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Sample records for human oral keratinocytes

  1. Induction of Apoptosis in Human Oral Keratinocyte by Doxorubicin.

    PubMed

    Sakagami, Hiroshi; Okudaira, Noriyuki; Masuda, Yoshiko; Amano, Osamu; Yokose, Satoshi; Kanda, Yumiko; Suguro, Madoka; Natori, Takenori; Oizumi, Hiroshi; Oizumi, Takaaki

    2017-03-01

    We have previously reported that doxorubicin (DXR) showed much higher cytotoxicity against human oral squamous cell carcinoma cell lines compared to normal human mesenchymal normal oral cells (gingival fibroblast, periodontal ligament fibroblast, pulp cell), yielding high tumor-specificity. However, we unexpectedly found that doxorubicin showed potent cytotoxicity against human normal oral keratinocytes and primary gingival epithelial cells. In the present study, we investigated the reproducibility, underlining mechanisms and generality of this unexpected finding. Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, fine cell structure by transmission electron microscopy and apoptosis induction by western blot analysis. Doxorubicin induced keratinocyte toxicity, regardless of cell density and concentration of FBS in the culture medium. Doxorubicin induced apoptosis (characterized by the loss of cell surface microvilli, chromatin condensation, nuclear fragmentation and caspase-3 activation) in keratinocytes. A total of 11 anticancer drugs showed similar keratinocyte toxicity. Alkaline extract of the leaves of Sasa senanensis Rehder partially alleviated the DXR-induced keratinocyte cytotoxicity by promoting cell growth. The present study suggested that oral keratinocyte toxicity is a novel adverse effect of most anticancer agents. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  2. Adrenergic Signaling in Human Oral Keratinocytes and Wound Repair

    PubMed Central

    Steenhuis, P.; Huntley, R.E.; Gurenko, Z.; Yin, L.; Dale, B.A.; Fazel, N.; Isseroff, R.R.

    2011-01-01

    Catecholamines are present in saliva, but their influence on oral epithelium is not understood. Because psychological stress increases salivary catecholamines and impairs oral mucosal wound healing, we sought to determine if epithelial adrenergic signaling could link these two findings. We found that cultured human oral keratinocytes (HOK) express the α2B- and β2-adrenergic receptors (ARs). Exposure of HOK to either epinephrine or the β-AR agonist, isoproterenol, reduced migratory speed and decreased in vitro scratch wound healing. Incubation with the β-AR antagonist timolol reversed the catecholamine-induced effects, indicating that the observed response is mediated by β-AR. Epinephrine treatment decreased phosphorylation of the mitogen-activated protein kinases (MAPK) ERK1/2 and p38; these decreases were also reversed with timolol. Cultured HOK express enzymes of the epinephrine synthetic pathway, and generate epinephrine. These findings demonstrate that stress-induced elevations of salivary catecholamines signal through MAPK pathways, and result in impaired oral keratinocyte migration required for healing. PMID:21127260

  3. 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

  4. 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.

  5. Comparison of two cellular harvesting methods for primary human oral culture of keratinocytes.

    PubMed

    Klingbeil, Ma Fátima Guarizo; Herson, Marisa Roma; Cristo, Elier Broche; dos Santos Pinto, Décio; Yoshito, Daniele; Mathor, Monica Beatriz

    2009-08-01

    The possibility of obtaining transplantable oral epithelia opens new perspectives for oral treatments. Most of them are surgical, resulting in mucosal failures. As reconstructive material this in vitro epithelia would be also useful for other parts of the human body. Many researchers still use controversial methods; therefore it was evaluated and compared the efficiency of the enzymatic and direct explant methods to obtain oral keratinocytes. To this project oral epithelia fragments were used. This work compared: time needed for cell obtainment, best cell amount, life-span and epithelia forming cell capacity. The results showed the possibility to obtain keratinocytes from a small oral fragment and we could verify the advantages and peculiar restrictions. We concluded that under our conditions the enzymatic method showed the best results: in the cells obtaining time needed, cell amount and life-span. Both methods showed the same capacity to form in vitro epithelia.

  6. Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells.

    PubMed

    Mehalick, Leslie A; Poulsen, Christopher; Fischer, Carol L; Lanzel, Emily A; Bates, Amber M; Walters, Katherine S; Cavanaugh, Joseph E; Guthmiller, Janet M; Johnson, Georgia K; Wertz, Philip W; Brogden, Kim A

    2015-12-01

    Long-chain bases, found in the oral cavity, have potent antimicrobial activity against oral pathogens. In an article associated with this dataset, Poulson and colleagues determined the cytotoxicities of long-chain bases (sphingosine, dihydrosphingosine, and phytosphingosine) for human oral gingival epithelial (GE) keratinocytes, oral gingival fibroblasts (GF), dendritic cells (DC), and squamous cell carcinoma (SCC) cell lines [1]. Poulson and colleagues found that GE keratinocytes were more resistant to long-chain bases as compared to GF, DC, and SCC cell lines [1]. In this study, we assess the susceptibility of DC to lower concentrations of long chain bases. 0.2-10.0 µM long-chain bases and GML were not cytotoxic to DC; 40.0-80.0 µM long-chain bases, but not GML, were cytotoxic for DC; and 80.0 µM long-chain bases were cytotoxic to DC and induced cellular damage and death in less than 20 mins. Overall, the LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections.

  7. Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells

    PubMed Central

    Mehalick, Leslie A.; Poulsen, Christopher; Fischer, Carol L.; Lanzel, Emily A.; Bates, Amber M.; Walters, Katherine S.; Cavanaugh, Joseph E.; Guthmiller, Janet M.; Johnson, Georgia K.; Wertz, Philip W.; Brogden, Kim A.

    2015-01-01

    Long-chain bases, found in the oral cavity, have potent antimicrobial activity against oral pathogens. In an article associated with this dataset, Poulson and colleagues determined the cytotoxicities of long-chain bases (sphingosine, dihydrosphingosine, and phytosphingosine) for human oral gingival epithelial (GE) keratinocytes, oral gingival fibroblasts (GF), dendritic cells (DC), and squamous cell carcinoma (SCC) cell lines [1]. Poulson and colleagues found that GE keratinocytes were more resistant to long-chain bases as compared to GF, DC, and SCC cell lines [1]. In this study, we assess the susceptibility of DC to lower concentrations of long chain bases. 0.2–10.0 µM long-chain bases and GML were not cytotoxic to DC; 40.0–80.0 µM long-chain bases, but not GML, were cytotoxic for DC; and 80.0 µM long-chain bases were cytotoxic to DC and induced cellular damage and death in less than 20 mins. Overall, the LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections. PMID:26550599

  8. Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells

    PubMed Central

    Poulsen, Christopher; Mehalick, Leslie A.; Fischer, Carol L.; Lanzel, Emily A.; Bates, Amber M.; Walters, Katherine S.; Cavanaugh, Joseph E.; Guthmiller, Janet M.; Johnson, Georgia K.; Wertz, Philip W.; Brogden, Kim A.

    2015-01-01

    Long-chain bases are present in the oral cavity. Previously we determined that sphingosine, dihydrosphingosine, and phytosphingosine have potent antimicrobial activity against oral pathogens. Here, we determined the cytotoxicities of long-chain bases for oral cells, an important step in considering their potential as antimicrobial agents for oral infections. This information would clearly help in establishing prophylactic or therapeutic doses. To assess this, human oral gingival epithelial (GE) keratinocytes, oral gingival fibroblasts (GF), and dendritic cells (DC) were exposed to 10.0-640.0 µM long-chain bases and glycerol monolaurate (GML). The effects of long-chain bases on cell metabolism (conversion of resazurin to resorufin), membrane permeability (uptake of propridium iodide or SYTOX-Green), release of cellular contents (LDH), and cell morphology (confocal microscopy) were all determined. GE keratinocytes were more resistant to long-chain bases as compared to GF and DC, which were more susceptible. For DC, 0.2 to 10.0 µM long-chain bases and GML were not cytotoxic; 40.0 to 80.0 µM long-chain bases, but not GML, were cytotoxic; and 80.0 µM long-chain bases induced cellular damage and death in less than 20 minutes. The LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections. PMID:26005054

  9. Cytotoxicity and terminal differentiation of human oral keratinocyte by indium ions from a silver-palladium-gold-indium dental alloy.

    PubMed

    Lee, Jung-Hwan; Seo, Sang-Hee; Lee, Sang-Bae; Om, Ji-Yeon; Kim, Kwang-Mahn; Kim, Kyoung-Nam

    2015-02-01

    Dental alloys containing indium (In) have been used in dental restoration for two decades; however, no study has investigated the biological effects of In ions, which may be released in the oral cavity, on human oral keratinocytes. The objective of the present study was to investigate the biological effects of In ions on human oral keratinocyte after confirming their release from a silver-palladium-gold-indium (Ag-Pd-Au-In) dental alloy. As a corrosion assay, a static immersion tests were performed by detecting the released ions in the corrosion solution from the Ag-Pd-Au-In dental alloy using inductively coupled plasma atomic emission spectroscopy. The cytotoxicity and biological effects of In ions were then studied with In compounds in three human oral keratinocyte cell lines: immortalized human oral keratinocyte (IHOK), HSC-2, and SCC-15. Higher concentrations of In and Cu ions were detected in Ag-Pd-Au-In (P<0.05) than in Ag-Pd-Au, and AgCl deposition occurred on the surface of Ag-Pd-Au-In after a 7-day corrosion test due to its low corrosion resistance. At high concentrations, In ions induced cytotoxicity; however, at low concentrations (∼0.8In(3+)mM), terminal differentiation was observed in human oral keratinocytes. Intracellular ROS was revealed to be a key component of In-induced terminal differentiation. In ions were released from dental alloys containing In, and high concentrations of In ions resulted in cytotoxicity, whereas low concentrations induced the terminal differentiation of human oral keratinocytes via increased intracellular ROS. Therefore, dental alloys containing In must be biologically evaluated for their safe use. Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  10. A simple and reliable technique for culturing of human oral keratinocytes and fibroblasts.

    PubMed

    Reid, C B; Cloos, J; Snow, G B; Braakhuis, B J

    1997-07-01

    Cultures of non-malignant oral cells are needed for many applications in the field of oral biology. We describe an efficient and rapid method for the culture of both keratinocytes and fibroblasts isolated from oral tissue, uvulas from patients undergoing reconstructive surgery and non-cancerous tissue from oral cancer patients. High cell yields with a high purity could be obtained. The technique is described in detail with respect to isolation of the cell types, the best way to propagate for over 5 passages and to measure cell proliferation in a colorimetric assay. Culture was successful in over 80% of cases for keratinocytes and over 63% for fibroblasts. Problems encountered were the risk of microbiological infection, the size of the specimen and the type of culture medium used. The potential applications of these primary cell cultures are mentioned.

  11. The expression of O(6) -methylguanine-DNA methyltransferase in human oral keratinocytes stimulated with arecoline.

    PubMed

    Lee, Shiuan-Shinn; Tsai, Chung-Hung; Yu, Cheng-Chia; Ho, Yung-Chuan; Hsu, Hsin-I; Chang, Yu-Chao

    2013-09-01

    O(6) -methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that can protect cells from carcinogenic effects of alkylating agents by removing adducts from the O(6) position of guanine. Evidences indicated that areca quid chewing may increase the risk of oral squamous cell carcinoma (OSCC). This study was to investigate the role of MGMT expression in OSCCs and the normal oral tissues. Thirty-two OSCCs from areca quid chewers and ten normal oral tissue biopsy samples without areca quid chewing were analyzed by the immunohistochemistry for MGMT. Primary human oral keratinocytes (HOKs) were challenged with arecoline, the major alkaloid of areca nut, by Western blot. Nicotine, an important component of cigarette smoke, was added to find the possible regulatory mechanisms. Significant association was observed between low MGMT expression and advanced clinical stage of OSCCs and lymph node metastasis (P = 0.03). MGMT expression was significantly higher in patients only chewing areca quid than patients both chewing areca quid and smoking (P = 0.028). Arecoline was found to elevate MGMT expression in a dose- and time-dependent manner. The addition of nicotine was found to enhance arecoline-induced MGMT expression. Our results indicate that MGMT could be used clinically as a predictive marker for tumor processing, the potential for lymph node metastasis as well as advanced clinical stage. MGMT expression was significantly upregulated by arecoline in HOKs. Nicotine has a synergistic effect of arecoline-induced MGMT expression. The cigarette smoking may act synergistically in the pathogenesis of OSCC in areca quid chewers via the upregulation of MGMT. © 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. cDNA microarray analysis of human keratinocytes cells of patients submitted to chemoradiotherapy and oral photobiomodulation therapy: pilot study.

    PubMed

    Antunes, Heliton S; Wajnberg, Gabriel; Pinho, Marcos B; Jorge, Natasha Andressa Nogueira; de Moraes, Joyce Luana Melo; Stefanoff, Claudio Gustavo; Herchenhorn, Daniel; Araújo, Carlos M M; Viégas, Celia Maria Pais; Rampini, Mariana P; Dias, Fernando L; de Araujo-Souza, Patricia Savio; Passetti, Fabio; Ferreira, Carlos G

    2017-08-24

    Oral mucositis is an acute toxicity that occurs in patients submitted to chemoradiotherapy to treat head and neck squamous cell carcinoma. In this study, we evaluated differences in gene expression in the keratinocytes of the oral mucosa of patients treated with photobiomodulation therapy and tried to associate the molecular mechanisms with clinical findings. From June 2009 to December 2010, 27 patients were included in a randomized double-blind pilot study. Buccal smears from 13 patients were obtained at days 1 and 10 of chemoradiotherapy, and overall gene expression of samples from both dates were analyzed by complementary DNA (cDNA) microarray. In addition, samples from other 14 patients were also collected at D1 and D10 of chemoradiotherapy for subsequent validation of cDNA microarray findings by qPCR. The expression array analysis identified 105 upregulated and 60 downregulated genes in our post-treatment samples when compared with controls. Among the upregulated genes with the highest fold change, it was interesting to observe the presence of genes related to keratinocyte differentiation. Among downregulated genes were observed genes related to cytotoxicity and immune response. The results indicate that genes known to be induced during differentiation of human epidermal keratinocytes were upregulated while genes associated with cytotoxicity and immune response were downregulated in the laser group. These results support previous clinical findings indicating that the lower incidence of oral mucositis associated with photobiomodulation therapy might be correlated to the activation of genes involved in keratinocyte differentiation.

  13. 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.

  14. Cytotoxicity and chromosome aberrations in normal human oral keratinocytes induced by chemical carcinogens: Comparison of inter-individual variations.

    PubMed

    Tsutsui, T; Kawamoto, Y; Suzuki, N; Gladen, B C; Barrett, J C

    1991-01-01

    Normal human keratinocytes from the oral cavity were cultured in vitro in serum-free medium. Cultures from different individuals were established, and the responses of the cells to different chemicals were compared. The cells, grown at clonal densities, were treated separately with an alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine; MNNG), two arsenical salts (sodium arsenate or sodium arsenite), sodium fluoride or two polyaromatic hydrocarbons (benzo[a]pyrene or 7,12-dimethylbenz[a]-anthracene). There were no significant differences in the colony-forming efficiencies (22.8 +/- 4.2%) of control (untreated) cells from five different individuals. At selected doses, each of the chemicals reduced the colony-forming efficiencies of the treated cells. The cytotoxicity of most of the chemicals did not differ significantly among cells derived from different individuals, with the exception of sodium arsenate at two doses and sodium fluoride at the highest dose tested. Induction of chromosome aberrations by MNNG, sodium arsenite, sodium arsenate and sodium flouride was analysed with cells derived from up to nine individuals. There was little difference in the inducibilities of chromosome aberrations among cultured keratinocytes from different donors. Treatment of cells from nine donors with one dose of sodium fluoride revealed a statistically significant inter-individual variation. These findings provide a model system to study the effects of carcinogens on the target cells for oral cancers. The results can be compared with findings for cells from other epithelial tissues, since the culture conditions support the growth of keratinocytes regardless of origin. Little inter-individual variation was observed in the response of oral keratinocytes to the chemicals examined.

  15. The influence of bisphosphonates on viability, migration, and apoptosis of human oral keratinocytes--in vitro study.

    PubMed

    Pabst, Andreas M; Ziebart, Thomas; Koch, Felix P; Taylor, Katherine Y; Al-Nawas, Bilal; Walter, Christian

    2012-02-01

    Bisphosphonate-associated osteonecrosis of the jaw (BP-ONJ) is one of the most often seen side effects in patients treated with bisphosphonates, presenting clinically as a non-healing wound. One theory of BP-ONJ etiology describes a negative effect on soft tissues, especially on keratinocytes, which play an important role in oral wound healing and oral soft tissue regeneration. A high cell viability of keratinocytes, which can migrate to the affected location, is essential for wound healing. The aim of this in vitro study was to investigate the effect of differently potent bisphosphonates on human oral keratinocytes (HOK).Three nitrogen-containing bisphosphonates (ibandronate, pamidronate, and zoledronate) and one non-nitrogen-containing bisphosphonate (clodronate) were compared concerning their potency on cell viability (calcein assay and MTT assay), migration ability (Boyden chamber migration assay and scratch wound proliferation assay), and apoptosis (TUNEL assay) of HOK.The nitrogen-containing bisphosphonates, particularly highly potent pamidronate and zoledronate preparations, had a strong negative influence on cell viability, migration ability, and apoptosis of HOK. The non-nitrogen-containing clodronate even increased cell viability in higher concentrations.This study demonstrates that bisphosphonates have a strong influence on HOK on different cellular levels like cell viability, migration ability, and apoptosis rate. The results support the theory that BP-ONJ is a multifactorially caused disease.Furthermore, this in vitro study confirms the theory that perioperative interruption of bisphosphonate application during dental surgical procedures might be feasible to promote better tissue regeneration and wound healing.

  16. Intrinsic Differences between Oral and Skin Keratinocytes

    PubMed Central

    Turabelidze, Anna; Guo, Shujuan; Chung, Allison Yen; Chen, Lin; Dai, Yang; Marucha, Phillip T.; DiPietro, Luisa A.

    2014-01-01

    Keratinocytes cover both the skin and some oral mucosa, but the morphology of each tissue and the behavior of the keratinocytes from these two sites are different. One significant dissimilarity between the two sites is the response to injury. Oral mucosal wounds heal faster and with less inflammation than equivalent cutaneous wounds. We hypothesized that oral and skin keratinocytes might have intrinsic differences at baseline as well as in the response to injury, and that such differences would be reflected in gene expression profiles. PMID:25198578

  17. Loss of heterozygosity on chromosome 7q in in vitro-immortalized human oral keratinocyte cell lines.

    PubMed

    Arai, Yasuhito; Shigeeda, Nobumasa; Uchida, Minoru; Tsutsui, Takeki

    2002-08-01

    Loss of heterozygosity in two in vitro-immortalized human oral keratinocyte cell lines was analysed by polymerase chain reaction using 42 polymorphic microsatellite markers on chromosomes 4, 6, 7 and 15. These chromosomes are regarded as candidates for harbouring genes involved in the immortalization of human cells or tumour-suppressor genes in several tumours, including oral cancers, and karyotypic analysis has revealed that both cell lines have non-random alterations in these chromosomes. No allele losses were detected at any informative loci on chromosomes 4 and 6 in the cell lines, including genomic regions adjacent to putative human tumour-suppressor genes and putative senescence genes. When analysed for loss of heterozygosity on chromosomes 7 and 15, allele losses common to both cell lines were detected in the regions at 7q11.2, 7q21.1-21.3 and 7q31.1. High frequencies of loss of heterozygosity on chromosome 7q in at least two distinct regions, particularly centred around 7q31, are observed in a variety of tumours, including oral squamous-cell carcinoma, suggesting that multiple genes involved in immortalization of these cell lines might be present on chromosome 7q.

  18. Effects of transforming growth factor beta-1 on growth-regulatory genes in tumour-derived human oral keratinocytes.

    PubMed Central

    Paterson, I. C.; Patel, V.; Sandy, J. R.; Prime, S. S.; Yeudall, W. A.

    1995-01-01

    This study examined the effect of transforming growth factor beta-1 (TGF-beta 1) on c-myc, RB1, junB and p53 expression together with pRb phosphorylation, in carcinoma-derived and normal human oral keratinocytes with a range of inhibitory responses to this ligand. Amplification of c-myc was observed in eight of eight tumour-derived cell lines and resulted in corresponding mRNA expression. The down-regulation of c-myc expression by TGF-beta 1 predominantly reflected growth inhibition by TGF-beta 1, but in two of eight tumour-derived cell lines which were partially responsive to TGF-beta 1 c-myc expression was unaltered by this ligand. While RB1 mRNA levels were unaltered by TGF-beta 1, the ligand caused the accumulation of the underphosphorylated form of the Rb protein in all cells irrespective of TGF-beta 1-induced growth arrest. junB expression was up-regulated by TGF-beta 1 in cells with a range of growth inhibitory responses. All cells contained mutant p53. TGF-beta 1 did not affect p53 mRNA expression in both tumour-derived and normal keratinocytes and there was no alteration in p53 protein levels in keratinocytes expressing stable p53 protein following TGF-beta 1 treatment. The data indicate that TGF-beta-induced growth control can exist independently of the presence of mutant p53 and the control of Rb phosphorylation and c-myc down-regulation. It may be that TGF-beta growth inhibition occurs via multiple mechanisms and that the loss of one pathway during tumour progression does not necessarily result in the abrogation of TGF-beta-induced growth control. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7547241

  19. Human Papillomavirus Promotes Epstein-Barr Virus Maintenance and Lytic Reactivation in Immortalized Oral Keratinocytes

    PubMed Central

    Makielski, Kathleen R.; Lee, Denis; Lorenz, Laurel D.; Nawandar, Dhananjay M.; Chiu, Ya- Fang; Kenney, Shannon C.; Lambert, Paul F.

    2016-01-01

    Epstein-Barr virus and human papillomaviruses are human tumor viruses that infect and replicate in upper aerodigestive tract epithelia and cause head and neck cancers. The productive phases of both viruses are tied to stratified epithelia highlighting the possibility that these viruses may affect each other’s life cycles. Our lab has established an in vitro model system to test the effects of EBV and HPV co-infection in stratified squamous oral epithelial cells. Our results indicate that HPV increases maintenance of the EBV genome in the co-infected cells and promotes lytic reactivation of EBV in upper layers of stratified epithelium. Expression of the HPV oncogenes E6 and E7 were found to be necessary and sufficient to account for HPV-mediated lytic reactivation of EBV. Our findings indicate that HPV increases the capacity of epithelial cells to support the EBV life cycle, which could in turn increase EBV-mediated pathogenesis in the oral cavity. PMID:27179345

  20. Fenretinide Perturbs Focal Adhesion Kinase in Premalignant and Malignant Human Oral Keratinocytes. Fenretinide's Chemopreventive Mechanisms Include ECM Interactions.

    PubMed

    Han, Byungdo B; Li, Suyang; Tong, Meng; Holpuch, Andrew S; Spinney, Richard; Wang, Daren; Border, Michael B; Liu, Zhongfa; Sarode, Sachin; Pei, Ping; Schwendeman, Steven P; Mallery, Susan R

    2015-05-01

    The membrane-associated protein, focal adhesion kinase (FAK), modulates cell-extracellular matrix interactions and also conveys prosurvival and proliferative signals. Notably, increased intraepithelial FAK levels accompany transformation of premalignant oral intraepithelial neoplasia (OIN) to oral squamous cell carcinoma (OSCC). OIN chemoprevention is a patient-centric, optimal strategy to prevent OSCC's comorbidities and mortality. The cancer chemopreventive and synthetic vitamin A derivative, fenretinide, has demonstrated protein-binding capacities, for example, mTOR- and retinol-binding protein interactions. These studies used a continuum of human oral keratinocytes (normal-HPV E6/E7-transduced-OSCC) to assess potential fenretinide-FAK drug protein interactions and functional consequences on cellular growth regulation and motility. Molecular modeling studies demonstrated that fenretinide has approximately 200-fold greater binding affinity relative to the natural ligand (ATP) at FAK's kinase domain. Fenretinide also shows intermediate binding at FAK's FERM domain and interacts at the ATP-binding site of the closest FAK analogue, PYK2. Fenretinide significantly suppressed proliferation via induction of apoptosis and G2-M cell-cycle blockade. Fenretinide-treated cells also demonstrated F-actin disruption, significant inhibition of both directed migration and invasion of a synthetic basement membrane, and decreased phosphorylation of growth-promoting kinases. A commercially available FAK inhibitor did not suppress cell invasion. Notably, although FAK's FERM domain directs cell invasion, FAK inhibitors target the kinase domain. In addition, FAK-specific siRNA-treated cells showed an intermediate cell migration capacity; data which suggest cocontribution of the established migrating-enhancing PYK2. Our data imply that fenretinide is uniquely capable of disrupting FAK's and PYK2's prosurvival and mobility-enhancing effects and further extend fenretinide

  1. Bmi-1 extends the life span of normal human oral keratinocytes by inhibiting the TGF-{beta} signaling

    SciTech Connect

    Kim, Reuben H.; Lieberman, Mark B.; Lee, Rachel; Shin, Ki-Hyuk; Mehrazarin, Shebli; Oh, Ju-Eun; Park, No-Hee; Kang, Mo K.

    2010-10-01

    We previously demonstrated that Bmi-1 extended the in vitro life span of normal human oral keratinocytes (NHOK). We now report that the prolonged life span of NHOK by Bmi-1 is, in part, due to inhibition of the TGF-{beta} signaling pathway. Serial subculture of NHOK resulted in replicative senescence and terminal differentiation and activation of TGF-{beta} signaling pathway. This was accompanied with enhanced intracellular and secreted TGF-{beta}1 levels, phosphorylation of Smad2/3, and increased expression of p15{sup INK4B} and p57{sup KIP2}. An ectopic expression of Bmi-1 in NHOK (HOK/Bmi-1) decreased the level of intracellular and secreted TGF-{beta}1 induced dephosphorylation of Smad2/3, and diminished the level of p15{sup INK4B} and p57{sup KIP2}. Moreover, Bmi-1 expression led to the inhibition of TGF-{beta}-responsive promoter activity in a dose-specific manner. Knockdown of Bmi-1 in rapidly proliferating HOK/Bmi-1 and cancer cells increased the level of phosphorylated Smad2/3, p15{sup INK4B}, and p57{sup KIP2}. In addition, an exposure of senescent NHOK to TGF-{beta} receptor I kinase inhibitor or anti-TGF-{beta} antibody resulted in enhanced replicative potential of cells. Taken together, these data suggest that Bmi-1 suppresses senescence of cells by inhibiting the TGF-{beta} signaling pathway in NHOK.

  2. Bmi-1 extends the lifespan of normal human oral keratinocytes by inhibiting the TGF-β signaling

    PubMed Central

    Kim, Reuben H.; Lieberman, Mark B.; Lee, Rachel; Shin, Ki-Hyuk; Mehrazarin, Shebli; Oh, Ju-Eun; Park, No-Hee; Kang, Mo K.

    2010-01-01

    We previously demonstrated that Bmi-1 extended the in vitro life span of normal human oral keratinocytes (NHOK). We now report that the prolonged life span of NHOK by Bmi-1 is, in part, due to inhibition of the TGF-β signaling pathway. Serial subculture of NHOK resulted in replicative senescence and terminal differentiation, and activation of TGF-β signaling pathway. This was accompanied with enhanced intracellular and secreted TGF-β1 levels, phosphorylation of Smad2/3, and increased expression of p15INK4B and p57KIP2. An ectopic expression of Bmi-1 in NHOK (HOK/Bmi-1) decreased intracellular and secreted TGF-β1 level, induced dephosphorylation of Smad2/3, and diminished the level of p15INK4B and p57KIP2. Moreover, Bmi-1 expression led to inhibition of TGF-β-responsive promoter activity in dose-specific manner. Knockdown of Bmi-1 in rapidly proliferating HOK/Bmi-1 and cancer cells increased the level of phosphorylated Smad2/3, p15INK4B and p57KIP2. In addition, an exposure of senescent NHOK to TGF-β receptor I kinase inhibitor or anti-TGF-β antibody resulted in enhanced replicative potential of cells. Taken together, these data suggest that Bmi-1 suppresses senescence of cells by inhibiting the TGF-β signaling pathway in NHOK. PMID:20630502

  3. The influence of geranylgeraniol on human oral keratinocytes after bisphosphonate treatment: An in vitro study.

    PubMed

    Pabst, Andreas Max; Krüger, Maximilian; Ziebart, Thomas; Jacobs, Collin; Sagheb, Keyvan; Walter, Christian

    2015-06-01

    This in vitro study analyzed the influence of geranylgeraniol (GGOH) on human oral keratinocytes (HOK) after exposure to bisphosphonates. HOK were incubated with four different bisphosphonates (clodronate, ibandronate, pamidronate, zoledronate) in two experimental set-ups: with and without GGOH. MTT and PrestoBlue assays were used to analyze HOK cell viability. HOK migration ability was examined with Boyden and Scratch assays, and Tunel and ToxiLight assays were used to detect the HOK apoptosis rate. No significant differences between the experimental set-ups, with and without GGOH, could be found for clodronate (p each >0.3). For the nitrogen-containing bisphosphonates, negative effects could be shown in the experimental set-ups without GGOH in all assays. In the GGOH experimental set-ups, the levels of HOK cell viability were significantly increased (MTT: p each ≤0.001; PrestoBlue: p each ≤0.012). The HOK migration ability was also greater (Boyden: p each <0.001; Scratch: p each ≤0.015). Regarding the apoptosis rate, reduced numbers of apoptotic HOK in the Tunel assay (p each <0.001) and decreased adenylate kinase release in the ToxiLight assay (p each ≤0.002) were observed. GGOH reversed the negative effects of bisphosphonates on HOK. These findings provide evidence that GGOH could be a promising treatment option for BP-ONJ.

  4. Reduction of radiochemotherapy-induced early oral mucositis by recombinant human keratinocyte growth factor (palifermin): Experimental studies in mice

    SciTech Connect

    Doerr, Wolfgang . E-mail: doerr@rcs.urz.tu-dresden.de; Baessler, Stefan; Reichel, Sandra; Spekl, Kathrin

    2005-07-01

    Purpose: To study the effect of recombinant human keratinocyte growth factor (rHuKGF or palifermin) on oral mucositis induced by radiochemotherapy in a mouse model. Methods and Materials: Cis-diamminedichloroplatinum (cisplatin) and/or 5-fluorouracil were given before single dose irradiation, combined with palifermin before or after the treatment, or both. Daily fractionated irradiation for 2 weeks was followed by graded test doses. With additional chemotherapy in Week 1, palifermin was given before radiotherapy and at the end of the first week, or additionally at the end of Week 2. Radiochemotherapy in Week 2 was combined with palifermin at the end of Weeks 1 and 2, Weeks 1, 2, and 3, or additionally before radiotherapy. Ulceration of mouse tongue mucosa was analyzed as the endpoint. Results: The dose associated with ulcer induction in 50% of the mice (ED{sub 50}) for single-dose irradiation was 11.5 {+-} 0.7 Gy. Palifermin increased the ED{sub 50} to about 19 Gy in all protocols tested. Similar values were observed when chemotherapy was added before irradiation. With fractionated irradiation, palifermin increased the ED{sub 50} for test irradiation from 5.7 {+-} 1.5 Gy to 12-15 Gy, depending on the administration protocol. With chemotherapy in Week 1, two palifermin injections had no significant effect, but a third injection increased the ED{sub 50} to 13 Gy. With chemotherapy in Week 2, all palifermin protocols resulted in ED{sub 50} values of 13-14 Gy. Conclusion: A marked increase in oral mucosal radiation tolerance by palifermin was found, which was preserved in combinations with chemotherapy using cisplatin and/or 5-fluorouracil.

  5. Modulation of TLR2 protein expression by a miR-105 in human oral keratinocytes

    EPA Science Inventory

    Mammalian biological processes such as inflammation, involve regulation of hundreds of genes controlling onset and termination. MicroRNAs (miRNAs) can translationally repress target mRNAs and can regulate innate immune responses. Our model system comprised primary human keratinoc...

  6. Modulation of TLR2 protein expression by a miR-105 in human oral keratinocytes

    EPA Science Inventory

    Mammalian biological processes such as inflammation, involve regulation of hundreds of genes controlling onset and termination. MicroRNAs (miRNAs) can translationally repress target mRNAs and can regulate innate immune responses. Our model system comprised primary human keratinoc...

  7. γ-tocotrienol prevents 5-FU-induced reactive oxygen species production in human oral keratinocytes through the stabilization of 5-FU-induced activation of Nrf2

    PubMed Central

    TAKANO, HIDEYUKI; MOMOTA, YUKIHIRO; KANI, KOUICHI; AOTA, KEIKO; YAMAMURA, YOSHIKO; YAMANOI, TOMOKO; AZUMA, MASAYUKI

    2015-01-01

    Chemotherapy-induced oral mucositis is a common adverse event in patients with oral squamous cell carcinoma, and is initiated through a variety of mechanisms, including the generation of reactive oxygen species (ROS). In this study, we examined the preventive effect of γ-tocotrienol on the 5-FU-induced ROS production in human oral keratinocytes (RT7). We treated RT7 cells with 5-FU and γ-tocotrienol at concentrations of 10 μg/ml and 10 nM, respectively. When cells were treated with 5-FU alone, significant growth inhibition was observed as compared to untreated cells. This inhibition was, in part, due to the ROS generated by 5-FU treatment, because N-acetyl cysteine (NAC), a ROS scavenger, significantly ameliorated the growth of RT7 cells. γ-tocotrienol showed no cytotoxic effect on the growth of RT7 cells. Simultaneous treatment of cells with these agents resulted in the significant recovery of cell growth, owing to the suppression of ROS generation by γ-tocotrienol. Whereas 5-FU stimulated the expression of NF-E2-related factor 2 (Nrf2) protein in the nucleus up to 12 h after treatment of RT7 cells, γ-tocotrienol had no obvious effect on the expression of nuclear Nrf2 protein. Of note, the combined treatment with both agents stabilized the 5-FU-induced nuclear Nrf2 protein expression until 24 h after treatment. In addition, expression of Nrf2-dependent antioxidant genes, such as heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO-1), was significantly augmented by treatment of cells with both agents. These findings suggest that γ-tocotrienol could prevent 5-FU-induced ROS generation by stabilizing Nrf2 activation, thereby leading to ROS detoxification and cell survival in human oral keratinocytes. PMID:25625649

  8. Tissue engineering of composite grafts: Cocultivation of human oral keratinocytes and human osteoblast-like cells on laminin-coated polycarbonate membranes and equine collagen membranes under different culture conditions.

    PubMed

    Glaum, R; Wiedmann-Al-Ahmad, M; Huebner, U; Schmelzeisen, R

    2010-05-01

    In complex craniomaxillofacial defects, the simultaneous reconstruction of hard and soft tissue is often necessary. Until now, oral keratinocytes and osteoblast-like cells have not been cocultivated on the same carrier. For the first time, the cocultivation of human oral keratinocytes and human osteoblast-like cells has been investigated in this study. Different carriers (laminin-coated polycarbonate and equine collagen membranes) and various culture conditions were examined. Human oral keratinocytes and human osteoblast-like cells from five patients were isolated from tissue samples, seeded on the opposite sides of the carriers and cultivated for 1 and 2 weeks under static conditions in an incubator and in a perfusion chamber. Proliferation and morphology of the cells were analyzed by EZ4U-tests, light microscopy, and scanning electron microscopy. Cocultivation of both cell-types seeded on one carrier was possible. Quantitative and qualitative growth was significantly better on collagen membranes when compared with laminin-coated polycarbonate membranes independent of the culture conditions. Using perfusion culture in comparison to static culture, the increase of cell proliferation after 2 weeks of cultivation when compared with the proliferation after 1 week was significantly lower, independent of the carriers used. In conclusion, the contemporaneous cultivation of human oral keratinocytes and human osteoblast-like cells on the same carrier is possible, a prerequisite for planned in vivo studies. As carrier collagen is superior to laminin-coated polycarbonate membranes. Regarding the development over time, the increase of proliferation rate is lower in perfusion culture. Examinations of cellular differentiation over time under various culture conditions will be subject of further investigations. Copyright 2009 Wiley Periodicals, Inc.

  9. 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.

  10. Bisphosphonates Inhibit Expression of p63 by Oral Keratinocytes

    PubMed Central

    Scheller, E.L.; Baldwin, C.M.; Kuo, S.; D’Silva, N.J.; Feinberg, S.E.; Krebsbach, P.H.; Edwards, P.C.

    2011-01-01

    Osteonecrosis of the jaw (ONJ), a side-effect of bisphosphonate therapy, is characterized by exposed bone that fails to heal within eight weeks. Healing time of oral epithelial wounds is decreased in the presence of amino-bisphosphonates; however, the mechanism remains unknown. We examined human tissue from individuals with ONJ and non-bisphosphonate-treated controlindividuals to identify changes in oral epithelium and connective tissue. Oral and intravenous bisphosphonate-treated ONJ sites had reduced numbers of basal epithelial progenitor cells, as demonstrated by a 13.8 ± 1.1% and 31.9 ± 5.8% reduction of p63 expression, respectively. No significant differences in proliferation rates, vessel density, or macrophage number were noted. In vitro treatment of clonal and primary oral keratinocytes with zoledronic acid (ZA) inhibited p63, and expression was rescued by the addition of mevalonate pathway intermediates. In addition, both ZA treatment and p63 shRNA knock-down impaired formation of 3D Ex Vivo Produced Oral Mucosa Equivalents (EVPOME) and closure of an in vitro scratch assay. Analysis of our data suggests that bisphosphonate treatment may delay oral epithelial healing by interfering with p63-positive progenitor cells in the basal layer of the oral epithelium in a mevalonate-pathway-dependent manner. This delay in healing may increase the likelihood of osteonecrosis developing in already-compromised bone. PMID:21551338

  11. The Effect of Lipoaspirates on Human Keratinocytes.

    PubMed

    Kim, Bong-Sung; Gaul, Charel; Paul, Nora E; Dewor, Manfred; Stromps, Jan-Philipp; Hwang, Soo Seok; Nourbakhsh, Mahtab; Bernhagen, Jürgen; Rennekampff, Hans-Oliver; Pallua, Norbert

    2016-09-01

    One increasingly important trend in plastic, reconstructive, and aesthetic surgery is the use of fat grafts to improve cutaneous wound healing. In clinical practice, lipoaspirates (adipose tissue harvested by liposuction) are re-injected in a procedure called lipofilling. Previous studies, however, mainly evaluated the regenerative effect of isolated adipocytes, adipose-derived stem cells, and excised en bloc adipose tissue on keratinocytes, whereas no study to date has examined the effect of lipoaspirates. The authors aimed to investigate differences in the regenerative property of en bloc adipose tissue and lipoaspirates on keratinocytes. Human keratinocytes, lipoaspirates, and en bloc adipose tissue from 36 healthy donors were isolated. In vitro proliferation, differentiation, migration, stratification, and wound healing of keratinocyte monolayers were measured. Furthermore, secreted levels of VEGF, bFGF, IGF-1, MMP-9, and MIF were detected by ELISA. Migration, proliferation, and wound healing of keratinocytes were increased by lipoaspirates. Interestingly, the effect of lipoaspirates on keratinocyte proliferation was significantly higher than by en bloc adipose tissue after 5 days. The differentiation of keratinocytes was equally attenuated by lipoaspirates and en bloc adipose tissue. Stratification of keratinocyte layers was enhanced by lipoaspirates and en bloc fat when compared to controls. Lipoaspirates secrete higher levels of bFGF, whereas higher levels of VEGF and IGF-1 are released by en bloc adipose tissue. We show that lipoaspirates and en bloc adipose tissue have a regenerative effect on keratinocytes. One reason for the higher effect of lipoaspirates on keratinocyte proliferation may be the secretion of different cytokines. © 2016 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com.

  12. Influence of CAD/CAM all-ceramic materials on cell viability, migration ability and adenylate kinase release of human gingival fibroblasts and oral keratinocytes.

    PubMed

    Pabst, A M; Walter, C; Grassmann, L; Weyhrauch, M; Brüllmann, D D; Ziebart, T; Scheller, H; Lehmann, K M

    2014-05-01

    The aim of this study was to analyze the influence of four CAD/CAM all-ceramic materials on cell viability, migration ability and adenylate kinase (ADK) release of human gingival fibroblasts (HGF) and oral keratinocytes (HOK). HGF and HOK were cultured on disc-shaped CAD/CAM all-ceramic materials (e.max CAD LT, e.max CAD HT, Empress CAD and Mark II) and on discs made of tissue culture polystyrene surface (TCPS) serving as control. Cell viability was analyzed by using an MTT assay, and migration ability was investigated by a scratch assay. A ToxiLight assay has been performed to analyze the effect of all-ceramic materials on ADK release and cell apoptosis. At MTT assay for HGF, no significant decrease of cell viability could be detected at all points of measurement (p each > 0.05), while HOK demonstrated a significant decrease in cell viability especially on Empress CAD and Mark II at each point of measurement (p each < 0.001). Scratch assay demonstrated an increased migration ability for HGF on e.max CAD HT, Empress CAD and Mark II (p each < 0.001), whereas HOK showed a significantly decreased migration ability on all tested materials at all points of measurement (between -36 % and -71 %; p each < 0.001). At ToxiLight assay, only small cytotoxic effects of the all-ceramic materials could be investigated. This study disclosed significant differences in cell viability and migration ability of HGF and HOK on CAD/CAM all-ceramic materials. CAD/CAM all-ceramic materials can influence oral cell lines responsible for soft tissue creation which may affect the esthetic outcome.

  13. Baicalin Downregulates Porphyromonas gingivalis Lipopolysaccharide-Upregulated IL-6 and IL-8 Expression in Human Oral Keratinocytes by Negative Regulation of TLR Signaling

    PubMed Central

    Luo, Wei; Wang, Cun-Yu; Jin, Lijian

    2012-01-01

    Periodontal (gum) disease is one of the main global oral health burdens and severe periodontal disease (periodontitis) is a leading cause of tooth loss in adults globally. It also increases the risk of cardiovascular disease and diabetes mellitus. Porphyromonas gingivalis lipopolysaccharide (LPS) is a key virulent attribute that significantly contributes to periodontal pathogenesis. Baicalin is a flavonoid from Scutellaria radix, an herb commonly used in traditional Chinese medicine for treating inflammatory diseases. The present study examined the modulatory effect of baicalin on P. gingivalis LPS-induced expression of IL-6 and IL-8 in human oral keratinocytes (HOKs). Cells were pre-treated with baicalin (0–80 µM) for 24 h, and subsequently treated with P. gingivalis LPS at 10 µg/ml with or without baicalin for 3 h. IL-6 and IL-8 transcripts and proteins were detected by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The expression of nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) proteins was analyzed by western blot. A panel of genes related to toll-like receptor (TLR) signaling was examined by PCR array. We found that baicalin significantly downregulated P. gingivalis LPS-stimulated expression of IL-6 and IL-8, and inhibited P. gingivalis LPS-activated NF-κB, p38 MAPK and JNK. Furthermore, baicalin markedly downregulated P. gingivalis LPS-induced expression of genes associated with TLR signaling. In conclusion, the present study shows that baicalin may significantly downregulate P. gingivalis LPS-upregulated expression of IL-6 and IL-8 in HOKs via negative regulation of TLR signaling. PMID:23239998

  14. Protective effects of antioxidants against smokeless tobacco-induced oxidative stress and modulation of Bcl-2 and p53 genes in human oral keratinocytes.

    PubMed

    Bagchi, M; Kuszynski, C A; Balmoori, J; Joshi, S S; Stohs, S J; Bagchi, D

    2001-08-01

    The oral use of chewing tobacco has greatly increased in recent years, and this usage is associated with cancers of the mouth, lip, nasal cavities, esophagus and gut. Oral cancer accounts for 3% of all cancers in U.S.A. and is the seventh most common cancer. Previous studies in our laboratory have demonstrated the protective abilities of a novel IH636 grape seed proanthocyanidin extract (GSPE) against reactive oxygen species both in vitro and in vivo models, and provided significantly better protection as compared to vitamins C, E and beta-carotene. In the recent past, we have demonstrated smokeless tobacco (STE)-induced oxidative stress, apoptotic cell death in a primary culture of normal human oral keratinocytes (NHOK), and have compared the protective abilities of vitamins C and E, singly and in combination, and GSPE in this pathobiology [Free Rad. Biol. Med., 26, 992-1000 (1999)]. In the present study, we have assessed the protective role of vitamins C and E, and GSPE against STE-induced modulation of intracellular oxidized states in NHOK cells as demonstrated by laser scanning confocal microscopy. Approximately 11%, 26%, 28% and 50% protection were observed following incubation with vitamin C, vitamin E, a combination of vitamins C plus E, and GSPE, respectively. DNA fragmentation was assessed as an index of oxidative DNA damage and similar results were observed. Furthermore, the cellular viability and functional roles of Bcl-2, p53 and c-myc genes were assessed in STE-induced oxidative stress in NHOK cells. NHOK cells were treated with STE (0-200 micrograms/ml) for 24 h and changes in the expression of Bcl-2, p53 and c-myc genes were measured by reverse transcriptase-polymerase chain reaction (RT-PCR), and the protective effect of GSPE was assessed. Approximately a 2.0-fold increase in p53 gene expression was observed following incubation of the oral keratinocytes with 100 micrograms/ml of STE, beyond which the expression of p53 decreased, confirming

  15. Cytotoxicity and anti-inflammatory effects of zinc ions and eugenol during setting of ZOE in immortalized human oral keratinocytes grown as three-dimensional spheroids.

    PubMed

    Lee, Jung-Hwan; Lee, Hae-Hyoung; Kim, Kyoung-Nam; Kim, Kwang-Mahn

    2016-05-01

    The objective of this study is to assess the cytotoxic and anti-inflammatory effects of ZOE cement during setting in two-dimensional (2D) or three-dimensional (3D) cultures of immortalized human oral keratinocytes (IHOKs) with determining the extract components responsible for these effects. Extracts of mixed ZOE at different stages of setting were analyzed by a digital pH meter, ICP-MS, and GC-MS. Serial concentrations of extract and their mixture of ZnCl2, ZnSO4·H2O, and eugenol liquid were added to the 2D and 3D IHOK cultures to determine the half maximal effective concentration in investigating the cause of cytotoxicity by means of WST assay and to investigate mRNA expression levels of inflammatory cytokines by RT-PCR. Zn(2+) and eugenol (4-19 ppm) were detected in the extracts. In the early setting stage, significant cytotoxicity was observed in the 2D and 3D IHOK cultures (P<0.05). The EC50 of Zn(2+) from ZnCl2 was 5-44 ppm in both cultures, whereas the EC50 of eugenol was not detectable under 100 ppm. Along with the lower levels of inflammatory cytokine gene expressions in the extract, treatment of the 2D IHOKs with Zn(2+) alone and treatment of the 3D IHOKs with Zn(2+) plus eugenol resulted in significantly lower expression levels of IL-1β, IL-6, and IL-8 (P<0.05). The cytotoxic effect of ZOE on IHOKs was greater during the setting stage owing to the presence of Zn(2+). The anti-inflammatory response to ZOE was induced by a combination of Zn(2+) and eugenol. Cytotoxic and anti-inflammatory effects differed between the 2D and 3D IHOK cultures. Copyright © 2016. Published by Elsevier Ltd.

  16. Streptococci-human papilloma virus interaction with ethanol exposure leads to keratinocyte damage.

    PubMed

    Schwartz, Joel; Pavlova, Sylvia; Kolokythas, Antonia; Lugakingira, Mulokozi; Tao, Lin; Miloro, Michael

    2012-08-01

    Ethanol, human papilloma virus (HPV), and poor oral hygiene are risk factors that have been attributed to oral carcinogenesis. Streptococci sp and HPV infections are common in the head and neck, often associated with sexual activity. Although HPV is linked to head and neck squamous cell carcinoma, it is unclear whether there is a similar role for Streptococci sp. This cell study examines whether Streptococci sp and HPV-16 with exposure to ethyl alcohol (ETOH) can act as cofactors in the malignant transformation of oral keratinocytes. ETOH (0.1%-20% vol/vol) was used to investigate Streptococci sp attachment with immortalized E6-expressing HPV/HOK-16B cells, human oral buccal keratinocytes, and foreskin keratinocytes. Streptococci sp (Streptococci mutans [LT11]) and various strains of acetaldehyde (AA) producer and nonproducer Streptococcus salivarius (110-1, 109-2, 101-7, and 107-1) and a lactic acid producer bacterium, Lactobacillus rhamnosus (24-1 and 25-2), were examined for interactions with keratinocytes by use of a green dye (percent of cells with colonies after 24 hours). Carcinogens, AA, malondialdehyde, DNA damage, and proliferation (5'-bromo-2-deoxyuridine) among keratinocytes were also quantified. AA and malondialdehyde production from permissible Streptococci sp significantly increased with attachment to keratinocytes, whereas L rhamnosus did not significantly attach to keratinocytes. This attachment was associated with enhanced levels of AA adduct formation, proliferation (5'-bromo-2-deoxyuridine incorporation), and enhanced migration through integrin-coated basement membrane by HPV oral keratinocytes, which are characteristics of a malignant phenotype. These cell studies suggest that oral Streptococci sp and HPV (HPV-16) cooperate to transform oral keratinocytes after low-level ETOH (1%) exposure. These results appear to suggest a significant clinical interaction, but further validation is warranted. Copyright © 2012 American Association of Oral and

  17. Novel method for proliferation of oral keratinocyte stem cells.

    PubMed

    Calenic, B; Paun, I A; van Staden, R I; Didilescu, A; Petre, A; Dinescu, M; Greabu, M

    2014-12-01

    Stem cell-based tissue engineering offers clear advantages over conventional normal cell approaches. Owing to their specific characteristics, oral keratinocyte stem cells represent an attractive solution for therapeutic applications. However, when cultured in vitro, these cells lose their unique properties, acquiring a limited capacity for self-renewal, and differentiate rapidly into normal functional keratinocytes. The main aim of the present study was to develop an in-vitro method for the expansion of oral keratinocyte stem cells using a biomaterial approach. Oral keratinocyte stem cells were isolated based on the identification of two surface markers - integrin α6β4 and CD71 - using a magnetic method. The cells were cultured on specific substrates formed from blends of polymers: poly(lactide-co-glycolide) (PLGA); poly(lactide-co-glycolide) + polyurethane (PLGA + PU); and poly(lactide-co-glycolide) + extracellular matrix (PLGA + ECM). The polymers were deposited using a laser-based technique - matrix-assisted pulsed laser evaporation. The cells were analyzed for cell size, cell proliferation, colony-forming efficiency, cell adhesion markers (such as E-cadherin and beta 1 integrin), keratinocyte stem cells and differentiation markers. The methods included ELISAs, immunofluorescence and atomic force microscopy imaging. After 14 d in culture, cells seeded on PLGA + PU stained positive for p63, cd44H, cytokeratin 19 and integrin α6β4 and negative for involucrin, cytokeratin 14 and cytokeratin 10. The levels of adhesion molecules were significantly increased in cells grown on PLGA + PU: at 14 d the E-cadherin levels were 5.4 ± 0.2 ng/mL (for cells grown on PLGA + PU) vs. 4.1 ± 0.4 ng/mL (for cells grown on control medium) (n = 5, p < 0.05 Bonferroni). Oral keratinocyte stem cells grown on PLGA + PU had the highest colony-forming efficiency and proliferation rate, together with the smallest cell size, compared with cells grown on

  18. Epithelial expression of keratinocytes growth factor in oral precancer lesions.

    PubMed

    Jimson, Sudha; Murali, S; Zunt, Susan L; Goldblatt, Lawrence I; Srinivasan, Mythily

    2016-01-01

    Keratinocyte growth factor (KGF) is a potent epithelial mitogen that acts by binding the KGF receptors (KGFRs) expressed on epithelial cells and regulates proliferation and differentiation. The objective of this study was to investigate the expression of KGF in the epithelium in oral precancer. Archival tissues of oral submucous fibrosis (SMF) and leukoplakia were assessed for epithelial KGF expression by immunohistochemistry and real-time quantitative polymerase chain reaction. KGF was predominantly expressed in the basal and parabasal cells in the epithelium of SMF tissues. KGF transcript in the epithelial cells increased with increasing severity of epithelial dysplasia in oral leukoplakia. Although widely reported as a product secreted by the mesenchymal cells, our data suggest that the KGF is also expressed in oral epithelial cells much like the expression in ovarian epithelial cells. Based on the localization of KGF in cells at the epithelial mesenchymal junction and that of the reported presence of KGFR in oral keratinocytes, a potential mechanism involving paracrine and autocrine interactions of KGF and KGFR in early stages of oral precancer is postulated.

  19. Epithelial expression of keratinocytes growth factor in oral precancer lesions

    PubMed Central

    Jimson, Sudha; Murali, S.; Zunt, Susan L.; Goldblatt, Lawrence I.; Srinivasan, Mythily

    2016-01-01

    Background: Keratinocyte growth factor (KGF) is a potent epithelial mitogen that acts by binding the KGF receptors (KGFRs) expressed on epithelial cells and regulates proliferation and differentiation. The objective of this study was to investigate the expression of KGF in the epithelium in oral precancer. Materials and Methods: Archival tissues of oral submucous fibrosis (SMF) and leukoplakia were assessed for epithelial KGF expression by immunohistochemistry and real-time quantitative polymerase chain reaction. Results: KGF was predominantly expressed in the basal and parabasal cells in the epithelium of SMF tissues. KGF transcript in the epithelial cells increased with increasing severity of epithelial dysplasia in oral leukoplakia. Conclusion: Although widely reported as a product secreted by the mesenchymal cells, our data suggest that the KGF is also expressed in oral epithelial cells much like the expression in ovarian epithelial cells. Based on the localization of KGF in cells at the epithelial mesenchymal junction and that of the reported presence of KGFR in oral keratinocytes, a potential mechanism involving paracrine and autocrine interactions of KGF and KGFR in early stages of oral precancer is postulated. PMID:27274338

  20. Conversion from human haematopoietic stem cells to keratinocytes requires keratinocyte secretory factors.

    PubMed

    Fujita, Y; Inokuma, D; Abe, R; Sasaki, M; Nakamura, H; Shimizu, T; Shimizu, H

    2012-08-01

    Recent studies have reported that bone-marrow-derived stem cells (BMSCs), including haematopoietic stem cells (HSCs) and mesenchymal stromal cells, differentiate in order to regenerate various cellular lineages. Based on these findings, it is known that BMSCs can be used clinically to treat various disorders, such as myocardial infarction and neurotraumatic injuries. However, the mechanism of HSC conversion into organ cells is incompletely understood. The mechanism is suspected to involve direct cell-cell interaction between BMSCs, damaged organ cells, and paracrine-regulated soluble factors from the organ, but to date, there have been no investigations into which of these are essential for keratinocyte differentiation from HSCs. To elucidate the mechanism and necessary conditions for HSC differentiation into keratinocytes in vitro. We cultured human (h)HSCs under various conditions to try to elucidate the mechanism and necessary conditions for hHSCs to differentiate into keratinocytes. hHSCs cocultured with mouse keratinocytes induced expression of human keratin 14 and transglutaminase I. Only 0.1% of the differentiated keratinocytes possessed multiple nuclei indicating cell fusion. Coculture of hHSCs with fixed murine keratinocytes (predicted to stabilize cellular components) failed to induce conversion into keratinocytes. Conversely, keratinocyte-conditioned medium from both human and mouse keratinocytes was found to mediate hHSC conversion into keratinocytes. Human HSCs are capable of differentiation into keratinocytes, and cell fusion is extremely rare. This differentiating is mediated by the plasma environment rather than by direct cell-cell interactions. © The Author(s). CED © 2012 British Association of Dermatologists.

  1. Tissue-specific regulation of CXCL9/10/11 chemokines in keratinocytes: Implications for oral inflammatory disease

    PubMed Central

    Marshall, Alison; Celentano, Antonio; Cirillo, Nicola; McCullough, Michael; Porter, Stephen

    2017-01-01

    The IFN-γ-inducible chemokines CXCL9, CXCL10, and CXCL11 play a key role in many inflammatory conditions, particularly those mediated by T cells. Therefore, the production of these chemokines in peripheral tissues could be instrumental in the pathophysiology of tissue-specific immunological diseases such as oral lichen planus (OLP). In the present study, we assessed the production of keratinocyte-derived CXCL9/10/11 under basal and inflammatory conditions and investigated whether these chemokines were involved in the pathogenesis of OLP. We used semi-quantitative PCR, ELISA, chemotaxis assays, and fluorescence-activated cell sorting (FACS) to assess the expression and functional role of CXCL9/10/11 in oral keratinocytes (three strains of normal human oral keratinocytes (NHOK), and the H357 oral cancer cell line) in the presence or absence of IFN-γ. CXCL9/10/11 were also assessed in tissues from normal patients and those with oral lichen planus (OLP). The time course study in oral keratinocytes treated with IFN-γ showed that expression of CXCL9/10/11 chemokines was significantly enhanced by IFN-γ in a time-dependent manner. In particular, CXCL10, a prominent chemokine that was overexpressed by IFN-γ-stimulated NHOK, was able to effectively recruit CD4 lymphocytes, mainly CD4+CD45RA- cells. Significantly higher levels of CXCL9/10/11 were found in tissues from patients with OLP compared to normal oral mucosa. Taken together, the results demonstrate that normal oral keratinocytes produce chemotactic molecules that mediate T cell recruitment. This study furthers understanding of chemokine production in oral keratinocytes and their role in the pathophysiology of oral mucosa, with particular relevance to OLP. PMID:28253295

  2. Methicillin-resistant Staphylococcus aureus adaptation to human keratinocytes.

    PubMed

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

    2015-04-21

    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. Human skin is a major site of staphylococcal infection, and keratinocytes actively participate in eradication of these pathogens. We demonstrate that methicillin-resistant Staphylococcus aureus (MRSA) is ingested by keratinocytes and activates caspase-1-mediated clearance through pyroptosis. Toxin-deficient MRSA mutants are selected within keratinocytes that fail to induce caspase-1 activity and keratinocyte-mediated clearance. These intracellular staphylococci induce autophagy that enhances their intracellular survival by diminishing

  3. 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.

  4. Modulation of calprotectin in human keratinocytes by keratinocyte growth factor and interleukin-1alpha.

    PubMed

    Bando, Mika; Hiroshima, Yuka; Kataoka, Masatoshi; Herzberg, Mark C; Ross, Karen F; Shinohara, Yasuo; Yamamoto, Takenori; Nagata, Toshihiko; Kido, Jun-ichi

    2010-01-01

    Calprotectin is an antimicrobial complex composed of the S100A8 and S100A9 protein family subunits. Contributing to innate immunity, calprotectin expression is increased by interleukin-1alpha (IL-1alpha), which modulates keratinocyte differentiation. Keratinocyte growth factor (KGF) is produced by mesenchymal cells and has a mitogenic activity for epithelial cells. In this study, we investigated the effect of KGF on calprotectin expression in keratinocytes and modulation by IL-1alpha. Human keratinocytes were cultured with KGF in the presence or absence of a KGF receptor (KGFR) inhibitor or mitogen-activated protein kinase (MAPK) inhibitors. Calprotectin (S100A8/S100A9) expression was determined by northern blotting and enzyme-linked immunosorbent assay, respectively, whereas MAPK phosphorylation was analyzed by western blot analysis. KGF significantly decreased the expression of S100A8/S100A9-specific mRNAs and calprotectin protein. In the presence of KGF, KGFR inhibitor or extracellular-regulated kinase inhibitor restored KGF-downregulated expression of S100A8/S100A9. KGF increased IL-1alpha expression in keratinocytes, whereas IL-1alpha increased KGF expression in fibroblasts. Cocultured fibroblast and keratinocytes showed lower S100A8/S100A9 mRNA expression than keratinocytes alone in the presence or absence of IL-1alpha or KGF. These results suggest that fibroblast-derived KGF reduces or restricts calprotectin expression in keratinocytes, which supports our hypothesis that calprotectin expression in keratinocytes is modulated by factors associated with epithelial-mesenchymal interactions.

  5. 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

  6. 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.

  7. SLURP-1 and -2 in normal, immortalized and malignant oral keratinocytes.

    PubMed

    Arredondo, Juan; Chernyavsky, Alexander I; Grando, Sergei A

    2007-05-30

    The secreted mammalian Ly-6/urokinase plasminogen activator receptor-related proteins (SLURP)-1 and -2 are produced by keratinocytes comprising the mucocutaneous epithelium. They regulate in autocrine and paracrine ways cell growth and differentiation through the nicotinic acetylcholine receptors (nAChRs) expressed on the plasma membrane. Keratinocyte nAChRs are targeted by tobacco-derived carcinogenic nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) that can induce tumorigenic transformation of Het-1A keratinocytes. In this study we asked if SLURPs could abolish tumorigenic effects of nitrosamines. Preincubation with either recombinant SLURP-1 or -2 in both cases considerably reduced the number of colonies in soft agar, and the number of tumor nodules >0.5 cm in diameter in Nu/Nu mice produced by Het-1A cells treated with nitrosamines. The levels of SLURP-1 and -2 mRNA transcripts in nitrosamine-transformed Het-1A cells as well as in the tumor cell lines SCC-25 and FaDu were significantly (p<0.05) less compared to normal gingival keratinocytes, which are probably the major source of the secreted SLURPs found in a sample of human saliva. The expression of SLURPs was decreased due to gene silencing of different nAChR alpha subunits with small hairpin RNA, suggesting that a positive feedback regulation is altered in malignant cells. Thus, SLURP-1 and -2 are efficient autocrine and paracrine ligands of keratinocyte nAChRs capable of preventing tobacco nitrosamine-induced malignant transformation of oral cells. These "proof-of-concept" preliminary results have salient clinical implications.

  8. Arecoline and oral keratinocytes may affect the collagen metabolism of fibroblasts.

    PubMed

    Xia, Li; Tian-You, Ling; Yi-Jun, Gao; Dong-Sheng, Tang; Wen-Hui, Li

    2009-05-01

    The characteristic of oral submucous fibrosis (OSF) is related with the disturbance of synthesis and degradation of extracellular matrix. Arecoline, the areca nut (betel nut) component of betel quid, plays a major role in pathogenesis of OSF. But the exact mechanism how arecoline influences the collagen metabolism is unclear. Oral keratinocytes and fibroblasts were cocultured and keratinocytes were pre-treated by arecoline. Fibroblasts alone, fibroblasts stimulated by arecoline, fibroblasts cocultured with keratinocytes and fibroblasts cocultured with keratinocyte pre-treated by arecoline were included as the four groups in the present study. The concentration of collagen, the content and activity of matrix metalloproteinase (MMP) and the concentration of tissue inhibitor of metalloproteinase (TIMP) were assessed. The collagen production of fibroblasts decreased when cocultured with keratinocytes; when cocultured with keratinocytes pre-treated by arecoline, fibroblasts produced more soluble collagen than non-pretreated coculture group. MMP-9 was produced only in coculture groups. There was no significant difference in the two coculture groups. The activation ratio of pro-MMP-2 in arecoline pre-treated keratinocytes-fibroblasts coculture group was significantly higher than that of non-coculture groups, but no significant difference existed in the two coculture groups. TIMP-1 produced by arecoline pre-treated keratinocytes-fibroblasts coculture group was significantly higher than those by the other three groups. TIMP-1 and the interaction of oral keratinocytes and fibroblasts play important role in pathogenesis of OSF.

  9. Enhancement of UVB-induced apoptosis by apigenin in human keratinocytes and organotypic keratinocyte cultures.

    PubMed

    Abu-Yousif, Adnan O; Smith, Kimberly A; Getsios, Spiro; Green, Kathleen J; Van Dross, Rukiyah T; Pelling, Jill C

    2008-04-15

    Topical application of the bioflavonoid 4',5,7-trihydroxyflavone (apigenin) to mouse skin effectively reduces the incidence and size of skin tumors caused by UVB exposure. The ability to act as a chemopreventive compound indicates that apigenin treatment alters the molecular events initiated by UVB exposure; however, the effects of apigenin treatment on UVB-irradiated keratinocytes are not fully understood. In the present study, we have used three models of human keratinocytes to study the effect of apigenin treatment on UVB-induced apoptosis: HaCaT human keratinocyte cells, primary keratinocyte cultures isolated from human neonatal foreskin, and human organotypic keratinocyte cultures. Each keratinocyte model was exposed to a moderate dose of UVB (300-1,000 J/m(2)), then treated with apigenin (0-50 micromol/L), and harvested to assess apoptosis by Western blot analysis for poly(ADP)ribose polymerase cleavage, annexin-V staining by flow cytometry, and/or the presence of sunburn cells. Apigenin treatment enhanced UVB-induced apoptosis >2-fold in each of the models tested. When keratinocytes were exposed to UVB, apigenin treatment stimulated changes in Bax localization and increased the release of cytochrome c from the mitochondria compared with UVB exposure alone. Overexpression of the antiapoptotic protein Bcl-2 and expression of a dominant-negative form of Fas-associated death domain led to a reduction in the ability of apigenin to enhance UVB-induced apoptosis. These results suggest that enhancement of UVB-induced apoptosis by apigenin treatment involves both the intrinsic and extrinsic apoptotic pathways. The ability of apigenin to enhance UVB-induced apoptosis may explain, in part, the photochemopreventive effects of apigenin.

  10. Substrate Stiffness Affects Human Keratinocyte Colony Formation

    PubMed Central

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

    2015-01-01

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

  11. [Effects of arecoline and nicotine on the expression of hTERT in oral keratinocytes].

    PubMed

    Gao, Yi-jun; Ling, Tian-you; Yin, Xiao-min; Li, Xia; Huang, Yan

    2007-01-01

    To investigate the effects of arecoline and nicotine on the expression of human telomerase reverse transcriptase (hTERT) mRNA and protein in cultured normal human oral keratinocytes (KC). The experiments were divided into arecoline group, arecoline/nicotine group and control group. The hTERT mRNA and protein expression of KC was examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Arecoline could induce the hTERT mRNA and protein expression of KC in a dose dependent manner, the hTERT mRNA and protein expression of KC was higher in 0.030, 0.060, 0.090 g/L arecoline group than control group (P < 0.001). Nicotine (0.025 g/L) increased hTERT mRNA and protein expression of KC induced by arecoline. Arecoline could increase the expression of hTERT mRNA and protein in oral keratinocytes. Nicotine had a synergistic effect on arecoline. hTERT over-expression induced by arecoline and nicotine may play an important role in the malignant transformation of oral submucous fibrosis.

  12. Keratinocyte growth factor protected cultured human keratinocytes exposed to oxidative stress.

    PubMed

    Gragnani, Alfredo; Rossi, Marina Bertelli; Albuquerque, Janne Cely Medeiros; Brito, Gabriela Soares Silva; Aloise, Antonio Carlos; Ferreira, Lydia Masako

    2010-02-01

    To evaluate effects of oxidative stress and supplementation of keratinocyte growth factor (KGF) on cultivated human keratinocytes. Oxidative stress was produced through addition of hydrogen peroxide (H(2)O(2)) to the culture medium. Cultivated human keratinocytes were divided in 4 groups: Group control (G C), Group KGF (G KGF), Group H(2)O(2) (G H(2)O(2)), Group H(2)O(2) and KGF (G H(2)O(2)-KGF). Each experiment was accomplished with the same lineage cultivated keratinocytes, in triplicate. Cell viability was evaluated by trypan blue exclusion assay. The results showed that the culture medium supplemented with KGF presented a small rate of cell viability when compared to cells only in culture medium (p<0,001). It demonstrated that only the growth factor does not have protector effects for cells in vitro. However, in front of the oxidative stress produced by addition of hydrogen peroxide to the medium, KGF showed a beneficial effect, protecting cells when compared to the group that suffered hydrogen peroxide action but had not been exposed to KGF (p<0,001). KGF determined protection to the primary human keratinocytes exposed to oxidative stress.

  13. Betel-derived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis.

    PubMed

    Moutasim, Karwan A; Jenei, Veronika; Sapienza, Karen; Marsh, Daniel; Weinreb, Paul H; Violette, Shelia M; Lewis, Mark P; Marshall, John F; Fortune, Farida; Tilakaratne, Waninayaka M; Hart, Ian R; Thomas, Gareth J

    2011-02-01

    Oral submucous fibrosis (OSF) is a premalignant, fibrosing disorder of the mouth, pharynx, and oesophagus, with a malignant transformation rate of 7-13%. OSF is strongly associated with areca (betel) nut chewing and worldwide, over 5 million people are affected. As αvβ6 integrin is capable of promoting both tissue fibrosis and carcinoma invasion, we examined its expression in fibroepithelial hyperplasia and OSF. αvβ6 was markedly up-regulated in OSF, with high expression detected in 22 of 41 cases (p < 0.001). We investigated the functional role of αvβ6 using oral keratinocyte-derived cells genetically modified to express high αvβ6 (VB6), and also NTERT-immortalized oral keratinocytes, which express low αvβ6 (OKF6/TERT-1). VB6 cells showed significant αvβ6-dependent activation of TGF-β1, which induced transdifferentiation of oral fibroblasts into myofibroblasts and resulted in up-regulation of genes associated with tissue fibrosis. These experimental in vitro findings were confirmed using human clinical samples, where we showed that the stroma of OSF contained myofibroblasts and that TGF-β1-dependent Smad signalling was detectable both in keratinocytes and in myofibroblasts. We also found that arecoline, the major alkaloid of areca nuts, up-regulated keratinocyte αvβ6 expression. This was modulated through the M(4) muscarinic acetylcholine receptor and was suppressed by the M(4) antagonist, tropicamide. Arecoline-dependent αvβ6 up-regulation promoted keratinocyte migration and induced invasion, raising the possibility that this mechanism may support malignant transformation. Over 80% of OSF-related oral cancers examined had moderate/high αvβ6 expression. These data suggest that the pathogenesis of OSF may be epithelial-driven and involve arecoline-dependent up-regulation of αvβ6 integrin.

  14. 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.

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

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

  16. 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.

  17. N-acetyl cysteine protects human oral keratinocytes from Bis-GMA-induced apoptosis and cell cycle arrest by inhibiting reactive oxygen species-mediated mitochondrial dysfunction and the PI3K/Akt pathway.

    PubMed

    Zhu, Yu; Gu, Ying-xin; Mo, Jia-ji; Shi, Jun-yu; Qiao, Shi-chong; Lai, Hong-chang

    2015-12-01

    Bisphenol-A-glycidyl methacrylate (Bis-GMA) released from dental resin materials causes various toxic effects on gingival epithelium. Thus the underlying mechanisms of its cytotoxicity should be elucidated for safety use. One potential cause of cell damage is the generation of reactive oxygen species (ROS) beyond the capacity of a balanced redox regulation. In this study, we found that exposure of human oral keratinocytes (HOKs) to Bis-GMA caused apoptosis and G1/S cell cycle arrest in parallel with an increased ROS level. Moreover, Bis-GMA induced a depletion of mitochondrial membrane potential, an increase in the Bax/Bcl-2 ratio, an activation of caspase-3 and altered expressions of cell cycle-related proteins (p21, PCNA, cyclinD1). Furthermore, the co-treatment of the ROS scavenger N-acetyl cysteine (NAC) obviously attenuated Bis-GMA-induced toxicity. Here we also evaluated the effects of Bis-GMA on the ROS-related PI3k/Akt pathway. We found that Bis-GMA inhibited the phosphorylation of Akt, whereas the amount of phosphorylated Akt was reverted to the control level in the presence of NAC. Our findings suggested that the toxic effects of Bis-GMA were related to ROS production and the antioxidant NAC effectively reduced Bis-GMA-mediated cytotoxicity. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Evaluation of microRNA expression in head and neck squamous cell carcinoma cell lines and in primary culture of oral keratinocytes.

    PubMed

    Andreghetto, Flavia Maziero; Klingbeil, Maria Fatima Guarizo; Soares, Renata Machado; Sitnik, Roberta; Pinto Junior, Décio Dos Santos; Mathor, Monica Beatriz; Nunes, Fabio Daumas; Severino, Patricia

    2011-12-01

    Functional in vitro studies are fundamental to understand the role of microRNAs, small non coding RNA molecules that function as post-transcriptional regulators, in cancer. The objective of this study was to determine the applicability of head and neck squamous cell carcinoma cell lines and human oral keratinocytes as models for functional studies on microRNAs previously identified as deregulated in head and neck squamous cell carcinomas. The expression level of four microRNAs was assessed in cell lines and in primary cultures of oral keratinocytes using specific real-time polymerase chain reactions. The identity of oral squamous cell carcinoma cell lines was confirmed by means of STR (short tandem repeats) profiling. The possible impact of feeder-layer gene expression in global microRNA expression results from keratinocyte primary culture was also evaluated. Significant differences in microRNA gene expression were observed among squamous cell carcinoma cell lines, particularly among cells lines from distinct subsites, as well as between primary culture of human keratinocytes and immortalized keratinocyte cell lines. Primary cultures of human keratinocytes and diverse tumor cell lines are relatively easy to obtain. However, each cell model possesses a characteristic phenotype; whereas one may be useful for a specific study, it may be inappropriate for another. Therefore, it is imperative that suitable cell lines are cautiously selected for functional studies in cancer.

  19. 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.

  20. [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.

  1. Polarized Integrin Mediates Human Keratinocyte Adhesion to Basal Lamina

    NASA Astrophysics Data System (ADS)

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

    1990-09-01

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

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

    PubMed

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

    2016-10-01

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

  3. AMPK regulation of the growth of cultured human keratinocytes

    SciTech Connect

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

    2006-10-20

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

  4. Proliferating cell nuclear antigen/cyclin in cultured human keratinocytes.

    PubMed

    Okada, N; Miyagawa, S; Steinberg, M L; Yoshikawa, K

    1990-09-01

    Expression of proliferating cell nuclear antigen (PCNA)/cyclin in cultured human keratinocytes was studied using an antibody from an SLE patient as the reagent. By indirect immunofluorescence staining, SV40-transformed human keratinocytes expressed PCNA/cyclin in 40-45% of the cells as a nulcear granular fluorescence. After synchronization of these cells, their nuclear distribution pattern during the S phase was sequential and showed a clear correlation with DNA synthesis. Primary cultured keratinocytes grown in high Ca+ medium expressed PCNA/cyclin in 10-15% of the cells with a similar staining pattern. These positively stained cells were confined to the basal and immediate suprabasal layers of the stratified culture sheet. The keratinocytes disaggregated by trypsin were separated according to cell size through a screen of Nitex monofilament cloth. The cells smaller than 15 microns in diameter synthesized abundant PCNA/cyclin, while the larger cells expressed very low levels. These results indicate that the expression of PCNA/cyclin correlates with DNA synthesis in cultured keratinocytes, but is not associated with their differentiation process.

  5. [The effect of retrovirus-mediated hTRT transfection into cultured oral keratinocytes].

    PubMed

    Huang, Ji-yan; Liu, Wei; Zhou, Zeng-tong; Zhou, Hai-wen

    2014-06-01

    Human telomerase reverse transcriptase (hTRT) was transfected into cultured oral keratinocytes (OKC) mediated by pBABE-tert recombined retrovirus to investigate the effect on OKC lifespan. pBABE-tert recombined retrovirus loaded with hTRT gene was amplified by transfected PT67 cells, and then transfected into cultured OKC in vitro. The positive clones of OKC were separated by puromycin and subcultured. Telomerase activity was analyzed by telomerase PCR-ELISA and PCR-PAGE. The hTRT positive clones of OKC showed telomerase expression, with extending lifespan to 8-9 passages. The hTRT transfected OKC can prolong doubly lifespan but not be immortalized, which indicates that cellular immortality mechanism is complicated and multi-controled. Telomerase activity is the key for cell immortalization but not the only impact factor.

  6. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Hyaluronan Does Not Regulate Human Epidermal Keratinocyte Proliferation and Differentiation*

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

    Albers, K.M.

    1985-01-01

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

  9. 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

  10. 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

  11. 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.

  12. Human embryonic stem cells derived keratinocyte as an in vitro research model for the study of immune response.

    PubMed

    Kidwai, Fahad Karim; Jokhun, Doorgesh Sharma; Movahednia, Mohammad Mehdi; Yeo, Jin Fei; Tan, Kai Soo; Cao, Tong

    2013-09-01

    The innate immune response (IMR) is critical for the oral mucosa due to their continuous exposure to various oral pathogens. Keratinocytes play important role in IMR. Therefore, to date, keratinocytes from different sources have been used as in vitro research model for the study of IMR. However, current keratinocyte research models are hampered by the limited supply, patients' dependency and batch to batch variation. Therefore, in this study, we demonstrated the use of human embryonic stem cells (hESCs) derived keratinocytes (H9-Kert) as an alternative research model for the study of IMR. The expression kinetics of toll-like receptor (TLR) 2, TLR 4, interleukin (IL) -6, IL-8, inducible nitric oxide synthase (iNOS) and tumour necrosis factor-alpha (TNF-α), in H9-Kert and immortalized human keratinocyte cell line (HaCaT) were analysed at mRNA levels by both reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. The activation of the inflammatory transcription factor nuclear factor kappa-b (NFĸB) was assayed in these cells by transiently transfecting the cells with NFĸB reporter plasmid. Activation of NFĸB following treatment with heat-killed Porphyromonas gingivalis (P. gingivalis), an oral pathogen, was determined by assaying for the reporter, secreted alkaline phosphatase activity. The expression of TLRs, cytokines and activation of NFĸB following bacterial stimulation showed in both H9-Kert and the widely used HaCaT keratinocyte cell line was similar. Overall, our results support the potential application of hESCs as an alternative limitless cell source for primary keratinocytes which can be used as consistent and dependable research tool with minimum variations and no donor's dependency. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. 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.

  14. 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

  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. 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.

  17. 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.

  18. Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocytes regulate its proper differentiation in the absence of dermis

    PubMed Central

    1988-01-01

    Human keratinocytes isolated from a skin biopsy and cultured in vitro reconstitute a stratified squamous epithelium suitable for grafting on burned patients. Melanocytes coisolated from the same skin biopsy also proliferate under these culture conditions and maintain differentiated functions (i.e., synthesize melanin granules, regularly intersperse in the basal layer of the cultured epidermis, and transfer melanosomes in the cytoplasm of contiguous keratinocytes) (De Luca, M., A. T. Franzi, F. D'Anna, A. Zicca, E. Albanese, S. Bondanza, and R. Cancedda. 1988. Eur. J. Cell Biol. 46:176-180). Isolated melanocytes in culture grow in the presence of specific growth factors with a mean population doubling time of 4-10 d. In this paper we show that (a) human keratinocytes and oral epithelial cells possess strong and specific melanocyte growth stimulating activity (doubling time, 24 h); (b) melanocyte growth is not autonomous but requires close keratinocyte contact and is regulated to maintain a physiological melanocytes/keratinocytes ratiol and (c) pure skin keratinocytes, but not oral epithelial cells, have all the information required for the proper physiological location and differentiation of melanocytes in the epidermis. PMID:2460471

  19. IL-22 mediates the oral mucosal wound healing via STAT3 in keratinocytes.

    PubMed

    Yu, Ran; Ding, Yumei; Zhu, Lijuan; Qu, Yinying; Zhang, Chenguang; Liu, Lin; Chen, Lili

    2016-12-01

    Wounds are common in the oral cavity. During wound healing, several cytokines are released, which are probably helpful in providing wound debridement, removal of damaged tissues and microbes. Most of the target cells of IL-22 are epithelial cells, which play an important role in mucosa immunity. The function of IL-22 in oral diseases is not well understood. We investigated the expression level of IL-22, collagen I and p-stat3 (Tyr705) via a mice tongue wound model in vivo and detected the effect of IL-22 on the expression of MMP-1, type I collagen and p-stat3 in keratinocytes. IL-22 and p-stat3 were associated with wound healing, and STAT3 was activated when the keratinocytes or the tongue tissue were stimulated by IL-22. In addition, IL-22 could mediate gene expression involved in wounds involving keratinocytes, such as type I collagen and MMP-1, which may contribute to scarless healing. Our study suggests that IL-22 mediates wound healing via STAT3 in keratinocytes. This study reveals a new role for IL-22 in mediating wound healing. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector.

    PubMed

    Nanba, Daisuke; Matsushita, Natsuki; Toki, Fujio; Higashiyama, Shigeki

    2013-10-18

    The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment. In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry. Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene. This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine.

  1. Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector

    PubMed Central

    2013-01-01

    Introduction The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment. Methods In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry. Results Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene. Conclusions This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine. PMID:24406242

  2. 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.

  3. Melanoma cells influence the differentiation pattern of human epidermal keratinocytes.

    PubMed

    Kodet, Ondřej; Lacina, Lukáš; Krejčí, Eliška; Dvořánková, Barbora; Grim, Miloš; Štork, Jiří; Kodetová, Daniela; Vlček, Čestmír; Šáchová, Jana; Kolář, Michal; Strnad, Hynek; Smetana, Karel

    2015-01-05

    Nodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome. Similarly to other tumors, permissive microenvironment is essential for melanoma progression. Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue. Here, we study the effect of melanoma cells on human primary keratinocytes (HPK). Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC). Comparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed. Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively. Differentially expressed candidate genes were verified by RT-qPCR. Biological activity of candidate proteins was assessed on cultured HPK. Epidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases. This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10. We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK. This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies. We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes. We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro. This interaction further highlights the role of intercellular interactions in melanoma. The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.

  4. Potassium channel in the mitochondria of human keratinocytes.

    PubMed

    Toczyłowska-Mamińska, Renata; Olszewska, Anna; Laskowski, Michał; Bednarczyk, Piotr; Skowronek, Krzysztof; Szewczyk, Adam

    2014-03-01

    The activation of mitochondrial potassium channels induces cytoprotection in various cell types. Hence, the identification of ion channels present in the inner mitochondrial membrane of keratinocytes is important in distinguishing possible protective mechanisms in these cells. In this paper, inner membrane mitochondrial ion channels of the human keratinocyte HaCaT cell line were investigated using a patch-clamp technique. We observed potassium-selective channel activity with a conductance of 83 pS at positive voltages. The I-V curve indicates that the observed channel has rectifying properties. Moreover, the channel activity was inhibited by acidic pH and 1 mM lidocaine. Using reverse transcriptase-PCR, we found an mRNA transcript for the TASK-3 (tandem pore domain acid-sensitive K channels) channel. We observed co-localization of the TASK-3 protein and a mitochondrial marker in the mitochondria of HaCaT cells. Additionally, we showed that TASK-3 knockdown HaCaT cells markedly decreased viability after UVB radiation exposure compared with control cells. In summary, the single-channel activity and properties of a mitochondrial potassium channel in a keratinocyte HaCaT cell line have been described.

  5. 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.

  6. Human Keratinocyte Cultures in the Investigation of Early Steps of Human Papillomavirus Infection

    PubMed Central

    Griffin, Laura M.; Cicchini, Louis; Xu, Tao; Pyeon, Dohun

    2014-01-01

    Summary Human papillomaviruses (HPVs) are non-enveloped DNA viruses that are highly tropic for mucosal and cutaneous epithelia. The HPV life cycle is tightly linked to epithelial cell differentiation, where HPVs only infect the basal proliferating keratinocytes, and progeny virus assembly and release only occurs in differentiated upper-layer keratinocytes. Therefore, human keratinocyte monolayer cultures provide a useful model to study the early stages of HPV infection. However, previous reports have shown some conflicting results of virus-host interactions during HPV entry, which may be partly attributable to the different cell culture models used to examine these steps of HPV infection. Thus, there is a need to have a standardized in vitro model system to study virus-host interactions during HPV entry. Here, we describe the 3 most widely accepted keratinocyte models for studying HPV infection: primary human foreskin keratinocytes (HFK), normal immortalized keratinocytes (NIKS), and transformed HaCaT keratinocytes. We also describe methods to genetically manipulate these cells, enabling the study of candidate host genes that may be important during HPV infection. Lastly, we will outline simple and robust methods to assay HPV infectivity, which can be used to determine whether knockdown or overexpression of a particular gene affects HPV entry. PMID:24281871

  7. Regulation of oxidative-stress responsive genes by arecoline in human keratinocytes.

    PubMed

    Thangjam, G S; Kondaiah, P

    2009-10-01

    Arecoline, an arecanut alkaloid present in the saliva of betel quid chewers, has been implicated in the pathogenesis of a variety of inflammatory oral diseases, including oral submucous fibrosis and periodontitis. To understand the molecular basis of arecoline action in epithelial changes associated with these diseases, we investigated the effects of arecoline on human keratinocytes with respect to cell growth regulation and the expression of stress-responsive genes. Human keratinocyte cells (of the HaCaT cell line) were treated with arecoline, following which cell viability was assessed using the Trypan Blue dye-exclusion assay, cell growth and proliferation were analyzed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and 5-bromo-2-deoxyuridine incorporation assays, cell cycle arrest and generation of reactive oxygen species were examined using flow cytometry, and gene expression changes were investigated using the reverse transcription-polymerase chain reaction technique. The role of oxidative stress, muscarinic acetylcholine receptor and mitogen-activated protein kinase (MAPK) pathways were studied using specific inhibitors. Western blot analysis was performed to study p38 MAPK activation. Arecoline induced the generation of reactive oxygen species and cell cycle arrest at the G1/G0 phase in HaCaT cells without affecting the expression of p21/Cip1. Arecoline-induced epithelial cell death at higher concentrations was caused by oxidative trauma without eliciting apoptosis. Sublethal concentrations of arecoline upregulated the expression of the following stress-responsive genes: heme oxygenase-1; ferritin light chain; glucose-6-phosphate dehydrogenase; glutamate-cysteine ligase catalytic subunit; and glutathione reductase. Additionally, there was a dose-dependent induction of interleukin-1alfa mRNA by arecoline via oxidative stress and p38 MAPK activation. Our data highlight the role of oxidative stress in arecoline-mediated cell death

  8. In Vitro Toxicity of Aluminum Nanoparticles in Human Keratinocytes

    DTIC Science & Technology

    2008-03-01

    Cell Line The HaCaT cell line consists of immortalized adult human keratinocytes developed by the German Cancer Research Center (DKFZ, Heidelberg...IL-8 using the R & D Systems®, Inc. assay kits would help validate the results of this research. The HaCaT cell line used in this study did not... HaCaT cell line . 1. Vacuum excess liquid from cell culture flasks. 2. Wash cells with 5 mL of 1X PBS. 3. Vacuum liquid from flask.

  9. Snail up-regulates pro-inflammatory mediators and inhibits differentiation in oral keratinocytes

    PubMed Central

    Lyons, J. Guy; Patel, Vyomesh; Roue, Naomi C.; Fok, Sandra Y.; Soon, Lilian L.; Halliday, Gary M.; Gutkind, J. Silvio

    2008-01-01

    The transcriptional repressor, Snail2, is over-expressed in head and neck squamous cell carcinomas (HNSCCs) relative to non-malignant head and neck mucosal epithelium, and in locally recurrent relative to non-recurrent HNSCCs. We investigated the mechanisms by which Snails might contribute to the pathogenesis of HNSCCs using cell biological and molecular analyses. Oral keratinocytes that expressed Snails acquired an enhanced ability to attract monocytes and to invade a dense interstitial collagen matrix. They were also found to up-regulate production of pro-inflammatory cytokines and cyclooxygenase-2 (COX2), which have previously been shown to correlate with malignancy. Induction of nuclear factor-kappa B transcriptional activity by Snails was weak and not sufficient to account for the elevated levels of COX2, interleukin-6, interleukin-8 or CXCL1. In addition, expression of Snails in oral keratinocytes impaired desquamation in vitro and strongly repressed expression of both ELF3 and matriptase-1, which play important roles in the terminal differentiation of keratinocytes. Re-expression of matriptase-1 in Snail-expressing cells partially rescued desquamation. This implicates Snails as contributing to malignancy both at the early stages, by impeding terminal differentiation, and at later stages, when invasion and inflammation are important. PMID:18559496

  10. 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 K; Bronner, Marianne E; Andreadis, Stelios T

    2017-05-01

    During development, neural crest (NC) cells are induced by signaling events at the neural plate border of all vertebrate embryos. Initially arising within the central nervous system, NC 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 (KC), in response to fibroblast growth factor 2 and insulin like growth factor 1 signals, can be reprogrammed toward a NC fate. Genome-wide transcriptome analyses show that keratinocyte-derived NC cells are similar to those derived from human embryonic stem cells. Moreover, they give rise in vitro and in vivo to NC derivatives such as peripheral neurons, melanocytes, Schwann cells and mesenchymal cells (osteocytes, chondrocytes, adipocytes, and smooth muscle cells). By demonstrating that human keratin-14+ KC can form NC cells, even from clones of single cells, our results have important implications in stem cell biology and regenerative medicine. Stem Cells 2017;35:1402-1415. © 2017 AlphaMed Press.

  11. 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

  12. Vandetanib-induced phototoxicity in human keratinocytes NCTC-2544.

    PubMed

    Salvador, Alessia; Vedaldi, Daniela; Brun, Paola; Dall'Acqua, Stefano

    2014-08-01

    The phototoxicity of the new anticancer drug vandetanib was evaluated using human keratinocyte cell line, NCTC-2544. This study was started since many clinical cases of vandetanib photosensitizing reactions were recently reported in literature. Vandetanib induces a clear drop in human keratinocytes viability after cell irradiation in concentration and UV-A dose dependent mode. Since vandetanib can photolyze with the formation of two main photoproducts after UV-A exposure, the contribution of these new species was also evaluated. These two photoproducts did not have a main role in the phototoxicity of their parent drug. In our opinion, the main hypothesis for the vandetanib phototoxic potential is the formation of a very reactive specie, such as an aryl radical, which can react promptly with different targets inside the cells. In fact, a massive DNA photodamage was detected both in the in vitro DNA photocleavage experiments, and in cells. Moreover, vandetanib was able to photoinduce lipid peroxidation and protein oxidations. Vandetanib photoinduced cell death by apoptosis with the involvement of mitochondria and lysosomes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. 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

  14. 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.

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

    PubMed

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

    2015-01-01

    The cytokinesis-block micronucleus (MN) assay was used to assess the genotoxicity of low doses of different types of space radiation. Normal human primary keratinocytes and immortalized keratinocytes grown in 2D monolayers each were exposed to graded doses of 0.3 or 1.0 GeV/n silicon ions or similar energies of iron ions. The frequencies of induced MN were determined and compared to γ-ray data. RBE(max) values ranged from 1.6 to 3.9 for primary keratinocytes and from 2.4 to 6.3 for immortalized keratinocytes. At low radiation doses ≤ 0.4 Gy, 0.3 GeV/n iron ions were the most effective at inducing MN in normal keratinocytes. An "over-kill effect" was observed for 0.3 GeV/n iron ions at higher doses, wherein 1.0 GeV/n iron ions were most efficient in inducing MN. In immortalized keratinocytes, 0.3 GeV/n iron ions produced MN with greater frequency than 1.0 GeV/n iron ions, except at the highest dose tested. MN formation was higher in immortalized keratinocytes than in normal keratinocytes for all doses and radiation qualities investigated. MN induction was also assessed in human keratinocytes cultured in 3D to simulate the complex architecture of human skin. RBE values for MN formation in 3D were reduced for normal keratinocytes exposed to iron ions, but were elevated for immortalized keratinocytes. Overall, MN induction was significantly lower in keratinocytes cultured in 3D than in 2D. Together, the results suggest that tissue architecture and immortalization status modulate the genotoxic response to space radiation, perhaps via alterations in DNA repair fidelity. © 2014 Wiley Periodicals, Inc.

  16. Protective effect of geranylgeranylacetone against radiation-induced delayed effects on human keratinocytes.

    PubMed

    Isoir, Muriel; Roque, Telma; Squiban, Claire; Milliat, Fabien; Mondon, Philippe; Mas-Chamberlin, Claire; Benderitter, Marc; Guipaud, Olivier; Tamarat, Radia

    2013-02-01

    Skin exposure to ionizing radiation affects the normal wound healing process. We investigated the beneficial effects of a pharmacological treatment with geranylgeranylacetone (GGA) on keratinocytes using in vitro scratch wound injury assay in nonirradiated and irradiated conditions. Irradiation affected the wound closure of keratinocytes 24 h after scratch injury, whereas re-epithelialization was markedly accelerated after GGA treatment when compared to nontreated keratinocytes. We demonstrated that GGA treatment increased migration of human epidermal keratinocytes and this migratory property was not related to RhoA signaling. Interestingly, Western blot analysis revealed that GGA treatment down-regulated caspase 3 active form expression and up-regulated the activated phenotype by inducing both keratin 6 (K6) expression and interleukin-1β (IL-1β) release without modification of the differentiate phenotype. Finally, the proteomic profiling was performed on keratinocytes, showing that global protein changes occurred after irradiation of keratinocytes treated or untreated with GGA.

  17. Ceramides stimulate caspase-14 expression in human keratinocytes

    PubMed Central

    Jiang, Yan J.; Kim, Peggy; Uchida, Yoshikazu; Elias, Peter M.; Bikle, Daniel D.; Grunfeld, Carl; Feingold, Kenneth R.

    2014-01-01

    Caspase-14 is an enzyme that is expressed predominantly in cornifying epithelia and catalyses the degradation of profilaggrin. Additionally, caspase-14 plays an important role in the terminal differentiation of keratinocytes. However, how caspase-14 expression is regulated remains largely unknown. Here we demonstrate that ceramides (C2-Cer and C6-Cer), but not other sphingolipids (C8-glucosylceramides, sphinganine, sphingosine-1-phosphate or ceramide-1-phosphate), increase caspase-14 expression (mRNA and protein) in cultured human keratinocytes in a dose- and time-dependent manner. Inhibitors of glucosylceramide synthase and ceramidase increase endogenous ceramide levels and also increase caspase-14 expression, indicating an important regulatory role for ceramides and suggesting that the conversion of ceramides to other metabolites is not required. The increase in caspase-14 expression induced by ceramides is first seen at 16 h and requires new protein synthesis, suggesting that the ceramide-induced increase is likely an indirect effect. Furthermore, ceramides increase caspase-14 gene expression primarily by increasing transcription. Blocking de novo synthesis of ceramides does not affect caspase-14 expression, suggesting that basal expression is not dependent on ceramide levels. These studies show that ceramides, an important structural lipid, stimulate caspase-14 expression providing a mechanism for coordinately regulating the formation of lipid lamellar membranes with the formation of corneocytes. PMID:23362869

  18. Oral keratinocyte responses to nickel-based dental casting alloys in vitro.

    PubMed

    Wylie, C M; Davenport, A J; Cooper, P R; Shelton, R M

    2010-09-01

    Adverse reactions of oral mucosa to nickel-based dental casting alloys are probably due to corrosion metal ion release. We exposed H400 oral keratinocytes to two Ni-based dental alloys (Matchmate and Dsign10) as well as NiCl( 2) (1-40 microg/mL Ni(2+)). Alloy derived Ni(2+) media concentrations were determined. Direct culture on both alloys resulted in inhibited growth with a greater effect observed for Dsign10 (higher ion release). Indirect exposure of cells to conditioned media from Dsign10 negatively affected cell numbers (approximately 64% of control by 6 days) and morphology while Matchmate-derived media did not. Exposure to increasing NiCl(2) negatively affected cell growth and morphology, and the Granulocyte-macrophage colony-stimulating factor (GM-CSF) transcript was significantly up-regulated in cells following direct and indirect exposure to Dsign10. NiCl(2) exposure up-regulated all cytokine transcripts at 1 day. At day 6, IL-1beta and IL-8 transcripts were suppressed while GM-CSF and IL-11 increased with Ni(2+) dose. Accumulation of Ni(2+) ions from alloys in oral tissues may affect keratinocyte viability and chronic inflammation.

  19. The ultrastructural surface morphology of oral cancer cells and keratinocytes after exposure to chitosan

    NASA Astrophysics Data System (ADS)

    Fatimah; Sarsito, A. S.; Wimardhani, Y. S.

    2017-08-01

    Low-molecular-weight chitosan (LMWC) has the same selective cytotoxic effects on oral cancer cells as cisplatin. The cell deaths caused by the anticancer characteristics of chitosan show that apoptosis is not the death pathway of the primary cells involved. The interactions between LMWC and the cells need to be explored. The objective of this study was to compare the ultrastructural morphology of oral Squamous Cell Carcinoma (SCC Ca)-922 and noncancer keratinocyte HaCaT cell lines after exposure to LMWC and cisplatin. The cells were treated with LMWC and cisplatin, and their ultrastructural morphology was analyzed using scanning electron micrographs. Features of early apoptosis, seen as the loss of microvilli, were detected in the LMWC-exposed Ca9-22 cells, and there was a material surrounding the cells. In contrast, the LMWC-exposed HaCaT cells showed no changes related to apoptosis. The results were the opposite when cisplatin was used. This study confirms that there are differences in the ultrastructural surface morphology of LMWC-exposed and cisplatin-exposed oral cancer cells and keratinocytes that could be correlated with their biological activity.

  20. Porphyromonas gingivalis induces CCR5-dependent transfer of infectious HIV-1 from oral keratinocytes to permissive cells

    PubMed Central

    Giacaman, Rodrigo A; Asrani, Anil C; Gebhard, Kristin H; Dietrich, Elizabeth A; Vacharaksa, Anjalee; Ross, Karen F; Herzberg, Mark C

    2008-01-01

    Background Systemic infection with HIV occurs infrequently through the oral route. The frequency of occurrence may be increased by concomitant bacterial infection of the oral tissues, since co-infection and inflammation of some cell types increases HIV-1 replication. A putative periodontal pathogen, Porphyromonas gingivalis selectively up-regulates expression of the HIV-1 coreceptor CCR5 on oral keratinocytes. We, therefore, hypothesized that P. gingivalis modulates the outcome of HIV infection in oral epithelial cells. Results Oral and tonsil epithelial cells were pre-incubated with P. gingivalis, and inoculated with either an X4- or R5-type HIV-1. Between 6 and 48 hours post-inoculation, P. gingivalis selectively increased the infectivity of R5-tropic HIV-1 from oral and tonsil keratinocytes; infectivity of X4-tropic HIV-1 remained unchanged. Oral keratinocytes appeared to harbor infectious HIV-1, with no evidence of productive infection. HIV-1 was harbored at highest levels during the first 6 hours after HIV exposure and decreased to barely detectable levels at 48 hours. HIV did not appear to co-localize with P. gingivalis, which increased selective R5-tropic HIV-1 trans infection from keratinocytes to permissive cells. When CCR5 was selectively blocked, HIV-1 trans infection was reduced. Conclusion P. gingivalis up-regulation of CCR5 increases trans infection of harbored R5-tropic HIV-1 from oral keratinocytes to permissive cells. Oral infections such as periodontitis may, therefore, increase risk for oral infection and dissemination of R5-tropic HIV-1. PMID:18371227

  1. Rotation is the primary motion of paired human epidermal keratinocytes.

    PubMed

    Tate, Sota; Imai, Matome; Matsushita, Natsuki; Nishimura, Emi K; Higashiyama, Shigeki; Nanba, Daisuke

    2015-09-01

    Collective motion of keratinocytes is involved in morphogenesis, homeostasis, and wound healing of the epidermis. Yet how the collective motion of keratinocytes emerges from the behavior of individual cells is still largely unknown. The aim of this study was to find the cellular behavior that links single and collective motion of keratinocytes. We investigated the behavior of two-cell colonies of HaCaT keratinocytes by a combination of time-lapse imaging and image processing. The two-cell colonies of HaCaT cells were formed as a contacted pair of keratinocyte clones. Image analysis and cell culture experiments revealed that the rotational speed of two-cell colonies was positively associated with their proliferative capacity. α6 integrin was required for the rotational motion of two-cell keratinocyte colonies. We also confirmed that two-cell colonies of keratinocytes predominantly exhibited the rotational, but not translational, motion, two modes of motion in a contact pair of rotating objects. The rotational motion is the primary motion of two-cell keratinocyte colonies and its speed is positively associated with their proliferative capacity. This study suggests that the assembly of rotating keratinocytes generates the collective motion of proliferative keratinocytes during morphogenesis and wound healing of the epidermis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  2. Muscarinic acetylcholine receptors regulating cell cycle progression are expressed in human gingival keratinocytes.

    PubMed

    Arredondo, J; Hall, L L; Ndoye, A; Chernyavsky, A I; Jolkovsky, D L; Grando, S A

    2003-02-01

    We have previously reported the presence in human gingival keratinocytes (GKC) of choline acetyltransferase, the acetylcholine (ACh) synthesizing enzyme, acetylcholinesterase, the ACh degrading enzyme, and alpha 3, alpha 5, alpha 7, beta 2 as well as alpha 9 nicotinic ACh receptor subunits. To expand the knowledge about the role of ACh in oral biology, we investigated the presence of the muscarinic ACh receptor (mAChR) subtypes in GKC. RT-PCR demonstrated the presence of m2, m3, m4, and m5 mRNA transcripts. Synthesis of the respective proteins was verified by immunoblotting with the subtype-specific antibodies that revealed receptor bands at the expected molecular weights. The antibodies mapped mAChR subtypes in the epithelium of human attached gingiva and also visualized them on the cell membrane of cultured GKC. The whole cell radioligand binding assay revealed that GKC have specific binding sites for the muscarinic ligand [3H]quinuclidinyl benzilate, Bmax = 222.9 fmol/106 cells with a Kd of 62.95 pM. The downstream coupling of the mAChRs to regulation of cell cycle progression in GKC was studied using quantitative RT-PCR and immunoblotting assays. Incubation of GKC for 24 h with 10 micro m muscarine increased relative amounts of Ki-67, PCNA and p53 mRNAs and PCNA, cyclin D1, p21 and p53 proteins. These effects were abolished in the presence of 50 micro m atropine. The finding in GKC of mAChRs coupled to regulation of the cell cycle progression demonstrate further the structure/function of the non-neuronal cholinergic system operating in human oral epithelium. The results obtained in this study help clarify the role for keratinocyte ACh axis in the physiologic control of oral gingival homeostasis.

  3. Co-culture of healthy human keratinocytes and T-cells promotes keratinocyte chemokine production and RORγt-positive IL-17 producing T-cell populations.

    PubMed

    Peters, Jorieke H; Tjabringa, Geuranne S; Fasse, Esther; de Oliveira, Vivian L; Schalkwijk, Joost; Koenen, Hans J P M; Joosten, Irma

    2013-01-01

    Both keratinocytes and T-cells are crucial players in cutaneous immune responses. We hypothesized that direct interactions between keratinocytes and T-cell subsets could shape the nature or strength of the local immune response. We investigated direct interactions between keratinocytes and T-cell subsets, focused on keratinocyte chemokine production and T-cell phenotype and cytokine production. A newly developed in vitro serum free co-culture model using primary keratinocytes and T-cells subsets from healthy human donors was used. Keratinocyte chemokine production was analyzed with luminex, T-cell phenotype and cytokine production were analyzed with flow cytometry. Our data show that upon co-culture with CD4(pos) or CD8(pos) T-cells primary human keratinocytes increased production of functionally active chemokines CCL2, CCL20 and CXCL10 and that regulatory T-cells did not regulate keratinocyte chemokine production. Next to that, we found that keratinocytes skewed CD4(pos) and CD8(pos) T-cell populations toward an IL-17(pos) CCR6(pos) RORγt(pos) phenotype in a cell-cell contact independent manner, and that Treg were able to decrease the absolute number of IL-17 producing T-cells in keratinocyte/T-cell co-cultures. Correspondingly, freshly isolated skin-derived T-cell populations contained relatively high percentages of IL-17(pos) cells. We provide evidence that keratinocyte/T-cell communication may regulate leukocyte influx in the skin, and that keratinocytes enrich T-cell populations for Th17/Tc17 cells. Accumulation of Th17/Tc17 cells, but not chemokine production, appears under the control of regulatory T-cells. Dysregulation of these processes may well contribute to the pathophysiology of inflammatory skin diseases. Copyright © 2012. Published by Elsevier Ireland Ltd.

  4. Effects of ozone in normal human epidermal keratinocytes.

    PubMed

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

    2013-05-01

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

  5. 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

  6. Serum of patients with oral pemphigus vulgaris impairs keratinocyte wound repair in vitro: a time-lapse study on the efficacy of methylprednisolone and pyridostigmine bromide.

    PubMed

    Lanza, A; Stellavato, A; Heulfe, I; Landi, C; Gombos, F; Cirillo, N

    2009-10-01

    Pemphigus vulgaris (PV) is an autoimmune blistering disease affecting primarily oral mucosa and skin. Among the drugs used for the therapy of pemphigus, both methylprednisolone (MP) and pyridostigmine bromide (PBr) can prevent acantholysis in vitro. However, their putative therapeutic properties in regenerating PV-like lesions and promoting the healing process still remain to be demonstrated. To address this issue, here we have developed a model for studying the process of epithelial cleft regeneration in PV by artificially wounding keratinocyte monolayers. The experimental model was established by scratching confluent monolayers to simulate the epithelial cleft; then, wound regeneration in the presence of submaximal concentrations of PV sera was studied by time-lapse microscopy, with or without the addition of MP and PBr in the culture medium. Pemphigus vulgaris serum inhibited epithelial cleft repair of wounded monolayers. Indeed, in the presence of 10% (v/v) PV serum, keratinocytes reached only 2% confluence within 72 h vs an almost complete healing of controls. When administered together with PV sera, MP significantly (P < 0.01) enhanced wound fill by 30% after 72 h. PV-associated wound repair was significantly (P < 0.05) ameliorated by PBr by 24 h and keratinocytes reached 20% confluence after 72 h. Interestingly, neither MP nor PBr could accelerate wound healing when compared with untreated control monolayers. In PV, MP and PBr exert their curative effects in part by enhancing the regeneration properties of keratinocytes. Indeed, our data suggest that both drugs can specifically counterbalance the detrimental effects of PV serum on keratinocyte wound healing. These findings provide an explanation for the efficacy of MP and PBr in the treatment of PV lesions in human skin and oral mucosa.

  7. 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

  8. P-cadherin controls the differentiation of oral keratinocytes by regulating cytokeratin 1/10 expression via C/EBP-beta-mediated signaling.

    PubMed

    Bauer, Karin; Gosau, Martin; Bosserhoff, Anja; Reichert, Torsten; Bauer, Richard

    2012-12-01

    P-cadherin belongs to the family of Ca(2+)-dependent homophilic glycosylated cell adhesion molecules. In the normal oral epithelium it shows a strong expression in the basal cell layer which gradually decreases in the suprabasal cell layers. The exact role of P-cadherin during the development and homeostasis of the oral epithelium has not been elucidated, yet. Here, we show for the first time that P-cadherin controls differentiation by regulating cytokeratin (CK) 1/10 expression in primary oral keratinocytes (POK) from normal, but interestingly not in POKs from oral squamous cell carcinoma (OSCC) tissue. SiRNA knockdown of P-cadherin in normal POKs revealed a strong upregulation of CK1/10 expression on mRNA and protein level. In contrast, E-cadherin knockdown in normal oral keratinocytes did not show any influence on CK1/10 expression. Moreover, in comparison with normal control keratinocytes normal oral keratinocytes with reduced P-cadherin expression displayed an enhanced expression and a stronger nuclear staining of C/EBP-beta, a well-known regulator of CK1/10 expression in keratinocytes. Furthermore, after P-cadherin knockdown in normal POKs the promoter activity of a C/EBP-responsive luciferase construct was significantly higher than in normal POKs with regular P-cadherin expression. Additionally, we noticed a proliferation advantage in normal oral keratinocytes in contrast to keratinocytes with diminished P-cadherin expression. However, the inverted effect was seen in tumor derived primary oral keratinocytes. In summary, we show that P-cadherin contributes to the keratinocyte differentiation in the oral epithelium by influencing the CK1 and CK10 expression via C/EBP-beta-mediated signaling in normal but not in tumor derived oral keratinocytes from OSCC patients.

  9. Ultraviolet radiation alters choline phospholipid metabolism in human keratinocytes

    SciTech Connect

    DeLeo, V.; Scheide, S.; Meshulam, J.; Hanson, D.; Cardullo, A.

    1988-10-01

    Ultraviolet radiation B (UVB-290-320 nm) induces inflammation and hyperproliferation in human epidermis. This response is associated with the recovery from irradiated skin of inflammatory mediators derived from membrane phospholipids. We have previously reported that UVB stimulates the production of such mediators by human keratinocytes (HK) in culture. In these studies we examined the effect of UVB on the metabolism of choline containing phospholipids in HK prelabeled with (/sup 3/H) choline. UVB (400-1600J/m2) stimulated a dose dependent release of (/sup 3/H) choline from HK within minutes of irradiation. Examination of media extracts by paper chromatography revealed that the released (/sup 3/H) choline was predominately in the form of glycerophosphorylcholine. Examination of label remaining in membranes of cells after irradiation by acid precipitation and HPLC revealed that the origin of the released (/sup 3/H) choline was the membrane phosphatidylcholine/lysophosphatidylcholine. These data support a concept of UVB stimulation of both a phospholipase A (1 or 2) and a lysophospholipase. These UVB induced alterations of HK membrane phospholipid metabolism likely have profound effects on UVB-induced inflammation and control of cell growth in human skin.

  10. Increase developmental plasticity of human keratinocytes with gene suppression

    PubMed Central

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

    2011-01-01

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

  11. Asymmetric Migration of Human Keratinocytes under Mechanical Stretch and Cocultured Fibroblasts in a Wound Repair Model

    PubMed Central

    Lü, Dongyuan; Liu, Xiaofeng; Gao, Yuxin; Huo, Bo; Kang, Yingyong; Chen, Juan; Sun, Shujin; Chen, Li; Luo, Xiangdong; Long, Mian

    2013-01-01

    Keratinocyte migration during re-epithelization is crucial in wound healing under biochemical and biomechanical microenvironment. However, little is known about the underlying mechanisms whereby mechanical tension and cocultured fibroblasts or keratinocytes modulate the migration of keratinocytes or fibroblasts. Here we applied a tensile device together with a modified transwell assay to determine the lateral and transmembrane migration dynamics of human HaCaT keratinocytes or HF fibroblasts. A novel pattern of asymmetric migration was observed for keratinocytes when they were cocultured with non-contact fibroblasts, i.e., the accumulative distance of HaCaT cells was significantly higher when moving away from HF cells or migrating from down to up cross the membrane than that when moving close to HF cells or when migrating from up to down, whereas HF migration was symmetric. This asymmetric migration was mainly regulated by EGF derived from fibroblasts, but not transforming growth factor α or β1 production. Mechanical stretch subjected to fibroblasts fostered keratinocyte asymmetric migration by increasing EGF secretion, while no role of mechanical stretch was found for EGF secretion by keratinocytes. These results provided a new insight into understanding the regulating mechanisms of two- or three-dimensional migration of keratinocytes or fibroblasts along or across dermis and epidermis under biomechanical microenvironment. PMID:24086354

  12. Development of a Full-Thickness Human Gingiva Equivalent Constructed from Immortalized Keratinocytes and Fibroblasts

    PubMed Central

    Buskermolen, Jeroen K.; Reijnders, Christianne M.A.; Spiekstra, Sander W.; Steinberg, Thorsten; Kleverlaan, Cornelis J.; Feilzer, Albert J.; Bakker, Astrid D.

    2016-01-01

    Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro. For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines would solve these problems. The aim of this study was to develop fully differentiated human gingiva equivalents (GE) constructed entirely from cell lines, to compare them with the primary cell counterpart (Prim), and to test relevance in an in vitro wound healing assay. Reconstructed gingiva epithelium on a gingiva fibroblast-populated collagen hydrogel was constructed from cell lines (keratinocytes: TERT or HPV immortalized; fibroblasts: TERT immortalized) and compared to GE-Prim and native gingiva. GE were characterized by immunohistochemical staining for proliferation (Ki67), epithelial differentiation (K10, K13), and basement membrane (collagen type IV and laminin 5). To test functionality of GE-TERT, full-thickness wounds were introduced. Reepithelialization, fibroblast repopulation of hydrogel, metabolic activity (MTT assay), and (pro-)inflammatory cytokine release (enzyme-linked immunosorbent assay) were assessed during wound closure over 7 days. Significant differences in basal KC cytokine secretion (IL-1α, IL-18, and CXCL8) were only observed between KC-Prim and KC-HPV. When Fib-Prim and Fib-TERT were stimulated with TNF-α, no differences were observed regarding cytokine secretion (IL-6, CXCL8, and CCL2). GE-TERT histology, keratin, and basement membrane protein expression very closely represented native gingiva and GE-Prim. In contrast, the epithelium of GE made with HPV-immortalized KC was disorganized, showing suprabasal proliferating cells, limited keratinocyte differentiation, and the absence of basement membrane proteins. When a wound was introduced into the more physiologically relevant GE-TERT model, an immediate inflammatory response (IL-6, CCL2, and

  13. Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes

    PubMed Central

    Utheim, Tor Paaske; Islam, Rakibul; Fostad, Ida G.; Eidet, Jon R.; Sehic, Amer; Olstad, Ole K.; Dartt, Darlene A.; Messelt, Edward B.; Griffith, May; Pasovic, Lara

    2016-01-01

    Purpose Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed. Materials and Methods Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR. Results Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C. Conclusion HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell

  14. Interactions of aluminum nanoparticles with human epidermal keratinocytes.

    PubMed

    Monteiro-Riviere, Nancy A; Oldenburg, Steven J; Inman, Alfred O

    2010-04-01

    Aluminum nanoparticles (Al NP) have been used in applications as diverse as drug delivery, material surface coatings and an ingredient for solid rocket fuel in military explosives and artillery. Although Al NP are used in many civilian and military applications, the health and safety implications of these nanosize particles are not known. To understand the interactions and biological activity of Al NP in human cells, cultured human neonatal epidermal keratinocytes (HEK) were exposed for 24 h to 50 and 80 nm Al NP in concentrations from 4.0 to 0.0004 mg ml(-1) to assess the cytotoxicity and inflammatory potential. UV-Vis measurements and nanoparticle controls revealed that the Al NP interact with the assay dyes. Viability did not decrease in HEK exposed to both the 50 and the 80 nm Al NP at all treatment concentrations with MTT, CellTiter 96 AQueous One (96 AQ) and alamar Blue (aB) viability assays. The 96 AQ and aB assays interact with the Al NP less than MTT, and proved to be the best assays to use with these Al NP. TEM depicted Al NP localized within the cytoplasmic vacuoles of the cells. Cytokine data was variable, indicating possible nanoparticle interactions with the cytokine assays. These studies illustrate the difficulties involved in assessing the biological safety of nanomaterials such as Al NP due to media- and temperature-dependent particle agglomeration and nanoparticle interactions with biomarkers of cytotoxicity. Copyright (c) 2009 John Wiley & Sons, Ltd.

  15. 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

  16. 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.

  17. The effect of neurotensin in human keratinocytes--implication on impaired wound healing in diabetes.

    PubMed

    Moura, Liane I F; Cruz, Maria T; Carvalho, Eugénia

    2014-01-01

    Diabetic foot ulcers are an important complication of diabetes mellitus characterized by chronic, non-healing ulcers resulting from poor proliferation and migration of fibroblasts and keratinocytes, thus impairing a correct re-epithelialization of wounded tissues. This healing process can be modulated by neuropeptides released from peripheral nerves; however, little is known regarding the role of neurotensin (NT) as a modulator of human keratinocyte function under hyperglycemic conditions. Therefore, this work is focused on the effect of NT in human keratinocytes, under normal and hyperglycemic conditions at different functional levels, namely NT receptors, cytokine, and growth factor expression, as well as proliferation and migration. Human keratinocyte cells were maintained at either 10/30 mM glucose and treated with or without NT (10 nM). The results show that NT did not affect keratinocyte viability. In addition, NT and all NT receptor expression levels were significantly reduced by hyperglycemia; however, NT treatment stimulated expression of NT and neurotensin receptor 2 (NTR2) while neurotensin receptor 1 (NTR1) and neurotensin receptor 3 (NTR3) expression levels were unchanged. Keratinocyte proliferation was not affected by NT and hyperglycemia, while cell migration was reduced by NT treatment. These results demonstrated that hyperglycemic conditions strongly impaired endogenous NT and NTR2 expression in keratinocytes. Despite the addition of exogenous NT to stimulate the endogenous NT and NTR2 expression, these changes do not translate into functional modifications on keratinocytes, particularly in terms of migration, proliferation, and production of cytokines or growth factors. These results suggest that NT production by keratinocytes may exert a paracrine effect on other skin cells, namely fibroblasts, macrophages, and dendritic cells for correct wound healing.

  18. Upregulation of FOXM1 induces genomic instability in human epidermal keratinocytes

    PubMed Central

    2010-01-01

    Background The human cell cycle transcription factor FOXM1 is known to play a key role in regulating timely mitotic progression and accurate chromosomal segregation during cell division. Deregulation of FOXM1 has been linked to a majority of human cancers. We previously showed that FOXM1 was upregulated in basal cell carcinoma and recently reported that upregulation of FOXM1 precedes malignancy in a number of solid human cancer types including oral, oesophagus, lung, breast, kidney, bladder and uterus. This indicates that upregulation of FOXM1 may be an early molecular signal required for aberrant cell cycle and cancer initiation. Results The present study investigated the putative early mechanism of UVB and FOXM1 in skin cancer initiation. We have demonstrated that UVB dose-dependently increased FOXM1 protein levels through protein stabilisation and accumulation rather than de novo mRNA expression in human epidermal keratinocytes. FOXM1 upregulation in primary human keratinocytes triggered pro-apoptotic/DNA-damage checkpoint response genes such as p21, p38 MAPK, p53 and PARP, however, without causing significant cell cycle arrest or cell death. Using a high-resolution Affymetrix genome-wide single nucleotide polymorphism (SNP) mapping technique, we provided the evidence that FOXM1 upregulation in epidermal keratinocytes is sufficient to induce genomic instability, in the form of loss of heterozygosity (LOH) and copy number variations (CNV). FOXM1-induced genomic instability was significantly enhanced and accumulated with increasing cell passage and this instability was increased even further upon exposure to UVB resulting in whole chromosomal gain (7p21.3-7q36.3) and segmental LOH (6q25.1-6q25.3). Conclusion We hypothesise that prolonged and repeated UVB exposure selects for skin cells bearing stable FOXM1 protein causes aberrant cell cycle checkpoint thereby allowing ectopic cell cycle entry and subsequent genomic instability. The aberrant upregulation of FOXM1

  19. 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

  20. 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.

  1. Niacin restriction upregulates NADPH oxidase and ROS in human keratinocytes

    PubMed Central

    Benavente, Claudia A.; Jacobson, Elaine L.

    2008-01-01

    NAD+ is a substrate for many enzymes, including poly(ADP-ribose) polymerases and sirtuins, which are involved in fundamental cellular processes including DNA repair, stress responses, signaling, transcription, apoptosis, metabolism, differentiation, chromatin structure, and life span. Because these molecular processes are important early in cancer development, we developed a model to identify critical NAD-dependent pathways potentially important in early skin carcinogenesis. Removal of niacin from the cell culture medium allowed control of intracellular NAD. Unlike many non-immortalized human cells, HaCaT keratinocytes, which are immortalized and have a mutant p53 and aberrant NF-kB activity, become severely NAD depleted but divide indefinitely under these conditions. Niacin deficient HaCaTs develop a decreased growth rate due to an increase in apoptotic cells and an arrest in the G2/M phase of the cell cycle. Long- term survival mechanisms in niacin deficient HaCats involve accumulation of reactive oxygen species and increased DNA damage. These alterations result, at least in part, from increased expression and activity of NADPH oxidase, whose downstream effects can be reversed by nicotinamide or NADPH oxidase inhibitors. Our data support the hypothesis that glutamine is a likely alternative energy source during niacin deficiency and we suggest a model for NADPH generation important in ROS production. PMID:17997992

  2. Areca nut exposure increases secretion of tumor-promoting cytokines in gingival fibroblasts that trigger DNA damage in oral keratinocytes.

    PubMed

    Illeperuma, Rasika P; Kim, Do Kyeong; Park, Young Jin; Son, Hwa Kyung; Kim, Jue Young; Kim, Jinmi; Lee, Doo Young; Kim, Ki-Yeol; Jung, Da-Woon; Tilakaratne, Wanninayake M; Kim, Jin

    2015-12-01

    Molecular crosstalk between cancer cells and fibroblasts has been an emerging hot issue in understanding carcinogenesis. As oral submucous fibrosis (OSF) is an inflammatory fibrotic disease that can potentially transform into squamous cell carcinoma, OSF has been considered to be an appropriate model for studying the role of fibroblasts during early stage carcinogenesis. In this sense, this study aims at investigating whether areca nut (AN)-exposed fibroblasts cause DNA damage of epithelial cells. For this study, immortalized hNOF (hTERT-hNOF) was used. We found that the levels of GRO-α, IL-6 and IL-8 increased in AN-exposed fibroblasts. Cytokine secretion was reduced by antioxidants in AN-exposed fibroblasts. Increase in DNA double strand breaks (DSB) and 8-oxoG FITC-conjugate was observed in immortalized human oral keratinocytes (IHOK) after the treatment of cytokines or a conditioned medium derived from AN-exposed fibroblasts. Cytokine expression and DNA damage were also detected in OSF tissues. The DNA damage was reduced by neutralizing cytokines or antioxidant treatment. Generation of reactive oxygen species (ROS) and DNA damage response, triggered by cytokines, were abolished when NADPH oxidase (NOX) 1 and 4 were silenced in IHOK, indicating that cytokine-triggered DNA damage was caused by ROS generation through NOX1 and NOX4. Taken together, this study provided strong evidence that blocking ROS generation might be a rewarding approach for cancer prevention and intervention in OSF.

  3. Areca nut exposure increases secretion of tumor‐promoting cytokines in gingival fibroblasts that trigger DNA damage in oral keratinocytes

    PubMed Central

    Illeperuma, Rasika P.; Kim, Do Kyeong; Park, Young Jin; Son, Hwa Kyung; Kim, Jue Young; Kim, Jinmi; Lee, Doo Young; Kim, Ki‐Yeol; Jung, Da‐Woon; Tilakaratne, Wanninayake M.

    2015-01-01

    Molecular crosstalk between cancer cells and fibroblasts has been an emerging hot issue in understanding carcinogenesis. As oral submucous fibrosis (OSF) is an inflammatory fibrotic disease that can potentially transform into squamous cell carcinoma, OSF has been considered to be an appropriate model for studying the role of fibroblasts during early stage carcinogenesis. In this sense, this study aims at investigating whether areca nut (AN)‐exposed fibroblasts cause DNA damage of epithelial cells. For this study, immortalized hNOF (hTERT‐hNOF) was used. We found that the levels of GRO‐α, IL‐6 and IL‐8 increased in AN‐exposed fibroblasts. Cytokine secretion was reduced by antioxidants in AN‐exposed fibroblasts. Increase in DNA double strand breaks (DSB) and 8‐oxoG FITC‐conjugate was observed in immortalized human oral keratinocytes (IHOK) after the treatment of cytokines or a conditioned medium derived from AN‐exposed fibroblasts. Cytokine expression and DNA damage were also detected in OSF tissues. The DNA damage was reduced by neutralizing cytokines or antioxidant treatment. Generation of reactive oxygen species (ROS) and DNA damage response, triggered by cytokines, were abolished when NADPH oxidase (NOX) 1 and 4 were silenced in IHOK, indicating that cytokine‐triggered DNA damage was caused by ROS generation through NOX1 and NOX4. Taken together, this study provided strong evidence that blocking ROS generation might be a rewarding approach for cancer prevention and intervention in OSF. PMID:26076896

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. Effect of tumour-cell-derived or recombinant keratinocyte growth factor (KGF) on proliferation and radioresponse of human epithelial tumour cells (HNSCC) and normal keratinocytes in vitro.

    PubMed

    Hille, Andrea; Grüger, Susanne; Christiansen, Hans; Wolff, Hendrik A; Volkmer, Beate; Lehmann, Jörg; Dörr, Wolfgang; Rave-Fränk, Margret

    2010-05-01

    Purpose of this work was to test the effect of tumour-cell-derived keratinocyte growth factor (KGF) or recombinant KGF (palifermin) on cell proliferation and radiation response of human HNSCC cells and normal keratinocytes in vitro. Four tumour cell cultures derived from head and neck squamous cell carcinomas, primary keratinocytes, and immortalized keratinocytes were analysed. Fibroblasts, the natural source of KGF protein, served as controls. KGF expression was observed in primary and immortalized keratinocytes, fibroblasts, and in tumour cells, while significant KGF receptor expression was only found in keratinocytes. Recombinant KGF as well as tumour-cell-derived KGF caused a significant growth stimulation and radioprotection in keratinocytes, which was abolished by a neutralizing anti-KGF antibody. This indicates that tumour-cell-derived KGF is biologically active. In the tumour cell lines, no significant growth stimulation was induced by recombinant KGF, and the neutralizing antibody did not influence tumour cell growth or radiation response. Our results indicate that the normal, paracrine KGF regulatory mechanisms, which are based on KGF receptor expression, are lost in malignant cells, with the consequence of irresponsiveness of the tumour cells to exogenous KGF. In face of the amelioration of the radiation response of normal epithelia, demonstrated in various clinical and various preclinical animal studies, recombinant KGF represents a candidate for the selective protection of normal epithelia during radio(chemo) therapy of squamous cell carcinoma.

  11. 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.

  12. 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.

  13. Breast cancer resistance protein identifies clonogenic keratinocytes in human interfollicular epidermis.

    PubMed

    Ma, Dongrui; Chua, Alvin Wen Choong; Yang, Ennan; Teo, Peiyun; Ting, Yixin; Song, Colin; Lane, Ellen Birgitte; Lee, Seng Teik

    2015-03-24

    There is a practical need for the identification of robust cell-surface markers that can be used to enrich for living keratinocyte progenitor cells. Breast cancer resistance protein (ABCG2), a member of the ATP binding cassette (ABC) transporter family, is known to be a marker for stem/progenitor cells in many tissues and organs. We investigated the expression of ABCG2 protein in normal human epidermis to evaluate its potential as a cell surface marker for identifying and enriching for clonogenic epidermal keratinocytes outside the pilosebaceous tract. Immunofluorescence and immunoblotting studies of human skin showed that ABCG2 is expressed in a subset of basal layer cells in the epidermis. Flow cytometry analysis showed approximately 2-3% of keratinocytes in non-hair-bearing epidermis expressing ABCG2; this population also expresses p63, β1 and α6 integrins and keratin 14, but not CD34, CD71, C-kit or involucrin. The ABCG2-positive keratinocytes showed significantly higher colony forming efficiency when co-cultured with mouse 3T3 feeder cells, and more extensive long-term proliferation capacity in vitro, than did ABCG2-negative keratinocytes. Upon clonal analysis, most of the freshly isolated ABCG2-positive keratinocytes formed holoclones and were capable of generating a stratified differentiating epidermis in organotypic culture models. These data indicate that in skin, expression of the ABCG2 transporter is a characteristic of interfollicular keratinocyte progentior cells and suggest that ABCG2 may be useful for enriching keratinocyte stem cells in human interfollicular epidermis.

  14. 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.

  15. Recombinant human epidermal growth factor accelerates the proliferation of irradiated human fibroblasts and keratinocytes in vitro and in vivo.

    PubMed

    Ryu, Seung-Hee; Moon, Soo Young; Yang, Youn-Joo; Moon, Sun Rock; Hong, Joon Pio; Choi, Jene; Lee, Sang-Wook

    2009-11-01

    Irradiation causes the impaired proliferation of cells lining mucosal membranes. Epidermal growth factor (EGF) facilitates proliferation of various skin cells; however, the wound healing effects of EGF on radiation-damaged cells is less well known. To evaluate the effects of recombinant human EGF (rhEGF) on the proliferation of cells following irradiation, we tested two types of fibroblast cell lines and one keratinocyte cell line. The viable cell numbers were significantly increased by rhEGF treatment for 24 h immediately after 8 Gy of irradiation. The most effective dose of rhEGF was 10 nM in all cell lines used in this study. The percentage of BrdU-labeled cells was also significantly increased by rhEGF treatment. To evaluate the effects of rhEGF on radiation-induced oral mucosal damage in BALB/c mice, we systematically injected 1 mg/kg/day EGF for three days after 17 Gy of irradiation. Administered rhEGF ameliorated radiation-induced mucosal damage in vivo. rhEGF significantly increased the epithelial cell layer thickness and the proliferation of basal layer cells as detected by Ki-67 staining. Our results suggest that rhEGF can be a therapeutic treatment for radiation-induced wounds by stimulating the proliferation of fibroblasts and keratinocytes following irradiation.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. Locomotion of Human Skin Keratinocytes on Polystyrene, Fibrin, and Collagen Substrata and its Modification by Cell-to-Cell Contacts.

    PubMed

    Drukala, Justyna; Bandura, Laura; Cieślik, Kazimierz; Korohoda, Włodzimierz

    2001-11-01

    Epithelial wound repair assures the recovery of the epithelial barrier after wounding. During wound healing epithelial cells migrate to cover the wound surface. The presented experiments were carried out to compare the migration of human keratinocytes from primary and secondary culture on polystyrene, collagen, and fibrin glue used in clinical techniques. The images of migrating keratinocytes were recorded and analyzed using computer-aided methods. The results show that the character of the substrate strongly affects the speed and turning behavior of keratinocytes locomoting over it. The highest motile activity of human skin keratinocytes was found on fibrin glue substratum. It was found that locomotion of freely moving isolated cells was much faster than that of cell sheets. The autologous keratinocytes cultured in vitro were applied with fibrin glue to cover trophic wounds. The transplantation of human autologous keratinocyte suspension in fibrin glue upon long-lasting trophic wounds appeared to induce rapid and permanent wound healing.

  2. 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.

  3. 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.

  4. 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.

  5. Directed differentiation of human embryonic stem cells into keratinocyte progenitors in vitro: an attempt with promise of clinical use.

    PubMed

    Li, Hanqing; Zhou, Haiwen; Fu, Xin; Xiao, Ran

    2016-09-01

    Human embryonic stem cells (hESCs) can differentiate into all somatic lineages including stratified squamous epithelia. Thus, efficient methods are required to direct hESC differentiation to obtain a pure subpopulation for tissue engineering. The study aimed to assess the effects of retinoic acid (RA), bone morphogenetic protein-4 (BMP4), and ascorbic acid (AA) on the differentiation of hESCs into keratinocyte progenitors in vitro. The first media contained AA and BMP4; the second contained RA, AA, and BMP4; the third was commercial-defined keratinocyte serum-free medium, which was used to differentiate H9 hESCs (direct approach) or embryoid bodies (EBs) (indirect approach) into keratinocyte progenitors. Real-time RT-PCR, immunofluorescence, and flow-cytometry were used to characterize the differentiated cells. Cells induced by AA + BMP4 + RA showed the typical epithelial morphology, while cells induced by AA + BMP4 showed multiple appearances. CK14 and p63 messenger RNA (mRNA) expressions in the AA + BMP4 + RA-treated cells were higher than those of the AA + BMP4-treated cells (CK14: 22.4-fold; p63: 84.7-fold). Epithelial marker CK18 mRNA expressions at 14 d of differentiation and keratinocyte marker CK14 and transcription factor p63 mRNA expressions at 35 d of differentiation were higher in cells differentiated from hESCs compared with those differentiated from EBs (CK18 10.51 ± 3.26 vs. 6.67 ± 1.28; CK14 9.27 ± 3.61 vs. 5.32 ± 1.86; p63 0.73 ± 0.06 vs. 0.44 ± 0.12, all P < 0.05) After hESC induction by AA+BMP4+RA, CK14 mRNA expression was upregulated after day 21, peaking by 35 d of differentiation. Combined RA, BMP4, and AA could effectively induce differentiation of hESCs into keratinocyte progenitors in vitro. These keratinocytes could be used for oral mucosa and skin tissue engineering.

  6. 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.

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

    PubMed

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

    2016-07-08

    Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H(+) gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake. Copyright © 2016 DHC corporation. Published by Elsevier Inc. All rights reserved.

  8. Sunscreen penetration of human skin and related keratinocyte toxicity after topical application.

    PubMed

    Hayden, C G J; Cross, S E; Anderson, C; Saunders, N A; Roberts, M S

    2005-01-01

    Sunscreen skin penetration and safety assessment should be considered together in order to ensure that in vitro cytotoxicity studies examine relevant doses of these organic chemical UV filters to which viable epidermal cells are realistically exposed. In this study, we sought to determine whether sufficient topically applied sunscreens penetrated into human viable epidermis to put the local keratinocyte cell populations at risk of toxicity. The penetration and retention of five commonly used sunscreen agents (avobenzone, octinoxate, octocrylene, oxybenzone and padimate O) in human skin was evaluated after application in mineral oil to isolated human epidermal membranes. Sunscreen concentration-human keratinocyte culture response curves were then defined using changes in cell morphology and proliferation (DNA synthesis using radiolabelled thymidine uptake studies) as evidence of sunscreens causing toxicity. Following 24 h of human epidermal exposure to sunscreens, detectable amounts of all sunscreens were present in the stratum corneum and viable epidermis, with epidermal penetration most evident with oxybenzone. The concentrations of each sunscreen found in human viable epidermis after topical application, adjusting for skin partitioning and binding effects, were at least 5-fold lower, based on levels detected in viable epidermal cells, than those appearing to cause toxicity in cultured human keratinocytes. It is concluded that the human viable epidermal levels of sunscreens are too low to cause any significant toxicity to the underlying human keratinocytes. Copyright (c) 2005 S. Karger AG, Basel.

  9. 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

  10. 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.

  11. Keratinocyte growth factor and the expression of wound-healing-related genes in primary human keratinocytes from burn patients.

    PubMed

    Chomiski, Verônica; Gragnani, Alfredo; Bonucci, Jéssica; Correa, Silvana Aparecida Alves; Noronha, Samuel Marcos Ribeiro de; Ferreira, Lydia Masako

    2016-08-01

    To evaluate the effect of keratinocyte growth factor (KGF) treatment on the expression of wound-healing-related genes in cultured keratinocytes from burn patients. Keratinocytes were cultured and divided into 4 groups (n=4 in each group): TKB (KGF-treated keratinocytes from burn patients), UKB (untreated keratinocytes from burn patients), TKC (KGF-treated keratinocytes from controls), and UKC (untreated keratinocytes from controls). Gene expression analysis using quantitative polymerase chain reaction (qPCR) array was performed to compare (1) TKC versus UKC, (2) UKB versus UKC, (3) TKB versus UKC, (4) TKB versus UKB, (5) TKB versus TKC, and (6) UKB versus TKC. Comparison 1 showed one down-regulated and one up-regulated gene; comparisons 2 and 3 resulted in the same five down-regulated genes; comparison 4 had no significant difference in relative gene expression; comparison 5 showed 26 down-regulated and 7 up-regulated genes; and comparison 6 showed 25 down-regulated and 11 up-regulated genes. There was no differential expression of wound-healing-related genes in cultured primary keratinocytes from burn patients treated with keratinocyte growth factor.

  12. A review of the influence of growth factors and cytokines in in vitro human keratinocyte migration.

    PubMed

    Peplow, Philip V; Chatterjee, Marissa P

    2013-04-01

    Keratinocyte migration from the wound edge is a crucial step in the reepithelization of cutaneous wounds. Growth factors and cytokines, released from cells that invade the wound matrix, play an important role, and several in vitro assays have been performed to elucidate this. The purposes of this study were to review in vitro human studies on keratinocyte migration to identify those growth factors or cytokines that stimulate keratinocyte migration and whether these assays might serve as a screening procedure prior to testing combinations of growth factors or cytokines to promote wound closure in vivo. Research papers investigating effect of growth factors and cytokines on human keratinocyte migration in vitro were retrieved from library sources, PubMed databases, reference lists of papers, and searches of relevant journals. Fourteen different growth factors and cytokines enhanced migration in scratch wound assay and HGF together with TGF-β, and IGF-1 with EGF, were more stimulatory than either growth factor alone. HGF with TGF-β1 had a greater chemokinetic effect than either growth factor alone in transmigration assay. TGF-β1, FGF-7, FGF-2 and AGF were chemotactic to keratinocytes. EGF, TGF-α, IL-1α, IGF and MGSA enhanced cell migration on ECM proteins. Many growth factors and cytokines enhanced migration of keratinocytes in vitro, and certain combinations of growth factors were more stimulatory than either alone. These and other combinations that stimulate keratinocyte migration in vitro should be tested for effect on wound closure and repair in vivo. The scratch wound assay provides a useful, inexpensive and easy-to-perform screening method for testing individual or combinations of growth factors or cytokines, or growth factors combined with other modalities such as laser irradiation, prior to performing wound healing studies with laboratory animals. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. Influence of CAD/CAM zirconia for implant-abutment manufacturing on gingival fibroblasts and oral keratinocytes.

    PubMed

    Pabst, A M; Walter, C; Bell, A; Weyhrauch, M; Schmidtmann, I; Scheller, H; Lehmann, K M

    2016-06-01

    This study investigated the influence of three CAD/CAM zirconia ceramics for implant-abutment manufacturing on cell viability, migration ability, and cytotoxicity of human gingival fibroblasts (HGF) and oral keratinocytes (HOK) in vitro. HGF and HOK were cultured on zirconia ceramic disks (VITA In-Ceram YZ, Ivoclar IPS e.max ZirCAD, Sirona inCoris ZI) and on control disks made of tissue culture polystyrene. Cell viability was analyzed by a MTT assay. Migration ability was detected by a scratch assay. A ToxiLight assay was used to analyze the influence of the tested zirconia ceramics on adenylate kinase (ADK) release and cytotoxicity. At MTT assay, HGF showed an increased cell viability compared to the control after 9 and 12 days for all ceramics (p each ≤0.0002) while HOK demonstrated a decreased cell viability after 9 and 12 days for all ceramics (p each ≤0.0003). At scratch assay, HGF exhibited for all ceramics decreased relative distances of the scratch wound compared to the control from 24 to 48 h (p each <0.0001) with exception of VITA In-Ceram YZ after 48 h. HOK showed increased distances compared to the control for all ceramics after 48 h (p each <0.0001). At ToxiLight assay, a minimal cytotoxicity of the tested materials could be detected. Overall, significant influences of the investigated CAD/CAM zirconia ceramics on HGF and HOK could be shown. The analyzed zirconia ceramics could influence oral soft-tissue cells that might affect the esthetic outcome after implant placement using CAD/CAM zirconia abutments.

  19. 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.

  20. 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

  1. Insulin binding properties of normal and transformed human epidermal cultured keratinocytes

    SciTech Connect

    Verrando, P.; Ortonne, J.P.

    1985-10-01

    Insulin binding to its receptors was studied in cultured normal and transformed (A431 line) human epidermal keratinocytes. The specific binding was a temperature-dependent, saturable process. Normal keratinocytes possess a mean value of about 80,000 receptors per cell. Fifteen hours exposure of the cells to insulin lowered their receptor number (about 65% loss in available sites); these reappeared when the hormone was removed from the culture medium. In the A431 epidermoid carcinoma cell line, there is a net decrease in insulin binding (84% of the initial bound/free hormone ratio in comparison with normal cells) essentially related to a loss in receptor affinity for insulin. Thus, cultured human keratinocytes which express insulin receptors may be a useful tool in understanding skin pathology related to insulin disorders.

  2. Low-energy helium-neon laser irradiation increases the motility of cultured human keratinocytes

    SciTech Connect

    Haas, A.F.; Isseroff, R.R.; Wheeland, R.G.; Rood, P.A.; Graves, P.J. )

    1990-06-01

    Helium-neon (HeNe) laser irradiation is known to stimulate wound healing. We investigated whether the biostimulatory effects of HeNe irradiation result from enhancement of keratinocyte proliferation or motility. HeNe effects on keratinocyte motility were evaluated by irradiating a wounded culture with 0.8 J/cm2 3 times over a 20-h period. At 20 h post-irradiation, videocinemicroscopy and sequential quantitative measurements of the leading edge were taken over a 6-h period. There was a significant difference in migration of the leading edge in irradiated wounds compared to non-irradiated wounded controls (12.0 microns/h vs 4.0 microns/h, p less than 0.0001). To determine if the increase in migration observed in irradiated cultures resulted from a proliferative effect of HeNe irradiation, subconfluent human keratinocyte cultures were irradiated with single or multiple doses of different fluences of HeNe irradiation (0.4 to 7.2 J/cm2) and evaluated 72 h post-irradiation. Irradiated and non-irradiated keratinocyte cultures grown on a microporous membrane surface were co-cultured with irradiated and non-irradiated fibroblasts to determine if HeNe irradiation induced a paracrine effect on keratinocyte proliferation. No significant increase in keratinocyte proliferation was demonstrated in any of these treatments. The biostimulatory effects of HeNe irradiation may now be extended to include enhancement of keratinocyte motility in vitro; this may contribute to the efficacy of HeNe irradiation in wound healing.

  3. Exogenous hydrogen sulfide promotes cell proliferation and differentiation by modulating autophagy in human keratinocytes

    SciTech Connect

    Xie, Xin; Dai, Hui; Zhuang, Binyu; Chai, Li; Xie, Yanguang; Li, Yuzhen

    2016-04-08

    The effects and the underlying mechanisms of hydrogen sulfide (H{sub 2}S) on keratinocyte proliferation and differentiation are still less known. In the current study, we investigated the effects and the underlying mechanisms of exogenous H{sub 2}S on keratinocyte proliferation and differentiation. Human keratinocytes (HaCaT cells) were treated with various concentrations (0.05, 0.25, 0.5 and 1 mM) of sodium hydrosulfide (NaHS, a donor of H{sub 2}S) for 24 h. A CCK-8 assay was used to assess cell viability. Western blot analysis was performed to determine the expression levels of proteins associated with differentiation and autophagy. Transmission electron microscopy was performed to observe autophagic vacuoles, and flow cytometry was applied to evaluate apoptosis. NaHS promoted the viability, induced the differentiation, and enhanced autophagic activity in a dose-dependent manner in HaCaT cells but had no effect on cell apoptosis. Blockage of autophagy by ATG5 siRNA inhibited NaHS-induced cell proliferation and differentiation. The current study demonstrated that autophagy in response to exogenous H{sub 2}S treatment promoted keratinocyte proliferation and differentiation. Our results provide additional insights into the potential role of autophagy in keratinocyte proliferation and differentiation. - Highlights: • Exogenous H{sub 2}S promotes keratinocyte proliferation and differentiation. • The effects of H{sub 2}S on proliferation and differentiation is modulated by autophagy. • Exogenous H{sub 2}S has no effect on keratinocyte apoptosis.

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

    SciTech Connect

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

    2016-07-08

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

  5. The antimycotic drugs itraconazole and terbinafine hydrochloride induce the production of human β-defensin-3 in human keratinocytes.

    PubMed

    Kanda, Naoko; Kano, Rui; Ishikawa, Takeko; Watanabe, Shinichi

    2011-04-01

    The antimicrobial peptide human β-defensin-3 (hBD-3) is produced by epidermal keratinocytes, and exhibits broad killing activity against bacteria or fungi. Prostaglandin D(2) enhances hBD-3 production in human keratinocytes by stimulating a transcription factor, activator protein-1 via chemoattractant receptor-homologous molecule expressed on T helper 2 cells (CRTH2). Prostaglandin H(2), a precursor of prostaglandin D(2) can be converted to thromboxane A(2). Certain antimycotic drugs act on keratinocytes and modulate their production of chemokines. In this in vitro study, we examined the effects of antimycotics on hBD-3 production in human keratinocytes. Antimycotics itraconazole and terbinafine hydrochloride increased hBD-3 secretion and mRNA levels in parallel to the enhanced activity of activator protein-1, expression and phosphorylation of activator protein-1 component, c-Fos, but fluconazole was ineffective. These effects were abrogated by CRTH2 antagonist. Itraconazole and terbinafine hydrochloride increased prostaglandin D(2) release from keratinocytes and reduced the release of thromboxane B(2), a thromboxane A(2) metabolite. The conditioned medium from itraconazole or terbinafine hydrochloride-treated keratinocytes inhibited the growth of Candida albicans dependently on hBD-3. These results suggest that itraconazole and terbinafine hydrochloride may enhance c-Fos expression and phosphorylation, activator protein-1 activity and hBD-3 production by increasing prostaglandin D(2) release from keratinocytes. These antimycotic drugs may suppress thromboxane A(2) synthesis and redirect the conversion of prostaglandin H(2) towards prostaglandin D(2). The induction of hBD-3 in keratinocytes is another possible mechanism for the antimycrobial effects of these drugs, which may augment the cutaneous defense activity against infection.

  6. 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.

  7. 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.

  8. Induction of proteins and mRNAs after uv irradiation of human epidermal keratinocytes

    SciTech Connect

    Kartasova, T.; Ponec, M.; van de Putte, P.

    1988-02-01

    uv sensitivity of cultured human epidermal keratinocytes was analyzed at different growth conditions and compared with the sensitivity of dermal fibroblasts derived from the same skin specimen. No significant differences in survival curves were found between these two cell types, although keratinocytes grown under standard conditions were slightly more resistant to uv irradiation than fibroblasts. The extracellular concentration of calcium appeared to be critical not only in the regulation of keratinocyte proliferation and differentiation, but also in the uv sensitivity of these cells: keratinocytes grown under conditions which favor cell proliferation (low calcium concentration) are more resistant to uv irradiation than those grown under conditions favoring differentiation (high calcium concentration). Two-dimensional protein gel electrophoresis was used to detect a possible effect of uv irradiation on the accumulation of specific mRNAs in the cytoplasm and/or on the synthesis of specific proteins. Proteins were pulse labeled in vivo with (/sup 35/S)methionine or synthesized in vitro in rabbit reticulocyte lysates on mRNA isolated from keratinocytes that were irradiated with different uv doses at different periods of time prior to isolation. Alterations in expression were demonstrated for several proteins in both in vivo and in vitro experiments.

  9. 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

  10. Exogenous hydrogen sulfide promotes cell proliferation and differentiation by modulating autophagy in human keratinocytes.

    PubMed

    Xie, Xin; Dai, Hui; Zhuang, Binyu; Chai, Li; Xie, Yanguang; Li, Yuzhen

    2016-04-08

    The effects and the underlying mechanisms of hydrogen sulfide (H2S) on keratinocyte proliferation and differentiation are still less known. In the current study, we investigated the effects and the underlying mechanisms of exogenous H2S on keratinocyte proliferation and differentiation. Human keratinocytes (HaCaT cells) were treated with various concentrations (0.05, 0.25, 0.5 and 1 mM) of sodium hydrosulfide (NaHS, a donor of H2S) for 24 h. A CCK-8 assay was used to assess cell viability. Western blot analysis was performed to determine the expression levels of proteins associated with differentiation and autophagy. Transmission electron microscopy was performed to observe autophagic vacuoles, and flow cytometry was applied to evaluate apoptosis. NaHS promoted the viability, induced the differentiation, and enhanced autophagic activity in a dose-dependent manner in HaCaT cells but had no effect on cell apoptosis. Blockage of autophagy by ATG5 siRNA inhibited NaHS-induced cell proliferation and differentiation. The current study demonstrated that autophagy in response to exogenous H2S treatment promoted keratinocyte proliferation and differentiation. Our results provide additional insights into the potential role of autophagy in keratinocyte proliferation and differentiation. Copyright © 2016. Published by Elsevier Inc.

  11. 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.

  12. 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.

  13. 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.

  14. 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...

  15. 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...

  16. Antileukoprotease in human skin: an antibiotic peptide constitutively produced by keratinocytes.

    PubMed

    Wiedow, O; Harder, J; Bartels, J; Streit, V; Christophers, E

    1998-07-30

    Antileukoprotease (ALP), also known as mucous protease inhibitor or secretory leukoprotease inhibitor, resembles one of the major antiproteases present in human body fluids. It is capable of preventing proteolytic degradation of extracellular matrix proteins by neutrophil-derived serine proteases. ALP was isolated from human callus and detected in supernatants of cultured human primary keratinocytes. ALP mRNA was constitutively expressed in keratinocytes and the expression was not significantly affected by TNF alpha or Interferon gamma stimulation. In microbicidal assays recombinant ALP exhibited antimicrobial activity against several human skin associated microorganisms like P. aeruginosa, S. aureus, S. epidermidis, and C. albicans, indicating that ALP may actively participate in mechanisms allowing homeostasis of bacterial and yeast colonization on human skin. Thus, ALP represents a major soluble serine protease inhibitor and antimicrobial agent expressed in human skin and seems to contribute to the high resistance of the epidermis against proteolysis and infections.

  17. 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.

  18. Nicotinamide enhances repair of ultraviolet radiation-induced DNA damage in human keratinocytes and ex vivo skin.

    PubMed

    Surjana, Devita; Halliday, Gary M; Damian, Diona L

    2013-05-01

    Nicotinamide (vitamin B3) protects from ultraviolet (UV) radiation-induced carcinogenesis in mice and from UV-induced immunosuppression in mice and humans. Recent double-blinded randomized controlled Phase 2 studies in heavily sun-damaged individuals have shown that oral nicotinamide significantly reduces premalignant actinic keratoses, and may reduce new non-melanoma skin cancers. Nicotinamide is a precursor of nicotinamide adenine dinucleotide (NAD(+)), an essential coenzyme in adenosine triphosphate (ATP) production. Previously, we showed that nicotinamide prevents UV-induced ATP decline in HaCaT keratinocytes. Energy-dependent DNA repair is a key determinant of cellular survival after exposure to DNA-damaging agents such as UV radiation. Hence, in this study we investigated whether nicotinamide protection from cellular energy loss influences DNA repair. We treated HaCaT keratinocytes with nicotinamide and exposed them to low-dose solar-simulated UV (ssUV). Excision repair was quantified using an assay of unscheduled DNA synthesis. Nicotinamide increased both the proportion of cells undergoing excision repair and the repair rate in each cell. We then investigated ssUV-induced cyclobutane pyrimidine dimers (CPDs) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxoG) formation and repair by comet assay in keratinocytes and with immunohistochemistry in human skin. Nicotinamide reduced CPDs and 8oxoG in both models and the reduction appeared to be due to enhancement of DNA repair. These results show that nicotinamide enhances two different pathways for repair of UV-induced photolesions, supporting nicotinamide's potential as an inexpensive, convenient and non-toxic agent for skin cancer chemoprevention.

  19. 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.

  20. 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.

  1. Formaldehyde solutions in simulated sweat increase human melanoma but not normal human keratinocyte cells proliferation.

    PubMed

    Rizzi, M; Cravello, B; Tonello, S; Renò, F

    2016-12-01

    Our skin is in close contact with clothes most of the time thus risking potentially noxious chemicals contact. One of the potentially harmful manufacturing by-products that can be released by textiles when sweating is formaldehyde, used as an anti-crease treatment. As it is known to be carcinogenic to humans and a potent skin sensitizer, the aim of this study was to investigate its effects on both normal human keratinocytes (HaCaT cells) and on a highly invasive malignant melanoma cell line (SK-MEL-28) in order to contribute to the definition of safety cut-off to be applied to the production processes. Formaldehyde concentrations below the commonly accepted limits (10-50μM) were obtained by diluting formaldehyde in simulated sweat (UNI EN ISO 105-E04). The effects on cell proliferation were evaluated by cell counting, while ERK pathway activation was evaluated by western blot. Low concentrations of formaldehyde (10μM) in both acidic and alkaline simulated sweat were able to increase malignant melanoma cell proliferation, while not affecting normal keratinocytes. Melanoma proliferation increase was greater in acidic (pH=5.5) than in alkaline (pH=8) conditions. Moreover, formaldehyde stimulation was able to induce ERK pathway activation. The data obtained suggest the need for an even increasing attention to the potentially harmful effects of textile manufacturing by-products. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. 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.

  3. Omega-3 polyunsaturated fatty acids selectively inhibit growth in neoplastic oral keratinocytes by differentially activating ERK1/2

    PubMed Central

    Parkinson, Eric Kenneth

    2013-01-01

    The long-chain omega-3 polyunsaturated fatty acids (n-3 PUFAs)—eicosapentaenoic acid (EPA) and its metabolite docosahexaenoic acid (DHA)—inhibit cancer formation in vivo, but their mechanism of action is unclear. Extracellular signal-regulated kinase 1/2 (ERK1/2) activation and inhibition have both been associated with the induction of tumour cell apoptosis by n-3 PUFAs. We show here that low doses of EPA, in particular, inhibited the growth of premalignant and malignant keratinocytes more than the growth of normal counterparts by a combination of cell cycle arrest and apoptosis. The growth inhibition of the oral squamous cell carcinoma (SCC) lines, but not normal keratinocytes, by both n-3 PUFAs was associated with epidermal growth factor receptor (EGFR) autophosphorylation, a sustained phosphorylation of ERK1/2 and its downstream target p90RSK but not with phosphorylation of the PI3 kinase target Akt. Inhibition of EGFR with either the EGFR kinase inhibitor AG1478 or an EGFR-blocking antibody inhibited ERK1/2 phosphorylation, and the blocking antibody partially antagonized growth inhibition by EPA but not by DHA. DHA generated more reactive oxygen species and activated more c-jun N-terminal kinase than EPA, potentially explaining its increased toxicity to normal keratinocytes. Our results show that, in part, EPA specifically inhibits SCC growth and development by creating a sustained signalling imbalance to amplify the EGFR/ERK/p90RSK pathway in neoplastic keratinocytes to a supraoptimal level, supporting the chemopreventive potential of EPA, whose toxicity to normal cells might be reduced further by blocking its metabolism to DHA. Furthermore, ERK1/2 phosphorylation may have potential as a biomarker of n-3 PUFA function in vivo. PMID:23892603

  4. 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.

  5. 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.

  6. Role of human papillomavirus in oral squamous cell carcinoma and oral potentially malignant disorders: A review of the literature

    PubMed Central

    Gupta, Shikha; Gupta, Sunita

    2015-01-01

    Human papillomaviruses (HPVs) are epitheliotropic viruses with an affinity for keratinocytes and are principally found in the anogenital tract, urethra, skin, larynx, tracheobronchial and oral mucosa. On the basis of high, but variable frequency of HPV in oral squamous cell carcinoma (OSCC), malignant potential of HPV infection has been hypothesized but not definitely confirmed. The aim of this review was to highlight the genomic structure and possible mechanism of infection and carcinogenesis by HPV in the oral mucosa and to review the frequency of HPV prevalence in OSCC and oral potentially malignant disorders. A computer database search was performed through the use of PubMed from 1994 to 2014. Search keywords used were: HPV and oral cancer, HPV and oral leukoplakia, HPV and oral lichen planus, HPV and OSCC, HPV and verrucous carcinoma, HPV and proliferative verrucous leukoplakia, HPV and oral papilloma. PMID:26097339

  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. © 2013 Elsevier Inc. All rights reserved.

  8. Upregulation of genes orchestrating keratinocyte differentiation, including the novel marker gene ID2, by contact sensitizers in human bulge-derived keratinocytes.

    PubMed

    Yoshikawa, Yoshie; Sasahara, Yusuke; Kitano, Yukio; Kanazawa, Nozomi; Shima, Hiroki; Hashimoto-Tamaoki, Tomoko

    2010-01-01

    In the epidermis, keratinocytes are involved in physical and first-line immune protection of the host. In this study, we analyzed the molecular responses to certain contact sensitizers (2,4-dinitrochlorobenzene and NiSO(4)) and irritants (sodium dodecyl sulfate and benzalkonium chloride) in cultured human keratinocytes from the bulge region of a plucked hair follicle (bulge-derived keratinocytes [BDKs]) and compared these molecular responses to those with the human monocytic leukemia cell line, THP-1. The BDKs, individually established without invasive biopsies, showed high reactivity to these stimulants. As a primary response to the contact sensitizers, the NRF2-mediated signaling pathway was upregulated in BDKs and THP-1. The expression of IL1B and IL8 genes was not induced by the irritants but by the sensitizers in THP-1. However, the expression of the IL1B and IL8 genes was induced at higher levels by the irritants in BDKs than by the sensitizers. Many genes orchestrating keratinocyte differentiation, including ID2, were significantly upregulated in response to the sensitizers in BDKs but not those in THP-1. The use of the ID2 gene to discriminate between sensitizers and irritants might be effective as a novel marker for application during in vitro sensitization with BDKs.

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

    PubMed

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

    2014-12-01

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

  10. 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

  11. 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.

  12. SET/I2PP2A overexpression induces phenotypic, molecular, and metabolic alterations in an oral keratinocyte cell line.

    PubMed

    Sobral, Lays M; Coletta, Ricardo D; Alberici, Luciane C; Curti, Carlos; Leopoldino, Andréia M

    2017-09-01

    The multifunctional SET/I2PP2A protein is known to be overexpressed in head and neck squamous cell carcinoma. However, SET has been reported to have apparently conflicting roles in promoting cancer cell survival under oxidative stress conditions and preventing invasion and metastasis, complicating efforts to understand the contribution of SET to carcinogenesis. In the present study, we overexpressed SETin a spontaneously immortalized oral keratinocyte cell line (NOK-SI SET) and demonstrated that SET upregulation alone was sufficient to transform cells. In comparison with NOK-SI cells, NOK-SI SET cells demonstrated increased levels of phosphorylated Akt, c-Myc and inactive/phosphorylated Rb, together with decreased total Rb protein levels. In addition, NOK-SI SET cells presented the following: (a) a spindle-cell shape morphology compared with the polygonal morphology of NOK-SI cells; (b) loss of mesenchymal stem cell markers CD44 and CD73, and epithelial cell markers CD71 and integrin α6/β4; (c) the ability to form xenograft tumors in nude mice; and (d) increased mitochondrial respiration accompanied by decreased ROSlevels. Overall, our results show that SEToverexpression promotes morphological and oncogenic cell transformation of an oral keratinocyte cell. © 2017 Federation of European Biochemical Societies.

  13. Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor

    SciTech Connect

    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. - Highlights: • Pro-inflammatory cytokines induced VEGF production in normal human

  14. 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

  15. Infection of human keratinocytes by Streptococcus dysgalactiae subspecies dysgalactiae isolated from milk of the bovine udder.

    PubMed

    Roma-Rodrigues, Catarina; Alves-Barroco, Cynthia; Raposo, Luís R; Costa, Mafalda N; Fortunato, Elvira; Baptista, Pedro Viana; Fernandes, Alexandra R; Santos-Sanches, Ilda

    2016-04-01

    Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) are considered exclusive animal pathogens; however, a putative zoonotic upper limb cellulitis, a prosthetic joint infection and an infective endocarditis were described in humans. To unravel if bovine SDSD isolates are able to infect human cells, the adherence and internalization to human primary keratinocytes of two bovine SDSD strains isolated from milk collected from udder were analyzed. Bacterial adhesion assays and confocal microscopy indicate a high adherence and internalization of SDSD isolates to human cells, suggesting for the first time the ability of bovine isolates to infect human cells.

  16. Effects of extracellular calcium on the growth-differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes.

    PubMed

    Micallef, Ludovic; Belaubre, Françoise; Pinon, Aline; Jayat-Vignoles, Chantal; Delage, Christiane; Charveron, Marie; Simon, Alain

    2009-02-01

    The keratinocyte growth and differentiation switch, tightly regulated by several mechanisms, is generally associated with decreased proliferation, cell cycle arrest in G0/G1 phase and expression of epidermal differentiation markers, such as keratin 1 (K1), keratin 10 (K10) and involucrin. In vitro, the spontaneously immortalized human keratinocyte cell line HaCaT is often used as a model to study keratinocyte functions. Comparative differentiation studies between HaCaT cells and normal human keratinocytes (NHK) over an extended time-period have rarely been reported. Therefore, we studied their switch from a proliferating to a differentiated state over 13 days. As culture conditions involved changes in cellular responses, cells were cultured in a specific medium for keratinocyte growth and differentiation was induced by increasing extracellular calcium concentration from 0.09 to 1.2 mm. In NHK, addition of calcium-induced morphological changes and concomitant decreased proliferation. For HaCaT cells, calcium addition resulted in morphological changes, but in an unexpected manner, cells were more proliferative than when cultured at low calcium levels. HaCaT cell hyperproliferation correlated with cell cycle analysis, showing an accumulation in S/G2-M phases. Furthermore, RT-PCR and western blot analysis revealed a delay in the expression of the differentiation markers K1, K10 and involucrin in HaCaT cells compared with NHK. In conclusion, even though calcium-induced differentiation was not associated with a decreased cell proliferation, HaCaT cells conserved properties characteristic of differentiation.

  17. 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.

  18. 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

  19. 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

  20. Extremely low frequency electromagnetic field enhances human keratinocyte cell growth and decreases proinflammatory chemokine production.

    PubMed

    Vianale, G; Reale, M; Amerio, P; Stefanachi, M; Di Luzio, S; Muraro, R

    2008-06-01

    Proliferation and differentiation of keratinocytes are central processes in tissue regeneration after injury. Chemokines, produced by a wide range of cell types including keratinocytes, play a regulatory role in inflammatory skin diseases. Several studies have shown that an electromagnetic field (EMF) can influence both inflammatory processes and repair mechanisms including wound healing on different tissue models. To elucidate the effect of extremely low frequency EMF (ELF-EMF) on keratinocyte proliferation and production of chemokines [RANTES, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1 alpha and interleukin (IL)-8] in order to evaluate a potential therapeutic use of magnetic fields. The human keratinocyte cell line HaCaT was exposed at 1 mT, 50 Hz for different lengths of time and compared with unexposed control cells. Cell growth and viability were evaluated at different exposure times by cell count and trypan blue exclusion. Chemokine production and expression were analysed by enzyme-linked immunosorbent assay (ELISA) and by real-time polymerase chain reaction. Total NF-kappaB p65 was quantified by ELISA. Significantly increased growth rates were observed after 48 h of EMF exposure as compared with control cells, while no difference in cell viabilities were detected. Gene expression and release of RANTES, MCP-1, MIP-1 alpha and IL-8 were significantly reduced after 72 h of exposure. NF-kappaB levels became almost undetectable after only 1 h of EMF exposure, and were inversely correlated with cell density. Our results show that ELF-EMF modulates chemokine production and keratinocyte growth through inhibition of the NF-kappaB signalling pathway and thus may inhibit inflammatory processes. ELF-EMF could represent an additional therapeutic approach in the treatment of skin injury.

  1. Activation of TRPV4 strengthens the tight-junction barrier in human epidermal keratinocytes.

    PubMed

    Akazawa, Y; Yuki, T; Yoshida, H; Sugiyama, Y; Inoue, S

    2013-01-01

    The transient receptor potential cation channel, subfamily V (TRPV), is expressed in the epidermis and considered to be a sensor of extrinsic stimuli such as temperature and other physical or chemical factors. In this study, we examined whether or not the activation of TRPVs by their agonists alters the epidermal tight junction (TJ) function in cultured human epidermal keratinocytes. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that mRNA for TRPV1, 3 and 4 were expressed in differentiated keratinocytes in which TJs had formed. Stimulation of the keratinocytes with a TRPV4 agonist (4α-phorbol 12, 13-didecanoate, 4α-PDD) strengthened the TJ-associated barrier, analyzed by means of transepithelial electric resistance measurements and flux measurements of the paracellular tracer. Stimulation with TRPV1 and TRPV3 agonists did not have the same result. Simultaneously, the 4α-PDD-stimulated keratinocytes showed an upregulation of TJ structural proteins, occludin and claudin-4, and TJ regulatory factors, phospho-atypical PKCζ/ι. It was also observed that the amounts of occludin and phospho-atypical PKCζ/ι complex were higher in 4α-PDD stimulated keratinocytes. In conclusion, we demonstrated that the activation of TRPV4 strengthened the TJ-associated barrier of epidermal cells. It was also suggested that the upregulation of TJ structural proteins and/or the posttranslational modification of TJ structural proteins by phospho-atypical PKCζ/ι are responsible for the enhancement of TJ function. Our study supports the hypothesis that TJs change their function in response to a change in the external environment sensed through TRPVs. Copyright © 2012 S. Karger AG, Basel.

  2. 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.

  3. COP1 contributes to UVB-induced signaling in human keratinocytes.

    PubMed

    Kinyó, Agnes; Kiss-László, Zsuzsanna; Hambalkó, Szabolcs; Bebes, Attila; Kiss, Mária; Széll, Márta; Bata-Csörgo, Zsuzsanna; Nagy, Ferenc; Kemény, Lajos

    2010-02-01

    UVB irradiation has been shown to trigger a broad range of changes in gene expression in human skin; however, factors governing these events are still not well understood. In this study, we show that human constitutive photomorphogenic protein-1 (huCOP1), an E3 ligase, contributes to the orchestration of UVB response of keratinocytes. Accordingly, our data show that (i) huCOP1 protein is expressed both in the nucleus and in the cytoplasm of cultured keratinocytes, (ii) UVB reduces the levels of the huCOP1 mRNA and protein, and (iii) induces changes in the subcellular localization of huCOP1. Finally, we show that gene-specific silencing of huCOP1 induces the accumulation of the tumor suppressor p53 protein, which is further increased after UVB irradiation.

  4. Photoprotective Effect of Carpomitra costata Extract against Ultraviolet B-Induced Oxidative Damage in Human Keratinocytes.

    PubMed

    Zheng, J; Hewage, S R K Madduma; Piao, Mei Jing; Kang, Kyoung Ah; Han, X; Kang, H K; Yoo, E S; Koh, Y S; Lee, N H; Ko, C S; Lee, J C; Ko, Mi Hee; Hyuna, Jin Won

    2016-01-01

    Natural marine products show various biological properties such as antiphotoaging, antioxidant, anticancer, and anti-inflammation. This study evaluated the protective effects of the brown alga Carpomitra costata (Stackhouse) Batters (Sporochnaceae) against ultraviolet B (UVB)-provoked damage in human HaCaT keratinocytes. C. costata extract (CCE) effectively reduced superoxide anion, hydroxyl radical, and UVB-stimulated intracellular reactive oxygen species (ROS) levels. CCE also restored the expression and activity of UVB-suppressed antioxidant enzymes. Furthermore, CCE decreased UVB-triggered oxidative damage to cellular components including DNA, protein, and lipid and defended the cells against mitochondrial membrane depolarization-medicated apoptosis. The results of this study indicate that CCE can safeguard human keratinocytes against UVB-induced cellular damage via a potent antioxidant mechanism. CCE may find utility as part of a therapeutic arsenal against the damaging effects of UVB radiation on the skin.

  5. Transforming growth factor-beta stimulates the expression of fibronectin by human keratinocytes.

    PubMed

    Wikner, N E; Persichitte, K A; Baskin, J B; Nielsen, L D; Clark, R A

    1988-09-01

    Transforming growth factor beta (TGF-beta) is a 25-kD protein which has regulatory activity over a variety of cell types. It is distinct from epidermal growth factor (EGF) and EGF analogs, and exerts its action via a distinct receptor. Its effect on proliferation or differentiation can be positive or negative depending on the cell type and the presence of other growth factors. It also modulates the expression of cellular products. TGF-beta causes fibroblasts to increase their production of the extracellular matrix components, fibronectin and collagen. Human keratinocytes (HK) are known to have TGF-beta receptors. We wished to study the effect of TGF-beta on the production of extracellular matrix proteins by human keratinocytes in culture. Human keratinocytes were grown in serum-free defined medium (MCDB-153) to about 70% confluence. Following a 16-h incubation in medium lacking EGF and TGF-beta, cells were incubated for 12 h in medium containing varying concentrations of EGF and TGF-beta. Cells were then labeled with 35S-methionine for 10 h in the same conditions. Labeled proteins from the medium were analyzed by SDS-PAGE and autoradiography. TGF-beta at 10 ng/ml induced a sixfold increase in the secretion of fibronectin, as well as an unidentified 50-kD protein. Thrombospondin production was also increased, but not over a generalized twofold increase in the production of all other proteins. EGF, at 10 ng/ml, caused a smaller additive effect. TGF-beta may be an important stimulator of extracellular matrix production by human keratinocytes.

  6. α-Fetoprotein as a modulator of the pro-inflammatory response of human keratinocytes

    PubMed Central

    Potapovich, AI; Pastore, S; Kostyuk, VA; Lulli, D; Mariani, V; De Luca, C; Dudich, EI; Korkina, LG

    2009-01-01

    Background and purpose: The immunomodulatory effects of α-fetoprotein (AFP) on lymphocytes and macrophages have been described in vitro and in vivo. Recombinant forms of human AFP have been proposed as potential therapeutic entities for the treatment of autoimmune diseases. We examined the effects of embryonic and recombinant human AFP on the spontaneous, UVA- and cytokine-induced pro-inflammatory responses of human keratinocytes. Experimental approach: Cultures of primary and immortalized human keratinocytes (HaCaT) and human blood T lymphocytes were used. The effects of AFP on cytokine expression were studied by bioplexed elisa and quantitative reverse transcriptase polymerase chain reaction assay. Kinase and nuclear factor kappa B (NFκB) phosphorylation were quantified by intracellular elisa. Nuclear activator protein 1 and NFκB DNA binding activity was measured by specific assays. Nitric oxide and H2O2 production and redox status were assessed by fluorescent probe and biochemical methods. Key results: All forms of AFP enhanced baseline expression of cytokines, chemokines and growth factors. AFP dose-dependently increased tumour necrosis factor alpha-stimulated granulocyte macrophage colony stimulating factor and interleukin 8 expression and decreased tumour necrosis factor alpha-induced monocyte chemotactic protein 1 and IP-10 (interferon gamma-produced protein of 10 kDa) expression. AFP induced a marked activator protein 1 activation in human keratinocytes. AFP also increased H2O2 and modulated nitrite/nitrate levels in non-stimulated keratinocytes whereas it did not affect these parameters or cytokine release from UVA-stimulated cells. Phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Akt1 but not NFκB was activated by AFP alone or by its combination with UVA. Conclusions and implications: Exogenous AFP induces activation of human keratinocytes, with de novo expression of a number of pro-inflammatory mediators and modulation of their

  7. Analyzing the Human Papillomavirus (HPV) Life Cycle in Primary Keratinocytes with a Quantitative Colony-Forming Assay.

    PubMed

    Lace, Michael J; Turek, Lubomir P; Anson, James R; Haugen, Thomas H

    2014-05-01

    Papillomavirus genomes replicate as extrachromosomal plasmids within infected keratinocytes, requiring the regulated expression of early viral gene products to initially amplify the viral genomes and subvert cell growth checkpoints as part of a complex path to immortalization. Building on contemporary keratinocyte transfection and culture systems, the methods described in this unit form a detailed approach to analyzing critical events in the human papillomavirus (HPV) life cycle, utilizing physiologic levels of viral gene products expressed from their native promoter(s) in the natural host cells for HPV infection. A quantitative colony-forming assay permits comparison of the capacities of various transfected HPV types and mutant HPV genomes to initially form colonies and immortalize human keratinocytes. In conjunction with additional methods, these protocols enable examination of genomic stability, viral and cellular gene expression, viral integration, and differentiation patterns influenced by HPV persistence in clonal human keratinocytes that effectively mimic early events in HPV infection. Copyright © 2014 John Wiley & Sons, Inc.

  8. Brd4 Activates Early Viral Transcription upon Human Papillomavirus 18 Infection of Primary Keratinocytes

    PubMed Central

    McKinney, Caleb C.; Kim, Min Jung; Chen, Dan

    2016-01-01

    ABSTRACT  Human papillomaviruses (HPVs) replicate in the cutaneous and mucosal epithelia, and the infectious cycle is synchronous with the differentiation program of the host keratinocytes. The virus initially infects dividing cells in the lower layers of the epithelium, where it establishes a persistent infection. The viral genome is maintained as a low-copy-number, extrachromosomal element in these proliferating cells but switches to the late stage of the life cycle in differentiated cells. The cellular chromatin adaptor protein Brd4 is involved in several stages and processes of the viral life cycle. In concert with the viral transcriptional regulator E2, Brd4 can repress transcription from the early viral promoter. Brd4 and E2 form a complex with the viral genome that associates with host chromosomes to partition the viral genome in dividing cells; Brd4 also localizes to active sites of productive HPV DNA replication. However, because of the difficulties in producing HPV viral particles, the role of Brd4 in modulating viral transcription and replication at the initial stage of infection is unclear. In this study, we have used an HPV18 quasivirus-based genome delivery system to assess the role of Brd4 in the initial infectivity of primary human keratinocytes. We show that, upon infection of primary human keratinocytes with HPV18 quasivirus, Brd4 activates viral transcription and replication. Furthermore, this activation is independent of the functional interaction between Brd4 and the HPV18 E2 protein. PMID:27879331

  9. Malignant progression of an SV40-transformed human epidermal keratinocyte cell line.

    PubMed Central

    Brown, K. W.; Gallimore, P. H.

    1987-01-01

    Human foetal keratinocytes were transfected with a recombinant plasmid (pSV6-1) which contained an origin defective SV40 genome. The resulting transformed cell line had many properties in common with previously described SV40-transformed keratinocytes, including expression of simple epithelial-type keratins. It was non-tumourigenic in nude mice at early passages, forming small benign cysts, however, after approximately 46 in vitro passages, these transformed keratinocytes formed invasive squamous cell carcinomas in athymic nude mice. Several in vitro changes were associated with this acquisition of tumourigenicity (a) an alteration in cellular morphology, (b) development of a cytogenetically marked clone and (c) loss of cell surface fibronectin. The loss of fibronectin was also observed in vivo; cysts formed by SV6-1 Bam/HFK produced human fibronectin whereas tumours did not, although both tumours and cysts were laminin- and keratin-positive. These results indicate that the spontaneous development of secondary events in immortalised human cells may lead to the acquisition of a malignant phenotype. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:2447927

  10. 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.

  11. A Sensitive Sensor Cell Line for the Detection of Oxidative Stress Responses in Cultured Human Keratinocytes

    PubMed Central

    Hofmann, Ute; Priem, Melanie; Bartzsch, Christine; Winckler, Thomas; Feller, Karl-Heinz

    2014-01-01

    In the progress of allergic and irritant contact dermatitis, chemicals that cause the generation of reactive oxygen species trigger a heat shock response in keratinocytes. In this study, an optical sensor cell line based on cultured human keratinocytes (HaCaT cells) expressing green fluorescent protein (GFP) under the control of the stress-inducible HSP70B' promoter were constructed. Exposure of HaCaT sensor cells to 25 μM cadmium, a model substance for oxidative stress induction, provoked a 1.7-fold increase in total glutathione and a ∼300-fold induction of transcript level of the gene coding for heat shock protein HSP70B'. An extract of Arnica montana flowers resulted in a strong induction of the HSP70B' gene and a pronounced decrease of total glutathione in keratinocytes. The HSP70B' promoter-based sensor cells conveniently detected cadmium-induced stress using GFP fluorescence as read-out with a limit of detection of 6 μM cadmium. In addition the sensor cells responded to exposure of cells to A. montana extract with induction of GFP fluorescence. Thus, the HaCaT sensor cells provide a means for the automated detection of the compromised redox status of keratinocytes as an early indicator of the development of human skin disorders and could be applied for the prediction of skin irritation in more complex in vitro 3D human skin models and in the development of micro-total analysis systems (μTAS) that may be utilized in dermatology, toxicology, pharmacology and drug screenings. PMID:24967604

  12. Histamine suppresses epidermal keratinocyte differentiation and impairs skin barrier function in a human skin model

    PubMed Central

    Gschwandtner, M; Mildner, M; Mlitz, V; Gruber, F; Eckhart, L; Werfel, T; Gutzmer, R; Elias, P M; Tschachler, E

    2013-01-01

    Background Defects in keratinocyte differentiation and skin barrier are important features of inflammatory skin diseases like atopic dermatitis. Mast cells and their main mediator histamine are abundant in inflamed skin and thus may contribute to disease pathogenesis. Methods Human primary keratinocytes were cultured under differentiation-promoting conditions in the presence and absence of histamine, histamine receptor agonists and antagonists. The expression of differentiation-associated genes and epidermal junction proteins was quantified by real-time PCR, Western blot, and immunofluorescence labeling. The barrier function of human skin models was tested by the application of biotin as tracer molecule. Results The addition of histamine to human keratinocyte cultures and organotypic skin models reduced the expression of the differentiation-associated proteins keratin 1/10, filaggrin, and loricrin by 80–95%. Moreover, the addition of histamine to skin models resulted in the loss of the granular layer and thinning of the epidermis and stratum corneum by 50%. The histamine receptor H1R agonist, 2-pyridylethylamine, suppressed keratinocyte differentiation to the same extent as did histamine. Correspondingly, cetirizine, an antagonist of H1R, virtually abrogated the effect of histamine. The expression of tight junction proteins zona occludens-1, occludin, claudin-1, and claudin-4, as well as that of desmosomal junction proteins corneodesmosin and desmoglein-1, was down-regulated by histamine. The tracer molecule biotin readily penetrated the tight junction barrier of skin cultures grown in the presence of histamine, while their diffusion was completely blocked in nontreated controls. Conclusions Our findings suggest a new mechanism by which mast cell activation and histamine release contribute to skin barrier defects in inflammatory skin diseases. PMID:23157658

  13. A sensitive sensor cell line for the detection of oxidative stress responses in cultured human keratinocytes.

    PubMed

    Hofmann, Ute; Priem, Melanie; Bartzsch, Christine; Winckler, Thomas; Feller, Karl-Heinz

    2014-06-25

    In the progress of allergic and irritant contact dermatitis, chemicals that cause the generation of reactive oxygen species trigger a heat shock response in keratinocytes. In this study, an optical sensor cell line based on cultured human keratinocytes (HaCaT cells) expressing green fluorescent protein (GFP) under the control of the stress-inducible HSP70B' promoter were constructed. Exposure of HaCaT sensor cells to 25 µM cadmium, a model substance for oxidative stress induction, provoked a 1.7-fold increase in total glutathione and a ~300-fold induction of transcript level of the gene coding for heat shock protein HSP70B'. An extract of Arnica montana flowers resulted in a strong induction of the HSP70B' gene and a pronounced decrease of total glutathione in keratinocytes. The HSP70B' promoter-based sensor cells conveniently detected cadmium-induced stress using GFP fluorescence as read-out with a limit of detection of 6 µM cadmium. In addition the sensor cells responded to exposure of cells to A. montana extract with induction of GFP fluorescence. Thus, the HaCaT sensor cells provide a means for the automated detection of the compromised redox status of keratinocytes as an early indicator of the development of human skin disorders and could be applied for the prediction of skin irritation in more complex in vitro 3D human skin models and in the development of micro-total analysis systems (µTAS) that may be utilized in dermatology, toxicology, pharmacology and drug screenings.

  14. 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.

  15. Impact of AQP3 inducer treatment on cultured human keratinocytes, ex vivo human skin and volunteers.

    PubMed

    Garcia, N; Gondran, C; Menon, G; Mur, L; Oberto, G; Guerif, Y; Dal Farra, C; Domloge, N

    2011-10-01

    One of the main functions of the skin is to protect the organism against environmental threats, such as thermal stress. Aquaporin-3 (AQP3) facilitates water and glycerol transport across cell membranes and therefore regulates osmotic balance in different situations of stress. This mechanism seems to be particularly important for the resistance of different organisms to cold stress. Consequently, we were interested in investigating the effect of cold and osmotic stress on AQP3 expression in normal human keratinocytes. We developed a new active ingredient to stimulate aquaporins in skin and demonstrated the partial restoration of AQP3 expression in keratinocytes transfected with AQP3 siRNA. Moreover, we examined the effect of cold stress on cell morphology and the impact of a pre-treatment with the active ingredient. Our results indicated that induction of AQP3 helped maintain a correct organization of the actin cytoskeleton, preserving cell morphology and preventing cells from rounding. Immunofluorescent staining revealed cytoplasmic localization of AQP3 and its translocation to the cell membrane following osmotic stress. Histological ex vivo studies of skin under different conditions, such as cold environment and tape-stripping, indicated that increase in AQP3 expression appears to be involved in skin protection and showed that the pattern of AQP3 expression was more enhanced in the active ingredient-treated samples. In vivo confocal microscopy by Vivascope showed a generally healthier appearance of the skin in the treated areas. These results attest to the potential value of the active ingredient in optimizing environmental stress resistance and protecting the skin from stratum corneum damage.

  16. Ultraviolet radiation induces changes in membrane metabolism of human keratinocytes in culture

    SciTech Connect

    De Leo, V.A.; Horlick, H.; Hanson, D.; Eisinger, M.; Harber, L.C.

    1984-11-01

    Human keratinocytes in culture were prelabeled with (/sup 3/H)arachidonic acid (AA) and then exposed to ultraviolet B radiation. Irradiated cells released labeled AA metabolites into media in a dose-dependent manner when compared to sham-irradiated cells. The response began immediately and continued for 24 h. Extracts from media were examined by high-performance liquid chromatography for identification of specific AA metabolites. Irradiated cells were stimulated to produce prostaglandin-like material (PGE2 and PGF2 alpha). These findings support the concept that the cell membrane of keratinocytes participates directly or indirectly in initiating the sunburn response. It is also felt that the metabolites formed following injury to the membrane are an integral component in the mediation of that response.

  17. EPA attenuates ultraviolet radiation-induced downregulation of aquaporin-3 in human keratinocytes.

    PubMed

    Jeon, Byoung-Kook; Kang, Moon-Kyung; Lee, Ghang-Tai; Lee, Kun-Kuk; Lee, Ho-Sub; Woo, Won-Hong; Mun, Yeun-Ja

    2015-08-01

    Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid (ω-3 PUFA) that protects against photodamage and photocarcinogenesis in mammals. Aquaporin-3 (AQP3) is a water/glycerol transport protein that is found in basal layer keratinocytes. In this study, we have investigated the protective effect of EPA against ultraviolet B (UVB)-induced AQP3 downregulation in human keratinocytes. EPA treatment was found to increase AQP3 gene and protein expression in human epidermal keratinocytes (HaCaT). Using a specific inhibitor, we observed that the effect of EPA on AQP3 expression was mediated by extracellular signal-regulated kinase (ERK) activation. UVB radiation induced AQP3 downregulation in HaCaT cells, and it was found that EPA treatment attenuated UVB-induced AQP3 reduction and the associated cell death. UVB-induced downregulation of AQP3 was blocked by EPA and p38 inhibitor SB203580. Collectively, the present results show that EPA increased AQP3 expression and that this led to a reduction UVB-induced photodamage.

  18. Novel protein in human epidermal keratinocytes: regulation of expression during differentiation

    SciTech Connect

    Kartasova, T.; van Muijen, G.N.; van Pelt-Heerschap, H.; van de Putte, P.

    1988-05-01

    Recently, two groups of cDNA clones have been isolated from human epidermal keratinocytes; the clones correspond to genes whose expression is stimulated by exposure of the cells to UV light or treatment with 4-nitroquinoline 1-oxide or 12-O-tetradecanoylphorbol 13-acetate. The proteins predicted by the nucleotide sequence of both groups of cDNAs are small (8 to 10 kilodaltons), are exceptionally rich in proline, glutamine, and cysteine, and contain repeating elements with a common sequence, PK PEPC. These proteins were designated sprI and sprII (small, proline rich). Here we describe the characterization of the sprIa protein, which is encoded by one of the group 1 cDNAs. The expression of this protein during keratinocyte differentiation in vitro and the distribution of the sprIa protein in some human tissues was studied by using a specific rabbit antiserum directed against a synthetic polypeptide corresponding to the 30 amino acids of the C-terminal region of the sprIa gene product. The results indicate that the expression of the sprIa protein is stimulated during keratinocyte differentiation both in vitro and in vivo.

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

    PubMed

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

    2012-11-01

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

  20. The Herbal Bitter Drug Gentiana lutea Modulates Lipid Synthesis in Human Keratinocytes In Vitro and In Vivo

    PubMed Central

    Haarhaus, Birgit; Seiwerth, Jasmin; Cawelius, Anja; Schwabe, Kay; Quirin, Karl-Werner; Schempp, Christoph M.

    2017-01-01

    Gentiana lutea is a herbal bitter drug that is used to enhance gastrointestinal motility and secretion. Recently we have shown that amarogentin, a characteristic bitter compound of Gentiana lutea extract (GE), binds to the bitter taste receptors TAS2R1 and TAS2R38 in human keratinocytes, and stimulates the synthesis of epidermal barrier proteins. Here, we wondered if GE also modulates lipid synthesis in human keratinocytes. To address this issue, human primary keratinocytes were incubated for 6 days with GE. Nile Red labeling revealed that GE significantly increased lipid synthesis in keratinocytes. Similarly, gas chromatography with flame ionization detector indicated that GE increases the amount of triglycerides in keratinocytes. GE induced the expression of epidermal ceramide synthase 3, but not sphingomyelinase. Lipid synthesis, as well as ceramide synthase 3 expression, could be specifically blocked by inhibitors of the p38 MAPK and PPARγ signaling pathway. To assess if GE also modulates lipid synthesis in vivo, we performed a proof of concept half side comparison on the volar forearms of 33 volunteers. In comparison to placebo, GE significantly increased the lipid content of the treated skin areas, as measured with a sebumeter. Thus, GE enhances lipid synthesis in human keratinocytes that is essential for building an intact epidermal barrier. Therefore, GE might be used to improve skin disorders with an impaired epidermal barrier, e.g., very dry skin and atopic eczema. PMID:28829355

  1. A synthetic sandalwood odorant induces wound-healing processes in human keratinocytes via the olfactory receptor OR2AT4.

    PubMed

    Busse, Daniela; Kudella, Philipp; Grüning, Nana-Maria; Gisselmann, Günter; Ständer, Sonja; Luger, Thomas; Jacobsen, Frank; Steinsträßer, Lars; Paus, Ralf; Gkogkolou, Paraskevi; Böhm, Markus; Hatt, Hanns; Benecke, Heike

    2014-11-01

    As the outermost barrier of the body, the skin is exposed to multiple environmental factors, including temperature, humidity, mechanical stress, and chemical stimuli such as odorants that are often used in cosmetic articles. Keratinocytes, the major cell type of the epidermal layer, express a variety of different sensory receptors that enable them to react to various environmental stimuli and process information in the skin. Here we report the identification of a novel type of chemoreceptors in human keratinocytes, the olfactory receptors (ORs). We cloned and functionally expressed the cutaneous OR, OR2AT4, and identified Sandalore, a synthetic sandalwood odorant, as an agonist of this receptor. Sandalore induces strong Ca(2+) signals in cultured human keratinocytes, which are mediated by OR2AT4, as demonstrated by receptor knockdown experiments using RNA interference. The activation of OR2AT4 induces a cAMP-dependent pathway and phosphorylation of extracellular signal-regulated kinases (Erk1/2) and p38 mitogen-activated protein kinases (p38 MAPK). Moreover, the long-term stimulation of keratinocytes with Sandalore positively affected cell proliferation and migration, and regeneration of keratinocyte monolayers in an in vitro wound scratch assay. These findings combined with our studies on human skin organ cultures strongly indicate that the OR 2AT4 is involved in human keratinocyte re-epithelialization during wound-healing processes.

  2. The Herbal Bitter Drug Gentiana lutea Modulates Lipid Synthesis in Human Keratinocytes In Vitro and In Vivo.

    PubMed

    Wölfle, Ute; Haarhaus, Birgit; Seiwerth, Jasmin; Cawelius, Anja; Schwabe, Kay; Quirin, Karl-Werner; Schempp, Christoph M

    2017-08-22

    Gentiana lutea is a herbal bitter drug that is used to enhance gastrointestinal motility and secretion. Recently we have shown that amarogentin, a characteristic bitter compound of Gentiana lutea extract (GE), binds to the bitter taste receptors TAS2R1 and TAS2R38 in human keratinocytes, and stimulates the synthesis of epidermal barrier proteins. Here, we wondered if GE also modulates lipid synthesis in human keratinocytes. To address this issue, human primary keratinocytes were incubated for 6 days with GE. Nile Red labeling revealed that GE significantly increased lipid synthesis in keratinocytes. Similarly, gas chromatography with flame ionization detector indicated that GE increases the amount of triglycerides in keratinocytes. GE induced the expression of epidermal ceramide synthase 3, but not sphingomyelinase. Lipid synthesis, as well as ceramide synthase 3 expression, could be specifically blocked by inhibitors of the p38 MAPK and PPARγ signaling pathway. To assess if GE also modulates lipid synthesis in vivo, we performed a proof of concept half side comparison on the volar forearms of 33 volunteers. In comparison to placebo, GE significantly increased the lipid content of the treated skin areas, as measured with a sebumeter. Thus, GE enhances lipid synthesis in human keratinocytes that is essential for building an intact epidermal barrier. Therefore, GE might be used to improve skin disorders with an impaired epidermal barrier, e.g., very dry skin and atopic eczema.

  3. Entry Pathways of Herpes Simplex Virus Type 1 into Human Keratinocytes Are Dynamin- and Cholesterol-Dependent

    PubMed Central

    Hsu, Mei-Ju; Rixon, Frazer J.; Knebel-Mörsdorf, Dagmar

    2011-01-01

    Herpes simplex virus type 1 (HSV-1) can enter cells via endocytic pathways or direct fusion at the plasma membrane depending on the cell line and receptor(s). Most studies into virus entry have used cultured fibroblasts but since keratinocytes represent the primary entry site for HSV-1 infection in its human host, we initiated studies to characterize the entry pathway of HSV-1 into human keratinocytes. Electron microscopy studies visualized free capsids in the cytoplasm and enveloped virus particles in vesicles suggesting viral uptake both by direct fusion at the plasma membrane and by endocytic vesicles. The ratio of the two entry modes differed in primary human keratinocytes and in the keratinocyte cell line HaCaT. Inhibitor studies further support a role for endocytosis during HSV-1 entry. Infection was inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin, which demonstrates the requirement for host cholesterol during virus entry. Since the dynamin-specific inhibitor dynasore and overexpression of a dominant-negative dynamin mutant blocked infection, we conclude that the entry pathways into keratinocytes are dynamin-mediated. Electron microscopy studies confirmed that virus uptake is completely blocked when the GTPase activity of dynamin is inhibited. Ex vivo infection of murine epidermis that was treated with dynasore further supports the essential role of dynamin during entry into the epithelium. Thus, we conclude that HSV-1 can enter human keratinocytes by alternative entry pathways that require dynamin and host cholesterol. PMID:22022400

  4. Mitochondrial and lipogenic effects of vitamin D on differentiating and proliferating human keratinocytes.

    PubMed

    Consiglio, Marco; Viano, Marta; Casarin, Stefania; Castagnoli, Carlotta; Pescarmona, Gianpiero; Silvagno, Francesca

    2015-10-01

    Even in cells that are resistant to the differentiating effects of vitamin D, the activated vitamin D receptor (VDR) can downregulate the mitochondrial respiratory chain and sustain cell growth through enhancing the activity of biosynthetic pathways. The aim of this study was to investigate whether vitamin D is effective also in modulating mitochondria and biosynthetic metabolism of differentiating cells. We compared the effect of vitamin D on two cellular models: the primary human keratinocytes, differentiating and sensitive to the genomic action of VDR, and the human keratinocyte cell line HaCaT, characterized by a rapid growth and resistance to vitamin D. We analysed the nuclear translocation and features of VDR, the effects of vitamin D on mitochondrial transcription and the consequences on lipid biosynthetic fate. We found that the negative modulation of respiratory chain is a general mechanism of action of vitamin D, but at high doses, the HaCaT cells became resistant to mitochondrial effects by upregulating the catabolic enzyme CYP24 hydroxylase. In differentiating keratinocytes, vitamin D treatment promoted intracellular lipid deposition, likewise the inhibitor of respiratory chain stigmatellin, whereas in proliferating HaCaT, this biosynthetic pathway was not inducible by the hormone. By linking the results on respiratory chain and lipid accumulation, we conclude that vitamin D, by suppressing respiratory chain transcription in all keratinocytes, is able to support both the proliferation and the specialized metabolism of differentiating cells. Through mitochondrial control, vitamin D can have an essential role in all the metabolic phenotypes occurring in healthy and diseased skin.

  5. Low dose chronic treatment of human keratinocytes with inorganic arsenic causes hyperproliferation and altered protein phosphorylation

    SciTech Connect

    Steinberg, M.L.; Su, L.; Snow, E.T. |

    1997-10-01

    Chronic exposure to arsenate [As(V)] or arsenite [As(III)] causes hyperproliferation of normal and SV40-transformed human epidermal keratinocytes. Line 327 SV40-infected human keratinocytes were grown in the presence of either As(III) or As(V) (0.01 to 10 {mu}M) in complete medium for seven days prior to harvesting and counting. Both As(III) and As(V) were cytotoxic at micromolar concentrations, however submicromolar arsenic caused a significant increase in cell growth. Cell numbers in cultures exposed to As(V) were increased more than 186% relative to controls, and an even larger stimulation in cell growth was observed after treatment with 50 nM As(III). Normal non-SV40 T-antigen. Preliminary cell cycle analysis using unselected, log-phase cultures of arsenic-treated keratinocytes shows an increased proportion of cells in S- and G2/M-phase. Isoelectric focusing of phosphotyrosine-containing proteins from cells labeled with {sup 32}P-inorganic phosphate showed that the hyperproliferation of keratinocytes grown in low concentrations of arsenic is accompanied by altered tyrosine-specific protein phosphorylation. A number of phosphorylated proteins were observed in As-treated cells that were not observed in the controls; and minor bands at IEPs of 3.0, 4.2, 7.2, 7.5 and 8.2. These results, together with the lack of direct enzyme inhibition by arsenic shown by Su et al., this volume, suggest that arsenic-induced skin lesions and carcinogenesis may be the result of altered cell cycle control rather than DNA damage or reduced DNA repair.

  6. PCB153 reduces telomerase activity and telomere length in immortalized human skin keratinocytes (HaCaT) but not in human foreskin keratinocytes (NFK)

    SciTech Connect

    Senthilkumar, P.K.; Robertson, L.W.; Ludewig, G.

    2012-02-15

    Polychlorinated biphenyls (PCBs), ubiquitous environmental pollutants, are characterized by long term-persistence in the environment, bioaccumulation, and biomagnification in the food chain. Exposure to PCBs may cause various diseases, affecting many cellular processes. Deregulation of the telomerase and the telomere complex leads to several biological disorders. We investigated the hypothesis that PCB153 modulates telomerase activity, telomeres and reactive oxygen species resulting in the deregulation of cell growth. Exponentially growing immortal human skin keratinocytes (HaCaT) and normal human foreskin keratinocytes (NFK) were incubated with PCB153 for 48 and 24 days, respectively, and telomerase activity, telomere length, superoxide level, cell growth, and cell cycle distribution were determined. In HaCaT cells exposure to PCB153 significantly reduced telomerase activity, telomere length, cell growth and increased intracellular superoxide levels from day 6 to day 48, suggesting that superoxide may be one of the factors regulating telomerase activity, telomere length and cell growth compared to untreated control cells. Results with NFK cells showed no shortening of telomere length but reduced cell growth and increased superoxide levels in PCB153-treated cells compared to untreated controls. As expected, basal levels of telomerase activity were almost undetectable, which made a quantitative comparison of treated and control groups impossible. The significant down regulation of telomerase activity and reduction of telomere length by PCB153 in HaCaT cells suggest that any cell type with significant telomerase activity, like stem cells, may be at risk of premature telomere shortening with potential adverse health effects for the affected organism. -- Highlights: ► Human immortal (HaCaT) and primary (NFK) keratinocytes were exposed to PCB153. ► PCB153 significantly reduced telomerase activity and telomere length in HaCaT. ► No effect on telomere length and

  7. 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

  8. Exploratory Study on the Stability Characteristics of Commercial Human Keratinocytes

    DTIC Science & Technology

    1990-04-01

    RHE) and human living skin equivalent (LSE) organotypic model systems in protocols for vesicant research. JSA: C-A-304. Skin Protection. Task Area...conducted to assess their utility for developing organotypic skin models for vesicant research. It has been shown that such in vitro models (e.g...reconstructed human epidermis [RHE) and living skin equivalents [LSE]) require normal keracinocytes (basal cells; stratum germinativum) that can prcliferate

  9. Reference genes for gene expression analysis in proliferating and differentiating human keratinocytes.

    PubMed

    Lanzafame, Manuela; Botta, Elena; Teson, Massimo; Fortugno, Paola; Zambruno, Giovanna; Stefanini, Miria; Orioli, Donata

    2015-04-01

    Abnormalities in keratinocyte growth and differentiation have a pathogenic significance in many skin disorders and result in gene expression alterations detectable by quantitative real-time RT-PCR (qRT-PCR). Relative quantification based on endogenous control (EC) genes is the commonly adopted approach, and the use of multiple reference genes from independent pathways is considered a best practice guideline, unless fully validated EC genes are available. The literature on optimal reference genes during in vitro calcium-induced differentiation of normal human epidermal keratinocytes (NHEK) is inconsistent. In many studies, the expression of target genes is compared to that of housekeeping genes whose expression, however, significantly varies during keratinocyte differentiation. Here, we report the results of our investigations on the expression stability of 15 candidate EC genes, including those commonly used as reference in expression analysis by qRT-PCR, during NHEK calcium-induced differentiation. We demonstrate that YWHAZ and UBC are extremely stable genes, and therefore, they represent optimal EC genes for expression studies in proliferating and calcium-induced differentiating NHEK. Furthermore, we demonstrate that YWHAZ/14-3-3-zeta is a suitable reference for quantitative comparison of both transcript and protein levels.

  10. Slug regulates integrin expression and cell proliferation in human epidermal keratinocytes.

    PubMed

    Turner, Frances E; Broad, Simon; Khanim, Farhat L; Jeanes, Alexa; Talma, Sonia; Hughes, Sharon; Tselepis, Chris; Hotchin, Neil A

    2006-07-28

    The human epidermis is a self-renewing epithelial tissue composed of several layers of keratinocytes. Within the epidermis there exists a complex array of cell adhesion structures, and many of the cellular events within the epidermis (differentiation, proliferation, and migration) require that these adhesion structures be remodeled. The link between cell adhesion, proliferation, and differentiation within the epidermis is well established, and in particular, there is strong evidence to link the process of terminal differentiation to integrin adhesion molecule expression and function. In this paper, we have analyzed the role of a transcriptional repressor called Slug in the regulation of adhesion molecule expression and function in epidermal keratinocytes. We report that activation of Slug, which is expressed predominantly in the basal layer of the epidermis, results in down-regulation of a number of cell adhesion molecules, including E-cadherin, and several integrins, including alpha3, beta1, and beta4. We demonstrate that Slug binds to the alpha3 promoter and that repression of alpha3 transcription by Slug is dependent on an E-box sequence within the promoter. This reduction in integrin expression is reflected in decreased cell adhesion to fibronectin and laminin-5. Despite the reduction in integrin expression and function, we do not observe any increase in differentiation. We do, however, find that activation of Slug results in a significant reduction in keratinocyte proliferation.

  11. Date seed oil inhibits hydrogen peroxide-induced oxidative stress in human epidermal keratinocytes.

    PubMed

    Ines, Dammak; Sonia, Boudaya; Fatma, Ben Abdallah; Souhail, Besbes; Hamadi, Attia; Hamida, Turki; Basma, Hentati

    2010-03-01

    Oxidative stress has been implicated in various skin diseases through the generation of reactive oxygen species and the depletion of endogenous antioxidant systems. The administration of antioxidants is reportedly helpful, notably to enhance the healing process. To protect the skin against oxidative damages, we have studied the effect of new oil: "date seed oil" (DSO). This oil, may serve as a potential source of natural antioxidants such as phenols and tocopherols. Here, we report the protective effect of DSO against hydrogen peroxide (H(2)O(2))-induced oxidative stress in terms of lipid peroxidation, depletion of endogenous antioxidant defense enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) using normal human epidermal keratinocytes (NHEK). In the investigated model system, DSO has significant chemoprotective effect, by inhibition of damage caused by H(2)O(2) compared with cells without such addition endowing with a radical scavenging ability. Treatment of NHEK with DSO inhibited H(2)O(2)-induced lipid peroxidation. In addition, this oil inhibited H(2)O(2)-induced depletion of antioxidant defense components, such as SOD, CAT and GPx. Our findings demonstrate that DSO is an efficient extract that is able to prevent keratinocytes oxidative damage induced by H(2)O(2) exposure and may thus be a potential promising candidate, as a chemopreventive agent, in the development of keratinocytes-related pathologies.

  12. Constitutive phenolics of Harpephyllum caffrum (Anacardiaceae) and their biological effects on human keratinocytes.

    PubMed

    Nawwar, Mahmoud; Hussein, Sahar; Ayoub, Nahla; Hashim, Amani; El-Sharawy, Reham; Lindequist, Urlike; Harms, Manualle; Wende, Kristian

    2011-12-01

    Assessment of the UV protecting potential of an aqueous methanol leaf extract of Harpephyllum caffrum proved that it possesses a distinct radical scavenging effect and inhibits the production of the proinflammatory cytokine IL-6 by human keratinocytes (HaCaT cells) following UV radiation. Phytochemical investigation of this extract led to isolation and structural determination of the hitherto unknown phenolics, kaempferol 3-O-(2″-sulphatogalactopyranoside), its quercetin analogue and 3-methoxyellagic acid 4-O-galactopyranoside in addition to 18 known phenolic compounds. The structures were determined by spectroscopic and conventional methods of analysis. Flavonoid sulphatoglycosides which have been rarely found in nature were major phenolic constituents of this plant, and this is the first report of the isolation of any of them from Anacardiaceae. The extract was found to diminish UV phototoxic reaction of keratinocytes. However, the isolated kaempferol sulphatogalactopyranoside did not interact with UVB triggered IL-6 production of HaCaT keratinocytes. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Protective effects of catalase overexpression on UVB-induced apoptosis in normal human keratinocytes.

    PubMed

    Rezvani, Hamid Reza; Mazurier, Frédéric; Cario-André, Muriel; Pain, Catherine; Ged, Cécile; Taïeb, Alain; de Verneuil, Hubert

    2006-06-30

    UV-induced apoptosis in keratinocytes is a highly complex process in which various molecular pathways are involved. These include the extrinsic pathway via triggering of death receptors and the intrinsic pathway via DNA damage and reactive oxygen species (ROS) formation. In this study we investigated the effect of catalase and CuZn-superoxide dismutase (SOD) overexpression on apoptosis induced by UVB exposure at room temperature or 4 degrees C on normal human keratinocytes. Irradiation at low temperature reduced UV-induced apoptosis by 40% in normal keratinocytes independently of any change in p53 and with a decrease in caspase-8 activation. Catalase overexpression decreased apoptosis by 40% with a reduction of caspase-9 activation accompanied by a decrease in p53. Keeping cells at low temperature and catalase overexpression had additive effects. CuZn-SOD overexpression had no significant effect on UVB-induced apoptosis. UVB induced an increase in ROS levels at two distinct stages: immediately following irradiation and around 3 h after irradiation. Catalase overexpression inhibited only the late increase in ROS levels. We conclude that catalase overexpression has a protective role against UVB irradiation by preventing DNA damage mediated by the late ROS increase.

  14. 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.

  15. The effect of the plasma needle on the human keratinocytes related to the wound healing process

    NASA Astrophysics Data System (ADS)

    Korolov, Ihor; Fazekas, Barbara; Széll, Márta; Kemény, Lajos; Kutasi, Kinga

    2016-01-01

    In the present study we aim to verify the influence of a non-thermal atmospheric pressure plasma on the wound healing process. In this process the major contributors are the keratinocytes, which migrate to fill in the gap created by the wound. Therefore, we performed the direct treatment of HPV-immortalized human keratinocytes, protected by a layer of phosphate buffered saline (PBS) solution, with the glow discharge generated in flowing helium by a plasma needle. To mimick a wound, a 4 mm scratch was performed on the cell culture (scratch assay). We conducted two types of experiments: (i) cell proliferation and (ii) wound-healing model experiments. The plasma needle configuration, the plasma treatment conditions and the thickness of the protecting PBS layer were set based on viability experiments. The proliferation studies showed that short, 5-10 s, and low power treatments, such as 18 W and 20 W input power, could positively influence the cell proliferation when keratinocytes were protected by PBS. On the other hand, the plasma treatment of cell medium covered keratinocytes resulted in the decrease of proliferation. The wound-healing model (scratch assay) studies showed, that there was a maximum in the wound reduction as a function of the input power and treatment time, namely, at 18 W and 5 s. Furthermore, the wound reduction strongly depended on the treated cell—PBS interaction time. To mimic an infected wound, the scratch assay was covered with a 1× {{10}9} cfu ml-1 Propionibacterium acnes suspension. The plasma treatment of this infected assay resulted in closing of the scratch, while in the non-treated assay the wound did not close at all.

  16. Detection of pesticide effects in human keratinocytes by means of Raman microspectroscopy

    NASA Astrophysics Data System (ADS)

    Perna, G.; Lasalvia, M.; Castro, A.; Mezzenga, E.; L'Abbate, N.; Biagi, P. F.; Capozzi, V.

    2009-08-01

    Biochemical modifications of single cells of human keratinocytes exposed to different concentrations of chlorpyriphos solutions were detected by means of Raman microspectroscopy. The modifications mainly consist of breakdown of amide linkage between aminoacids. Fragmentation of DNA bases and single aminoacids occurs as well. The protein and DNA damage are already detectable at a chlorpyriphos concentration value (10-6M), which is at least three orders of magnitude lower than the concentration estimated as cytotoxic (10-3M). Raman microspectroscopy technique results to be a very sensitive technique to detect molecular changes even in single human cells exposed to toxic pesticide.

  17. The expressions of ABCC4 and ABCG2 xenobiotic transporters in human keratinocytes are proliferation-related.

    PubMed

    Bebes, Attila; Kis, Kornélia; Nagy, Tünde; Kurunczi, Anita; Polyánka, Hilda; Bata-Csörgo, Zsuzsanna; Kemény, Lajos; Dobozy, Attila; Széll, Márta

    2012-01-01

    Xenobiotic transporters of the ATP-binding cassette (ABC) protein superfamily play important roles in maintaining the biochemical barrier of various tissues, but their precise functions in the skin are not yet known. Screening of the expressions of the known xenobiotic transporter genes in two in vitro keratinocyte differentiation models revealed that the ABCC4 and ABCG2 transporters are highly expressed in proliferating keratinocytes, their expressions decreasing along with differentiation. Abrogation of the ABCC4 and ABCG2 protein functions by siRNA-mediated silencing and chemical inhibition did not affect the proliferation of HaCaT cells. In contrast, disruption of the ABCG2 function had no effect on normal human epidermal keratinocyte proliferation, while the inhibition of ABCC-type transporters by probenecid resulted in a striking decrease in the proliferation of the cells. These results indicate that, besides their possible therapy-modulating effects, xenobiotic transporters may contribute significantly to other keratinocyte functions, such as cell proliferation.

  18. 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

  19. PCB153 reduces telomerase activity and telomere length in immortalized human skin keratinocytes (HaCaT) but not in human foreskin keratinocytes (NFK).

    PubMed

    Senthilkumar, P K; Robertson, L W; Ludewig, G

    2012-02-15

    Polychlorinated biphenyls (PCBs), ubiquitous environmental pollutants, are characterized by long term-persistence in the environment, bioaccumulation, and biomagnification in the food chain. Exposure to PCBs may cause various diseases, affecting many cellular processes. Deregulation of the telomerase and the telomere complex leads to several biological disorders. We investigated the hypothesis that PCB153 modulates telomerase activity, telomeres and reactive oxygen species resulting in the deregulation of cell growth. Exponentially growing immortal human skin keratinocytes (HaCaT) and normal human foreskin keratinocytes (NFK) were incubated with PCB153 for 48 and 24days, respectively, and telomerase activity, telomere length, superoxide level, cell growth, and cell cycle distribution were determined. In HaCaT cells exposure to PCB153 significantly reduced telomerase activity, telomere length, cell growth and increased intracellular superoxide levels from day 6 to day 48, suggesting that superoxide may be one of the factors regulating telomerase activity, telomere length and cell growth compared to untreated control cells. Results with NFK cells showed no shortening of telomere length but reduced cell growth and increased superoxide levels in PCB153-treated cells compared to untreated controls. As expected, basal levels of telomerase activity were almost undetectable, which made a quantitative comparison of treated and control groups impossible. The significant down regulation of telomerase activity and reduction of telomere length by PCB153 in HaCaT cells suggest that any cell type with significant telomerase activity, like stem cells, may be at risk of premature telomere shortening with potential adverse health effects for the affected organism.

  20. The αE(CD103)β7 integrin interacts with oral and skin keratinocytes in an E-cadherin-independent manner*

    PubMed Central

    Jenkinson, Sarah E; Whawell, Simon A; Swales, Brenka M; Corps, Elaine M; Kilshaw, Peter J; Farthing, Paula M

    2011-01-01

    The integrin αE(CD103)β7 (αEβ7) is expressed by intraepithelial lymphocytes, dendritic cells and regulatory T cells. It plays an important role in the mucosal immune system by retaining lymphocytes within the epithelium and is involved in graft rejection, immunity against tumours and the generation of gut-homing effector cells. In gut and breast, the ligand for αEβ7 is E-cadherin but in human oral mucosa and skin, there is evidence that lymphocytes use an alternative, unknown, ligand. In the present study, the I domain of the human αE subunit, which contains the E-cadherin-binding site, was locked in a highly active, ‘open’ and an inactive, ‘closed’ conformation by the introduction of disulphide bonds and these domains were expressed as IgG Fc fusion proteins. αE fusion proteins recognize E-cadherin, the only known ligand for αEβ7. This interaction was inhibited by an antibody that blocks the αE-binding site on E-cadherin and by the omission of Mn2+, which is essential for integrin function in vitro. The locked ‘open’ conformation of αE adhered to human oral and skin keratinocytes, including the E-cadherin-negative H376 cell line, and this was not inhibited by blocking antibody against the αEβ7-binding site on E-cadherin, providing further evidence for the existence of an alternative ligand for αEβ7 in skin and oral mucosa. The interaction with E-cadherin and the alternative ligand was Mn2+ dependent and mediated by the metal ion-dependent coordination site (MIDAS) of the locked ‘open’αE I domain, independently of the β7 subunit. PMID:20875079

  1. The αE(CD103)β7 integrin interacts with oral and skin keratinocytes in an E-cadherin-independent manner*.

    PubMed

    Jenkinson, Sarah E; Whawell, Simon A; Swales, Brenka M; Corps, Elaine M; Kilshaw, Peter J; Farthing, Paula M

    2011-02-01

    The integrin αE(CD103)β7 (αEβ7) is expressed by intraepithelial lymphocytes, dendritic cells and regulatory T cells. It plays an important role in the mucosal immune system by retaining lymphocytes within the epithelium and is involved in graft rejection, immunity against tumours and the generation of gut-homing effector cells. In gut and breast, the ligand for αEβ7 is E-cadherin but in human oral mucosa and skin, there is evidence that lymphocytes use an alternative, unknown, ligand. In the present study, the I domain of the human αE subunit, which contains the E-cadherin-binding site, was locked in a highly active, 'open' and an inactive, 'closed' conformation by the introduction of disulphide bonds and these domains were expressed as IgG Fc fusion proteins. αE fusion proteins recognize E-cadherin, the only known ligand for αEβ7. This interaction was inhibited by an antibody that blocks the αE-binding site on E-cadherin and by the omission of Mn(2+) , which is essential for integrin function in vitro. The locked 'open' conformation of αE adhered to human oral and skin keratinocytes, including the E-cadherin-negative H376 cell line, and this was not inhibited by blocking antibody against the αEβ7-binding site on E-cadherin, providing further evidence for the existence of an alternative ligand for αEβ7 in skin and oral mucosa. The interaction with E-cadherin and the alternative ligand was Mn(2+) dependent and mediated by the metal ion-dependent coordination site (MIDAS) of the locked 'open'αE I domain, independently of the β7 subunit.

  2. miR-125b inhibits keratinocyte proliferation and promotes keratinocyte apoptosis in oral lichen planus by targeting MMP-2 expression through PI3K/Akt/mTOR pathway.

    PubMed

    Wang, Jing; Luo, Hong; Xiao, Yan; Wang, Luyao

    2016-05-01

    Oral lichen planus (OLP) is a chronic inflammatory mucosal disease that involves the degeneration of keratinocytes. However, the etiology and mechanisms of OLP pathogenesis have not been fully elucidated. In this study, we used keratinocytes HaCaT stimulated with lipopolysaccharide (LPS) to mimic a local OLP immune environment, and investigated the regulatory role of miR-125b in keratinocyte proliferation and apoptosis under OLP conditions. Immunohistochemical analysis and quantitative real-time PCR (qRT-PCR) assay showed that MMP-2 expression was up-regulated and miR-125b expression was down-regulated in both OLP mucosa tissues and LPS-incubated HaCaT cells. Western blot analysis indicated that miR-125b overexpression suppressed LPS-induced MMP-2 expression in HaCaT cells. Molecularly, our results confirmed that MMP-2 is a target gene of miR-125b in HaCaT cells. The effect of miR-125b on cell proliferation was revealed by CCK-8 assay, BrdU assay and cell cycle analysis, which illustrated that miR-125b overexpression impeded LPS-induced HaCaT cell proliferation. Flow cytometry analysis further demonstrated that miR-125b overexpression promoted HaCaT cell apoptosis. Moreover, these effects were involved in PI3K/Akt/mTOR activation, as miR-125b overexpression inhibited LPS-enhanced expression of p-Akt and p-mTOR proteins. Taken together, these data confirm that miR-125b might inhibit keratinocyte proliferation and promote keratinocyte apoptosis in OLP pathogenesis by targeting MMP-2 through PI3K/Akt/mTOR pathway. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  3. 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.

  4. Ultraviolet Radiation Increases the Toxicity of Pyrene, 1-Aminopyrene and 1-Hydroxypyrene to Human Keratinocytes

    PubMed Central

    Ekunwe, Stephen I. N.; Hunter, Rochelle D.; Hwang, Huey-Min

    2005-01-01

    Over the past several years, a great deal of interest has been focused on the harmful effects of ultraviolet (UV) radiation to human skin. UV light has been implicated in aging, sunburn and skin cancer. Few studies, however, have been done to determine the effects that UV light, in conjunction with other environmental contaminants, may have on human skin. Polycyclic Aromatic Hydrocarbons (PAHs) are a class of compounds that have been reported to be toxic, mutagenic and carcinogenic to many eukaryotic organisms. UV light is also known to increase the toxicity of PAHs through photo-activation and photo-modification. The purpose of this study was to assess the effects of UV-A irradiated pyrene (Pyr), 1-aminopyrene (1-AP) and 1-hydroxypyrene (1-HP) on human keratinocytes, the skin primary site of UV irradiated PAH exposure. Our findings indicate that simultaneous treatment of human keratinocyte cell line, HaCaT, with 1.0μg/ml pyrene, 1-AP or 1-HP and 3.9 J/cm2/min UV-A light resulted in significant inhibition of cell proliferation. Approximately 100% of the cells died in the case of UV-A irradiated 1-AP and 1-HP. In the case of UV-A irradiated pyrene, more than 70% of the cells died, indicating that UV-A is able to transform these PAHs into more harmful intermediates. PMID:16708424

  5. Immortalization of human foreskin keratinocytes by various human papillomavirus DNAs corresponds to their association with cervical carcinoma

    SciTech Connect

    Woodworth, C.D.; Doniger, J.; DiPaolo, J.A.

    1989-01-01

    Normal human foreskin keratinocytes cotransfected with the neomycin resistance gene and recombinant human papillomavirus (HPV) DNAs (types 16, 18, 31, and 33) that have a high or moderate association with cervical malignancy acquired immortality and contained integrated and transcriptionally active viral genomes. Only transcripts from the intact E6 and E7 genes were detected in at least one cell line, suggesting that one or both of these genes are responsible for immortalization. Recombinant HPV DNAs with low or no oncogenic potential for cervical cancer (HPV1a, -5, -6b, and -11) induced small G418-resistant colonies that senesced as did the nontransfected cells. These colonies contained only episomal virus DNA; therefore, integration of HPV sequences is important for immortalization of keratinocytes. This study suggests that the virus-encoded immortalization function contributes to the pathogenesis of cervical carcinoma.

  6. MMP- and TIMP-secretion by human cutaneous keratinocytes and fibroblasts--impact of coculture and hydration.

    PubMed

    Tandara, Andrea A; Mustoe, Thomas A

    2011-01-01

    Epithelial-mesenchymal interactions are important in wound healing and scarring, but are difficult to study in vitro. We have previously reported on an in vitro keratinocyte-fibroblast coculture system exploring these interactions and found that coculture modifies the levels of cytokines they secrete. The same coculture model was used to study changes in MMP- and TIMP-activity. We hypothesised that the previously shown decrease of collagen is partly due to increased MMPs. Adult human cutaneous keratinocytes and fibroblasts were cocultured under serum-free conditions. Keratinocytes were either kept at the air-liquid interface or hydrated. The conditioned medium was submitted to a multiplex sandwich enzyme-linked immunosorbent assay including gelatinases, collagenases, stromelysins, and tissue inhibitors of metalloproteinases. Collagen content was determined by western blot. Zymography depicted the gelatinases in conditioned media. For confirmation of the coculture results fibroblasts were treated with conditioned media from keratinocyte monocultures as well. MMP-1, MMP-9, and MMP-10 were mainly secreted by keratinocytes, whereas MMP-2, TIMP-1 and -2 by fibroblasts. MMP-13 was secreted by both cell types at comparable levels. Collagenases, gelatinases, MMP-3, and TIMPs increased significantly in cocultures compared to monocultures. Hydration of keratinocytes revealed a significant increase of MMP-3 and MMP-2, and a decrease of TIMP-2. Paracrine interactions between keratinocytes and fibroblasts modify strongly MMPs and TIMPs, whereas hydration of keratinocytes had a smaller impact in this context. The observed changes may be in part responsible for reduced collagen in coculture-conditioned media. The present coculture experiments reemphasise the role of epidermis in controlling scarring.

  7. UVB exposure of a humanized skin model reveals unexpected dynamic of keratinocyte proliferation and Wnt inhibitor balancing.

    PubMed

    Michalczyk, Teresa; Biedermann, Thomas; Böttcher-Haberzeth, Sophie; Klar, Agnes S; Meuli, Martin; Reichmann, Ernst

    2017-07-17

    We developed human dermo-epidermal skin substitutes which are presently applied in phase I and II clinical trials. Here we used these very same skin equivalents containing melanocytes, named MelSkin, as an experimental skin model. We investigated the effects of UVB irradiation on the skin grafts transplanted on immune-compromised rats. The irradiation induces a strong wound healing response going along with massive proliferation of basal keratinocytes, basically quiescent under non-irradiated, homeostatic conditions. As a consequence of UVB irradiation, the initially clearly defined basal keratinocyte (mono)layer expands into about three layers of keratinocytes, all of which still express the basal keratinocyte marker Keratin15. In contrast, epidermal melanocytes remain quiescent under these circumstances. Moreover, the Wnt inhibitors Dickkopf 3 and Wif1 are downregulated upon UVB irradiation in basal keratinocytes, whereas melanocytes continue to express Wnt inhibitors. These findings suggest that there is 1) a suprabasal population, proliferating in the homeostatic state, hence maintaining the integrity of the epidermis, and 2) a basal, usually quiescent keratinocyte population that is induced to massively proliferate upon irradiation. Importantly, the finding that MelSkin responds in a physiological fashion to UVB is of paramount importance in light of the planned clinical application. This article is protected by copyright. All rights reserved.

  8. Effect of Surface Coating on the Toxicity of Silver Nanomaterials on Human Skin Keratinocytes.

    PubMed

    Lu, Wentong; Senapati, Dulal; Wang, Shuguang; Tovmachenko, Oleg; Singh, Anant Kumar; Yu, Hongtao; Ray, Paresh Chandra

    2010-02-25

    As nanotechnology field continues to develop, assessing nanoparticle toxicity is very important for advancing nanoparticles for daily life application. In this Letter, we report the effect of surface coating on cyto, geno and photo-toxicity of silver nanomaterials of different shapes on human skin HaCaT keratinocytes. We found that the citrate coated colloidal silver nanoparticles at 100 µg/mL level are not geno-, cyto- and phtotoxic. On the other hand, citrate coated powder form of the silver nanoparticles are toxic. We have demonstrated that coating of the silver nanoparticles with a biodegradable polymer prevents the toxicity of the powder. Toxicity mechanism has been discussed.

  9. Effect of surface coating on the toxicity of silver nanomaterials on human skin keratinocytes

    NASA Astrophysics Data System (ADS)

    Lu, Wentong; Senapati, Dulal; Wang, Shuguang; Tovmachenko, Oleg; Singh, Anant Kumar; Yu, Hongtao; Ray, Paresh Chandra

    2010-02-01

    As nanotechnology field continues to develop, assessing nanoparticle toxicity is very important for advancing nanoparticles for daily life application. In this Letter, we report the effect of surface coating on cyto, geno and photo-toxicity of silver nanomaterials of different shapes on human skin HaCaT keratinocytes. We found that the citrate coated colloidal silver nanoparticles at 100 μg/mL level are not geno-, cyto- and phtotoxic. On the other hand, citrate coated powder form of the silver nanoparticles are toxic. We have demonstrated that coating of the silver nanoparticles with a biodegradable polymer prevents the toxicity of the powder. Toxicity mechanism has been discussed.

  10. Shuttling of the autoantigen La between nucleus and cell surface after uv irradiation of human keratinocytes

    SciTech Connect

    Bachmann, M.; Chang, S.; Slor, H.; Kukulies, J.; Mueller, W.E. )

    1990-12-01

    During the past years we have established that the nuclear autoantigen La shuttles between the nucleus and the cytoplasm in tumor cells after inhibition of transcription or virus infection. We reinvestigated this shuttling using primary human keratinocytes from both healthy donors and patients with xeroderma pigmentosum. Ultraviolet irradiation resulted in both an inhibition of transcription and a translocation of La protein from the nucleus to the cytoplasm. After a prolonged inhibition of transcription La protein relocated into the nucleus and assembled with nuclear storage regions. The uv-induced shuttling included a translocation to the cell surface, where La protein colocalized with epidermal growth factor receptors.

  11. Mycobacterium ulcerans mycolactone interferes with adhesion, migration and proliferation of primary human keratinocytes and HaCaT cell line.

    PubMed

    Graziola, Francesca; Colombo, Elena; Tiberio, Rossana; Leigheb, Giorgio; Bozzo, Chiarella

    2017-04-01

    The pathogenicity of Mycobacterium ulcerans (Buruli ulcer) is closely associated with the secretion of exotoxin mycolactone. The cytotoxicity of mycolactone has been linked to its apoptogenic activity. We explored if low mycolactone concentrations, which are not able to induce apoptosis, can influence other essential activities on two primary human keratinocyte populations, keratinocyte stem cells (KSC) and transit amplifying cells (TAC), and on a human keratinocyte line, HaCaT. We demonstrated that 0.01 and 0.1 ng/ml mycolactone A/B are not able to induce apoptosis in primary human keratinocytes, but interfere with KSC wound repair. Moreover, the same toxin concentrations reduce cell proliferation of KSC and TAC and their ability to adhere to type IV collagen. HaCaT cells are more resistant to the toxin; nevertheless, they show a delayed woud repair when treated with 1 and 10 ng/ml mycolactone A/B. Moreover, these sub-apoptotic concentrations affect their ability to proliferate and adhere to collagen IV. Wound healing is a complex mechanism, which occurs "in vivo" as the outcome of many co-ordinated events. Sub-apoptotic mycolactone concentrations can affect essential mechanisms, which are required to achieve wound repair, such as adhesion, migration and proliferation of human keratinocytes.

  12. The Haemophilus ducreyi serum resistance antigen DsrA confers attachment to human keratinocytes.

    PubMed

    Cole, Leah E; Kawula, Thomas H; Toffer, Kristen L; Elkins, Christopher

    2002-11-01

    Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. H. ducreyi serum resistance protein A (DsrA) is a member of a family of multifunctional outer membrane proteins that are involved in resistance to killing by human serum complement. The members of this family include YadA of Yersinia species, the UspA proteins of Moraxella catarrhalis, and the Eib proteins of Escherichia coli. The role of YadA, UspA1, and UspA2H as eukaryotic cell adhesins and the function of UspA2 as a vitronectin binder led to our investigation of the cell adhesion and vitronectin binding properties of DsrA. We found that DsrA was a keratinocyte-specific adhesin as it was necessary and sufficient for attachment to HaCaT cells, a keratinocyte cell line, but was not required for attachment to HS27 cells, a fibroblast cell line. We also found that DsrA was specifically responsible for the ability of H. ducreyi to bind vitronectin. We then theorized that DsrA might use vitronectin as a bridge to bind to human cells, but this hypothesis proved to be untrue as eliminating HaCaT cell binding of vitronectin with a monoclonal antibody specific to integrin alpha(v)beta(5) did not affect the attachment of H. ducreyi to HaCaT cells. Finally, we wanted to examine the importance of keratinocyte adhesion in chancroid pathogenesis so we tested the wild-type and dsrA mutant strains of H. ducreyi in our swine models of chancroid pathogenesis. The dsrA mutant was less virulent than the wild type in both the normal and immune cell-depleted swine models of chancroid infection.

  13. Coriandrum sativum L. protects human keratinocytes from oxidative stress by regulating oxidative defense systems.

    PubMed

    Park, G; Kim, H G; Kim, Y O; Park, S H; Kim, S Y; Oh, M S

    2012-01-01

    Oxidative radicals are major environmental causes of human skin damage. Oxidative defense factors, including nuclear factor erythroid-derived 2-related factor 2 (Nrf2), are centrally involved in repairing skin cells or protecting them from oxidative damage. Coriandrum sativum L. (coriander; CS) is a commonly consumed food and a traditional phytomedicine in Asia and Europe. In this study, we examined the protective effects of a standardized CS leaf extract against oxidative stress in human HaCaT keratinocytes. CS significantly and dose-dependently protected cells against reduced cell viability caused by H2O2-induced damage, as assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Other assays demonstrated that CS protected HaCaT cells by increasing the levels of glutathione and activities of oxidative defense enzymes, such as superoxide dismutase and catalase. Moreover, it increased the expression of activated Nrf2, which plays a crucial role in protecting skin cells against oxidative stress. These results suggest that CS protects human keratinocytes from H2O2-induced oxidative stress through antioxidant effects. Copyright © 2012 S. Karger AG, Basel.

  14. Paracrine Activity from Adipose-Derived Stem Cells on In Vitro Wound Healing in Human Tympanic Membrane Keratinocytes.

    PubMed

    Ong, Huan Ting; Redmond, Sharon L; Marano, Robert J; Atlas, Marcus D; von Unge, Magnus; Aabel, Peder; Dilley, Rodney J

    2017-03-15

    Stem cell therapies for tympanic membrane repair have shown initial experimental success using mesenchymal stem cells in rat models to promote healing; however, the mechanisms providing this benefit are not known. We investigated in vitro the paracrine effects of human adipose-derived stem cells (ADSCs) on wound healing mechanisms for human tympanic membrane-derived keratinocytes (hTM) and immortalized human keratinocytes (HaCaT). ADSC conditioned media (CMADSC) were assessed for paracrine activity on keratinocyte proliferation and migration, with hypoxic conditions for ADSC culture used to generate contrasting effects on cytokine gene expression. Keratinocytes cultured in CMADSC showed a significant increase in cell number compared to serum-free cultures and further significant increases in hypoxic CMADSC. Assessment of ADSC gene expression on a cytokine array showed a range of wound healing cytokines expressed and under stringent hypoxic and serum-free conditions was upregulated (VEGF A, MMP9, Tissue Factor, PAI-1) or downregulated (CXCL5, CCL7, TNF-α). Several of these may contribute to the activity of conditioned media on the keratinocytes with potential applications in TM perforation repair. VEGFA protein was confirmed by immunoassay to be increased in conditioned media. Together with gene regulation associated with hypoxia in ADSCs, this study has provided several strong leads for a stem cell-derived approach to TM wound healing.

  15. Interaction of urokinase A chain with the receptor of human keratinocytes stimulates release of urokinase-like plasminogen activator

    SciTech Connect

    Fibbi, G.; Magnelli, L.; Pucci, M.; Del Rosso, M. )

    1990-03-01

    On the basis of a fibrinolytic assay with {sup 125}I-fibrin, zymography, and immunoprobing with anti-human urokinase antibody, the authors have observed that the in vitro established NCTC human keratinocyte cell line releases into the culture medium a 54,000-Da plasminogen activator which is indistinguishable from human urokinase. Only the early release following the washing of keratinocyte monolayers is accounted for by secretion of preformed enzyme, while late secretory events require the de novo synthesis of urokinase. The released enzyme can interact by autocriny with its own receptor present on keratinocytes. The addition to the keratinocyte culture medium of the urokinase A chain can stimulate a concentration-dependent urokinase oversecretion, which is not paralleled by oversecretion of plasminogen activator inhibitor-1. Since stimulation of urokinase production can be obtained by an A chain concentration which was previously shown to be efficient in inducing keratinocyte mobilization in an in vitro migration model system, they hypothesize that this mechanism may be important in vivo during the process of wound repair.

  16. Staphylococcus aureus Biofilm and Planktonic cultures differentially impact gene expression, mapk phosphorylation, and cytokine production in human keratinocytes.

    PubMed

    Secor, Patrick R; James, Garth A; Fleckman, Philip; Olerud, John E; McInnerney, Kate; Stewart, Philip S

    2011-06-21

    Many chronic diseases, such as non-healing wounds are characterized by prolonged inflammation and respond poorly to conventional treatment. Bacterial biofilms are a major impediment to wound healing. Persistent infection of the skin allows the formation of complex bacterial communities termed biofilm. Bacteria living in biofilms are phenotypically distinct from their planktonic counterparts and are orders of magnitude more resistant to antibiotics, host immune response, and environmental stress. Staphylococcus aureus is prevalent in cutaneous infections such as chronic wounds and is an important human pathogen. The impact of S. aureus soluble products in biofilm-conditioned medium (BCM) or in planktonic-conditioned medium (PCM) on human keratinocytes was investigated. Proteomic analysis of BCM and PCM revealed differential protein compositions with PCM containing several enzymes involved in glycolysis. Global gene expression of keratinocytes exposed to biofilm and planktonic S. aureus was analyzed after four hours of exposure. Gene ontology terms associated with responses to bacteria, inflammation, apoptosis, chemotaxis, and signal transduction were enriched in BCM treated keratinocytes. Several transcripts encoding cytokines were also upregulated by BCM after four hours. ELISA analysis of cytokines confirmed microarray results at four hours and revealed that after 24 hours of exposure, S. aureus biofilm induced sustained low level cytokine production compared to near exponential increases of cytokines in planktonic treated keratinocytes. The reduction in cytokines produced by keratinocytes exposed to biofilm was accompanied by suppressed phosphorylation of MAPKs. Chemical inhibition of MAPKs did not drastically reduce cytokine production in BCM-treated keratinocytes suggesting that the majority of cytokine production is mediated through MAPK-independent mechanisms. Collectively the results indicate that S. aureus biofilms induce a distinct inflammatory response

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

    SciTech Connect

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

    1985-08-01

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

  18. Protection from UVB Toxicity in Human Keratinocytes by Thailand Native Herbs Extracts.

    PubMed

    Thongrakard, Visa; Ruangrungsi, Nijsiri; Ekkapongpisit, Maneerat; Isidoro, Ciro; Tencomnao, Tewin

    2014-01-01

    Thai traditional medicine employs a wide range of indigenous herbs in the forms of tincture or tea for the cure of skin and systemic inflammatory diseases. The protection by Thai plants extracts against UVB DNA damage and cytotoxicity was investigated in human keratinocytes. Petroleum ether, dichloromethane and ethanol extracts were prepared from 15 Thai herb species, and the total phenolic and flavonoid contents, the antioxidant and UV-absorbing properties were assessed by standard procedures. Cytoprotective effects were evaluated on the basis of cell survival, caspase-3 activity and pyrimidine dimers determination. High total phenolic and flavonoid contents were found in the ethanol and dichloromethane fractions. Dichloromethane extract of turmeric was shown to possess the highest antioxidant activity. The maximum UV absorptions were found in the ethanol extract of turmeric and in the dichloromethane extract of ginger. These extracts stimulated the synthesis of Thioredoxin 1, an antioxidant protein, and could protect human HaCaT keratinocytes from UV-induced DNA damage and cytotoxicity. The present data support the utilization of turmeric and ginger extracts in anti-UV cosmetic pharmaceuticals. © 2013 The American Society of Photobiology.

  19. Production of human papillomavirus type 16 virions in a keratinocyte cell line.

    PubMed

    Sterling, J; Stanley, M; Gatward, G; Minson, T

    1990-12-01

    Human papillomavirus type 16 (HPV-16) is strongly associated with carcinoma of the cervix, but the complete life cycle of the virus cannot be studied because no experimental system is available in which HPV-16 progeny are produced, and there is currently no source of HPV-16 virus particles. Most cell lines that harbor HPV-16 DNA contain the viral genome as integrated or concatenated DNA in which open reading frames are disrupted or deleted, but a human cervical keratinocyte cell line has been described which maintains HPV-16 DNA in monomeric episomal form (M.A. Stanley, H.M. Brown, M.W. Appleby, and A.C. Minson, Int. J. Cancer 43:672-676, 1989). This cell line was induced to form a stratified differentiating epithelium by grafting onto nude mice. Long-term grafts displayed the histological features of a low-grade cervical dysplasia, and terminally differentiated cells contained amplified levels of HPV-16 DNA, virus capsid antigen, and virus particles. This experimental system appears to permit the completion of the HPV-16 life cycle in virus-containing keratinocytes.

  20. Long-wave ultraviolet light induces phospholipase activation in cultured human epidermal keratinocytes

    SciTech Connect

    Hanson, D.; DeLeo, V. )

    1990-08-01

    Long wave ultraviolet radiation (UVA) has been shown to play an important role in the overall response of skin to solar radiation, including sunburn, tanning, premature aging, and non-melanoma skin cancer. UVA induction of inflammation in human skin is thought to be mediated by membrane lipid derived products. In order to investigate the mechanism of this response we examined the effect of UVA on phospholipid metabolism of human epidermal keratinocytes in culture. Keratinocytes were grown in serum free low calcium medium. The cells were prelabeled with (3H) arachidonic acid or (3H) choline and irradiated with UVA (Honle 2002-Hg vapor lamp). Identification and quantitation of specific membrane phospholipid-derived components was achieved using high-performance liquid chromatography, paper chromatography, and radioimmunoassay. UVA resulted in a linear dose dependent release of (3H) arachidonic acid into medium between 1 and 20 joule/cm2. This response was inhibited in an oxygen-reduced environment. The radiolabel released was predominantly free arachidonate and cyclooxygenase metabolites. Cyclooxygenase metabolites prostaglandin E2 and prostacyclin derivative, 6-keto-prostaglandin F1a, were stimulated following UVA irradiation, but the lipoxygenase metabolite, leukotriene B was not detected. Maximal release was measured immediately after irradiation and changed little over 24 h post-irradiation. UVA stimulated an increase of (3H) choline metabolites glycerophosphorylcholine and phosphorylcholine in media extracts suggesting UVA activation of phospholipase C and phospholipase A2 or diacylglyceride lipase.

  1. HPV-18 confers resistance to TNF-{alpha} in organotypic cultures of human keratinocytes

    SciTech Connect

    Boccardo, Enrique . E-mail: eboccardo@ludwig.org.br; Noya, Francisco; Broker, Thomas R.; Chow, Louise T.; Villa, Luisa L.

    2004-10-25

    The proinflammatory cytokine tumor necrosis factor-alpha (TNF-{alpha}) inhibits normal keratinocytes proliferation. However, many human papillomavirus (HPV)-immortalized or transformed cell lines are resistant to TNF-{alpha} antiproliferative effect. The present study analyzes the effects of TNF-{alpha} on organotypic cultures of primary human keratinocytes (PHKs) that express HPV-18 oncogenes. Raft cultures prepared with PHKs acutely transfected with HPV-18 whole genome or infected with recombinant retroviruses containing only E6/E7 or E7 were treated with 2 nM TNF-{alpha}. While BrdU incorporation into basal/parabasal cells of normal PHKs cultures was markedly inhibited by TNF-{alpha} cultures transfected with HPV-18 whole genome showed proliferation in all cell strata. Furthermore, BrdU incorporation into cultures expressing E6/E7 or E7 was not significantly reduced, indicating that E7 alone confers partial resistance to TNF-{alpha}. Besides, TNF-{alpha} treatment did not alter p16{sup ink4a}, p21{sup cip1}, p27{sup kip1}, or cyclin E levels, but did reduce cyclin A and PCNA levels in sensitive cells.

  2. Apoptosis and accidental cell death in cultured human keratinocytes after thermal injury.

    PubMed

    Matylevitch, N P; Schuschereba, S T; Mata, J R; Gilligan, G R; Lawlor, D F; Goodwin, C W; Bowman, P D

    1998-08-01

    The respective roles of apoptosis and accidental cell death after thermal injury were evaluated in normal human epidermal keratinocytes. By coupling the LIVE/DEAD fluorescence viability assay with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and ultrastructural morphology, these two processes could be distinguished. Cells were grown on glass coverslips with a microgrid pattern so that the results of several staining procedures performed sequentially could be visualized in the same cells after heating at temperatures of up to 72 degrees C for 1 second. After exposure to temperatures of 58 to 59 degrees C, cells died predominantly by apoptosis; viable cells became TUNEL positive, indicating degradation of DNA. After exposure to temperatures of 60 to 66 degrees C, both TUNEL-positive viable cells and TUNEL-positive nonviable cells were observed, indicating that apoptosis and accidental cell death were occurring simultaneously. Cells died almost immediately after exposure to temperatures above 72 degrees C, presumably from heat fixation. The fluorescent mitochondrial probe MitoTracker Orange indicated that cells undergoing apoptosis became TUNEL positive before loss of mitochondrial function. Nucleosomal fragmentation of DNA analyzed by enzyme-linked immunosorbent assay and gel electrophoresis occurred after exposure to temperatures of 58 to 59 degrees C. The characteristic morphological findings of cells undergoing apoptosis, by transmission electron microscopy, included cellular shrinkage, cytoplasmic budding, and relatively intact mitochondria. Depending on temperature and time of exposure, normal human epidermal keratinocytes may die by apoptosis, accidental cell death, or heat fixation.

  3. 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.

  4. Comparative removal of pyrimidine dimers from human epidermal keratinocytes in vivo and in vitro

    SciTech Connect

    Reusch, M.K.; Meager, K.; Leadon, S.A.; Hanawalt, P.C.

    1988-10-01

    We have compared the kinetics for repair of UV-induced cyclobutane pyrimidine dimers in the DNA of keratinocytes in human skin and in cell culture. A small area of the buttocks of volunteers was exposed to UVB-irradiation and biopsies were taken at various time intervals. Epidermal keratinocytes in culture from the same subjects were exposed to UVC with doses chosen to elicit comparable yields of dimers in cellular DNA. The initial density of pyrimidine dimers and the kinetics of their removal were assessed utilizing the dimer-specific T4 endonuclease V and sedimentation of the unlabeled DNA through alkaline sucrose gradients. The position of DNA in the gradients was determined using a monoclonal antibody against random sequences of single-stranded DNA in a sensitive immunoassay. The initial dimer frequency was 3.9-6.7 per 10(8) daltons DNA. About 40% of the dimers were removed within 1 h, 70% in 6 h, and 90% in 24 h for both in vivo and in vitro samples. The early rapid removal phase may represent preferential repair of actively transcribed genes. Our findings reaffirm the usefulness and applicability of cell culture systems to model in vivo repair phenomena. The use of monoclonal antibodies to detect single-stranded DNA in alkaline sucrose gradients may be of value in a variety of studies involving human tissues in which it is not possible to use radioactive labeling of the DNA for the analysis.

  5. Additive Effects of Millimeter Waves and 2-Deoxyglucose Co-Exposure on the Human Keratinocyte Transcriptome

    PubMed Central

    Soubere Mahamoud, Yonis; Aite, Meziane; Martin, Catherine; Zhadobov, Maxim; Sauleau, Ronan; Le Dréan, Yves

    2016-01-01

    Millimeter Waves (MMW) will be used in the next-generation of high-speed wireless technologies, especially in future Ultra-Broadband small cells in 5G cellular networks. Therefore, their biocompatibilities must be evaluated prior to their massive deployment. Using a microarray-based approach, we analyzed modifications to the whole genome of a human keratinocyte model that was exposed at 60.4 GHz-MMW at an incident power density (IPD) of 20 mW/cm2 for 3 hours in athermic conditions. No keratinocyte transcriptome modifications were observed. We tested the effects of MMWs on cell metabolism by co-treating MMW-exposed cells with a glycolysis inhibitor, 2-deoxyglucose (2dG, 20 mM for 3 hours), and whole genome expression was evaluated along with the ATP content. We found that the 2dG treatment decreased the cellular ATP content and induced a high modification in the transcriptome (632 coding genes). The affected genes were associated with transcriptional repression, cellular communication and endoplasmic reticulum homeostasis. The MMW/2dG co-treatment did not alter the keratinocyte ATP content, but it did slightly alter the transcriptome, which reflected the capacity of MMW to interfere with the bioenergetic stress response. The RT-PCR-based validation confirmed 6 MMW-sensitive genes (SOCS3, SPRY2, TRIB1, FAM46A, CSRNP1 and PPP1R15A) during the 2dG treatment. These 6 genes encoded transcription factors or inhibitors of cytokine pathways, which raised questions regarding the potential impact of long-term or chronic MMW exposure on metabolically stressed cells. PMID:27529420

  6. Reciprocal regulation of p63 by C/EBP delta in human keratinocytes

    PubMed Central

    Borrelli, Serena; Testoni, Barbara; Callari, Maurizio; Alotto, Daniela; Castagnoli, Carlotta; Romano, Rose-Anne; Sinha, Satrajit; Viganò, Alessandra M; Mantovani, Roberto

    2007-01-01

    Background Genetic experiments have clarified that p63 is a key transcription factor governing the establishment and maintenance of multilayered epithelia. Key to our understanding of p63 strategy is the identification of target genes. We perfomed an RNAi screening in keratinocytes for p63, followed by profiling analysis. Results C/EBPδ, member of a family with known roles in differentiation pathways, emerged as a gene repressed by p63. We validated C/EBPδ as a primary target of ΔNp63α by RT-PCR and ChIP location analysis in HaCaT and primary cells. C/EBPδ is differentially expressed in stratification of human skin and it is up-regulated upon differentiation of HaCaT and primary keratinocytes. It is bound to and activates the ΔNp63 promoter. Overexpression of C/EBPδ leads to alteration in the normal profile of p63 isoforms, with the emergence of ΔNp63β and γ, and of the TA isoforms, with different kinetics. In addition, there are changes in the expression of most p63 targets. Inactivation of C/EBPδ leads to gene expression modifications, in part due to the concomitant repression of ΔNp63α. Finally, C/EBPδ is found on the p63 targets in vivo by ChIP analysis, indicating that coregulation is direct. Conclusion Our data highlight a coherent cross-talk between these two transcription factors in keratinocytes and a large sharing of common transcriptional targets. PMID:17903252

  7. Fibroblast Growth Factor Type 2 Signaling Is Critical for DNA Repair in Human Keratinocyte Stem Cells

    PubMed Central

    Harfouche, Ghida; Vaigot, Pierre; Rachidi, Walid; Rigaud, Odile; Moratille, Sandra; Marie, Mélanie; Lemaitre, Gilles; Fortunel, Nicolas O; Martin, Michèle T

    2010-01-01

    Tissue stem cells must be endowed with superior maintenance and repair systems to ensure genomic stability over multiple generations, which would be less necessary in more differentiated cells. We previously reported that human keratinocyte stem cells were more resistant to ionizing radiation toxicity than their direct progeny, the keratinocyte progenitor cells. In the present study we addressed the mechanisms underlying this difference. Investigations of DNA repair showed that both single and double DNA strand breaks were repaired more rapidly and more efficiently in stem cells than in progenitors. As cell signaling is a key regulatory step in the management of DNA damage, a gene profiling study was performed. Data revealed that several genes of the fibroblast growth factor type 2 (FGF2) signaling pathway were induced by DNA damage in stem cells and not in progenitors. Furthermore, an increased content of the FGF2 protein was found in irradiated stem cells, both for the secreted and the cellular forms of the protein. To examine the role of endogenous FGF2 in DNA repair, stem cells were exposed to FGF2 pathway inhibitors. Blocking the FGF2 receptor (FGF receptor 1) or the kinase (Ras-mitogen-activated protein kinase 1) resulted in a inhibition of single and double DNA strand-break repair in the keratinocyte stem cells. Moreover, supplementing the progenitor cells with exogenous FGF2 activated their DNA repair. We propose that, apart from its well-known role as a strong mitogen and prosurvival factor, FGF2 helps to maintain genomic integrity in stem cells by activating stress-induced DNA repair. Stem Cells 2010; 28:1639–1648. PMID:20681019

  8. Fibroblast growth factor type 2 signaling is critical for DNA repair in human keratinocyte stem cells.

    PubMed

    Harfouche, Ghida; Vaigot, Pierre; Rachidi, Walid; Rigaud, Odile; Moratille, Sandra; Marie, Mélanie; Lemaitre, Gilles; Fortunel, Nicolas O; Martin, Michèle T

    2010-09-01

    Tissue stem cells must be endowed with superior maintenance and repair systems to ensure genomic stability over multiple generations, which would be less necessary in more differentiated cells. We previously reported that human keratinocyte stem cells were more resistant to ionizing radiation toxicity than their direct progeny, the keratinocyte progenitor cells. In the present study we addressed the mechanisms underlying this difference. Investigations of DNA repair showed that both single and double DNA strand breaks were repaired more rapidly and more efficiently in stem cells than in progenitors. As cell signaling is a key regulatory step in the management of DNA damage, a gene profiling study was performed. Data revealed that several genes of the fibroblast growth factor type 2 (FGF2) signaling pathway were induced by DNA damage in stem cells and not in progenitors. Furthermore, an increased content of the FGF2 protein was found in irradiated stem cells, both for the secreted and the cellular forms of the protein. To examine the role of endogenous FGF2 in DNA repair, stem cells were exposed to FGF2 pathway inhibitors. Blocking the FGF2 receptor (FGF receptor 1) or the kinase (Ras-mitogen-activated protein kinase 1) resulted in a inhibition of single and double DNA strand-break repair in the keratinocyte stem cells. Moreover, supplementing the progenitor cells with exogenous FGF2 activated their DNA repair. We propose that, apart from its well-known role as a strong mitogen and prosurvival factor, FGF2 helps to maintain genomic integrity in stem cells by activating stress-induced DNA repair.

  9. Pseudomonas-Derived Ceramidase Induces Production of Inflammatory Mediators from Human Keratinocytes via Sphingosine-1-Phosphate

    PubMed Central

    Oizumi, Ami; Nakayama, Hitoshi; Okino, Nozomu; Iwahara, Chihiro; Kina, Katsunari; Matsumoto, Ryo; Ogawa, Hideoki; Takamori, Kenji; Ito, Makoto; Suga, Yasushi; Iwabuchi, Kazuhisa

    2014-01-01

    Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD). A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P) stimulated the production of inflammatory mediators such as TNF-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed “3D keratinocytes”), which form a stratum corneum. PaCDase alone did not affect TNF-α gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-α, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-α, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. Collectively, these findings suggest that (i) 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii) S1P induces the production of TNF

  10. Evaluation of the toxicological risk to humans of caulerpenyne using human hematopoietic progenitors, melanocytes, and keratinocytes in culture.

    PubMed

    Parent-Massin, D; Fournier, V; Amade, P; Lemée, R; Durand-Clément, M; Delescluse, C; Pesando, D

    1996-01-01

    The extensive growth of Caulerpa taxifolia in the Mediterranean sea produces important quantities of bioactive secondary metabolites unable to enter the food chain. The cytotoxic effects of caulerpenyne, the major secondary metabolite from C. taxifolia, was studied in different in vitro models: skin cells, primary cultures of melanocytes and keratinocytes, immortalized keratinocytes (HaCaT and HESV), and bone marrow cells (hematopoietic progenitors CFU-GM). Typical dose-response curves from neutral red uptake and MTT assays were recorded in all models with IC50 ranging from 6 to 24 microM. Hematopoietic progenitors were more sensitive to caulerpenyne than melanocyte and keratinocyte cell lines, which could be due to their higher proliferative rate. The distribution of aggregates in colonies, macroclusters, and microclusters of hematopoietic progenitors was also altered in the presence of caulerpenyne. From our evaluation of the caulerpenyne concentrations required to result in cellular toxicity, the risks of cutaneous and/or food intoxication to humans may be considered minimal.

  11. Platelet lysate induces in vitro wound healing of human keratinocytes associated with a strong proinflammatory response.

    PubMed

    El Backly, Rania; Ulivi, Valentina; Tonachini, Laura; Cancedda, Ranieri; Descalzi, Fiorella; Mastrogiacomo, Maddalena

    2011-07-01

    Platelet lysates (PL), which are derived from platelets, are cocktails of growth factors and cytokines that can promote tissue regeneration. Until today, most studies have focused on growth factor content of platelets rather than on their potential as a reservoir of mediators and cytokines. Taking advantage of an in vitro scratch assay performed under both normal and inflammatory conditions, in the present work, we report that at physiologic concentrations, PL enhanced wound closure rates of NCTC 2544 human keratinocytes. This effect was clearly detectable 6 h after wounding. Moreover, PL induced a strong cell actin cytoskeletal re-organization that persisted up to 24 h. The accelerated wound closure promoted by PL, in either presence or absence of serum, was associated with a high expression of the inflammatory cytokine interleukin-8. Further, after 24 h PL treatment, confluent keratinocytes also expressed low amounts of interleukin-8 and of the antimicrobial peptide neutrophil gelatinase-associated lipocalin, which dramatically increased under inflammatory conditions. These effects were associated with activation of the inflammatory pathways, p38 mitogen-activated protein kinase, and NF-κB. Our findings support the concept that platelet-derived preparations could accelerate regeneration of difficult-to-heal wounds by triggering an inflammatory cascade and having an antimicrobial role.

  12. Cysteine Prevents the Reduction in Keratin Synthesis Induced by Iron Deficiency in Human Keratinocytes.

    PubMed

    Miniaci, Maria Concetta; Irace, Carlo; Capuozzo, Antonella; Piccolo, Marialuisa; Di Pascale, Antonio; Russo, Annapina; Lippiello, Pellegrino; Lepre, Fabio; Russo, Giulia; Santamaria, Rita

    2016-02-01

    L-cysteine is currently recognized as a conditionally essential sulphur amino acid. Besides contributing to many biological pathways, cysteine is a key component of the keratin protein by its ability to form disulfide bridges that confer strength and rigidity to the protein. In addition to cysteine, iron represents another critical factor in regulating keratins expression in epidermal tissues, as well as in hair follicle growth and maturation. By focusing on human keratinocytes, the aim of this study was to evaluate the effect of cysteine supplementation as nutraceutical on keratin biosynthesis, as well as to get an insight on the interplay of cysteine availability and cellular iron status in regulating keratins expression in vitro. Herein we demonstrate that cysteine promotes a significant up-regulation of keratins expression as a result of de novo protein synthesis, while the lack of iron impairs keratin expression. Interestingly, cysteine supplementation counteracts the adverse effect of iron deficiency on cellular keratin expression. This effect was likely mediated by the up-regulation of transferrin receptor and ferritin, the main cellular proteins involved in iron homeostasis, at last affecting the labile iron pool. In this manner, cysteine may also enhance the metabolic iron availability for DNA synthesis without creating a detrimental condition of iron overload. To the best of our knowledge, this is one of the first study in an in vitro keratinocyte model providing evidence that cysteine and iron cooperate for keratins expression, indicative of their central role in maintaining healthy epithelia.

  13. Analyses of the secondary particle radiation and the DNA damage it causes to human keratinocytes

    SciTech Connect

    Lebel E.; Rusek A.; Sivertz, M.; Yip, K.; Thompson, K.; Tafrov, S.

    2011-11-22

    High-energy protons, and high mass and energy ions, along with the secondary particles they produce, are the main contributors to the radiation hazard during space explorations. Skin, particularly the epidermis, consisting mainly of keratinocytes with potential for proliferation and malignant transformation, absorbs the majority of the radiation dose. Therefore, we used normal human keratinocytes to investigate and quantify the DNA damage caused by secondary radiation. Its manifestation depends on the presence of retinol in the serum-free media, and is regulated by phosphatidylinositol 3-kinases. We simulated the generation of secondary radiation after the impact of protons and iron ions on an aluminum shield. We also measured the intensity and the type of the resulting secondary particles at two sample locations; our findings agreed well with our predictions. We showed that secondary particles inflict DNA damage to different extents, depending on the type of primary radiation. Low-energy protons produce fewer secondary particles and cause less DNA damage than do high-energy protons. However, both generate fewer secondary particles and inflict less DNA damage than do high mass and energy ions. The majority of cells repaired the initial damage, as denoted by the presence of 53BPI foci, within the first 24 hours after exposure, but some cells maintained the 53BP1 foci longer.

  14. Analyses of the Secondary Particle Radiation and the DNA Damage it Causes to Human Keratinocytes

    SciTech Connect

    Lebel E. A.; Tafrov S.; Rusek, A.; Sivertz, M. B.; Yip, K.; Thompson, K. H.

    2011-11-01

    High-energy protons, and high mass and energy ions, along with the secondary particles they produce, are the main contributors to the radiation hazard during space explorations. Skin, particularly the epidermis, consisting mainly of keratinocytes with potential for proliferation and malignant transformation, absorbs the majority of the radiation dose. Therefore, we used normal human keratinocytes to investigate and quantify the DNA damage caused by secondary radiation. Its manifestation depends on the presence of retinol in the serum-free media, and is regulated by phosphatidylinositol 3-kinases. We simulated the generation of secondary radiation after the impact of protons and iron ions on an aluminum shield. We also measured the intensity and the type of the resulting secondary particles at two sample locations; our findings agreed well with our predictions. We showed that secondary particles inflict DNA damage to different extents, depending on the type of primary radiation. Low-energy protons produce fewer secondary particles and cause less DNA damage than do high-energy protons. However, both generate fewer secondary particles and inflict less DNA damage than do high mass and energy ions. The majority of cells repaired the initial damage, as denoted by the presence of 53BPI foci, within the first 24 hours after exposure, but some cells maintained the 53BP1 foci longer.

  15. Solar Simulated Ultraviolet Radiation Induces Global Histone Hypoacetylation in Human Keratinocytes.

    PubMed

    Zhang, Xiaoru; Kluz, Thomas; Gesumaria, Lisa; Matsui, Mary S; Costa, Max; Sun, Hong

    2016-01-01

    Ultraviolet radiation (UVR) from sunlight is the primary effector of skin DNA damage. Chromatin remodeling and histone post-translational modification (PTM) are critical factors in repairing DNA damage and maintaining genomic integrity, however, the dynamic changes of histone marks in response to solar UVR are not well characterized. Here we report global changes in histone PTMs induced by solar simulated UVR (ssUVR). A decrease in lysine acetylation of histones H3 and H4, particularly at positions of H3 lysine 9, lysine 56, H4 lysine 5, and lysine 16, was found in human keratinocytes exposed to ssUVR. These acetylation changes were highly associated with ssUVR in a dose-dependent and time-specific manner. Interestingly, H4K16ac, a mark that is crucial for higher order chromatin structure, exhibited a persistent reduction by ssUVR that was transmitted through multiple cell divisions. In addition, the enzymatic activities of histone acetyltransferases were significantly reduced in irradiated cells, which may account for decreased global acetylation. Moreover, depletion of histone deacetylase SIRT1 in keratinocytes rescued ssUVR-induced H4K16 hypoacetylation. These results indicate that ssUVR affects both HDAC and HAT activities, leading to reduced histone acetylation.

  16. Phevalin (aureusimine B) production by Staphylococcus aureus biofilm and impacts on human keratinocyte gene expression.

    PubMed

    Secor, Patrick R; Jennings, Laura K; James, Garth A; Kirker, Kelly R; Pulcini, Elinor Delancey; McInnerney, Kate; Gerlach, Robin; Livinghouse, Tom; Hilmer, Jonathan K; Bothner, Brian; Fleckman, Philip; Olerud, John E; Stewart, Philip S

    2012-01-01

    Staphylococcus aureus biofilms are associated with chronic skin infections and are orders of magnitude more resistant to antimicrobials and host responses. S. aureus contains conserved nonribosomal peptide synthetases that produce the cyclic dipeptides tyrvalin and phevalin (aureusimine A and B, respectively). The biological function of these compounds has been speculated to be involved in virulence factor gene expression in S. aureus, protease inhibition in eukaryotic cells, and interspecies bacterial communication. However, the exact biological role of these compounds is unknown. Here, we report that S. aureus biofilms produce greater amounts of phevalin than their planktonic counterparts. Phevalin had no obvious impact on the extracellular metabolome of S. aureus as measured by high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. When administered to human keratinocytes, phevalin had a modest effect on gene expression. However, conditioned medium from S. aureus spiked with phevalin amplified differences in keratinocyte gene expression compared to conditioned medium alone. Phevalin may be exploited as potential biomarker and/or therapeutic target for chronic, S. aureus biofilm-based infections.

  17. Solar Simulated Ultraviolet Radiation Induces Global Histone Hypoacetylation in Human Keratinocytes

    PubMed Central

    Zhang, Xiaoru; Kluz, Thomas; Gesumaria, Lisa; Matsui, Mary S.; Costa, Max; Sun, Hong

    2016-01-01

    Ultraviolet radiation (UVR) from sunlight is the primary effector of skin DNA damage. Chromatin remodeling and histone post-translational modification (PTM) are critical factors in repairing DNA damage and maintaining genomic integrity, however, the dynamic changes of histone marks in response to solar UVR are not well characterized. Here we report global changes in histone PTMs induced by solar simulated UVR (ssUVR). A decrease in lysine acetylation of histones H3 and H4, particularly at positions of H3 lysine 9, lysine 56, H4 lysine 5, and lysine 16, was found in human keratinocytes exposed to ssUVR. These acetylation changes were highly associated with ssUVR in a dose-dependent and time-specific manner. Interestingly, H4K16ac, a mark that is crucial for higher order chromatin structure, exhibited a persistent reduction by ssUVR that was transmitted through multiple cell divisions. In addition, the enzymatic activities of histone acetyltransferases were significantly reduced in irradiated cells, which may account for decreased global acetylation. Moreover, depletion of histone deacetylase SIRT1 in keratinocytes rescued ssUVR-induced H4K16 hypoacetylation. These results indicate that ssUVR affects both HDAC and HAT activities, leading to reduced histone acetylation. PMID:26918332

  18. IKKα regulates human keratinocyte migration through surveillance of the redox environment

    PubMed Central

    Lisse, Thomas S.

    2017-01-01

    ABSTRACT Although the functions of H2O2 in epidermal wound repair are conserved throughout evolution, the underlying signaling mechanisms are largely unknown. In this study we used human keratinocytes (HEK001) to investigate H2O2-dependent wound repair mechanisms. Scratch wounding led to H2O2 production in two or three cell layers at the wound margin within ∼30 min and subsequent cysteine modification of proteins via sulfenylation. Intriguingly, exogenous H2O2 treatment resulted in preferential sulfenylation of keratinocytes that adopted a migratory phenotype and detached from neighboring cells, suggesting that one of the primary functions of H2O2 is to stimulate signaling factors involved in cell migration. Based on previous findings that revealed epidermal growth factor receptor (EGFR) involvement in H2O2-dependent cell migration, we analyzed oxidation of a candidate upstream target, the inhibitor of κB kinase α (IKKα; encoded by CHUK), as a mechanism of action. We show that IKKα is sulfenylated at a conserved cysteine residue in the kinase domain, which correlates with de-repression of EGF promoter activity and increased EGF expression. Thus, this indicates that IKKα promotes migration through dynamic interactions with the EGF promoter depending on the redox state within cells. PMID:28122935

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

    PubMed

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

    2017-02-01

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

  20. Distinct Histone Modifications Modulate DEFB1 Expression in Human Vaginal Keratinocytes in Response to Lactobacillus spp.

    PubMed

    Lee, Jaehyouk; Jang, Ara; Kim, Jin Wook; Han, Jun Hyun; Chun, Byung Hee; Jung, Hye Su; Jeon, Che Ok; Myung, Soon Chul

    2017-05-15

    Vaginal commensal lactobacilli are considered to contribute significantly to the control of vaginal microbiota by competing with other microflora for adherence to the vaginal epithelium and by producing antimicrobial compounds. However, the molecular mechanisms of symbiotic prokaryotic-eukaryotic communication in the vaginal ecosystem remain poorly understood. Here, we showed that both DNA methylation and histone modifications were associated with expression of the DEFB1 gene, which encodes the antimicrobial peptide human β-defensin-1, in vaginal keratinocyte VK2/E6E7 cells. We investigated whether exposure to Lactobacillus gasseri and Lactobacillus reuteri would trigger the epigenetic modulation of DEFB1 expression in VK2/E6E7 cells in a bacterial species-dependent manner. While enhanced expression of DEFB1 was observed when VK2/E6E7 cells were exposed to L. gasseri, treatment with L. reuteri resulted in reduced DEFB1 expression. Moreover, L. gasseri stimulated the recruitment of active histone marks and, in contrast, L. reuteri led to the decrease of active histone marks at the DEFB1 promoter. It was remarkable that distinct histone modifications within the same promoter region of DEFB1 were mediated by L. gasseri and L. reuteri. Therefore, our study suggested that one of the underlying mechanisms of DEFB1 expression in the vaginal ecosystem might be associated with the epigenetic crosstalk between individual Lactobacillus spp. and vaginal keratinocytes.

  1. Effect of Porcine Placenta Extract from Subcritical Water Extraction on Photodamage in Human Keratinocytes

    PubMed Central

    Han, Bok Kyung; Choi, Hyeon-Son; Hong, Yang Hee; Jung, Eun Young

    2015-01-01

    The objective of this study was to evaluated the photoprotective effects of porcine placenta extract (PPE) on ultraviolet B (UVB)-induced oxidative stress in human keratinocytes (HaCaT) to evaluate its functional activities as a skin food ingredient. PPE prepared by subcritical water extraction was termed SPE, and subsequently digested by enzymes to prepare E-SPE. Increased intracellular reactive oxygen species (ROS) levels (192.0%) induced by UVB were decreased by SPE and E-SPE. SPE had more effective ROS scavenging activity than E-SPE treatment. UVB treatment increased expression of tissue inhibitor of metalloproteinase 1 (TIMP-1), and this elevated expression was decreased by E-SPE treatment. High-dose treatment with E-SPE (50 and 100 µg/mL) reduced TIMP-1 expression levels of UVB-C (control) to 33.5 and 34.6%, respectively. In contrast, at low SPE doses (1 and 10 µg/mL), the treatment slightly decreased TIMP-1 expression levels to 73.3% and 71.3% of UVB-C, respectively. In conclusion, the present study demonstrated the protective effect of SPE and E-SPE against UVB damage in keratinocytes via ROS scavenging, down-regulating MMP-2 expression and up-regulating TIMP-1 expression. This highlights the potential for SPE as an ingredient in the preparation of functional food against photoaging. PMID:26761824

  2. Immortalization of primary human keratinocytes by the helix–loop–helix protein, Id-1

    PubMed Central

    Alani, Rhoda M.; Hasskarl, Jens; Grace, Miranda; Hernandez, Maria-Clementia; Israel, Mark A.; Münger, Karl

    1999-01-01

    Basic helix–loop–helix (bHLH) DNA-binding proteins have been demonstrated to regulate tissue-specific transcription within multiple cell lineages. The Id family of helix–loop–helix proteins does not possess a basic DNA-binding domain and functions as a negative regulator of bHLH proteins. Overexpression of Id proteins within a variety of cell types has been shown to inhibit their ability to differentiate under appropriate conditions. We demonstrate that ectopic expression of Id-1 leads to activation of telomerase activity and immortalization of primary human keratinocytes. These immortalized cells have a decreased capacity to differentiate as well as activate phosphorylation of the retinoblastoma protein. Additionally, these cells acquire an impaired p53-mediated DNA-damage response as a late event in immortalization. We conclude that bHLH proteins play a pivotal role in regulating normal keratinocyte growth and differentiation, which can be disrupted by the immortalizing functions of Id-1 through activation of telomerase activity and inactivation of the retinoblastoma protein. PMID:10449746

  3. Regulation of tight junction permeability by sodium caprate in human keratinocytes and reconstructed epidermis

    SciTech Connect

    Kurasawa, Masumi; Kuroda, Shohei; Kida, Naoko; Murata, Michiyo; Oba, Ai; Yamamoto, Takuya; Sasaki, Hiroyuki

    2009-04-03

    Tight junctions (TJs) restrict paracellular flux of water and solutes in epithelia and endothelia. In epidermis, the physiological role of TJs is not fully understood. In this study, sodium caprate (C10), which dilates intestinal TJs, was applied to cultured human epidermal keratinocytes and reconstructed human epidermis to investigate the effects of C10 on epidermal TJs. C10 treatment decreased transepithelial electrical resistance and increased paracellular permeability, although Western blots showed that the expression of TJ-related transmembrane proteins was not decreased. The effects of C10 were reversible. Immunofluorescence microscopy and immuno-replica electron microscopy showed that the localization of TJ strands were disintegrated, concomitant with the dispersion and/or disappearance of TJ-related molecules from the cell surface. These findings suggest that C10 impairs barrier function by physically disrupting TJ conformation in the epidermis. Furthermore, these results also show that proper localization of the molecules on the cellular membrane is important for TJ barrier function.

  4. Hesperidin Attenuates Ultraviolet B-Induced Apoptosis by Mitigating Oxidative Stress in Human Keratinocytes

    PubMed Central

    Hewage, Susara Ruwan Kumara Madduma; Piao, Mei Jing; Kang, Kyoung Ah; Ryu, Yea Seong; Han, Xia; Oh, Min Chang; Jung, Uhee; Kim, In Gyu; Hyun, Jin Won

    2016-01-01

    Human skin cells undergo pathophysiological processes via generation of reactive oxygen species (ROS) upon excessive exposure to ultraviolet B (UVB) radiation. This study investigated the ability of hesperidin (C28H34O15) to prevent apoptosis due to oxidative stress generated through UVB-induced ROS. Hesperidin significantly scavenged ROS generated by UVB radiation, attenuated the oxidation of cellular macromolecules, established mitochondrial membrane polarization, and prevented the release of cytochrome c into the cytosol. Hesperidin downregulated expression of caspase-9, caspase-3, and Bcl-2-associated X protein, and upregulated expression of B-cell lymphoma 2. Hesperidin absorbed wavelengths of light within the UVB range. In summary, hesperidin shielded human keratinocytes from UVB radiation-induced damage and apoptosis via its antioxidant and UVB absorption properties. PMID:26797112

  5. Differential effects of selenite and selenate on human melanocytes, keratinocytes, and melanoma cells.

    PubMed

    Bandura, Laura; Drukala, Justyna; Wolnicka-Glubisz, Agnieszka; Björnstedt, Mikael; Korohoda, Wlodzimierz

    2005-04-01

    Among the substances that attracted the attention of oncologists in recent years are selenium-containing compounds, both inorganic and organic. Several epidemiological studies have shown an inverse correlation between selenium intake and cancer incidence. In the experiments reported here, we compared the effects of 2 inorganic selenium-containing salts that differed in the level of selenium oxidation, selenite IV and selenate VI. We tested the effects of these 2 compounds on cell survival and growth, cell cycle processing, cell morphology, cytoskeleton, and lipid peroxidation in 3 human skin cell types: normal keratinocytes, melanocytes, and human melanoma cell line HTB140. The different effects of selenite and selenate on the viability, growth, and morphology of normal cells and tumor cells are reported and provide a base for future research and treatment of some neoplastic diseases. The attention is paid to cell apoptosis induced by selenite and not by selenate, and the effects of tested substances on thioredoxin reductase system are postulated.

  6. Dectin-1 activation induces proliferation and migration of human keratinocytes enhancing wound re-epithelialization.

    PubMed

    van den Berg, Linda M; Zijlstra-Willems, Esther M; Richters, Cornelia D; Ulrich, Magda M W; Geijtenbeek, Teunis B H

    2014-01-01

    Beta-glucans in temporary wound dressings have immuno-stimulatory capacities and have been shown to enhance wound healing in burn patients. Curdlan is a 1,3-linked bacterial/fungal derived beta-glucan that induces inflammatory responses via the C-type lectin receptor dectin-1 on dendritic cells (DCs). Here we investigated the effect of beta-glucan curdlan and the role of dectin-1 expressed by keratinocytes (KCs) in wound healing. Curdlan enhanced migration, proliferation and wound closure of human KCs in a dectin-1 dependent manner, both in vitro and ex vivo. Our data suggest that curdlan induces human KC proliferation and migration and could therefore be used in creams to enhance wound healing.

  7. Nuclear DNA damage-triggered NLRP3 inflammasome activation promotes UVB-induced inflammatory responses in human keratinocytes

    SciTech Connect

    Hasegawa, Tatsuya Nakashima, Masaya; Suzuki, Yoshiharu

    2016-08-26

    Ultraviolet (UV) radiation in sunlight can result in DNA damage and an inflammatory reaction of the skin commonly known as sunburn, which in turn can lead to cutaneous tissue disorders. However, little has been known about how UV-induced DNA damage mediates the release of inflammatory mediators from keratinocytes. Here, we show that UVB radiation intensity-dependently increases NLRP3 gene expression and IL-1β production in human keratinocytes. Knockdown of NLRP3 with siRNA suppresses UVB-induced production of not only IL-1β, but also other inflammatory mediators, including IL-1α, IL-6, TNF-α, and PGE{sub 2}. In addition, inhibition of DNA damage repair by knockdown of XPA, which is a major component of the nucleotide excision repair system, causes accumulation of cyclobutane pyrimidine dimer (CPD) and activation of NLRP3 inflammasome. In vivo immunofluorescence analysis confirmed that NLRP3 expression is also elevated in UV-irradiated human epidermis. Overall, our findings indicate that UVB-induced DNA damage initiates NLRP3 inflammasome activation, leading to release of various inflammatory mediators from human keratinocytes. - Highlights: • UVB radiation induces NLRP3 inflammasome activation in human keratinocytes. • NLRP3 knockdown suppresses production of UVB-induced inflammatory mediators. • UVB-induced DNA damage triggers NLRP3 inflammasome activation. • NLRP3 expression in human epidermis is elevated in response to UV radiation.

  8. Keratinocytes and fibroblasts in a human skin equivalent model enhance melanocyte survival and melanin synthesis after ultraviolet irradiation.

    PubMed

    Archambault, M; Yaar, M; Gilchrest, B A

    1995-05-01

    To investigate paracrine effects of fibroblasts and keratinocytes on melanocyte behavior after ultraviolet (UV) irradiation, we compared an in vitro skin equivalent model with melanocyte cultures. Human melanocytes were maintained alone in monolayer cultures or on dermal equivalents with or without keratinocytes and were irradiated daily with solar-simulated light. After seven daily UV irradiations, monolayer melanocytes displayed dose-dependent increases in cellular damage. In contrast, melanocytes on dermal equivalents survived strikingly better. Moreover, UV-irradiated skin equivalent melanocytes became highly dendritic as compared with sham-irradiated cells, closely mimicking their morphology in UV-irradiated skin. In addition, in skin equivalents melanocytes migrated from the center to the periphery of the keratinocyte layer after UV irradiation. Melanin production per culture, as measured by 14C-dihydroxyphenylalanine incorporation, was consistently higher in skin equivalent melanocytes than in monolayer melanocytes from the same donor, and it was highest in melanocytes from skin equivalents containing both keratinocytes and fibroblasts. Our data strongly suggest that fibroblasts and keratinocytes modulate melanocyte function in skin. The skin equivalent is a valuable model for investigating paracrine effects on melanocytes after UV irradiation.

  9. 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.

  10. Rho kinase inhibitor Y-27632 prolongs the life span of adult human keratinocytes, enhances skin equivalent development, and facilitates lentiviral transduction.

    PubMed

    van den Bogaard, Ellen H; Rodijk-Olthuis, Diana; Jansen, Patrick A M; van Vlijmen-Willems, Ivonne M J J; van Erp, Piet E; Joosten, Irma; Zeeuwen, Patrick L J M; Schalkwijk, Joost

    2012-09-01

    The use of tissue-engineered human skin equivalents (HSE) for fundamental research and industrial application requires the expansion of keratinocytes from a limited number of skin biopsies donated by adult healthy volunteers or patients. A pharmacological inhibitor of Rho-associated protein kinases, Y-27632, was recently reported to immortalize neonatal human foreskin keratinocytes. Here, we investigated the potential use of Y-27632 to expand human adult keratinocytes and evaluated its effects on HSE development and in vitro gene delivery assays. Y-27632 was found to significantly increase the life span of human adult keratinocytes (up to five to eight passages). The epidermal morphology of HSEs generated from high-passage, Y-27632-treated keratinocytes resembled the native epidermis and was improved by supplementing Y-27632 during the submerged phase of HSE development. In addition, Y-27632-treated keratinocytes responded normally to inflammatory stimuli, and could be used to generate HSEs with a psoriatic phenotype, upon stimulation with relevant cytokines. Furthermore, Y-27632 significantly enhanced both lentiviral transduction efficiency of primary adult keratinocytes and epidermal morphology of HSEs generated thereof. Our study indicates that Y-27632 is a potentially powerful tool that is used for a variety of applications of adult human keratinocytes.

  11. Human papillomavirus in oral lesions.

    PubMed

    González, Joaquín V; Gutiérrez, Rafael A; Keszler, Alicia; Colacino, Maria del Carmen; Alonio, Lidia V; Teyssie, Angelica R; Picconi, Maria Alejandra

    2007-01-01

    Growing evidence suggests a role for human papillomavirus (HPV) in oral cancer; however its involvement is still controversial. This study evaluates the frequency of HPV DNA in a variety of oral lesions in patients from Argentina. A total of 77 oral tissue samples from 66 patients were selected (cases); the clinical-histopathological diagnoses corresponded to: 11 HPV- associated benign lesions, 8 non-HPV associated benign lesions, 33 premalignant lesions and 25 cancers. Sixty exfoliated cell samples from normal oral mucosa were used as controls. HPV detection and typing were performed by polymerase chain reaction (PCR) using primers MY09, 11, combined with RFLP or alternatively PCR using primers GP5+, 6+ combined with dot blot hybridization. HPV was detected in 91.0% of HPV- associated benign lesions, 14.3% of non-HPV associated benign lesions, 51.5% of preneoplasias and 60.0% of cancers. No control sample tested HPV positive. In benign HPV- associated lesions, 30.0% of HPV positive samples harbored high-risk types, while in preneoplastic lesions the value rose to 59.9%. In cancer lesions, HPV detection in verrucous carcinoma was 88.9% and in squamous cell carcinoma 43.8%, with high-risk type rates of 75.5% and 85.6%, respectively. The high HPV frequency detected in preneoplastic and neoplastic lesions supports an HPV etiological role in at least a subset of oral cancers.

  12. Synergistic phototoxic effects of glycolic acid in a human keratinocyte cell line (HaCaT).

    PubMed

    Lai, Wan-Wen; Hsiao, Yu-Ping; Chung, Jing-Gung; Wei, Yau-Huei; Cheng, Ya-Wen; Yang, Jen-Hung

    2011-12-01

    Glycolic acid (GA) has been widely used in cosmetic agents and superficial chemical peeling in recent years. It has long been concerned that UV irradiation would enhance the photosensitivity of GA on human skin. Therefore, it is mandatory to explore the biologic effects of concomitant exposure of GA and UV irradiation in human keratinocytes. The aim of the study is to explore the effects of concomitant exposure of GA and UVB in a human keratinocyte cell line (HaCaT). We used HaCaT to investigate the effects of GA (5mM), UVB (50mJ/cm(2)), and co-treatment with GA and UVB (GA+UVB) in human keratinocytes. We used a phase contrast microscope to observe morphological changes of the cells, and employed flow cytometry to detect cell viability, cell cycle, and mitochondrial membrane potential (MMP), and intracellular reactive oxygen species (ROS) levels. Cell damage was detected by DAPI stain, and Western blot was used to detect the activities of apoptosis- related and cycle checkpoint-related proteins such as Bax, Bcl-2, caspases-3, -4, -9, Endo G, AIF, and p21, p27, p53, cdk2, cyclin E, cyclin A. We found that either GA or UVB alone had inhibitory effect on cell proliferation, and co-treatment with GA and UVB had synergistic anti-proliferative effect. GA alone did not affect the cell cycle, and UVB induced HaCaT cells accumulated at S phase, and co-treatment with GA and UVB arrested cells at S phase more prominently. Moreover, all the treatment with GA, UVB, and GA+UVB in HaCaT cells induced apoptosis. We further demonstrated that GA had synergistic apoptotic effect in human keratinocytes. GA and UVB both had effects on the decline of MMP and increase of ROS release, and GA had synergistic increase in the level of ROS in UVB-treated HaCaT cells. Besides, co-treatment with GA and UVB had synergistic effect on apoptosis through the over-expressions of Bax, p21, p53, caspases-3, -4, -9, Endo G and AIF, and confocal microscopy disclosed translocation of AIF and Endo G from

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

    PubMed

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

    2017-01-01

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

  14. Chromium III histidinate exposure modulates antioxidant gene expression in HaCaT human keratinocytes exposed to oxidative stress

    USDA-ARS?s Scientific Manuscript database

    While the toxicity of hexavalent chromium is well established, trivalent Cr (Cr(III)) is an essential nutrient involved in insulin and glucose homeostasis. Recently, antioxidant effects of chromium (III) histidinate (Cr(III)His) were reported in HaCaT human keratinocytes exposed to oxidative stress...

  15. EFFECT OF ARSENICALS ON THE EXPRESSION OF CELL CYCLE PROTEINS AND EARLY SIGNALING EVENTS IN PRIMARY HUMAN KERATINOCYTES.

    EPA Science Inventory

    Effect of Arsenicals on the Expression of Cell Cycle Proteins and Early Signaling Events in Primary Human Keratinocytes.

    Mudipalli, A, Owen R. D. and R. J. Preston, Environmental Carcinogenesis Division, USEPA, RTP, NC 27711.

    Environmental exposure to arsenic is a m...

  16. EFFECT OF ARSENICALS ON THE EXPRESSION OF CELL CYCLE PROTEINS AND EARLY SIGNALING EVENTS IN PRIMARY HUMAN KERATINOCYTES.

    EPA Science Inventory

    Effect of Arsenicals on the Expression of Cell Cycle Proteins and Early Signaling Events in Primary Human Keratinocytes.

    Mudipalli, A, Owen R. D. and R. J. Preston, Environmental Carcinogenesis Division, USEPA, RTP, NC 27711.

    Environmental exposure to arsenic is a m...

  17. Luteolin Inhibits Human Keratinocyte Activation and Decreases NF-κB Induction That Is Increased in Psoriatic Skin

    PubMed Central

    Weng, Zuyi; Patel, Arti B.; Vasiadi, Magdalini; Therianou, Anastasia; Theoharides, Theoharis C.

    2014-01-01

    Psoriasis (Ps) is an autoimmune disease characterized by keratinocyte hyperproliferation and chronic inflammation, with increased expression of tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF). Anti-TNF biologic agents are effective in treating Ps, but are associated with increased risk of infections and blood malignancies. Moreover, keratinocyte hyperproliferation and activation have yet to be addressed. Flavonoids, such as luteolin, are natural compounds with potent anti-inflammatory properties, but their actions on keratinocytes remain unknown. We show that TNF (50 ng/mL) triggers significant production of inflammatory mediators interleukin-6, interleukin-8 and VEGF from both human HaCaT and primary keratinocytes. Pretreatment with the flavonoid luteolin (10–100 µM) significantly inhibits mRNA expression and release of all three mediators in a concentration-dependent manner. More importantly, luteolin decreases TNF-induced phosphorylation, nuclear translocation and DNA binding of the nuclear factor-kappa B (NF-κB) typically involved in inflammatory mediator transcription. We also report that luteolin reduces TNF-induced mRNA expression of two genes (NFKB1 and RELA) encoding two NF-κB subunits (NF-κB p50 and NF-κB p65, respectively). Interestingly, we show that gene expression of RELA is increased in human psoriatic skin. Keratinocyte proliferation, which is a characteristic feature of psoriatic skin, is effectively reduced by luteolin in HaCaT cells, but not in primary keratinocytes. Finally, luteolin does not affect intracellular ATP production or viability. Appropriate formulations of luteolin and related flavones may be promising candidates to be developed into local and systemic treatments for Ps and other inflammatory skin diseases. PMID:24587411

  18. 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.

  19. Calcium spirulan derived from Spirulina platensis inhibits herpes simplex virus 1 attachment to human keratinocytes and protects against herpes labialis.

    PubMed

    Mader, Julia; Gallo, Antonio; Schommartz, Tim; Handke, Wiebke; Nagel, Claus-Henning; Günther, Patrick; Brune, Wolfram; Reich, Kristian

    2016-01-01

    Chronic infections with herpes simplex virus (HSV) type 1 are highly prevalent in populations worldwide and cause recurrent oral lesions in up to 40% of infected subjects. We investigated the antiviral activity of a defined Spirulina platensis microalga extract and of purified calcium spirulan (Ca-SP), a sulfated polysaccharide contained therein. The inhibitory effects of HSV-1 were assessed by using a plaque reduction assay and quantitative PCR in a susceptible mammalian epithelial cell line and confirmed in human keratinocytes. Time-of-addition and attachment experiments and fluorescence detection of the HSV-1 tegument protein VP16 were used to analyze the mechanism of HSV-1 inhibition. Effects of Ca-SP on Kaposi sarcoma-associated herpesvirus/human herpes virus 8 replication and uptake of the ORF45 tegument protein were tested in human retinal pigment epithelial cells. In an observational trial the prophylactic effects of topically applied Ca-SP were compared with those of systemic and topical nucleoside analogues in 198 volunteers with recurrent herpes labialis receiving permanent lip makeup. Ca-SP inhibited HSV-1 infection in vitro with a potency at least comparable to that of acyclovir by blocking viral attachment and penetration into host cells. Ca-SP also inhibited entry of Kaposi sarcoma-associated herpesvirus/human herpes virus 8. In the clinical model of herpes exacerbation, the prophylactic effect of a Ca-SP and microalgae extract containing cream was superior to that of acyclovir cream. These data indicate a potential clinical use of Ca-SP containing Spirulina species extract for the prophylactic treatment of herpes labialis and suggest possible activity of Ca-SP against infections caused by other herpesviruses. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  20. Squamous epitheliotropism of Enterovirus A71 in human epidermis and oral mucosa.

    PubMed

    Phyu, Win Kyaw; Ong, Kien Chai; Kong, Chee Kwan; Alizan, Abdul Khalil; Ramanujam, Tindivanam Muthurangam; Wong, Kum Thong

    2017-03-21

    Hand-foot-and-mouth disease is a self-limiting paediatric infectious disease commonly caused by Enterovirus A71 (Genus: Enterovirus, Family: Picornaviridae). Typical lesions in and around the hands, feet, oral cavity and other places may rarely be complicated by acute flaccid paralysis and acute encephalomyelitis. Although virus is readily cultured from skin vesicles and oral secretions, the cellular target/s of Enterovirus A71 in human skin and oral mucosa are unknown. In Enterovirus A71-infected human skin and oral mucosa organotypic cultures derived from the prepuce and lip biopsies, focal viral antigens and viral RNA were localized to cytoplasm of epidermal and mucosal squamous cells as early as 2 days post-infection. Viral antigens/RNA were associated with cytoplasmic vacuolation and cellular necrosis. Infected primary prepuce epidermal keratinocyte cultures showed cytopathic effects with concomitant detection of viral antigens from 2 days post-infection. Supernatant and/or tissue homogenates from prepuce skin organotypic cultures and primary prepuce keratinocyte cultures showed viral titres consistent with active viral replication. Our data strongly support Enterovirus A71 squamous epitheliotropism in the human epidermis and oral mucosa, and suggest that these organs are important primary and/or secondary viral replication sites that contribute significantly to oral and cutaneous viral shedding resulting in person-to-person transmission, and viraemia, which could lead to neuroinvasion.

  1. Squamous epitheliotropism of Enterovirus A71 in human epidermis and oral mucosa

    PubMed Central

    Phyu, Win Kyaw; Ong, Kien Chai; Kong, Chee Kwan; Alizan, Abdul Khalil; Ramanujam, Tindivanam Muthurangam; Wong, Kum Thong

    2017-01-01

    Hand-foot-and-mouth disease is a self-limiting paediatric infectious disease commonly caused by Enterovirus A71 (Genus: Enterovirus, Family: Picornaviridae). Typical lesions in and around the hands, feet, oral cavity and other places may rarely be complicated by acute flaccid paralysis and acute encephalomyelitis. Although virus is readily cultured from skin vesicles and oral secretions, the cellular target/s of Enterovirus A71 in human skin and oral mucosa are unknown. In Enterovirus A71-infected human skin and oral mucosa organotypic cultures derived from the prepuce and lip biopsies, focal viral antigens and viral RNA were localized to cytoplasm of epidermal and mucosal squamous cells as early as 2 days post-infection. Viral antigens/RNA were associated with cytoplasmic vacuolation and cellular necrosis. Infected primary prepuce epidermal keratinocyte cultures showed cytopathic effects with concomitant detection of viral antigens from 2 days post-infection. Supernatant and/or tissue homogenates from prepuce skin organotypic cultures and primary prepuce keratinocyte cultures showed viral titres consistent with active viral replication. Our data strongly support Enterovirus A71 squamous epitheliotropism in the human epidermis and oral mucosa, and suggest that these organs are important primary and/or secondary viral replication sites that contribute significantly to oral and cutaneous viral shedding resulting in person-to-person transmission, and viraemia, which could lead to neuroinvasion. PMID:28322333

  2. Co-Regulation of p16INK4a and Migratory Genes in Culture Conditions that Lead to Premature Senescence in Human Keratinocytes

    PubMed Central

    Darbro, Benjamin W.; Schneider, Galen B.; Klingelhutz, Aloysius J.

    2007-01-01

    Cellular stasis, also known as telomere-independent senescence, prevents many epithelial cells from becoming immortalized by telomerase alone. As human keratinocytes age in culture, protein levels of the tumor suppressor p16INK4a continue to increase, resulting in growth arrest independent of telomere length. Differences in culture conditions have been shown to modulate both p16INK4a expression and replicative capacity of human keratinocytes; however, the mechanism of p16INK4a induction under these conditions is unknown. Using multiple primary keratinocyte cell strains, we verified a delay in p16INK4a induction and an extended lifespan of human keratinocytes when grown in co-culture with post-mitotic fibroblast feeder cells as compared with keratinocytes grown on tissue culture plastic alone. Evaluation of gene expression levels in the two culture conditions by microarray analysis, and subsequent validation, demonstrated that keratinocytes cultured on plastic alone had significantly increased expression of many genes involved in keratinocyte migration and reduced expression levels of genes involved in keratinocyte differentiation. Higher levels of p16INK4a expression were present in cells that also displayed increased amounts of autophosphorylated focal adhesion kinase and urokinase plaminogen activator receptor (uPAR), both markers of keratinocyte migration. Furthermore, when tyrosine phosphorylation or urokinase-type plasminogen activator (uPA)/uPAR function was inhibited, both keratinocyte migration and p16INK4a expression were reduced. Our results indicate that keratinocytes cultured in the absence of feeder cells exhibit a migratory phenotype and suggest that p16INK4a is selectively induced under these conditions by a mechanism involving tyrosine kinase activity and the urokinase plasminogen activation system. PMID:16117791

  3. Arsenic exposure disrupts epigenetic regulation of SIRT1 in human keratinocytes

    SciTech Connect

    Herbert, Katharine J.; Holloway, Adele; Cook, Anthony L.; Chin, Suyin P.; Snow, Elizabeth T.

    2014-11-15

    Arsenic is an environmental toxin which increases skin cancer risk for exposed populations worldwide; however the underlying biomolecular mechanism for arsenic-induced carcinogenesis is complex and poorly defined. Recent investigations show that histone deacetylase and DNA methyltransferase activity is impaired, and epigenetic patterns of gene regulation are consistently altered in cancers associated with arsenic exposure. Expression of the histone deacetylase SIRT1 is altered in solid tumours and haematological malignancies; however its role in arsenic-induced pathology is unknown. In this study we investigated the effect of arsenic on epigenetic regulation of SIRT1 and its targeting microRNA, miR-34a in primary human keratinocytes. Acetylation of histone H4 at lysine 16 (H4K16) increased in keratinocytes exposed to 0.5 μM arsenite [As(III)]; and this was associated with chromatin remodelling at the miR-34a promoter. Moreover, although SIRT1 protein initially increased in these As(III)-exposed cells, after 24 days expression was not significantly different from untreated controls. Extended exposure to low-dose As(III) (0.5 μM; > 5 weeks) compromised the pattern of CpG methylation at SIRT1 and miR-34a gene promoters, and this was associated with altered expression for both genes. We have found that arsenic alters epigenetic regulation of SIRT1 expression via structural reorganisation of chromatin at the miR-34a gene promoter in the initial 24 h of exposure; and over time, through shifts in miR-34a and SIRT1 gene methylation. Taken together, this investigation demonstrates that arsenic produces cumulative disruptions to epigenetic regulation of miR-34a expression, and this is associated with impaired coordination of SIRT1 functional activity. - Highlights: • Submicromolar arsenic concentrations disrupt SIRT1 activity and expression in human keratinocytes. • Arsenic-induced chromatin remodelling at the miR-34a gene promoter is associated with hyperacetylation

  4. Cobalt Oxide Nanoparticles: Behavior towards Intact and Impaired Human Skin and Keratinocytes Toxicity

    PubMed Central

    Mauro, Marcella; Crosera, Matteo; Pelin, Marco; Florio, Chiara; Bellomo, Francesca; Adami, Gianpiero; Apostoli, Piero; De Palma, Giuseppe; Bovenzi, Massimo; Campanini, Marco; Larese Filon, Francesca

    2015-01-01

    Skin absorption and toxicity on keratinocytes of cobalt oxide nanoparticles (Co3O4NPs) have been investigated. Co3O4NPs are commonly used in industrial products and biomedicine. There is evidence that these nanoparticles can cause membrane damage and genotoxicity in vitro, but no data are available on their skin absorption and cytotoxicity on keratinocytes. Two independent 24 h in vitro experiments were performed using Franz diffusion cells, using intact (experiment 1) and needle-abraded human skin (experiment 2). Co3O4NPs at a concentration of 1000 mg/L in physiological solution were used as donor phase. Cobalt content was evaluated by Inductively Coupled–Mass Spectroscopy. Co permeation through the skin was demonstrated after 24 h only when damaged skin protocol was used (57 ± 38 ng·cm−2), while no significant differences were shown between blank cells (0.92 ± 0.03 ng cm−2) and those with intact skin (1.08 ± 0.20 ng·cm−2). To further investigate Co3O4NPs toxicity, human-derived HaCaT keratinocytes were exposed to Co3O4NPs and cytotoxicity evaluated by MTT, Alamarblue® and propidium iodide (PI) uptake assays. The results indicate that a long exposure time (i.e., seven days) was necessary to induce a concentration-dependent cell viability reduction (EC50 values: 1.3 × 10−4 M, 95% CL = 0.8–1.9 × 10−4 M, MTT essay; 3.7 × 10−5 M, 95% CI = 2.2–6.1 × 10−5 M, AlamarBlue® assay) that seems to be associated to necrotic events (EC50 value: 1.3 × 10−4 M, 95% CL = 0.9–1.9 × 10−4 M, PI assay). This study demonstrated that Co3O4NPs can penetrate only damaged skin and is cytotoxic for HaCat cells after long term exposure. PMID:26193294

  5. Cobalt Oxide Nanoparticles: Behavior towards Intact and Impaired Human Skin and Keratinocytes Toxicity.

    PubMed

    Mauro, Marcella; Crosera, Matteo; Pelin, Marco; Florio, Chiara; Bellomo, Francesca; Adami, Gianpiero; Apostoli, Piero; De Palma, Giuseppe; Bovenzi, Massimo; Campanini, Marco; Filon, Francesca Larese

    2015-07-17

    Skin absorption and toxicity on keratinocytes of cobalt oxide nanoparticles (Co3O4NPs) have been investigated. Co3O4NPs are commonly used in industrial products and biomedicine. There is evidence that these nanoparticles can cause membrane damage and genotoxicity in vitro, but no data are available on their skin absorption and cytotoxicity on keratinocytes. Two independent 24 h in vitro experiments were performed using Franz diffusion cells, using intact (experiment 1) and needle-abraded human skin (experiment 2). Co3O4NPs at a concentration of 1000 mg/L in physiological solution were used as donor phase. Cobalt content was evaluated by Inductively Coupled-Mass Spectroscopy. Co permeation through the skin was demonstrated after 24 h only when damaged skin protocol was used (57 ± 38 ng·cm⁻²), while no significant differences were shown between blank cells (0.92 ± 0.03 ng cm⁻²) and those with intact skin (1.08 ± 0.20 ng·cm⁻²). To further investigate Co3O4NPs toxicity, human-derived HaCaT keratinocytes were exposed to Co3O4NPs and cytotoxicity evaluated by MTT, Alamarblue and propidium iodide (PI) uptake assays. The results indicate that a long exposure time (i.e., seven days) was necessary to induce a concentration-dependent cell viability reduction (EC50 values: 1.3 × 10-4 M, 95% CL = 0.8-1.9 × 10⁻⁴ M, MTT essay; 3.7 × 10⁻⁵ M, 95% CI = 2.2-6.1 × 10⁻⁵ M, AlamarBlue assay) that seems to be associated to necrotic events (EC50 value: 1.3 × 10⁻⁴ M, 95% CL = 0.9-1.9 × 10⁻⁴ M, PI assay). This study demonstrated that Co3O4NPs can penetrate only damaged skin and is cytotoxic for HaCat cells after long term exposure.

  6. Enhancement of major histocompatibility class I protein synthesis by DNA damage in cultured human fibroblasts and keratinocytes

    SciTech Connect

    Lambert, M.E.; Ronai, Z.A.; Weinstein, I.B.; Garrels, J.I.

    1989-02-01

    Exposure of primary human fibroblasts or simian virus 40-transformed human keratinocytes to several different classes of DNA damage, including UV light C (254 nm), resulted in a rapid increase in the expression of human major histocompatibility class I (MHC-I) proteins. MHC-I induction was also detected after exposure to low doses of the protein synthesis inhibitor cycloheximide, suggesting that MHC-I induction by DNA damage may be a component in a derepressible cellular SOS pathway.

  7. The phototumorigenic fluoroquinolone lomefloxacin photosensitizes pyrimidine dimer formation in human keratinocytes in vitro.

    PubMed

    Traynor, N J; Gibbs, N K

    1999-12-01

    The fluoroquinolone antibiotic lomefloxacin is phototoxic, photogenotoxic, photomutagenic and photosensitizes tumorigenesis in mouse skin. We have used T4 endonuclease V to demonstrate that lomefloxacin photosensitizes pyrimidine dimer formation in a human keratinocyte line (HaCaT). A possible mechanism for this effect would be triplet-triplet energy transfer. However, there is indirect evidence that the lomefloxacin triplet yield is very low, making this reaction less likely. The finding that lomefloxacin photosensitizes production of highly mutagenic pyrimidine dimers correlates with its ability to initiate skin tumor formation in mice. Until the potential of other fluoroquinolones to photosensitize dimer formation is explored it may be unadvisable to prescribe these antibiotics to patients with defective DNA repair capacity (e.g. xeroderma pigmentosum).

  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. Chromosomal loci of 50 human keratinocyte cDNAs assigned by fluorescence in situ hybridization

    SciTech Connect

    Morishima, Yohich; Ariyama, Takeshi; Yamanishi, Kiyofumi

    1995-07-20

    The chromosomal loci of expressed genes provide useful information for a candidate gene approach to the genes responsible for genetic diseases. A large set of randomly isolated cDNAs catalogued by partial sequencing can serve as a resource for accessing and isolating these disease genes. Using fluorescence in situ hybridization, we examined the chromosomal loci of 217 human keratinocyte-derived cDNAs, with independent novel sequence tags at the 3{prime} end region. Among them, we determined the loci of 50 cDNAs. Single-pass sequencing of these from the 5{prime} ends indicated that 39 cDNAs still can be produced for new genes. These cDNAs with identified chromosomal loci are powerful tools that can be used to help elucidate the genes responsible for hereditary skin disorders. 42 refs., 3 figs., 2 tabs.

  10. TELOMERE AND TELOMERASE MODULATION BY BERGAMOT POLYPHENOLIC FRACTION IN EXPERIMENTAL PHOTOAGEING IN HUMAN KERATINOCYTES.

    PubMed

    Nisticò, S; Ehrlich, J; Gliozzi, M; Maiuolo, J; Del Duca, E; Muscoli, C; Mollace, V

    2015-01-01

    Photoageing represents the addition of extrinsic chronic ultraviolet radiation-induced damage on intrinsic ageing and accounts for most age-associated changes in skin appearance. In this study, we evaluated the effect of 38% BPF, a highly concentrated extract of the bergamot fruit (Citrus bergamia) on UVB-induced photoageing by examining inflammatory cytokine expression, telomere length/telomerase alterations and cellular viability in human immortalized HaCaT keratinocytes. Our results suggest that 38% BPF protects HaCaT cells against UVB-induced oxidative stress and markers of photoageing in a dose-dependent manner and could be a useful supplement in skin care products. Together with antioxidant properties, BPF, a highly concentrated extract of the bergamot fruit, appears to modulate basic cellular signal transduction pathways leading to anti-proliferative, anti-aging and immune modulating responses.

  11. 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.

  12. Nifedipine prevents sodium caprate-induced barrier dysfunction in human epidermal keratinocyte cultures.

    PubMed

    Uchino, Yoshihiro; Matsumoto, Junichi; Watanabe, Takuya; Hamabashiri, Masato; Tsuchiya, Takashi; Kimura, Ikuya; Yamauchi, Atsushi; Kataoka, Yasufumi

    2015-01-01

    Tight junctions (TJs) of the epidermis play an important role in maintaining the epidermal barrier. TJ breakdown is associated with skin problems, such as wrinkles and transepidermal water loss (TEWL). Clinical studies have reported that topical nifedipine is effective in reducing the depth of wrinkles and improving TEWL. However, it remains unknown whether nifedipine influences the TJ function in the epidermis. In the present study, we investigated the effect of nifedipine on epidermal barrier dysfunction in normal human epidermal keratinocytes (NHEKs) treated with sodium caprate (C10), a TJ inhibitor. Nifedipine reversed the C10-decreased transepithelial electrical resistance values as a measure of disruption of the epidermal barrier. Immunocytochemical observations revealed that nifedipine improved the C10-induced irregular arrangement of claudin-1, a key protein in TJs. Taken together, these findings suggest that nifedipine prevents epidermal barrier dysfunction, at least in part, by reconstituting the irregular claudin-1 localization at TJs in C10-treated NHEKs.

  13. Human hair follicle and interfollicular keratinocyte reactivity to mouse HPV16-transformed cells: an in vitro study.

    PubMed

    Smetana, Karel; Dvoránková, Barbora; Lacina, Lukás; Cada, Zdenek; Vonka, Vladimír

    2008-07-01

    The role of stem cells in cancer formation and spreading has been established. As with normal tissue, the cancer stem cells need a special microenvironment to support their growth. This microenvironment may be represented by the tumor stroma. One of the possible ways of tumor stromal formation is the epithelial-mesenchymal transition of tumor epithelium. Following this mechanism, stromal cells must share the basic genetic alterations with the tumor cells. In an attempt to create a system capable of testing some aspects of the mesenchymal cell-keratinocyte interactions, we studied the effects of the fibroblastoid mouse TC-1 cells that were prepared by the introduction of human papillomavirus type 16 (HPV16) genes E6 and E7 to lung epithelial cells on the phenotype of normal human interfollicular and hair follicle keratinocytes. From this point of view, they may resemble stromal cells formed by the epithelial-mesenchymal transition of cells from HPV-induced squamous cell carcinoma. In contrast to 3T3 murine embryonic fibroblasts which were used as control cells, TC-1 cells influenced not only the size of the keratinocytes and the shape of their colonies, but also induced the expression of keratins 8 and 19 and vimentin. In conclusion, TC-1 cells exhibited a marked biological activity by influencing the behavior of the normal human follicular and intefollicular keratinocytes. This observation is compatible with the hypothesis that stromal cells play an important role in tumor progression and spreading.

  14. Characterization of a Human Skin Equivalent Model to Study the Effects of Ultraviolet B Radiation on Keratinocytes

    PubMed Central

    Van Lonkhuyzen, Derek R.; Dawson, Rebecca A.; Kimlin, Michael G.; Upton, Zee

    2014-01-01

    The incidences of skin cancers resulting from chronic ultraviolet radiation (UVR) exposure are on the incline in both Australia and globally. Hence, the cellular and molecular pathways that are associated with UVR-induced photocarcinogenesis need to be urgently elucidated, in order to develop more robust preventative and treatment strategies against skin cancers. In vitro investigations into the effects of UVR (in particular, the highly mutagenic UVB wavelength) have, to date, mainly involved the use of cell culture and animal models. However, these models possess biological disparities to native skin, which, to some extent, have limited their relevance to the in vivo situation. To address this, we characterized a three-dimensional, tissue-engineered human skin equivalent (HSE) model (consisting of primary human keratinocytes cultured on a dermal-derived scaffold) as a representation of a more physiologically relevant platform to study keratinocyte responses to UVB. Significantly, we demonstrate that this model retains several important epidermal properties of native skin. Moreover, UVB irradiation of the HSE constructs was shown to induce key markers of photodamage in the HSE keratinocytes, including the formation of cyclobutane pyrimidine dimers, the activation of apoptotic pathways, the accumulation of p53, and the secretion of inflammatory cytokines. Importantly, we also demonstrate that the UVB-exposed HSE constructs retain the capacity for epidermal repair and regeneration after photodamage. Together, our results demonstrate the potential of this skin equivalent model as a tool to study various aspects of the acute responses of human keratinocytes to UVB radiation damage. PMID:24219750

  15. 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.

  16. ATP signalling is crucial for the response of human keratinocytes to mechanical stimulation by hypo-osmotic shock.

    PubMed

    Azorin, Nathalie; Raoux, Matthieu; Rodat-Despoix, Lise; Merrot, Thierry; Delmas, Patrick; Crest, Marcel

    2011-05-01

    Touch is detected through receptors located in the skin and the activation of channels in sensory nerve fibres. Epidermal keratinocytes themselves, however, may sense mechanical stimulus and contribute to skin sensation. Here, we showed that the mechanical stimulation of human keratinocytes by hypo-osmotic shock releases adenosine triphosphate (ATP) and increases intracellular calcium. We demonstrated that the release of ATP was found to be calcium independent because emptying the intracellular calcium stores did not cause ATP release; ATP release was still observed in the absence of external calcium and it persisted on chelating cytosolic calcium. On the other hand, the released ATP activated purinergic receptors and mobilized intracellular calcium stores. The resulting depletion of stored calcium led to the activation of capacitative calcium entry. Increase in cytosolic calcium concentration was blocked by the purinergic receptor blocker suramin, phospholipase C inhibitor and apyrase, which hydrolyses ATP. Collectively, our data demonstrate that human keratinocytes are mechanically activated by hypo-osmotic shock, leading first to the release of ATP, which in turn stimulates purinergic receptors, resulting in the mobilization of intracellular calcium and capacitative calcium entry. These results emphasize the crucial role of ATP signalling in the transduction of mechanical stimuli in human keratinocytes.

  17. [Generation of a substitute for human oral mucosa and verification of its viability by tissue-engineering].

    PubMed

    Marañés Gálvez, C; Liceras Liceras, E; Alaminos, M; Fernández Valadés, R; Ruiz Montes, A M; Garzón, I; Sánchez-Quevedo, M C; Campos, A

    2011-01-01

    Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues. This way, tissue ingineering techniques may provide a source of autologous tissues available for transplant in these patients. In this work, we have developed a new model for artificial oral mucosa generated by tissue engineering using a fibrin-agarosa scaffold. For that purpose, we have generated primary cultures of human oral mucosa fibroblasts and keratinocytes from small biopsies of normal mucosa oral using enzymatic treatments. Then, we have determined the viability of cultured cells by electron probe quantitative X-ray microanalysis, and we have demonstrated that most of the cells in the primary cultures were alive and hd high K/Na ratios. Once cell viability was determined, we used cultured fibroblasts and keratinocytes to develop an artificial oral mucosa construct by using a fibrin-agarosa extracellular matrix and a sequential culture technique using porous culture inserts. Histological analysis of the artificial tissues showed high similarities with normal oral mucosa controls. The epithelium of the oral substitutes had several layers, with desmosomes and apical microvilli and microplicae. Both the controls and de oral mucosa substitutes showed high suprabasal expression of cytokeratin 13 and low expression of cytokeratin 10. All these results suggest that our model of oral mucosa using fibrin-agarose scaffolds show several similarities with native human oral mucosa.

  18. Human allogeneic keratinocytes cultured on acellular xenodermis: the use in healing of burns and other skin defects.

    PubMed

    Matousková, Eva; Broz, Ludomír; Stolbová, Vlasta; Klein, Leo; Konigová, Radana; Veselý, Pavel

    2006-01-01

    The tissue engineered skin should be composed of both dermal and epidermal layers. We combined cultured human allogeneic keratinocytes with acellular xenodermis prepared from pig xenografts. The resulting composite skin was termed recombined human/pig skin (RHPS), and could be cultured in both, undifferentiated and differentiated phenotype. The undifferentiated RHPS was grown submerged and formed 1-2 layers of keratinocytes. The differentiated phenotype (D-RHPS) was grown at the air-liquid interface and formed 5-20 cell layers similar to the normal epidermis, including the granular and horny layers. Undifferentiated RHPS has skin-like consistency and has been successfully used for treatment of burns and skin defects using "upside-down" application. Donor sites and deep dermal burn wounds prepared by tangential excision or deep dermabrasion grafted with RHPS healed in the course of about one week after keratinocyte transplantation. Simple acellular xenodermis without keratinocytes can also be used as temporary cover for donor sites, small to medium leg ulcers and other skin defects. Xenodermis can be fully sterilized and stored at the room temperature.

  19. alpha-Melanocyte-stimulating hormone, MSH 11-13 KPV and adrenocorticotropic hormone signalling in human keratinocyte cells.

    PubMed

    Elliott, Richard J; Szabo, Marika; Wagner, Mark J; Kemp, E Helen; MacNeil, Sheila; Haycock, John W

    2004-04-01

    alpha-MSH signals by binding to the melanocortin-1 receptor (MC-1R) and elevating cyclic AMP in several different cells. The anti-inflammatory properties of this peptide are also believed to be cyclic AMP dependent. The carboxyl terminal tripeptides of alpha-MSH (KPV / KP-D-V) are the smallest minimal sequences reported to prevent inflammation but it is not known if they operate via MC-1R or cyclic AMP. The aim of this study was to examine the intracellular signalling of key MSH and ACTH peptides in human keratinotocytes. No elevation in cyclic AMP was detected in either HaCaT or normal human keratinocytes in response to alpha-MSH, KPV or ACTH peptides. Rapid and acute intracellular calcium, however, were observed in HaCaT keratinocytes in response to alpha-MSH (10(-15)-10(-7) M), KPV (10(-15)-10(-7) M), KP-D-V (10(-15)-10(-7) M) and ACTH (10(-15)-10(-7) M), but only in the presence of PIA, an adenosine agonist that inhibits the cyclic AMP pathway. Normal keratinocytes responded to all the above peptides but in addition responded to ACTH 1-17 (10(-13)-10(-7) M) in contrast to the HaCaT keratinocytes. Stable transfection of Chinese hamster ovary cells with the MC-1 receptor showed that alpha-MSH and the KPV peptides elevated intracellular calcium.

  20. Stable vitiligo treated by a combination of low-dose oral pulse betamethasone and autologous, noncultured melanocyte-keratinocyte cell transplantation.

    PubMed

    Mulekar, Sanjeev V

    2006-04-01

    Various surgical procedures are in use to treat stable vitiligo. The possibility of Koebner phenomenon always exists with surgical treatment. Partial or complete failure to repigment is observed in spite of clinical stability. To evaluate the usefulness of a combination treatment of low-dose oral betamethasone and melanocyte-keratinocyte transplantation. Oral betamethasone was given to patients who failed to respond either completely or partially to melanocyte-keratinocyte cell transplantation, and the procedure was repeated for previously treated and nontreated area. A simpler and modified method described by Mulekar was performed. Seventeen patients with vitiligo vulgaris and eight patients with segmental vitiligo were retransplanted after giving oral betamethasone for 2 to 10 months after the initial procedure. Two patients of vitiligo vulgaris and one of segmental vitiligo failed to respond completely even after repeat transplantation. Fifteen vulgaris and seven segmental patients showed excellent to good repigmentation after repeat transplantation. Combined treatment of oral betamethasone and melanocyte cell transplantation has a potential to produce complete repigmentation in patients with large vitiliginous areas.

  1. 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.

  2. The genetic basis of human keratinocyte immortalisation in squamous cell carcinoma development: the role of telomerase reactivation.

    PubMed

    Parkinson, E K; Newbold, R F; Keith, W N

    1997-04-01

    Normal human keratinocytes have a finite replicative lifespan which culminates in senescence. Chromosomal telomere length may act as a mediator of replicative senescence, signalling cell cycle arrest in G1 when one or more telomeres become too short. Telomeric attrition in normal keratinocytes may be due to inadequate levels of telomerase activity and possibly also to oxidative damage. In advanced squamous cell carcinoma replicative senescence breaks down to yield immortal variants, in which several dominantly acting genes are functionally compromised, including p53 and the cyclin D-Cdk4/6 inhibitor CDKN2A/p16. The increased activity of both of these proteins would be expected to contribute to the G1 arrest in senescence and we have shown that levels of p16 are dramatically increased in senescent keratinocytes. In addition, two other genes which control a cell cycle G1 checkpoint independently of p53 and pRb appear dysfunctional. These genes are uncloned but map to chromosome 4q and 7q31.1 and appear to represent senescence complementation groups B and D, respectively. In immortal neoplastic keratinocytes, telomerase is strongly upregulated and there is evidence for a suppressor of the enzyme on the short arm of chromosome 3 mapping to 3p21.2-p21.3. We have also mapped the human telomerase RNA gene to 3q26.3 and found it to be overrepresented or amplified in a proportion of squamous cell tumours and cell lines. These observations may explain why isochromosome 3q is so common in human squamous carcinoma. None of these genetic alterations are seen in carcinomas which senesce and suggest that multiple genetic alterations are required for keratinocyte immortality.

  3. Inhibition of Inflammatory and Proliferative Responses of Human Keratinocytes Exposed to the Sesquiterpene Lactones Dehydrocostuslactone and Costunolide

    PubMed Central

    Scarponi, Claudia; Butturini, Elena; Sestito, Rosanna; Madonna, Stefania; Cavani, Andrea; Mariotto, Sofia; Albanesi, Cristina

    2014-01-01

    The imbalance of the intracellular redox state and, in particular, of the glutathione (GSH)/GSH disulfide couple homeostasis, is involved in the pathogenesis of a number of diseases. In many skin diseases, including psoriasis, oxidative stress plays an important role, as demonstrated by the observation that treatments leading to increase of the local levels of oxidant species ameliorate the disease. Recently, dehydrocostuslactone (DCE) and costunolide (CS), two terpenes naturally occurring in many plants, have been found to exert various anti-inflammatory and pro-apoptotic effects on different human cell types. These compounds decrease the level of the intracellular GSH by direct interaction with it, and, therefore, can alter cellular redox state. DCE and CS can trigger S-glutathionylation of various substrates, including the transcription factor STAT3 and JAK1/2 proteins. In the present study, we investigated on the potential role of DCE and CS in regulating inflammatory and proliferative responses of human keratinocytes to cytokines. We demonstrated that DCE and CS decreased intracellular GSH levels in human keratinocytes, as well as inhibited STAT3 and STAT1 phosphorylation and activation triggered by IL-22 or IFN-γ, respectively. Consequently, DCE and CS decreased the IL-22- and IFN-γ-induced expression of inflammatory and regulatory genes in keratinocytes, including CCL2, CXCL10, ICAM-1 and SOCS3. DCE and CS also inhibited proliferation and cell-cycle progression-related gene expression, as well as they promoted cell cycle arrest and apoptosis. In parallel, DCE and CS activated the anti-inflammatory EGFR and ERK1/2 molecules in keratinocytes, and, thus, wound healing in an in vitro injury model. In light of our findings, we can hypothesize that the employment of DCE and CS in psoriasis could efficiently counteract the pro-inflammatory effects of IFN-γ and IL-22 on keratinocytes, revert the apoptosis-resistant phenotype, as well as inhibit hyperproliferation

  4. Biological interactions of quantum dot nanoparticles in skin and in human epidermal keratinocytes

    SciTech Connect

    Zhang, Leshuai W.; Yu, William W.; Colvin, Vicki L.; Monteiro-Riviere, Nancy A.

    2008-04-15

    Quantum dots nanoparticles have novel optical properties for biomedical applications and electronics, but little is known about their skin permeability and interaction with cells. QD621 are nail-shaped nanoparticles that contain a cadmium/selenide core with a cadmium sulfide shell coated with polyethylene glycol (PEG) and are soluble in water. QD were topically applied to porcine skin flow-through diffusion cells to assess penetration at 1 {mu}M, 2 {mu}M and 10 {mu}M for 24 h. QD were also studied in human epidermal keratinocytes (HEK) to determine cellular uptake, cytotoxicity and inflammatory potential. Confocal microscopy depicted the penetration of QD621 through the uppermost stratum corneum (SC) layers of the epidermis and fluorescence was found primarily in the SC and near hair follicles. QD were found in the intercellular lipid bilayers of the SC by transmission electron microscopy (TEM). Inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis for cadmium (Cd) and fluorescence for QD both did not detect Cd nor fluorescence signal in the perfusate at any time point or concentration. In HEK, viability decreased significantly (p < 0.05) from 1.25 nM to 10nM after 24 h and 48 h. There was a significant increase in IL-6 at 1.25 nM to 10 nM, while IL-8 increased from 2.5nM to 10nM after 24 h and 48 h. TEM of HEK treated with 10 nM of QD621 at 24 h depicted QD in cytoplasmic vacuoles and at the periphery of the cell membranes. These results indicate that porcine skin penetration of QD621 is minimal and limited primarily to the outer SC layers, yet if the skin were damaged allowing direct QD exposure to skin or keratinocytes, an inflammatory response could be initiated.

  5. Organization and formation of the tight junction system in human epidermis and cultured keratinocytes.

    PubMed

    Brandner, Johanna M; Kief, Sabine; Grund, Christine; Rendl, Michael; Houdek, Pia; Kuhn, Caecilia; Tschachler, Erwin; Franke, Werner W; Moll, Ingrid

    2002-05-01

    Occludin and several proteins of the claudin family have been identiried in simple epithelia and in endothelia as major and structure-determining transmembrane proteins clustered in the barrier-forming tight junctions (TJ), where they are associated with a variety of TJ plaque proteins, including protein ZO-1. To examine whether TJ also occur in the squamous stratified epithelium of the interfollicular human epidermis we have applied several microscopic and biochemical techniques. Using RT-PCR techniques, we have identiried mRNAs encoding protein ZO-1, occludin and claudins 1, 4, 7, 8, 11, 12, and 17 in both tissues, skin and cultured keratinocytes, whereas claudins i and 10 have only been detected in skin tissue. By immunocytochemistry we have localized claudin-1, occludin and protein ZO-1 in distinct plasma membrane structures representing cell-cell attachment zones. While claudin-1 occurs in plasma membranes of all living cell layers, protein ZO-1 is concentrated in or even restricted to the uppermost layers, and occludin is often detected only in the stratum granulosum. Using electron microscopy, typical TJ structures ("kissing points") as well as some other apparently related junctional structures have been detected in the stratum granulosum, interspersed between desmosomes. Modes and patterns of TJ formation have also been studied in experimental model systems, e.g., during wound healing and stratification as well as in keratinocyte cultures during Ca2+-induced stratification. We conclude that the epidermis contains in the stratum granulosum a continuous zonula occludens-equivalent structure with typical TJ morphology and molecular composition, characterized by colocalization of occludin, claudins and TJ plaque proteins. In addition, cell-cell contact structures and certain TJ proteins can also be detected in other epidermal cell layers in specific cell contacts. The pattern of formation and possible functions of epidermal TJ and related structures are

  6. Multiple biological effects of inhibitors of arachidonic acid metabolism on human keratinocytes.

    PubMed

    Pacherník, Jirí; Hampl, Ales; Soucek, Karel; Kovaríková, Martina; Andrysík, Zdenek; Hofmanová, Jirina; Kozubík, Alois

    2002-02-01

    Various compounds that inhibit processing of arachidonic acid (AA) are being intensively tested for their antitumour activity. However, the mechanisms responsible for such activity remain rather elusive. To approach this issue, we examined the effects of several structurally different inhibitors of AA metabolism in the human keratinocyte HaCaT cell line. Several parameters were determined in HaCaT cells exposed to increasing concentrations of the inhibitors for 24 and/or 48 h. These included (1) oxidoreductase activity, total protein mass and cell cycle distribution to assess cell proliferation, (2) degradation of PARP protein to assess apoptosis, and (3) cell morphology, distribution of F-actin and expression of cytokeratins and E-cadherin to evaluate changes in differentiation status. While eicosatetraynoic acid (ETYA), nordihydroguaiaretic acid (NDGA), esculetin and MK-886 reduced proliferation of HaCaT cells, the cyclooxygenase inhibitors indomethacin and piroxicam had no such effects. Esculetin and NDGA arrested cells in S phase, and ETYA and MK-886 delayed cell progression through G(1) phase. Higher concentrations of NDGA, MK886 and/or ETYA caused cleavage of PARP. No changes in the expression of cytokeratins and E-cadherin were observed upon treatment with any of the inhibitors. However, esculetin induced redistribution of F-actin accompanied by increased cell adhesion and size. Our findings indicate that, in addition to their ability to inhibit cell proliferation and to induce apoptosis, lipoxygenase inhibitors and/or ETYA may also elicit other important physiological responses in HaCaT keratinocytes.

  7. Retinoic acid stimulates essential fatty acid-supplemented human keratinocytes in culture.

    PubMed

    Marcelo, C L; Dunham, W R

    1997-05-01

    The effect of all-trans retinoic acid on the proliferation of essential fatty acid (EFA)-deficient and of EFA-supplemented adult human keratinocytes was investigated. EFA-deficient cell strains were supplied with one of four different fatty acid-supplemented media at the P0 to P1 passage. All-trans retinoic acid at 0.5 or 1.0 microM was added to the cultures at the P1 to P2 passage. At passage P3, and 3 and 7 d thereafter, the cell growth rate was determined. The fatty acid content of cultures grown in each medium was measured using gas chromatography. All the EFA media "normalized" the cellular fatty acid composition and drastically decreased the cell number and total DNA and protein of the cultures. All-trans retinoic acid at 1 microM prevented the loss of cell viability and growth usually associated with EFA supplementation but did not affect the control (EFA deficient) or 18:1 fatty acid-supplemented cultures. All-trans retinoic acid at 1 microM altered the fatty acid content of the EFA-supplemented cultures. A statistically significant increase in 14:0, 14:1, 16:1, 18:1, and 20:4 fatty acids occurred, whereas the amounts of 18:0 and 18:2 fatty acids decreased. The largest changes were in 16:1 fatty acid (8-14%) and 18:2 fatty acid (12-5%). All-trans retinoic acid at 0.5 microM also affected both cell growth and fatty acid composition without induction of the CRABP II message. These studies demonstrate that all-trans retinoic acid stimulates the growth of EFA-supplemented keratinocyte cultures while also altering the fatty acid composition of the cells.

  8. 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.

  9. The Effect of Calcipotriol on the Expression of Human β Defensin-2 and LL-37 in Cultured Human Keratinocytes

    PubMed Central

    Kim, Beom Joon; Rho, Yong Kwan; Lee, Hye In; Jeong, Mi Sook; Li, Kapsok; Seo, Seong Jun; Kim, Myeung Nam; Hong, Chang Kwun

    2009-01-01

    Background. Vitamin D has been reported to regulate innate immunity by controlling the expression of antimicrobial peptides (AMPs). Objective. We investigated the effect of calcipotriol on the expression of AMPs in human cultured keratinocytes. Methods. Keratinocytes were treated with lipopolysaccharide (LPS), TNF-α, Calcipotriol and irradiated with UVB, cultured, and harvested. To assess the expression of human beta defensin-2 and LL-37 in the control group, not exposed to any stimulants, the experimental group was treated with LPS, TNF-α, or UVB, and another group was treated again with calcipotriol; reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemical staining were performed. Results. In the experimental group treated with LPS, UVB irradiation, and TNF-α, the expression of β-defensin and LL-37 was increased more than in the control group and then decreased in the experimental group treated with calcipotriol. Conclusions. Calcipotriol suppressed HBD-2 and LL-37, which were stimulated by UVB, LPS, and TNF-α. PMID:20182640

  10. Characterization of Electronic Cigarette Aerosol and Its Induction of Oxidative Stress Response in Oral Keratinocytes

    PubMed Central

    Zhao, Tongke; Shu, Shi; Chang, Chong Hyun; Messadi, Diana; Xia, Tian; Zhu, Yifang; Hu, Shen

    2016-01-01

    In this study, we have generated and characterized Electronic Cigarette (EC) aerosols using a combination of advanced technologies. In the gas phase, the particle number concentration (PNC) of EC aerosols was found to be positively correlated with puff duration whereas the PNC and size distribution may vary with different flavors and nicotine strength. In the liquid phase (water or cell culture media), the size of EC nanoparticles appeared to be significantly larger than those in the gas phase, which might be due to aggregation of nanoparticles in the liquid phase. By using in vitro high-throughput cytotoxicity assays, we have demonstrated that EC aerosols significantly decrease intracellular levels of glutathione in NHOKs in a dose-dependent fashion resulting in cytotoxicity. These findings suggest that EC aerosols cause cytotoxicity to oral epithelial cells in vitro, and the underlying molecular mechanisms may be or at least partially due to oxidative stress induced by toxic substances (e.g., nanoparticles and chemicals) present in EC aerosols. PMID:27223106

  11. Modulation of the pro-inflammatory cytokines and matrix metalloproteinases production in co-cultivated human keratinocytes and melanocytes.

    PubMed

    Decean, H; Perde-Schrepler, M; Tatomir, C; Fischer-Fodor, E; Brie, I; Virag, P

    2013-10-01

    The human epidermis exerts immunoregulatory functions through the variety of cytokines and other molecules elaborated by keratinocytes and melanocytes. Their constitutive production is very low; however, considerably increased upon stimulation. In vivo, keratinocytes and melanocytes have a typical exposure in the skin, referred as melanocyte epidermal unit. In the present study we co-cultivated these cells in vitro proposing to elucidate some communication links in close cell-to-cell association. We assessed the amounts of IL-6, IL-8, and matrix metalloproteinases (MMP-2 and MMP-9) in individually and co-cultured cells, exposed or not to UVB radiation. Normal human epidermal keratinocytes and melanocytes were grown in specific media and supplements. Cells were exposed to UVB radiation (100 mJ/cm(2)) to create comparable stress to the environmental one. Cytokines were determined with ELISA and confirmed with Western blot and metalloproteinases with gel zimography. Pure cultures of keratinocytes and melanocytes released low amounts of cytokines and metalloproteinases, these secretions being enhanced by UVB irradiation. In co-cultures, the cell-to-cell proximity triggered signals which markedly augmented the cytokines' secretions, whereas metalloproteinases were down-regulated. UVB irradiation did not influence either of these secretions in co-cultures. Concurrently with the highest levels of the pro-inflammatory cytokines, MMP-9 was up-regulated creating pro-inflammatory conditions and premises for changes in cellular survival, differentiation and phenotype. A complex network of interactions occurred between keratinocytes and melanocytes in co-cultures, resulting in modulated pro-inflammatory cytokines and metalloproteinases productions. Therefore, any disturbances in the microenvironmental signaling system and its molecular constituents may result in inflammation or even tumorigenesis in the epidermis.

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

    PubMed Central

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

    2014-01-01

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

  13. Trehalose, sucrose and raffinose are novel activators of autophagy in human keratinocytes through an mTOR-independent pathway

    PubMed Central

    Chen, Xu; Li, Min; Li, Li; Xu, Song; Huang, Dan; Ju, Mei; Huang, Ju; Chen, Kun; Gu, Heng

    2016-01-01

    Trehalose is a natural disaccharide that is found in a diverse range of organisms but not in mammals. Autophagy is a process which mediates the sequestration, lysosomal delivery and degradation of proteins and organelles. Studies have shown that trehalose exerts beneficial effects through inducing autophagy in mammalian cells. However, whether trehalose or other saccharides can activate autophagy in keratinocytes is unknown. Here, we found that trehalose treatment increased the LC3-I to LC3-II conversion, acridine orange-stained vacuoles and GFP-LC3B (LC3B protein tagged with green fluorescent protein) puncta in the HaCaT human keratinocyte cell line, indicating autophagy induction. Trehalose-induced autophagy was also observed in primary keratinocytes and the A431 epidermal cancer cell line. mTOR signalling was not affected by trehalose treatment, suggesting that trehalose induced autophagy through an mTOR-independent pathway. mTOR-independent autophagy induction was also observed in HaCaT and HeLa cells treated with sucrose or raffinose but not in glucose, maltose or sorbitol treated HaCaT cells, indicating that autophagy induction was not a general property of saccharides. Finally, although trehalose treatment had an inhibitory effect on cell proliferation, it had a cytoprotective effect on cells exposed to UVB radiation. Our study provides new insight into the saccharide-mediated regulation of autophagy in keratinocytes. PMID:27328819

  14. Lipophilic prodrugs of amino acids and vitamin E as osmolytes for the compensation of hyperosmotic stress in human keratinocytes.

    PubMed

    Graf, Rüdiger; Kock, Michael; Bock, Andreas; Schubert-Zsilavecz, Manfred; Steinhilber, Dieter; Kaufmann, Roland; Gassenmeier, Thomas; Beschmann, Heike; Bernd, August; Kippenberger, Stefan

    2009-04-01

    Skin keratinocytes are subjected to changing osmotic conditions and evolved counteracting mechanisms. Particularly, the expression of osmolyte transporters serves for the maintenance of cell volume in a hypertonic environment. In this study, we show that hyperosmotic stress significantly decreases the proliferation in HaCaT keratinocytes. Supplementation of the culture medium with the amino acids glycine, sarcosine, betaine, taurine and proline restored the proliferation indicating osmoprotective properties of these substances. Amino acids are highly polar molecules and therefore unable to penetrate into deeper epidermal layers after topical application. Thus, we utilized a prodrug concept in which the tested amino acids are coupled to a lipophilic moiety. Ethyl glycinate as a first model compound also showed an osmoprotective effect. In addition, improved penetration of the glycine derivative into deeper epidermal layers could be demonstrated. The prodrug concept was further developed by using the lipid soluble antioxidant alpha-tocopherol as a lipophilic moiety. The derivatives d,l-alpha-tocopheryl-(mono-) glycinate (TMG) and d,l-alpha-tocopheryl-(mono-) prolinate caused an increase in proliferation of HaCaT keratinocytes under salt stress and a decrease in apoptosis induced by hypertonic conditions. Furthermore, the osmoprotective effect of d,l-TMG could be corroborated in normal human keratinocytes. Therefore, it seems feasible that amino acids and their lipophilic derivatives may help to improve the osmotic balance and the hydration of skin. Clinical and cosmetic indications such as atopic eczema, UV exposed skin or aged skin may benefit from this new concept.

  15. [Effect of aloe vera polysaccharide on the release of cytokines and nitric oxide in cultured human keratinocytes].

    PubMed

    Chen, Xiao-dong; Huang, Li-ying; Wu, Bo-yu; Jiang, Qiong; Wang, Zhong-cheng; Lin, Xin-hua

    2005-05-01

    To investigate the effects of polysaccharide extracted from Aloe Barbadensis on the release of cytokines and nitric oxide (NO) in cultured human keratinocytes. The levels of transforming growth factor-alpha (TGF-alpha), TGF-beta1, interleukin-1beta (IL-1beta), IL-6, IL-8, tumor necrosis factor (TNF) and NO in the supernatants of keratinocyte culture in which culture media containing 25, 50, 100, 200, 400 microg/ml, respectively of aloe polysaccharide were assayed. In the control group equal volume of media without the polysaccharide was used. Compared with control group, the levels of TGF-alpha, TGF-beta1, IL-1beta, IL-6, IL-8 and TNF in the supernatants of cultured keratinocytes were significantly higher when aloe polysaccharide was added (P<0.05 or P<0.01), and they were positively correlated to the concentration of aloe polysaccharide (P<0.01). However, aloe polysaccharide markedly decreased the level of NO in a dose dependent manner (P<0.01). Aloe polysaccharide could promote keratinocytes to secrete TGF-alpha, TGF-beta1, IL-1beta, IL-6, IL-8 and TNF, and inhibit the release of NO.

  16. Translational repression protects human keratinocytes from UVB-induced apoptosis through a discordant eIF2 kinase stress response

    PubMed Central

    Collier, Ann E.; Wek, Ronald C.; Spandau, Dan F

    2015-01-01

    This study delineates the mechanisms by which ultraviolet B (UVB) regulates protein synthesis in human keratinocytes and the importance of translational control in cell survival. Translation initiation is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2~P), which causes decreased global protein synthesis coincident with enhanced translation of selected stress-related transcripts, such as ATF4. ATF4 is a transcriptional activator of the Integrated Stress Response (ISR), which has cytoprotective functions as well as apoptotic signals through the downstream transcriptional regulator CHOP (GADD153/DDIT3). We determined that UVB irradiation is a potent inducer of eIF2~P in keratinocytes, leading to decreased levels of translation initiation. However, expression of ATF4 or CHOP was not induced by UVB as compared to traditional ISR activators. The rationale for this discordant response is that ATF4 mRNA is reduced by UVB, and despite its ability to be preferentially translated there are diminished levels of available transcript. Forced expression of ATF4 and CHOP protein prior to UVB irradiation significantly enhanced apoptosis, suggesting that this portion of the ISR is deleterious in keratinocytes following UVB. Inhibition of eIF2~P and translational control reduced viability following UVB, which was alleviated by cycloheximide, indicating that translation repression through eIF2~P is central to keratinocyte survival. PMID:25950825

  17. Kallikrein-related Peptidase 5 Functions in Proteolytic Processing of Profilaggrin in Cultured Human Keratinocytes*

    PubMed Central

    Sakabe, Jun-ichi; Yamamoto, Mami; Hirakawa, Satoshi; Motoyama, Akira; Ohta, Isao; Tatsuno, Kazuki; Ito, Taisuke; Kabashima, Kenji; Hibino, Toshihiko; Tokura, Yoshiki

    2013-01-01

    Filaggrin protein is synthesized in the stratum granulosum of the skin and contributes to the formation of the human skin barrier. Profilaggrin is cleaved by proteolytic enzymes and converted to functional filaggrin, but its processing mechanism remains not fully elucidated. Kallikrein-related peptidase 5 (KLK5) is a major serine protease found in the skin, which is secreted from lamellar granules following its expression in the stratum granulosum and activated in the extracellular space of the stratum corneum. Here, we searched for profilaggrin-processing protease(s) by partial purification of epidermal extracts and found KLK5 as a possible candidate. We used high performance liquid chromatography coupled with electrospray tandem mass spectrometry to show that KLK5 cleaves profilaggrin. Furthermore, based on a proximity ligation assay, immunohistochemistry, and immunoelectron microscopy analysis, we reveal that KLK5 and profilaggrin co-localize in the stratum granulosum in human epidermis. KLK5 knockdown in normal cultured human epidermal keratinocytes resulted in higher levels of profilaggrin, indicating that KLK5 potentially functions in profilaggrin cleavage. PMID:23629652

  18. S100A11, an Dual Mediator for Growth Regulation of Human Keratinocytes

    PubMed Central

    Sakaguchi, Masakiyo; Sonegawa, Hiroyuki; Murata, Hitoshi; Kitazoe, Midori; Futami, Jun-ichiro; Kataoka, Ken; Yamada, Hidenori

    2008-01-01

    We previously revealed a novel signal pathway involving S100A11 for inhibition of the growth of normal human keratinocytes (NHK) caused by high Ca++ or transforming growth factor β. Exposure to either agent resulted in transfer of S100A11 to nuclei, where it induced p21WAF1. In contrast, S100A11 has been shown to be overexpressed in many human cancers. To address this apparent discrepancy, we analyzed possible new functions of S100A11, and we provide herein evidence that 1) S100A11 is actively secreted by NHK; 2) extracellular S100A11 acts on NHK to enhance the production of epidermal growth factor family proteins, resulting in growth stimulation; 3) receptor for advanced glycation end products, nuclear factor-κB, Akt, and cAMP response element-binding protein are involved in the S100A11-triggered signal transduction; and 4) production and secretion of S100A11 are markedly enhanced in human squamous cancer cells. These findings indicate that S100A11 plays a dual role in growth regulation of epithelial cells. PMID:17978094

  19. Raman microspectroscopy discrimination of single human keratinocytes exposed at low dose of pesticide

    NASA Astrophysics Data System (ADS)

    Perna, Giuseppe; Lasalvia, Maria; Capozzi, Vito

    2012-02-01

    Raman spectroscopy is a useful technique for early diagnosis of cellular damage related to the exposure at toxic chemicals, because biochemical changes related to action mechanism of chemicals can be detected in Raman spectra. In this investigation Raman microspectroscopy has been used, in correlation with the principal component analysis method, to detect biochemical changes occurring in cultured human cells as a consequence of exposure at a commercial pesticide. Cultured human keratinocyte cells have been exposed at increasing concentrations of pesticide for 24 h. Viability tests indicated that the cells vitality is almost completely preserved when the concentration of active ingredient of pesticide is very low (5 × 10-8 M, about two orders of magnitude lower than the cytotoxic concentration at 24 h exposure). Nonetheless, the analysis of Raman spectra allows to state that a biochemical change occurs: it involves mainly the protein linkages between aminoacids (amide I bonds) and, at a minor amount, lipids. On the whole, principal components analysis is able to classify into two separate clusters the control and exposed human cells.

  20. Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung.

    PubMed Central

    Simonet, W S; DeRose, M L; Bucay, N; Nguyen, H Q; Wert, S E; Zhou, L; Ulich, T R; Thomason, A; Danilenko, D M; Whitsett, J A

    1995-01-01

    Expression of human keratinocyte growth factor (KGF/FGF-7) was directed to epithelial cells of the developing embryonic lung of transgenic mice disrupting normal pulmonary morphogenesis during the pseudoglandular stage of development. By embryonic day 15.5(E15.5), lungs of transgenic surfactant protein C (SP-C)-KGF mice resembled those of humans with pulmonary cystadenoma. Lungs were cystic, filling the thoracic cavity, and were composed of numerous dilated saccules lined with glycogen-containing columnar epithelial cells. The normal distribution of SP-C proprotein in the distal regions of respiratory tubules was disrupted. Columnar epithelial cells lining the papillary structures stained variably and weakly for this distal respiratory cell marker. Mesenchymal components were preserved in the transgenic mouse lungs, yet the architectural relationship of the epithelium to the mesenchyme was altered. SP-C-KGF transgenic mice failed to survive gestation to term, dying before E17.5. Culturing mouse fetal lung explants in the presence of recombinant human KGF also disrupted branching morphogenesis and resulted in similar cystic malformation of the lung. Thus, it appears that precise temporal and spatial expression of KGF is likely to play a crucial role in the control of branching morphogenesis during fetal lung development. Images Fig. 1 Fig. 2 Fig. 3 PMID:8618921

  1. Sacha Inchi Oil (Plukenetia volubilis L.), effect on adherence of Staphylococus aureus to human skin explant and keratinocytes in vitro.

    PubMed

    Gonzalez-Aspajo, German; Belkhelfa, Haouaria; Haddioui-Hbabi, Laïla; Bourdy, Geneviève; Deharo, Eric

    2015-08-02

    Plukenetia volubilis L. (Euphorbiaceae) is a domesticated vine distributed from the high-altitude Andean rain forest to the lowlands of the Peruvian Amazon. Oil from the cold-pressed seeds, sold under the commercial name of Sacha Inchi Oil (SIO) is actually much in favour because it contains a high percentage of omega 3 and omega 6, and is hence used as a dietary supplement. SIO is also used traditionally for skin care, in order to maintain skin softness, and for the treatment of wounds, insect bites and skin infections, in a tropical context where the skin is frequently damaged. This study was designed in order to verify whether the traditional use of SIO for skin care would have any impact on Staphylococcus aureus growth and skin adherence, as S. aureus is involved in many skin pathologies (impetigo, folliculitis, furuncles and subcutaneous abscesses) being one if the main pathogens that can be found on the skin. Therefore, our objective was to assess SIO bactericidal activity and interference with adherence to human skin explants and the keratinocyte cell line. Cytotoxicity on that cells was also determined. The activity of SIO was compared to coconut oil (CocO), which is widely used for skin care but has different unsaturated fatty acids contents. Laboratory testing with certified oil, determined antibacterial activity against radio labelled S. aureus. Cytotoxic effects were measured with XTT on keratinocyte cells and with neutral red on human skin explants; phenol was used as cytotoxic control. Adherence assays were carried out by mixing H3-labelled S. aureus bacteria with keratinocyte cells and human skin explants, incubated with oils 2h before (to determine the inhibition of adherence, assimilated to a preventive effect) or 2h after the contact of the biological material with S. aureus (to assess the detachment of the bacteria, assimilated to a curative effect). Residual radioactivity measured after washings made it possible to determine the adherence

  2. Interaction of Mycobacterium leprae with the HaCaT human keratinocyte cell line: new frontiers in the cellular immunology of leprosy.

    PubMed

    Lyrio, Eloah C D; Campos-Souza, Ivy C; Corrêa, Luiz C D; Lechuga, Guilherme C; Verícimo, Maurício; Castro, Helena C; Bourguignon, Saulo C; Côrte-Real, Suzana; Ratcliffe, Norman; Declercq, Wim; Santos, Dilvani O

    2015-07-01

    Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae affecting the skin and peripheral nerves. Despite M. leprae invasion of the skin and keratinocytes importance in innate immunity, the interaction of these cells in vitro during M. leprae infection is poorly understood. Conventional and fluorescence optical microscopy, transmission electronic microscopy, flow cytometry and ELISA were used to study the in vitro interaction of M. leprae with the HaCaT human keratinocyte cell line. Keratinocytes uptake of M. leprae is described, and modulation of the surface expression of CD80 and CD209, cathelicidin expression and TNF-α and IL-1β production of human keratinocytes are compared with dendritic cells and macrophages during M. leprae interaction. This study demonstrated that M. leprae interaction with human keratinocytes enhanced expression of cathelicidin and greatly increased TNF-α production. The highest spontaneous expression of cathelicidin was by dendritic cells which are less susceptible to M. leprae infection. In contrast, keratinocytes displayed low spontaneous cathelicidin expression and were more susceptible to M. leprae infection than dendritic cells. The results show, for the first time, an active role for keratinocytes during infection by irradiated whole cells of M. leprae and the effect of vitamin D on this process. They also suggest that therapies which target cathelicidin modulation may provide novel approaches for treatment of leprosy.

  3. Interleukin 6 is Expressed in High Levels in Psoriatic Skin and Stimulates Proliferation of Cultured Human Keratinocytes

    NASA Astrophysics Data System (ADS)

    Grossman, Rachel M.; Krueger, James; Yourish, Debra; Granelli-Piperno, Angela; Murphy, Daniel P.; May, Lester T.; Kupper, Thomas S.; Sehgal, Pravinkumar B.; Gottlieb, Alice B.

    1989-08-01

    Psoriasis is a common papulosquamous skin disease. The histopathology is characterized by epidermal hyperplasia and inflammation. Recent studies suggest that keratinocyte proliferation and inflammation in psoriasis are manifestations of the same underlying pathological process. Interleukin 6 (IL-6), a cytokine that is a major mediator of the host response to tissue injury and infection, is produced by both keratinocytes and leukocytes in culture. IL-6 expression was studied in psoriatic plaques by immunoperoxidase staining with two different polyclonal anti-recombinant IL-6 antisera and by in situ nucleic acid hybridization with IL-6 cRNA probes. Epidermal and dermal cells in active psoriatic plaques from 35 psoriasis patients stained heavily for IL-6 as compared with nonlesional skin and with plaques after treatment with antimetabolic and antiinflammatory agents. Absorption of the anti-recombinant IL-6 antisera with purified fibroblast-derived IL-6 or with recombinant IL-6, but not bovine serum albumin, removed the immunostaining. Increased levels of IL-6 were detected in the plasma of patients with active psoriasis (mean 3 ng/ml) by using two different bioassays. IL-6 production by proliferating keratinocytes was suggested by IL-6-specific immunostaining in cultured normal and psoriatic keratinocytes and by the detection of mRNA specific for IL-6 in psoriatic epidermis by in situ hybridization. IL-6 stimulated the proliferation of cultured, normal human keratinocytes as assessed by two different assays. Thus, IL-6 could directly contribute to the epidermal hyperplasia seen in psoriatic epithelium as well as affect the function of dermal inflammatory cells.

  4. Claudin-1 Binder Enhances Epidermal Permeability in a Human Keratinocyte Model.

    PubMed

    Nakajima, Misaki; Nagase, Shotaro; Iida, Manami; Takeda, Shuji; Yamashita, Mayo; Watari, Akihiro; Shirasago, Yoshitaka; Fukasawa, Masayoshi; Takeda, Hiroyuki; Sawasaki, Tatsuya; Yagi, Kiyohito; Kondoh, Masuo

    2015-09-01

    Tight junctions (TJs) are complex biochemical structures that seal the intercellular space and prevent the free movement of solutes across epithelial cell sheets. Modulating the TJ seal is a promising option for increasing the transdermal absorption of drugs. Within TJs, the binding of the claudin (CLDN) family of tetratransmembrane proteins through cis- and trans-interactions is an integral part of seal formation. Because epidermal TJs contain CLDN-1 and CLDN-4, a binder for these CLDNs may be a useful modulator of the permeability of the epidermal barrier. Here, we investigated whether m19, which can bind to CLDN-1/-4 (also CLDN-2/-5), modulates the integrity of epidermal TJs and the permeability of cell sheets to solutes. Treatment of normal human epidermal keratinocytes (NHEKs) with the CLDN binder reduced the integrity of TJs. A CLDN-1-specific binder (a monoclonal antibody, clone 7A5) also weakened the TJ seal in NHEKs. Although m19 attenuated the TJ barrier in human intestinal epithelial cells (Caco-2), 7A5 did not. Treatment of NHEKs with 7A5 enhanced permeation of a paracellular permeation marker. These findings indicate that CLDN-1 is a potential target for modulating the permeability of the epidermis, and that our CLDN-1 binder is a promising candidate molecule for development as a dermal absorption enhancer.

  5. Photoprotective Activity of Vulpinic and Gyrophoric Acids Toward Ultraviolet B-Induced Damage in Human Keratinocytes.

    PubMed

    Varol, Mehmet; Türk, Ayşen; Candan, Mehmet; Tay, Turgay; Koparal, Ayşe Tansu

    2016-01-01

    Vulpinic and gyrophoric acids are known as ultraviolet filters for natural lichen populations because of their chemical structures. However, to the best of our knowledge, there has been no reference to their cosmetic potential for skin protection against ultraviolet B (UVB)-induced damage and, consequently, we propose to highlight their photoprotective profiles in human keratinocytes (HaCaT). Therefore, vulpinic acid and gyrophoric acid were isolated from acetone extracts of Letharia vulpina and Xanthoparmelia pokornyi, respectively. Their photoprotective activities on irradiated HaCaT cells and destructive effects on non-irradiated HaCaT cells were compared through in vitro experimentation: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays, 4',6-diamino-2-phenylindole and tetramethylrhodamine B isothiocyanate-phalloidin staining protocols. Both of the lichen substances effectively prevented cytotoxic, apoptotic and cytoskeleton alterative activities of 2.5 J/cm(2) UVB in a dose-dependent manner. Moreover, vulpinic and gyrophoric acids showed no toxic, apoptotic or cytoskeleton alterative effects on non-irradiated HaCaT cells, except at high doses (≥400 μM) of gyrophoric acid. The findings suggest that vulpinic and gyrophoric acids can be promising cosmetic ingredients to photo-protect human skin cells and should therefore be further investigated by in vitro and in vivo multiple bioassays.

  6. Activation of endogenous opioid gene expression in human keratinocytes and fibroblasts by pulsed radiofrequency energy fields

    PubMed Central

    Moffett, John; Fray, Linley M; Kubat, Nicole J

    2012-01-01

    Background Pulsed radiofrequency energy (PRFE) fields are being used increasingly for the treatment of pain arising from dermal trauma. However, despite their increased use, little is known about the biological and molecular mechanism(s) responsible for PRFE-mediated analgesia. In general, current therapeutics used for analgesia target either endogenous factors involved in inflammation, or act on endogenous opioid pathways. Methods and Results Using cultured human dermal fibroblasts (HDF) and human epidermal keratinocytes (HEK), we investigated the effect of PRFE treatment on factors, which are involved in modulating peripheral analgesia in vivo. We found that PRFE treatment did not inhibit cyclooxygenase enzyme activity, but instead had a positive effect on levels of endogenous opioid precursor mRNA (proenkephalin, pro-opiomelanocortin, prodynorphin) and corresponding opioid peptide. In HEK cells, increases in opioid mRNA were dependent, at least in part, on endothelin-1. In HDF cells, additional pathways also appear to be involved. PRFE treatment was also followed by changes in endogenous expression of several cytokines, including increased levels of interleukin-10 mRNA and decreased levels of interleukin-1β mRNA in both cell types. Conclusion These findings provide a new insight into the molecular mechanism underlying PRFE-mediated analgesia reported in the clinical setting. PMID:23055776

  7. Effects of silver nanoparticles on human dermal fibroblasts and epidermal keratinocytes.

    PubMed

    Galandáková, A; Franková, J; Ambrožová, N; Habartová, K; Pivodová, V; Zálešák, B; Šafářová, K; Smékalová, M; Ulrichová, J

    2016-09-01

    Biomedical application of silver nanoparticles (AgNPs) has been rapidly increasing. Owing to their strong antimicrobial activity, AgNPs are used in dermatology in the treatment of wounds and burns. However, recent evidence for their cytotoxicity gives rise to safety concerns. This study was undertaken as a part of an ongoing programme in our laboratory to develop a topical agent for wound healing. Here, we investigated the potential toxicity of AgNPs using normal human dermal fibroblasts (NHDF) and normal human epidermal keratinocytes (NHEK) with the aim of comparing the effects of AgNPs and ionic silver (Ag-I). Besides the effect of AgNPs and Ag-I on cell viability, the inflammatory response and DNA damage in AgNPs and Ag-I-treated cells were examined. The results showed that Ag-I were significantly more toxic than AgNPs both on NHDF and NHEK. Non-cytotoxic concentrations of AgNPs and Ag-I did not induce DNA strand breaks and did not affect inflammatory markers, except for a transient increase in interleukin 6 levels in Ag-I-treated NHDF. The results showed that AgNPs are more suitable for the intended application as a topical agent for wound healing up to the concentration 25 µg/mL.

  8. Baicalin Scavenged Reactive Oxygen Species and Protected Human Keratinocytes Against UVB-induced Cytotoxicity.

    PubMed

    Chang, Wen-Shin; Lin, En-Yuan; Hsu, Shih-Wei; Hu, Pei-Shin; Chuang, Chin-Liang; Liao, Cheng-Hsi; Fu, Chun-Kai; Su, Chung-Hao; Gong, Chi-Li; Hsiao, Chieh-Lun; Bau, DA-Tian; Tsai, Chia-Wen

    Ultraviolet B (UVB), with a wavelength of 280-320 nm, represents one of the most important environmental factors for skin disorders, including sunburn, hyperpigmentation, solar keratosis, solar elastosis and skin cancer. Therefore, protection against excessive UVA-induced damage is useful for prevention of sunburn and other human diseases. Baicalin, a major component of traditional Chinese medicine Scutellaria baicalensis, has been reported to possess antioxidant and cytostatic capacities. In this study, we examined whether baicalin is also capable of protecting human keratinocytes from UVB irradiation. The results showed that baicalin effectively scavenged reactive oxygen species (ROS) elevated within 4 h after UVB radiation and reversed the UVB-suppressed cell viability and UVB-induced apoptosis after 24 h. Our results demonstrated the utility of baicalin to complement the contributions of traditional Chinese medicine in UVB-induced damage to skin and suggested their potential application as pharmaceutical agents in long-term sun-shining injury prevention. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  9. Activation of endogenous opioid gene expression in human keratinocytes and fibroblasts by pulsed radiofrequency energy fields.

    PubMed

    Moffett, John; Fray, Linley M; Kubat, Nicole J

    2012-01-01

    Pulsed radiofrequency energy (PRFE) fields are being used increasingly for the treatment of pain arising from dermal trauma. However, despite their increased use, little is known about the biological and molecular mechanism(s) responsible for PRFE-mediated analgesia. In general, current therapeutics used for analgesia target either endogenous factors involved in inflammation, or act on endogenous opioid pathways. Using cultured human dermal fibroblasts (HDF) and human epidermal keratinocytes (HEK), we investigated the effect of PRFE treatment on factors, which are involved in modulating peripheral analgesia in vivo. We found that PRFE treatment did not inhibit cyclooxygenase enzyme activity, but instead had a positive effect on levels of endogenous opioid precursor mRNA (proenkephalin, pro-opiomelanocortin, prodynorphin) and corresponding opioid peptide. In HEK cells, increases in opioid mRNA were dependent, at least in part, on endothelin-1. In HDF cells, additional pathways also appear to be involved. PRFE treatment was also followed by changes in endogenous expression of several cytokines, including increased levels of interleukin-10 mRNA and decreased levels of interleukin-1β mRNA in both cell types. These findings provide a new insight into the molecular mechanism underlying PRFE-mediated analgesia reported in the clinical setting.

  10. 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

  11. Violet Light Down-Regulates the Expression of Specific Differentiation Markers through Rhodopsin in Normal Human Epidermal Keratinocytes

    PubMed Central

    Kim, Hyoung-June; Son, Eui Dong; Jung, Ji-Yong; Choi, Hyun; Lee, Tae Ryong; Shin, Dong Wook

    2013-01-01

    Several recent reports have demonstrated that photoreceptors are expressed in human skin. The rod and cone photoreceptor-like proteins are expressed in human skin and rhodopsin, long wavelength-opsin, and short wavelength-opsin are also present in cultured murine melanocytes. Furthermore, the photopigment rhodopsin is expressed in human melanocytes and is involved in ultraviolet A phototransduction which induces early melanin synthesis. In this study, we investigated whether rhodopsin is expressed and plays any physiological roles in the normal human epidermal keratinocytes (NHEKs). We found that rhodopsin was expressed and localized on the plasma membrane in NHEKs, and only violet light among several wavelengths within the visible range significantly increased the expression of rhodopsin mRNA. We further found that rhodopsin over-expression decreased the mRNA expression levels of keratinocyte differentiation markers, such as keratin-1 and keratin-10, and violet light also decreased the mRNA expression levels of keratinocyte differentiation markers and these decreased expression levels were recovered by a rhodopsin-directed siRNA. Moreover, we further demonstrated that violet light significantly decreased the phosphorylation levels of cAMP responsive element-binding protein (CREB) and that it more effectively decreased the phosphorylation of CREB when rhodopsin was over-expressed. In addition, we observed that pertussis toxin, a Gαi protein inhibitor, restored the rhodopsin-induced decrease in the differentiation markers in NHEKs. Taken together, these results suggest that rhodopsin down-regulates the expression levels of specific keratinocyte differentiation markers via the Gαi signaling pathway in NHEKs. PMID:24069221

  12. An "ice-cold" TR(i)P to skin biology: the role of TRPA1 in human epidermal keratinocytes.

    PubMed

    Bíró, Tamás; Kovács, László

    2009-09-01

    Recent studies have suggested the expression of numerous heat-sensitive transient receptor potential (TRP) ion channels in non-neuronal cell populations of the skin. In this issue, Atoyan et al. provide evidence that the noxious cold-activated TRPA1 is widely expressed in various human cutaneous cells and that it may be directly involved in the regulation of keratinocyte proliferation and differentiation and in cutaneous inflammatory responses.

  13. Impact on Autophagy and Ultraviolet B Induced Responses of Treatment with the MTOR Inhibitors Rapamycin, Everolimus, Torin 1, and pp242 in Human Keratinocytes

    PubMed Central

    Xu, Song; Li, Li; Li, Min; Zhang, Mengli

    2017-01-01

    The mechanistic target of Rapamycin (MTOR) protein is a crucial signaling regulator in mammalian cells that is extensively involved in cellular biology. The function of MTOR signaling in keratinocytes remains unclear. In this study, we detected the MTOR signaling and autophagy response in the human keratinocyte cell line HaCaT and human epidermal keratinocytes treated with MTOR inhibitors. Moreover, we detected the impact of MTOR inhibitors on keratinocytes exposed to the common carcinogenic stressors ultraviolet B (UVB) and UVA radiation. As a result, keratinocytes were sensitive to the MTOR inhibitors Rapamycin, everolimus, Torin 1, and pp242, but the regulation of MTOR downstream signaling was distinct. Next, autophagy induction only was observed in HaCaT cells treated with Rapamycin. Furthermore, we found that MTOR signaling was insensitive to UVB but sensitive to UVA radiation. UVB treatment also had no impact on the inhibition of MTOR signaling by MTOR inhibitors. Finally, MTOR inhibition by Rapamycin, everolimus, or pp242 did not affect the series of biological events in keratinocytes exposed to UVB, including the downregulation of BiP and PERK, activation of Histone H2A and JNK, and cleavage of caspase-3 and PARP. Our study demonstrated that MTOR inhibition in keratinocytes cannot always induce autophagy, and the MTOR pathway does not play a central role in the UVB triggered cellular response. PMID:28400912

  14. 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.

  15. Comparative toxicity of mono- and bifunctional alkylating homologues of sulphur mustard in human skin keratinocytes.

    PubMed

    Sawyer, Thomas W; McNeely, Karin; Louis, Kristen; Lecavalier, Pierre; Song, Yanfeng; Villanueva, Mercy; Clewley, Robin

    2017-03-08

    Sulphur mustard (bis(2-chloroethyl) sulphide; agent H) is a vesicant chemical warfare (CW) agent whose mechanism of action is not known with any certainty and for which there are no effective antidotes. It has a pronounced latent period before signs and symptoms of poisoning appear which it shares with the nitrogen mustards, and that differentiates it from other classes of vesicant agents. Sulphur mustard, the sulphur mustard CW agents Q (1,2-bis(2-chloroethylthio) ethane) and T (1,1 bis(2-chloroethylthioethyl) ether), the H partial hydrolysis product hemi-sulphur mustard (2-chloroethyl 2-hydroxyethyl sulphide; HSM), and the commercially available 2-chloroethyl ethyl sulphide (CEES) were characterized with respect to their toxicity in first passage cultures of proliferating human skin keratinocytes, the target cell of H-induced skin vesication. Agents H and T were equitoxic and half as toxic as agent Q. Hemi-sulphur mustard and CEES were approximately six times and seventeen times, respectively less cytotoxic than H. 2-Chloroethyl ethyl sulphide was only slightly less toxic in confluent cultures compared to actively proliferating cells. In contrast, the toxicity of H, Q, T and HSM significantly decreased as the cultures became confluent, paralleling the decreasing sensitivity of skin keratinocytes to H as they leave the basement membrane of the skin. The toxicity of CEES was maximal by 24h. In contrast, the maximal toxicity of the other four agents occurred at 48h, mirroring the latent period observed for these agents in vivo. The markedly different characteristics of toxicity between CEES and the other four test compounds indicate that it is likely that different mechanisms of action are operative between them. Caution should therefore be taken when interpreting the results of studies utilizing CEES as a simulant for the mechanistic study of H, or in the elucidation of medical countermeasures against this CW agent. It is also notable that the toxicity

  16. The HIV-1 viral synapse signals human foreskin keratinocytes to secrete thymic stromal lymphopoietin facilitating HIV-1 foreskin entry.

    PubMed

    Zhou, Z; Xu, L; Sennepin, A; Federici, C; Ganor, Y; Tudor, D; Damotte, D; Barry Delongchamps, N; Zerbib, M; Bomsel, M

    2017-04-26

    The complexity of signal transduction resulting from the contact of human immunodeficiency virus type 1 (HIV-1)-infected cells and mucosal cells has hampered our comprehension of HIV-1 mucosal entry. Such process is driven efficiently only by viral synapse contacts, whereas cell-free HIV-1 remains poorly infectious. Using CD4(+) T-cells expressing only HIV-1 envelope inoculated on human adult foreskin tissues, we designed methodologies to identify the signals transduced in foreskin keratinocytes following HIV-1-envelope-dependent viral synapse formation. We find that the viral synapse activates the MyD88-independent TLR-4-nuclear factor (NfκB) signaling pathway in keratinocytes and the subsequent secretion of cytokines including thymic stromal lymphopoietin (TSLP), a cytokine linking innate and T-helper type 2-adaptive immune responses. Moreover, the viral synapse upregulates the non-coding microRNA miR-375, known to control TSLP, and transfection of keratinocytes with anti-miR-375 blocks significantly TSLP secretion. Thus, the secretion of TSLP by keratinocytes is induced by the viral synapse in a miR-375 controlled manner. At the tissue level, these signals translate into the epidermal redistribution of Langerhans cells and formation of conjugates with T-cells, recapitulating the initial events observed in human foreskin infection by HIV-1. These results open new possibilities for designing strategies to block mucosal HIV-1 transmission, the major pathway by which HIV-1 spreads worldwide.Mucosal Immunology advance online publication 26 April 2017; doi:10.1038/mi.2017.23.

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

    SciTech Connect

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

    1995-05-01

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

  18. 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

  19. Protection against 2-chloroethyl ethyl sulfide (CEES) - induced cytotoxicity in human keratinocytes by an inducer of the glutathione detoxification pathway

    SciTech Connect

    Abel, Erika L.; Bubel, Jennifer D.; Simper, Melissa S.; Powell, Leslie; McClellan, S. Alex; Andreeff, Michael; MacLeod, Michael C.; DiGiovanni, John

    2011-09-01

    Sulfur mustard (SM or mustard gas) was first used as a chemical warfare agent almost 100 years ago. Due to its toxic effects on the eyes, lungs, and skin, and the relative ease with which it may be synthesized, mustard gas remains a potential chemical threat to the present day. SM exposed skin develops fluid filled bullae resulting from potent cytotoxicity of cells lining the basement membrane of the epidermis. Currently, there are no antidotes for SM exposure; therefore, chemopreventive measures for first responders following an SM attack are needed. Glutathione (GSH) is known to have a protective effect against SM toxicity, and detoxification of SM is believed to occur, in part, via GSH conjugation. Therefore, we screened 6 potential chemopreventive agents for ability to induce GSH synthesis and protect cultured human keratinocytes against the SM analog, 2-chloroethyl ethyl sulfide (CEES). Using NCTC2544 human keratinocytes, we found that both sulforaphane and methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) stimulated nuclear localization of Nrf2 and induced expression of the GSH synthesis gene, GCLM. Additionally, we found that treatment with CDDO-Me elevated reduced GSH content of NCTC2544 cells and preserved their viability by {approx} 3-fold following exposure to CEES. Our data also suggested that CDDO-Me may act additively with 2,6-dithiopurine (DTP), a nucleophilic scavenging agent, to increase the viability of keratinocytes exposed to CEES. These results suggest that CDDO-Me is a promising chemopreventive agent for SM toxicity in the skin. - Highlights: > CDDO-Me treatment increased intracellular GSH in human keratinocytes. > CDDO-Me increased cell viability following exposure to the half-mustard, CEES. > The cytoprotective effect of CDDO-Me was likely due to scavenging with endogenous GSH.

  20. 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.

  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. Early effects of Staphylococcus aureus biofilm secreted products on inflammatory responses of human epithelial keratinocytes

    PubMed Central

    2014-01-01

    Background Chronic wounds such as diabetic foot ulcers, pressure ulcers, and venous leg ulcers contribute to a considerable amount of mortality in the U.S. annually. The inability of these wounds to heal has now been associated with the presence of microbial biofilms. The aim of this study was to determine if products secreted by S. aureus biofilms play an active role in chronic wounds by promoting inflammation, which is a hallmark of chronic wounds. Methods In vitro experiments were conducted to examine changes in gene expression profiles and inflammatory response of human epithelial keratinocytes (HEKa) exposed to products secreted by S. aureus grown in biofilms or products secreted by S. aureus grown planktonically. Results After only two hours of exposure, gene expression microarray data showed marked differences in inflammatory, apoptotic, and nitric oxide responses between HEKa cells exposed to S. aureus biofilm conditioned media (BCM) and HEKa cells exposed to S. aureus planktonic conditioned media (PCM). As early as 4 hours post exposure, ELISA results showed significant increases in IL-6, IL-8, TNFα, and CXCL2 production by HEKa cells exposed to BCM compared to HEKa cells exposed to PCM or controls. Nitric oxide assay data also showed significant increases in nitric oxide production by HEKa cells treated with BCM compared to HEKa cells treated with PCM, or controls. Conclusions Taken together, these results support and extend previous findings that indicate products secreted by S. aureus biofilms directly contribute to the chronic inflammation associated with chronic wounds. PMID:24936153

  3. Glutathione peroxidase-1 inhibits UVA-induced AP-2{alpha} expression in human keratinocytes

    SciTech Connect

    Yu Lei; Venkataraman, Sujatha; Coleman, Mitchell C.; Spitz, Douglas R.; Wertz, Philip W.; Domann, Frederick E. . E-mail: frederick-domann@uiowa.edu

    2006-12-29

    In this study, we found a role for H{sub 2}O{sub 2} in UVA-induced AP-2{alpha} expression in the HaCaT human keratinocyte cell line. UVA irradiation not only increased AP-2{alpha}, but also caused accumulation of H{sub 2}O{sub 2} in the cell culture media, and H{sub 2}O{sub 2} by itself could induce the expression of AP-2{alpha}. By catalyzing the removal of H{sub 2}O{sub 2} from cells through over-expression of GPx-1, induction of AP-2{alpha} expression by UVA was abolished. Induction of transcription factor AP-2{alpha} by UVA had been previously shown to be mediated through the second messenger ceramide. We found that not only UVA irradiation, but also H{sub 2}O{sub 2} by itself caused increases of ceramide in HaCaT cells, and C2-ceramide added to cells induced the AP-2{alpha} signaling pathway. Finally, forced expression of GPx-1 eliminated UVA-induced ceramide accumulation as well as AP-2{alpha} expression. Taken together, these findings suggest that GPx-1 inhibits UVA-induced AP-2{alpha} expression by suppressing the accumulation of H{sub 2}O{sub 2}.

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

    NASA Astrophysics Data System (ADS)

    Werrlein, Robert; Madren-Whalley, Janna S.

    2002-06-01

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

  5. Photodynamic effects of 31 different phthalocyanines on a human keratinocyte cell line.

    PubMed

    Jančula, Daniel; Maršálek, Blahoslav; Babica, Pavel

    2013-10-01

    Phthalocyanines (Pcs, colored macromolecular compounds with the ability to generate singlet oxygen) represent a promising group of photosensitizers due to their intense absorption in the red and UV portion of the spectrum which leads to their excitation. In order to characterize possible toxic effects associated with eventual practical use and application of these chemicals, we employed an in vitro cell culture model to evaluate cytotoxic effects of 31 different phthalocyanines using neutral red uptake assay. An immortalized human keratinocyte cell line HaCaT was exposed to the tested chemicals for 2 or 24h, either with or without illumination in the last 60 min of the exposure period. After 2- or 24-h exposure without illumination, no cytotoxic effects or weak cytotoxic effects were induced by any Pc under the study and EC50 values could not be obtained within the tested concentration ranges (1.25-20 mg L(-1) or 0.625-10 mg L(-1)). On the other hand, exposure to phthalocyanines under illumination induced a significant cytotoxic effect. The most pronounced cytotoxicity was elicited by Pcs previously shown to have high positive charge densities at peripheral parts of substituent groups, which is most likely the factor responsible for the binding of Pc to negatively charged membranes on the cell surface and thus guaranteeing the tight connection necessary for the singlet oxygen attack on the cell surface.

  6. Effects of different terahertz frequencies on gene expression in human keratinocytes

    NASA Astrophysics Data System (ADS)

    Echchgadda, Ibtissam; Cerna, Cesario Z.; Sloan, Mark A.; Elam, David P.; Ibey, Bennet L.

    2015-03-01

    In recent years, a surge in the development of many terahertz (THz) sensing and imaging technologies occurred leading to increased use in military and civil operations. Therefore, understanding the biological effects associated with exposures to this radiation is becoming increasingly important. Previous studies have speculated that cells exposed to different frequencies of THz radiation may exhibit differential responses. However, empirical studies to confirm such differences have not been performed. The question of whether cells exposed to different THz frequencies exhibited specific biological responses remains unclear. In this study, we exposed human keratinocytes to a THz laser tuned to several different THz frequencies using our recently developed THz exposure system. This system consists of an optically pumped molecular gas THz laser source coupled to a modified cell culture incubator permitting THz radiation exposures under controlled standard tissue culture conditions. For all frequencies, we matched the THz exposure duration and irradiance. During THz exposure, we monitored the power as DC voltage-logged values (LabVIEW™ IV log). To determine the temperature changes by THz exposure, we collected temperature readings from the unexposed and THz-exposed cells using thermocouples. We assessed cellular viability after exposure using MTT colorimetric assays. We compared the changes in gene expression profiles using messenger RNA (mRNA) microarrays, and we identified the THz-induced signaling pathways for each frequency using bioinformatics. Our data provide valuable new insights that give a comparative picture of the genes and intracellular signaling pathways triggered in cells exposed to THz radiation at different frequencies.

  7. A mint purified extract protects human keratinocytes from short-term, chemically induced oxidative stress.

    PubMed

    Berselli, Patrizia Valeria Rita; Zava, Stefania; Montorfano, Gigliola; Corsetto, Paola Antonia; Krzyzanowska, Justyna; Oleszek, Wieslaw; Berra, Bruno; Rizzo, Angela Maria

    2010-11-10

    Oxidative stress is strictly correlated to the pathogenesis of many diseases, and a diet rich in fruits and vegetables, or adequately integrated, is currently considered to be a protective and preventive factor. This study aimed to analyze the efficacy of a 1 h preincubation with the highest nontoxic dose of a characterized Mentha longifolia extract (80 μg/mL) in protecting human keratinocytes (NCTC2544) from chemically induced oxidative stress (500 μM H2O2 for 2, 16, and 24 h). As reference synthetic pure compounds rosmarinic acid (360.31 μg/mL), a major mint phenolic constituent, and resveratrol (31.95 mg/mL), a well-known antioxidant, were used. Cellular viability was significantly protected by mint, which limited protein and DNA damage, decreased lipid peroxidation, and preserved glutathione and superoxide dismutase activity in the shorter phases of oxidative stress induction, in extents comparable to or better than those of pure compounds. These data suggest that mint use as only a flavoring has to be revised, taking into consideration its enrichment in foodstuff and cosmetics.

  8. In vitro evaluation of the effects of yttria-alumina-silica microspheres on human keratinocyte cells.

    PubMed

    Radu, T; Chiriac, M T; Popescu, O; Simon, V; Simon, S

    2013-02-01

    The behavior of yttria-alumina-silica spray-dried microspheres was investigated in vitro on a human keratinocyte cell line, first to exclude their cytotoxicity. The HaCaT cells were chosen due to their well-characterized phenotype and their phagocytic ability. Microscopic analysis and cell viability tests showed no negative effect of the microspheres on cells morphology and behavior. Scanning electron microscopy and transmission electron microscopy results evidenced the cellular internalization of the microspheres at 48 h after their incubation with cultured cells. The shape, size distribution, structure, composition, and chemical states of the elements on samples surface were analyzed by SEM, transmission electron microscopy, x-ray diffraction, and x-ray photoelectron spectroscopy, because these properties could influence their internalization by cells. The yttrium distribution on the microspheres surface was indicated by fluorescence microscopy imaging. The microspheres dimension and shape inside the cells was in accordance with their dimension and shape before incubation. The microspheres seemed captured and engulfed by the cells in native form and appeared resistant to degradation over the first 48 h. Most of the analyzed cells took up more microspheres, suggesting that the microspheres were actively phagocytosed by the cells and accumulated within the cytoplasm. X-ray photoelectron spectroscopy results on Al and Si atomic environments denoted Al-O-Si crosslinks, which improve the surface protection to corrosion. Copyright © 2012 Wiley Periodicals, Inc.

  9. 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.

  10. 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.

  11. Quantification and Characterization of UVB-Induced Mitochondrial Fragmentation in Normal Primary Human Keratinocytes

    PubMed Central

    Jugé, Romain; Breugnot, Josselin; Da Silva, Célia; Bordes, Sylvie; Closs, Brigitte; Aouacheria, Abdel

    2016-01-01

    UV irradiation is a major environmental factor causing skin dryness, aging and cancer. UVB in particular triggers cumulative DNA damage, oxidative stress and mitochondrial dysfunction. The objective of our study was to provide both qualitative and quantitative analysis of how mitochondria respond to UVB irradiation in normal human epidermal keratinocytes (NHEK) of healthy donors, with the rationale that monitoring mitochondrial shape will give an indication of cell population fitness and enable the screening of bioactive agents with UVB-protective properties. Our results show that NHEK undergo dose-dependent mitochondrial fragmentation after exposure to UVB. In order to obtain a quantitative measure of this phenomenon, we implemented a novel tool for automated quantification of mitochondrial morphology in live cells based on confocal microscopy and computational calculations of mitochondrial shape descriptors. This method was used to substantiate the effects on mitochondrial morphology of UVB irradiation and of knocking-down the mitochondrial fission-mediating GTPase Dynamin-related protein 1 (DRP1). Our data further indicate that all the major mitochondrial dynamic proteins are expressed in NHEK but that their level changes were stronger after mitochondrial uncoupler treatment than following UVB irradiation or DRP1 knock-down. Our system and procedures might be of interest for the identification of cosmetic or dermatologic UVB-protective agents. PMID:27731355

  12. Impact of blue LED irradiation on proliferation and gene expression of cultured human keratinocytes

    NASA Astrophysics Data System (ADS)

    Becker, Anja; Sticht, Carsten; Dweep, Harsh; van Abeelen, Frank A.; Gretz, Norbert; Oversluizen, Gerrit

    2015-03-01

    Blue light is known for its anti-microbial, anti-proliferative and anti-inflammatory effects. Furthermore, it is already used for the treatment of neonatal jaundice and acne. However, little is known about the exact mechanisms of action on gene expression level. The aim of this study was to assess the impact of blue LED irradiation on the proliferation and gene expression in immortalized human keratinocytes (HaCaT) in vitro. Furthermore its safety was assessed. XTT-tests revealed a decrease in cell proliferation in blue light irradiated cells depending on the duration of light irradiation. Moreover, gene expression analysis demonstrated deregulated genes already 3 hours after blue light irradiation. 24 hours after blue light irradiation the effects seemed to be even more pronounced. The oxidative stress response was significantly increased, pointing to increased ROS production due to blue light, as well as steroid hormone biosynthesis. Downregulated pathways or biological processes were connected to anti-inflammatory response. Interestingly, also the melanoma pathway contained significantly downregulated genes 24 hours after blue light irradiation, which stands in accordance to literature that blue light can also inhibit proliferation in cancer cells. First tests with melanoma cells revealed a decrease in cell proliferation after blue light irradiation. In conclusion, blue light irradiation might open avenues to new therapeutic regimens; at least blue light seems to have no effect that induces cancer growth or formation.

  13. Niacin protects against UVB radiation-induced apoptosis in cultured human skin keratinocytes

    PubMed Central

    LIN, FUQUAN; XU, WEN; GUAN, CUIPING; ZHOU, MIAONI; HONG, WEISONG; FU, LIFANG; LIU, DONGYIN; XU, AIE

    2012-01-01

    Niacin and its related derivatives have been shown to have effects on cellular activities. However, the molecular mechanism of its reduced immunosuppressive effects and photoprotective effects remains unclear. In this study, we investigated the molecular mechanism of the photoprotective effect of niacin in ultraviolet (UV)-irradiated human skin keratinocytes (HaCaT cells). We found that niacin effectively suppressed the UV-induced cell death and cell apoptosis of HaCaT cells. Existing data have shown that AKT activation is involved in the cell survival process. Yet, the potential mechanism of niacin in protection against UV-induced skin damage has thus far not fully been eluvidated. We observed that niacin pretreatment enhances UV induced activation of AKT (Ser473 phosphorylation) as well as that of the downstream signal mTOR (S6 and 4E-BP1 phosphorylation). The PI3K/AKT inhibitor, LY294002, and the mTOR inhibitor, rapamycin, largely neutralized the protective effects of niacin, suggesting that AKT and downstream signaling mTOR/S6 activation are necessary for the niacin-induced protective effects against UV-induced cell death and cell apoptosis. Collectively, our data suggest that niacin may be utilized to prevent UV-induced skin damage and provide a novel mechanism of its photoprotective effects against the UV radiation of sunlight by modulating both AKT and downstream mTOR signaling pathways. PMID:22246168

  14. Phototoxicity of phenylenediamine hair dye chemicals in Salmonella typhimurium TA102 and human skin keratinocytes.

    PubMed

    Mosley-Foreman, Charity; Choi, Jaehwa; Wang, Shuguang; Yu, Hongtao

    2008-12-01

    Phenylenediamines (PD) are dye precursors used to manufacture hair dyes. The three PDs, 1,2-,1,3-, and 1,4-PD and three chlorinated PDs, 4-chloro-1,2-PD, 4-chloro-1,3-PD, and 4,5-dichloro-1,2-PD were studied for their mutagenic effect in Salmonella typhimurium TA 102, cytotoxicity in human skin keratinocyte cells, and for DNA cleavage. The results show that all six compounds are not toxic/mutagenic in TA 102 bacteria or skin cells, and do not cause DNA cleavage in PhiX 174 phage DNA. If the same tests are carried out by exposing them to light irradiation concurrently, all three chlorinated PDs cause mutation in TA 102 bacteria and single strand cleavage in PhiX174 phage DNA. This indicates that chlorination of the PDs makes these compounds more photochemically active and produces reactive species that cause DNA damage and mutation. For the photocytotoxicity test in skin cells, it appears there is no such structure-activity relationship. Two chlorinated PDs and two non-chlorinated PDs are cytotoxic at a fairly high concentration (1000microM) upon exposure to light irradiation.

  15. Protective effects of a red orange extract on UVB-induced damage in human keratinocytes.

    PubMed

    Cimino, Francesco; Cristani, Mariateresa; Saija, Antonina; Bonina, Franco Paolo; Virgili, Fabio

    2007-01-01

    UV light is considered one of the major etiological factor in skin aging, cancer and also to systemic impairment such as immunosuppression. Increased production of reactive oxygen species (ROS) and oxidative stress condition are known to play a central role in initiating and driving the signalling events that lead to cellular response following UV irradiation. In the present study we have investigated the photoprotective activity of a standardized extract from red orange (ROE), obtained from three red orange varieties and containing as main active principles phenolic compounds (anthocyanins, flavanones and hydroxycinnamic acids) and ascorbic acid. The aim of this study was to evaluate the efficacy of ROE in modulating cellular responses to UVB in human keratinocytes (HaCaT). Our data indicate that ROE is potentially able to efficiently counteract UVB-induced response, and in particular some events associated to inflammation and apoptosis, such as NF-kB and AP-1 translocation and procaspase-3 cleavage. This activity is probably due to a block of cellular oxidative stress-related events. Thus we can propose ROE as a useful natural standardised extract in skin photoprotection with promising applications in the field of dermatology.

  16. Hydrogen sulfide modulates the release of nitric oxide and VEGF in human keratinocytes.

    PubMed

    Merighi, Stefania; Gessi, Stefania; Varani, Katia; Fazzi, Debora; Borea, Pier Andrea

    2012-11-01

    Hydrogen sulfide (H(2)S) is a novel signaling molecule with both pro- or anti-inflammatory effect. The present study aimed to: (i) characterize the in vitro effects of H(2)S on human keratinocyte's proliferation and death; (ii) investigate the ability of H(2)S to modulate VEGF and NO production; (iii) examine the intracellular signaling pathways involved in VEGF and NO modulatory effect. We found that exogenous application of H(2)S (NaHS and GYY4137 as H(2)S donors) significantly enhances NO through increase of iNOS, in a manner Akt-dependent. The increment in NO down-regulates ERK1/2 activation thereby resulting in the decrease of VEGF release. We suggest that H(2)S-releasing agents may be promising therapeutics for chronic inflammatory disorders of the skin, i.e. psoriasis, in which NO increases as well as anti-VEGF treatments have been suggested to be novel effective approaches.

  17. Low-concentration hydrogen peroxide can upregulate keratinocyte intracellular calcium and PAR-2 expression in a human keratinocyte-melanocyte co-culture system.

    PubMed

    Li, Jian; Tang, Lu-Yan; Fu, Wen-Wen; Yuan, Jin; Sheng, You-Yu; Yang, Qin-Ping

    2016-12-01

    Hydrogen peroxide (H2O2) may have a biphasic effect on melanin synthesis and melanosome transfer. High H2O2 concentrations are involved in impaired melanosome transfer in vitiligo. However, low H2O2 concentration promotes the beneficial proliferation and migration of melanocytes. The aim of this study was to explore low H2O2 and its mechanism in melanosome transfer, protease-activated receptor-2 (PAR-2) expression and calcium balance. Melanosomes were fluorescein-labeled for clear visualization of their transfer. The expression of protease-activated receptor-2 (PAR-2) in keratinocytes was determined by western blot analysis. Flow cytometry was employed to evaluate the effects of H2O2 on calcium levels in keratinocytes. Fluorescence microscopy showed the upregulation of melanosome transfer into keratinocytes following 0.3 mM H2O2 treatment in the co-cultures rather than in the untreated control groups, which was associated with higher expression of PAR-2 protein and increased calcium concentration. The addition of a PAR-2 antagonist inhibited the positive activity of H2O2 and calcium flow in keratinocytes. When calcium flow was blocked by a calcium chelator, the addition of H2O2 did not increase the PAR-2 expression level in keratinocytes, therefore, inhibiting dendrite formation and melanosome transfer. Low H2O2 concentration promotes melanosome transfer with increased PAR-2 expression level and calcium concentration in keratinocytes. In addition, the interaction between melanocytes and keratinocytes is more beneficial to enhance calcium levels in keratinocytes which mediate melanin transfer. Moreover, low H2O2 concentration promotes dendrite formation, in which extracellular calcium and Par-2 were involved.

  18. Overabundance of Putative Cancer Stem Cells in Human Skin Keratinocyte Cells Malignantly Transformed by Arsenic

    PubMed Central

    Sun, Yang; Tokar, Erik J.; Waalkes, Michael P.

    2012-01-01

    Arsenic is a human skin carcinogen. Cancer is probably a disease driven by stem cells (SCs), and SCs are likely a key target during arsenic oncogenesis. In utero arsenic exposure predisposes mice to skin cancers that overproduce cancer SCs (CSCs) and have distorted CSC signaling and population dynamics. Therefore, we hypothesized CSC accumulation may occur during arsenic-induced malignant transformation in vitro of human skin keratinocytes. Thus, the HaCaT cell line, malignantly transformed by arsenite (100nM, 30 weeks; termed As-TM cells) in prior work, was further studied for the quantity and nature of SCs after this transformation. SCs were isolated from passage-matched control and As-TM cells by a magnetic bead system that enriches for CD34-positive cells. There were 2.5 times more SCs isolated from As-TM cells than control. Holoclone production from As-TM putative CSCs was 2.5-fold higher by 1 week and 3.5-fold higher by 2 weeks than control SCs. Potential malignant phenotype was assessed in isolated SC/CSCs. Transcript level of SC/CSC markers were elevated in both isolated As-TM CSCs and control SCs compared with parental cells, but compared with control SCs, As-TM putative CSCs had elevated CD34, K5, K14, K15, and K19 transcripts and dramatically stronger staining for p63, Rac1, K5, Notch1, and K19. As-TM putative CSCs also showed markedly elevated MMP-9 secretion and colony formation, indicators of cancer phenotype, even compared with total population of As-TM cells. Thus, malignant phenotype is particularly pronounced in CSCs after arsenic-induced transformation of human skin cells and occurs concurrently with a potential overproduction of these cells. PMID:22011395

  19. Comparative toxicities of bismuth oxybromide and titanium dioxide exposure on human skin keratinocyte cells.

    PubMed

    Gao, Xiaoya; Wang, Yawen; Peng, Shiqi; Yue, Bin; Fan, Caimei; Chen, Weiyi; Li, Xiaona

    2015-09-01

    Nano-sized bismuth oxybromide (BiOBr) particles are being considered for applications within the semiconductor industry. However, little is known about their potential impact on human health. In this study, we comparatively investigated the cytotoxicity of BiOBr and titanium dioxide (TiO2) nanoparticles (NPs) using human skin keratinocyte cell line (HaCaT) as a research model. Results indicate that lamellar-shaped BiOBr (length: 200 nm, width: 150 nm, and an average thickness: around 15 nm) has less toxic effects on cell viability and intracellular organelles than TiO2 (P25) NPs. BiOBr mainly induced late cell apoptosis, while for TiO2, both early apoptosis and late apoptosis were involved. Cell cycle arrest was found in cells on both NPs exposure, and more prominent in TiO2-treated cells. More cellular uptake was achieved after TiO2 exposure, particularly at 10 μg mL(-1), presence of TiO2 resulted in more than 2-fold increase in cellular granularity compared with BiOBr. Furthermore, TiO2 had a high potential to generate intracellular reactive oxygen species (ROS) in cells, where a 2.7-fold increase in TiO2 group and 2.0-fold increase in BiOBr group at the same concentration of 25 μg mL(-1). Higher cellular uptake and ROS stimulation should contribute to the more hazards of TiO2 than BiOBr NPs. This knowledge is a crucial component in the environmental and human hazard assessment of BiOBr and TiO2 NPs.

  20. Dynamic changes in nicotinamide pyridine dinucleotide content in normal human epidermal keratinocytes and their effect on retinoic acid biosynthesis

    SciTech Connect

    Pinkas-Sarafova, Adriana . E-mail: apinkassaraf@notes.cc.sunysb.edu; Markova, N.G. . E-mail: nmarkova@notes.cc.sunysb.edu; Simon, M. . E-mail: marsimon@notes.cc.sunysb.edu

    2005-10-21

    The function of many enzymes that regulate metabolism and transcription depends critically on the nicotinamide pyridine dinucleotides. To understand the role of NAD(P)(H) in physiology and pathophysiology, it is imperative to estimate both their amount and ratios in a given cell type. In human epidermis and in cultured epidermal keratinocytes, we found that the total dinucleotide content is in the low millimolar range. The dinucleotide pattern changes during proliferation and maturation of keratinocytes in culture. Differences in the concentrations of NAD(P)(H) of 1.5- to 12-fold were observed. This resulted in alteration of the NAD(P)H/NAD(P) ratio, which could impact the differential regulation of both transcriptional and metabolic processes. In support of this notion, we provide evidence that the two-step oxidation of retinol to retinoic acid, a nuclear hormone critical for epidermal homeostasis, can be regulated by the relative physiological amounts of the pyridine dinucleotides.

  1. Enhanced G2 chromatid radiosensitivity, an early stage in the neoplastic transformation of human epidermal keratinocytes in culture

    SciTech Connect

    Gantt, R.; Sanford, K.K.; Parshad, R.; Price, F.M.; Peterson, W.D. Jr.; Rhim, J.S.

    1987-03-01

    A deficiency in DNA repair, manifest as enhanced chromatid radiosensitivity during the G2 phase of the cell cycle, together with a proliferative stimulus such as that provided by active oncogenes may be necessary and sufficient for the malignant neoplastic transformation of human keratinocytes in culture. Normal epidermal keratinocytes established as continuous cell lines by transfection with pSV3-neo or infection with adeno 12-SV40 hybrid virus developed enhanced G2 chromatid radiosensitivity after 18 passages in culture. In contrast to cells from primary or secondary culture, these cells could be transformed to malignant neoplastic cells by infection with Kirsten murine sarcoma virus containing the Ki-ras oncogene or in one line by the chemical carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine; both of these agents produced a marked proliferative response. Cytological heterogeneity and karyotypic instability characterized the cells during their progression to neoplasia. These results are interpreted in terms of a mechanism for neoplastic transformation.

  2. Gracilaria bursa-pastoris (Gmelin) Silva extract attenuates ultraviolet B radiation-induced oxidative stress in human keratinocytes.

    PubMed

    Piao, M J; Kim, K C; Zheng, J; Yao, C W; Cha, J W; Kang, H K; Yoo, E S; Koh, Y S; Ko, M H; Lee, N H; Hyun, Jin Won

    2014-01-01

    The purpose of this study was to assess the protective effects of an ethanol extract derived from the red alga Gracilaria bursa-pastoris (Gmelin) Silva (GBE) on ultraviolet B (UVB)-irradiated human HaCaT keratinocytes. GBE exhibited scavenging activity against intracellular reactive oxygen species that were induced by either hydrogen peroxide or UVB radiation. In addition, both the superoxide anion and the hydroxyl radical were scavenged by GBE in cell-free systems. GBE absorbed light in the UVB range (280-320 nm) of the electromagnetic spectrum and lessened the extent of UVB-induced oxidative damage to cellular lipids, proteins, and DNA. Finally, GBE-treated keratinocytes showed a reduction in UVB-induced apoptosis, as exemplified by fewer apoptotic bodies. These results suggest that GBE exerts cytoprotective actions against UVB-stimulated oxidative stress by scavenging ROS and absorbing UVB rays, thereby attenuating injury to cellular constituents and preventing cell death.

  3. Protective effects of phenolic fraction of blue honeysuckle fruits against UVA-induced damage to human keratinocytes.

    PubMed

    Svobodová, Alena; Rambousková, Jana; Walterová, Daniela; Vostálová, Jitka

    2008-06-01

    In this study, the UVA (photo)protective activity of the phenolic fraction of L. caerulea fruits (PFLC) was assessed in human keratinocytes HaCaT. The keratinocytes were pre- or post-treated with PFLC (1-250 mg/l) and exposed to UVA irradiation (10-30 J/cm(2)). The results showed that both pre- and post-treatment with PFLC significantly suppressed UVA-induced ROS production, which was also revealed as a decrease in intracellular lipid peroxidation and elevation of reduced glutathione. Protection was concentration-dependent with a maximum at 50 mg/l. These results suggest that PFLC attenuates UVA-induced oxidative stress by reduction of ROS generation and ROS-mediated damage. For this reason, PFLC has potentially skin-protective functions against the deleterious effects of sunlight.

  4. Tumorigenic transformation of murine keratinocytes by the E5 genes of bovine papillomavirus type 1 and human papillomavirus type 16.

    PubMed Central

    Leptak, C; Ramon y Cajal, S; Kulke, R; Horwitz, B H; Riese, D J; Dotto, G P; DiMaio, D

    1991-01-01

    To examine the biological properties of the bovine papillomavirus type 1 (BPV) and human papillomavirus type 16 (HPV16) E5 genes, each was cloned separately into a retroviral expression vector and helper-free recombinant viruses were generated in packaging cell lines. The BPV E5 retroviruses efficiently caused morphologic and tumorigenic transformation of cultured lines of murine fibroblasts, whereas the HPV16 E5 viruses were inactive in these assays. In contrast, infection of the p117 established line of murine epidermal keratinocytes with either the BPV or the HPV16 E5 retrovirus resulted in the generation of tumorigenic cells. Pam212 murine keratinocytes were also transformed to tumorigenicity by the HPV16 E5 gene but not by the gene carrying a frameshift mutation. These results establish that the HPV16 E5 gene is a transforming gene in cells related to its normal host epithelial cells. Images PMID:1658398

  5. Clinical and biochemical studies support smokeless tobacco’s carcinogenic potential in the human oral cavity

    PubMed Central

    Mallery, Susan R.; Tong, Meng; Michaels, Gregory C.; Kiyani, Amber R.; Hecht, Stephen S.

    2014-01-01

    In 2007, International Agency for Cancer Research presented compelling evidence that linked smokeless tobacco use to the development of human oral cancer. While these findings imply vigorous local carcinogen metabolism, little is known regarding levels and distribution of Phase I, II and drug egress enzymes in human oral mucosa. In the study presented here, we integrated clinical data, imaging and histopathologic analyses of an oral squamous cell carcinoma that arose at the site of smokeless tobacco quid placement in a patient. Immunoblot and immunohistochemical (IHC) analyses were employed to identify tumor and normal human oral mucosal smokeless tobacco-associated metabolic activation and detoxification enzymes. Human oral epithelium contains every known Phase I enzyme associated with nitrosamine oxidative bioactivation with ~2 fold inter-donor differences in protein levels. Previous studies have confirmed ~3.5 fold inter-donor variations in intraepithelial Phase II enzymes. Unlike the superficially located enzymes in non-replicating esophageal surface epithelium, IHC studies confirmed oral mucosal nitrosamine metabolizing enzymes reside in the basilar and suprabasilar region which notably is the site of ongoing keratinocyte DNA replication. Clearly, variations in product composition, nitrosamine metabolism and exposure duration will modulate clinical outcomes. The data presented here form a coherent picture consistent with the abundant experimental data that links tobacco-specific nitrosamines to human oral cancer. PMID:24265177

  6. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

    SciTech Connect

    Chen, Yi; Pirisi, Lucia; Creek, Kim E.

    2013-09-15

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski.

  7. 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.

  8. Six1 overexpression at early stages of HPV16-mediated transformation of human keratinocytes promotes differentiation resistance and EMT

    SciTech Connect

    Xu, Hanwen; Pirisi, Lucia; Creek, Kim E.

    2015-01-01

    Previous studies in our laboratory discovered that SIX1 mRNA expression increased during in vitro progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we explored the role of Six1 at early stages of HPV16-mediated transformation by overexpressing Six1 in HKc/HPV16. We found that Six1 overexpression in HKc/HPV16 increased cell proliferation and promoted cell migration and invasion by inducing epithelial–mesenchymal transition (EMT). Moreover, the overexpression of Six1 in HKc/HPV16 resulted in resistance to serum and calcium-induced differentiation, which is the hallmark of the HKc/DR phenotype. Activation of MAPK in HKc/HPV16 overexpressing Six1 is linked to resistance to calcium-induced differentiation. In conclusion, this study determined that Six1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation of human keratinocytes. - Highlights: • Six1 expression increases during HPV16-mediated transformation. • Six1 overexpression causes differentiation resistance in HPV16-immortalized cells. • Six1 overexpression in HPV16-immortalized keratinocytes activates MAPK. • Activation of MAPK promotes EMT and differentiation resistance. • Six1 overexpression reduces Smad-dependent TGF-β signaling.

  9. Influence of acidic pH on keratinocyte function and re-epithelialisation of human in vitro wounds.

    PubMed

    Lönnqvist, Susanna; Emanuelsson, Peter; Kratz, Gunnar

    2015-01-01

    Chronic wounds are one of the greatest challenges for the healthcare system. Today, a plethora of dressings are used in the treatment of these wounds, each with specific influence on the wound environment. Due to differences in the permeability of the dressings the use will result in differences in the pH balance in the wound bed. However, little is known about how changes in the pH in the wound environment affect the different phases of the healing process. The aim of the present study was to investigate the effects of acidic pH on the regeneration phase by studying keratinocyte function in vitro and re-epithelialisation in an in vitro model of human skin. In vitro assays showed reduced viability and migration rates in human keratinocytes when pH was lowered. Real time PCR revealed differential expression of genes related to wound healing and environmental impairment. Tissue culture showed no re-epithelialisation of wounds subjected to pH 5.0 and moderate re-epithelialisation at pH 6.0, compared to controls at pH 7.4. The results indicate that lowering pH down to pH 5.0 in wounds is counterproductive in aspect of keratinocyte function which is crucial for successful wound healing.

  10. Induced pluripotent stem cells from human hair follicle keratinocytes as a potential source for in vitro hair follicle cloning

    PubMed Central

    Lim, Sheng Jye; Ho, Shu Cheow; Mok, Pooi Ling; Tan, Kian Lee; Ong, Alan H.K.

    2016-01-01

    Background Human hair follicles are important for the renewal of new hairs and their development. The generation of induced pluripotent stem cells (iPSCs) from hair follicles is easy due to its accessibility and availability. The pluripotent cells derived from hair follicles not only have a higher tendency to re-differentiate into hair follicles, but are also more suited for growth in hair scalp tissue microenvironment. Methods In this study, human hair follicular keratinocytes were used to generate iPSCs, which were then further differentiated in vitro into keratinocytes. The derived iPSCs were characterised by using immunofluorescence staining, flow cytometry, and reverse-transcription PCR to check for its pluripotency markers expression. Results The iPSC clones expressed pluripotency markers such as TRA-1-60, TRA-1-81, SSEA4, OCT4, SOX2, NANOG, LEFTY, and GABRB. The well-formed three germ layers were observed during differentiation using iPSCs derived from hair follicles. The successful formation of keratioctyes from iPSCs was confirmed by the expression of cytokeratin 14 marker. Discussion Hair follicles represent a valuable keratinocytes source for in vitro hair cloning for use in treating hair balding or grafting in burn patients. Our significant findings in this report proved that hair follicles could be used to produce pluripotent stem cells and suggested that the genetic and micro-environmental elements of hair follicles might trigger higher and more efficient hair follicles re-differentiation. PMID:27867768

  11. 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.

  12. Toll-like receptor 2 (TLR2) mediates intracellular signalling in human keratinocytes in response to Malassezia furfur.

    PubMed

    Baroni, Adone; Orlando, Manuela; Donnarumma, Giovanna; Farro, Pietro; Iovene, Maria Rosaria; Tufano, Maria Antonietta; Buommino, Elisabetta

    2006-01-01

    Toll-like receptors (TLRs) are crucial players in the innate immune response to microbial invaders. The lipophilic yeast Malassezia furfur has been implicated in the triggering of scalp lesions in psoriasis. The aim of the present study was to assess the role of TLRs in the defence against M. furfur infection. The expression of the myeloid differentiation factor 88 (MyD88) gene, which is involved in the signalling pathway of many TLRs, was also analysed. In addition, a possible correlation of antimicrobial peptides of the beta-defensin family to TLRs was tested. Human keratinocytes infected with M. furfur and a variety of M. furfur-positive psoriatic skin biopsies were analysed by RT-PCR, for TLRs, MyD88, human beta-defensin 2 (HBD-2), HBD-3 and interleukin-8 (IL-8) mRNA expression. When keratinocytes were infected with M. furfur, an up-regulation for TLR2, MyD88, HBD-2, HBD-3 and IL-8 mRNA was demonstrated, compared to the untreated cells. The same results were obtained when psoriatic skin biopsies were analysed. The M. furfur-induced increase in HBD-2 and IL-8 gene expression is inhibited by anti-TLR2 neutralising antibodies, suggesting that TLR2 is involved in the M. furfur-induced expression of these molecules. These findings suggest the importance of TLRs in skin protection against fungi and the importance of keratinocytes as a component of innate immunity.

  13. 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

  14. 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.

  15. Xylan-regulated delivery of human keratinocyte growth factor-2 to the inflamed colon by the human anaerobic commensal bacterium Bacteroides ovatus.

    PubMed

    Hamady, Zaed Z R; Scott, Nigel; Farrar, Mark D; Lodge, J Peter A; Holland, Keith T; Whitehead, Terence; Carding, Simon R

    2010-04-01

    Human growth factors are potential therapeutic agents for various inflammatory disorders affecting the gastrointestinal tract. However, they are unstable when administered orally and systemic administration requires high doses increasing the risk of unwanted side effects. Live microorganism-based delivery systems can overcome these problems although they suffer from the inability to control heterologous protein production and there are concerns regarding biosafety and environmental contamination. To overcome these limitations we have developed a new live bacteria drug-delivery system using the human commensal gut bacterium Bacteroides ovatus engineered to secrete human growth factors in response to dietary xylan. The anaerobic nature of B ovatus provides an inherent biosafety feature. B ovatus strains expressing human keratinocyte growth factor-2, which plays a central role in intestinal epithelial homeostasis and repair (BO-KGF), were generated by homologous recombination and evaluated using the dextran sodium sulfate (DSS)-induced model of intestinal epithelial injury and colitis. In response to xylan BO-KGF produced biologically active KGF both in vitro and in vivo. In DSS treated mice administration of xylan and BO-KGF had a significant therapeutic effect in reducing weight loss, improving stool consistency, reducing rectal bleeding, accelerating healing of damaged epithelium, reducing inflammation and neutrophil infiltration, reducing expression of pro-inflammatory cytokines, and accelerating production of goblet cells. BO-KGF and xylan treatment also had a marked prophylactic effect limiting the development of inflammation and disruption of the epithelial barrier. This novel, diet-regulated, live bacterial drug delivery system may be applicable to treating various bowel disorders.

  16. 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

  17. 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.

  18. Oral Human Papillomavirus Infection in Children.

    PubMed

    Ilea, Aranka; Boşca, Bianca; Miclăuş, Viorel; Rus, Vasile; Băbţan, Anida Maria; Mesaros, Anca; Crişan, Bogdan; Câmpian, Radu Septimiu

    2016-02-01

    Oral human papillomavirus infection is rare in children, but the presence of a villous lesion with slow but continuous growth concerns parents, who need information and therapeutic solutions from the physician. All these aspects are discussed based on a case report of a 9-year-old child with an oral human papillomavirus infection.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. A Novel Controllable Hydrogen Sulfide-Releasing Molecule Protects Human Skin Keratinocytes Against Methylglyoxal-Induced Injury and Dysfunction

    PubMed Central

    Yang, Chun-tao; Zhao, Yu; Xian, Ming; Li, Jian-hua; Dong, Qi; Bai, Hong-bo; Xu, Ji-de; Zhang, Mei-fen

    2014-01-01

    Background/Aim Delayed wound healing is a common skin complication of diabetes, which is associated with keratinocyte injury and dysfunction. Levels of methylglyoxal (MGO), an α-dicarbonyl compound, are elevated in diabetic skin tissue and plasma, while levels of hydrogen sulfide (H2S), a critical gaseous signaling molecule, are reduced. Interestingly, the gas has shown dermal protection in our previous study. To date, there is no evidence demonstrating whether MGO affects keratinocyte viability and function or H2S donation abolishes these effects and improves MGO-related impairment of wound healing. The current study was conducted to examine the effects of MGO on the injury and function in human skin keratinocytes and then to evaluate the protective action of a novel H2S-releasing molecule. Methods An N-mercapto-based H2S donor (NSHD)-1 was synthesized and its ability to release H2S was observed in cell medium and cells, respectively. HaCaT cells, a cell line of human skin keratinocyte, were exposed to MGO to establish an in vitro diabetic wound healing model. NSHD-1 was added to the cells before MGO exposure and the improvement of cell function was observed in respect of cellular viability, apoptosis, oxidative stress, mitochondrial membrane potential (MMP) and behavioral function. Results Treatment with MGO decreased cell viability, induced cellular apoptosis, increased intracellular reactive oxygen species (ROS) content and depressed MMP in HaCaT cells. The treatment also damaged cell behavioral function, characterized by decreased cellular adhesion and migration. The synthesized H2S-releasing molecule, NSHD-1, was able to increase H2S levels in both cell medium and cells. Importantly, pretreatment with NSHD-1 inhibited MGO-induced decreases in cell viability and MMP, increases in apoptosis and ROS accumulation in HaCaT cells. The pretreatment was also able to improve adhesion and migration function. Conclusion These results demonstrate that the novel

  4. Efficient KRT14 Targeting and Functional Characterization of Transplanted Human Keratinocytes for the Treatment of Epidermolysis Bullosa Simplex

    PubMed Central

    Petek, Lisa M; Fleckman, Philip; Miller, Daniel G

    2010-01-01

    Inherited skin blistering conditions collectively named epidermolysis bullosa (EB) cause significant morbidity and mortality due to the compromise of the skin's barrier function, the pain of blisters, inflammation, and in some cases scaring and cancer. The simplex form of EB is usually caused by dominantly inherited mutations in KRT5 or KRT14. These mutations result in the production of proteins with dominant-negative activity that disrupt polymerization of intermediate filaments in the basal keratinocyte layer and result in a weak epidermal–dermal junction. The genome of adeno-associated virus (AAV) vectors can recombine with chromosomal sequence so that mutations can be corrected, or production of proteins with dominant-negative activity can be disrupted. We demonstrate a clinically feasible strategy for efficient targeting of the KRT14 gene in normal and EB-affected human keratinocytes. Using a gene-targeting vector with promoter trap design, targeted alteration of one allele of KRT14 occurred in 100% of transduced cells and transduction frequencies ranged from 0.1 to 0.6% of total cells. EBS patient keratinocytes with precise modifications of the mutant allele are preferentially recovered from targeted cell populations. Single epidermal stem cell clones produced histologically normal skin grafts after transplantation to athymic mice and could generate a sufficient number of cells to transplant the entire skin surface of an individual. PMID:20571545

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

    PubMed

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

    2015-01-01

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

  6. Typha latifolia L. fruit polysaccharides induce the differentiation and stimulate the proliferation of human keratinocytes in vitro.

    PubMed

    Gescher, Kirsten; Deters, Alexandra M

    2011-09-01

    In Northern America Typha latifolia L. (Typhaceae) fruits are used for more than 4000 years for treatment of skin disorders, burns and as wound dressing to absorb the ichors. The following studies attempted to characterize water-soluble polysaccharides from aqueous Typha latifolia extracts and to investigate the influence of the polymers on cell physiology of human dermal fibroblasts (NHDF) and epidermal keratinocytes (NHEK). Water-soluble raw polysaccharides (RPS) were isolated from Typha latifolia fruits and fractionated by anion exchange chromatography (AEC) and size exclusion chromatography (GPC). Fractions obtained were characterized concerning monosaccharide composition by HPAEC-PAD. The bioactivity of the polysaccharides was investigated on cell viability, proliferation, differentiation and gene expression NHDF of NHEK. RPS was fractionated into 5 heterodisperse fractions (TL1-TL5). The polysaccharides were composed mainly of glucose (more than 50% in RPS and TL4), galactose, xylose, mannose, glucuronic acid, galacturonic acid, arabinose, ribose, fucose, rhamnose, and fructose with differing amounts concerning to RPS and AEC-fractions. Proteins were detected in the RPS (10%) and to a less extend in TL1-TL3 (1-3%). TL1-TL3 significantly increased the proliferation of keratinocytes, whereas TL4 was shown to be a potent inductor of the early differentiation process of keratinocytes. Gene expression analysis supported these results since Smad3 and PKC-α, known to be part of signal pathways leading to cell differentiation, were significantly up regulated. Effects on fibroblasts were not observed, indicating cell specific activity of the polysaccharides. The results clearly indicate a rationale for the traditional use of Typha latifolia fruits extracts for wound healing to the strong stimulatory activity of the polysaccharides on keratinocytes proliferation and early differentiation, major activities necessary for potent wound-healing agents. Copyright © 2011

  7. Azelaic acid modulates the inflammatory response in normal human keratinocytes through PPARgamma activation.

    PubMed

    Mastrofrancesco, Arianna; Ottaviani, Monica; Aspite, Nicaela; Cardinali, Giorgia; Izzo, Enzo; Graupe, Klaus; Zouboulis, Christos C; Camera, Emanuela; Picardo, Mauro

    2010-09-01

    Azelaic acid (AzA), a nine-carbon dicarboxylic acid, is an agent for the topical treatment of acne. It has also been shown to be effective in rosacea; however, the mechanism of action has not been clarified. Because inflammation is a common feature of both conditions, we investigated the effects of azelaic acid on the inflammatory response of normal human keratinocytes to ultraviolet B light, which is a photosensitizer agent in rosacea. AzA, at 20 mM, a concentration achievable following topical application of a 15% gel, suppresses ultraviolet B light-induced interleukins-1beta, -6 and tumor necrosis factor-alpha mRNA expression and protein secretion. Mechanistically, azelaic acid significantly reduced the ultraviolet B light-induced nuclear translocation of nuclear factor kB p65 subunit and the phosphorylation of the p38 mitogen and stress-activated protein kinase. Moreover, as peroxisome proliferators-activated receptor gamma, (PPARgamma) which has a crucial role in the control of inflammation, is activated by fatty acids and products of lipid peroxidation, we further investigated the effect of azelaic acid on the expression of this nuclear receptor. AzA induced peroxisome proliferators-activated receptor-gamma mRNA and its transcriptional activity. The PPARgamma antagonist GW9662 abrogated the inhibitory effects of AzA on the UVB-induced pro-inflammatory cytokines release and on the cell proliferation. Our study provides new insights into the molecular mechanisms of the activity of azelaic acid and lands additional evidences for its therapeutic effects on inflammatory skin diseases, such as rosacea.

  8. Potential Wound Healing Activities of Galla Rhois in Human Fibroblasts and Keratinocytes.

    PubMed

    Park, Hyo-Hyun; Park, Na-Young; Kim, Sun-Gun; Jeong, Kyu-Tae; Lee, Eu-Jin; Lee, Eunkyung

    2015-01-01

    Wound healing is a complex process orchestrated by the regeneration of the epithelium and the remodeling of the extracellular matrix through processes like collagen deposition. Galla Rhois has been widely used in traditional Korean medicine for its various pharmacological effects, including an anticoccidial effect, however, little is known about its healing activity. The purpose of this study was to determine the effects of Galla Rhois ethanol extract (GRE) on wound healing activities, including H2O2-induced oxidative stress, cell migration, and lactate dehydrogenase (LDH) release assays using human keratinocyte (HaCaT) and dermal fibroblasts (CCD-986SK). In addition, total soluble collagen deposition and collagen gene expression for Type I and III collagen were evaluated in CCD-986SK. Total tannin and flavonoid contents for GRE were measured. GRE induced a significant increase in the number and migration of cells, along with a decrease in cell death and LDH release. In addition, it also induced the over-expression of collagen Type I and III mRNA and caused increased synthesis of total soluble collagen. The contents of total tannin and flavonoid for GRE were 55.7% ([Formula: see text][Formula: see text]mg/g) and 62.9% ([Formula: see text][Formula: see text]mg/g), respectively. The results suggest that GRE can cause accelerated wound healing by increasing cell survival, proliferation, migration, and collagen synthesis along with a potential anti-oxidant property. This evidence provides novel insight into natural therapy for tissue injury.

  9. Induction of cyclin D1 by submicromolar concentrations of arsenite in human epidermal keratinocytes

    SciTech Connect

    Hwang, B.-J.; Utti, Charles; Steinberg, Mark . E-mail: marste@sci.ccny.cuny.edu

    2006-12-01

    Arsenic is a prevalent environmental carcinogen but arsenic is not directly mutagenic and the mechanism by which arsenite brings about oncogenic transformation is poorly understood. To gain insight into the oncogenic properties of arsenic, we studied the expression of cyclin D1 in cultured human epidermal keratinocytes treated with submicromolar concentrations of sodium arsenite. Arsenite at concentrations between 200 and 800 nM over a 3-day period brought about an increase in cell growth rate. Uptake of the vital stain, neutral red, arsenite at 200 and 400 nM concentrations brought about a parallel increase in cell viability over the same treatment period. Analysis of cell cycle parameters by flow cytometry showed that the growth stimulation was accompanied by a concomitant shift from the G1 into the S/G2 cell cycle compartment in the arsenite-treated cells. Real-time PCR analysis of cyclin D1 transcription showed that there was an induction of more than three-fold in cells exposed to 400 nM arsenite for 3 days. Quantitation of cyclin D levels in Western blots showed that arsenite treatment caused a time-dependent induction of cyclin D proteins representing an induction of about 2.0-fold after a 7 day treatment period. Electrophoretic mobility shift assays (EMSA) showed that arsenite also stimulated binding of the transcription factors, AP1 and CREBP to their respective binding motifs within 3 days. This supports a mechanism of oncogenesis based on persistent upregulation of D type cyclins leading to a concomitant loss of G1/S checkpoint control.

  10. Pretreatment of human epidermal keratinocytes in vitro with ethacrynic Acid reduces sulfur mustard cytotoxicity.

    PubMed

    Gross, Clark L; Nipwoda, Mary T; Nealley, Eric W; Smith, William J

    2004-01-01

    Sulfur mustard (SM) is a potent alkylating agent, profoundly cytotoxic, and a powerful vesicant. SM reacts quite extensively with glutathione (GSH) and forms GSH conjugates, which are presumably excreted through the mercapturic acid pathway in mammals. It is unknown whether any enzymes, such as the glutathione-S-transferases (GST), are involved in this detoxification of SM by the formation of conjugates. A prototypic inhibitor (ethacrynic acid, EAA) and a prototypic inducer (Oltipraz, OLT) of GSH-S-transferase, have been used as pretreatment compounds in human epidermal keratinocytes (HEK) to investigate the effect of enzyme levels on cytotoxicity following SM challenge from 50 muM to 300 muM. Pretreatment of HEK for 24 h with EAA doubled survival against 200 muM SM (36% viability in non-pretreated cells vs. 81% in EAA-pretreated cells) and quadrupled survival (17% viability in non-pretreated controls vs. 71% in EAA-pretreated cells), while OLT pretreatment had no effect on cytotoxicity at either SM dose. The role of GST in SM cytotoxicity could not be tested because of the lack of an effect on modulation of GST activities by these 2 drugs. Cellular levels of GSH were increased 250-300% over control values using EAA pretreatment, while OLT pretreatment did not lead to any increase in GSH. Pretreatment of HEK with buthionine sulfoximine (BSO), a known depleter of glutathione levels, reduced glutathione levels and increased cytotoxicity. This large increase in GSH appears to be solely responsible for the enhanced survivability of EAA-pretreated HEK.

  11. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. UVB radiation induces an increase in intracellular zinc in human epidermal keratinocytes.

    PubMed

    Stork, Christian J; Martorano, Lisa M; Li, Yang V

    2010-10-01

    Ultraviolet (UV) radiation is known to cause oxidative stress, inflammation, DNA damage and apoptotic cell death; however, many details of these malign mechanism have yet to be elucidated. In this study, the exposure of adult human epidermal keratinocytes (HEKa) with UVB (>100 mJ/cm(2)) resulted in the significant increase of intracellular zinc that was released from its storage and was detected by fluorescent zinc indicators. Toxicity testing revealed that UVB-induced zinc release in HEKa is associated with HEKa cell death. Cells that showed elevated intracellular zinc fluorescence upon UVB exposure were also stained by propidium iodide (PI), a traditional viability indicator whose fluorescent signal is as a result of its intercalating with DNA fragments and is unaffected by zinc concentration, showing significant colocalization [Pearson's correlation coefficients r=0.956 (n=6)]. The cytotoxicity of zinc was also determined by an MTT assay after applying the exogenous zinc (ZnCl2) along with its ionophore pyrithione (20 microM) into HEKa culture medium. A significant reduction in cell viability as a function of both zinc concentration and exposure time was observed. The treatments of 1, 10 and 100 microM ZnCl2 with pyrithione demonstrated 2.3, 60 and 84% cell deaths, respectively (control 0.5%) after 30 min. ZnCl2 (100 microM) was also found to induce complete HEKa death after 1 h. Thus, the present study demonstrates that UVB irradiation-induced increased zinc is detrimental to HEKa viability, and zinc may be a necessary step in UVB-induced cell death signaling pathways.

  13. Protective mechanism of morin against ultraviolet B-induced cellular senescence in human keratinocyte stem cells.

    PubMed

    Lee, Jienny; Shin, Yeun-Kyung; Song, Jae-Young; Lee, Kyung-Woo

    2014-01-01

    Ultraviolet-B (UVB) irradiation is a major inducer of DNA damage in the epidermis. Here we investigated the protective mechanism of polyphenolic phytonutrient, morin against UVB-induced DNA damage in human keratinocyte stem cells (KSC). After confirming the characteristics of the KSC, we examined the protective ability of morin against the cell damage of KSC under UVB irradiation condition. As a result, morin significantly inhibited the UVB-induced damage to KSC. These inhibitory effects by morin were also confirmed by the senescence-associated beta-galactosidase and alkaline comet assays. Next, we monitored the effects of morin on the UVB-induced production of inflammatory cytokines. Morin significantly decreased the production of tumor necrosis factor-α, interleukin-1β, and interleukin-6 in the UVB-irradiated KSC. Also, morin significantly inhibited the UVB-induced phosphorylation of ataxia telangiectasia mutated (ATM), serine threonine kinase checkpoint kinase 2, tumor suppressor protein 53 (p53), c-Jun N-terminal kinase/stress-activated protein kinase, p38/mitogen-activated protein kinase, S6 ribosomal protein, and histone 2A family member X in KSC. Furthermore, while UVB irradiation induced p53 reporter activation in KSC, morin significantly inhibited UVB-induced p53 reporter activation in KSC. In addition, mouse double minute 2 homolog (MDM2, p53 E3 ubiquitin protein ligase) inhibitor significantly increased the p53 reporter activation in the UVB-irradiated KSC, but morin decreased the MDM2 inhibitor-mediated increase in p53 reporter activation. On the contrary, ATM inhibitor did not affect the protective effect of morin in UVB irradiation-induced p53 reporter activation. Collectively, these findings suggest that morin could effectively enrich the p53 specific ligasing ability of MDM2 in UVB irradiation-induced p53 activation.

  14. Factors affecting ultraviolet-A photon emission from β-irradiated human keratinocyte cells.

    PubMed

    Le, M; Mothersill, C E; Seymour, C B; Ahmad, S B; Armstrong, A; Rainbow, A J; McNeill, F E

    2015-08-21

    The luminescence intensity of 340±5 nm photons emitted from HaCaT (human keratinocyte) cells was investigated using a single-photon-counting system during cellular exposure to (90)Y β-particles. Multiple factors were assessed to determine their influence upon the quantity and pattern of photon emission from β-irradiated cells. Exposure of 1 x 10(4) cells/5 mL to 703 μCi resulted in maximum UVA photoemission at 44.8 x 10(3)±2.5 x 10(3) counts per second (cps) from live HaCaT cells (background: 1-5 cps); a 16-fold increase above cell-free controls. Significant biophoton emission was achieved only upon stimulation and was also dependent upon presence of cells. UVA luminescence was measured for (90)Y activities 14 to 703 μCi where a positive relationship between photoemission and (90)Y activity was observed. Irradiation of live HaCaT cells plated at various densities produced a distinct pattern of emission whereby luminescence increased up to a maximum at 1 x 10(4) cells/5 mL and thereafter decreased. However, this result was not observed in the dead cell population. Both live and dead HaCaT cells were irradiated and were found to demonstrate different rates of photon emission at low β activities (⩽400 μCi). Dead cells exhibited greater photon emission rates than live cells which may be attributable to metabolic processes taking place to modulate the photoemissive effect. The results indicate that photon emission from HaCaT cells is perturbed by external stimulation, is dependent upon the activity of radiation delivered, the density of irradiated cells, and cell viability. It is postulated that biophoton emission may be modulated by a biological or metabolic process.

  15. Factors affecting ultraviolet-A photon emission from β-irradiated human keratinocyte cells

    NASA Astrophysics Data System (ADS)

    Le, M.; Mothersill, C. E.; Seymour, C. B.; Ahmad, S. B.; Armstrong, A.; Rainbow, A. J.; McNeill, F. E.

    2015-08-01

    The luminescence intensity of 340+/- 5 nm photons emitted from HaCaT (human keratinocyte) cells was investigated using a single-photon-counting system during cellular exposure to 90Y β-particles. Multiple factors were assessed to determine their influence upon the quantity and pattern of photon emission from β-irradiated cells. Exposure of 1× {{10}4} cells/5 mL to 703 μCi resulted in maximum UVA photoemission at 44.8× {{10}3}+/- 2.5× {{10}3} counts per second (cps) from live HaCaT cells (background: 1-5 cps); a 16-fold increase above cell-free controls. Significant biophoton emission was achieved only upon stimulation and was also dependent upon presence of cells. UVA luminescence was measured for 90Y activities 14 to 703 μCi where a positive relationship between photoemission and 90Y activity was observed. Irradiation of live HaCaT cells plated at various densities produced a distinct pattern of emission whereby luminescence increased up to a maximum at 1× {{10}4} cells/5 mL and thereafter decreased. However, this result was not observed in the dead cell population. Both live and dead HaCaT cells were irradiated and were found to demonstrate different rates of photon emission at low β activities (⩽400 μCi). Dead cells exhibited greater photon emission rates than live cells which may be attributable to metabolic processes taking place to modulate the photoemissive effect. The results indicate that photon emission from HaCaT cells is perturbed by external stimulation, is dependent upon the activity of radiation delivered, the density of irradiated cells, and cell viability. It is postulated that biophoton emission may be modulated by a biological or metabolic process.

  16. Human papillomavirus type 59 immortalized keratinocytes express late viral proteins and infectious virus after calcium stimulation.

    PubMed

    Lehr, Elizabeth E; Qadadri, Brahim; Brown, Calla R; Brown, Darron R

    2003-09-30

    Human papillomavirus type 59 (HPV 59) is an oncogenic type related to HPV 18. HPV 59 was recently propagated in the athymic mouse xenograft system. A continuous keratinocyte cell line infected with HPV 59 was created from a foreskin xenograft grown in an athymic mouse. Cells were cultured beyond passage 50. The cells were highly pleomorphic, containing numerous abnormally shaped nuclei and mitotic figures. HPV 59 sequences were detected in the cells by DNA in situ hybridization in a diffuse nuclear distribution. Southern blots were consistent with an episomal state of HPV 59 DNA at approximately 50 copies per cell. Analysis of the cells using a PCR/reverse blot strip assay, which amplifies a portion of the L1 open reading frame, was strongly positive. Differentiation of cells in monolayers was induced by growth in F medium containing 2 mM calcium chloride for 10 days. Cells were harvested as a single tissue-like sheet, and histologic analysis revealed a four-to-six cell-thick layer. Transcripts encoding involucrin, a cornified envelope protein, and the E1/E4 and E1/E4/L1 viral transcripts were detected after several days of growth in F medium containing 2 mM calcium chloride. The E1/E4 and L1 proteins were detected by immunohistochemical analysis, and virus particles were seen in electron micrographs in a subset of differentiated cells. An extract of differentiated cells was prepared by vigorous sonication and was used to infect foreskin fragments. These fragments were implanted into athymic mice. HPV 59 was detected in the foreskin xenografts removed 4 months later by DNA in situ hybridization and PCR/reverse blot assay. Thus, the complete viral growth cycle, including production on infectious virus, was demonstrated in the HPV 59 immortalized cells grown in a simple culture system.

  17. Substrate roughness induces the development of defective E-cadherin junctions in human gingival keratinocytes

    PubMed Central

    2017-01-01

    Purpose The entry of bacteria or harmful substances through the epithelial seal of human gingival keratinocytes (HGKs) in the junctional epithelium (JE) is blocked by specialized intercellular junctions such as E-cadherin junctions (ECJs). However, the influence of roughened substrates, which may occur due to apical migration of the JE, root planing, or peri-implantitis, on the development of the ECJs of HGKs remains largely unknown. Methods HGKs were cultured on substrates with varying levels of roughness, which were prepared by rubbing hydrophobic polystyrene dishes with silicon carbide papers. The activity of c-Jun N-terminal kinase (JNK) was inhibited with SP600125 or by transfection with JNK short hairpin RNA. The development of intercellular junctions was analyzed using scanning electron microscopy or confocal laser scanning microscopy after immunohistochemical staining of the cells for E-cadherin. The expression level of phospho-JNK was assessed by immunoblotting. Results HGKs developed tight intercellular junctions devoid of wide intercellular gaps on smooth substrates and on rough substrates with low-nanometer dimensions (average roughness [Ra]=121.3±13.4 nm), although the ECJs of HGKs on rough substrates with low-nanometer dimensions developed later than those of HGKs on smooth substrates. In contrast, HGKs developed short intercellular junctions with wide intercellular gaps on rough substrates with mid- or high-nanometer dimensions (Ra=505.3±115.3 nm, 867.0±168.6 nm). Notably, the stability of the ECJs was low on the rough substrates, as demonstrated by the rapid destruction of the cell junction following calcium depletion. Inhibition of JNK activity promoted ECJ development in HGKs. JNK was closely associated with cortical actin in the regulation of ECJs in HGKs. Conclusions These results indicate that on rough substrates with nanometer dimensions, the ECJs of HGKs develop slowly or defectively, and that this effect can be reversed by inhibiting JNK

  18. The osmolyte strategy of normal human keratinocytes in maintaining cell homeostasis.

    PubMed

    Warskulat, Ulrich; Reinen, Andrea; Grether-Beck, Susanne; Krutmann, Jean; Häussinger, Dieter

    2004-09-01

    Compatible organic osmolytes, such as betaine, myoinositol, and taurine, are involved in cell volume homeostasis as well as in cell protection, for example, against oxidative stress. This so-called osmolyte strategy requires the expression of specific osmolyte transporting systems such as the betaine/gamma-amino-n-butyric acid (GABA) transporter, the sodium-dependent myoinositol transporter and the taurine transporter (TAUT). In contrast to liver, kidney, and neural cells, nothing is known about osmolytes in the skin. Here we report that primary normal human keratinocytes (NHK) express mRNA specific for the betaine/GABA transporter, for the sodium-dependent myoinositol transporter and for the TAUT. In comparison to normoosmotic (305 mosmol per L) controls, a 3-5-fold induction of mRNA expression for the betaine/GABA-, the sodium-dependent myoinositol- and the TAUT was observed within 6-24 h after hyperosmotic exposure (405 mosmol per L). Expression of osmolyte transporters was associated with an increased uptake of radiolabeled osmolytes. Conversely, hypoosmotic (205 mosmol per L) stimulation induced significant efflux of these osmolytes. Exposure to ultraviolet B (290-315 nm) or ultraviolet A (340-400 nm) radiation, which are major sources of oxidative stress in skin, significantly stimulated osmolyte uptake. Increased osmolyte uptake was associated with upregulation of mRNA steady-state levels for osmolyte transporters in irradiated cells. These studies demonstrate that NHK possess an osmolyte strategy, which is important for their capacity to maintain cell volume homeostasis and seems to be part of their response to UV radiation.

  19. Exploration of the functional hierarchy of the basal layer of human epidermis at the single-cell level using parallel clonal microcultures of keratinocytes.

    PubMed

    Fortunel, Nicolas O; Cadio, Emmanuelle; Vaigot, Pierre; Chadli, Loubna; Moratille, Sandra; Bouet, Stéphan; Roméo, Paul-Henri; Martin, Michèle T

    2010-04-01

    The basal layer of human epidermis contains both stem cells and keratinocyte progenitors. Because of this cellular heterogeneity, the development of methods suitable for investigations at a clonal level is dramatically needed. Here, we describe a new method that allows multi-parallel clonal cultures of basal keratinocytes. Immediately after extraction from tissue samples, cells are sorted by flow cytometry based on their high integrin-alpha 6 expression and plated individually in microculture wells. This automated cell deposition process enables large-scale characterization of primary clonogenic capacities. The resulting clonal growth profile provided a precise assessment of basal keratinocyte hierarchy, as the size distribution of 14-day-old clones ranged from abortive to highly proliferative clones containing 1.7 x 10(5) keratinocytes (17.4 cell doublings). Importantly, these 14-day-old primary clones could be used to generate three-dimensional reconstructed epidermis with the progeny of a single cell. In long-term cultures, a fraction of highly proliferative clones could sustain extensive expansion of >100 population doublings over 14 weeks and exhibited long-term epidermis reconstruction potency, thus fulfilling candidate stem cell functional criteria. In summary, parallel clonal microcultures provide a relevant model for single-cell studies on interfollicular keratinocytes, which could be also used in other epithelial models, including hair follicle and cornea. The data obtained using this system support the hierarchical model of basal keratinocyte organization in human interfollicular epidermis.

  20. Nicotinamide attenuates aquaporin 3 overexpression induced by retinoic acid through inhibition of EGFR/ERK in cultured human skin keratinocytes.

    PubMed

    Song, Xiuzu; Xu, Aie; Pan, Wei; Wallin, Brittany; Kivlin, Rebecca; Lu, Shan; Cao, Cong; Bi, Zhigang; Wan, Yinsheng

    2008-08-01

    The most common adverse effects that are related to all-trans retinoic acid (atRA) treatment are irritation and dryness of the skin. atRA therapy is reported to impair barrier function as achieved by trans-epidermal water loss (TEWL). Treatment with nicotinamide prior to initiation of atRA therapy provides additional barrier protection and thus reduces susceptibility of retinoic acid. Our previous studies showed that atRA upregulates aquaporin 3 (AQP3) in cultured human skin keratinocytes and fibroblasts. Others have demonstrated that in atopic dermatitis, overexpression of AQP3 is linked to elevated TEWL and that nicotinamide treatment reduces skin TEWL. In this study, we observed that while atRA upregulates AQP3 expression in cultured human skin keratinocytes (HaCaT cells), nicotinamide attenuates the effect of atRA in a concentration-dependent manner. atRA treatment induces EGFR and ERK activation. PD153035, an EGFR inhibitor, and U0126, an ERK inhibitor, inhibit atRA-induced upregulation of AQP3. Nicotinamide also inhibits atRA-induced activation of EGFR/ERK signal transduction and decreases water permeability by downregulating AQP3 expression. Collectively, our results indicate that the effect of atRA on AQP3 expression is at least partly mediated by EGFR/ERK signaling in cultured human skin keratinocytes. Nicotinamide attenuates atRA-induced AQP3 expression through inhibition of EGFR/ERK signal transduction and eventually decreases water permeability and water loss. Our study provides insights into the molecular mechanism through which nicotinamide reverses the side effects of dryness in human skin after treatment with atRA.

  1. Roles of the PDZ-binding motif of HPV 16 E6 protein in oncogenic transformation of human cervical keratinocytes.

    PubMed

    Yoshimatsu, Yuki; Nakahara, Tomomi; Tanaka, Katsuyuki; Inagawa, Yuki; Narisawa-Saito, Mako; Yugawa, Takashi; Ohno, Shin-Ichi; Fujita, Masatoshi; Nakagama, Hitoshi; Kiyono, Tohru

    2017-07-01

    The high-risk human papillomavirus E6 proteins have been shown to interact with and lead to degradation of PDZ-domain-containing proteins through its carboxy-terminal motif. This PDZ-binding motif plays important roles in transformation of cultured cells and carcinogenesis of E6-transgenic mice. However, its biological effects on the natural host cells have not been elucidated. We have examined its roles in an in vitro carcinogenesis model for cervical cancer, in which E6 and E7 together with activated HRAS (HRAS(G)(12V) ) can induce tumorigenic transformation of normal human cervical keratinocytes. In this model, E6Δ151 mutant, which is defective in binding to PDZ domains, almost lost tumorigenic ability, whereas E6SAT mutant, which is defective in p53 degradation showed activity close to wild-type E6. Interestingly, we found decreased expression of PAR3 in E6-expressing cells independently of E6AP, which has not been previously recognized. Therefore, we knocked down several PDZ-domain containing proteins including PAR3 in human cervical keratinocytes expressing E7, HRAS(G)(12V) and E6Δ151 to examine whether depletion of these proteins can restore the tumorigenic ability. Single knockdown of SCRIB, MAGI1 or PAR3 significantly but partially restored the tumorigenic ability. The combinatorial knockdown of SCRIB and MAGI1 cooperatively restored the tumorigenic ability, and additional depletion of PAR3 further enhanced the tumorigenic ability surpassing that induced by wild-type E6. These data highlight the importance of the carboxy-terminal motif of the E6 protein and downregulation of PAR3 in tumorigenic transformation of human cervical keratinocytes. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  2. Photo-Oxidation Products of Skin Surface Squalene Mediate Metabolic and Inflammatory Responses to Solar UV in Human Keratinocytes

    PubMed Central

    Kostyuk, Vladimir; Potapovich, Alla; Stancato, Andrea; De Luca, Chiara; Lulli, Daniela; Pastore, Saveria; Korkina, Liudmila

    2012-01-01

    The study aimed to identify endogenous lipid mediators of metabolic and inflammatory responses of human keratinocytes to solar UV irradiation. Physiologically relevant doses of solar simulated UVA+UVB were applied to human skin surface lipids (SSL) or to primary cultures of normal human epidermal keratinocytes (NHEK). The decay of photo-sensitive lipid-soluble components, alpha-tocopherol, squalene (Sq), and cholesterol in SSL was analysed and products of squalene photo-oxidation (SqPx) were quantitatively isolated from irradiated SSL. When administered directly to NHEK, low-dose solar UVA+UVB induced time-dependent inflammatory and metabolic responses. To mimic UVA+UVB action, NHEK were exposed to intact or photo-oxidised SSL, Sq or SqPx, 4-hydroxy-2-nonenal (4-HNE), and the product of tryptophan photo-oxidation 6-formylindolo[3,2-b]carbazole (FICZ). FICZ activated exclusively metabolic responses characteristic for UV, i.e. the aryl hydrocarbon receptor (AhR) machinery and downstream CYP1A1/CYP1B1 gene expression, while 4-HNE slightly stimulated inflammatory UV markers IL-6, COX-2, and iNOS genes. On contrast, SqPx induced the majority of metabolic and inflammatory responses characteristic for UVA+UVB, acting via AhR, EGFR, and G-protein-coupled arachidonic acid receptor (G2A). Conclusions/Significance Our findings indicate that Sq could be a primary sensor of solar UV irradiation in human SSL, and products of its photo-oxidation mediate/induce metabolic and inflammatory responses of keratinocytes to UVA+UVB, which could be relevant for skin inflammation in the sun-exposed oily skin. PMID:22952984

  3. Effects of UVB radiation on 4-hydroxy-2-trans-nonenal metabolism and toxicity in human keratinocytes.

    PubMed

    Aldini, Giancarlo; Granata, Paola; Marinello, Cristina; Beretta, Giangiacomo; Carini, Marina; Facino, Roberto Maffei

    2007-03-01

    Endogenous lipid peroxidation (LPO)-derived aldehydes accumulate in human skin after photoexposure and contribute to the development of skin cytotoxicity and cancer. This study employed LC-ESI-MS and HPLC-UV-DAD techniques to investigate the effect of UVB radiation on the biotransformation and detoxification of the prototype aldehyde 4-hydroxy-2-trans-nonenal (HNE) using the human keratinocyte cell line (NCTC 2544). In parallel we followed the keratinocytes' cytotoxic response to HNE through morphological analysis and cell viability assay. In UVB-unstressed keratinocytes, even a supraphysiological dose of the aldehyde (200 microM) was rapidly and completely cleared in metabolized form (free and GSH-conjugated metabolites) from the cell, with no signs of cytotoxicity. By contrast, UVB preexposure already at 1 MED (50 mJ/cm2, the minimal erythemal dose in humans) markedly impaired HNE metabolism. After 2 h of incubation, the relative amount of GSH-conjugated adducts dose-dependently dropped from 44% (unirradiated cells) to 22% at 3 MED as a consequence of UVB-induced GSH depletion (no impairment of GST A4.4 nor of G6PD activities was observed). The levels of free metabolites, 1,4-dihydroxy-trans-nonene (DHN) and 4-hydroxy-trans-2-nonenoic acid (HNA), were modified (+30% DHN, -22% HNA) only at 3 MED, in parallel to the AR and ALDH enzyme activity modulation. In addition, a dose-dependent increase of unmodified HNE was found in the extracellular medium, paralleled by a significant fraction of the HNE-incubated dose not recovered at the intra- or extracellular level. The impairment of HNE metabolism paralleled a dramatic cytotoxic response. These results provide a reasonable explanation for the massive accumulation of carbonyl toxins in human skin in vivo after photoexposure and shed light on the detrimental effects of UVB radiation in the presence of unmetabolized LPO metabolites.

  4. Epidemiology of oral human papillomavirus infection

    PubMed Central

    Chung, Christine H.; Bagheri, Ashley; D'Souza, Gypsyamber

    2013-01-01

    Human papillomavirus (HPV) infection is known to cause a subset of oropharyngeal cancers. Data regarding oral HPV infection is limited but emerging. HPV infection of the genital tract has been more thoroughly researched and helps inform our understanding of oral HPV infection. In this article we review current data on HPV prevalence, natural history, mode of acquisition, and risk factors for oral HPV infection. PMID:24080455

  5. Oral Administration of p-Hydroxycinnamic Acid Attenuates Atopic Dermatitis by Downregulating Th1 and Th2 Cytokine Production and Keratinocyte Activation

    PubMed Central

    Lee, Hyun-Su; Choi, Eun-Ju; Lee, Kyung-Sik; Kim, Hye-Ran; Na, Bo-Ra; Kwon, Min-Sung; Jeong, Gil-Saeng; Choi, Hyun Gyu; Choi, Eun Young; Jun, Chang-Duk

    2016-01-01

    Atopic dermatitis (AD) is a complex disease that is caused by various factors, including environmental change, genetic defects, and immune imbalance. We previously showed that p-hydroxycinnamic acid (HCA) isolated from the roots of Curcuma longa inhibits T-cell activation without inducing cell death. Here, we demonstrated that oral administration of HCA in a mouse model of ear AD attenuates the following local and systemic AD manifestations: ear thickening, immune-cell infiltration, production of AD-promoting immunoregulatory cytokines in ear tissues, increased spleen and draining lymph node size and weight, increased pro-inflammatory cytokine production by draining lymph nodes, and elevated serum immunoglobulin production. HCA treatment of CD4+ T cells in vitro suppressed their proliferation and differentiation into Th1 or Th2 and their Th1 and Th2 cytokine production. HCA treatment of keratinocytes lowered their production of the pro-inflammatory cytokines that drive either Th1 or Th2 responses in AD. Thus, HCA may be of therapeutic potential for AD as it acts by suppressing keratinocyte activation and downregulating T-cell differentiation and cytokine production. PMID:26959360

  6. A hyperosmotic stimulus elevates intracellular calcium and inhibits proliferation of a human keratinocyte cell line.

    PubMed

    Dascalu, A; Matithyou, A; Oron, Y; Korenstein, R

    2000-10-01

    Occlusion has previously been used to treat psoriatic plaques and was shown to improve the condition. We investigated the consequences of applying a mechanical stress, in vitro, on the HaCaT keratinocyte cell line. A mechanical load applied to cells can be mimicked by a hyperosmotic stimulus. Exposure of HaCaT keratinocytes to different hyperosmotic solutions (final osmolarity in the range 350-600 mOsm, produced by sucrose addition) resulted in an inhibition of cell proliferation after 96 h of treatment. As keratinocyte maturation is regulated by calcium levels, we measured hyperosmotic-stimulus-induced changes of intracellular calcium ([Ca2+]i) by single-cell image analysis employing FURA-2/AM. The hyperosmotic stimulus produced a rapid transient 2.6-fold elevation of [Ca2+]i followed by a gradual decay to the basal level. The transients originated from extracellular as well as from intracellular calcium pools and did not respond to voltage-sensitive calcium channel blockers. The hyperosmotic stimulus was shown to increase the cellular expression of involucrin, a differentiation marker, following 72 h of incubation, as measured by flow cytometry. Treatment of cells with the [Ca2+]i chelator BAPTA/AM almost completely blocked the [Ca2+]i elevation, but did not alter cellular growth or the induction of differentiation observed after hyperosmotic stimulus. It is suggested that treatment of keratinocytes with hyperosmotic stimulus can induce short-time effects (calcium transients) as well as long-term cellular maturation.

  7. 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