal junction. A high-throughput screening device was developed by our laboratory that allows for the simultaneous investigation of the conjugation of individual extracellular matrix proteins (e.g. collagen type I, collagen type IV, laminin, or fibronectin) as well as their effect on keratinocyte attachment, on the surface of an implantable collagen membrane. Fluorescence microscopy coupled with quantitative digital image analyses indicated that the extracellular matrix proteins adsorbed to the collagen-GAG membranes in a dose-dependent manner. To determine the relationship between extracellular matrix protein signaling cues and keratinocyte attachment, cells were seeded on protein-conjugated collagen-GAG membranes and a tetrazolium-based colorimetric assay was used to quantify viable keratinocyte attachment. Our results indicate that keratinocyte attachment was significantly enhanced on the surfaces of collagen membranes that were conjugated with fibronectin and type IV collagen. These findings define a set of design parameters that will enhance keratinocyte binding efficiency on the surface of collagen membranes and ultimately improve the rate of epithelialization for dermal equivalents.

  7. Regulation of protein kinase D during differentiation and proliferation of primary mouse keratinocytes.

    PubMed

    Ernest Dodd, M; Ristich, Vladimir L; Ray, Sagarika; Lober, Robert M; Bollag, Wendy B

    2005-08-01

    Diseased skin often exhibits a deregulated program of the keratinocyte maturation necessary for epidermal stratification and function. Protein kinase D (PKD), a serine/threonine kinase, is expressed in proliferating keratinocytes, and PKD activation occurs in response to mitogen stimulation in other cell types. We have proposed that PKD functions as a pro-proliferative and/or anti-differentiative signal in keratinocytes and hypothesized that differentiation inducers will downmodulate PKD to allow differentiation to proceed. Thus, changes in PKD levels, autophosphorylation, and activity were analyzed upon stimulation of differentiation and proliferation in primary mouse keratinocytes. Elevated extracellular calcium and acute 12-O-tetradecanoylphorbol-13-acetate (TPA) treatments induced differentiation and triggered a downmodulation of PKD levels, autophosphorylation at serine 916, and activity. Chronic TPA treatment stimulated proliferation and resulted in a recovery of PKD levels, autophosphorylation, and activity. Immunohistochemical analysis demonstrated PKD localization predominantly in the proliferative basal layer of mouse epidermis. Co-expression studies revealed a pro-proliferative, anti-differentiative effect of PKD on keratinocyte maturation as monitored by increased and decreased promoter activities of keratin 5, a proliferative marker, and involucrin, a differentiative marker, respectively. This work describes the inverse regulation of PKD during keratinocyte differentiation and proliferation and the pro-proliferative/anti-differentiative effects of PKD co-expression on keratinocyte maturation.

  8. Comparison of the enzymatic and explant methods for the culture of keratinocytes isolated from human foreskin

    PubMed Central

    ORAZIZADEH, MAHMOUD; HASHEMITABAR, MAHMOUD; BAHRAMZADEH, SOMAYEH; DEHBASHI, FRESHTEH NEJAD; SAREMY, SADEGH

    2015-01-01

    Currently, culture and growth keratinocytes are important stages in achieving a reliable and reproducible skin tissue. In the present study, two different methods, enzymatic and explant methods, for keratinocytes isolation from human foreskin were compared. Foreskins were cut into 2–3 mm pieces and placed in trypsin at 4°C overnight for separation of the epidermis from the dermis. Subsequently, these samples were divided into two groups: i) Keratinocytes separated from the epidermis by trypsin and ii) by the explant method. These keratinocytes were divided into two groups: i) With no feeder layer and ii) onto a type I collagen scaffold. The cells were evaluated using immunocytochemistry and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) staining. In the enzymatic treatment, after 7–10 days no attached cells were found in the cell culture dishes. In the explant method, keratinocytes were separated after ~24 h, attached rapidly and formed big colonies into a collagen scaffold. In the absence of a feeder layer, small colonies were developed with rapid loss of proliferation within 2–3 days. Keratinocytes showed positive immunoreactivity for the pan-cytokeratin marker and keratinocytes' nuclei were clearly observed. This method could be applied and developed as a component of skin substitutes to treat burns and wounds and also in laboratory testing. PMID:26137227

  9. Lactobacillus rhamnosus GG Lysate Increases Re-Epithelialization of Keratinocyte Scratch Assays by Promoting Migration.

    PubMed

    Mohammedsaeed, Walaa; Cruickshank, Sheena; McBain, Andrew J; O'Neill, Catherine A

    2015-11-05

    A limited number of studies have investigated the potential of probiotics to promote wound healing in the digestive tract. The aim of the current investigation was to determine whether probiotic bacteria or their extracts could be beneficial in cutaneous wound healing. A keratinocyte monolayer scratch assay was used to assess re-epithelialization; which comprises keratinocyte proliferation and migration. Primary human keratinocyte monolayers were scratched then exposed to lysates of Lactobacillus (L) rhamnosus GG, L. reuteri, L. plantarum or L. fermentum. Re-epithelialization of treated monolayers was compared to that of untreated controls. Lysates of L. rhamnosus GG and L. reuteri significantly increased the rate of re-epithelialization, with L. rhamnosus GG being the most efficacious. L. reuteri increased keratinocyte proliferation while L. rhamnosus GG lysate significantly increased proliferation and migration. Microarray analysis of L. rhamnosus GG treated scratches showed increased expression of multiple genes including the chemokine CXCL2 and its receptor CXCR2. These are involved in normal wound healing where they stimulate keratinocyte proliferation and/or migration. Increased protein expression of both CXCL2 and CXCR2 were confirmed by ELISA and immunoblotting. These data demonstrate that L. rhamnosus GG lysate accelerates re-epithelialization of keratinocyte scratch assays, potentially via chemokine receptor pairs that induce keratinocyte migration.

  10. Clasp2 ensures mitotic fidelity and prevents differentiation of epidermal keratinocytes

    PubMed Central

    Shahbazi, Marta N.; Peña-Jimenez, Daniel; Antonucci, Francesca; Drosten, Matthias

    2017-01-01

    ABSTRACT Epidermal homeostasis is tightly controlled by a balancing act of self-renewal or terminal differentiation of proliferating basal keratinocytes. An increase in DNA content as a consequence of a mitotic block is a recognized mechanism underlying keratinocyte differentiation, but the molecular mechanisms involved in this process are not yet fully understood. Using cultured primary keratinocytes, here we report that the expression of the mammalian microtubule and kinetochore-associated protein Clasp2 is intimately associated with the basal proliferative makeup of keratinocytes, and its deficiency leads to premature differentiation. Clasp2-deficient keratinocytes exhibit increased centrosomal numbers and numerous mitotic alterations, including multipolar spindles and chromosomal misalignments that overall result in mitotic stress and a high DNA content. Such mitotic block prompts premature keratinocyte differentiation in a p53-dependent manner in the absence of cell death. Our findings reveal a new role for Clasp2 in governing keratinocyte undifferentiated features and highlight the presence of surveillance mechanisms that prevent cell cycle entry in cells that have alterations in the DNA content. PMID:28069833

  11. Actomyosin contractility provokes contact inhibition in E-cadherin-ligated keratinocytes.

    PubMed

    Hirata, Hiroaki; Samsonov, Mikhail; Sokabe, Masahiro

    2017-04-13

    Confluence-dependent inhibition of epithelial cell proliferation, termed contact inhibition, is crucial for epithelial homeostasis and organ size control. Here we report that among epithelial cells, keratinocytes, which compose the stratified epithelium in the skin, possess a unique, actomyosin-dependent mechanism for contact inhibition. We have observed that under actomyosin-inhibited conditions, cell-cell contact itself through E-cadherin promotes proliferation of keratinocytes. Actomyosin activity in confluent keratinocytes, however, inhibits nuclear localization of β-catenin and YAP, and causes attenuation of β-catenin- and YAP-driven cell proliferation. Confluent keratinocytes develop E-cadherin-mediated punctate adhesion complexes, to which radial actin cables are connected. Eliminating the actin-to-E-cadherin linkage by depleting α-catenin increases proliferation of confluent keratinocytes. By contrast, enforced activation of RhoA-regulated actomyosin or external application of pulling force to ligated E-cadherin attenuates their proliferation, suggesting that tensile stress at E-cadherin-mediated adhesion complexes inhibits proliferation of confluent keratinocytes. Our results highlight actomyosin contractility as a crucial factor that provokes confluence-dependent inhibition of keratinocyte proliferation.

  12. Keratinocyte differentiation is regulated by the Rho and ROCK signaling pathway.

    PubMed

    McMullan, Rachel; Lax, Siân; Robertson, Vicki H; Radford, David J; Broad, Simon; Watt, Fiona M; Rowles, Alison; Croft, Daniel R; Olson, Michael F; Hotchin, Neil A

    2003-12-16

    The epidermis comprises multiple layers of specialized epithelial cells called keratinocytes. As cells are lost from the outermost epidermal layers, they are replaced through terminal differentiation, in which keratinocytes of the basal layer cease proliferating, migrate upwards, and eventually reach the outermost cornified layers. Normal homeostasis of the epidermis requires that the balance between proliferation and differentiation be tightly regulated. The GTP binding protein RhoA plays a fundamental role in the regulation of the actin cytoskeleton and in the adhesion events that are critically important to normal tissue homeostasis. Two central mediators of the signals from RhoA are the ROCK serine/threonine kinases ROCK-I and ROCK-II. We have analyzed ROCK's role in the regulation of epidermal keratinocyte function by using a pharmacological inhibitor and expressing conditionally active or inactive forms of ROCK-II in primary human keratinocytes. We report that blocking ROCK function results in inhibition of keratinocyte terminal differentiation and an increase in cell proliferation. In contrast, activation of ROCK-II in keratinocytes results in cell cycle arrest and an increase in the expression of a number of genes associated with terminal differentiation. Thus, these results indicate that ROCK plays a critical role in regulating the balance between proliferation and differentiation in human keratinocytes.

  13. HuCOP1 contributes to the regulation of DNA repair in keratinocytes.

    PubMed

    Fazekas, B; Carty, M P; Németh, I; Kemény, L; Széll, M; Ádám, É

    2017-03-01

    We have previously demonstrated that the E3 ligase Human Constitutive Photomorphogenic Protein (huCOP1) is expressed in human keratinocytes and negatively regulates p53. The MutS homolog 2 (MSH2) protein plays a central role in DNA MMR mechanism and is implicated in the cellular response to anticancer agents, such as cisplatin. Our aim was to clarify whether huCOP1 plays a role in DNA MMR by affecting MSH2 protein level in human keratinocytes. To define the role of huCOP1 in DNA mismatch repair, we determined whether huCOP1 affects MSH2 abundance. MSH2 protein level was detected by immunocytochemical staining using a keratinocyte cell line in which the expression level of huCOP1 was stably decreased (siCOP1). To investigate whether huCOP1 silencing influences cisplatin-induced cell death, control and siCOP1 keratinocyte cells were treated with increasing concentrations of cisplatin and cell viability was recorded after 48 and 96 h. Stable silencing of huCOP1 in human keratinocytes resulted in a reduced level of MSH2 protein. huCOP1 silencing also sensitized keratinocytes to the interstrand crosslinking inducer cisplatin. Our results indicate that decreased huCOP1 correlates with lower MSH2 levels. These protein level changes lead to increased sensitivity toward cisplatin treatment, implicating that huCOP1 plays a positive role in maintaining genome integrity in human keratinocytes.

  14. Fir honeydew honey flavonoids inhibit TNF-α-induced MMP-9 expression in human keratinocytes: a new action of honey in wound healing.

    PubMed

    Majtan, Juraj; Bohova, Jana; Garcia-Villalba, Rocio; Tomas-Barberan, Francisco A; Madakova, Zuzana; Majtan, Tomas; Majtan, Viktor; Klaudiny, Jaroslav

    2013-09-01

    Matrix metalloproteinase-9 (MMP-9) appears to be a major protease responsible for the degradation of matrix and growth-promoting agents in chronic wounds. Honey has been successfully used for treating non-healing wounds associated with infections. However, the mechanisms of its action at the cellular level have remained poorly understood. The aim of this study was to investigate the effect of fir honeydew honey on TNF-α-induced MMP-9 expression and secretion from human keratinocytes (HaCaT) and to identify the honey component(s) responsible for a discovered effect. A C18 solid-phase column was used for preparation of honey aqueous extract (HAE). Expression and production of MMP-9 by HaCaT cells were determined by reverse transcription-PCR, gelatine zymography and Western blot analysis using a polyclonal antibody against MMP-9. We found that HAE inhibited TNF-α-induced production of MMP-9 in keratinocytes in a dose-dependent manner at both the mRNA and protein levels. Apigenin and kaempferol, identified flavonoids in HAE, markedly inhibited MMP-9 production from HaCaT and epidermal keratinocytes. Taken together, fir honeydew honey, which contains certain flavonoids, prevents TNF-α-induced proteolytic activity in cutaneous inflammation. Thus, our findings provide clear evidence that honey may serve as a natural treatment for dermatological problems associated with a persistent inflammation.

  15. Differential effects of detergents on keratinocyte gene expression.

    PubMed

    van Ruissen, F; Le, M; Carroll, J M; van der Valk, P G; Schalkwijk, J

    1998-04-01

    We have studied the effect of various detergents on keratinocyte gene expression in vitro, using an anionic detergent (sodium dodecyl sulfate), a cationic detergent cetyltrimethylammoniumbromide (CTAB), and two nonionic detergents, Nonidet P-40 and Tween-20. We measured the effect of these detergents on direct cellular toxicity (lactate dehydrogenase release), on the expression of markers for normal differentiation (cytokeratin 1 and involucrin expression), and on disturbed keratinocyte differentiation (SKALP) by northern blot analysis. As reported in other studies, large differences were noted in direct cellular toxicity. In a culture model that mimics normal epidermal differentiation we found that low concentrations of sodium dodecyl sulfate could induce the expression of SKALP, a proteinase inhibitor that is not normally expressed in human epidermis but is found in hyperproliferative skin. Sodium dodecyl sulfate caused upregulation of involucrin and downregulation of cytokeratin 1 expression, which is associated with the hyperproliferative/inflammatory epidermal phenotype found in psoriasis, wound healing, and skin irritation. These changes were not induced after treatment of cultures with CTAB, Triton X-100, and Nonidet-P40. This effect appeared to be specific for the class of anionic detergents because sodium dodecyl benzene sulfonate and sodium laurate also induced SKALP expression. These in vitro findings showed only a partial correlation with the potential of different detergents to induce clinical, biophysical, and cell biologic changes in vivo in human skin. Both sodium dodecyl sulfate and CTAB were found to cause induction and upregulation of SKALP and involucrin at low doses following a 24 h patch test, whereas high concentrations of Triton X-100 did not. Sodium dodecyl sulfate induced higher rates of transepidermal water loss, whereas CTAB treated skin showed more signs of cellular toxicity. We conclude that the action of anionic detergents on

  16. Bioinformatics approach for choosing the correct reference genes when studying gene expression in human keratinocytes.

    PubMed

    Beer, Lucian; Mlitz, Veronika; Gschwandtner, Maria; Berger, Tanja; Narzt, Marie-Sophie; Gruber, Florian; Brunner, Patrick M; Tschachler, Erwin; Mildner, Michael

    2015-10-01

    Reverse transcription polymerase chain reaction (qRT-PCR) has become a mainstay in many areas of skin research. To enable quantitative analysis, it is necessary to analyse expression of reference genes (RGs) for normalization of target gene expression. The selection of reliable RGs therefore has an important impact on the experimental outcome. In this study, we aimed to identify and validate the best suited RGs for qRT-PCR in human primary keratinocytes (KCs) over a broad range of experimental conditions using the novel bioinformatics tool 'RefGenes', which is based on a manually curated database of published microarray data. Expression of 6 RGs identified by RefGenes software and 12 commonly used RGs were validated by qRT-PCR. We assessed whether these 18 markers fulfilled the requirements for a valid RG by the comprehensive ranking of four bioinformatics tools and the coefficient of variation (CV). In an overall ranking, we found GUSB to be the most stably expressed RG, whereas the expression values of the commonly used RGs, GAPDH and B2M were significantly affected by varying experimental conditions. Our results identify RefGenes as a powerful tool for the identification of valid RGs and suggest GUSB as the most reliable RG for KCs.

  17. Two Mechanisms Regulate Keratin K15 Expression In Keratinocytes: Role of PKC/AP-1 and FOXM1 Mediated Signalling

    PubMed Central

    Bose, Amrita; Teh, Muy-Teck; Hutchison, Iain L.; Wan, Hong; Leigh, Irene M.; Waseem, Ahmad

    2012-01-01

    Background Keratin 15 (K15) is a type I keratin that is used as a marker of stem cells. Its expression is restricted to the basal layer of stratified epithelia, and the bulge in hair follicles. However, in certain clinical situations including oral lichen planus, K15 is induced in suprabasal layers, which is inconsistent with the role of a stem cell marker. This study provides insights into the mechanisms of K15 expression in the basal and differentiating keratinocytes. Methodology/Principal Findings Human keratinocytes were differentiated by three different methods; suspension in methylcellulose, high cell density and treatment with phorbol ester. The expression of mRNA was determined by quantitative PCR and protein by western blotting and immunostaining. Keratinocytes in suspension suppressed β1-integrin expression, induced differentiation-specific markers and K15, whereas FOXM1 (a cell cycle regulated protein) and K14 were downregulated. Rescuing β1-integrin by either fibronectin or the arginine-glycine-aspartate peptide suppressed K15 but induced K14 and FOXM1 expression. Specific inhibition of PKCδ, by siRNA, and AP-1 transcription factor, by TAM67 (dominant negative c-Jun), suppressed K15 expression, suggesting that PKC/AP-1 pathway plays a role in the differentiation-specific expression of K15. The basal cell-specific K15 expression may involve FOXM1 because ectopic expression of the latter is known to induce K15. Using chromatin immunoprecipitation, we have identified a single FOXM1 binding motif in the K15 promoter. Conclusions/Significance The data suggests that K15 is induced during terminal differentiation mediated by the down regulation of β1-integrin. However, this cannot be the mechanism of basal/stem cell-specific K15 expression in stratified epithelia, because basal keratinocytes do not undergo terminal differentiation. We propose that there are two mechanisms regulating K15 expression in stratified epithelia; differentiation-specific involving

  18. T-plastin expression downstream to the calcineurin/NFAT pathway is involved in keratinocyte migration.

    PubMed

    Brun, Cécilia; Demeaux, Agathe; Guaddachi, Frédéric; Jean-Louis, Francette; Oddos, Thierry; Bagot, Martine; Bensussan, Armand; Jauliac, Sébastien; Michel, Laurence

    2014-01-01

    Cutaneous wound healing requires keratinocyte proliferation, migration and differentiation to restore the barrier function of the skin. The calcineurin/nuclear factor of activated-T-cell (NFAT) signaling pathway has been recently shown to be involved in keratinocyte growth, differentiation and migration. It is induced by an increased intracellular calcium rate and its inhibition results in decreased capacities of keratinocytes to migrate. Nevertheless, the link between calcineurin activation and keratinocyte migration remains unknown. Recently, Orai1, a pore subunit of a store-operated calcium channel that favors calcium influx, was shown to play a critical role to control proliferation and migration of basal keratinocytes. Of interest, the actin-bundling T-plastin is crucial in cell motility through cross-linking to actin filament and its synthesis was shown to be induced by calcium influx and regulated by the calcineurin/NFAT pathway in tumor Sezary cells. We investigated herein the role of the calcineurin/NFAT pathway-dependent T-plastin in keratinocyte migration, by quantifying T-plastin expression in keratinocytes and by analyzing their migration under calcineurin inhibition or knockdown of NFAT2 or T-plastin. We did confirm the role of the calcineurin/NFAT pathway in keratinocyte migration as shown by their decreased capacities to migrate after FK506 treatment or siNFAT2 transfection in both scratching and Boyden assays. The expression of NFAT2 and T-plastin in keratinocytes was decreased under FK506 treatment, suggesting that T-plastin plays a role in keratinocyte migration downstream to the calcineurin/NFAT pathway. Accordingly, siRNA knockdown of T-plastin expression also decreased their migration capacities. Actin lamellipodia formation as well as FAK and β6-integrin expression were also significantly decreased after treatment with FK506 or siRNA, reinforcing that NFAT2-dependent T-plastin expression plays a role in keratinocyte migration. These results

  19. [Current Topics on Vitamin D. The vitamin D functions in keratinocytes and its therapeutic applications].

    PubMed

    Sawatsubashi, Shun

    2015-03-01

    1,25 (OH) 2D and calcium have been shown to promote epidermal keratinocyte differentiation and prevent proliferation. These prodifferentiation and antiproliferative effects of 1,25 (OH) 2D have led to its clinical use in the treatment of psoriasis. However, the mechanism of vitamin D action on keratinocytes remains largely unknown. While the actions of calcium and the vitamin D receptor signaling pathways on epidermal keratinocyte differentiation are redundant, their effects on the hair follicle are not. In this review, we discuss how the vitamin D and its receptor contribute to skin and hair follicle homeostasis.

  20. Normal human oral keratinocytes demonstrate abnormal DNA end joining activity during replicative senescence.

    PubMed

    Kang, Mo K; Shin, Ki-Hyuk; Yip, Felix K; Park, No-Hee

    2005-04-01

    Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of cellular genetic integrity. DSBs are repaired by cellular end joining activity, which could proceed with varying degrees of accuracy. Abnormal end joining may lead to an accumulation of mutations and contribute to genetic instability and cellular aging. In the present study, we compared the efficiency and accuracy of end joining activities in exponentially replicating and senescing normal human oral keratinocytes (NHOK). We developed an in vitro end joining assay utilizing a plasmid linearized with a unique EcoR I or EcoR V restriction site. The efficiency of end joining was determined by PCR with primers that could amplify the fragment containing the end joining site. The accuracy of end joining was assessed by determining whether the original EcoR I site was restored after end joining. Both replicating and senescing cultures of NHOK yielded a similar level of end joining efficiency, which was noted by the similar intensity of PCR amplification. However, the frequency of end joining errors was significantly elevated in NHOK during replicative senescence. Senescing NHOK could thus accumulate abnormal end joining products, which might contribute to cellular aging and cancer.

  1. Wound healing in the alpha2beta1 integrin-deficient mouse: altered keratinocyte biology and dysregulated matrix metalloproteinase expression.

    PubMed

    Grenache, David G; Zhang, Zhonghua; Wells, Laura E; Santoro, Samuel A; Davidson, Jeffrey M; Zutter, Mary M

    2007-02-01

    The alpha2beta1 integrin, a collagen/laminin receptor, is expressed at high level in the basal cell layer of the epidermis. To define the role of the alpha2beta1 integrin in wound healing, wound repair was extensively evaluated in wild-type and alpha2-null mice in vivo. In addition, the impact of alpha2beta1 integrin-deficiency on the function of primary murine keratinocytes in vitro was analyzed. Our in vivo findings demonstrate that genetic deletion of the alpha2beta1 integrin does not significantly alter the rate of re-epithelialization, collagen deposition, or tensile strength during wound closure in mice. In marked contrast to the observed similarities in wound healing, deletion of the alpha2beta1 integrin resulted in a dramatic increase in neoangiogenesis in the wound microenvironment. In contrast to in vivo studies, primary keratinocytes from alpha2-null mice adhered poorly and displayed impaired migration on type I collagen in vitro. We demonstrate that alpha2beta1 integrin-ligation negatively regulates expression of genes including matrix metalloproteinases both in vivo and in vitro. Furthermore, the changes in gene expression could potentially account for relatively normal wound healing in the alpha2-deficient mouse and our recent observation that suggests an antiangiogenic role for the alpha2beta1 integrin in vivo.

  2. Keratinocyte growth factor induces pancreatic ductal epithelial proliferation.

    PubMed

    Yi, E S; Yin, S; Harclerode, D L; Bedoya, A; Bikhazi, N B; Housley, R M; Aukerman, S L; Morris, C F; Pierce, G F; Ulich, T R

    1994-07-01

    Keratinocyte growth factor (KGF) causes a proliferation of pancreatic ductal epithelial cells in adult rats after daily systemic administration for 1 to 2 weeks. Even before the proliferation of intralobular ducts is histologically evident, KGF also induces proliferating cell nuclear antigen expression within the ductal epithelium of intercalated, intralobular, and interlobular ducts. KGF also causes incorporation of 5-bromodeoxyuridine in ductal epithelial cells. Epithelial cell proliferation is histologically most prominent at the level of the intralobular ducts adjacent to and within the islets of Langerhans. Pancreatic ductal proliferation is not histologically apparent in rats sacrificed 7 to 10 days after the cessation of KGF administration. The pancreatic hormones insulin, glucagon, somatostatin, and pancreatic polypeptide are normally distributed within islets that demonstrate intrainsular ductal proliferation. The proliferating ductal epithelium does not show endocrine differentiation as evidenced by the lack of immunoreactivity for pancreatic hormones. KGF is a potent in vivo mitogen for pancreatic ductal epithelial cells.

  3. Effects of ozone in normal human epidermal keratinocytes.

    PubMed

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

    2013-05-01

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

  4. Adducin Is Required for Desmosomal Cohesion in Keratinocytes*

    PubMed Central

    Rötzer, Vera; Breit, Andreas; Waschke, Jens; Spindler, Volker

    2014-01-01

    Adducin is a protein organizing the cortical actin cytoskeleton and a target of RhoA and PKC signaling. However, the role for intercellular cohesion is unknown. We found that adducin silencing induced disruption of the actin cytoskeleton, reduced intercellular adhesion of human keratinocytes, and decreased the levels of the desmosomal adhesion molecule desmoglein (Dsg)3 by reducing its membrane incorporation. Because loss of cell cohesion and Dsg3 depletion is observed in the autoantibody-mediated blistering skin disease pemphigus vulgaris (PV), we applied antibody fractions of PV patients. A rapid phosphorylation of adducin at serine 726 was detected in response to these autoantibodies. To mechanistically link autoantibody binding and adducin phosphorylation, we evaluated the role of several disease-relevant signaling molecules. Adducin phosphorylation at serine 726 was dependent on Ca2+ influx and PKC but occurred independent of p38 MAPK and PKA. Adducin phosphorylation is protective, because phosphorylation-deficient mutants resulted in loss of cell cohesion and Dsg3 fragmentation. Thus, PKC elicits both positive and negative effects on cell adhesion, since its contribution to cell dissociation in pemphigus is well established. We additionally evaluated the effect of RhoA on adducin phosphorylation because RhoA activation was shown to block pemphigus autoantibody-induced cell dissociation. Our data demonstrate that the protective effect of RhoA activation was dependent on the presence of adducin and its phosphorylation at serine 726. These experiments provide novel mechanisms for regulation of desmosomal adhesion by RhoA- and PKC-mediated adducin phosphorylation in keratinocytes. PMID:24711455

  5. Ratite oils promote keratinocyte cell growth and inhibit leukocyte activation.

    PubMed

    Bennett, Darin C; Leung, Gigi; Wang, Eddy; Ma, Sam; Lo, Blanche K K; McElwee, Kevin J; Cheng, Kimberly M

    2015-09-01

    Traditionally, native Australian aborigines have used emu oil for the treatment of inflammation and to accelerate wound healing. Studies on mice suggest that topically applied emu oil may have anti-inflammatory properties and may promote wound healing. We investigated the effects of ratite oils (6 emu, 3 ostrich, 1 rhea) on immortalized human keratinocytes (HaCaT cells) in vitro by culturing the cells in media with oil concentrations of 0%, 0.5%, and 1.0%. Peking duck, tea tree, and olive oils were used as comparative controls. The same oils at 0.5% concentration were evaluated for their influence on peripheral blood mononuclear cell (PBMC) survival over 48 hr and their ability to inhibit IFNγ production in PBMCs activated by phytohemagglutinin (PHA) in ELISpot assays. Compared to no oil control, significantly shorter population doubling time durations were observed for HaCaT cells cultured in emu oil (1.51×faster), ostrich oil (1.46×faster), and rhea oil (1.64×faster). Tea tree oil demonstrated significant antiproliferative activity and olive oil significantly prolonged (1.35×slower) cell population doubling time. In contrast, almost all oils, particularly tea tree oil, significantly reduced PBMC viability. Different oils had different levels of inhibitory effect on IFNγ production with individual emu, ostrich, rhea, and duck oil samples conferring full inhibition. This preliminary investigation suggests that emu oil might promote wound healing by accelerating the growth rate of keratinocytes. Combined with anti-inflammatory properties, ratite oil may serve as a useful component in bandages and ointments for the treatment of wounds and inflammatory skin conditions.

  6. Regulation of p53 during senescence in normal human keratinocytes

    PubMed Central

    Kim, Reuben H; Kang, Mo K; Kim, Terresa; Yang, Paul; Bae, Susan; Williams, Drake W; Phung, Samantha; Shin, Ki-Hyuk; Hong, Christine; Park, No-Hee

    2015-01-01

    p53, the guardian of the genome, is a tumor suppressor protein and critical for the genomic integrity of the cells. Many studies have shown that intracellular level of p53 is enhanced during replicative senescence in normal fibroblasts, and the enhanced level of p53 is viewed as the cause of senescence. Here, we report that, unlike in normal fibroblasts, the level of intracellular p53 reduces during replicative senescence and oncogene-induced senescence (OIS) in normal human keratinocytes (NHKs). We found that the intracellular p53 level was also decreased in age-dependent manner in normal human epithelial tissues. Senescent NHKs exhibited an enhanced level of p16INK4A, induced G2 cell cycle arrest, and lowered the p53 expression and transactivation activity. We found that low level of p53 in senescent NHKs was due to reduced transcription of p53. The methylation status at the p53 promoter was not altered during senescence, but senescent NHKs exhibited notably lower level of acetylated histone 3 (H3) at the p53 promoter in comparison with rapidly proliferating cells. Moreover, p53 knockdown in rapidly proliferating NHKs resulted in the disruption of fidelity in repaired DNA. Taken together, our study demonstrates that p53 level is diminished during replicative senescence and OIS and that such diminution is associated with H3 deacetylation at the p53 promoter. The reduced intracellular p53 level in keratinocytes of the elderly could be a contributing factor for more frequent development of epithelial cancer in the elderly because of the loss of genomic integrity of cells. PMID:26138448

  7. Adducin is required for desmosomal cohesion in keratinocytes.

    PubMed

    Rötzer, Vera; Breit, Andreas; Waschke, Jens; Spindler, Volker

    2014-05-23

    Adducin is a protein organizing the cortical actin cytoskeleton and a target of RhoA and PKC signaling. However, the role for intercellular cohesion is unknown. We found that adducin silencing induced disruption of the actin cytoskeleton, reduced intercellular adhesion of human keratinocytes, and decreased the levels of the desmosomal adhesion molecule desmoglein (Dsg)3 by reducing its membrane incorporation. Because loss of cell cohesion and Dsg3 depletion is observed in the autoantibody-mediated blistering skin disease pemphigus vulgaris (PV), we applied antibody fractions of PV patients. A rapid phosphorylation of adducin at serine 726 was detected in response to these autoantibodies. To mechanistically link autoantibody binding and adducin phosphorylation, we evaluated the role of several disease-relevant signaling molecules. Adducin phosphorylation at serine 726 was dependent on Ca(2+) influx and PKC but occurred independent of p38 MAPK and PKA. Adducin phosphorylation is protective, because phosphorylation-deficient mutants resulted in loss of cell cohesion and Dsg3 fragmentation. Thus, PKC elicits both positive and negative effects on cell adhesion, since its contribution to cell dissociation in pemphigus is well established. We additionally evaluated the effect of RhoA on adducin phosphorylation because RhoA activation was shown to block pemphigus autoantibody-induced cell dissociation. Our data demonstrate that the protective effect of RhoA activation was dependent on the presence of adducin and its phosphorylation at serine 726. These experiments provide novel mechanisms for regulation of desmosomal adhesion by RhoA- and PKC-mediated adducin phosphorylation in keratinocytes.

  8. Ratite oils promote keratinocyte cell growth and inhibit leukocyte activation

    PubMed Central

    Bennett, Darin C.; Leung, Gigi; Wang, Eddy; Ma, Sam; Lo, Blanche K. K.; McElwee, Kevin J.; Cheng, Kimberly M.

    2015-01-01

    Traditionally, native Australian aborigines have used emu oil for the treatment of inflammation and to accelerate wound healing. Studies on mice suggest that topically applied emu oil may have anti-inflammatory properties and may promote wound healing. We investigated the effects of ratite oils (6 emu, 3 ostrich, 1 rhea) on immortalized human keratinocytes (HaCaT cells) in vitro by culturing the cells in media with oil concentrations of 0%, 0.5%, and 1.0%. Peking duck, tea tree, and olive oils were used as comparative controls. The same oils at 0.5% concentration were evaluated for their influence on peripheral blood mononuclear cell (PBMC) survival over 48 hr and their ability to inhibit IFNγ production in PBMCs activated by phytohemagglutinin (PHA) in ELISpot assays. Compared to no oil control, significantly shorter population doubling time durations were observed for HaCaT cells cultured in emu oil (1.51 × faster), ostrich oil (1.46 × faster), and rhea oil (1.64 × faster). Tea tree oil demonstrated significant antiproliferative activity and olive oil significantly prolonged (1.35 × slower) cell population doubling time. In contrast, almost all oils, particularly tea tree oil, significantly reduced PBMC viability. Different oils had different levels of inhibitory effect on IFNγ production with individual emu, ostrich, rhea, and duck oil samples conferring full inhibition. This preliminary investigation suggests that emu oil might promote wound healing by accelerating the growth rate of keratinocytes. Combined with anti-inflammatory properties, ratite oil may serve as a useful component in bandages and ointments for the treatment of wounds and inflammatory skin conditions. PMID:26217022

  9. Fibronectin and alpha5 integrin regulate keratinocyte cell cycling. A mechanism for increased fibronectin potentiation of T cell lymphokine-driven keratinocyte hyperproliferation in psoriasis.

    PubMed Central

    Bata-Csorgo, Z; Cooper, K D; Ting, K M; Voorhees, J J; Hammerberg, C

    1998-01-01

    In addition to being T lymphocyte-driven, psoriasis may be due in part to abnormal integrin expression. Normal-appearing (uninvolved) skin from psoriatic patients was examined to determine whether altered fibronectin or its receptor expression is detectable before development of psoriatic lesions. In contrast to skin from normal subjects, we detect by immunofluorescence the abnormal presence of plasma fibronectin in the basal cell layer of the epidermis of psoriatic uninvolved skin. Furthermore, increased fibronectin exposure superinduces the in vitro cell cycle induction and expansion of psoriatic nonlesional keratinocytes in response to a cocktail of T cell lymphokines. Fibronectin alone also appeared to increase cell cycle entry among uninvolved but not normal keratinocytes. Concordantly, the alpha5 integrin fibronectin receptor, but not alpha2 or alpha3, is overexpressed in the in vivo nonlesional psoriatic epidermis. The involvement of alpha5beta1 in the early outgrowth of clonogenic keratinocytes in the ex vivo culture was demonstrated by the ability of anti-alpha5 mAb to inhibit keratinocyte growth on fibronectin. Thus, the fibronectin receptor appears to be one of the components required for the development of the hyperresponsiveness of psoriatic keratinocytes to signals for proliferation provided by lymphokines produced by intralesional T lymphocytes in psoriasis. PMID:9525994

  10. Impact of Hyperglycemia and Low Oxygen Tension on Adipose-Derived Stem Cells Compared with Dermal Fibroblasts and Keratinocytes: Importance for Wound Healing in Type 2 Diabetes

    PubMed Central

    Lafosse, Aurore; Dufeys, Cécile; Beauloye, Christophe; Horman, Sandrine; Dufrane, Denis

    2016-01-01

    Aim Adipose-derived stem cells (ASC) are currently proposed for wound healing in those with type 2 diabetes mellitus (T2DM). Therefore, this study investigated the impact of diabetes on adipose tissue in relation to ASC isolation, proliferation, and growth factor release and the impact of hyperglycemia and low oxygen tension (found in diabetic wounds) on dermal fibroblasts, keratinocytes, and ASC in vitro. Methods Different sequences of hypoxia and hyperglycemia were applied in vitro to ASC from nondiabetic (n = 8) or T2DM patients (n = 4) to study cell survival, proliferation, and growth factor release. Comparisons of dermal fibroblasts (n = 8) and keratinocytes (primary lineage) were made. Results No significant difference of isolation and proliferation capacities was found in ASC from nondiabetic and diabetic humans. Hypoxia and hyperglycemia did not impact cell viability and proliferation. Keratinocyte Growth Factor release was significantly lower in diabetic ASC than in nondiabetic ASC group in each condition, while Vascular Endothelial Growth Factor release was not affected by the diabetic origin. Nondiabetic ASC exposition to hypoxia (0.1% oxygen) combined with hyperglycemia (25mM glucose), resulted in a significant increase in VEGF secretion (+64%, p<0.05) with no deleterious impact on KGF release in comparison to physiological conditions (5% oxygen and 5 mM glucose). Stromal cell-Derived Factor-1α (-93%, p<0.001) and KGF (-20%, p<0.05) secretion by DF decreased in these conditions. Conclusions A better profile of growth factor secretion (regarding wound healing) was found in vitro for ASC in hyperglycemia coupled with hypoxia in comparison to dermal fibroblasts and keratinocytes. Interestingly, ASC from T2DM donors demonstrated cellular growth rates and survival (in hypoxia and hyperglycemic conditions) similar to those of healthy ASC (from normoglycemic donors); however, KGF secretion was significantly depleted in ASC obtained from T2DM patients. This

  11. The extracellular adherence protein (Eap) of Staphylococcus aureus acts as a proliferation and migration repressing factor that alters the cell morphology of keratinocytes.

    PubMed

    Eisenbeis, Janina; Peisker, Henrik; Backes, Christian S; Bur, Stephanie; Hölters, Sebastian; Thewes, Nicolas; Greiner, Markus; Junker, Christian; Schwarz, Eva C; Hoth, Markus; Junker, Kerstin; Preissner, Klaus T; Jacobs, Karin; Herrmann, Mathias; Bischoff, Markus

    2017-02-01

    Staphyloccocus aureus is a major human pathogen and a common cause for superficial and deep seated wound infections. The pathogen is equipped with a large arsenal of virulence factors, which facilitate attachment to various eukaryotic cell structures and modulate the host immune response. One of these factors is the extracellular adherence protein Eap, a member of the "secretable expanded repertoire adhesive molecules" (SERAM) protein family that possesses adhesive and immune modulatory properties. The secreted protein was previously shown to impair wound healing by interfering with host defense and neovascularization. However, its impact on keratinocyte proliferation and migration, two major steps in the re-epithelialization process of wounds, is not known. Here, we report that Eap affects the proliferation and migration capacities of keratinocytes by altering their morphology and adhesive properties. In particular, treatment of non-confluent HaCaT cell cultures with Eap resulted in cell morphology changes as well as a significant reduction in cell proliferation and migration. Eap-treated HaCaT cells changed their appearance from an oblong via a trapezoid to an astral-like shape, accompanied by decreases in cell volume and cell stiffness, and exhibited significantly increased cell adhesion. Eap had a similar influence on endothelial and cancer cells, indicative for a general effect of Eap on eukaryotic cell morphology and functions. Specifically, Eap was found to interfere with growth factor-stimulated activation of the mitogen-activated protein kinase (MAPK) pathway that is known to be responsible for cell shape modulation, induction of proliferation and migration of epithelial cells. Western blot analyses revealed that Eap blocked the phosphorylation of extracellular signal-regulated kinase 1 and 2 (Erk1/2) in keratinocyte growth factor (KGF)-stimulated HaCaT cells. Together, these data add another antagonistic mechanism of Eap in wound healing, whereby the

  12. Biosimilar Insulins: How Similar is Similar?

    PubMed Central

    Heinemann, Lutz; Hompesch, Marcus

    2011-01-01

    Biosimilar insulins (BIs) are viewed as commercially attractive products by a number of companies. In order to obtain approval in the European Union or the United States, where there is not a single BI currently on the market, a manufacturer needs to demonstrate that a given BI has a safety and efficacy profile that is similar to that of the “original” insulin formulation that is already on the market. As trivial as this may appear at first glance, it is not trivial at all for a good number of reasons that will be discussed in this commentary. As with protein manufacturing, modifications in the structure of the insulin molecule can take place (which can have serious consequences for the biological effects induced), so a rigid and careful assessment is absolutely necessary. The example of Marvel's failed application with the European Medicines Agency provides insights into the regulatory and clinical challenges surrounding the matter of BI. Although a challenging BI approval process might be regarded as a hurdle to keep companies out of certain markets, it is fair to say that the potential safety and efficacy issues surrounding BI are substantial and relevant and do warrant a careful and evidence-driven approval process. PMID:21722590

  13. Curcumin Protects Human Keratinocytes against Inorganic Arsenite-Induced Acute Cytotoxicity through an NRF2-Dependent Mechanism

    PubMed Central

    Zhao, Rui; Yang, Bei; Wang, Linlin; Xue, Peng; Deng, Baocheng; Zhang, Guohua; Jiang, Shukun; Zhang, Miao; Liu, Min; Pi, Jingbo; Guan, Dawei

    2013-01-01

    Human exposure to inorganic arsenic leads to various dermal disorders, including hyperkeratosis and skin cancer. Curcumin is demonstrated to induce remarkable antioxidant activity in a variety of cells and tissues. The present study aimed at identifying curcumin as a potent activator of nuclear factor erythroid 2-related factor 2 (NRF2) and demonstrating its protective effect against inorganic arsenite- (iAs3+-) induced cytotoxicity in human keratinocytes. We found that curcumin led to nuclear accumulation of NRF2 protein and increased the expression of antioxidant response element- (ARE-) regulated genes in HaCaT keratinocytes in concentration- and time-dependent manners. High concentration of curcumin (20 μM) also increased protein expression of long isoforms of NRF1. Treatment with low concentrations of curcumin (2.5 or 5 μM) effectively increased the viability and survival of HaCaT cells against iAs3+-induced cytotoxicity as assessed by the MTT assay and flow cytometry and also attenuated iAs3+-induced expression of cleaved caspase-3 and cleaved PARP protein. Selective knockdown of NRF2 or KEAP1 by lentiviral shRNAs significantly diminished the cytoprotection conferred by curcumin, suggesting that the protection against iAs3+-induced cytotoxicity is dependent on the activation of NRF2. Our results provided a proof of the concept of using curcumin to activate the NRF2 pathway to alleviate arsenic-induced dermal damage. PMID:23710286

  14. Nerve signaling regulates basal keratinocyte proliferation in the blastema apical epithelial cap in the axolotl (Ambystoma mexicanum).

    PubMed

    Satoh, Akira; Bryant, Susan V; Gardiner, David M

    2012-06-15

    The ability of adult vertebrates to repair tissue damage is widespread and impressive; however, the ability to regenerate structurally complex organs such as the limb is limited largely to the salamanders. The fact that most of the tissues of the limb can regenerate has led investigators to question and identify the barriers to organ regeneration. From studies in the salamander, it is known that one of the earliest steps required for successful regeneration involves signaling between nerves and the wound epithelium/apical epithelial cap (AEC). In this study we confirm an earlier report that the keratinocytes of the AEC acquire their function coincident with exiting the cell cycle. We have discovered that this unique, coordinated behavior is regulated by nerve signaling and is associated with the presence of gap junctions between the basal keratinocytes of the AEC. Disruption of nerve signaling results in a loss of gap junction protein, the reentry of the cells into the cell cycle, and regenerative failure. Finally, coordinated exit from the cell cycle appears to be a conserved behavior of populations of cells that function as signaling centers during both development and regeneration.

  15. Phylogenetic profiling and gene expression studies implicate a primary role of PSORS1C2 in terminal differentiation of keratinocytes.

    PubMed

    Abbas Zadeh, Salman; Mlitz, Veronika; Lachner, Julia; Golabi, Bahar; Mildner, Michael; Pammer, Johannes; Tschachler, Erwin; Eckhart, Leopold

    2017-04-01

    PSORS1C2 is a gene located between coiled-coil alpha-helical rod protein 1 (CCHCR1) and corneodesmosin (CDSN) within the psoriasis susceptibility locus 1 (PSORS1). Here, we performed a comparative genomics analysis of the as-yet incompletely characterized PSORS1C2 gene and determined its expression pattern in human tissues. In contrast to CCHCR1, which is common to all vertebrates investigated, PSORS1C2 and CDSN are present exclusively in mammals, indicating that the latter genes have originated after the evolutionary divergence of mammals and reptiles. CDSN is conserved in aquatic mammals, whereas PSORS1C2 orthologs contain gene-inactivating frame shift mutations in whales and dolphins, in which the epidermal differentiation programme has degenerated. Reverse-transcription PCR screening demonstrated that, in human tissues, PSORS1C2 is expressed principally in the epidermis and weakly in the thymus. PSORS1C2 mRNA was strongly upregulated during terminal differentiation of human keratinocytes in vitro. Immunohistochemistry revealed exclusive expression of PSORS1C2 in the granular layer of the epidermis and in cornifying epithelial cells of Hassall's corpuscles of the thymus. In summary, our results identify PSORS1C2 as a keratinocyte cornification-associated protein that has originated in evolutionarily basal mammals and has undergone gene inactivation in association with the loss of the skin barrier function in aquatic mammals.

  16. Keratinocytes exposed to ultraviolet radiation reveal three down-regulated genes with potential function in differentiation and cell cycle control.

    PubMed

    Pötter, T; Göhde, W; Wedemeyer, N; Köhnlein, W

    2000-08-01

    The incidence of skin cancer is increasing in epidemic proportion. Although solar UV radiation is known to be the major risk factor, much information is lacking about the molecular mechanisms leading to skin cancer. To gain a deeper insight into these mechanisms, we have examined cells of a human keratinocyte cell line (HaCat) after exposure to 0.16 minimal erythema doses of UVB radiation. This dose led to an S-phase delay that was reversible 22 h postirradiation. To examine gene expression 10 h after UV irradiation, a nonradioactive differential display was employed. Three genes were identified as being down-regulated significantly. The first encodes for topoisomerase-IIbeta-binding protein 1 (expression level 5% 6 h after irradiation). This protein is associated with human topoisomerase IIbeta and appears to be necessary for DNA replication during the onset of S phase. The second gene product has previously been reported to be involved in differentiation and is therefore known as differentiation-dependent A4 protein (28% 8 h after irradiation). The third gene is XPO1 (also known as CRM1) (5% 8 h after irradiation), whose protein is involved in nuclear export of mRNA molecules. Differential expression of these genes after UV irradiation has not been reported. Because of their potential involvement in cell cycle control and differentiation, these proteins could be important for understanding the reaction of keratinocytes after exposure to UV radiation.

  17. MicroRNA 203 expression in keratinocytes is dependent on regulation of p53 levels by E6.

    PubMed

    McKenna, Declan J; McDade, Simon S; Patel, Daksha; McCance, Dennis J

    2010-10-01

    A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miRNA 203 (miR-203), which has previously been shown to play an important role in epithelial cell biology by regulating p63 levels. We investigated how expression of human papillomavirus type 16 (HPV16) oncoproteins E6 and E7 affected miR-203 expression during proliferation and differentiation of HFKs. We demonstrated that miR-203 expression is reduced in HFKs where p53 function is compromised, either by the viral oncoprotein E6 or by knockout of p53 using short hairpin RNAs (p53i). We show that the induction of miR-203 observed during calcium-induced differentiation of HFKs is significantly reduced in HFKs expressing E6 and in p53i HFKs. Induction of miR-203 in response to DNA damage is also reduced in the absence of p53. We report that proliferation of HFKs is dependent on the level of miR-203 expression and that overexpression of miR-203 can reduce overproliferation in E6/E7-expressing and p53i HFKs. In summary, these results indicate that expression of miR-203 is dependent on p53, which may explain how expression of HPV16 E6 can disrupt the balance between proliferation and differentiation, as well as the response to DNA damage, in keratinocytes.

  18. Molecular similarity measures.

    PubMed

    Maggiora, Gerald M; Shanmugasundaram, Veerabahu

    2011-01-01

    Molecular similarity is a pervasive concept in chemistry. It is essential to many aspects of chemical reasoning and analysis and is perhaps the fundamental assumption underlying medicinal chemistry. Dissimilarity, the complement of similarity, also plays a major role in a growing number of applications of molecular diversity in combinatorial chemistry, high-throughput screening, and related fields. How molecular information is represented, called the representation problem, is important to the type of molecular similarity analysis (MSA) that can be carried out in any given situation. In this work, four types of mathematical structure are used to represent molecular information: sets, graphs, vectors, and functions. Molecular similarity is a pairwise relationship that induces structure into sets of molecules, giving rise to the concept of chemical space. Although all three concepts - molecular similarity, molecular representation, and chemical space - are treated in this chapter, the emphasis is on molecular similarity measures. Similarity measures, also called similarity coefficients or indices, are functions that map pairs of compatible molecular representations that are of the same mathematical form into real numbers usually, but not always, lying on the unit interval. This chapter presents a somewhat pedagogical discussion of many types of molecular similarity measures, their strengths and limitations, and their relationship to one another. An expanded account of the material on chemical spaces presented in the first edition of this book is also provided. It includes a discussion of the topography of activity landscapes and the role that activity cliffs in these landscapes play in structure-activity studies.

  19. Molecular similarity measures.

    PubMed

    Maggiora, Gerald M; Shanmugasundaram, Veerabahu

    2004-01-01

    Molecular similarity is a pervasive concept in chemistry. It is essential to many aspects of chemical reasoning and analysis and is perhaps the fundamental assumption underlying medicinal chemistry. Dissimilarity, the complement of similarity, also plays a major role in a growing number of applications of molecular diversity in combinatorial chemistry, high-throughput screening, and related fields. How molecular information is represented, called the representation problem, is important to the type of molecular similarity analysis (MSA) that can be carried out in any given situation. In this work, four types of mathematical structure are used to represent molecular information: sets, graphs, vectors, and functions. Molecular similarity is a pairwise relationship that induces structure into sets of molecules, giving rise to the concept of a chemistry space. Although all three concepts molecular similarity, molecular representation, and chemistry space are treated in this chapter, the emphasis is on molecular similarity measures. Similarity measures, also called similarity coefficients or indices, are functions that map pairs of compatible molecular representations, that is, representations of the same mathematical form, into real numbers usually, but not always, lying on the unit interval. This chapter presents a somewhat pedagogical discussion of many types of molecular similarity measures, their strengths and limitations, and their relationship to one another.

  20. Biological effects of glycolic acid on dermal matrix metabolism mediated by dermal fibroblasts and epidermal keratinocytes.

    PubMed

    Okano, Yuri; Abe, Yumiko; Masaki, Hitoshi; Santhanam, Uma; Ichihashi, Masamitsu; Funasaka, Yoko

    2003-01-01

    Glycolic acid (GA), one of the alpha-hydroxy acids, is widely used as an agent for chemical peeling. Although there are several reports about the clinical effects of GA in the literature, its biological mechanism remains mostly unclear, and there are only a few reports about its effects on skin rejuvenation mediated by keratinocytes. The aim of this study was to investigate the effect of GA on the dermal matrix metabolism of keratinocytes and fibroblasts using in vitro and ex vivo systems. Our study shows that GA not only directly accelerates collagen synthesis by fibroblasts, but it also modulates matrix degradation and collagen synthesis through keratinocyte-released cytokines. We confirm that IL-1alpha is one of the primary mediators for matrix degradation released from keratinocytes after GA treatment. These results suggest that GA contributes to the recovery of photodamaged skin through various actions, depending on the skin cell type.

  1. Promoting effect of lactoferrin on barrier function and epithelial differentiation of human keratinocytes.

    PubMed

    Uchida, Ryo; Aoki, Reiji; Aoki-Yoshida, Ayako; Tajima, Atsushi; Takayama, Yoshiharu

    2017-02-01

    The purpose of this study was to elucidate the effects of bovine lactoferrin on keratinocyte differentiation and barrier function. Addition of bovine lactoferrin to differentiating HaCaT human keratinocytes led to increased transepithelial electrical resistance (TER), a marker of epithelial barrier function. This elevation was followed by upregulation of two differentiation markers, involucrin and filaggrin. The expression level of sterol regulatory element-binding protein-1 was also enhanced by bovine lactoferrin. The lactoferrin-induced upregulation of involucrin and filaggrin expression were confirmed in normal human epidermal keratinocytes (NHEK). Treatment with SB203580, a p38 mitogen-activated protein kinase (MAPK) α inhibitor, impaired the upregulation of involucrin and filaggrin expression in response to lactoferrin. The elevation of p38 MAPK phosphorylation was further enhanced by lactoferrin in the initial stage of differentiation of HaCaT keratinocytes. The findings suggest that bovine lactoferrin promotes epithelial differentiation by a p38-MAPK-dependent mechanism.

  2. Aloe-emodin inhibits proliferation of adult human keratinocytes in vitro.

    PubMed

    Popadic, Dusan; Savic, Emina; Ramic, Zorica; Djordjevic, Vladimir; Trajkovic, Vladimir; Medenica, Ljiljana; Popadic, Svetlana

    2012-01-01

    Aloe-emodin (AE) is a plant-derived hydroxyanthraquinone with several biological activities. It is present in a variety of skin-conditioning agents containing aloe extracts, but its influence on keratinocyte growth was not examined so far. We investigated the influence of AE on human keratinocyte proliferation and apoptosis in vitro. AE significantly inhibited proliferation of cultivated human keratinocytes at 5 μM concentration, as revealed by incorporation of radioactive thymidine. The antiproliferative effect of AE was accompanied with induction of apoptosis, but not necrosis, as demonstrated by flow cytometric analysis and lactate dehydrogenase release assay. Based on the half maximal inhibitory concentration values, we demonstrated that AE may impair proliferation of keratinocytes at concentrations far below the industry standards for commercial products containing aloe extracts. Therefore, further research of AE effects on the human skin and proper labeling of products are necessary for maximizing benefits from aloe extracts and to avoid undesired responses.

  3. Tyrosine phosphorylation is an early and specific event involved in primary keratinocyte differentiation.

    PubMed Central

    Filvaroff, E; Stern, D F; Dotto, G P

    1990-01-01

    Very little is known about early molecular events triggering epithelial cell differentiation. We have examined the possible role of tyrosine phosphorylation in this process, as observed in cultures of primary mouse keratinocytes after exposure to calcium or 12-O-tetradecanoylphorbol-13-acetate (TPA). Immunoblotting with phosphotyrosine-specific antibodies as well as direct phosphoamino acid analysis revealed that induction of tyrosine phosphorylation occurs as a very early and specific event in keratinocyte differentiation. Very little or no induction of tyrosine phosphorylation was observed in a keratinocyte cell line resistant to the differentiating effects of calcium. Treatment of cells with tyrosine kinase inhibitors prevented induction of tyrosine phosphorylation by calcium and TPA and interfered with the differentiative effects of these agents. These results suggest that specific activation of tyrosine kinase(s) may play an important regulatory role in keratinocyte differentiation. Images PMID:1689456

  4. Potassium channel openers stimulate DNA synthesis in mouse epidermal keratinocyte and whole hair follicle cultures.

    PubMed

    Harmon, C S; Lutz, D; Ducote, J

    1993-01-01

    We have conducted studies using primary mouse epidermal keratinocyte and whole hair follicle cultures to investigate the mechanism of the hypertrichotic activity of potassium channel openers. In a time course study, the extent of stimulation of epidermal keratinocyte DNA synthesis by minoxidil increased as the rate of DNA synthesis in control cultures declined. Minoxidil stimulation of DNA synthesis in 7-day cultures required prolonged (> 1 day) exposure to the agent. Pinacidil and diazoxide also stimulated DNA synthesis in mouse epidermal keratinocyte cultures. In addition, minoxidil, pinacidil, diazoxide, and cromakalim stimulated DNA synthesis in whole-organ cultures of mouse hair follicles. These results suggest that potassium channel openers retard the loss of proliferative activity of differentiating keratinocytes and support the hypothesis that these agents stimulate hair growth through a direct effect on hair follicles.

  5. Expression, topography, and function of integrin receptors are severely altered in keratinocytes from involved and uninvolved psoriatic skin.

    PubMed Central

    Pellegrini, G; De Luca, M; Orecchia, G; Balzac, F; Cremona, O; Savoia, P; Cancedda, R; Marchisio, P C

    1992-01-01

    Psoriasis is a hyperproliferative cutaneous disease of unknown etiology and etiopathogenesis. Alteration of keratinocyte adhesiveness to basal lamina has been proposed as the initial disturbance leading to poorly controlled proliferation. Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are segregated to discrete membrane domains. In this paper, the expression and function of integrins in psoriatic keratinocytes were examined, both in vivo and in vitro. We found that: (a) in psoriatic keratinocytes the integrin heterodimers alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4 have lost their polarized distribution on the plasma membrane; (b) the role of these integrins in mediating keratinocyte adhesion in vitro is altered; (c) psoriatic keratinocytes form focal contacts containing both beta 1 and beta 4 integrins. In normal adult keratinocytes the alpha 5 beta 1 fibronectin receptor is poorly expressed and diffusely distributed on the basal keratinocyte plasma membrane and is not organized in defined adhesive structures. In contrast, psoriatic keratinocytes show a clear fibronectin receptor staining in vivo, and organize alpha 5 beta 1 in typical focal contacts in vitro without any obvious increase of its expression and synthesis. These multiple alterations of integrins are also present in uninvolved keratinocytes from psoriatic patients, suggesting a key role for altered integrin-mediated adhesion in the pathogenesis of this disease. Images PMID:1534817

  6. Expression, topography, and function of integrin receptors are severely altered in keratinocytes from involved and uninvolved psoriatic skin.

    PubMed

    Pellegrini, G; De Luca, M; Orecchia, G; Balzac, F; Cremona, O; Savoia, P; Cancedda, R; Marchisio, P C

    1992-06-01

    Psoriasis is a hyperproliferative cutaneous disease of unknown etiology and etiopathogenesis. Alteration of keratinocyte adhesiveness to basal lamina has been proposed as the initial disturbance leading to poorly controlled proliferation. Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are segregated to discrete membrane domains. In this paper, the expression and function of integrins in psoriatic keratinocytes were examined, both in vivo and in vitro. We found that: (a) in psoriatic keratinocytes the integrin heterodimers alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4 have lost their polarized distribution on the plasma membrane; (b) the role of these integrins in mediating keratinocyte adhesion in vitro is altered; (c) psoriatic keratinocytes form focal contacts containing both beta 1 and beta 4 integrins. In normal adult keratinocytes the alpha 5 beta 1 fibronectin receptor is poorly expressed and diffusely distributed on the basal keratinocyte plasma membrane and is not organized in defined adhesive structures. In contrast, psoriatic keratinocytes show a clear fibronectin receptor staining in vivo, and organize alpha 5 beta 1 in typical focal contacts in vitro without any obvious increase of its expression and synthesis. These multiple alterations of integrins are also present in uninvolved keratinocytes from psoriatic patients, suggesting a key role for altered integrin-mediated adhesion in the pathogenesis of this disease.

  7. Transcriptional profiling defines the roles of ERK and p38 kinases in epidermal keratinocytes.

    PubMed

    Gazel, Alix; Nijhawan, Rajiv I; Walsh, Rebecca; Blumenberg, Miroslav

    2008-05-01

    Epidermal keratinocytes respond to extracellular influences by activating cytoplasmic signal transduction pathways that change gene expression. Using pathway-specific transcriptional profiling, we identified the genes regulated by two such pathways, p38 and ERK. These pathways are at the fulcrum of epidermal differentiation, proliferative and inflammatory skin diseases. We used SB203580 and PD98059 as specific inhibitors and Affymetrix Hu133Av2 microarrays, to identify the genes regulated after 1, 4, 24, and 48 h and compared them to genes regulated by JNK. Unexpectedly, inhibition of MAPK pathways is compensated by activation of the NFkappaB pathway and suppression of the DUSP enzymes. Both pathways promote epidermal differentiation; however, there is a surprising disconnect between the expression of steroid synthesis enzymes and differentiation markers. The p38 pathway induces the expression of extracellular matrix and proliferation-associated genes, while suppressing microtubule-associated genes. The ERK pathway induces nuclear envelope and mRNA splicing proteins, while suppressing steroid synthesis and mitochondrial energy production enzymes. Transcription factors SRY, c-FOS, and N-Myc are the principal targets of the p38 pathway, Elk-1 SAP1 and HLH2 of ERK, while FREAC-4, ARNT and USF are shared. The results suggest a list of targets potentially useful in therapeutic interventions in cutaneous diseases and wound healing.

  8. Ultraviolet-induced transformation of keratinocytes: possible involvement of long interspersed element-1 reverse transcriptase.

    PubMed

    Banerjee, Gautam; Gupta, Nishma; Tiwari, Jyoti; Raman, Govindarajan

    2005-02-01

    The normal human keratinocyte cell line, HaCaT, was transformed using multiple doses of ultraviolet (UV)A+B (UVA, 150-200 mJ/cm(2) and UVB, 15-20 mJ/cm(2) x 6). Malignant transformation was confirmed by upregulation of Cyclin D1 (mRNA) and formation of colonies on soft agar. To identify the genes involved in this transformation process, we have done rapid amplification of polymorphic DNA using RNA from unexposed and multiple-exposed cells. Six percent PAGE showed several differentially regulated genes in exposed cells compared with unexposed cells. Total 19 genes were identified, cloned and sequenced. Three of these 19 cloned genes showed 99% homology at both DNA and protein levels to a stretch of 540 bp (180 aa) of long interspersed element (LINE)-1 reverse transcriptase (RT) open reading frame (ORF-2). Colonies from soft agar showed upregulation of this gene compared with non-colonized (lawn on soft agar) cells as detected by RT-PCR. This data implicates LINE-1 RT (ORF-2) in UV-induced malignancy and can possibly be used as a marker for the diagnosis of UV-induced skin cancer.

  9. Essential role of the keratinocyte-specific endonuclease DNase1L2 in the removal of nuclear DNA from hair and nails.

    PubMed

    Fischer, Heinz; Szabo, Sandra; Scherz, Jennifer; Jaeger, Karin; Rossiter, Heidemarie; Buchberger, Maria; Ghannadan, Minoo; Hermann, Marcela; Theussl, Hans-Christian; Tobin, Desmond J; Wagner, Erwin F; Tschachler, Erwin; Eckhart, Leopold

    2011-06-01

    Degradation of nuclear DNA is a hallmark of programmed cell death. Epidermal keratinocytes die in the course of cornification to function as the dead building blocks of the cornified layer of the epidermis, nails, and hair. Here, we investigated the mechanism and physiological function of DNA degradation during cornification in vivo. Targeted deletion of the keratinocyte-specific endonuclease DNase1-like 2 (DNase1L2) in the mouse resulted in the aberrant retention of DNA in hair and nails, as well as in epithelia of the tongue and the esophagus. In contrast to our previous studies in human keratinocytes, ablation of DNase1L2 did not compromise the cornified layer of the epidermis. Quantitative PCRs showed that the amount of nuclear DNA was dramatically increased in both hair and nails, and that mitochondrial DNA was increased in the nails of DNase1L2-deficient mice. The presence of nuclear DNA disturbed the normal arrangement of structural proteins in hair corneocytes and caused a significant decrease in the resistance of hair to mechanical stress. These data identify DNase1L2 as an essential and specific regulator of programmed cell death in skin appendages, and demonstrate that the breakdown of nuclear DNA is crucial for establishing the full mechanical stability of hair.

  10. Transforming growth factor-beta modulates plasminogen activator activity and plasminogen activator inhibitor type-1 expression in human keratinocytes in vitro.

    PubMed

    Wikner, N E; Elder, J T; Persichitte, K A; Mink, P; Clark, R A

    1990-11-01

    Transforming growth factor beta (TGF-beta) is a multifunctional mediator with effects on cellular growth, differentiation, and extracellular matrix (ECM) metabolism. Because TGF-beta stimulates fibronectin expression in cultured human keratinocytes, we wished to determine whether it might also affect ECM degradation through the plasminogen activator (PA)-plasminogen activator inhibitor (PAI) system. Immunofluorescence of human keratinocytes using a monospecific antiserum to type 1 PAI (PAI-1) showed enhanced cellular and ECM staining when they were cultured in the presence of TGF-beta. The antiserum also identified an Mr 50,000 protein in conditioned media that was markedly enhanced by TGF-beta. A corresponding stimulation of PAI-1 mRNA was demonstrated by quantitative RNA blot analysis. Total plasminogen activating activity of conditioned medium was markedly decreased by TGF-beta. Zymography showed this to be at least partially due to decreased secreted urokinase activity. TGF-beta may play an important role in stabilizing the provisional matrix synthesized by keratinocytes in healing wounds.

  11. Modulation of keratinocyte expression of antioxidants by 4-hydroxynonenal, a lipid peroxidation end product

    SciTech Connect

    Zheng, Ruijin; Heck, Diane E.; Mishin, Vladimir; Black, Adrienne T.; Shakarjian, Michael P.; Kong, Ah-Ng Tony; Laskin, Debra L.; Laskin, Jeffrey D.

    2014-03-01

    4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86–98 fold within 6 h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-dependent increases in mRNA and protein expression which were maximum after 6 h with 30 μM. 4-HNE stimulated keratinocyte Erk1/2, JNK and p38 MAP kinases, as well as PI3 kinase. Inhibition of these enzymes suppressed 4-HNE-induced HO-1 mRNA and protein expression. 4-HNE also activated Nrf2 by inducing its translocation to the nucleus. 4-HNE was markedly less effective in inducing HO-1 mRNA and protein in keratinocytes from Nrf2 −/− mice, when compared to wild type mice, indicating that Nrf2 also regulates 4-HNE-induced signaling. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that 4-HNE-induced HO-1 is localized in keratinocyte caveolae. Treatment of the cells with methyl-β-cyclodextrin, which disrupts caveolar structure, suppressed 4-HNE-induced HO-1. These findings indicate that 4-HNE modulates expression of antioxidant enzymes in keratinocytes, and that this can occur by different mechanisms. Changes in expression of keratinocyte antioxidants may be important in protecting the skin from oxidative stress. - Highlights: • Lipid peroxidation generates 4-hydroxynonenal, a reactive aldehyde. • 4-HNE induces antioxidant proteins in mouse keratinocytes. • Induction of

  12. 3D co-cultures of keratinocytes and melanocytes and cytoprotective effects on keratinocytes against reactive oxygen species by insect virus-derived protein microcrystals.

    PubMed

    Shimabukuro, Junji; Yamaoka, Ayako; Murata, Ken-Ichi; Kotani, Eiji; Hirano, Tomoko; Nakajima, Yumiko; Matsumoto, Goichi; Mori, Hajime

    2014-09-01

    Stable protein microcrystals called polyhedra are produced by certain insect viruses. Cytokines, such as fibroblast growth factors (FGFs), can be immobilized within polyhedra. Here, we investigated three-dimensional (3D) co-cultures of keratinocytes and melanocytes on collagen gel containing FGF-2 and FGF-7 polyhedra. Melanocytes were observed to reside at the base of the 3D cell culture and melanin was also typically observed in the lower layer. The 3D cell culture model with FGF-2 and FGF-7 polyhedra was a useful in vitro model of the epidermis due to effective melanogenesis, proliferation and differentiation of keratinocytes. FGF-7 polyhedra showed a potent cytoprotective effect when keratinocytes were treated with menadione, which is a generator of reactive oxygen species. The cytoprotective effect was activated by the inositol triphosphate kinase-Akt pathway leading to upregulation of the antioxidant enzymes superoxide dismutase and peroxiredoxin 6.

  13. Macelignan inhibits melanosome transfer mediated by protease-activated receptor-2 in keratinocytes.

    PubMed

    Choi, Eun-Jung; Kang, Young-Gyu; Kim, Jaekyung; Hwang, Jae-Kwan

    2011-01-01

    Skin pigmentation is the result of melanosome transfer from melanocytes to keratinocytes. Protease-activated receptor-2 (PAR-2) is a key mediator of melanosome transfer, which occurs as the melanocyte extends its dendrite toward surrounding keratinocytes that take up melanosomes by phagocytosis. We investigated the effects of macelignan isolated from Myristica fragrans HOUTT. (nutmeg) on melanosome transfer and the regulation of PAR-2 in human keratinocytes (HaCaT). HaCaT cells stimulated by the PAR-2-activating peptide Ser-Leu-Ile-Gly-Arg-Leu-NH₂ (SLIGRL) were treated with macelignan; PAR-2 expression was then determined by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry. We evaluated the effects of macelignan on calcium mobilization and keratinocyte phagocytosis. In addition, B16F10 melanoma cells and keratinocytes were co-cultured to assess the effects of macelignan on prostaglandin E₂ (PGE₂) secretion and subsequent dendrite formation. Macelignan decreased HaCaT PAR-2 mRNA and protein levels in a dose-dependent manner. Furthermore, macelignan markedly reduced intracellular calcium mobilization and significantly downregulated keratinocyte phagocytosis, as shown by decreased ingestion of Escherichia coli bioparticles and fluorescent microspheres. In co-culture experiments, macelignan reduced keratinocyte PGE₂ secretion, thereby preventing dendrite formation in B16F10 melanoma cells compared with SLIGRL-treated controls. Macelignan inhibits melanosome transfer by downregulating PAR-2, thereby reducing keratinocyte phagocytosis and PGE₂ secretion, which in turn inhibits dendrite formation in B16F10 melanoma cells. Taken together, our findings suggest that macelignan could be used as a natural depigmenting agent to ameliorate hyperpigmentation.

  14. Specificity in stress response: epidermal keratinocytes exhibit specialized UV-responsive signal transduction pathways.

    PubMed

    Adachi, Makoto; Gazel, Alix; Pintucci, Giuseppe; Shuck, Alyssa; Shifteh, Shiva; Ginsburg, Dov; Rao, Laxmi S; Kaneko, Takehiko; Freedberg, Irwin M; Tamaki, Kunihiko; Blumenberg, Miroslav

    2003-10-01

    UV light, a paradigmatic initiator of cell stress, invokes responses that include signal transduction, activation of transcription factors, and changes in gene expression. Consequently, in epidermal keratinocytes, its principal and frequent natural target, UV regulates transcription of a distinctive set of genes. Hypothesizing that UV activates distinctive epidermal signal transduction pathways, we compared the UV-responsive activation of the JNK and NFkappaB pathways in keratinocytes, with the activation of the same pathways by other agents and in other cell types. Using of inhibitors and antisense oligonucleotides, we found that in keratinocytes only UVB/UVC activate JNK, while in other cell types UVA, heat shock, and oxidative stress do as well. Keratinocytes express JNK-1 and JNK-3, which is unexpected because JNK-3 expression is considered brain-specific. In keratinocytes, ERK1, ERK2, and p38 are activated by growth factors, but not by UV. UVB/UVC in keratinocytes activates Elk1 and AP1 exclusively through the JNK pathway. JNKK1 is essential for UVB/UVC activation of JNK in keratinocytes in vitro and in human skin in vivo. In contrast, in HeLa cells, used as a control, crosstalk among signal transduction pathways allows considerable laxity. In parallel, UVB/UVC and TNFalpha activate the NFkappaB pathway via distinct mechanisms, as shown using antisense oligonucleotides targeted against IKKbeta, the active subunit of IKK. This implies a specific UVB/UVC responsive signal transduction pathway independent from other pathways. Our results suggest that in epidermal keratinocytes specific signal transduction pathways respond to UV light. Based on these findings, we propose that the UV light is not a genetic stress response inducer in these cells, but a specific agent to which epidermis developed highly specialized responses.

  15. Cooperative response of keratinocytes and melanocytes to UV radiation during PUVA therapy

    NASA Astrophysics Data System (ADS)

    Stolnitz, Mikhail M.; Baskakov, Pavel V.; Peshkova, Anna Y.

    1999-03-01

    The mathematical model of processes in UV-irradiated furocoumarin-sensitized epidermis is presented taking into account the mutual influence of keratinocytes and melanocytes populations. The model describes epidermis as a hierarchical structure on tissue (keratinocytes-melanocytes cooperation, melanin screen formation), cellular (proliferation and differentiation, transitions between subpopulations), subcellular (cell movement on mitotic cycle, generation, maturing and migration of melanosomes), and molecular (melanin synthesis, processes of DNA damage and repair, molecular signal transduction) levels.

  16. The Gender Similarities Hypothesis

    ERIC Educational Resources Information Center

    Hyde, Janet Shibley

    2005-01-01

    The differences model, which argues that males and females are vastly different psychologically, dominates the popular media. Here, the author advances a very different view, the gender similarities hypothesis, which holds that males and females are similar on most, but not all, psychological variables. Results from a review of 46 meta-analyses…

  17. CHOLINERGIC REGULATION OF KERATINOCYTE INNATE IMMUNITY AND PERMEABILITY BARRIER INTEGRITY: NEW PERSPECTIVES IN EPIDERMAL IMMUNITY AND DISEASE

    PubMed Central

    Curtis, Brenda J.; Radek, Katherine A.

    2015-01-01

    Several cutaneous inflammatory diseases and their clinical phenotypes are recapitulated in animal models of skin disease. However, the identification of shared pathways for disease progression is limited by the ability to delineate the complex biochemical processes fundamental for development of the disease. Identifying common signaling pathways that contribute to cutaneous inflammation and immune function will facilitate better scientific and therapeutic strategies to span a variety of inflammatory skin diseases. Aberrant antimicrobial peptide (AMP) expression and activity is one mechanism behind the development and severity of several inflammatory skin diseases and directly influences the susceptibility of skin to microbial infections. Our studies have recently exposed a newly identified pathway for negative regulation of AMPs in the skin by the cholinergic anti-inflammatory pathway via acetylcholine (ACh). The role of ACh in AMP regulation of immune and permeability barrier function in keratinocytes is reviewed, and the importance for a better comprehension of cutaneous disease progression by cholinergic signaling is discussed. PMID:21918536

  18. Role for WNT16B in human epidermal keratinocyte proliferation and differentiation.

    PubMed

    Teh, Muy-Teck; Blaydon, Diana; Ghali, Lucy R; Briggs, Victoria; Edmunds, Scott; Pantazi, Eleni; Barnes, Michael R; Leigh, Irene M; Kelsell, David P; Philpott, Michael P

    2007-01-15

    WNT signalling regulates a variety of cell functions including cell fate, polarity, and differentiation via the canonical or beta-catenin stabilisation pathway and/or the planar cell polarity or non-canonical pathway. We have previously demonstrated that two isoforms (A and B) from the WNT16 locus have differential expression in various adult human tissues. In this study we show that WNT16B but not WNT16A isoform was upregulated in basal cell carcinomas compared with normal skin. We further investigated the cellular and molecular functions of WNT16B in primary human epidermal keratinocytes and a keratinocyte cell line. Cellular expression of WNT16B neither stabilised beta-catenin nor activated the lymphoid enhancer factor or T-cell factor transcriptional reporter in primary keratinocytes. WNT16B activated the Jun-N-terminal kinase cascade suggesting the activation of a non-canonical WNT signalling pathway. Constitutive expression of WNT16B significantly enhanced the rate of cell proliferation and prolonged clonogenicity in primary keratinocytes. Silencing WNT16B by RNA interference reduced keratinocyte proliferation. Furthermore, overexpression of WNT16B induced a hyperproliferation phenotype in an organotypical culture system. This work presents the first evidence that WNT16B activates human keratinocyte proliferation possibly via a beta-catenin-independent non-canonical WNT transduction pathway.

  19. Ligand targeting of EphA2 enhances keratinocyte adhesion and differentiation via desmoglein 1.

    PubMed

    Lin, Samantha; Gordon, Kristin; Kaplan, Nihal; Getsios, Spiro

    2010-11-15

    EphA2 is a receptor tyrosine kinase that is engaged and activated by membrane-linked ephrin-A ligands residing on adjacent cell surfaces. Ligand targeting of EphA2 has been implicated in epithelial growth regulation by inhibiting the extracellular signal-regulated kinase 1/2 (Erk1/2)-mitogen activated protein kinase (MAPK) pathway. Although contact-dependent EphA2 activation was required for dampening Erk1/2-MAPK signaling after a calcium switch in primary human epidermal keratinocytes, the loss of this receptor did not prevent exit from the cell cycle. Incubating keratinocytes with a soluble ephrin-A1-Fc peptide mimetic to target EphA2 further increased receptor activation leading to its down-regulation. Moreover, soluble ligand targeting of EphA2 restricted the lateral expansion of epidermal cell colonies without limiting proliferation in these primary cultures. Rather, ephrin-A1-Fc peptide treatment promoted epidermal cell colony compaction and stratification in a manner that was associated with increased keratinocyte differentiation. The ligand-dependent increase in keratinocyte adhesion and differentiation relied largely upon the up-regulation of desmoglein 1, a desmosomal cadherin that maintains the integrity and differentiated state of suprabasal keratinocytes in the epidermis. These data suggest that keratinocytes expressing EphA2 in the basal layer may respond to ephrin-A1-based cues from their neighbors to facilitate entry into a terminal differentiation pathway.

  20. Overexpression of connexin26 in the basal keratinocytes reduces sensitivity to tumor promoter TPA.

    PubMed

    Wang, Xiao; Ramirez, Angel; Budunova, Irina

    2010-07-01

    Connexin 26 is important in keratinocyte proliferation, differentiation and skin pathologies. Cx26 is barely expressed in normal adult epidermis, but its expression is induced during wound healing, psoriasis, and skin hyperplasia stimulated by tumor promoters. In hyperplastic proliferating epidermis, Cx26 is co-expressed with Cx43 typical for basal and suprabasal keratinocytes. As Cx26 and Cx43 can not form permeable gap junctions, their co-expression may alter the gap junctional communication between keratinocytes and induce proliferation. To test the effect of persistent co-expression of Cx26 and Cx43 in epidermis, we generated transgenic mice using keratin5 promoter to target Cx26 to basal Cx43-positive keratinocytes. We evaluated the effect of ectopic Cx26 on keratinocyte proliferation and differentiation in normal and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-treated skin. The ectopic Cx26 expression in epidermis did not significantly affect skin development, keratinocyte differentiation and proliferation in newborn and adult skin. Unexpectedly, the proliferative effect of tumor promoter TPA was strongly decreased in epidermis of K5.Cx26 transgenics. This correlated with significant down-regulation of TPA-induced activity of protein kinase C (PKC) in K5.Cx26 mice.

  1. The antimicrobial protein REG3A regulates keratinocyte proliferation and differentiation after skin injury.

    PubMed

    Lai, Yuping; Li, Dongqing; Li, Changwei; Muehleisen, Beda; Radek, Katherine A; Park, Hyun Jeong; Jiang, Ziwei; Li, Zhiheng; Lei, Hu; Quan, Yanchun; Zhang, Tian; Wu, Yelin; Kotol, Paul; Morizane, Shin; Hata, Tissa R; Iwatsuki, Keiji; Tang, Ce; Gallo, Richard L

    2012-07-27

    Epithelial keratinocyte proliferation is an essential element of wound repair, and abnormal epithelial proliferation is an intrinsic element in the skin disorder psoriasis. The factors that trigger epithelial proliferation in these inflammatory processes are incompletely understood. Here we have shown that regenerating islet-derived protein 3-alpha (REG3A) is highly expressed in keratinocytes during psoriasis and wound repair and in imiquimod-induced psoriatic skin lesions. The expression of REG3A by keratinocytes is induced by interleukin-17 (IL-17) via activation of keratinocyte-encoded IL-17 receptor A (IL-17RA) and feeds back on keratinocytes to inhibit terminal differentiation and increase cell proliferation by binding to exostosin-like 3 (EXTL3) followed by activation of phosphatidylinositol 3 kinase (PI3K) and the kinase AKT. These findings reveal that REG3A, a secreted intestinal antimicrobial protein, can promote skin keratinocyte proliferation and can be induced by IL-17. This observation suggests that REG3A may mediate the epidermal hyperproliferation observed in normal wound repair and in psoriasis.

  2. In vitro investigations on the effect of dermal fibroblasts on keratinocyte responses to ultraviolet B radiation.

    PubMed

    Fernandez, Tara L; Van Lonkhuyzen, Derek R; Dawson, Rebecca A; Kimlin, Michael G; Upton, Zee

    2014-01-01

    Exposure to ultraviolet radiation is closely linked to the development of skin cancers in humans. The ultraviolet B (UVB) radiation wavelength (280-320 nm), in particular, causes DNA damage in epidermal keratinocytes, which are linked to the generation of signature premalignant mutations. Interactions between dermal fibroblasts and keratinocytes play a role in epidermal repair and regeneration after UVB-induced damage. To investigate these processes, established two and three-dimensional culture models were utilized to study the impact of fibroblast-keratinocyte crosstalk during the acute UVB response. Using a coculture system it was observed that fibroblasts enhanced keratinocyte survival and the repair of cyclobutane pyrimidine dimers (CPDs) after UVB radiation exposure. These findings were also mirrored in irradiated human skin coculture models employed in this study. Fibroblast coculture was shown to play a role in the expression and activation of members of the apoptotic cascade, including caspase-3 and Bad. Interestingly, the expression and phosphorylation of p53, a key player in the regulation of keratinocyte cell fate postirradiation, was also shown to be influenced by fibroblast-produced factors. This study highlights the importance of synergistic interactions between fibroblasts and keratinocytes in maintaining a functional epidermis while promoting repair and regeneration following UVB radiation-induced damage.

  3. Notch Cooperates with Survivin to Maintain Stemness and to Stimulate Proliferation in Human Keratinocytes during Ageing

    PubMed Central

    Palazzo, Elisabetta; Morandi, Paolo; Lotti, Roberta; Saltari, Annalisa; Truzzi, Francesca; Schnebert, Sylvianne; Dumas, Marc; Marconi, Alessandra; Pincelli, Carlo

    2015-01-01

    The Notch signaling pathway orchestrates cell fate by either inducing cell differentiation or maintaining cells in an undifferentiated state. This study aims to evaluate Notch expression and function in normal human keratinocytes. Notch1 is expressed in all epidermal layers, though to a different degree of intensity, with a dramatic decrease during ageing. Notch1 intracellular domain (N1ICD) levels are decreased during transit from keratinocyte stem cells (KSC) to transit amplifying (TA) cells, mimicking survivin expression in samples from donors of all ages. Calcium markedly reduces N1ICD levels in keratinocytes. N1ICD overexpression induces the up-regulation of survivin and the down-regulation of keratin 10 and involucrin, while increasing the S phase of the cell cycle. On the other hand, Notch1 inhibition (DAPT) dose-dependently decreases survivin, stimulates differentiation, and reduces keratinocyte proliferation in samples from donors of all ages. Silencing Notch downgrades survivin and increases keratin 10. In addition, Notch1 inhibition decreases survivin levels and proliferation both in KSC and TA cells. Finally, while survivin overexpression decreases keratinocyte differentiation and increases N1ICD expression both in KSC and TA cells, silencing survivin results in N1ICD down-regulation and an increase in differentiation markers. These results suggest that the Notch1/survivin crosstalk contributes to the maintenance of stemness in human keratinocytes. PMID:26540052

  4. Erk5 Controls Slug Expression and Keratinocyte Activation during Wound Healing

    PubMed Central

    Arnoux, Valerie; Nassour, Mayssaa; L'Helgoualc'h, Annie; Hipskind, Robert A.

    2008-01-01

    Reepithelialization during cutaneous wound healing involves numerous signals that result in basal keratinocyte activation, spreading, and migration, all linked to a loosening of cell–cell adhesion structures. The transcription factor Slug is required for this process, and EGF treatment of human keratinocytes induced activating phosphorylation of Erk5 that coincides with slug transcription. Accordingly, ectopic activation of Erk5 led to increased Slug mRNA levels and faster wound healing, whereas keratinocyte migration was totally blocked by Erk5 pathway inhibition. Expression of a shRNA specific for Erk5 strongly diminished Erk5 levels in keratinocytes and significantly decreased their motility response to EGF, along with induction of Slug expression. These Erk5-deprived keratinocytes showed an altered, more compact morphology, along with disruption of desmosome organization. Accordingly, they displayed an altered ability to form cell aggregates. These results implicate a novel EGFR/Erk5/Slug pathway in the control of cytoskeleton organization and cell motility in keratinocytes treated with EGF. PMID:18716062

  5. Lipid deregulation in UV irradiated skin cells: Role of 25-hydroxycholesterol in keratinocyte differentiation during photoaging.

    PubMed

    Olivier, Elodie; Dutot, Mélody; Regazzetti, Anne; Dargère, Delphine; Auzeil, Nicolas; Laprévote, Olivier; Rat, Patrice

    2016-05-18

    Skin photoaging due to UV irradiation is a degenerative process that appears more and more as a growing concern. Lipids, including oxysterols, are involved in degenerative processes; as skin cells contain various lipids, the aim of our study was to evaluate first, changes in keratinocyte lipid levels induced by UV exposure and second, cellular effects of oxysterols in cell morphology and several hallmarks of keratinocyte differentiation. Our mass spectrometry results demonstrated that UV irradiation induces changes in lipid profile of cultured keratinocytes; in particular, ceramides and oxysterols, specifically 25-hydroxycholesterol (25-OH), were increased. Using holography and confocal microscopy analyses, we highlighted cell thickening and cytoskeletal disruption after incubation of keratinocytes with 25-OH. These alterations were associated with keratinocyte differentiation patterns: autophagy stimulation and intracellular calcium increase as measured by cytofluorometry, and increased involucrin level detected by immunocytochemistry. To conclude, oxysterol deregulation could be considered as a common marker of degenerative disorders. During photoaging, 25-OH seems to play a key role inducing morphological changes and keratinocyte differentiation.

  6. A Two-Stepped Culture Method for Efficient Production of Trichogenic Keratinocytes

    PubMed Central

    Chan, Chih-Chieh; Fan, Sabrina Mai-Yi; Wang, Wei-Hung; Mu, Yi-Fen

    2015-01-01

    Successful hair follicle (HF) neogenesis in adult life depends on the existence of both capable dermal cells and competent epidermal keratinocytes that recapitulate embryonic organogenesis through epithelial–mesenchymal interaction. In tissue engineering, the maintenance of trichogenic potential of adult epidermal cells, while expanding them remains a challenging issue. We found that although HF outer root sheath keratinocytes could be expanded for more than 100 passages as clonogenic cells without losing the proliferative potential with a 3T3J2 fibroblast feeder layer, these keratinocytes were unable to form new HFs when combined with inductive HF dermal papilla (DP) cells. However, when these high-passage keratinocytes were cocultured with HF DP cells for 4 days in vitro, they regained the trichogenic ability to form new HFs after transplantation. We found that the short-term coculture with DP cells enhanced both Wnt/β-catenin signaling, a signaling cascade key to HF development, and upregulated the expression of HF-specific genes, including K6, K16, K17, and K75, in keratinocytes, indicating that these cells were poised toward a HF fate. Hence, efficient production of trichogenic keratinocytes can be obtained by a two-stepped procedure with initial cell expansion with a 3T3J2 fibroblast feeder followed by short-term coculture with DP cells. PMID:25951188

  7. Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor

    SciTech Connect

    Matsumoto, K.; Hashimoto, K.; Yoshikawa, K.; Nakamura, T. )

    1991-09-01

    Effect of hepatocyte growth factor (HGF) on normal human epidermal keratinocytes cultured under conditions of low Ca2+ (0.1 mM, growth-promoting condition) and physiological Ca2+ (1.8 mM, differentiation-promoting condition) was investigated. In low Ca2+, HGF markedly enhanced the migration of keratinocytes while it suppressed cell growth and DNA synthesis in a dose-dependent manner. In contrast, HGF enhanced the migration, cell growth, and DNA synthesis of keratinocytes cultured under conditions of physiological Ca2+. The maximal stimulation of DNA synthesis (2.4-fold stimulation) in physiological Ca2+ was seen at 2.5-5 ng/ml HGF and the stimulatory effect of HGF was suppressed by transforming growth factor-beta 1. Analysis of the HGF receptor using 125I-HGF as a ligand showed that human keratinocytes expressed a single class of specific, saturable receptor for HGF in both low and physiological Ca2+ conditions, exhibiting a Kd = 17.3 pM and approximately 690 binding sites/cell under physiological Ca2+. Thus, HGF is a potent factor which enhances growth and migration of normal human keratinocytes under conditions of physiological Ca2+. HGF may play an important role in epidermal tissue repair as it enhances both the migration and growth of keratinocytes.

  8. Evidence for multiple bone resorption-stimulating factors produced by normal human keratinocytes in culture.

    PubMed

    Fried, R M; Voelkel, E F; Rice, R H; Levine, L; Tashjian, A H

    1988-06-01

    Conditioned medium from cultured normal human foreskin keratinocytes enhanced the release of calcium from neonatal mouse calvaria in organ culture. Unfractionated keratinocyte-conditioned medium (KCM) stimulated bone resorption in a dose-dependent manner, but it did not increase the concentration of prostaglandin E2 (PGE2) in the bone culture medium until a maximal dose of KCM for resorption was used. Furthermore, inhibitors of PGE2 synthesis, indomethacin, ibuprofen, and piroxicam, did not inhibit KCM-induced calcium release. High concentrations of KCM increased cAMP production by calvaria in the presence of isobutylmethylxanthine, but the increase was small compared with that produced by a dose of bovine PTH that caused a similar level of bone resorption. The bone resorption-stimulating activity of KCM was not lost after incubation at 56 C for 60 min, but it was lost after heating at 100 C for 10 min. Fractionation of KCM by gel filtration chromatography revealed two distinct peaks of bone resorption-stimulating activity. One peak, KCMI, caused a significant increase in bone resorption at 2 micrograms protein/ml. KCMI did not increase medium PGE2, and inhibition of PGE2 synthesis in bone had no effect on KCMI-induced bone resorption. KCMI failed to increase cAMP production by human osteosarcoma SaOS-2 cells. Another peak, KCMII, caused a dose-dependent increase in bone resorption, and a significant increase in medium calcium was noted at a 20-fold lower concentration (0.1 microgram protein/ml) than with KCMI. In contrast to KCMI, the increase in bone resorption stimulated by KCMII was accompanied by a parallel increase in the production of PGE2, and inhibition of PGE2 synthesis completely inhibited the bone resorption-stimulating activity of KCMII. KCMII also caused an increase in cAMP production by SaOS-2 cells. We conclude that KCM contains at least two distinct bone resorption-stimulating factors, one of which acts via a PG-mediated mechanism and the other by

  9. Xenobiotic metabolism capacities of human skin in comparison with a 3D epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: activating enzymes (Phase I).

    PubMed

    Götz, Christine; Pfeiffer, Roland; Tigges, Julia; Blatz, Veronika; Jäckh, Christine; Freytag, Eva-Maria; Fabian, Eric; Landsiedel, Robert; Merk, Hans F; Krutmann, Jean; Edwards, Robert J; Pease, Camilla; Goebel, Carsten; Hewitt, Nicola; Fritsche, Ellen

    2012-05-01

    Skin is important for the absorption and metabolism of exposed chemicals such as cosmetics or pharmaceuticals. The Seventh Amendment to the EU Cosmetics Directive prohibits the use of animals for cosmetic testing for certain endpoints, such as genotoxicity; therefore, there is an urgent need to understand the xenobiotic metabolizing capacities of human skin and to compare these activities with reconstructed 3D skin models developed to replace animal testing. We have measured Phase I enzyme activities of cytochrome P450 (CYP) and cyclooxygenase (COX) in ex vivo human skin, the 3D skin model EpiDerm™ (EPI-200), immortalized keratinocyte-based cell lines and primary normal human epidermal keratinocytes. Our data demonstrate that basal CYP enzyme activities are very low in whole human skin and EPI-200 as well as keratinocytes. In addition, activities in monolayer cells differed from organotypic tissues after induction. COX activity was similar in skin, EPI-200 and NHEK cells, but was significantly lower in immortalized keratinocytes. Hence, the 3D model EPI-200 might represent a more suitable model for dermatotoxicological studies. Altogether, these data help to better understand skin metabolism and expand the knowledge of in vitro alternatives used for dermatotoxicity testing.

  10. Loss of Desmoglein Binding Is Not Sufficient for Keratinocyte Dissociation in Pemphigus.

    PubMed

    Vielmuth, Franziska; Waschke, Jens; Spindler, Volker

    2015-12-01

    Pemphigus vulgaris (PV) is a severe autoimmune disease in which autoantibodies against the desmosomal cell adhesion molecules desmoglein (Dsg) 1 and Dsg3 cause intraepidermal blister formation. Mechanistically, the fundamental question is still unresolved whether loss of cell cohesion is a result of (1) direct inhibition of Dsg interaction by autoantibodies or (2) intracellular signaling events, which are altered in response to antibody binding and finally cause desmosome destabilization. We used atomic force microscopy (AFM) to perform Dsg3 adhesion measurements on living keratinocytes to investigate the contributions of direct inhibition and signaling to loss of cell cohesion after autoantibody treatment. Dsg3 binding was rapidly blocked following antibody exposure under conditions where no depletion of surface Dsg3 was detectable, demonstrating direct inhibition of Dsg3 interaction. Inhibition of p38MAPK, a central signaling molecule in PV pathogenesis, abrogated loss of cell cohesion, but had a minor effect on loss of Dsg3 binding. Similarly, the cholesterol-depleting agent methyl-β-cyclodextrin (β-MCD) fully blocked cell dissociation, but did not restore Dsg3 interactions or prevent the activation of p38MAPK. These results demonstrate that inhibition of Dsg3 binding is not sufficient to cause loss of cell cohesion, but rather alters signaling events which, in lipid raft-dependent manner, induce cell dissociation.

  11. Keratinocyte Growth Factor Causes Cystic Dilation of the Mammary Glands of Mice

    PubMed Central

    Yi, Eunhee S.; Bedoya, Adriana A.; Lee, Hyesun; Kim, Seokhyun; Housley, Regina M.; Aukerman, Sharon L.; Tarpley, John E.; Starnes, Charles; Yin, Songmei; Pierce, Glenn F.; Ulich, Thomas R.

    1994-01-01

    Keratinocyte growth factor (KGF) is a paracrine mediator of epithelial cell proliferation that has been reported to induce marked proliferation of mammary epithelium in rats. In this study, systemic administration of KGF into naive and oophorectomized mice causes mammary gland proliferation, as evidenced histologically by the appearance of cysts lined by a single layer of epithelium and by hyperplastic epithelium. Whole mount preparations of the mammary glands reveal that the histologically noted cysts are actually ducts that are dilated along much of their length. The histology of the mammary glands of KGF-treated mice is similar to the histology of fibrocystic disease in the buman female breast. The response in mice differs significantly from the appearance of the mammary glands in KGF-treated rats in which ductal epithelial proliferation is most prominent. Estrogen and progesterone when administered in combination but not alone cause the development of numerous endbuds in the mouse mammary gland. KGF in estrogen- and progesterone-pretreated mice causes the growth of dilated ducts, hyperplastic epithelium within ducts and endbuds, and a fibrous metamorphosis of periductal adipose tissue. The mammary epithelial hyperplasia caused by KGF is rapidly reversible in both mice and rats after cessation of KGF treatment. The spectrum of KGF-, estrogen-, and progesterone-induced mammary histopathology in mice provides a model for the study of fibrocystic and hyperplastic breast disease. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6 PMID:7977634

  12. The chemopreventive bioflavonoid apigenin modulates signal transduction pathways in keratinocyte and colon carcinoma cell lines.

    PubMed

    Van Dross, Rukiyah; Xue, Yue; Knudson, Alexandra; Pelling, Jill C

    2003-11-01

    Apigenin is a nonmutagenic chemopreventive agent found in fruits and green vegetables. In this study, we used two different epithelial cell lines (308 mouse keratinocytes and HCT116 colon carcinoma cells) to determine the effect of apigenin on the mitogen-activated protein kinase (MAPK) cascade. Apigenin induced a dose-dependent phosphorylation of both extracellular signal-regulated protein kinase (ERK) and p38 kinase but had little effect on the phosphorylation of c-jun amino terminal kinase (JNK). We used immunoprecipitation-coupled kinase assays to show that apigenin increased the kinase activity of ERK and p38 but not JNK. Consistent with these results, we found that apigenin induced a 7.4-fold induction in the phosphorylation of Elk, the downstream phosphorylation target of ERK kinase. Similarly, apigenin induced a 3.2-fold induction in the phosphorylation of activating transcription factor-2, the downstream phosphorylation target of p38 kinase. Little change was observed in the phosphorylation of c-jun, the phosphorylation target of JNK. These data suggest that part of the chemopreventive activity of apigenin may be mediated by its ability to modulate the MAPK cascade.

  13. Towards personalized medicine: leveraging patient similarity and drug similarity analytics.

    PubMed

    Zhang, Ping; Wang, Fei; Hu, Jianying; Sorrentino, Robert

    2014-01-01

    The rapid adoption of electronic health records (EHR) provides a comprehensive source for exploratory and predictive analytic to support clinical decision-making. In this paper, we investigate how to utilize EHR to tailor treatments to individual patients based on their likelihood to respond to a therapy. We construct a heterogeneous graph which includes two domains (patients and drugs) and encodes three relationships (patient similarity, drug similarity, and patient-drug prior associations). We describe a novel approach for performing a label propagation procedure to spread the label information representing the effectiveness of different drugs for different patients over this heterogeneous graph. The proposed method has been applied on a real-world EHR dataset to help identify personalized treatments for hypercholesterolemia. The experimental results demonstrate the effectiveness of the approach and suggest that the combination of appropriate patient similarity and drug similarity analytics could lead to actionable insights for personalized medicine. Particularly, by leveraging drug similarity in combination with patient similarity, our method could perform well even on new or rarely used drugs for which there are few records of known past performance.

  14. The gender similarities hypothesis.

    PubMed

    Hyde, Janet Shibley

    2005-09-01

    The differences model, which argues that males and females are vastly different psychologically, dominates the popular media. Here, the author advances a very different view, the gender similarities hypothesis, which holds that males and females are similar on most, but not all, psychological variables. Results from a review of 46 meta-analyses support the gender similarities hypothesis. Gender differences can vary substantially in magnitude at different ages and depend on the context in which measurement occurs. Overinflated claims of gender differences carry substantial costs in areas such as the workplace and relationships.

  15. Loss of nuclear receptor RXRα in epidermal keratinocytes promotes the formation of Cdk4-activated invasive melanomas.

    PubMed

    Hyter, Stephen; Bajaj, Gaurav; Liang, Xiaobo; Barbacid, Mariano; Ganguli-Indra, Gitali; Indra, Arup Kumar

    2010-10-01

    Keratinocytes contribute to melanocyte transformation by affecting their microenvironment, in part through the secretion of paracrine factors. Here we report a loss of expression of nuclear receptor RXRα in epidermal keratinocytes during human melanoma progression. In the absence of keratinocytic RXRα, in combination with mutant Cdk4, cutaneous melanoma was generated that metastasized to lymph nodes in a bigenic mouse model. Expression of several keratinocyte-derived mitogenic growth factors (Et-1, Hgf, Scf, α-MSH and Fgf 2 ) was elevated in skin of bigenic mice, whereas Fas, E-cadherin and Pten, implicated in apoptosis, cellular invasion and melanomagenesis, respectively, were downregulated within the microdissected melanocytic tumors. We demonstrated that RXRα is recruited on the proximal promoter of both Et-1 and Hgf, possibly directly regulating their transcription in keratinocytes. These studies demonstrate the contribution of keratinocytic paracrine signaling during the cellular transformation and malignant conversion of melanocytes.

  16. Additive Similarity Trees

    ERIC Educational Resources Information Center

    Sattath, Shmuel; Tversky, Amos

    1977-01-01

    Tree representations of similarity data are investigated. Hierarchical clustering is critically examined, and a more general procedure, called the additive tree, is presented. The additive tree representation is then compared to multidimensional scaling. (Author/JKS)

  17. Modulation of NFAT-5, an outlying member of the NFAT family, in human keratinocytes and skin

    PubMed Central

    Al-Daraji, Wael I; Afolayan, John; Zelger, Bettina G; Abdellaoui, Adel; Zelger, Bernhard

    2009-01-01

    Background Cyclosporin A (CsA) and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor Nuclear Factor of Activated T cells (NFAT). NFAT compose a family of transcription factors that are turned on during T cell activation. Aims To study the expression of NFAT-5 mRNA and protein in normal human keratinocytes and to investigate the cellular and subcellular pattern of expression of NFAT-5 in normal human skin and psoriasis, and analyze effects of different agonists and ultraviolet radiation on NFAT-5 in normal human skin. Methods Tissue cultures, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), Western analysis, immunostaining, confocal microscopy. Results Sequencing of RT-PCR products confirmed the identity of the product that showed 100 % homology with the predicted NFAT-5 sequence. anti-NFAT-5 mainly detected a single band in cultured keratinocytes and dermal fibroblasts using Western analysis. Immunohistochemistry showed that epidermal keratinocytes and dermal fibroblasts in normal human and psoriatic skin express NFAT-5. NFAT-5 showed predominantly nuclear localization in epidermal keratinocytes and dermal fibroblasts within five normal adult skin biopsies. Our data also suggest that UV irradiation reduces NFAT-5 nuclear localization within the epidermis. Unlike NFAT 1-4, NFAT-5/TonEBP was localized to both nucleus and cytoplasm of cultured keratinocytes. Cyclosporin A induces nuclear membrane translocation of NFAT-5 in cultured keratinocytes and raffinose (a hypertonicity inducing agent) induces more nuclear localization of NFAT-5 compared to untreated cells. In addition, differentiation-promoting agonists that induce sustained rise in intracellular calcium did not result in changes in NFAT-5 localization in cultured keratinocytes. Conclusion These studies provide the first observation of expression of NFAT-5/TonEBP mRNA protein in

  18. Transient Receptor Potential Vanilloid 4 Ion Channel Functions as a Pruriceptor in Epidermal Keratinocytes to Evoke Histaminergic Itch*

    PubMed Central

    Chen, Yong; Fang, Quan; Wang, Zilong; Zhang, Jennifer Y.; MacLeod, Amanda S.; Hall, Russell P.; Liedtke, Wolfgang B.

    2016-01-01

    TRPV4 ion channels function in epidermal keratinocytes and in innervating sensory neurons; however, the contribution of the channel in either cell to neurosensory function remains to be elucidated. We recently reported TRPV4 as a critical component of the keratinocyte machinery that responds to ultraviolet B (UVB) and functions critically to convert the keratinocyte into a pain-generator cell after excess UVB exposure. One key mechanism in keratinocytes was increased expression and secretion of endothelin-1, which is also a known pruritogen. Here we address the question of whether TRPV4 in skin keratinocytes functions in itch, as a particular form of “forefront” signaling in non-neural cells. Our results support this novel concept based on attenuated scratching behavior in response to histaminergic (histamine, compound 48/80, endothelin-1), not non-histaminergic (chloroquine) pruritogens in Trpv4 keratinocyte-specific and inducible knock-out mice. We demonstrate that keratinocytes rely on TRPV4 for calcium influx in response to histaminergic pruritogens. TRPV4 activation in keratinocytes evokes phosphorylation of mitogen-activated protein kinase, ERK, for histaminergic pruritogens. This finding is relevant because we observed robust anti-pruritic effects with topical applications of selective inhibitors for TRPV4 and also for MEK, the kinase upstream of ERK, suggesting that calcium influx via TRPV4 in keratinocytes leads to ERK-phosphorylation, which in turn rapidly converts the keratinocyte into an organismal itch-generator cell. In support of this concept we found that scratching behavior, evoked by direct intradermal activation of TRPV4, was critically dependent on TRPV4 expression in keratinocytes. Thus, TRPV4 functions as a pruriceptor-TRP in skin keratinocytes in histaminergic itch, a novel basic concept with translational-medical relevance. PMID:26961876

  19. Ethanol Extract of Cirsium japonicum var. ussuriense Kitamura Exhibits the Activation of Nuclear Factor Erythroid 2-Related Factor 2-dependent Antioxidant Response Element and Protects Human Keratinocyte HaCaT Cells Against Oxidative DNA Damage

    PubMed Central

    Yoo, Ok-Kyung; Choi, Bu Young; Park, Jin-Oh; Lee, Ji-Won; Park, Byoung-Kwon; Joo, Chul Gue; Heo, Hyo-Jung; Keum, Young-Sam

    2016-01-01

    Keratinocytes are constantly exposed to extracellular insults, such as ultraviolet B, toxic chemicals and mechanical stress, all of which can facilitate the aging of keratinocytes via the generation of intracellular reactive oxygen species (ROS). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays a critical role in protecting keratinocytes against oxidants and xenobiotics by binding to the antioxidant response element (ARE), a cis-acting element existing in the promoter of most phase II cytoprotective genes. In the present study, we have attempted to find novel ethanol extract(s) of indigenous plants of Jeju island, Korea that can activate the Nrf2/ARE-dependent gene expression in human keratinocyte HaCaT cells. As a result, we identified that ethanol extract of Cirsium japonicum var. ussuriense Kitamura (ECJUK) elicited strong stimulatory effect on the ARE-dependent gene expression. Supporting this observation, we found that ECJUK induced the expression of Nrf2, hemoxygenase-1, and NAD(P)H:quinone oxidoreductase-1 and this event was correlated with Akt1 phosphorylation. We also found that ECJUK increased the intracellular reduced glutathione level and suppressed 12-O-tetradecanoylphorbol acetate-induced 8-hydroxyguanosine formation without affecting the overall viability. Collectively, our results provide evidence that ECJUK can protect against oxidative stress-mediated damages through the activation of Nrf2/ARE-dependent phase II cytoprotective gene expression. PMID:27051652

  20. Non-thermal dielectric-barrier discharge plasma damages human keratinocytes by inducing oxidative stress.

    PubMed

    Kim, Ki Cheon; Piao, Mei Jing; Madduma Hewage, Susara Ruwan Kumara; Han, Xia; Kang, Kyoung Ah; Jo, Jin Oh; Mok, Young Sun; Shin, Jennifer H; Park, Yeunsoo; Yoo, Suk Jae; Hyun, Jin Won

    2016-01-01

    The aim of this study was to identify the mechanisms through which dielectric-barrier discharge plasma damages human keratinocytes (HaCaT cells) through the induction of oxidative stress. For this purpose, the cells were exposed to surface dielectric-barrier discharge plasma in 70% oxygen and 30% argon. We noted that cell viability was decreased following exposure of the cells to plasma in a time-dependent manner, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The levels of intracellular reactive oxygen species (ROS) were determined using 2',7'-dichlorodihydrofluorescein diacetate and dihydroethidium was used to monitor superoxide anion production. Plasma induced the generation of ROS, including superoxide anions, hydrogen peroxide and hydroxyl radicals. N-acetyl cysteine, which is an antioxidant, prevented the decrease in cell viability caused by exposure to plasma. ROS generated by exposure to plasma resulted in damage to various cellular components, including lipid membrane peroxidation, DNA breaks and protein carbonylation, which was detected by measuring the levels of 8-isoprostane and diphenyl-1-pyrenylphosphine assay, comet assay and protein carbonyl formation. These results suggest that plasma exerts cytotoxic effects by causing oxidative stress-induced damage to cellular components.

  1. Non-thermal dielectric-barrier discharge plasma damages human keratinocytes by inducing oxidative stress

    PubMed Central

    KIM, KI CHEON; PIAO, MEI JING; HEWAGE, SUSARA RUWAN KUMARA MADDUMA; HAN, XIA; KANG, KYOUNG AH; JO, JIN OH; MOK, YOUNG SUN; SHIN, JENNIFER H.; PARK, YEUNSOO; YOO, SUK JAE; HYUN, JIN WON

    2016-01-01

    The aim of this study was to identify the mechanisms through which dielectric-barrier discharge plasma damages human keratinocytes (HaCaT cells) through the induction of oxidative stress. For this purpose, the cells were exposed to surface dielectric-barrier discharge plasma in 70% oxygen and 30% argon. We noted that cell viability was decreased following exposure of the cells to plasma in a time-dependent manner, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The levels of intracellular reactive oxygen species (ROS) were determined using 2′,7′-dichlorodihydro-fluorescein diacetate and dihydroethidium was used to monitor superoxide anion production. Plasma induced the generation of ROS, including superoxide anions, hydrogen peroxide and hydroxyl radicals. N-acetyl cysteine, which is an antioxidant, prevented the decrease in cell viability caused by exposure to plasma. ROS generated by exposure to plasma resulted in damage to various cellular components, including lipid membrane peroxidation, DNA breaks and protein carbonylation, which was detected by measuring the levels of 8-isoprostane and diphenyl-1-pyrenylphosphine assay, comet assay and protein carbonyl formation. These results suggest that plasma exerts cytotoxic effects by causing oxidative stress-induced damage to cellular components. PMID:26573561

  2. Blockage of JNK pathway enhances arsenic trioxide-induced apoptosis in human keratinocytes

    SciTech Connect

    Huang, H.-S.; Liu, Z.-M.; Hong, D.-Y.

    2010-04-15

    Arsenic is well known as a carcinogen predisposing humans to some severe diseases and also as an effective medicine for treating acute promyelocytic leukemia, syphilis, and psoriasis. Multiple active mechanisms, including cell cycle arrest and apoptosis, have been proposed in therapy; however, the opposing effects of arsenic remain controversial. Our previous study found that arsenic trioxide (ATO)-induced activation of p21{sup WAF1/CIP1} (p21) led to A431 cell death through the antagonistic effects of the signaling of ERK1/2 and JNK1. In the current study, the inhibitory effects of JNK1 on ATO-induced p21 expression were explored. Over-expression of JNK1 in A431 cells could inhibit p21 expression, which was associated with HDAC1 and TGIF. Using the GST pull-down assay and fluorescence resonance energy transfer analysis, N-terminal domain (amino acids 1-108) of TGIF, critical to its binding with c-Jun, was found. Using reporter assays, requirement of the C-terminal domain (amino acids 138-272) of TGIF to suppress ATO-induced p21 expression was observed. Thus, the domains of TGIF that carried out its inhibitory effects on p21 were identified. Finally, treatment with JNK inhibitor SP600125 could enhance ATO-induced apoptosis of HaCaT keratinocytes by using flow cytometry.

  3. Cobalt toxicity: chemical and radiological combined effects on HaCaT keratinocyte cell line.

    PubMed

    Gault, N; Sandre, C; Poncy, J-L; Moulin, C; Lefaix, J-L; Bresson, C

    2010-02-01

    Cobalt (Co) is an essential trace element well known as a constituent of vitamin B(12), but different compounds of Co are also described as highly toxic and/or radiotoxic for individuals or the environment. In nuclear power plants, (58)Co and (60)Co are radioactive isotopes of cobalt present as activation products of stable Co and Ni used in alloys. Skin exposure is a current occupational risk in the hard metal and nuclear industries. As biochemical and molecular cobalt-induced toxicological mechanisms are not fully identified, we investigated cobalt toxicity in a model human keratinocyte cell line, HaCaT. In this study, we propose a model to determine the in vitro chemical impact on cell viability of a soluble form of cobalt (CoCl(2)) with or without gamma-ray doses to mimic contamination by (60)Co, to elucidate the mechanisms of cobalt intracellular chemical and radiological toxicity. Intracellular cobalt concentration was determined after HaCaT cell contamination and chemical toxicity was evaluated in terms of cellular viability and clonogenic survival. We investigated damage to DNA in HaCaT cells by combined treatment with chemical cobalt and a moderate gamma-ray dose. Additive effects of cobalt and irradiation were demonstrated. The underlying mechanism of cobalt toxicity is not clearly established, but our results seem to indicate that the toxicity of Co(II) and of irradiation arises from production of reactive oxygen species.

  4. Streptolysin S Promotes Programmed Cell Death and Enhances Inflammatory Signaling in Epithelial Keratinocytes during Group A Streptococcus Infection

    PubMed Central

    Flaherty, Rebecca A.; Puricelli, Jessica M.; Higashi, Dustin L.; Park, Claudia J.

    2015-01-01

    Streptococcus pyogenes, or group A Streptococcus (GAS), is a pathogen that causes a multitude of human diseases from pharyngitis to severe infections such as toxic shock syndrome and necrotizing fasciitis. One of the primary virulence factors produced by GAS is the peptide toxin streptolysin S (SLS). In addition to its well-recognized role as a cytolysin, recent evidence has indicated that SLS may influence host cell signaling pathways at sublytic concentrations during infection. We employed an antibody array-based approach to comprehensively identify global host cell changes in human epithelial keratinocytes in response to the SLS toxin. We identified key SLS-dependent host responses, including the initiation of specific programmed cell death and inflammatory cascades with concomitant downregulation of Akt-mediated cytoprotection. Significant signaling responses identified by our array analysis were confirmed using biochemical and protein identification methods. To further demonstrate that the observed SLS-dependent host signaling changes were mediated primarily by the secreted toxin, we designed a Transwell infection system in which direct bacterial attachment to host cells was prevented, while secreted factors were allowed access to host cells. The results using this approach were consistent with our direct infection studies and reveal that SLS is a bacterial toxin that does not require bacterial attachment to host cells for activity. In light of these findings, we propose that the production of SLS by GAS during skin infection promotes invasive outcomes by triggering programmed cell death and inflammatory cascades in host cells to breach the keratinocyte barrier for dissemination into deeper tissues. PMID:26238711

  5. Oxygen tension changes the rate of migration of human skin keratinocytes in an age-related manner.

    PubMed

    Ross, Caitlin; Alston, Myrissa; Bickenbach, Jackie R; Aykin-Burns, Nukhet

    2011-01-01

    Migration of keratinocytes to re-epithelialize wounds is a key step in dermal wound healing. In aged human skin, wound healing rates decrease and cellular damage by reactive oxygen species (ROS) accumulates. The relationship between age, ROS and human skin keratinocyte migration is not clearly understood. In this study, 4% and 21% oxygen tensions were used to modify levels of ROS produced by metabolism to model low and high oxidative stress conditions. When migration of keratinocytes from young and old primary skin was compared using an in vitro scratch assay, old keratinocytes migrated faster in high oxygen tension than did young keratinocytes, whereas young keratinocytes migrated faster in low oxygen tension. Although all young and old cells at the scratch margins showed intense increases in dihydroethidium oxidation immediately after scratching, the old keratinocytes grown at 21% oxygen demonstrated a greater decrease in the DHE oxidation following scratching and migrated the fastest. These results show that old and young keratinocytes respond to oxygen tension differently and support the hypothesis that keratinocyte migration is affected by the capacity to remove ROS.

  6. Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.

    PubMed

    Tarafder, Abul K; Bolasco, Giulia; Correia, Maria S; Pereira, Francisco J C; Iannone, Lucio; Hume, Alistair N; Kirkpatrick, Niall; Picardo, Mauro; Torrisi, Maria R; Rodrigues, Inês P; Ramalho, José S; Futter, Clare E; Barral, Duarte C; Seabra, Miguel C

    2014-04-01

    The transfer of melanin from melanocytes to keratinocytes is a crucial process underlying maintenance of skin pigmentation and photoprotection against UV damage. Here, we present evidence supporting coupled exocytosis of the melanin core, or melanocore, by melanocytes and subsequent endocytosis by keratinocytes as a predominant mechanism of melanin transfer. Electron microscopy analysis of human skin samples revealed three lines of evidence supporting this: (1) the presence of melanocores in the extracellular space; (2) within keratinocytes, melanin was surrounded by a single membrane; and (3) this membrane lacked the melanosomal membrane protein tyrosinase-related protein 1 (TYRP1). Moreover, co-culture of melanocytes and keratinocytes suggests that melanin exocytosis is specifically induced by keratinocytes. Furthermore, depletion of Rab11b, but not Rab27a, caused a marked decrease in both keratinocyte-stimulated melanin exocytosis and transfer to keratinocytes. Thus, we propose that the predominant mechanism of melanin transfer is keratinocyte-induced exocytosis, mediated by Rab11b through remodeling of the melanosome membrane, followed by subsequent endocytosis by keratinocytes.

  7. Tungsten carbide cobalt nanoparticles exert hypoxia-like effects on the gene expression level in human keratinocytes

    PubMed Central

    2010-01-01

    Background Tungsten carbide (WC) and tungsten carbide cobalt (WC-Co) nanoparticles are of occupational health relevance because of the increasing usage in hard metal industries. Earlier studies showed an enhanced toxic potential for WC-Co compared to WC or cobalt ions alone. Therefore, we investigated the impact of these particles, compared to cobalt ions applied as CoCl2, on the global gene expression level in human keratinocytes (HaCaT) in vitro. Results WC nanoparticles exerted very little effects on the transcriptomic level after 3 hours and 3 days of exposure. In contrast, WC-Co nanoparticles caused significant transcriptional changes that were similar to those provoked by CoCl2. However, CoCl2 exerted even more pronounced changes in the transcription patterns. Gene set enrichment analyses revealed that the differentially expressed genes were related to hypoxia response, carbohydrate metabolism, endocrine pathways, and targets of several transcription factors. The role of the transcription factor HIF1 (hypoxia inducible factor 1) is particularly highlighted and aspects of downstream events as well as the role of other transcription factors related to cobalt toxicity are considered. Conclusions This study provides extensive data useful for the understanding of nanoparticle and cobalt toxicity. It shows that WC nanoparticles caused low transcriptional responses while WC-Co nanoparticles are able to exert responses similar to that of free cobalt ions, particularly the induction of hypoxia-like effects via interactions with HIF1α in human keratinocytes. However, the enhanced toxicity of WC-Co particles compared to CoCl2 could not be explained by differences in gene transcription. PMID:20105288

  8. Forkhead Box C1 Regulates Human Primary Keratinocyte Terminal Differentiation

    PubMed Central

    Deng, Liehua; Yang, Hengwen; Zhu, Leqing; Wang, Xiao; Edwards, Michael G.; Richers, Brittany; Leung, Donald Y. M.

    2016-01-01

    The epidermis serves as a critical protective barrier between the internal and external environment of the human body. Its remarkable barrier function is established through the keratinocyte (KC) terminal differentiation program. The transcription factors specifically regulating terminal differentiation remain largely unknown. Using a RNA-sequencing (RNA-seq) profiling approach, we found that forkhead box c 1 (FOXC1) was significantly up-regulated in human normal primary KC during the course of differentiation. This observation was validated in human normal primary KC from several different donors and human skin biopsies. Silencing FOXC1 in human normal primary KC undergoing differentiation led to significant down-regulation of late terminal differentiation genes markers including epidermal differentiation complex genes, keratinization genes, sphingolipid/ceramide metabolic process genes and epidermal specific cell-cell adhesion genes. We further demonstrated that FOXC1 works down-stream of ZNF750 and KLF4, and upstream of GRHL3. Thus, this study defines FOXC1 as a regulator specific for KC terminal differentiation and establishes its potential position in the genetic regulatory network. PMID:27907090

  9. Human Beta-papillomavirus infection and keratinocyte carcinomas.

    PubMed

    Quint, Koen D; Genders, Roel E; de Koning, Maurits N C; Borgogna, Cinzia; Gariglio, Marisa; Bouwes Bavinck, Jan Nico; Doorbar, John; Feltkamp, Mariet C

    2015-01-01

    Although the role of oncogenic human Alpha-papillomaviruses (HPVs) in the development of mucosal carcinomas at different body sites (eg cervix, anus, oropharynx) is fully recognized, a role for HPV in keratinocyte carcinomas (KCs; basal and squamous cell carcinomas) of the skin is not yet clear. KCs are the most common cancers in Caucasians, with the major risk factor being ultraviolet (UV) light exposure. A possible role for Beta-HPV types (BetaPV) in the development of KC was suggested several decades ago, supported by a number of epidemiological studies. Our current review summarizes the recent molecular and histopathological evidence in support of a causal association between BetaPV and the development of KC, and outlines the suspected synergistic effect of viral gene expression with UV radiation and immune suppression. Further insights into the molecular pathways and protein interactions used by BetaPV and the host cell is likely to extend our understanding of the role of BetaPV in KC.

  10. Vanillin protects human keratinocyte stem cells against ultraviolet B irradiation.

    PubMed

    Lee, Jienny; Cho, Jae Youl; Lee, Sang Yeol; Lee, Kyung-Woo; Lee, Jongsung; Song, Jae-Young

    2014-01-01

    Ultraviolet-B (UVB) irradiation is one of major factors which induce cellular damages in the epidermis. We investigated protective effects and mechanisms of vanillin, a main constituent of vanilla beans, against UVB-induced cellular damages in keratinocyte stem cells (KSC). Here, vanillin significantly attenuated UVB irradiation-induced cytotoxicity. The vanillin effects were also demonstrated by the results of the senescence-associated β-galactosidase and alkaline comet assays. In addition, vanillin induced production of pro-inflammatory cytokines. Attempts to elucidate a possible mechanism underlying the vanillin-mediated effects revealed that vanillin significantly reduced UVB-induced phosphorylation of ataxia telangiectasia mutated (ATM), serine threonine kinase checkpoint kinase 2 (Chk2), tumor suppressor protein 53 (p53), p38/mitogen-activated protein kinase (p38), c-Jun N-terminal kinase/stress-activated protein kinase (JNK), S6 ribosomal protein (S6RP), and histone 2A family member X (H2A.X). UVB-induced activation of p53 luciferase reporter was also significantly inhibited by vanillin. In addition, while ATM inhibitor had no effect on the vanillin effects, mouse double minute 2 homolog (MDM2) inhibitor significantly attenuated suppressive effects of vanillin on UVB-induced activation of p53 reporter in KSC. Taken together, these findings suggest that vanillin protects KSC from UVB irradiation and its effects may occur through the suppression of downstream step of MDM2 in UVB irradiation-induced p53 activation.

  11. Keratinocyte stem cells and the targets for nonmelanoma skin cancer.

    PubMed

    Singh, Ashok; Park, Heuijoon; Kangsamaksin, Thaned; Singh, Anupama; Readio, Nyssa; Morris, Rebecca J

    2012-01-01

    The mammalian skin is a complex dynamic organ composed of thin multilayered epidermis and a thick underlying connective tissue layer dermis. The epidermis undergoes continuous renewal throughout life. The stems cells uniquely express particular surface markers utilized for their identification, isolation and localization in specific niches in epidermis as well as hair follicles (HFs). The two stage skin carcinogenesis model involves stepwise accumulation of genetic alterations and ultimately leading to malignancy. Whereas early research on skin carcinogenesis focused on the molecular nature of carcinogens and tumor promoters, more recent studies have focused on the identification of the target cells and tumor promoting cells for both chemical and physical carcinogens and promoters. Recent studies support the hypothesis that keratinocyte stem cells are the targets in skin carcinogenesis. In this review, we discuss briefly the localization of stem cells in the epidermis and HFs, and review the possibility that skin papillomas and carcinomas are derived from stem cells, as well as from other cells in the cutaneous epithelium whose stem cell properties are not well known.

  12. Keratinocyte gene therapy for systemic diseases. Circulating interleukin 10 released from gene-transferred keratinocytes inhibits contact hypersensitivity at distant areas of the skin.

    PubMed Central

    Meng, X; Sawamura, D; Tamai, K; Hanada, K; Ishida, H; Hashimoto, I

    1998-01-01

    This study has examined the systemic effects of a circulating gene product, human interleukin 10 (IL-10), released from transduced keratinocytes. IL-10 is an anti-inflammatory cytokine which has an inhibitory effect on contact hypersensitivity (CHS). An expression vector (phIL-10) was constructed for human IL-10 and was injected into the dorsal skin of hairless rats. Local expression of IL-10 mRNA and protein was detected by reverse-transcriptase polymerase chain reaction and immunohistochemical staining, respectively. Enzyme-linked immunosorbent assay showed that the amount of IL-10 in the local keratinocytes and in the circulation increased with the dose of phIL-10 transferred. To determine whether circulating IL-10 could inhibit the effector phase of CHS at a distant area of the skin, various doses of phIL-10 were injected into the dorsal skin of sensitized rats before challenge on the ears. Our results showed that the degree of swelling of the ears of phIL-10- treated rats was significantly lower than that in the negative control animals. These results suggest that IL-10 released from transduced keratinocytes can enter the bloodstream and cause biological effects at distant areas of the skin. This study demonstrates that it may be possible to treat systemic disease using keratinocyte gene therapy. PMID:9502789

  13. Transcription factor 7-like 1 dysregulates keratinocyte differentiation through upregulating lipocalin 2

    PubMed Central

    Xu, M; Zhang, Y; Cheng, H; Liu, Y; Zou, X; Zhan, N; Xiao, S; Xia, Y

    2016-01-01

    Recent studies strongly suggested that transcription factor 7-like 1 (Tcf7l1, also known as Tcf3) is involved in the differentiation of several types of cells, and demonstrated that Tcf7l1 modulates keratinocytes physiologically through regulating lipocalin 2 (LCN2), a key regulator of cell differentiation. To reveal the potential role of Tcf7l1 in the dysregulation of keratinocyte differentiation, both Tcf7l1 and LCN2 were determined in a variety of skin disorders. The in vitro effect of Tcf7l1 on keratinocyte differentiation was studied by culturing SCC-13 cells, and the human foreskin keratinocytes (HFKs) that were transfected with vectors for overexpressing human papillomavirus E6/E7 or Tcf7l1 genes. We found that both Tcf7l1 and LCN2 were highly expressed in those diseases characterized by defective keratinocyte differentiation (especially psoriasis vulgaris, condyloma acuminatum, squamous cell carcinoma, etc). Moreover, compared with control HFKs, SCC-13 cells and E6/E7-harboring HFKs expressed more Tcf7l1 and LCN2. Tcf7l1 siRNA transfection decreased LCN2 but increased involucrin and loricrin in HFKs under calcium stimuli. Conversely, Tcf7l1 overexpression in SCC-13 cells or vector-transfected HFKs induced lower involucrin and loricrin expression and less keratinocyte apoptosis, both of which, however, were partially abrogated by LCN2 siRNA or neutralizing anti-LCN2 antibody. Interestingly, the Tcf7l1 expression in HFKs correlated positively with the MMP-2 level, and the inhibition of MMP-2 decreased the LCN2 level and even attenuated the effect of Tcf7l1 on LCN2 expression. Therefore, Tcf7l1 dysregulates keratinocyte differentiation, possibly through upregulating the LCN2 pathway in an MMP-2 mediated manner. Elucidating the interaction between Tcf7l1 and LCN2 may help understand disordered cell differentiation in some skin diseases. PMID:27551519

  14. Keratinocytes contribute intrinsically to psoriasis upon loss of Tnip1 function.

    PubMed

    Ippagunta, Sirish K; Gangwar, Ruchika; Finkelstein, David; Vogel, Peter; Pelletier, Stephane; Gingras, Sebastien; Redecke, Vanessa; Häcker, Hans

    2016-10-11

    Psoriasis is a chronic inflammatory skin disease with a clear genetic contribution, characterized by keratinocyte proliferation and immune cell infiltration. Various closely interacting cell types, including innate immune cells, T cells, and keratinocytes, are known to contribute to inflammation. Innate immune cells most likely initiate the inflammatory process by secretion of IL-23. IL-23 mediates expansion of T helper 17 (Th17) cells, whose effector functions, including IL-17A, activate keratinocytes. Keratinocyte activation in turn results in cell proliferation and chemokine expression, the latter of which fuels the inflammatory process through further immune cell recruitment. One question that remains largely unanswered is how genetic susceptibility contributes to this process and, specifically, which cell type causes disease due to psoriasis-specific genetic alterations. Here we describe a mouse model based on the human psoriasis susceptibility locus TNIP1, also referred to as ABIN1, whose gene product is a negative regulator of various inflammatory signaling pathways, including the Toll-like receptor pathway in innate immune cells. We find that Tnip1-deficient mice recapitulate major features of psoriasis on pathological, genomic, and therapeutic levels. Different genetic approaches, including tissue-specific gene deletion and the use of various inflammatory triggers, reveal that Tnip1 controls not only immune cells, but also keratinocyte biology. Loss of Tnip1 in keratinocytes leads to deregulation of IL-17-induced gene expression and exaggerated chemokine production in vitro and overt psoriasis-like inflammation in vivo. Together, the data establish Tnip1 as a critical regulator of IL-17 biology and reveal a causal role of keratinocytes in the pathogenesis of psoriasis.

  15. Overexpressed TRPV3 ion channels in skin keratinocytes modulate pain sensitivity via prostaglandin E2

    PubMed Central

    Huang, Susan M.; Lee, Hyosang; Chung, Man-Kyo; Park, Una; Yu, Yin Yin; Bradshaw, Heather B.; Coulombe, Pierre A.; Walker, J. Michael; Caterina, Michael J.

    2009-01-01

    The ability to sense changes in the environment is essential for survival because it permits responses such as withdrawal from noxious stimuli and regulation of body temperature. Keratinocytes, which occupy much of the skin epidermis, are situated at the interface between the external environment and the body's internal milieu, and have long been appreciated for their barrier function against external insults. The recent discovery of temperature-sensitive TRPV ion channels in keratinocytes has raised the possibility that these cells also actively participate in acute temperature and pain sensation. To address this notion, we generated and characterized transgenic mice that overexpress TRPV3 in epidermal keratinocytes under the control of the keratin 14 promoter. Compared to wild-type controls, keratinocytes overexpressing TRPV3 exhibited larger currents as well as augmented prostaglandin E2 (PGE2) release in response to two TRPV3 agonists, 2-aminoethoxydiphenyl borate (2APB) and heat. Thermal selection behavior and heat-evoked withdrawal behavior of naïve mice overexpressing TRPV3 were not consistently altered. Upon selective pharmacological inhibition of TRPV1 with JNJ-7203212, however, the keratinocyte-specific TRPV3 transgenic mice showed increased escape responses to noxious heat relative to their wild-type littermates. Co-administration of the cyclooxygenase inhibitor, ibuprofen, with the TRPV1 antagonist decreased inflammatory thermal hyperalgesia in transgenic but not wild-type animals. Our results reveal a previously undescribed mechanism for keratinocyte participation in thermal pain transduction through keratinocyte TRPV3 ion channels and the intercellular messenger PGE2. PMID:19091963

  16. The effects of microenvironment on wound healing by keratinocytes derived from mesenchymal stem cells.

    PubMed

    Lin, Yi-Han; Fu, Keng-Yen; Hong, Po-Da; Ma, Hsu; Liou, Nien-Hsien; Ma, Kuo-Hsing; Liu, Jiang-Chuan; Huang, Kun-Lun; Dai, Lien-Guo; Chang, Shun-Cheng; Yi-Hsin Chan, James; Chen, Shyi-Gen; Chen, Tim-Mo; Dai, Niann-Tzyy

    2013-12-01

    Embryonic stem cells (ESCs) are pluripotent cells that can differentiate into various cell types, including keratinocyte-like cells, within suitable microniches. In this study, we aimed to investigate the effects of culture media, cell coculture, and a tissue-engineering biocomposite on the differentiation of mouse ESCs (MESCs) into keratinocyte-like cells and applied these cells to a surgical skin wound model. MESCs from BALB/c mice (ESC26GJ), which were transfected using pCX-EGFP expressing green fluorescence, were used to track MESC-derived keratinocytes. Weak expression of the keratinocyte early marker Cytokeratin 14 (CK-14) was observed up to 12 days when MESCs were cultured in a keratinocyte culture medium on tissue culture plastic and on a gelatin/collagen/polycaprolactone (GCP) biocomposite. MESCs cocultured with human keratinocyte cells (HKCs) also expressed CK-14, but did not express CK-14 when cocultured with human fibroblast cells (HFCs). Furthermore, CK-14 expression was observed when MESCs were cocultured by seeding HKCs or HFCs on the same or opposite side of the GCP biocomposite. The highest CK-14 expression was observed by seeding MESCs and HKCs on the same side of the GCP composite and with HFCs on the opposite side. To verify the effectiveness of wound healing in vivo, adipose-derived stem cells were applied to treat surgical wounds in nude mice. An obvious epidermis multilayer and better collagen deposition during wound healing were observed, as assessed by Masson staining. This study demonstrated the potential of keratinocyte-like differentiation from mesenchymal stem cells for use in promoting wound closure and skin regeneration.

  17. Inhibitory effect of beta-thujaplicin on ultraviolet B-induced apoptosis in mouse keratinocytes.

    PubMed

    Baba, T; Nakano, H; Tamai, K; Sawamura, D; Hanada, K; Hashimoto, I; Arima, Y

    1998-01-01

    Sunburn cells are thought to represent ultraviolet B-induced apoptotic keratinocytes. It has been demonstrated that enzymatic and nonenzymatic antioxidants effectively suppress sunburn cell formation, indicating that reactive oxygen species may play a role in the progression of ultraviolet B-induced apoptosis. Metallothionein, a cytosol protein, has antioxidant activity, and overexpression of metallothionein has been reported to reduce the number of sunburn cells in mouse skin. We have also demonstrated that overexpression of metallothionein inhibits ultraviolet B-induced DNA ladder formation in mouse keratinocytes. These findings support the hypothesis that cellular metallothionein may play an important role in the inhibition of ultraviolet B-induced apoptosis in keratinocytes through its antioxidant activity. In the present study, we investigated the effects of beta-thujaplicin, an extract from the woods of Thuja plicata D. Don. and Chamaecyparis obtuse, Sieb. et Zucc., on ultraviolet B-induced apoptosis in keratinocytes and on metallothionein induction. Topical application of beta-thujaplicin decreased the number of ultraviolet B-mediated sunburn cells and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling-positive cells in mouse ear skin. Incubation with beta-thujaplicin suppressed ultraviolet B-induced DNA ladder formation in cultured mouse keratinocytes. Histochemical analysis showed that topical application of beta-thujaplicin induced metallothionein protein in mouse skin. Northern analysis and western blotting revealed significant induction of metallothionein mRNA and metallothionein protein, respectively, in beta-thujaplicin-treated cultured mouse keratinocytes. These findings indicate that beta-thujaplicin inhibits ultraviolet B-induced apoptosis in keratinocytes and strongly suggest that the inhibitory mechanism is due to the antioxidant activity of metallothionein induced by the agent.

  18. The Qualitative Similarity Hypothesis

    ERIC Educational Resources Information Center

    Paul, Peter V.; Lee, Chongmin

    2010-01-01

    Evidence is presented for the qualitative similarity hypothesis (QSH) with respect to children and adolescents who are d/Deaf or hard of hearing. The primary focus is on the development of English language and literacy skills, and some information is provided on the acquisition of English as a second language. The QSH is briefly discussed within…

  19. Quantifying Similarity in Seismic Polarizations

    NASA Astrophysics Data System (ADS)

    Eaton, D. W. S.; Jones, J. P.; Caffagni, E.

    2015-12-01

    Measuring similarity in seismic attributes can help identify tremor, low S/N signals, and converted or reflected phases, in addition to diagnosing site noise and sensor misalignment in arrays. Polarization analysis is a widely accepted method for studying the orientation and directional characteristics of seismic phases via. computed attributes, but similarity is ordinarily discussed using qualitative comparisons with reference values. Here we introduce a technique for quantitative polarization similarity that uses weighted histograms computed in short, overlapping time windows, drawing on methods adapted from the image processing and computer vision literature. Our method accounts for ambiguity in azimuth and incidence angle and variations in signal-to-noise (S/N) ratio. Using records of the Mw=8.3 Sea of Okhotsk earthquake from CNSN broadband sensors in British Columbia and Yukon Territory, Canada, and vertical borehole array data from a monitoring experiment at Hoadley gas field, central Alberta, Canada, we demonstrate that our method is robust to station spacing. Discrete wavelet analysis extends polarization similarity to the time-frequency domain in a straightforward way. Because histogram distance metrics are bounded by [0 1], clustering allows empirical time-frequency separation of seismic phase arrivals on single-station three-component records. Array processing for automatic seismic phase classification may be possible using subspace clustering of polarization similarity, but efficient algorithms are required to reduce the dimensionality.

  20. What difference reveals about similarity.

    PubMed

    Sagi, Eyal; Gentner, Dedre; Lovett, Andrew

    2012-08-01

    Detecting that two images are different is faster for highly dissimilar images than for highly similar images. Paradoxically, we showed that the reverse occurs when people are asked to describe how two images differ--that is, to state a difference between two images. Following structure-mapping theory, we propose that this disassociation arises from the multistage nature of the comparison process. Detecting that two images are different can be done in the initial (local-matching) stage, but only for pairs with low overlap; thus, "different" responses are faster for low-similarity than for high-similarity pairs. In contrast, identifying a specific difference generally requires a full structural alignment of the two images, and this alignment process is faster for high-similarity pairs. We described four experiments that demonstrate this dissociation and show that the results can be simulated using the Structure-Mapping Engine. These results pose a significant challenge for nonstructural accounts of similarity comparison and suggest that structural alignment processes play a significant role in visual comparison.

  1. Development of a Full-Thickness Human Skin Equivalent In Vitro Model Derived from TERT-Immortalized Keratinocytes and Fibroblasts

    PubMed Central

    Reijnders, Christianne M.A.; van Lier, Amanda; Roffel, Sanne; Kramer, Duco; Scheper, Rik J.

    2015-01-01

    Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve these limitations. The aim was to develop a fully differentiated HSE constructed entirely from human skin cell lines, which could be applied for in vitro wound-healing assays. Skin equivalents were constructed from human TERT-immortalized keratinocytes and fibroblasts (TERT-HSE) and compared with native skin and primary HSEs. HSEs were characterized by hematoxylin–eosin and immunohistochemical stainings with markers for epidermal proliferation and differentiation, basement membrane (BM), fibroblasts, and the extracellular matrix (ECM). Ultrastructure was determined with electron microscopy. To test the functionality of the TERT-HSE, burn and cold injuries were applied, followed by immunohistochemical stainings, measurement of reepithelialization, and determination of secreted wound-healing mediators. The TERT-HSE was composed of a fully differentiated epidermis and a fibroblast-populated dermis comparable to native skin and primary HSE. The epidermis consisted of proliferating keratinocytes within the basal layer, followed by multiple spinous layers, a granular layer, and cornified layers. Within the TERT-HSE, the membrane junctions such as corneosomes, desmosomes, and hemidesmosomes were well developed as shown by ultrastructure pictures. Furthermore, the BM consisted of a lamina lucida and lamina densa comparable to native skin. The dermal matrix of the TERT-HSE was more similar to native skin than the primary construct, since collagen III, an ECM marker, was present in TERT-HSEs and absent in primary HSEs. After wounding, the TERT-HSE was able to reepithelialize and secrete inflammatory wound-healing mediators. In conclusion, the novel TERT-HSE, constructed entirely

  2. Development of a Full-Thickness Human Skin Equivalent In Vitro Model Derived from TERT-Immortalized Keratinocytes and Fibroblasts.

    PubMed

    Reijnders, Christianne M A; van Lier, Amanda; Roffel, Sanne; Kramer, Duco; Scheper, Rik J; Gibbs, Susan

    2015-09-01

    Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve these limitations. The aim was to develop a fully differentiated HSE constructed entirely from human skin cell lines, which could be applied for in vitro wound-healing assays. Skin equivalents were constructed from human TERT-immortalized keratinocytes and fibroblasts (TERT-HSE) and compared with native skin and primary HSEs. HSEs were characterized by hematoxylin-eosin and immunohistochemical stainings with markers for epidermal proliferation and differentiation, basement membrane (BM), fibroblasts, and the extracellular matrix (ECM). Ultrastructure was determined with electron microscopy. To test the functionality of the TERT-HSE, burn and cold injuries were applied, followed by immunohistochemical stainings, measurement of reepithelialization, and determination of secreted wound-healing mediators. The TERT-HSE was composed of a fully differentiated epidermis and a fibroblast-populated dermis comparable to native skin and primary HSE. The epidermis consisted of proliferating keratinocytes within the basal layer, followed by multiple spinous layers, a granular layer, and cornified layers. Within the TERT-HSE, the membrane junctions such as corneosomes, desmosomes, and hemidesmosomes were well developed as shown by ultrastructure pictures. Furthermore, the BM consisted of a lamina lucida and lamina densa comparable to native skin. The dermal matrix of the TERT-HSE was more similar to native skin than the primary construct, since collagen III, an ECM marker, was present in TERT-HSEs and absent in primary HSEs. After wounding, the TERT-HSE was able to reepithelialize and secrete inflammatory wound-healing mediators. In conclusion, the novel TERT-HSE, constructed entirely

  3. Influence of Physiologic Folate Deficiency on Human Papillomavirus Type 16 (HPV16)-harboring Human Keratinocytes in Vitro and in Vivo*

    PubMed Central

    Xiao, Suhong; Tang, Ying-Sheng; Khan, Rehana A.; Zhang, Yonghua; Kusumanchi, Praveen; Stabler, Sally P.; Jayaram, Hiremagalur N.; Antony, Aśok C.

    2012-01-01

    Although HPV16 transforms infected epithelial tissues to cancer in the presence of several co-factors, there is insufficient molecular evidence that poor nutrition has any such role. Because physiological folate deficiency led to the intracellular homocysteinylation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) and activated a nutrition-sensitive (homocysteine-responsive) posttranscriptional RNA operon that included interaction with HPV16 L2 mRNA, we investigated the functional consequences of folate deficiency on HPV16 in immortalized HPV16-harboring human (BC-1-Ep/SL) keratinocytes and HPV16-organotypic rafts. Although homocysteinylated hnRNP-E1 interacted with HPV16 L2 mRNA cis-element, it also specifically bound another HPV16 57-nucleotide poly(U)-rich cis-element in the early polyadenylation element (upstream of L2̂L1 genes) with greater affinity. Together, these interactions led to a profound reduction of both L1 and L2 mRNA and proteins without effects on HPV16 E6 and E7 in vitro, and in cultured keratinocyte monolayers and HPV16-low folate-organotypic rafts developed in physiological low folate medium. In addition, HPV16-low folate-organotypic rafts contained fewer HPV16 viral particles, a similar HPV16 DNA viral load, and a much greater extent of integration of HPV16 DNA into genomic DNA when compared with HPV16-high folate-organotypic rafts. Subcutaneous implantation of 18-day old HPV16-low folate-organotypic rafts into folate-replete immunodeficient mice transformed this benign keratinocyte-derived raft tissue into an aggressive HPV16-induced cancer within 12 weeks. Collectively, these studies establish a likely molecular linkage between poor folate nutrition and HPV16 and predict that nutritional folate and/or vitamin-B12 deficiency, which are both common worldwide, will alter the natural history of HPV16 infections and also warrant serious consideration as reversible co-factors in oncogenic transformation of HPV16-infected tissues to cancer

  4. Influence of physiologic folate deficiency on human papillomavirus type 16 (HPV16)-harboring human keratinocytes in vitro and in vivo.

    PubMed

    Xiao, Suhong; Tang, Ying-Sheng; Khan, Rehana A; Zhang, Yonghua; Kusumanchi, Praveen; Stabler, Sally P; Jayaram, Hiremagalur N; Antony, Asok C

    2012-04-06

    Although HPV16 transforms infected epithelial tissues to cancer in the presence of several co-factors, there is insufficient molecular evidence that poor nutrition has any such role. Because physiological folate deficiency led to the intracellular homocysteinylation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) and activated a nutrition-sensitive (homocysteine-responsive) posttranscriptional RNA operon that included interaction with HPV16 L2 mRNA, we investigated the functional consequences of folate deficiency on HPV16 in immortalized HPV16-harboring human (BC-1-Ep/SL) keratinocytes and HPV16-organotypic rafts. Although homocysteinylated hnRNP-E1 interacted with HPV16 L2 mRNA cis-element, it also specifically bound another HPV16 57-nucleotide poly(U)-rich cis-element in the early polyadenylation element (upstream of L2L1 genes) with greater affinity. Together, these interactions led to a profound reduction of both L1 and L2 mRNA and proteins without effects on HPV16 E6 and E7 in vitro, and in cultured keratinocyte monolayers and HPV16-low folate-organotypic rafts developed in physiological low folate medium. In addition, HPV16-low folate-organotypic rafts contained fewer HPV16 viral particles, a similar HPV16 DNA viral load, and a much greater extent of integration of HPV16 DNA into genomic DNA when compared with HPV16-high folate-organotypic rafts. Subcutaneous implantation of 18-day old HPV16-low folate-organotypic rafts into folate-replete immunodeficient mice transformed this benign keratinocyte-derived raft tissue into an aggressive HPV16-induced cancer within 12 weeks. Collectively, these studies establish a likely molecular linkage between poor folate nutrition and HPV16 and predict that nutritional folate and/or vitamin-B(12) deficiency, which are both common worldwide, will alter the natural history of HPV16 infections and also warrant serious consideration as reversible co-factors in oncogenic transformation of HPV16-infected tissues to cancer.

  5. Human keratinocytes restrict chikungunya virus replication at a post-fusion step

    SciTech Connect

    Bernard, Eric; Simmons, Graham; Chazal, Nathalie; and others

    2015-02-15

    Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses. - Highlights: • Human keratinocytes support endocytosis of CHIKV and fusion of viral membranes. • CHIKV replication is blocked at a post entry step in these cells. • Infection upregulates type-I, –II and –III IFN genes expression. • Keratinocytes behave as immune sentinels against CHIKV.

  6. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts

    PubMed Central

    Ramos-Jerz, Maria del R.; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M.

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes. PMID:24371457

  7. Rictor/mTORC2 deficiency enhances keratinocyte stress tolerance via mitohormesis.

    PubMed

    Tassone, Beatrice; Saoncella, Stefania; Neri, Francesco; Ala, Ugo; Brusa, Davide; Magnuson, Mark A; Provero, Paolo; Oliviero, Salvatore; Riganti, Chiara; Calautti, Enzo

    2017-04-01

    How metabolic pathways required for epidermal tissue growth and remodeling influence the ability of keratinocytes to survive stressful conditions is still largely unknown. The mechanistic target of rapamycin complex 2 (mTORC2) regulates growth and metabolism of several tissues, but its functions in epidermal cells are poorly defined. Rictor is an adaptor protein essential for mTORC2 activity. To explore the roles of mTORC2 in the epidermis, we have conditionally deleted rictor in mice via K14-Cre-mediated homologous recombination and found that its deficiency causes moderate tissue hypoplasia, reduced keratinocyte proliferation and attenuated hyperplastic response to TPA. Noteworthy, rictor-deficient keratinocytes displayed increased lifespan, protection from senescence, and enhanced tolerance to cellular stressors such as growth factors deprivation, epirubicin and X-ray in vitro and radioresistance in vivo. Rictor-deficient keratinocytes exhibited changes in global gene expression profiles consistent with metabolic alterations and enhanced stress tolerance, a shift in cell catabolic processes from glycids and lipids to glutamine consumption and increased production of mitochondrial reactive oxygen species (ROS). Mechanistically, the resiliency of rictor-deficient epidermal cells relies on these ROS increases, indicating stress resistance via mitohormesis. Thus, our findings reveal a new link between metabolic changes and stress adaptation of keratinocytes centered on mTORC2 activity, with potential implications in skin aging and therapeutic resistance of epithelial tumors.

  8. Keratins as the main component for the mechanical integrity of keratinocytes.

    PubMed

    Ramms, Lena; Fabris, Gloria; Windoffer, Reinhard; Schwarz, Nicole; Springer, Ronald; Zhou, Chen; Lazar, Jaroslav; Stiefel, Simone; Hersch, Nils; Schnakenberg, Uwe; Magin, Thomas M; Leube, Rudolf E; Merkel, Rudolf; Hoffmann, Bernd

    2013-11-12

    Keratins are major components of the epithelial cytoskeleton and are believed to play a vital role for mechanical integrity at the cellular and tissue level. Keratinocytes as the main cell type of the epidermis express a differentiation-specific set of type I and type II keratins forming a stable network and are major contributors of keratinocyte mechanical properties. However, owing to compensatory keratin expression, the overall contribution of keratins to cell mechanics was difficult to examine in vivo on deletion of single keratin genes. To overcome this problem, we used keratinocytes lacking all keratins. The mechanical properties of these cells were analyzed by atomic force microscopy (AFM) and magnetic tweezers experiments. We found a strong and highly significant softening of keratin-deficient keratinocytes when analyzed by AFM on the cell body and above the nucleus. Magnetic tweezers experiments fully confirmed these results showing, in addition, high viscous contributions to magnetic bead displacement in keratin-lacking cells. Keratin loss neither affected actin or microtubule networks nor their overall protein concentration. Furthermore, depolymerization of actin preserves cell softening in the absence of keratin. On reexpression of the sole basal epidermal keratin pair K5/14, the keratin filament network was reestablished, and mechanical properties were restored almost to WT levels in both experimental setups. The data presented here demonstrate the importance of keratin filaments for mechanical resilience of keratinocytes and indicate that expression of a single keratin pair is sufficient for almost complete reconstitution of their mechanical properties.

  9. Hypoxia Regulates mTORC1-Mediated Keratinocyte Motility and Migration via the AMPK Pathway

    PubMed Central

    Yan, Tiantian; Zhang, Junhui; Tang, Di; Zhang, Xingyue; Jiang, Xupin; Zhao, Liping; Zhang, Qiong; Zhang, Dongxia; Huang, Yuesheng

    2017-01-01

    Keratinocyte migration, the initial event and rate-limiting step in wound healing, plays a vital role in restoration of the intact skin barrier, also known as re-epithelialization. After acute tissue injury, hypoxic microenvironment gradually develops and acts as an early stimulus to initiate the healing process. Although we have previously found that hypoxia induces keratinocyte migration, the underlying mechanism remains unknown. Here, we first observed that hypoxia increased mTORC1 activity. Recombinant lentivirus vector and Rapamycin were used for silencing mTORC1 in HaCaT cells and primary mouse keratinocytes (MKs). Using cell migration assay and a Zeiss chamber equipped with imaging system, we also demonstrated that mTORC1 downregulation reversed hypoxia-induced keratinocyte motility and lateral migration. Importantly, hypoxia-activated mTORC1 was accompanied by the AMPK downregulation, and we found that the AMPK pathway activators Metformin (Met) and 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) decreased the mTORC1 activity, cell motility and lateral migration. Thus, our results suggest that hypoxia regulates mTORC1-mediated keratinocyte motility and migration via the AMPK pathway. PMID:28068384

  10. ROCK inhibitor reduces Myc-induced apoptosis and mediates immortalization of human keratinocytes

    PubMed Central

    Dakic, Aleksandra; DiVito, Kyle; Fang, Shuang; Suprynowicz, Frank; Gaur, Anirudh; Li, Xin; Palechor-Ceron, Nancy; Simic, Vera; Choudhury, Sujata; Yu, Songtao; Simbulan-Rosenthal, Cynthia M.; Rosenthal, Dean; Schlegel, Richard; Liu, Xuefeng

    2016-01-01

    The Myc/Max/Mad network plays a critical role in cell proliferation, differentiation and apoptosis and c-Myc is overexpressed in many cancers, including HPV-positive cervical cancer cell lines. Despite the tolerance of cervical cancer keratinocytes to high Myc expression, we found that the solitary transduction of the Myc gene into primary cervical and foreskin keratinocytes induced rapid cell death. These findings suggested that the anti-apoptotic activity of E7 in cervical cancer cells might be responsible for negating the apoptotic activity of over-expressed Myc. Indeed, our earlier in vitro studies demonstrated that Myc and E7 synergize in the immortalization of keratinocytes. Since we previously postulated that E7 and the ROCK inhibitor, Y-27632, were members of the same functional pathway in cell immortalization, we tested whether Y-27632 would inhibit apoptosis induced by the over-expression of Myc. Our findings indicate that Y-27632 rapidly inhibited Myc-induced membrane blebbing and cellular apoptosis and, more generally, functioned as an inhibitor of extrinsic and intrinsic pathways of cell death. Most important, Y-27632 cooperated with Myc to immortalize keratinocytes efficiently, indicating that apoptosis is a major barrier to Myc-induced immortalization of keratinocytes. The anti-apoptotic activity of Y-27632 correlated with a reduction in p53 serine 15 phosphorylation and the consequent reduction in the expression of downstream target genes p21 and DAPK1, two genes involved in the induction of cell death. PMID:27556514

  11. A Glutathione-Nrf2-Thioredoxin Cross-Talk Ensures Keratinocyte Survival and Efficient Wound Repair

    PubMed Central

    Telorack, Michèle; Meyer, Michael; Ingold, Irina; Conrad, Marcus; Bloch, Wilhelm; Werner, Sabine

    2016-01-01

    The tripeptide glutathione is the most abundant cellular antioxidant with high medical relevance, and it is also required as a co-factor for various enzymes involved in the detoxification of reactive oxygen species and toxic compounds. However, its cell-type specific functions and its interaction with other cytoprotective molecules are largely unknown. Using a combination of mouse genetics, functional cell biology and pharmacology, we unraveled the function of glutathione in keratinocytes and its cross-talk with other antioxidant defense systems. Mice with keratinocyte-specific deficiency in glutamate cysteine ligase, which catalyzes the rate-limiting step in glutathione biosynthesis, showed a strong reduction in keratinocyte viability in vitro and in the skin in vivo. The cells died predominantly by apoptosis, but also showed features of ferroptosis and necroptosis. The increased cell death was associated with increased levels of reactive oxygen and nitrogen species, which caused DNA and mitochondrial damage. However, epidermal architecture, and even healing of excisional skin wounds were only mildly affected in the mutant mice. The cytoprotective transcription factor Nrf2 was strongly activated in glutathione-deficient keratinocytes, but additional loss of Nrf2 did not aggravate the phenotype, demonstrating that the cytoprotective effect of Nrf2 is glutathione dependent. However, we show that deficiency in glutathione biosynthesis is efficiently compensated in keratinocytes by the cysteine/cystine and thioredoxin systems. Therefore, our study highlights a remarkable antioxidant capacity of the epidermis that ensures skin integrity and efficient wound healing. PMID:26808544

  12. B-Myb enhances proliferation and suppresses differentiation of keratinocytes in three-dimensional cell culture.

    PubMed

    Maruyama, Hiroshi; Ishitsuka, Yosuke; Fujisawa, Yasuhiro; Furuta, Junichi; Sekido, Mitsuru; Kawachi, Yasuhiro

    2014-05-01

    B-Myb (Mybl2) is a member of the Myb gene family of transcription factors involved in the control of cell growth, differentiation, and apoptosis. The effects of B-Myb on keratinocyte proliferation and differentiation have not yet been clarified. The present study was performed to examine the role of B-Myb in proliferation and differentiation of the spontaneously immortalized human skin keratinocyte cell line HaCaT and normal human keratinocytes with formation of a stratified epidermoid structure in air-liquid interface three-dimensional culture. B-Myb was expressed specifically in undifferentiated normal keratinocytes and downregulated during differentiation. The constitutive overexpression of B-Myb in HaCaT cells during air exposure-induced differentiation resulted in an undifferentiated phenotype, i.e., thickening of the stratified layers, suppression of differentiation marker expression, and retention of proliferative activity with activation of cell cycle regulatory proteins in the S and G2/M phases. In contrast, suppression of B-Myb caused their downregulation and constrained proliferation with retention of differentiation capacity. These findings suggested that B-Myb plays an important role in maintenance of the undifferentiated phenotype of keratinocytes in the basal epidermal layer.

  13. Type IV collagen aggregates promote keratinocyte proliferation and formation of epidermal layer in human skin equivalents.

    PubMed

    Matsuura-Hachiya, Yuko; Arai, Koji Y; Muraguchi, Taichi; Sasaki, Tasuku; Nishiyama, Toshio

    2017-03-07

    Type IV collagen isolated from lens capsule without enzymatic treatment is known to form a gel under physiological condition and influences cellular activities. In case of human keratinocytes, the suppression of proliferation on reconstituted type IV collagen gels was reported in monolayer culture. In this study, we examined effects of type IV collagen isolated from porcine lens capsule on epidermal formation in human skin equivalents. Type IV collagen aggregates were prepared under the culture condition and the aggregates suppressed keratinocyte proliferation in monolayer culture as well as the culture on the gels. In human skin equivalents type IV collagen aggregates were reconstituted on the surface of contracted collagen gels containing human dermal fibroblasts and the keratinocytes were then cultured on the aggregates for 14 days. Interestingly, in human skin equivalents with type IV collagen aggregates, the BrdU-positive keratinocytes were increased and the thickness of the epidermal layer was around twice than that of control culture. Epidermal differentiation markers were expressed in the upper layer of the epidermis and the defined deposition of human basement membrane components were increased at the dermal-epidermal junction. These results indicate that the type IV collagen aggregates stimulate the proliferation of basal keratinocytes and improve the stratification of epidermal layers in human skin equivalents. This article is protected by copyright. All rights reserved.

  14. Air-stimulated ATP release from keratinocytes occurs through connexin hemichannels.

    PubMed

    Barr, Travis P; Albrecht, Phillip J; Hou, Quanzhi; Mongin, Alexander A; Strichartz, Gary R; Rice, Frank L

    2013-01-01

    Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins represent a novel, peripheral target for the treatment of chronic pain and dermatological disease.

  15. The stress caused by nitrite with titanium dioxide nanoparticles under UVA irradiation in human keratinocyte cell.

    PubMed

    Tu, Min; Huang, Yi; Li, Hai-Ling; Gao, Zhong-Hong

    2012-09-04

    Our previous work found that in the presence of nitrite, titanium dioxide nanoparticles can cause protein tyrosine nitration under UVA irradiation in vivo. In this paper, the human keratinocyte cells was used as a skin cell model to further study the photo-toxicity of titanium dioxide nanoparticles when nitrite was present. The results showed that nitrite increased the photo-toxicity of titanium dioxide in a dose-dependant manner, and generated protein tyrosine nitration in keratinocyte cells. Morphological study of keratinocyte cells suggested a specific apoptosis mediated by apoptosis inducing factor. It was also found the main target nitrated in cells was cystatin-A, which expressed abundantly in cytoplasm and functioned as a cysteine protease inhibitor. The stress induced by titanium dioxide with nitrite under UVA irradiation in human keratinocyte cells appeared to trigger the apoptosis inducing factor mediated cell death and lose the inhibition of active caspase by cystatin-A. We conclude that nitrite can bring new damage and stress to human keratinocyte cells with titanium dioxide nanoparticles under UVA irradiation.

  16. Assessment of in vitro cellular responses of monocytes and keratinocytes to tannic acid modified silver nanoparticles.

    PubMed

    Orlowski, Piotr; Krzyzowska, Malgorzata; Zdanowski, Robert; Winnicka, Anna; Nowakowska, Julita; Stankiewicz, Wanda; Tomaszewska, Emilia; Celichowski, Grzegorz; Grobelny, Jaroslaw

    2013-09-01

    Hydrolyzable tannins are known to exhibit diverse biological effects, which can be used in combination with silver nanoparticles (AgNPs). In this study, we tested toxic and inflammatory properties of tannic-acid modified 13, 33, 46 nm and unmodified 10-65 nm AgNPs using murine 291.03C keratinocyte and RAW 264.7 monocyte cell lines. Both cell lines exposed for 24h to 1-10 μg/ml of 13 nm, 33 nm, 46 nm and unmodified AgNPs showed dose-dependent toxicity and decreased cell proliferation. Only small-sized AgNPs induced production of ROS by monocytes, but not keratinocytes. Monocytes internalized large aggregates of 33, 46 nm and 10-65 nm AgNPs in cytoplasmic vacuoles, whereas keratinocytes accumulated less particles. AgNPs of 13 nm were localized ubiquitously within both cell types. The tested AgNPs strongly down-regulated production of tumor necrosis factor-α (TNF-α) by monocytes, whereas keratinocytes exposed to AgNPs showed an opposite effect. Unmodified but not tannic acid-modified AgNPs increased production of the pro-inflammatory MCP-1 by monocytes and keratinocytes. In summary, low inflammatory potential and lack of ROS production by tannic-acid modified AgNPs sized above 30 nm suggests that tannic acid modification of large silver nanoparticles may help to increase AgNPs biosafety.

  17. Regulation of migratory activity of human keratinocytes by topography of multiscale collagen-containing nanofibrous matrices.

    PubMed

    Fu, Xiaoling; Xu, Meng; Liu, Jie; Qi, Yanmei; Li, Shaohua; Wang, Hongjun

    2014-02-01

    Nanofibrous matrices hold great promise in skin wound repair partially due to their capability of recapturing the essential attributes of native extracellular matrix (ECM). With regard to limited studies on the effect of nanofibrous matrices on keratinocytes, the present study was aimed to understand how the topographical feature of nanofibrous matrices regulates keratinocyte motility by culturing keratinocytes on polycaprolactone (PCL)/collagen nanofibrous matrices (rough surface with fiber diameters of 331 ± 112 nm) or the matrices coated with a thin layer of collagen gel to form a secondary ultrafine fibrous network (smooth surface with ultrafine fiber diameters of 55 ± 26 nm). It was found that the PCL/collagen nanofibrous matrices alone did not stimulate cell migration, while collagen gel coating could significantly increase cell motility. Further studies demonstrated that the ultrafine fibrous network of collagen gel coating significantly activated integrin β1, Rac1 and Cdc42, facilitated the deposition of laminin-332 (formerly called laminin-5), and promoted the expression of active matrix metalloproteinases (MMPs) (i.e., MMP-2 and 9). Neutralization of integrin β1 activity abrogated the gel coating-induced keratinocyte migration. These findings provide important evidence on the role of topographical features of nanofibrous matrices in regulating the phenotypic alteration of keratinocytes and suggest the possible utility of collagen-containing nanofibrous matrices for skin regeneration especially in re-epithelialization.

  18. Guanine nucleotide exchange factor RABGEF1 regulates keratinocyte-intrinsic signaling to maintain skin homeostasis

    PubMed Central

    Marichal, Thomas; El Abbas, Sophie; Sibilano, Riccardo; Zurek, Oliwia; Reber, Laurent L.; Pirottin, Dimitri; Kim, Jinah; Chambon, Pierre; Roers, Axel; Antoine, Nadine; Kawakami, Yuko; Bureau, Fabrice; Tam, See-Ying; Tsai, Mindy

    2016-01-01

    Epidermal keratinocytes form a structural and immune barrier that is essential for skin homeostasis. However, the mechanisms that regulate epidermal barrier function are incompletely understood. Here we have found that keratinocyte-specific deletion of the gene encoding RAB guanine nucleotide exchange factor 1 (RABGEF1, also known as RABEX-5) severely impairs epidermal barrier function in mice and induces an allergic cutaneous and systemic phenotype. RABGEF1-deficient keratinocytes exhibited aberrant activation of the intrinsic IL-1R/MYD88/NF-κB signaling pathway and MYD88-dependent abnormalities in expression of structural proteins that contribute to skin barrier function. Moreover, ablation of MYD88 signaling in RABGEF1-deficient keratinocytes or deletion of Il1r1 restored skin homeostasis and prevented development of skin inflammation. We further demonstrated that epidermal RABGEF1 expression is reduced in skin lesions of humans diagnosed with either atopic dermatitis or allergic contact dermatitis as well as in an inducible mouse model of allergic dermatitis. Our findings reveal a key role for RABGEF1 in dampening keratinocyte-intrinsic MYD88 signaling and sustaining epidermal barrier function in mice, and suggest that dysregulation of RABGEF1 expression may contribute to epidermal barrier dysfunction in allergic skin disorders in mice and humans. Thus, RABGEF1-mediated regulation of IL-1R/MYD88 signaling might represent a potential therapeutic target. PMID:27820702

  19. Improvement of Human Keratinocyte Migration by a Redox Active Bioelectric Dressing

    PubMed Central

    Banerjee, Jaideep; Das Ghatak, Piya; Roy, Sashwati; Khanna, Savita; Sequin, Emily K.; Bellman, Karen; Dickinson, Bryan C.; Suri, Prerna; Subramaniam, Vish V.; Chang, Christopher J.; Sen, Chandan K.

    2014-01-01

    Exogenous application of an electric field can direct cell migration and improve wound healing; however clinical application of the therapy remains elusive due to lack of a suitable device and hence, limitations in understanding the molecular mechanisms. Here we report on a novel FDA approved redox-active Ag/Zn bioelectric dressing (BED) which generates electric fields. To develop a mechanistic understanding of how the BED may potentially influence wound re-epithelialization, we direct emphasis on understanding the influence of BED on human keratinocyte cell migration. Mapping of the electrical field generated by BED led to the observation that BED increases keratinocyte migration by three mechanisms: (i) generating hydrogen peroxide, known to be a potent driver of redox signaling, (ii) phosphorylation of redox-sensitive IGF1R directly implicated in cell migration, and (iii) reduction of protein thiols and increase in integrinαv expression, both of which are known to be drivers of cell migration. BED also increased keratinocyte mitochondrial membrane potential consistent with its ability to fuel an energy demanding migration process. Electric fields generated by a Ag/Zn BED can cross-talk with keratinocytes via redox-dependent processes improving keratinocyte migration, a critical event in wound re-epithelialization. PMID:24595050

  20. Expression and biological significance of Ca2+-activated ion channels in human keratinocytes.

    PubMed

    Koegel, H; Alzheimer, C

    2001-01-01

    In whole-cell recordings from HaCaT keratinocytes, ATP, bradykinin, and histamine caused a biphasic change of the membrane potential consisting of an initial transient depolarization, followed by a pronounced and long-lasting hyperpolarization. Flash photolysis of caged IP3 mimicked the agonist-induced voltage response, suggesting that intracellular Ca2+ release and subsequent opening of Ca2+-activated ion channels serve as the common transduction mechanism. In contrast, cAMP- and PKC-dependent pathways were not involved in the electrophysiological effects of the extracellular signaling molecules. The depolarization was predominantly mediated by a DIDS- and niflumic acid-sensitive Cl- current, whereas a charybdotoxin- and clotrimazole-sensitive K+ current underlay the prominent hyperpolarization. Consistent with the electrophysiological data, RT-PCR showed that HaCaT keratinocytes express two types of Ca2+-activated Cl- channels, CaCC2 and CaCC3 (CLCA2), as well as the Ca2+-activated K+ channel hSK4. That the pronounced hSK4-mediated hyperpolarization bears significance on the growth and differentiation properties of keratinocytes is suggested by RNase protection assays showing that hSK4 mRNA expression is strongly down-regulated under conditions that allow keratinocyte differentiation. hSK4 might thus play a role in linking changes in membrane potential to the biological fate of keratinocytes.

  1. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts.

    PubMed

    Ramos-Jerz, Maria Del R; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes.

  2. Neutrophils contact to plasma membrane of keratinocytes including desmosomal structures in canine pemphigus foliaceus.

    PubMed

    Yabuzoe, Atsushi; Nishifuji, Koji; Sekiguchi, Maiko; Shimizu, Atsushi; Momoi, Yasuyuki; Ishiko, Akira; Iwasaki, Toshiroh

    2008-08-01

    Pemphigus foliaceus (PF) is an autoimmune blistering skin disease that affects certain mammals including dogs. In canine PF, neutrophils are infiltrated intensely into pustular lesions including acantholytic cells, although neutrophilic infiltration is not characterized in human PF. The roles of the neutrophils in the cutaneous lesions of canine PF have not yet been understood. The purpose of this study was to characterize the ultrastructural features underlying the acantholysis with pustule formation in canine PF. Four dogs diagnosed as PF on the basis of clinical signs, histopathological findings, and direct and indirect immunofluorescence examinations were performed. Electron microscopy revealed that the acantholytic cells were adjacent to multiple neutrophils in the pustules. At the contact points between neutrophils and acantholytic keratinocytes, half-desmosomes of acantholytic keratinocytes with intact attachment plaques were observed within invaginations of neutrophils. Furthermore, on the surface of acantholytic cells in the pustules, neutrophil granules seemed to be secreted to the surface of acantholytic cells and to degenerate the half-desmosome structures. Neutrophils were also observed within the epidermis adjacent to the pustule. At the intercellular gap between two dissociated keratinocytes, neutrophils inserted its pseudopodia into the gap between the two half-desmosomes of keratinocytes. These findings taken together suggested that, at least in the areas where we analyzed ultrastructurally, neutrophils contact desmosomal structures and seem to play some parts in separation of keratinocytes and degeneration of split-desmosomes in pustules of dogs with PF.

  3. "Free-floating" desmosomes in lipoid proteinosis: an inherent defect in keratinocyte adhesion?

    PubMed

    Dyer, Jon A; Yu, Qian-Chun; Paller, Amy S

    2006-01-01

    The classic features of lipoid proteinosis - beadlike papules and hoarseness - result from the accumulation of hyaline material in the mucocutaneous dermis. However, the characteristic manifestation in children - erosive, crusted lesions that lead to scarring - is rarely discussed and poorly understood. Lipoid proteinosis results from mutations in extracellular matrix protein 1, but the function of this protein is largely unknown. We performed ultrastructural studies on lesional epidermis, cultured monolayer keratinocytes, and raft keratinocyte cultures from blistering lesions of a child with lipoid proteinosis. All sections showed the dissociation of relatively intact desmosomes from keratinocytes, with desmosomes that were "free-floating" in the intercellular spaces or attached by thin strands to the cell membrane. These changes were present in serial sections of both tissue and cultured keratinocytes, suggesting this observation to be an inherent feature of keratinocytes devoid of extracellular matrix protein 1, rather than an artifact. Although additional patients should be studied, the diminished appearance of the inner dense plaque - the region of attachment of keratin intermediate filaments to desmosomal proteins - provides preliminary evidence that extracellular matrix protein 1 may participate in attaching keratin intermediate filaments to desmosomal region protein(s).

  4. TSLP produced by keratinocytes promotes allergen sensitization through skin and thereby triggers atopic march in mice.

    PubMed

    Leyva-Castillo, Juan Manuel; Hener, Pierre; Jiang, Hua; Li, Mei

    2013-01-01

    Atopic dermatitis often precedes the development of asthma, a phenomenon known as "atopic march". An important role of allergen sensitization developed through barrier-defective skin has been recognized in the onset of atopic march; however, the underlying mechanism remains poorly understood. In this study, we use an experimental atopic march mouse model, in which the sensitization to allergen is achieved through barrier-impaired skin, followed by allergen challenge in the airway. By using thymic stromal lymphopoietin (TSLP)(iep-/-) mice in which the cytokine TSLP is selectively and inducibly ablated in epidermal keratinocytes, we demonstrate that keratinocytic TSLP, the expression of which is induced by skin barrier impairment, is essential for generating skin allergic inflammation and allergen-induced T helper type 2 response, for developing sensitization to allergen, and for triggering a subsequent allergic asthma. Furthermore, using TSLP(over) mice in which overexpression of keratinocytic TSLP is induced by skin topical application of MC903 (a vitamin D3 analog) in a dose-dependent manner, we show that keratinocytic TSLP levels are correlated with skin sensitization strength and asthma severity. Taken together, our study uncovers a crucial role of keratinocytic TSLP in the "atopic march" by promoting allergen sensitization occurring in barrier-impaired skin, which ultimately leads to allergic asthma.

  5. Human papillomavirus causes an angiogenic switch in keratinocytes which is sufficient to alter endothelial cell behavior

    SciTech Connect

    Chen, W.; Li, F.; Mead, L.; White, H.; Walker, J.; Ingram, D.A.; Roman, A.

    2007-10-10

    One of the requirements for tumor growth is the ability to recruit a blood supply, a process known as angiogenesis. Angiogenesis begins early in the progression of cervical disease from mild to severe dysplasia and on to invasive cancer. We have previously reported that expression of human papillomavirus type 16 E6 and E7 (HPV16 E6E7) proteins in primary foreskin keratinocytes (HFKs) decreases expression of two inhibitors and increases expression of two angiogenic inducers [Toussaint-Smith, E., Donner, D.B., Roman, A., 2004. Expression of human papillomavirus type 16 E6 and E7 oncoproteins in primary foreskin keratinocytes is sufficient to alter the expression of angiogenic factors. Oncogene 23, 2988-2995]. Here we report that HPV-induced early changes in the keratinocyte phenotype are sufficient to alter endothelial cell behavior both in vitro and in vivo. Conditioned media from HPV16 E6E7 expressing HFKs as well as from human cervical keratinocytes containing the intact HPV16 were able to stimulate proliferation and migration of human microvascular endothelial cells. In addition, introduction of the conditioned media into immunocompetent mice using a Matrigel plug model resulted in a clear angiogenic response. These novel data support the hypothesis that HPV proteins contribute not only to the uncontrolled keratinocyte growth seen following HPV infection but also to the angiogenic response needed for tumor formation.

  6. The Effect of Titanium Dioxide Nanoparticles on Keratinocyte Cell (KC) and Squamous Cell Carcinoma (SCC-13)

    NASA Astrophysics Data System (ADS)

    Lin, Chienhsiu; Simon, Marcia; Jurukovski, Vladimir; Lee, Wilson; Rafailovich, Miriam

    2009-03-01

    We have studied the effects of TiO2 nanoparticles on cell keratinocyte and SCC (Squamous Cell Carcinoma) cells. We found that the concentration of particles required to adversely affect the cells was many times higher for keratinocyte than SCC cells. Confocal microscope shows that the particles in keratinocyte culture are sequestered in membranes between the cell colonies. The particles penetrated into the cells in the case of the SCC cells. TEM images revealed very few particles in the keratinocyte, many more particles were observed sequestered in vacuole of the SCC cells. These results indicate that the keratinocyte layer behaves very different from the fibroblast layers which are much more sensate to TiO2 nanoparticle damage and may suggest a protection mechanism of the dermal tissue. The effect of UV exposure in the presence of DNA was also investigated. We found that adsorbed proteins, as well as grafted polymer provided a measure of protection against free radical formation. The effects of low level UV exposure when the particles are near in-vitro cell culture will be presented.

  7. Galectin-7 overexpression is associated with the apoptotic process in UVB-induced sunburn keratinocytes

    PubMed Central

    Bernerd, Francoise; Sarasin, Alain; Magnaldo, Thierry

    1999-01-01

    Galectin-7 is a β-galactoside binding protein specifically expressed in stratified epithelia and notably in epidermis, but barely detectable in epidermal tumors and absent from squamous carcinoma cell lines. Galectin-7 gene is an early transcriptional target of the tumor suppressor protein P53 [Polyak, K., Xia, Y., Zweier, J., Kinzler, K. & Vogelstein, B. (1997) Nature (London) 389, 300–305]. Because p53 transcriptional activity is increased by genotoxic stresses we have examined the possible effects of ultraviolet radiations (UVB) on galectin-7 expression in epidermal keratinocytes. The amounts of galectin-7 mRNA and protein are increased rapidly after UVB irradiation of epidermal keratinocytes. The increase of galectin-7 is parallel to P53 stabilization. UVB irradiation of skin reconstructed in vitro and of human skin ex vivo demonstrates that galectin-7 overexpression is associated with sunburn/apoptotic keratinocytes. Transfection of a galectin-7 expression vector results in a significant increase in terminal deoxynucleotidyltransferase-mediated UTP end labeling-positive keratinocytes. The present findings demonstrate a keratinocyte-specific protein involved in the UV-induced apoptosis, an essential process in the maintenance of epidermal homeostasis. PMID:10500176

  8. Paracoccidioides brasiliensis interacts with dermal dendritic cells and keratinocytes in human skin and oral mucosa lesions.

    PubMed

    Silva, Wellington Luiz Ferreira da; Pagliari, Carla; Duarte, Maria Irma Seixas; Sotto, Mirian N

    2016-05-01

    Paracoccidioidomycosis (PCM) is a systemic disease caused by the fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. In PCM the skin and oral mucosa are often affected. Dendritic cells and keratinocytes of the integument play a role in innate and adaptive immune response against pathogens, due to their function as antigen presenting cells. Aiming to verify the interaction of P. brasiliensis with these cell populations, we studied 52 skin and 47 oral mucosa samples taken from patients with proven diagnosis of PCM. The biopsies were subjected to immunohistochemical and/or immunofluorescence staining with anti-factor XIIIa (marker of dermal dendrocytes), anti-CD207 (marker of mature Langerhans cells), anti-pan cytokeratins (AE1-AE3) and anti-P. brasiliensis antibodies. Analyses with confocal laser microscopy were also performed for better visualization of the interaction between keratinocytes and the fungi. In sum, 42% of oral mucosa samples displayed yeast forms in Factor XIIIa dermal dendrocytes cytoplasm. Langerhans cells in skin and oral mucosa samples did not show yeast cells in their cytoplasm. In sum, 54% of skin and 60% of mucosal samples displayed yeast cells in the cytoplasm of keratinocytes. The parasitism of keratinocytes may represent a possible mechanism of evasion of the fungus to local immune mechanisms. Factor XIIIa dendrocytes and keratinocytes may be acting as antigen-presenting cells to fulfill the probably impaired function of Langerhans cells in skin and oral mucosa of human PCM.

  9. Loss of serum response factor in keratinocytes results in hyperproliferative skin disease in mice

    PubMed Central

    Koegel, Heidi; von Tobel, Lukas; Schäfer, Matthias; Alberti, Siegfried; Kremmer, Elisabeth; Mauch, Cornelia; Hohl, Daniel; Wang, Xiao-Jing; Beer, Hans-Dietmar; Bloch, Wilhelm; Nordheim, Alfred; Werner, Sabine

    2009-01-01

    The transcription factor serum response factor (SRF) plays a crucial role in the development of several organs. However, its role in the skin has not been explored. Here, we show that keratinocytes in normal human and mouse skin expressed high levels of SRF but that SRF expression was strongly downregulated in the hyperproliferative epidermis of wounded and psoriatic skin. Keratinocyte-specific deletion within the mouse SRF locus during embryonic development caused edema and skin blistering, and all animals died in utero. Postnatal loss of mouse SRF in keratinocytes resulted in the development of psoriasis-like skin lesions. These lesions were characterized by inflammation, hyperproliferation, and abnormal differentiation of keratinocytes as well as by disruption of the actin cytoskeleton. Ultrastructural analysis revealed markedly reduced cell-cell and cell-matrix contacts and loss of cell compaction in all epidermal layers. siRNA-mediated knockdown of SRF in primary human keratinocytes revealed that the cytoskeletal abnormalities and adhesion defects were a direct consequence of the loss of SRF. In contrast, the hyperproliferation observed in vivo was an indirect effect that was most likely a consequence of the inflammation. These results reveal that loss of SRF disrupts epidermal homeostasis and strongly suggest its involvement in the pathogenesis of hyperproliferative skin diseases, including psoriasis. PMID:19307725

  10. Effects of titanium dioxide nanoparticles on human keratinocytes.

    PubMed

    Wright, Clayton; Iyer, Anand Krishnan V; Wang, Liying; Wu, Nianqiang; Yakisich, Juan S; Rojanasakul, Yon; Azad, Neelam

    2017-01-01

    Titanium dioxide (TiO2) is a ubiquitous whitening compound widely used in topical products such as sunscreens, lotions and facial creams. The damaging health effects of TiO2 inhalation has been widely studied in rats, mice and humans showing oxidative stress increase, DNA damage, cell death and inflammatory gene upregulation in lung and throat cells; however, the effects on skin cells from long-term topical use of various products remain largely unknown. In this study, we assessed the effect of specific TiO2 nanoparticles (H2TiO7) on a human keratinocyte cell line (HaCaT). We performed a comparative analysis using three TiO2 particles varying in size (Fine, Ultrafine and H2TiO7) and analyzed their effects on HaCaTs. There is a clear dose-dependent increase in superoxide production, caspase 8 and 9 activity, and apoptosis in HaCaTs after treatment with all three forms of TiO2; however, there is no consistent effect on cell viability and proliferation with either of these TiO2 particles. While there is data suggesting UV exposure can enhance the carcinogenic effects of TiO2, we did not observe any significant effect of UV-C exposure combined with TiO2 treatment on HaCaTs. Furthermore, TiO2-treated cells showed minimal effects on VEGF upregulation and Wnt signaling pathway thereby showing no potential effect on angiogenesis and malignant transformation. Overall, we report here an increase in apoptosis, which may be caspase 8/Fas-dependent, and that the H2TiO7 nanoparticles, despite their smaller particle size, had no significant enhanced effect on HaCaT cells as compared to Fine and Ultrafine forms of TiO2.

  11. Effects of titanium dioxide nanoparticles on human keratinocytes

    PubMed Central

    Wright, Clayton; Iyer, Anand Krishnan V.; Wang, Liying; Wu, Nianqiang; Yakisich, Juan S.; Rojanasakul, Yon; Azad, Neelam

    2016-01-01

    Titanium dioxide (TiO2) is a ubiquitous whitening compound widely used in topical products such as sunscreens, lotions and facial creams. The damaging health effects of TiO2 inhalation has been widely studied in rats, mice and humans showing oxidative stress increase, DNA damage, cell death and inflammatory gene upregulation in lung and throat cells; however, the effects on skin cells from long-term topical use of various products remain largely unknown. In this study, we assessed the effect of specific TiO2 nanoparticles (H2TiO7) on a human keratinocyte cell line (HaCaT). We performed a comparative analysis using three TiO2 particles varying in size (Fine, Ultrafine and H2TiO7) and analyzed their effects on HaCaTs. There is a clear dose-dependent increase in superoxide production, caspase 8 and 9 activity, and apoptosis in HaCaTs after treatment with all three forms of TiO2; however, there is no consistent effect on cell viability and proliferation with either of these TiO2 particles. While there is data suggesting UV exposure can enhance the carcinogenic effects of TiO2, we did not observe any significant effect of UV-C exposure combined with TiO2 treatment on HaCaTs. Furthermore, TiO2-treated cells showed minimal effects on VEGF upregulation and Wnt signaling pathway thereby showing no potential effect on angiogenesis and malignant transformation. Overall, we report here an increase in apoptosis, which may be caspase 8/Fas-dependent, and that the H2TiO7 nanoparticles, despite their smaller particle size, had no significant enhanced effect on HaCaT cells as compared to Fine and Ultrafine forms of TiO2. PMID:27310834

  12. Bathing in carbon dioxide-enriched water alters protein expression in keratinocytes of skin tissue in rats

    NASA Astrophysics Data System (ADS)

    Kälsch, Julia; Pott, Leona L.; Takeda, Atsushi; Kumamoto, Hideo; Möllmann, Dorothe; Canbay, Ali; Sitek, Barbara; Baba, Hideo A.

    2016-10-01

    Beneficial effects of balneotherapy using naturally occurring carbonated water (CO2 enriched) have been known since the Middle Ages. Although this therapy is clinically applied for peripheral artery disease and skin disorder, the underlying mechanisms are not fully elucidated. Under controlled conditions, rats were bathed in either CO2-enriched water (CO2 content 1200 mg/L) or tap water, both at 37 °C, for 10 min daily over 4 weeks. Proliferation activity was assessed by Ki67 immunohistochemistry of the epidermis of the abdomen. The capillary density was assessed by immunodetection of isolectin-positive cells. Using cryo-fixed abdominal skin epidermis, follicle cells and stroma tissue containing capillaries were separately isolated by means of laser microdissection and subjected to proteomic analysis using label-free technique. Differentially expressed proteins were validated by immunohistochemistry. Proliferation activity of keratinocytes was not significantly different in the epidermis after bathing in CO2-enriched water, and also, capillary density did not change. Proteomic analysis revealed up to 36 significantly regulated proteins in the analyzed tissue. Based on the best expression profiles, ten proteins were selected for immunohistochemical validation. Only one protein, far upstream element binding protein 2 (FUBP2), was similarly downregulated in the epidermis after bathing in CO2-enriched water with both techniques. Low FUBP2 expression was associated with low c-Myc immune-expression in keratinocytes. Long-term bathing in CO2-enriched water showed a cellular protein response of epithelial cells in the epidermis which was detectable by two different methods. However, differences in proliferation activity or capillary density were not detected in the normal skin.

  13. Similar enzymes, different structures

    PubMed Central

    Tarasev, Michael; Kaddis, Catherine S.; Yin, Sheng; Loo, Joseph A.; Burgner, John; Ballou, David P.

    2007-01-01

    Phthalate dioxygenase (PDO) is a member of a class of bacterial oxygenases that contain both Rieske [2Fe-2S] and Fe(II) mononuclear centers. Recent crystal structures of several Rieske dioxygenases showed that they exist as α3β3 multimers with subunits arranged head-to-tail in α and β stacked planar consists of only α-subunits, remains to be solved. Although similar to other Rieske dioxygenases in many aspects, PDO was shown to differ in the mechanism of catalysis. Gel filtration and analytical centrifugation experiments, supplemented with mass spectrometric analysis (both ESI-MS and ESI-GEMMA), in this work showed a hexameric arrangement of subunits in the PDO multimer. Our proposed model for the subunit arrangement in PDO postulates two α3 planar rings one on top the other, similar to the α3β3 arrangement in other Rieske dioxygenases. Unlike other Rieske dioxygenases, this arrangement brings two Rieske and two mononuclear centers, all on separate subunits, into proximity, allowing their cooperation for catalysis. Potential reasons necessitating this unusual structural arrangement are discussed. PMID:17764654

  14. Platelets Regulate the Migration of Keratinocytes via Podoplanin/CLEC-2 Signaling during Cutaneous Wound Healing in Mice.

    PubMed

    Asai, Jun; Hirakawa, Satoshi; Sakabe, Jun-ichi; Kishida, Tsunao; Wada, Makoto; Nakamura, Naomi; Takenaka, Hideya; Mazda, Osam; Urano, Tetsumei; Suzuki-Inoue, Katsue; Tokura, Yoshiki; Katoh, Norito

    2016-01-01

    Podoplanin is an endogenous ligand for C-type lectin-like receptor 2 (CLEC-2), which is expressed on platelets. Recent evidence indicates that this specific marker of lymphatic endothelial cells is also expressed by keratinocytes at the edge of wounds. However, whether podoplanin or platelets play a role in keratinocyte activity during wound healing remains unknown. We evaluated the effect of podoplanin expression levels on keratinocyte motility using cultured primary normal human epidermal keratinocytes (NHEKs). Down-regulation of podoplanin in NHEKs via transfection with podoplanin siRNA inhibited their migration, indicating that podoplanin plays a mandatory role in this process. In addition, down-regulation of podoplanin was correlated with up-regulation of E-cadherin, suggesting that podoplanin-mediated stimulation of keratinocyte migration is associated with a loss of E-cadherin. Both the addition of platelets and treatment with CLEC-2 inhibited the migration of NHEKs. The down-regulation of RhoA activity and the up-regulation of E-cadherin in keratinocytes were also induced by CLEC-2. In conclusion, these results suggest that podoplanin/CLEC-2 signaling regulates keratinocyte migration via modulating E-cadherin expression through RhoA signaling. Altering the regulation of keratinocyte migration by podoplanin might be a novel therapeutic approach to improve wound healing.

  15. Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage

    PubMed Central

    Han, Xia; Piao, Mei Jing; Kim, Ki Cheon; Madduma Hewage, Susara Ruwan Kumara; Yoo, Eun Sook; Koh, Young Sang; Kang, Hee Kyoung; Shin, Jennifer H; Park, Yeunsoo; Yoo, Suk Jae; Chae, Sungwook; Hyun, Jin Won

    2015-01-01

    Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repressed UVB-facilitated programmed cell death in the keratinocytes, as evidenced by a reduction in apoptotic body formation, and nuclear fragmentation. Additionally, isorhamnetin suppressed the ability of UVB light to trigger mitochondrial dysfunction. Taken together, these results indicate that isorhamnetin has the potential to protect human keratinocytes against UVB-induced cell damage and death. PMID:26157553

  16. RIP2: A novel player in the regulation of keratinocyte proliferation and cutaneous wound repair?

    SciTech Connect

    Adams, Stephanie; Valchanova, Ralitsa S.; Munz, Barbara

    2010-03-10

    We could recently demonstrate an important role of receptor interacting protein 4 (RIP4) in the regulation of keratinocyte differentiation. Now, we analyzed a potential role of the RIP4 homolog RIP2 in keratinocytes. Specifically, we demonstrate here that rip2 expression is induced by scratch-wounding and after the induction of differentiation in these cells. Furthermore, serum growth factors and cytokines can induce rip2, with TNF-{alpha}-dependent induction being dependent on p38 MAPK. In addition, we demonstrate that scratch-induced upregulation of rip2 expression is completely blocked by the steroid dexamethasone. Since we also show that RIP2 is an important player in the regulation of keratinocyte proliferation, these data suggest that inhibition of rip2 upregulation after wounding might contribute to the reduced and delayed wound re-epithelialization phenotype seen in glucocorticoid-treated patients.

  17. Peptides from Tetraspanin CD9 Are Potent Inhibitors of Staphylococcus Aureus Adherence to Keratinocytes

    PubMed Central

    Ventress, Jennifer K.; Partridge, Lynda J.; Read, Robert C.; Cozens, Daniel; MacNeil, Sheila

    2016-01-01

    Staphylococcus aureus is one of the primary causative agents of skin and wound infections. As bacterial adherence is essential for infection, blocking this step can reduce invasion of host tissues by pathogens. An anti-adhesion therapy, based on a host membrane protein family, the tetraspanins, has been developed that can inhibit the adhesion of S. aureus to human cells. Synthetic peptides derived from a keratinocyte-expressed tetraspanin, CD9, were tested for anti-adhesive properties and at low nanomolar concentrations were shown to inhibit bacterial adhesion to cultured keratinocytes and to be effective in a tissue engineered model of human skin infection. These potential therapeutics had no effect on keratinocyte viability, migration or proliferation, indicating that they could be a valuable addition to current treatments for skin infection. PMID:27467693

  18. Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage.

    PubMed

    Han, Xia; Piao, Mei Jing; Kim, Ki Cheon; Madduma Hewage, Susara Ruwan Kumara; Yoo, Eun Sook; Koh, Young Sang; Kang, Hee Kyoung; Shin, Jennifer H; Park, Yeunsoo; Yoo, Suk Jae; Chae, Sungwook; Hyun, Jin Won

    2015-07-01

    Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repressed UVB-facilitated programmed cell death in the keratinocytes, as evidenced by a reduction in apoptotic body formation, and nuclear fragmentation. Additionally, isorhamnetin suppressed the ability of UVB light to trigger mitochondrial dysfunction. Taken together, these results indicate that isorhamnetin has the potential to protect human keratinocytes against UVB-induced cell damage and death.

  19. Phylogenetic metrics of community similarity.

    PubMed

    Ives, Anthony R; Helmus, Matthew R

    2010-11-01

    We derive a new metric of community similarity that takes into account the phylogenetic relatedness among species. This metric, phylogenetic community dissimilarity (PCD), can be partitioned into two components, a nonphylogenetic component that reflects shared species between communities (analogous to Sørensen' s similarity metric) and a phylogenetic component that reflects the evolutionary relationships among nonshared species. Therefore, even if a species is not shared between two communities, it will increase the similarity of the two communities if it is phylogenetically related to species in the other community. We illustrate PCD with data on fish and aquatic macrophyte communities from 59 temperate lakes. Dissimilarity between fish communities associated with environmental differences between lakes often has a phylogenetic component, whereas this is not the case for macrophyte communities. With simulations, we then compare PCD with two other metrics of phylogenetic community similarity, II(ST) and UniFrac. Of the three metrics, PCD was best at identifying environmental drivers of community dissimilarity, showing lower variability and greater statistical power. Thus, PCD is a statistically powerful metric that separates the effects of environmental drivers on compositional versus phylogenetic components of community structure.

  20. Laminin α5 in the keratinocyte basement membrane is required for epidermal-dermal intercommunication.

    PubMed

    Wegner, Jeannine; Loser, Karin; Apsite, Gunita; Nischt, Roswitha; Eckes, Beate; Krieg, Thomas; Werner, Sabine; Sorokin, Lydia

    2016-12-01

    Laminin α5 is broadly expressed in the epidermal basement membrane (BM) of mature mice and its elimination at this site (Lama5(Ker5) mouse) results in hyperproliferation of basal keratinocytes and a delay in hair follicle development, which correlated with upregulation of the dermally-derived laminin α2 and laminin α4 chains in the epidermal BM and of tenascin-C subjacent to the BM. In vitro studies revealed laminin 511 to be strongly adhesive for primary keratinocytes and that loss of laminin α5 does not result in cell autonomous defects in proliferation. Flow cytometry reveals that the loss of laminin α5 resulted in increased numbers of CD45(+), CD4(+) and CD11b(+) immune cells in the skin, which temporo-spatial analyses revealed were detectable only subsequent to the loss of laminin α5 and the appearance of the hyperproliferative keratinocyte phenotype. These findings indicate that immune cell changes are the consequence and not the cause of keratinocyte hyperproliferation. Loss of laminin α5 in the epidermal BM was also associated with changes in the expression of several dermally-derived growth factors involved in keratinocyte proliferation and hair follicle development in adult but not new born Lama5(Ker5) skin, including KGF, EGF and KGF-2. In situ binding of FGF-receptor-2α (IIIb)-Fc chimera (FGFR2IIIb) to mouse skin sections revealed decoration of several BMs, including the epidermal BM, which was absent in Lama5(Ker5) skin. This indicates reduced levels of FGFR2IIIb ligands, which include KGF and KGF-2, in the epidermal BM of adult Lama5(Ker5) skin. Our data suggest an initial inhibitory effect of laminin α5 on basal keratinocyte proliferation and migration, which is exacerbated by subsequent changes in growth factor expression by epidermal and dermal cells, implicating laminin α5 in epidermal-dermal intercommunication.

  1. Vaccinia Virus Induces Rapid Necrosis in Keratinocytes by a STAT3-Dependent Mechanism

    PubMed Central

    He, Yong; Fisher, Robert; Chowdhury, Soma; Sultana, Ishrat; Pereira, Claudia P.; Bray, Mike; Reed, Jennifer L.

    2014-01-01

    Rationale Humans with a dominant negative mutation in STAT3 are susceptible to severe skin infections, suggesting an essential role for STAT3 signaling in defense against cutaneous pathogens. Methods To focus on innate antiviral defenses in keratinocytes, we used a standard model of cutaneous infection of severe combined immunodeficient mice with the current smallpox vaccine, ACAM-2000. In parallel, early events post-infection with the smallpox vaccine ACAM-2000 were investigated in cultured keratinocytes of human and mouse origin. Results Mice treated topically with a STAT3 inhibitor (Stattic) developed larger vaccinia lesions with higher virus titers and died more rapidly than untreated controls. Cultured human and murine keratinocytes infected with ACAM-2000 underwent rapid necrosis, but when treated with Stattic or with inhibitors of RIP1 kinase or caspase-1, they survived longer, produced higher titers of virus, and showed reduced activation of type I interferon responses and inflammatory cytokines release. Treatment with inhibitors of RIP1 kinase and STAT3, but not caspase-1, also reduced the inflammatory response of keratinocytes to TLR ligands. Vaccinia growth properties in Vero cells, which are known to be defective in some antiviral responses, were unaffected by inhibition of RIP1K, caspase-1, or STAT3. Conclusions Our findings indicate that keratinocytes suppress the replication and spread of vaccinia virus by undergoing rapid programmed cell death, in a process requiring STAT3. These data offer a new framework for understanding susceptibility to skin infection in patients with STAT3 mutations. Interventions which promote prompt necroptosis/pyroptosis of infected keratinocytes may reduce risks associated with vaccination with live vaccinia virus. PMID:25419841

  2. Similarity transformed semiclassical dynamics

    NASA Astrophysics Data System (ADS)

    Van Voorhis, Troy; Heller, Eric J.

    2003-12-01

    In this article, we employ a recently discovered criterion for selecting important contributions to the semiclassical coherent state propagator [T. Van Voorhis and E. J. Heller, Phys. Rev. A 66, 050501 (2002)] to study the dynamics of many dimensional problems. We show that the dynamics are governed by a similarity transformed version of the standard classical Hamiltonian. In this light, our selection criterion amounts to using trajectories generated with the untransformed Hamiltonian as approximate initial conditions for the transformed boundary value problem. We apply the new selection scheme to some multidimensional Henon-Heiles problems and compare our results to those obtained with the more sophisticated Herman-Kluk approach. We find that the present technique gives near-quantitative agreement with the the standard results, but that the amount of computational effort is less than Herman-Kluk requires even when sophisticated integral smoothing techniques are employed in the latter.

  3. Endothelin-1 is a transcriptional target of p53 in epidermal keratinocytes and regulates UV induced melanocyte homeostasis

    PubMed Central

    Hyter, Stephen; Coleman, Daniel J.; Ganguli-Indra, Gitali; Merrill, Gary F.; Ma, Steven; Yanagisawa, Masashi; Indra, Arup K.

    2013-01-01

    Summary Keratinocytes contribute to melanocyte activity by influencing their microenvironment, in part, through secretion of paracrine factors. Here we discovered that p53 directly regulates Edn1 expression in epidermal keratinocytes and controls UV-induced melanocyte homeostasis. Selective ablation of EDN1 in murine epidermis (EDN1ep−/−) does not alter melanocyte homeostasis in newborn skin but decreases dermal melanocytes in adult skin. Results showed that keratinocytic EDN1 in a non-cell autonomous manner controls melanocyte proliferation, migration, DNA damage and apoptosis after UVB irradiation. Expression of other keratinocyte derived paracrine factors did not compensate for the loss of EDN1. Topical treatment with EDN1 receptor (EDNRB) antagonist BQ788 abrogated UV induced melanocyte activation and recapitulated the phenotype seen in EDN1ep−/− mice. Altogether, present studies establish an essential role of EDN1 in epidermal keratinocytes to mediate UV induced melanocyte homeostasis in vivo. PMID:23279852

  4. Regulation of Srpr Expression by miR-330-5p Controls Proliferation of Mouse Epidermal Keratinocyte

    PubMed Central

    Kim, Bong-Kyu; Yoo, Hye-In; Choi, Keonwoo; Lee, Ah-Reum; Yoon, Sungjoo Kim

    2016-01-01

    Srpr is a gene encoding α subunit of the signal recognition particle receptor which is involved in the targeting and translocation of nascent secretory and membrane proteins to the endoplasmic reticulum. Previous studies showed aberrant expression of Srpr in several cell types with abnormal growth rate. Although Srpr is expressed in various tissues including skin, the role of Srpr in keratinocytes and regulation of its expression by miRNAs have not been studied. In this study, we investigated the role of SRPR and regulation of its expression by miRNA in skin keratinocytes. We found that SRPR was highly expressed in epidermal keratinocytes and regulated keratinocyte proliferation by affecting cell cycle progression. We also demonstrated that miR-330-5p directly inhibits Srpr expression. These data suggest that miR-330-5p-mediated regulation of the SRPR level is needed for the regulation of proliferation of epidermal keratinocytes. PMID:27768721

  5. [Cultured epidermis in the treatment of leg ulcer: "edge effect" and correlation with keratinocyte proliferation index].

    PubMed

    Giannotti, V; Pimpinelli, N; Mariotti, V; Borgognoni, L; Reali, U M

    1990-04-01

    Cultured keratinocyte grafting (KG) of chronic leg ulcers produces an impressive stimulation of host epithelization by an effect on the edge of the ulcer, which starts to grow in rapidly ("edge effect"). In 5 patients with chronic leg ulcers treated by KG, we have studied the proliferation index of the epidermis of the edge and of the graft area and that of secondary culture keratinocytes. The aim of the study was to correlate this parameter with the clinical evolution of the treated lesions. We found a significant correlation between the proliferation index of the edge epidermis and the clinical evidence of "edge effect".

  6. Effects of low frequency pulsed electrical current on keratinocytes in vitro

    SciTech Connect

    Hinsenkamp, M.; Jercinovic, A.

    1997-05-01

    The effects of low frequency pulsed electrical current on epidermal repair in vitro were examined. Charge-balanced current stimuli proposed for chronic wound treatment were tested on skin keratinocytes cultured at an air-liquid interface on dead human dermis. Results imply that the balance between proliferation and differentiation in electrically treated samples is significantly modified in favor of differentiation. More advanced differentiation, shown through epidermal histology, was obtained in cultures exposed to electrical current, whereas the culture growth, the result of keratinocyte migration and proliferation, was greater in control samples.

  7. Nonlinear cellular dynamics of keratinocytes in normal and psoriatic epidermis under action of UV radiation

    NASA Astrophysics Data System (ADS)

    Stolnitz, Mikhail M.; Medvedev, Boris A.; Gribko, Tatyana V.

    2004-05-01

    The semi-phenomenological model of epidermal cell dynamics is submitted. The model takes into account three types of basal layer keratinocytes (stem, transient amplifying, terminally differentiated), distribution of first two types cells on mitotic cycle stages and resting states, keratinocytes-lymphocytes interactions that provide a positive feedback loop, influence of more differentiated cells on their progenitors that provide a negative feedback loop. Simplified model are developed and its stationary solutions are received. The opportunity of interpretation of some received modes as corresponding to various stages of psoriasis is discussed. Influence of UV-radiation on transitions between various modes of epidermis functioning is qualitatively analyzed.

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

    NASA Astrophysics Data System (ADS)

    Laporta, Robert F.; Taichman, Lorne B.

    1982-06-01

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

  9. Galangin (3,5,7-trihydroxyflavone) shields human keratinocytes from ultraviolet B-induced oxidative stress.

    PubMed

    Madduma Hewage, Susara Ruwan Kumara; Piao, Mei Jing; Kim, Ki Cheon; Cha, Ji Won; Han, Xia; Choi, Yung Hyun; Chae, Sungwook; Hyun, Jin Won

    2015-03-01

    Most skin damage caused by ultraviolet B (UVB) radiation is owing to the generation of reactive oxygen species. Phytochemicals can act as antioxidants against UVB-induced oxidative stress. This study investigated the protective effects of the flavone galangin against UVB-induced oxidative damage in human keratinocytes. Galangin efficiently scavenged free radicals and reduced UVB-induced damage to cellular macromolecules, such as DNA, lipids, and proteins. Furthermore, galangin rescued cells undergoing apoptosis induced by UVB radiation via recovering mitochondrial polarization and down-regulating apoptotic proteins. These results showed that galangin protects human keratinocytes against UVB radiation-induced cellular damage and apoptosis via its antioxidant effects.

  10. Benzoyl peroxide interferes with metabolic co-operation between cultured human epidermal keratinocytes

    SciTech Connect

    Lawrence, N.J.; Parkinson, E.K.; Emmerson, A.

    1984-03-01

    The ability of benzoyl peroxide to inhibit metabolic co-operation in rodent cell cultures may be relevant to its recently reported tumour promoting activity in mouse epidermis. We show here that non-toxic doses of this compound reduce metabolic co-operation between human epidermal keratinocytes to approximately 30% of that found in controls. The doses of benzoyl peroxide used did not affect keratinocyte morphology or their rate of attachment to the culture substratum. These results could be important as benzoyl peroxide is widely used in industry.

  11. Areca (betel) nut extract activates mitogen-activated protein kinases and NF-kappaB in oral keratinocytes.

    PubMed

    Lin, Shu-Chun; Lu, Suu-Yi; Lee, Szu-Ying; Lin, Chi-Yen; Chen, Chun-Hsien; Chang, Kuo-Wei

    2005-09-10

    Areca (betel) was recently proved a carcinogenic substance by the International Agency for Research on Cancer. However, the signaling impact of areca in oral keratinocyte is still obscure. Mitogen-activated protein kinase superfamilies, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinases (JNK) and p38, together with transcription factor NF-kappaB, are important signaling elements. We examined the activation of these signaling pathways in OECM-1 and SAS oral keratinocytes, treated with ripe areca nut extract (ANE). In both cells, a rapid increase in JNK1 activity at 0.5 hr was noted following treatment of ANE. ERK was profoundly activated during 0.5-2 hr in OECM-1 cells. Contrasting p38 activity was noted in these 2 cells. In both cells, ANE also activated NF-kappaB pathway in a biphasic manner, particularly for SAS cells. NF-kappaB was activated by approximately 2- to 4-fold at 0.5-1 hr and a plateau or slight decrease of activity existed between 1 and 6 hr. Later, another higher episode of NF-kappaB activity was raised. This was accompanied with the rapid degradation in cytosolic IkappaBalpha as well as an increase of nuclear NF-kappaB in both cells. ANE treatment did not activate epidermal growth factor receptor signaling system, but blockage of NF-kappaB activation rendered the suppression of ANE-modulated COX-2 upregulation in OECM-1. This study identified that ANE affected interactive signaling systems in oral keratonocytes that could be the pathogenetic basis for areca.

  12. TGF-α/HA complex promotes tympanic membrane keratinocyte migration and proliferation via ErbB1 receptor

    SciTech Connect

    Mei Teh, Bing; Redmond, Sharon L.; Shen, Yi; Atlas, Marcus D.; Marano, Robert J.; Dilley, Rodney J.

    2013-04-01

    Tympanic membrane perforations are common and represent a management challenge to clinicians. Current treatments for chronic perforations involve a graft surgery and require general anaesthesia, including associated costs and morbidities. Bioactive molecules (e.g. growth factors, cytokines) play an important role in promoting TM wound healing following perforation and the use of growth factors as a topical treatment for tympanic membrane perforations has been suggested as an alternative to surgery. However, the choice of bioactive molecules best suited to promote wound healing has yet to be identified. We investigated the effects of hyaluronic acid, vitronectin, TGF-α, IL-24 and their combinations on migration, proliferation and adhesion of cultured human tympanic membrane-derived keratinocytes (hTM), in addition to their possible mechanisms of action. We found that TGF-α, TGF-α/HA and TGF-α/IL-24 promoted wound healing by significantly increasing both migration and proliferation. TGF-α and/or HA treated cells showed comparable cell–cell adhesion whilst maintaining an epithelial cell phenotype. With the use of receptor binding inhibitors for ErbB1 (AG1478) and CD44 (BRIC235), we revealed that the activation of ErbB1 is required for TGF-α/HA-mediated migration and proliferation. These results suggest factors that may be incorporated into a tissue-engineered membrane or directly as topical treatment for tympanic membrane perforations and hence reduce the need for a surgery. - Highlights: ► TGF-α, TGF-α/HA and TGF-α/IL-24 improved hTM keratinocyte migration and proliferation. ► TGF-α and/or HA maintained epithelial cell phenotype. ► TGF-α/HA-mediated migration and proliferation requires activation of ErbB1 receptor.

  13. MicroRNA-23b-3p regulates human keratinocyte differentiation through repression of TGIF1 and activation of the TGF-ß-SMAD2 signalling pathway.

    PubMed

    Barbollat-Boutrand, Laetitia; Joly-Tonetti, Nicolas; Dos Santos, Morgan; Metral, Elodie; Boher, Aurélie; Masse, Ingrid; Berthier-Vergnes, Odile; Bertolino, Philippe; Damour, Odile; Lamartine, Jérôme

    2017-01-01

    MicroRNAs (miRNAs) are a class of short non-coding RNAs capable of repressing gene expression at the post-transcriptional level. miRNAs participate in the control of numerous cellular mechanisms, including skin homeostasis and epidermal differentiation. However, few miRNAs involved in these processes have been identified so far in human skin, and the gene networks they control remain largely unknown. Here, we focused on miR-23b-3p, a miRNA that is expressed during the late step of human keratinocyte differentiation. We report that miR-23b-3p silencing modulates epidermal differentiation in human skin reconstructs. The SMAD transcriptional corepressor TGIF1 was identified on bioinformatic analysis as a potential target of miR-23b-3p. Expression analysis and reporter gene assays confirmed direct regulation of TGIF1 expression by miR-23b-3p. Finally, we showed that miR-23-3p was able to activate TGF-ß signalling in human keratinocytes by increasing SMAD2 phosphorylation through TGIF1 repression. Taken together, these data identify miR-23b-3p as a new regulator of human epidermal differentiation in line with TGF-ß signalling.

  14. Bitter apricot essential oil induces apoptosis of human HaCaT keratinocytes.

    PubMed

    Li, Keyou; Yang, Wenhua; Li, Zhe; Jia, Wangwang; Li, Jiazhou; Zhang, Pengfei; Xiao, Tiancun

    2016-05-01

    Psoriasis is a chronic skin disease that affects approximately 2% of the world's population. Conventional therapeutic approaches are not effective or necessarily safe for treating symptoms due to the serious side effects and resistance to currently prescribed drugs. Traditionally, in oriental medicine, apricot seed (Semen Armeniacae amarum) is used to treat skin diseases. However, the underlying mechanism of action has not been systematically elucidated. In the present study, the anti-proliferative effect of bitter apricot essential oil (BAEO) on cultured HaCaT cells was evaluated and the mechanism of action investigated. BAEO was isolated by hydrodistillation, and gas chromatography-mass spectrometry (GC-MS) analysis identified benzaldehyde (75.35%), benzoic acid (6.21%) and mandelonitrile (5.38%). HaCaT cell growth, measured by sulforhodamine B assay (SRB), was inhibited by BAEO with an IC50 value of 142.45 μg/ml. Apoptosis of HaCaT cells treated with BAEO was detected by cell cycle, flow cytometry, and western blot analyses. These measurements revealed G0/G1 cell cycle arrest, elevated numbers of early and late stage apoptotic cells, and caspases-3/8/9 and PARP activation. Z-VAD-FMK, a broad-spectrum caspase inhibitor, attenuated BAEO-induced apoptosis. Also, increased Bax and decreased Bcl-2 levels suggest that BAEO-induced apoptosis is mediated through both death receptor and mitochondrial pathways. Moreover, reduced Rel/NF-κB levels suggest that BAEO-mediated apoptosis is also associated with inhibition of the NF-κB pathway. These data suggest that BAEO is a naturally occurring material that functions as a potent pro-apoptotic factor for human keratinocytes. Thus, it is a promising candidate to treat psoriasis.

  15. Differentiation of epidermal keratinocytes is dependent on glucosylceramide:ceramide processing.

    PubMed

    Amen, Nicole; Mathow, Daniel; Rabionet, Mariona; Sandhoff, Roger; Langbein, Lutz; Gretz, Norbert; Jäckel, Carsten; Gröne, Hermann-Josef; Jennemann, Richard

    2013-10-15

    Skin barrier function is primarily assigned to the outer epidermal layer, the stratum corneum (SC), mainly composed of corneocytes and lipid-enriched extracellular matrix. Epidermal ceramides (Cers) are essential barrier lipids, containing ultra-long-chain (ULC) fatty acids (FAs) with a unique ω-hydroxy group, which is necessary for binding to corneocyte proteins. In the SC, Cers are believed to derive from glucosylated intermediates, namely glucosylceramides (GlcCers), as surmised from human Gaucher's disease and related mouse models. Tamoxifen (TAM)-induced deletion of the endogenous GlcCer-synthesizing enzyme UDP-glucose:ceramide glucosyltransferase (UGCG) in keratin K14-positive cells resulted in epidermal GlcCer depletion. Although free extractable Cers were elevated in total epidermis and as well in SC, protein-bound Cers decreased significantly in Ugcg(f/fK14CreERT2) mice, indicating glucosylation to be required for regular Cer processing as well as arrangement and extrusion of lipid lamellae. The almost complete loss of protein-bound Cers led to a disruption of the water permeability barrier (WPB). UGCG-deficient mice developed an ichthyosis-like skin phenotype marked by impaired keratinocyte differentiation associated with delayed wound healing. Gene expression profiling of Ugcg-mutant skin revealed a subset of differentially expressed genes involved in lipid signaling and epidermal differentiation/proliferation, correlating to human skin diseases such as psoriasis and atopic dermatitis. Peroxisome proliferator-activated receptor beta/delta (PPARβ/δ), a Cer-sensitive transcription factor was identified as potential mediator of the altered gene sets.

  16. Tumor necrosis factor beta and ultraviolet radiation are potent regulators of human keratinocyte ICAM-1 expression

    SciTech Connect

    Krutmann, J.; Koeck, A.S.; Schauer, E.; Parlow, F.; Moeller, A.K.; Kapp, A.; Foerster, E.S.; Schoepf, E.L.; Luger, T.A. )

    1990-08-01

    Intercellular adhesion molecule-1 (ICAM-1) functions as a ligand of leukocyte function-associated antigen-1 (LFA-1), as well as a receptor for human picorna virus, and its regulation thus affects various immunologic and inflammatory reactions. The weak, constitutive ICAM-1 expression on human keratinocytes (KC) can be up-regulated by cytokines such as interferon-gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha). In order to further examine the regulation of KC ICAM-1 expression, normal human KC or epidermoid carcinoma cells (KB) were incubated with different cytokines and/or exposed to ultraviolet (UV) radiation. Subsequently, ICAM-1 expression was monitored cytofluorometrically using a monoclonal anti-ICAM-1 antibody. Stimulation of cells with recombinant human (rh) interleukin (IL) 1 alpha, rhIL-4, rhIL-5, rhIL-6, rh granulocyte/macrophage colony-stimulating factor (GM-CSF), rh interferon alpha (rhIFN alpha), and rh transforming growth factor beta (TGF beta) did not increase ICAM-1 surface expression. In contrast, rhTNF beta significantly up-regulated ICAM-1 expression in a time- and dose-dependent manner. Moreover, the combination of rhTNF beta with rhIFN gamma increased the percentage of ICAM-1-positive KC synergistically. This stimulatory effect of rhTNF beta was further confirmed by the demonstration that rhTNF beta was capable of markedly enhancing ICAM-1 mRNA expression in KC. Finally, exposure of KC in vitro to sublethal doses of UV radiation (0-100 J/m2) prior to cytokine (rhIFN tau, rhTNF alpha, rhTNF beta) stimulation inhibited ICAM-1 up-regulation in a dose-dependent fashion. These studies identify TNF beta and UV light as potent regulators of KC ICAM-1 expression, which may influence both attachment and detachment of leukocytes and possibly viruses to KC.

  17. Activity-relevant similarity values for fingerprints and implications for similarity searching

    PubMed Central

    Jasial, Swarit; Hu, Ye; Vogt, Martin; Bajorath, Jürgen

    2016-01-01

    A largely unsolved problem in chemoinformatics is the issue of how calculated compound similarity relates to activity similarity, which is central to many applications. In general, activity relationships are predicted from calculated similarity values. However, there is no solid scientific foundation to bridge between calculated molecular and observed activity similarity. Accordingly, the success rate of identifying new active compounds by similarity searching is limited. Although various attempts have been made to establish relationships between calculated fingerprint similarity values and biological activities, none of these has yielded generally applicable rules for similarity searching. In this study, we have addressed the question of molecular versus activity similarity in a more fundamental way. First, we have evaluated if activity-relevant similarity value ranges could in principle be identified for standard fingerprints and distinguished from similarity resulting from random compound comparisons. Then, we have analyzed if activity-relevant similarity values could be used to guide typical similarity search calculations aiming to identify active compounds in databases. It was found that activity-relevant similarity values can be identified as a characteristic feature of fingerprints. However, it was also shown that such values cannot be reliably used as thresholds for practical similarity search calculations. In addition, the analysis presented herein helped to rationalize differences in fingerprint search performance. PMID:27127620

  18. Identification and validation of reference genes for expression studies in human keratinocyte cell lines treated with and without interferon-γ - a method for qRT-PCR reference gene determination.

    PubMed

    Riemer, Angelika B; Keskin, Derin B; Reinherz, Ellis L

    2012-08-01

    Based on the exquisite sensitivity, reproducibility and wide dynamic range of quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR), it is currently the gold standard for gene expression studies. Target gene expression is calculated relative to a stably expressed reference gene. An ideal reference should be uniformly expressed during all experimental conditions within the given experimental system. However, no commonly applicable 'best' reference gene has been identified. Thus, endogenous controls must be determined for every experimental system. As no appropriate reference genes have been reported for immunological studies in keratinocytes, we aimed at identifying and validating a set of endogenous controls for these settings. An extensive validation of sixteen possible endogenous controls in a panel of 8 normal and transformed keratinocyte cell lines in experimental conditions with and without interferon-γ was performed. RNA and cDNA quality was stringently controlled. Candidate reference genes were assessed by TaqMan(®) qRT-PCR. Two different statistical algorithms were used to determine the most stably and reproducibly expressed housekeeping genes. mRNA abundance was compared and reference genes with widely different ranges of expression than possible target genes were excluded. Subsequent geNorm and NormFinder analyses identified GAPDH, PGK1, IPO8 and PPIA as the most stably expressed genes in the keratinocyte panel under the given experimental conditions. We conclude that the geometric means of expression values of these four genes represents a robust normalization factor for qRT-PCR analyses in interferon-γ-dependent gene expression studies in keratinocytes. The methodology and results herein may help other researchers by facilitating their choice of reference genes.

  19. Comparative evaluation of the antiproliferative effect of cyclosporin A and gamma-interferon on normal and HPV-transformed keratinocytes by cell counting, MTT assay and tritiated thymidine incorporation.

    PubMed

    Marionnet, A V; Lizard, G; Chardonnet, Y; Schmitt, D

    1997-02-01

    We compared three techniques, the MTT tetrazolium assay, cell counting, and tritiated thymidine ([3H]TdR) incorporation assay to measure the antiproliferative effect of cyclosporin A (CsA) and interferon-gamma (IFN-gamma) on normal human skin keratinocyte cultures (NHK) used at the second passage and human papilomavirus type 16- and 18-transformed cell lines (EK16 and EK18) exposed continuously to the drugs for 3 days. The three techniques showed that under CsA (0.5 and 8 micrograms/ml) and IFN-gamma (5 and 160 U/ml) treatments the cells remained viable and that the growth of keratinocytes was inhibited. For IFN-gamma, the MTT colorimetric assay consistently underestimated its growth inhibitory activity as compared to cell counting or [3H]TdR incorporation, whatever the cells used. For high doses of CsA, MTT and cell counting gave similar percentages, of inhibitory activity whatever the cells; MTT underestimated this activity as compared to [3H]TdR incorporation only in NHK and EK18 cells, whereas similar results were obtained with EK16 cells. In conclusion, this investigations shows that MTT sensitivity differed with the drug and also according to the keratinocyte cultures. The MTT test is clearly not appropriate for study of IFN-gamma treatment whatever the keratinocytes used. Such discrepancies indicate that the MTT test should be done with care on cultures to measure the effects of drugs on cell growth; the growth inhibition should be carefully considered and it would be best if two different methods were used.

  20. Identifying Hazards

    EPA Pesticide Factsheets

    The federal government has established a system of labeling hazardous materials to help identify the type of material and threat posed. Summaries of information on over 300 chemicals are maintained in the Envirofacts Master Chemical Integrator.

  1. DeltaNp63alpha repression of the Notch1 gene supports the proliferative capacity of normal human keratinocytes and cervical cancer cells.

    PubMed

    Yugawa, Takashi; Narisawa-Saito, Mako; Yoshimatsu, Yuki; Haga, Kei; Ohno, Shin-ichi; Egawa, Nagayasu; Fujita, Masatoshi; Kiyono, Tohru

    2010-05-15

    The p53 family member p63 is a master regulator of epithelial development. One of its isoforms, DeltaNp63alpha, is predominantly expressed in the basal cells of stratified epithelia and plays a fundamental role in control of regenerative potential and epithelial integrity. In contrast to p53, p63 is rarely mutated in human cancers, but it is frequently overexpressed in squamous cell carcinomas (SCC). However, its functional relevance to tumorigenesis remains largely unclear. We previously identified the Notch1 gene as a novel transcriptional target of p53. Here, we show that DeltaNp63alpha functions as a transcriptional repressor of the Notch1 gene through the p53-responsive element. Knockdown of p63 caused upregulation of Notch1 expression and marked reduction in proliferation and clonogenicity of both normal human keratinocytes and cervical cancer cell lines overexpressing DeltaNp63alpha. Concomitant silencing of Notch1 significantly rescued this phenotype, indicating the growth defect induced by p63 deficiency to be, at least in part, attributable to Notch1 function. Conversely, overexpression of DeltaNp63alpha decreased basal levels of Notch1, increased proliferative potential of normal human keratinocytes, and inhibited both p53-dependent and p53-independent induction of Notch1 and differentiation markers upon genotoxic stress and serum exposure, respectively. These results suggest that DeltaNp63alpha maintains the self-renewing capacity of normal human keratinocytes and cervical cancer cells partly through transcriptional repression of the Notch1 gene and imply a novel pathogenetical significance of frequently observed overexpression of DeltaNp63alpha together with p53 inactivation in SCCs.

  2. Insulin-like growth factor-binding protein-7 (IGFBP7) transcript: A-to-I editing events in normal and cancerous human keratinocytes.

    PubMed

    Hochberg, Malka; Gilead, Leon; Markel, Gal; Nemlich, Yael; Feiler, Yulia; Enk, Claes David; Denichenko, Polina; Karni, Rotem; Ingber, Arieh

    2013-08-01

    Non-melanoma skin cancers (NMSC) are the most common malignancies in caucasians worldwide. Insulin-like growth factor-binding protein-7 (IGFBP7) was suggested to function as a tumor suppressor gene in several cancers, and to play a role in the proliferation of keratinocytes. A-to-I RNA editing is a post-transcriptional mechanism frequently used to expand and diversify transcriptome and proteome repertoire in eukaryotic cells. A-to-I RNA editing can alter codons, substitute amino acids and affect protein sequence, structure, and function. Two editing sites were identified within the IGFBP7 transcript. To evaluate the expression and editing of IGFBP7 mRNA in NMSC compared to normal epidermis. We examined the expression and mRNA editing level of IGFBP7 in 22 basal cell carcinoma (BCC), 15 squamous cell carcinoma (SCC), and 18 normal epidermis samples that were surgically removed from patients by the Mohs Micrographic Surgery procedure. We studied the effect of IGFBP7 editing on an immortalized HaCaT keratinocyte cell model. IGFBP7 mRNA is over expressed in BCC and SCC compared to normal epidermis. Moreover, the IGFBP7 transcript is highly edited in normal epidermis, but its editing is significantly reduced in BCC and SCC. The edited form of IGFBP7 can inhibit proliferation and induce senescence in cultured keratinocytes. This study describes for the first time A-to-I editing in the coding sequence of a tumor suppressor gene in humans, and suggests that IGFBP7 editing serves as a fine-tuning mechanism to maintain the equilibrium between proliferation and senescence in normal skin.

  3. In vivo production of novel vitamin D2 hydroxy-derivatives by human placentas, epidermal keratinocytes, Caco-2 colon cells and the adrenal gland

    PubMed Central

    Slominski, Andrzej T.; Kim, Tae-Kang; Shehabi, Haleem Z.; Tang, Edith; Benson, Heather A. E.; Semak, Igor; Lin, Zongtao; Yates, Charles R.; Wang, Jin; Li, Wei; Tuckey, Robert C.

    2014-01-01

    We investigated the metabolism of vitamin D2 to hydroxyvitamin D2 metabolites ((OH)D2) by human placentas ex-utero, adrenal glands ex-vivo and cultured human epidermal keratinocytes and colonic Caco-2 cells, and identified 20(OH)D2, 17,20(OH)2D2, 1,20(OH)2D2, 25(OH)D2 and 1,25(OH)2D2 as products. Inhibition of product formation by 22R-hydroxycholesterol indicated involvement of CYP11A1 in 20- and 17-hydroxylation of vitamin D2, while use of ketoconazole indicated involvement of CYP27B1 in 1α-hydroxylation of products. Studies with purified human CYP11A1 confirmed the ability of this enzyme to convert vitamin D2 to 20(OH)D2 and 17,20(OH)2D2. In placentas and Caco-2 cells, production of 20(OH)D2 was higher than 25(OH)D2 while in human keratinocytes the production of 20(OH)D2 and 25(OH)D2 were comparable. HaCaT keratinocytes showed high accumulation of 1,20(OH)2D2 relative to 20(OH)D2 indicating substantial CYP27B1 activity. This is the first in vivo evidence for a novel pathway of vitamin D2 metabolism initiated by CYP11A1 and modified by CYP27B1, with the product profile showing tissue- and cell-type specificity. PMID:24382416

  4. In vivo production of novel vitamin D2 hydroxy-derivatives by human placentas, epidermal keratinocytes, Caco-2 colon cells and the adrenal gland.

    PubMed

    Slominski, Andrzej T; Kim, Tae-Kang; Shehabi, Haleem Z; Tang, Edith K Y; Benson, Heather A E; Semak, Igor; Lin, Zongtao; Yates, Charles R; Wang, Jin; Li, Wei; Tuckey, Robert C

    2014-03-05

    We investigated the metabolism of vitamin D2 to hydroxyvitamin D2 metabolites ((OH)D2) by human placentas ex-utero, adrenal glands ex-vivo and cultured human epidermal keratinocytes and colonic Caco-2 cells, and identified 20(OH)D2, 17,20(OH)₂D2, 1,20(OH)₂D2, 25(OH)D2 and 1,25(OH)₂D2 as products. Inhibition of product formation by 22R-hydroxycholesterol indicated involvement of CYP11A1 in 20- and 17-hydroxylation of vitamin D2, while use of ketoconazole indicated involvement of CYP27B1 in 1α-hydroxylation of products. Studies with purified human CYP11A1 confirmed the ability of this enzyme to convert vitamin D2 to 20(OH)D2 and 17,20(OH)₂D2. In placentas and Caco-2 cells, production of 20(OH)D2 was higher than 25(OH)D2 while in human keratinocytes the production of 20(OH)D2 and 25(OH)D2 were comparable. HaCaT keratinocytes showed high accumulation of 1,20(OH)₂D2 relative to 20(OH)D2 indicating substantial CYP27B1 activity. This is the first in vivo evidence for a novel pathway of vitamin D2 metabolism initiated by CYP11A1 and modified by CYP27B1, with the product profile showing tissue- and cell-type specificity.

  5. Toxicity Assessment of Six Titanium Dioxide Nanoparticles in Human Epidermal Keratinocytes

    EPA Science Inventory

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

  6. Effect of cultured autologous oral keratinocyte suspension in fibrin glue on oral wound healing in rabbits.

    PubMed

    Lis, G J; Zarzecka, J; Litwin, J A; Jasek, E; Cichocki, T; Zapała, J

    2012-09-01

    The effect of cultured autologous oral keratinocyte suspension in fibrin glue on the healing of surgically produced oral mucosal wounds was assessed in the rabbit model. Using the light microscope and a digital image analysis system, the epithelization parameters (marginal epithelization and percentage of wound re-epithelization) were measured in haematoxylin-eosin stained sections of the wound area and compared with those of wounds treated with fibrin glue alone and untreated ones. The epithelization was significantly higher in keratinocytes plus fibrin glue-treated wounds on postoperative days 3 and 7. No significant differences were observed on postoperative day 1, when the healing process had just begun, and on postoperative day 14, when re-epithelization was completed or nearly completed in all groups. The inflammatory infiltration of the wounded mucosa was weakest in keratinocyte-treated wounds and strongest in untreated wounds. In conclusion, suspension of cultured autologous oral keratinocytes in fibrin glue significantly accelerates oral wound healing in the rabbit model and could be beneficial in the treatment of oral wounds in patients.

  7. [Role of keratinocytes in preservation of oral mucosa epithelium integrity. Part I].

    PubMed

    Zapała, Jan; Zarzecka, Joanna; Drukała, Justyna

    2005-01-01

    Functions of oral mucosa epithelium in preservation of homeostasis have been presented. Characteristic features that distinguish epithelial cells from the other somatic cells influencing mechanical resistance of oral epithelium and creating selective chemical barrier have been described. The participation of keratinocytes in selected phases of wound healing process has been analyzed.

  8. CCN1, a Pro-Inflammatory Factor, Aggravates Psoriasis Skin Lesions by Promoting Keratinocyte Activation.

    PubMed

    Sun, Yue; Zhang, Jie; Zhou, Zhou; Wu, Pinru; Huo, Rongfen; Wang, Beiqing; Shen, Zhengyu; Li, Huidan; Zhai, Tianhang; Shen, Baihua; Chen, Xiangdong; Li, Ningli

    2015-11-01

    Psoriasis is a common chronic skin disease characterized by epidermal hyperplasia and inflammation. The pathogenesis of psoriasis is multifactorial and is not fully understood. Here we demonstrate that CCN1 (also called Cyr61, which is short for cysteine-rich 61), an extracellular matrix protein that is also considered a pro-inflammatory factor, is highly expressed in the lesional skin of psoriasis patients, as well as in that of imiquimod (IMQ)- and IL-23-treated psoriasis-like mice. Then we show that blocking CCN1 function in vivo attenuates epidermal hyperplasia and inflammation in psoriasis-like mice. Further, in primary cultured normal human keratinocytes and HaCaT (human keratinocyte cell line) cells, CCN1 promotes keratinocyte activation, including the proliferation and expression of immune-related molecules. Finally, we observe that integrin α6β1 is the receptor of CCN1 in keratinocytes, and CCN1 stimulation activates the downstream phosphoinositide-3 kinase/Akt/NF-κB signaling pathway. Taken together, our findings reveal that CCN1 has a critical role in psoriasis pathogenesis. Moreover, as CCN1 is a secreted extracellular matrix (ECM) protein, our study also provides evidence that ECM, which is involved in psoriatic pathogenesis, could be a potent target for psoriasis treatment.

  9. Matriptase regulates proliferation and early, but not terminal, differentiation of human keratinocytes.

    PubMed

    Chen, Ya-Wen; Wang, Jehng-Kang; Chou, Fen-Pai; Wu, Bai-Yao; Hsiao, Hui-Chung; Chiu, Han; Xu, Zhonghong; Baksh, Adrienne N H; Shi, Galen; Kaul, Malvika; Barndt, Robert; Shanmugam, Victoria K; Johnson, Michael D; Lin, Chen-Yong

    2014-02-01

    Genetic defects in matriptase are linked to two congenital ichthyoses: autosomal recessive ichthyosis with hypotrichosis (ARIH, OMIM 610765) and ichthyosis, follicular atrophoderma, hypotrichosis, and hypohidrosis (IFAH, OMIM 602400). Mouse models with matriptase deficiency indicate an involvement of matriptase in suprabasal keratinocytes in the maintenance of the epidermal barrier. In contrast to what has been reported for mouse skin, we show that in human skin matriptase is primarily expressed in the basal and spinous keratinocytes, but not in the more differentiated keratinocytes of the granular layer. In addition, matriptase zymogen activation was predominantly detected in the basal cells. Furthermore, by using skin organotypic cultures as a model system to monitor the course of human epidermal differentiation, we found elevated matriptase zymogen activation during early stages of epidermal differentiation, coupled with a loss of matriptase expression in the late stages of this process. We also show here that matriptase deficiency in HaCaT cells modestly reduces cell proliferation and temporally affects calcium-induced expression of differentiation markers. These collective data suggest that, unlike mouse matriptase, human matriptase may be involved in the regulation of keratinocyte growth and early differentiation, rather than terminal differentiation, providing mechanistic insights into the pathology of the two congenital ichthyoses: ARIH and IFAH.

  10. Influence of different buffers (HEPES/MOPS) on keratinocyte cell viability and microbial growth.

    PubMed

    Dias, Kássia de Carvalho; Barbugli, Paula Aboud; Vergani, Carlos Eduardo

    2016-06-01

    This study assessed the effect of the buffers 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 3-(N-morpholino) propanesulfonic acid (MOPS) on keratinocyte cell viability and microbial growth. It was observed that RPMI buffered with HEPES, supplemented with l-glutamine and sodium bicarbonate, can be used as a more suitable medium to promote co-culture.

  11. Expression of HLA-DR (Ia like) antigen on epidermal keratinocytes in human dermatoses.

    PubMed Central

    Lampert, I A

    1984-01-01

    Ia antigen (HLA-DR in man) has been demonstrated in keratinocytes in graft versus host disease. This study investigates the occurrence of HLA-DR in keratinocytes in the following dermatoses: eczematous dermatitis, discoid lupus erythematosus, with immunoglobulin and non-exposed skin from cases of systemic lupus erythematosus with immunoglobulin deposits, lichen planus, lichen simplex, bullous pemphigoid, pemphigus vulgaris, 'toxic erthema', tuberculid and chillblain. Keratinocyte staining was found in a variety of conditions. The unifying features of the instances of its occurrence was lymphoid infiltration and usually some focal evidence of keratinocyte damage. Thus in eczema the staining was mid-epidermal, while in discoid lupus erythematosus and lichen planus it was basal. HLA-DR staining was absent in bullous pemphigoid and pemphigus vulgaris, which is consistent with the hypothesis that in these conditions the damage is mediated by autoantibodies and complement in the absence of cellular immune attack. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:6204802

  12. Candida albicans phospholipomannan triggers inflammatory responses of human keratinocytes through Toll-like receptor 2.

    PubMed

    Li, Min; Chen, Qing; Shen, Yongnian; Liu, Weida

    2009-07-01

    The Toll-like receptors (TLRs) play an important role in the recognition of Candida albicans components and activation of innate immunity. Phospholipomannan (PLM), a glycolipid, is expressed at the surface of C. albicans cell wall, which acts as a member of the pathogen-associated molecular patterns family. In this study, we sought to clarify whether C. albicans-native PLM could induce an inflammation response in human keratinocytes and to determine the underlying mechanisms. Exposure of cultured human primary keratinocytes to PLM led to the increased gene expression and secretion of proinflammatory cytokines (IL-6) and chemokines (IL-8). PLM hydrolysed with beta-d-mannoside mannohydrolase failed to induce gene expression and secretion of IL-6 and IL-8. PLM up-regulated the mRNA and protein levels of TLR2, whereas the mRNA level of TLR4 was not altered. Keratinocytes challenged with PLM resulted in the activation of NF-kappaB and mitogen-activated protein kinase (MAPKs) including p38. Anti-TLR2 neutralizing antibody, NFkappaB and p38MAPK inhibitors blocked the PLM-induced secretion of IL-6, IL-8 in keratinocytes, but no such effect was observed in pretreatment with anti-TLR4-neutralizing antibody and lipopolysaccharide inhibitor (polymyxin B). These data suggest C. albicans-native PLM may contribute to the inflammatory responses of cutaneous candidiasis in the TLR2-NF-kappaB and p38MAPK signalling pathway dependent manner.

  13. Plakophilin-1 protects keratinocytes from pemphigus vulgaris IgG by forming calcium-independent desmosomes.

    PubMed

    Tucker, Dana K; Stahley, Sara N; Kowalczyk, Andrew P

    2014-04-01

    Plakophilin-1 (PKP-1) is an armadillo family protein critical for desmosomal adhesion and epidermal integrity. In the autoimmune skin-blistering disease pemphigus vulgaris (PV), autoantibodies (IgG) target the desmosomal cadherin desmoglein 3 (Dsg3) and compromise keratinocyte cell-cell adhesion. Here, we report that enhanced expression of PKP-1 protects keratinocytes from PV IgG-induced loss of cell-cell adhesion. PKP-1 prevents loss of Dsg3 and other desmosomal proteins from cell-cell borders and prevents alterations in desmosome ultrastructure in keratinocytes treated with PV IgG. Using a series of Dsg3 chimeras and deletion constructs, we find that PKP-1 clusters Dsg3 with the desmosomal plaque protein desmoplakin in a manner dependent on the plakoglobin-binding domain of the Dsg3 tail. Furthermore, PKP-1 expression transforms desmosome adhesion from a calcium-dependent to a calcium-independent and hyperadhesive state. These results demonstrate that manipulating the expression of a single desmosomal plaque protein can block the pathogenic effects of PV IgG on keratinocyte adhesion.

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

    SciTech Connect

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

    2011-08-26

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

  15. Thr308 determines Akt1 nuclear localization in insulin-stimulated keratinocytes

    SciTech Connect

    Goren, Itamar; Mueller, Elke; Pfeilschifter, Josef

    2008-07-18

    Here, we determined the localization and activation of protein kinase B (Akt) in acute cutaneous wound tissue in mice. Akt1 represented the major Akt isoform that was expressed and activated in wound margin keratinocytes and also in the cultured human keratinocyte line HaCaT. Mutation of Akt1 protein, exchanging the activation-essential Ser473 and Thr308 residues for inactive Ala or phosphorylation-mimicking Asp and Glu residues, revealed that phosphorylation of Ser473 represented an essential prerequisite for auto-phosphorylation of Thr308 within the Akt1 protein in keratinocytes. Moreover, cell culture experiments and transfection studies using Thr308 mutated Akt1 proteins demonstrated that phosphorylation of Akt1 at Thr308 appeared to selectively exclude the active kinase from the nucleus and direct the kinase to the cytoplasmic compartment in keratinocytes upon insulin stimulation. In summary, our data show that phosphorylation of Thr308 during insulin-mediated Akt1 activation is an essential prerequisite to exclude Akt1 from the nuclear compartment.

  16. miR-155 promotes cutaneous wound healing through enhanced keratinocytes migration by MMP-2.

    PubMed

    Yang, Longlong; Zheng, Zhao; Zhou, Qin; Bai, Xiaozhi; Fan, Lei; Yang, Chen; Su, Linlin; Hu, Dahai

    2017-04-01

    Inflammation, re-epithelization and tissue remodeling are three essential steps during wound healing. The re-epithelization process plays the most important role which mainly involves keratinocyte proliferation and migration. miR-155 has been reported to participate in cell migration and transformation, however, its function in skin wound healing is largely unknown. Here we hypothesize that overexpression of miR-155 at wound edges could accelerate wound healing mediated by enhanced keratinocyte migration. To test this hypothesis, direct local injection of miR-155 expression plasmid to wound edges was conducted to overexpress miR-155 in vivo. Results shown that miR-155 significantly promoted wound healing and re-epithelization compared to control, while did not affect wound contraction. Also, miR-155 overexpression accelerated primarily cultured keratinocyte migration in vitro, but had no effect on cell proliferation. Importantly, western blot analysis shown that MMP-2 was significantly upregulated whiles its inhibitor TIMP-1 downregulated after miR-155 treatment. Moreover, the use of ARP-101, an MMP-2 inhibitor, effectively attenuated the accelerative effects on cell migration induced by miR-155. Taken together, our results suggest that miR-155 has the promote effect on wound healing that is probably mediated by accelerating keratinocyte migration via upregulated MMP-2 level. This study provides a rationale for the therapeutic effect of miR-155 on wound healing.

  17. Inhibition of peroxisome proliferator-activated receptor (PPAR)-mediated keratinocyte differentiation by lipoxygenase inhibitors.

    PubMed Central

    Thuillier, Philippe; Brash, Alan R; Kehrer, James P; Stimmel, Julie B; Leesnitzer, Lisa M; Yang, Peiying; Newman, Robert A; Fischer, Susan M

    2002-01-01

    Lipoxygenase (LOX) metabolites from arachidonic acid and linoleic acid have been implicated in atherosclerosis, inflammation, keratinocyte differentiation and tumour progression. We previously showed that peroxisome proliferator-activated receptors (PPARs) play a role in keratinocyte differentiation and that the PPARalpha ligand 8S-hydroxyeicosatetraenoic acid is important in this process. We hypothesized that blocking LOX activity would block PPAR-mediated keratinocyte differentiation. Three LOX inhibitors, nordihydroguaiaretic acid, quercetin and morin, were studied for their effects on primary keratinocyte differentiation and PPAR activity. All three LOX inhibitors blocked calcium-induced expression of the differentiation marker keratin 1. In addition, activity of a PPAR-responsive element was inhibited in the presence of all three inhibitors, and this effect was mediated primarily through PPARalpha and PPARgamma. LOX inhibitors decreased the activity of a chimaeric PPAR-Gal4-ligand-binding domain reporter system and this effect was reversed by addition of PPAR ligands. Ligand-binding studies revealed that the LOX inhibitors bind directly to PPARs and demonstrate a novel mechanism for these inhibitors in altering PPAR-mediated gene expression. PMID:12069687

  18. Trichodysplasia spinulosa: a polyomavirus infection specifically targeting follicular keratinocytes in immunocompromised patients.

    PubMed

    Rouanet, J; Aubin, F; Gaboriaud, P; Berthon, P; Feltkamp, M C; Bessenay, L; Touzé, A; Nicol, J T J; Franck, F; D'Incan, M

    2016-03-01

    Trichodysplasia spinulosa (TS) is a rare skin disease, caused by a specific polyomavirus, occurring in immunocompromised patients. The pathophysiological mechanisms of TS are not yet fully understood. By using polymerase chain reaction and skin biopsy immunostaining we report evidence, in a paediatric case, of follicular keratinocytes being the primary target of trichodysplasia spinulosa-associated polyomavirus.

  19. Modulation of phospholipid metabolism in murine keratinocytes by tumor promoter, 12-O-tetradecanoylphorbol-13-acetate

    SciTech Connect

    Galey, C.I.; Ziboh, V.A.; Marcelo, C.L.; Voorhees, J.J.

    1985-10-01

    The possibility that phospholipid deacylation may be a critical event in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-associated effects on mouse skin prompted us to examine in vitro the effects of TPA on arachidonic acid metabolism in neonatal mouse keratinocytes. Three-day old neonatal keratinocytes were prelabeled with ( UC)arachidonic acid (( UC)AA) and ( UC) stearic acid (( UC)ST) and used to characterize the lipases that were activated when these cells were treated with TPA in culture. Data from these studies demonstrate that phosphatidylcholine (PC) and phosphatidylinositol (PI) are the major phospholipids that undergo early hydrolysis to release arachidonic acid when challenged by TPA. Of particular interest was the novel observation of the hydrolysis of UC-labeled PI in these keratinocytes, the accumulation of ( UC)1,2-diacylglyceride and the lack of the ( UC)diacylglyceride phosphorylation to form ( UC)phosphatidic acid. This lack of ( UC) phosphatidic accumulation implied that although TPA enhanced the hydrolysis of ( UC)PI resulting in increased ( UC)diacylglyceride it did not enhance the resynthesis of the ( UC)PI via the phosphorylation of the ( UC)diacylglyceride. Therefore, TPA probably is not involved in the turnover of PI in these cells but is involved in the activation of PC hydrolyzing phospholipase A2 and PI hydrolyzing phospholipase C in these keratinocytes releasing arachidonic acid which then undergoes oxygenation reactions to provide biologically active eicosanoids.

  20. Kinetin Improves Barrier Function of the Skin by Modulating Keratinocyte Differentiation Markers

    PubMed Central

    An, Sungkwan; Cha, Hwa Jun; Ko, Jung-Min; Han, Hyunjoo; Kim, Su Young; Kim, Kyung-Suk; Lee, Song Jeong; An, In-Sook; Kim, Sangwon; Youn, Hae Jeong

    2017-01-01

    Background Kinetin is a plant hormone that regulates growth and differentiation. Keratinocytes, the basic building blocks of the epidermis, function in maintaining the skin barrier. Objective We examined whether kinetin induces skin barrier functions in vitro and in vivo. Methods To evaluate the efficacy of kinetin at the cellular level, expression of keratinocyte differentiation markers was assessed. Moreover, we examined the clinical efficacy of kinetin by evaluating skin moisture, transepidermal water loss (TEWL), and skin surface roughness in patients who used kinetin-containing cream. We performed quantitative real-time polymerase chain reaction to measure the expression of keratinocyte differentiation markers in HaCaT cells following treatment. A clinical trial was performed to assess skin moisture, TEWL, and evenness of skin texture in subjects who used kinetin-containing cream for 4 weeks. Results Kinetin increased involucrin, and keratin 1 mRNA in HaCaT cells. Moreover, use of a kinetin-containing cream improved skin moisture and TEWL while decreasing roughness of skin texture. Conclusion Kinetin induced the expression of keratinocyte differentiation markers, suggesting that it may affect differentiation to improve skin moisture content, TEWL, and other signs of skin aging. Therefore, kinetin is a potential new component for use in cosmetics as an anti-aging agent that improves the barrier function of skin. PMID:28223740

  1. Role of dermatopontin in re-epithelialization: implications on keratinocyte migration and proliferation.

    PubMed

    Krishnaswamy, Venkat Raghavan; Korrapati, Purna Sai

    2014-12-09

    Re-epithelialization is a key event in wound healing and any impairment in that process is associated with various pathological conditions. Epidermal keratinocyte migration and proliferation during re-epithelialization is largely regulated by the cytokines and growth factors from the provisional matrix and dermis. Extracellular matrix consists of numerous growth factors which mediate cell migration via cell membrane receptors. Dermatopontin (DPT), a non-collagenous matrix protein highly expressed in dermis is known for its striking ability to promote cell adhesion. DPT also enhances the biological activity of transforming growth factor beta 1 which plays a central role in the process of wound healing. This study was designed to envisage the role of DPT in keratinocyte migration and proliferation along with its mRNA and protein expression pattern in epidermis. The results showed that DPT promotes keratinocyte migration in a dose dependant fashion but fail to induce proliferation. Further, PCR and immunodetection studies revealed that the mRNA and protein expression of DPT is considerably negligible in the epidermis in contrast to the dermis. To conclude, DPT has a profound role in wound healing specifically during re-epithelialization by promoting keratinocyte migration via paracrine action from the underlying dermis.

  2. Analysis of the response of human keratinocytes to Malassezia globosa and restricta strains.

    PubMed

    Donnarumma, Giovanna; Perfetto, Brunella; Paoletti, Iole; Oliviero, Giovanni; Clavaud, Cécile; Del Bufalo, Aurelia; Guéniche, Audrey; Jourdain, Roland; Tufano, Maria Antonietta; Breton, Lionel

    2014-10-01

    Malassezia spp. are saprophyte yeasts involved in skin diseases with different degrees of severity. The aim of our study was to analyze the response of human epidermal keratinocytes to Malassezia globosa and restricta strains evaluating the host defence mechanisms induced by Malassezia spp. colonization. Our results showed a different modulation of the inflammatory and immunomodulatory cytokine pathways obtained with the different strains of Malassezia tested. In addition, this expression is altered by blocking the TLR2 receptor. In comparison with M. furfur, M. globosa and restricta displayed an unexpected and striking cytotoxicity on keratinocytes. The differences observed could be related to the different modalities of interaction between keratinocytes and Malassezia strains, but also to their growth condition. Taken together, these results indicate that M. globosa or M. restricta colonization exert a different control on the cytokine inflammatory response activated in the human keratinocyte in which TLR2 might be involved. M. globosa and M. restricta may play a synergistic role in the exacerbation of skin diseases in which both are found.

  3. UVB-dependent changes in the expression of fast-responding early genes is modulated by huCOP1 in keratinocytes.

    PubMed

    Fazekas, B; Polyánka, H; Bebes, A; Tax, G; Szabó, K; Farkas, K; Kinyó, A; Nagy, F; Kemény, L; Széll, M; Ádám, É

    2014-11-01

    Ultraviolet (UV) B is the most prominent physical carcinogen in the environment leading to the development of various skin cancers. We have previously demonstrated that the human ortholog of the Arabidopsis thaliana constitutive photomorphogenesis 1 (COP1) protein, huCOP1, is expressed in keratinocytes in a UVB-regulated manner and is a negative regulator of p53 as a posttranslational modifier. However, it was not known whether huCOP1 plays a role in mediating the UVB-induced early transcriptional responses of human keratinocytes. In this study, we report that stable siRNA-mediated silencing of huCOP1 affects the UVB response of several genes within 2 h of irradiation, indicating that altered huCOP1 expression sensitizes the cells toward UVB. Pathway analysis identified a molecular network in which 13 of the 30 examined UVB-regulated genes were organized around three central proteins. Since the expression of the investigated genes was upregulated by UVB in the siCOP1 cell line, we hypothesize that huCOP1 is a repressor of the identified pathway. Several members of the network have been implicated previously in the pathogenesis of non-melanoma skin cancers; therefore, clarifying the role of huCOP1 in these skin diseases may have clinical relevance in the future.

  4. A derivatization-enhanced detection strategy in mass spectrometry: analysis of 4-hydroxybenzoates and their metabolites after keratinocytes are exposed to UV radiation

    PubMed Central

    Lee, Yi-Hsuan; Lin, Ying-Chi; Feng, Chia-Hsien; Tseng, Wei-Lung; Lu, Chi-Yu

    2017-01-01

    4-Hydroxybenzoate is a phenolic derivative of alkyl benzoates and is a widely used preservative in cosmetic and pharmaceutical products. The presence of 4-hydroxybenzoates in the human body may result from the use of pharmaceutical and personal care products. These compounds are also known to exhibit estrogenic and genotoxic activities. The potential adverse effects of these compounds include endocrine disruption, oxidative and DNA damage, contact dermatitis, and allergic reactions. This study used two mass spectrometry methods that are applicable when using a derivatization-enhanced detection strategy (DEDS) to screen 4-hydroxybenzoates and their metabolites. Chemical derivatization was used to enhance the detection of these compounds. To evaluate the metabolic process triggered by UV radiation, human keratinocyte HaCaT cells treated with these 4-hydroxybenzoates were further exposed to UVA, UVB and UVC radiation. Metabolites transformed by human keratinocytes in the chemical derivatization procedure were identified by a nano ultra-performance liquid chromatographic system (nanoUPLC) coupled with LTQ Orbitrap. The experiments confirmed the feasibility of this method for identifying 4-hydroxybenzoate metabolites and for high-throughput screening of 4-hydroxybenzoate in commercial products (50 samples) by the DEDS. PMID:28057923

  5. Non-coding Double-stranded RNA and Antimicrobial Peptide LL-37 Induce Growth Factor Expression from Keratinocytes and Endothelial Cells.

    PubMed

    Adase, Christopher A; Borkowski, Andrew W; Zhang, Ling-Juan; Williams, Michael R; Sato, Emi; Sanford, James A; Gallo, Richard L

    2016-05-27

    A critical function for skin is that when damaged it must simultaneously identify the nature of the injury, repair barrier function, and limit the intrusion of pathogenic organisms. These needs are carried out through the detection of damage-associated molecular patterns (DAMPs) and a response that includes secretion of cytokines, chemokines, growth factors, and antimicrobial peptides (AMPs). In this study, we analyzed how non-coding double-stranded RNA (dsRNAs) act as a DAMP in the skin and how the human cathelicidin AMP LL-37 might influence growth factor production in response to this DAMP. dsRNA alone significantly increased the expression of multiple growth factors in keratinocytes, endothelial cells, and fibroblasts. Furthermore, RNA sequencing transcriptome analysis found that multiple growth factors increase when cells are exposed to both LL-37 and dsRNA, a condition that mimics normal wounding. Quantitative PCR and/or ELISA validated that growth factors expressed by keratinocytes in these conditions included, but were not limited to, basic fibroblast growth factor (FGF2), heparin-binding EGF-like growth factor (HBEGF), vascular endothelial growth factor C (VEGFC), betacellulin (BTC), EGF, epiregulin (EREG), and other members of the transforming growth factor β superfamily. These results identify a novel role for DAMPs and AMPs in the stimulation of repair and highlight the complex interactions involved in the wound environment.

  6. Murine skin and vaginal mucosa are similarly susceptible to infection by pseudovirions of different papillomavirus classifications and species

    PubMed Central

    Handisurya, Alessandra; Day, Patricia M.; Thompson, Cynthia D.; Buck, Christopher B.; Kwak, Kihyuck; Roden, Richard B. S.; Lowy, Douglas R.; Schiller, John T.

    2013-01-01

    Depending upon viral genotype, productive papillomavirus infection and disease display preferential tropism for cutaneous or mucosal stratified squamous epithelia, although the mechanisms are unclear. To investigate papillomavirus entry tropism, we used reporter pseudovirions based on various cutaneous and mucosal papillomavirus species, including the recently identified murine papillomavirus. Pseudovirus transduction of BALB/c mice was examined using an improved murine skin infection protocol and a previously developed cervicovaginal challenge model. In the skin, HPV5, HPV6, HPV16, BPV1 and MusPV1 pseudovirions preferentially transduced keratinocytes at sites of trauma, similar to the genital tract. Skin infection, visualized by in vivo imaging using a luciferase reporter gene, peaked between days 2–3 and rapidly diminished for all pseudovirion types. Murine cutaneous and genital tissues were similarily permissive for pseudovirions of HPV types 5,6,8,16,18,26,45,51,58 and animal papillomaviruses BPV1 and MusPV1, implying that papillomavirus’ tissue and host tropism is governed primarily by post-entry regulatory events in the mouse. PMID:22985477

  7. Fibroblast Growth Factor-Peptide Improves Barrier Function and Proliferation in Human Keratinocytes After Radiation

    SciTech Connect

    Zhang Kunzhong; Tian Yeping; Yin Liangjie; Zhang Mei; Beck, Lisa A.; Zhang, Bingrong; Okunieff, Paul; Zhang Lurong; Vidyasagar, Sadasivan

    2011-09-01

    Purpose: Epidermal keratinocytes, which can be severely damaged after ionizing radiation (IR), are rapid turnover cells that function as a barrier, protecting the host from pathogenic invasion and fluid loss. We tested fibroblast growth factor-peptide (FGF-P), a small peptide derived from the receptor-binding domain of FGF-2, as a potential mitigator of radiation effects via proliferation and the barrier function of keratinocytes. Methods and Materials: Keratinocytes isolated from neonatal foreskin were grown on transwells. After being exposed to 0, 5, or 10 Gy IR, the cells were treated with a vehicle or FGF-P. The permeability of IR cells was assessed by using transepithelial electrical resistance (TEER) and a paracellular tracer flux of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) with Ussing chambers. The cell proliferation was measured with yellow tetrazolium salt (MTT) and tritiated thymidine ([{sup 3}H]-TdR) assays. The phosphorylation of extracellular signal-regulated kinases (ERK) was measured in an enzyme-linked immunosorbent (ELISA)-like assay, and the proteins related to tight junctions (TJ) and adherens junctions (AJ) were examined with Western blotting. We used a mouse model to assess the ability of FGF-P to promote the healing of skin {beta} burns created with a strontium applicator. Results: We found (1) FGF-P reduced the permeability of irradiated keratinocytes, as evidenced by increased TEER and decreased diffusion of FITC-BSA, both associated with the regulation of different proteins and levels of TJ and AJ; and (2) FGF-P enhanced the proliferation of irradiated keratinocytes, as evidenced by increased MTT activity and [{sup 3}H]-TdR incorporation, which was associated with activation of the ERK pathway; and (3) FGF-P promoted the healing of skin {beta} burns. Conclusions: FGF-P enhances the barrier function, including up-regulation of TJ proteins, increases proliferation of human keratinocytes, and accelerates the

  8. Contribution of Sp1 to Telomerase Expression and Activity in Skin Keratinocytes Cultured With a Feeder Layer.

    PubMed

    Bisson, Francis; Paquet, Claudie; Bourget, Jean-Michel; Zaniolo, Karine; Rochette, Patrick J; Landreville, Solange; Damour, Odile; Boudreau, François; Auger, François A; Guérin, Sylvain L; Germain, Lucie

    2015-02-01

    The growth of primary keratinocytes is improved by culturing them with a feeder layer. The aim of this study was to assess whether the feeder layer increases the lifespan of cultured epithelial cells by maintaining or improving telomerase activity and expression. The addition of an irradiated fibroblast feeder layer of either human or mouse origin (i3T3) helped maintain telomerase activity as well as expression of the transcription factor Sp1 in cultured keratinocytes. In contrast, senescence occurred earlier, together with a reduction of Sp1 expression and telomerase activity, in keratinocytes cultured without a feeder layer. Telomerase activity was consistently higher in keratinocytes grown on the three different feeder layers tested relative to cells grown without them. Suppression of Sp1 expression by RNA inhibition (RNAi) reduced both telomerase expression and activity in keratinocytes and also abolished their long-term growth capacity suggesting that Sp1 is a key regulator of both telomerase gene expression and cell cycle progression of primary cultured human skin keratinocytes. The results of the present study therefore suggest that the beneficial influence of the feeder layer relies on its ability to preserve telomerase activity in cultured human keratinocytes through the maintenance of stable levels of Sp1 expression.

  9. Galectin-3 protects keratinocytes from UVB-induced apoptosis by enhancing AKT activation and suppressing ERK activation

    PubMed Central

    Saegusa, Jun; Hsu, Daniel K.; Liu, Wei; Kuwabara, Ichiro; Kuwabara, Yasuko; Yu, Lan; Liu, Fu-Tong

    2009-01-01

    Keratinocytes undergo apoptosis in a variety of physiological and pathological conditions. Galectin-3 is a member of a family of β-galactoside-binding animal lectins expressed abundantly in keratinocytes and other epithelial cells. Here we have studied the regulatory role of galectin-3 in keratinocyte apoptosis by using cells from gene-targeted galectin-3 null (gal3−/−) mice. We showed that galectin-3 mRNA was transiently upregulated in ultraviolet-B (UVB)-irradiated wild-type keratinocytes. We found that gal3−/− keratinocytes were significantly more sensitive to apoptosis induced by UVB as well as various other stimuli, both in vitro and in vivo, than wild-type cells. Moreover, we demonstrated that increased apoptosis in gal3−/− keratinocytes was attributable to higher extracellular signal-regulated kinase (ERK) activation and lower AKT activation after UVB irradiation. We conclude that endogenous galectin-3 is an anti-apoptotic molecule in keratinocytes functioning by suppressing ERK activation and enhancing AKT activation and may play a role in the development of apoptosis-related skin diseases. PMID:18463681

  10. Eccrine sweat contains IL-1α, IL-1β and IL-31 and activates epidermal keratinocytes as a danger signal.

    PubMed

    Dai, Xiuju; Okazaki, Hidenori; Hanakawa, Yasushi; Murakami, Masamoto; Tohyama, Mikiko; Shirakata, Yuji; Sayama, Koji

    2013-01-01

    Eccrine sweat is secreted onto the skin's surface and is not harmful to normal skin, but can exacerbate eczematous lesions in atopic dermatitis. Although eccrine sweat contains a number of minerals, proteins, and proteolytic enzymes, how it causes skin inflammation is not clear. We hypothesized that it stimulates keratinocytes directly, as a danger signal. Eccrine sweat was collected from the arms of healthy volunteers after exercise, and levels of proinflammatory cytokines in the sweat were quantified by ELISA. We detected the presence of IL-1α, IL-1β, and high levels of IL-31 in sweat samples. To investigate whether sweat activates keratinocytes, normal human keratinocytes were stimulated with concentrated sweat. Western blot analysis demonstrated the activation of NF-κB, ERK, and JNK signaling in sweat-stimulated keratinocytes. Real-time PCR using total RNA and ELISA analysis of supernatants showed the upregulation of IL-8 and IL-1β by sweat. Furthermore, pretreatment with IL-1R antagonist blocked sweat-stimulated cytokine production and signal activation, indicating that bioactive IL-1 is a major factor in the activation of keratinocytes by sweat. Moreover, IL-31 seems to be another sweat stimulator that activates keratinocytes to produce inflammatory cytokine, CCL2. Sweat is secreted onto the skin's surface and does not come into contact with keratinocytes in normal skin. However, in skin with a defective cutaneous barrier, such as atopic dermatitis-affected skin, sweat cytokines can directly act on epidermal keratinocytes, resulting in their activation. In conclusion, eccrine sweat contains proinflammatory cytokines, IL-1 and IL-31, and activates epidermal keratinocytes as a danger signal.

  11. TCDD induces dermal accumulation of keratinocyte-derived matrix metalloproteinase-10 in an organotypic model of human skin

    SciTech Connect

    De Abrew, K. Nadira; Thomas-Virnig, Christina L.; Rasmussen, Cathy A.; Bolterstein, Elyse A.; Schlosser, Sandy J.; Allen-Hoffmann, B. Lynn

    2014-05-01

    The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization. These tissue remodeling events were preceded temporally by changes in expression of the extracellular matrix degrading enzyme, matrix metalloproteinase-10 (MMP-10). In organotypic cultures MMP-10 mRNA and protein were highly induced following TCDD treatment. Q-PCR and immunoblot results from TCDD-treated monolayer cultures, as well as indirect immunofluorescence and immunoblot analysis of TCDD-treated organotypic cultures, showed that MMP-10 was specifically contributed by the epidermal keratinocytes but not the dermal fibroblasts. Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment of organotypic cultures. TCDD-induced epidermal phenotypes in organotypic cultures were attenuated by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited in the basement membrane, the basal keratinocyte layer, and the cornified layer of TCDD-treated organotypic cultures. Our studies reveal a novel mechanism by which the epithelial–stromal microenvironment is altered in a tissue-specific manner thereby inducing structural and functional pathology in the interfollicular epidermis of human skin. - Highlights: • TCDD causes hyperkeratosis and basement membrane changes in a model of human skin. • TCDD induces MMP-10 expression in organotypic cultures

  12. Desmoglein 2 is less important than desmoglein 3 for keratinocyte cohesion.

    PubMed

    Hartlieb, Eva; Kempf, Bettina; Partilla, Miriam; Vigh, Balázs; Spindler, Volker; Waschke, Jens

    2013-01-01

    Desmosomes provide intercellular adhesive strength required for integrity of epithelial and some non-epithelial tissues. Within the epidermis, the cadherin-type adhesion molecules desmoglein (Dsg) 1-4 and desmocollin (Dsc) 1-3 build the adhesive core of desmosomes. In keratinocytes, several isoforms of these proteins are co-expressed. However, the contribution of specific isoforms to overall cell cohesion is unclear. Therefore, in this study we investigated the roles of Dsg2 and Dsg3, the latter of which is known to be essential for keratinocyte adhesion based on its autoantibody-induced loss of function in the autoimmune blistering skin disease pemphigus vulgaris (PV). The pathogenic PV antibody AK23, targeting the Dsg3 adhesive domain, led to profound loss of cell cohesion in human keratinocytes as revealed by the dispase-based dissociation assays. In contrast, an antibody against Dsg2 had no effect on cell cohesion although the Dsg2 antibody was demonstrated to interfere with Dsg2 transinteraction by single molecule atomic force microscopy and was effective to reduce cell cohesion in intestinal epithelial Caco-2 cells which express Dsg2 as the only Dsg isoform. To substantiate these findings, siRNA-mediated silencing of Dsg2 or Dsg3 was performed in keratinocytes. In contrast to Dsg3-depleted cells, Dsg2 knockdown reduced cell cohesion only under conditions of increased shear. These experiments indicate that specific desmosomal cadherins contribute differently to keratinocyte cohesion and that Dsg2 compared to Dsg3 is less important in this context.

  13. Keratinocyte proliferation, differentiation, and apoptosis-Differential mechanisms of regulation by curcumin, EGCG and apigenin

    SciTech Connect

    Balasubramanian, Sivaprakasam; Eckert, Richard L.

    2007-11-01

    We have proposed that it is important to examine the impact of chemopreventive agents on the function of normal human epidermal keratinocytes since these cells comprise the barrier that protects the body from a range of environmental insults. In this context, it is widely appreciated that cancer may be retarded by consumption or topical application of naturally occurring food-derived chemopreventive agents. Our studies show that (-)-epigallocatechin-3-gallate (EGCG), a green tea-derived polyphenol, acts to enhance the differentiation of normal human keratinocytes as evidenced by its ability to increase involucrin (hINV), transglutaminase type 1 (TG1) and caspase-14 gene expression. EGCG also stimulates keratinocyte morphological differentiation. These actions of EGCG are mediated via activation of a nPKC, Ras, MEKK1, MEK3, p38{delta}-ERK1/2 signaling cascade which leads to increased activator protein 1 (AP1) and CAATT enhancer binding protein (C/EBP) transcription factor expression, increased binding of these factors to DNA, and increased gene transcription. In contrast, apigenin, a dietary flavonoid derived from plants and vegetables, and curcumin, an agent derived from turmeric, inhibit differentiation by suppressing MAPK signal transduction and reducing API transcription factor level. Curcumin also acts to enhance apoptosis, although EGCG and apigenin do not stimulate apoptosis. In addition, all of these agents inhibit keratinocyte proliferation. These findings indicate that each of these diet-derived chemopreventive agents has a profound impact on normal human keratinocyte function and that they operate via distinct and sometimes opposing mechanisms. However, all are expected to act as chemopreventive agents.

  14. Bone morphogenetic protein signaling suppresses wound-induced skin repair by inhibiting keratinocyte proliferation and migration.

    PubMed

    Lewis, Christopher J; Mardaryev, Andrei N; Poterlowicz, Krzysztof; Sharova, Tatyana Y; Aziz, Ahmar; Sharpe, David T; Botchkareva, Natalia V; Sharov, Andrey A

    2014-03-01

    Bone morphogenetic protein (BMP) signaling plays a key role in the control of skin development and postnatal remodeling by regulating keratinocyte proliferation, differentiation, and apoptosis. To study the role of BMPs in wound-induced epidermal repair, we used transgenic mice overexpressing the BMP downstream component Smad1 under the control of a K14 promoter as an in vivo model, as well as ex vivo and in vitro assays. K14-caSmad1 (transgenic mice overexpressing a constitutively active form of Smad1 under K14 promoter) mice exhibited retarded wound healing associated with significant inhibition of proliferation and increased apoptosis in healing wound epithelium. Furthermore, microarray and quantitative real-time reverse-transcriptase-PCR (qRT-PCR) analyses revealed decreased expression of a number of cytoskeletal/cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myosin VA (Myo5a), in the epidermis of K14-caSmad1 mice versus wild-type (WT) controls during wound healing. BMP treatment significantly inhibited keratinocyte migration ex vivo, and primary keratinocytes of K14-caSmad1 mice showed retarded migration compared with WT controls. Finally, small interfering RNA (siRNA)-mediated silencing of BMPR-1B in primary mouse keratinocytes accelerated cell migration and was associated with increased expression of Krt16, Krt17, and Myo5a compared with controls. Thus, this study demonstrates that BMPs inhibit keratinocyte proliferation, cytoskeletal organization, and migration in regenerating skin epithelium during wound healing, and raises a possibility for using BMP antagonists for the management of chronic wounds.

  15. Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor.

    PubMed

    Bae, Ok-Nam; Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung; Kim, Eun-Sun; Jeong, Tae Cheon; Chun, Young-Jin; Lee, Ai-Young; Noh, Minsoo

    2015-03-01

    Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD.

  16. Proliferation and motility of HaCaT keratinocyte derivatives is enhanced by fibroblast nemosis

    SciTech Connect

    Raesaenen, Kati; Vaheri, Antti

    2010-06-10

    The role of paracrine tumor-stroma regulation in the progression of cancer is under intense investigation. Activated fibroblasts are key components of the tumor microenvironment providing the soluble factors mediating the regulation. Nemosis is an experimental model to study these parameters: formation of a multicellular spheroid activates fibroblasts and leads to increased production of soluble factors involved in the promotion of growth and motility. Role of nemosis was investigated in the tumorigenesis of HaCaT derivatives representing skin carcinoma progression. Conditioned medium from fibroblast spheroids increased proliferation rate of HaCaT derivatives. Expression of proliferation marker Ki-67 increased significantly in benign A5 and low-grade malignant II-4 cells, but did not further increase in the metastatic RT3 cells. Expression of p63, keratinocyte stem cell marker linked to cancer progression, was augmented by medium from nemotic fibroblasts; this increase was also seen in RT3 cells. Scratch-wound healing of the keratinocytes was enhanced in response to fibroblast nemosis. Neutralizing antibodies against growth factors inhibited wound healing to some extent; the response varied between benign and malignant keratinocytes. Migration and invasion were enhanced by conditioned medium from nemotic fibroblasts in benign and low-grade malignant cells. RT3 keratinocyte migration was further augmented, but invasion was not, indicating their intrinsic capacity to invade. Our data demonstrate that fibroblast nemosis increases proliferation and motility of HaCaT keratinocyte derivatives, and thus nemosis can be used as a model to study the role of soluble factors secreted by fibroblasts in tumor progression.

  17. SIRT1 inhibition restores apoptotic sensitivity in p53-mutated human keratinocytes

    SciTech Connect

    Herbert, Katharine J.; Cook, Anthony L. Snow, Elizabeth T.

    2014-06-15

    Mutations to the p53 gene are common in UV-exposed keratinocytes and contribute to apoptotic resistance in skin cancer. P53-dependent activity is modulated, in part, by a complex, self-limiting feedback loop imposed by miR-34a-mediated regulation of the lysine deacetylase, SIRT1. Expression of numerous microRNAs is dysregulated in squamous and basal cell carcinomas; however the contribution of specific microRNAs to the pathogenesis of skin cancer remains untested. Through use of RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors, this study explored the influence of p53 mutational status, SIRT1 activity and miR-34a levels on apoptotic sensitivity in primary (NHEK) and p53-mutated (HaCaT) keratinocyte cell lines. SIRT1 and p53 are overexpressed in p53-mutated keratinocytes, whilst miR-34a levels are 90% less in HaCaT cells. HaCaTs have impaired responses to p53/SIRT1/miR-34a axis manipulation which enhanced survival during exposure to the chemotherapeutic agent, camptothecin. Inhibition of SIRT1 activity in this cell line increased p53 acetylation and doubled camptothecin-induced cell death. Our results demonstrate that p53 mutations increase apoptotic resistance in keratinocytes by interfering with miR-34a-mediated regulation of SIRT1 expression. Thus, SIRT1 inhibitors may have a therapeutic potential for overcoming apoptotic resistance during skin cancer treatment. - Highlights: • Impaired microRNA biogenesis promotes apoptotic resistance in HaCaT keratinocytes. • TP53 mutations suppress miR-34a-mediated regulation of SIRT1 expression. • SIRT1 inhibition increases p53 acetylation in HaCaTs, restoring apoptosis.

  18. In ovo gene manipulation of melanocytes and their adjacent keratinocytes during skin pigmentation of chicken embryos.

    PubMed

    Murai, Hidetaka; Tadokoro, Ryosuke; Sakai, Ken-Ichiro; Takahashi, Yoshiko

    2015-04-01

    During skin pigmentation in avians and mammalians, melanin is synthesized in the melanocytes, and subsequently transferred to adjacently located keratinocytes, leading to a wide coverage of the body surface by melanin-containing cells. The behavior of melanocytes is influenced by keratinocytes shown mostly by in vitro studies. However, it has poorly been investigated how such intercellular cross-talk is regulated in vivo because of a lack of suitable experimental models. Using chicken embryos, we developed a method that enables in vivo gene manipulations of melanocytes and keratinocytes, where these cells are separately labeled by different genes. Two types of gene transfer techniques were combined: one was a retrovirus-mediated gene infection into the skin/keratinocytes, and the other was the in ovo DNA electroporation into neural crest cells, the origin of melanocytes. Since the Replication-Competent Avian sarcoma-leukosis virus long terminal repeat with Splice acceptor (RCAS) infection was available only for the White leghorn strain showing little pigmentation, melanocytes prepared from the Hypeco nera (pigmented) were back-transplanted into embryos of White leghorn. Prior to the transplantation, enhanced green fluorescent protein (EGFP)(+) Neo(r+) -electroporated melanocytes from Hypeco nera were selectively grown in G418-supplemented medium. In the skin of recipient White leghorn embryos infected with RCAS-mOrange, mOrange(+) keratinocytes and transplanted EGFP(+) melanocytes were frequently juxtaposed each other. High-resolution confocal microscopy also revealed that transplanted melanocytes exhibited normal behaviors regarding distribution patterns of melanocytes, dendrite morphology, and melanosome transfer. The method described in this study will serve as a useful tool to understand the mechanisms underlying intercellular regulations during skin pigmentation in vivo.

  19. Loss of flotillin expression results in weakened desmosomal adhesion and Pemphigus vulgaris-like localisation of desmoglein-3 in human keratinocytes

    PubMed Central

    Völlner, Frauke; Ali, Jawahir; Kurrle, Nina; Exner, Yvonne; Eming, Rüdiger; Hertl, Michael; Banning, Antje; Tikkanen, Ritva

    2016-01-01

    Desmosomes are adhesion plaques that mediate cell-cell adhesion in many tissues, including the epidermis, and generate mechanical resistance to tissues. The extracellular domains of desmosomal cadherin proteins, desmogleins and desmocollins, are required for the interaction with cadherins of the neighbouring cells, whereas their cytoplasmic tails associate with cytoplasmic proteins which mediate connection to intermediate filaments. Disruption of desmosomal adhesion by mutations, autoantibodies or bacterial toxins results in severe human disorders of e.g. the skin and the heart. Despite the vital role of desmosomes in various tissues, the details of their molecular assembly are not clear. We here show that the two members of the flotillin protein family directly interact with the cytoplasmic tails of desmogleins. Depletion of flotillins in human keratinocytes results in weakened desmosomal adhesion and reduced expression of desmoglein-3, most likely due to a reduction in the desmosomal pool due to increased turnover. In the absence of flotillins, desmoglein-3 shows an altered localisation pattern in the cell-cell junctions of keratinocytes, which is highly similar to the localisation observed upon treatment with pemphigus vulgaris autoantibodies. Thus, our data show that flotillins, which have previously been connected to the classical cadherins, are also of importance for the desmosomal cell adhesion. PMID:27346727

  20. Loss of flotillin expression results in weakened desmosomal adhesion and Pemphigus vulgaris-like localisation of desmoglein-3 in human keratinocytes.

    PubMed

    Völlner, Frauke; Ali, Jawahir; Kurrle, Nina; Exner, Yvonne; Eming, Rüdiger; Hertl, Michael; Banning, Antje; Tikkanen, Ritva

    2016-06-27

    Desmosomes are adhesion plaques that mediate cell-cell adhesion in many tissues, including the epidermis, and generate mechanical resistance to tissues. The extracellular domains of desmosomal cadherin proteins, desmogleins and desmocollins, are required for the interaction with cadherins of the neighbouring cells, whereas their cytoplasmic tails associate with cytoplasmic proteins which mediate connection to intermediate filaments. Disruption of desmosomal adhesion by mutations, autoantibodies or bacterial toxins results in severe human disorders of e.g. the skin and the heart. Despite the vital role of desmosomes in various tissues, the details of their molecular assembly are not clear. We here show that the two members of the flotillin protein family directly interact with the cytoplasmic tails of desmogleins. Depletion of flotillins in human keratinocytes results in weakened desmosomal adhesion and reduced expression of desmoglein-3, most likely due to a reduction in the desmosomal pool due to increased turnover. In the absence of flotillins, desmoglein-3 shows an altered localisation pattern in the cell-cell junctions of keratinocytes, which is highly similar to the localisation observed upon treatment with pemphigus vulgaris autoantibodies. Thus, our data show that flotillins, which have previously been connected to the classical cadherins, are also of importance for the desmosomal cell adhesion.

  1. Melanosome transfer to keratinocyte in the chicken embryonic skin is mediated by vesicle release associated with Rho-regulated membrane blebbing

    PubMed Central

    Tadokoro, Ryosuke; Murai, Hidetaka; Sakai, Ken-ichiro; Okui, Takahiro; Yokota, Yasuhiro; Takahashi, Yoshiko

    2016-01-01

    During skin pigmentation in amniotes, melanin synthesized in the melanocyte is transferred to keratinocytes by a particle called the melanosome. Previous studies, mostly using dissociated cultured cells, have proposed several different models that explain how the melanosome transfer is achieved. Here, using a technique that labels the plasma membrane of melanocytes within a three-dimensional system that mimics natural tissues, we have visualized the plasma membrane of melanocytes with EGFP in chicken embryonic skin. Confocal time-lapse microscopy reveals that the melanosome transfer is mediated, at least in part, by vesicles produced by plasma membrane. Unexpectedly, the vesicle release is accompanied by the membrane blebbing of melanocytes. Blebs that have encapsulated a melanosome are pinched off to become vesicles, and these melanosome-containing vesicles are finally engulfed by neighboring keratinocytes. For both the membrane blebbing and vesicle release, Rho small GTPase is essential. We further show that the membrane vesicle-mediated melanosome transfer plays a significant role in the skin pigmentation. Given that the skin pigmentation in inter-feather spaces in chickens is similar to that in inter-hair spaces of humans, our findings should have important consequences in cosmetic medicine. PMID:27910904

  2. LuSens: a keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification.

    PubMed

    Ramirez, Tzutzuy; Mehling, Annette; Kolle, Susanne N; Wruck, Christoph J; Teubner, Wera; Eltze, Tobias; Aumann, Alexandra; Urbisch, Daniel; van Ravenzwaay, Ben; Landsiedel, Robert

    2014-12-01

    Allergic contact dermatitis can develop following repeated exposure to allergenic substances. To date, hazard identification is still based on animal studies as non-animal alternatives have not yet gained global regulatory acceptance. Several non-animal methods addressing key-steps of the adverse outcome pathway (OECD, 2012) will most likely be needed to fully address this effect. Among the initial cellular events is the activation of keratinocytes and currently only one method, the KeratinoSens™, has been formally validated to address this event. In this study, a further method, the LuSens assay, that uses a human keratinocyte cell line harbouring a reporter gene construct composed of the antioxidant response element (ARE) of the rat NADPH:quinone oxidoreductase 1 gene and the luciferase gene. The assay was validated in house using a selection of 74 substances which included the LLNA performance standards. The predictivity of the LuSens assay for skin sensitization hazard identification was comparable to other non-animal methods, in particular to the KeratinoSens™. When used as part of a testing battery based on the OECD adverse outcome pathway for skin sensitization, a combination of the LuSens assay, the DPRA and a dendritic cell line activation test attained predictivities similar to that of the LLNA.

  3. Defective responses of transformed keratinocytes to terminal differentiation stimuli. Their role in epidermal tumour promotion by phorbol esters and by deep skin wounding.

    PubMed Central

    Parkinson, E. K.

    1985-01-01

    Epidermal tumourigenesis can be achieved in rodents by the application of a single subthreshold dose of a carcinogen (initiation) followed by repeated applications of a tumour promoter such as 12-0-tetradecanoyl phorbol, 13-acetate (TPA). TPA induces terminal differentiation in the majority of epidermal keratinocytes in vitro. However, transformed keratinocytes respond weakly to this terminal differentiation signal, and it is suggested that this property allows initiated cells and their progeny to obtain a selective advantage over their normal counterparts during promotion of papilloma formation by TPA. New data are reviewed which suggest that a putative wound hormone TGF-beta has similar differential effects on normal and transformed epithelial cells to those of TPA. It is proposed that the release of TGF-beta from platelets following deep skin wounding may be an explanation as to why wounding is a promoting stimulus but milder forms of epidermal injury are not. Weakly promoting hyperplasiogenic agents are also discussed within the context of a selection theory of tumour promotion. PMID:2415144

  4. Negative control of keratinocyte differentiation by Rho/CRIK signaling coupled with up-regulation of KyoT1/2 (FHL1) expression

    PubMed Central

    Grossi, Maddalena; Hiou-Feige, Agnès; Di Vignano, Alice Tommasi; Calautti, Enzo; Ostano, Paola; Lee, Sam; Chiorino, Giovanna; Dotto, G. Paolo

    2005-01-01

    Rho GTPases integrate control of cell structure and adhesion with downstream signaling events. In keratinocytes, RhoA is activated at early times of differentiation and plays an essential function in establishment of cell–cell adhesion. We report here that, surprisingly, Rho signaling suppresses downstream gene expression events associated with differentiation. Similar inhibitory effects are exerted by a specific Rho effector, CRIK (Citron kinase), which is selectively down-modulated with differentiation, thereby allowing the normal process to occur. The suppressing function of Rho/CRIK on differentiation is associated with induction of KyoT1/2, a LIM domain protein gene implicated in integrin-mediated processes and/or Notch signaling. Like activated Rho and CRIK, elevated KyoT1/2 expression suppresses differentiation. Thus, Rho signaling exerts an unexpectedly complex role in keratinocyte differentiation, which is coupled with induction of KyoT1/2, a LIM domain protein gene with a potentially important role in control of cell self renewal. PMID:16061799

  5. Effect of MC1R variant allele status on MSH-ligand induction of dopachrome tautomerase in melanocytes co-cultured with keratinocytes.

    PubMed

    Ainger, Stephen A; Wong, Shu S; Roberts, Donald W; Leonard, J Helen; Sturm, Richard A

    2011-08-01

    A co-culture system of melanocytic cell