Cell culture. The human keratinocyte cell lines HaCaT and HaCaT-RG were generously provided by P. Boukamp (German Cancer Research Center, Heidelberg, Germany); the squamous carcinoma cell lines SCC-1, SCC-6, SCC-17B, and SCC-74B were gifts from T. Carey (University of Michigan, Ann Arbor, MI); the squamous carcinoma cell lines SCC-012 and SCC-028 were kindly provided by D. Sidransky (Johns Hopkins University, Baltimore, MD). Human epidermal keratinocytes (HEK) and normal human dermal fibroblasts (NHDF) were obtained from the Vanderbilt Skin Disease Research core. HaCaT, HaCaT-RG, SCC-1, SCC-6, SCC-17B, SCC-74B, HeLa [American Type Culture Collection (ATCC), Manassas, VA], A-431 (ATCC), and NHDF were cultured in DMEM supplemented with 10% FCS and 1% penicillin-streptomycin. SCC-012 and SCC-028 cells were cultured in RPMI 1640 supplemented with 10% FCS and 1% penicillin-streptomycin. The A-549 human lung adenocarcinoma cell line (ATCC) was cultured in DMEM supplemented with 10% FCS, 10 μg/mL ...
Breast Tumor Kinase (Brk/PTK6) has a relatively limited expression profile in normal tissue. Its expression is restricted to epithelial cells that are differentiating such as those in the epidermis, and Brk expression appears to be absent from proliferating cells in normal tissue. Also, there is now some evidence to suggest that Brk plays a functional role in the differentiation of the keratinocytes in the epidermis. We have, therefore, investigated the role that Brk/PTK6 plays in normal human primary keratinocytes by suppressing protein levels using RNA interference. We show that as primary human keratinocytes are induced to differentiate in vitro, Brk levels decrease. Decreasing Brk protein levels lead to an increase in the number of cells with a permeable plasma membrane, a decrease in epidermal growth factor receptor (EGFR) and a parallel increase in keratin 10 levels, but classical markers of apoptosis or terminal differentiation are not affected. We propose Brk, Keratin 10 and EGFR are ...
Univ of Wisconsin School of Medicine & Public Health Introduction: Wound healing affects millions of people and the impact on the US economy is over $25B annually. We are interested in discovering novel strategies to enhance keratinocyte migration, proliferation, and differentiation in order to improve wound healing. Chrysin (5,7-dihydroxyflavone), a natural flavonoid found in the blue passion flower, has recently been shown to protect keratinocytes from UV-induced damage. We investigated the effect of chrysin on cell differentiation in primary human keratinocytes.. Methods: Primary human keratinocytes were isolated from neonatal foreskin. The basal (60 M calcium) EpiLife medium (Invitrogen, Carlsbad, CA) supplemented with human keratinocyte growth supplements (Invitrogen) was used for experiments. Chrysin was purchased from MP Biomedicals (Santa Ana, CA). Effetcs of chrysin on keratinocyte differentiation was investigated the presence of low (60 M) and high calcium (1 mM). Light microscopy was ...
Keratinocyte expression of A20/TNFAIP3 controls skin inflammation associated with atopic dermatitis and psoriasis: Keratinocytes are key players in chronic infl
During gestation the epidermis develops from a single layer of ectoderm into a layer of keratinocytes overlaid by a layer of periderm; this is followed by a progressive increase in the number of layers of keratinocytes, until finally the distinct granular and cornified layers characteristic of mature epidermis are formed. As part of our investigation into the function of the peanut lectin-binding glycoproteins of cultured human keratinocytes, we have examined their expression at different stages of human epidermal development. We found that the onset of expression of the glycoproteins coincided with the transition from a two- to a three-layered epidermis, both in vivo and in organ culture. In adult epidermis, the patterns of binding of peanut lectin and Limax flavus lectin are complementary, with peanut binding more strongly to suprabasal keratinocytes and Limax flavus lectin binding more strongly to cells in the basal layer. We found that the complementary pattern of binding of the two lectins ...
Keratinocytes are the primary constituents of human skin, the functional barrier between our bodies and the external environment. The balance between keratinocyte differentiation and self-renewal is crucial to skin homeostasis. Primary keratinocyte culture serves as a tractable model for understanding human epithelial cell differentiation as well as self-renewal.
Skin exposure to ultraviolet B (UVB) irradiation leads to the generation of reactive oxygen species (ROS). Excessive ROS cause aging of the skin via basement membrane/extracellular matrix degradation by matrix metalloproteinases (MMPs). We recently demonstrated that 3-bromo-4,5-dihydroxybenzaldehyde (BDB), a natural compound of red algae, had a photo-protective effect against UVB-induced oxidative stress in human keratinocytes. The present study focused on the effect of BDB on UVB-irradiated photo-aging in HaCaT keratinocytes and the underlying mechanism. BDB significantly impeded MMP-1 activation and expression, and abrogated the activation of mitogen-activated protein kinases and intracellular Ca2+ level in UVB-irradiated HaCaT cells. Moreover, BDB decreased the expression levels of c-Fos and phospho-c-Jun and the binding of activator protein-1 to the MMP-1 promoter induced by UVB irradiation. These results offer evidence that BDB is potentially useful for the prevention of UVB-irradiated skin damage.
One challenge of systems biology is the integration of new data into the preexisting, and then re-interpretation of the integrated data. Here we use readily available metaanalysis computational methods to integrate new data on the transcriptomic effects of EGF in primary human epidermal keratinocytes with preexisting transcriptomics data in keratinocytes and in EGF-treated non-epidermal cell types. We find that EGF promotes keratinocyte proliferation, attachment and motility and, surprisingly, induces DUSPs that attenuate the EGF signal. Our metaanalysis identified overlapping effects of EGF with those of IL-1 and IFNγ, activators of keratinocyte in inflammation and wound healing. We also identified the genes and pathways suppressed by EGF but induced by agents promoting epidermal differentiation. Metaanalysis comparison with the EGF effects in other cell types identified extensive similarities between responses in keratinocytes and in other epithelial cell types, but specific differences with the EGF
Interleukin (IL)-8 is a pro-inflammatory cytokine that has a direct effect on immune cells, including polymorphonuclear cells. Keratinocytes are a rich source of IL-8. However, there is little knowledge on the role of IL-8 in clinical wound healing and the direct biological effect of IL-8 on keratinocytes. In this study, the effect of recombinant human IL-8 (rhIL-8) on migration and adhesion was tested using HaCaT keratinocytes as a cell model. The cell functions were evaluated using impedance cell sensing. The expression of IL-8 receptor (IL-8R) transcripts in human skin and wounds (acute and chronic) was assessed using real-time transcript analysis. rhIL-8 significantly increased the migration of keratinocytes (3.5 +/- 0.3 for cells treated with IL-8 vs. 2.7 +/- 0.6 for controls; p=0.029). It is interesting to note that treatment of keratinocytes with IL-8 resulted in a marked shift in the responsive frequencies. IL-8 only resulted in a marginal increase in cell adhesion, which was ...
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Rat Keratinocyte Stem Cells Frozen Vial. This Product is also available as Plated Cells. T25 Plated Cells: $550.00. T75 Plated Cells: $750.00. T150 Plated Cells: $2500.00. T225 Plated Cells: $5500.00. This product also requires Celprogens Rat Keratinocyte Stem Cell Culture Extra-cellular Matrix. Cat# E55008-09 and Complete Media with Serum Cat# M55008-09S, and for serum free conditions Cat# M55008-09 is required provided the cells are weaned off the serum. as indicated in their specified protocol provided with purchase of these cells.. Source : Rat Skin. Positive Markers: Keratinocyte function (CD44), and profileration index (Ki67), keratin, CD71, CD34, keratinocyte derived chemokine (KC).. 120 Population Doublings.. Cells are only guaranteed with purchase of Celprogen Media, Extra Cellular Matrix, Trypsin EDTA, 1X PBS, and freezing media for the appropriate cell culture, for 30 days from the date of shipment.. ...
Rat Keratinocyte Stem Cells Frozen Vial. This Product is also available as Plated Cells. T25 Plated Cells: $550.00. T75 Plated Cells: $750.00. T150 Plated Cells: $2500.00. T225 Plated Cells: $5500.00. This product also requires Celprogens Rat Keratinocyte Stem Cell Culture Extra-cellular Matrix. Cat# E55008-09 and Complete Media with Serum Cat# M55008-09S, and for serum free conditions Cat# M55008-09 is required provided the cells are weaned off the serum. as indicated in their specified protocol provided with purchase of these cells.. Source : Rat Skin. Positive Markers: Keratinocyte function (CD44), and profileration index (Ki67), keratin, CD71, CD34, keratinocyte derived chemokine (KC).. 120 Population Doublings.. Cells are only guaranteed with purchase of Celprogen Media, Extra Cellular Matrix, Trypsin EDTA, 1X PBS, and freezing media for the appropriate cell culture, for 30 days from the date of shipment.. ...
Atopic eczema and psoriasis are common skin diseases. While it is well established that the pathogenesis of these diseases varies, both are characterized by impairment in epidermal barrier function and abnormal IL-17 expression in the skin and peripheral blood. Recent findings indicated that filaggrin is essential during barrier formation and its insufficiency underlies the pathogenesis of atopic eczema. Filaggrin downregulation has also been reported in psoriasis. It is clear that Th1/Th2 bias influences expression of the protein, but an analysis of the effects of interleukin-17 (IL-17) on the expression of the protein and profilaggrin-processing enzymes has not yet been reported. In addition, the effect of the cytokine on components of functional epidermal barrier, tight junctions and adhesion/desmosomal proteins, has not been elucidated. Keratinocytes were exposed to interleukin-17A, and microarray analysis was performed. Filaggrin protein level was assessed by western blot. We have observed a
TY - JOUR. T1 - Ca2+ waves in keratinocytes are transmitted to sensory neurons. T2 - The involvement of extracellular ATP and P2Y2 receptor activation. AU - Koizumi, Schuichi. AU - Fujishita, Kayoko. AU - Inoue, Kaori. AU - Shigemoto-Mogami, Yukari. AU - Tsuda, Makoto. AU - Inoue, Kazuhide. PY - 2004/6/1. Y1 - 2004/6/1. N2 - ATP acts as an intercellular messenger in a variety of cells. In the present study, we have characterized the propagation of Ca2+ waves mediated by extracellular ATP in cultured NHEKs (normal human epidermal keratinocytes) that were co-cultured with mouse DRG (dorsal root ganglion) neurons. Pharmacological characterization showed that NHEKs express functional metabotropic P2Y2 receptors. When a cell was gently stimulated with a glass pipette, an increase in [Ca2+]i (intracellular Ca2+ concentration) was observed, followed by the induction of propagating Ca2+ waves in neighbouring cells in an extracellular ATP-dependent manner. Using an ATP-imaging technique, the release and ...
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 ...
Efficient wound repair is essential for the maintenance of the integrity of the skin. The repair process is controlled by a variety of growth factors and cytokines, and their abnormal expression or activity can cause healing disorders. Here, we show that wound repair is severely delayed in mice lacking fibroblast growth factor receptors (FGFR) 1 and 2 in keratinocytes. As the underlying mechanism, we identified impaired wound contraction and a delay in re-epithelialization that resulted from impaired keratinocyte migration at the wound edge. Scratch wounding and transwell assays demonstrated that FGFR1/2-deficient keratinocytes had a reduced migration velocity and impaired directional persistence owing to inefficient formation and turnover of focal adhesions. Underlying this defect, we identified a significant reduction in the expression of major focal adhesion components in the absence of FGFR signaling, resulting in a general migratory deficiency. These results identify FGFs as key regulators ...
TY - JOUR. T1 - Inhibition of mTORC2 enhances UVB-induced apoptosis in keratinocytes through a mechanism dependent on the FOXO3a transcriptional target NOXA but independent of TRAIL. AU - Feehan, Robert P.. AU - Nelson, Amanda M.. AU - Shantz, Lisa M.. PY - 2018/12. Y1 - 2018/12. N2 - The primary cause of non-melanoma skin cancer (NMSC) is ultraviolet B (UVB) radiation. We have shown previously that mTORC2 inhibition sensitizes keratinocytes to UVB-induced apoptosis mediated by the transcription factor FOXO3a. FOXO3a is a key regulator of apoptosis and a tumor suppressor in several cancer types. Activation of FOXO3a promotes apoptosis through the coordinated expression of a variety of target genes, including TRAIL and NOXA. We hypothesized that in the setting of mTORC2 inhibition, the UVB-induced expression of these factors would lead to apoptosis in a FOXO3a-dependent manner. Using spontaneously immortalized human keratinocytes (HaCaT cells), we observed that both TRAIL and NOXA expression ...
Cindy: You might look at the work of and/or contact Drs. Kim Creek and Lucia Pirisi-Creek at the University of South Carolina School of Medicine. When last I interacted with them 2 years ago, their labs were doing primary keratinocyte cultures from human newborn foreskins on a weekly basis. courtland.yockey at mindspring.com ======== In article ,387D1B12.895B0221 at biomail.ucsd.edu,, cgb at biomail.ucsd.edu (Cindy Gustafson-Brown) wrote: I am interested in generating primary keratinocyte cell cultures and would appreciate any advice, references, protocols, and sources of reagents. Thanks, Cindy Gustafson-Brown ----------------------------------------------------------- Cindy Gustafson-Brown lab (858) 822-0568 or 0593 UCSD Biology fax (858) 534-5831 9500 Gilman Dr La Jolla, CA 92093-0366 ...
Productive infections by human papillomaviruses (HPVs) occur only in differentiated keratinocytes in squamous epithelia in which the HPV E7 protein reactivates the host DNA replication machinery to support viral DNA replication. In a fraction of the differentiated keratinocytes, E7 also posttranscriptionally induces p21cip1, which is distributed in a mutually exclusive manner with unscheduled cellular DNA synthesis. In this study, double immunofluorescence labeling unexpectedly revealed that E7 caused a concordant accumulation of both cyclin E and p21cip1 to high levels in patient papillomas and in organotypic cultures of primary human keratinocytes. The induction of cyclin E is mutually exclusive with unscheduled cellular DNA synthesis or abundant viral DNA. These novel virus-host interactions in differentiated keratinocytes are in contrast to previous observations made in submerged proliferating cultures, in which HPV E7 induces cyclin E and overcomes p21cip1 inhibition of S-phase entry. We ...
The human stem cell factor (SCF) is a crucial growth factor for mast cells in the dermis and for the melanocytes in the basal layers of the epidermis. SCF is produced, among others, by keratinocytes. This study examines the possible regulation of the expression of SCF from keratinocytes by all-trans retinoic acid (RA) and dexamethasone in vitro by the keratinocyte cell line HaCaT. The HaCaT-cells were incubated for 24 hours or 11 days, respectively, with one of the above mentioned substances (10 to the power of -5 M to 10 to the power of -9 M). The analysis of the number of HaCaT-cells, of the total SCF protein, its splice variants (mSCF, sSCF), the receptors of RA (RAR-alpha, -beta, -gamma), and of the dexamethasone (GR-alpha, -beta) was done by ELISA and RT-PCR. The following results were found: RA induces an increase of SCF, dexamethasone at a short incubation period a considerable increase of SCF, and at long-term incubation a strong decrease. The RA-receptors RA-alpha und -gamma expression ...
Hypoxia Regulates mTORC1-Mediated Keratinocyte Motility and Migration via the AMPK Pathway. . Biblioteca virtual para leer y descargar libros, documentos, trabajos y tesis universitarias en PDF. Material universiario, documentación y tareas realizadas por universitarios en nuestra biblioteca. Para descargar gratis y para leer online.
TGF-β isoforms are important signalling molecules in wound repair in the skin. Transforming growth factor β3 (TGF-β3) has been implicated in scarless healing. In both animal and human models the application of exogenous TGF-β3 causes a reduction in the inflammatory response and improves the architecture of the neodermis. Research into the influence of TGF-β on scarring has tended to focus on fibroblasts. However, keratinocytes play a major role in scarring both indirectly, as a result of their influence over the behaviour of fibroblasts and also by directly influencing wound contraction. Thus, experiments were carried out to investigate the influence of TGF-β3 on the behaviours of a keratinocyte cell line (HaCaT). Incubation with TGF-β3 increased cell spreading and appeared to reduce cell-surface contacts indicated by both SPR imaging and a detachment assay. TGF-β3 also caused a decreased cell alignment response to microcontact printed protein patterns, in part due to the deposition of ...
The productive program of the human papillomaviruses takes place in terminally differentiating squamous epithelia. In this chapter, we provide the protocols for robust production of HPV-18 in organotypic cultures of early passages of primary human keratinocytes. A critical step is the generation of genomic HPV plasmids in vivo by using Cre-loxP-mediated excisional recombination from a vector plasmid. We discuss the rationale for this approach. This system produces high yields of infectious virus and facilitates genetic analyses of HPV protein functions and their regulation in the context of recapitulated host tissue environment.. ...
TY - JOUR. T1 - Overexpression of bcl-2 protein inhibits terminal differentiation of oral keratinocytes in vitro. AU - Harada, Hidemitsu. AU - Mitsuyasu, Takeshi. AU - Seta, Yuji. AU - Maruoka, Yuka. AU - Toyoshima, Kuniaki. AU - Yasumoto, Shigeru. PY - 1998/1. Y1 - 1998/1. N2 - The bcl-2 proto-oncogene is a known inhibitor of apoptosis; in normal human stratified squamous epithelium, its expression is restricted to the basal cell layer. To investigate the functional role of bcl-2 protein in the process of differentiation of oral keratinocytes, bcl-2 expression vector was transfected into SCC-25 cells, which normally undergo squamous cell differentiation in vitro while expressing specific differentiation markers, e.g., keratin 10/11 and involucrin. In bcl-2 transfected SCC-25 cells, the expression of these differentiation markers was markedly suppressed. The bcl-2 proto-oncogene may play a critical role in opposing the commitment to terminal differentiation and apoptosis of oral ...
Molecular Cancer 2015, 14:1 doi:10.1186/1476-4598-14-1 Published: 5 January 2015 Article source: http://healthmedicinet.com/i/another-drug-is-approved-to-help-the-obese/ Melanoma cells influence the differentiation pattern of human epidermal keratinocytes
Chemically induced mouse skin carcinogenesis represents the most extensively utilized animal model to unravel the multistage nature of tumour development and to design novel therapeutic concepts of human epithelial neoplasia. We combined this tumour model with comprehensive gene expression analysis and could identify a large set of novel tumour-associated genes that have not been associated with epithelial skin cancer development yet. Expression data of selected genes were confirmed by semiquantitative and quantitative RT-PCR as well as in situ hybridization and immunofluorescence analysis on mouse tumour sections. Enhanced expression of genes identified in our screen was also demonstrated in mouse keratinocyte cell lines that form tumours in vivo. Self-organizing map clustering was performed to identify different kinetics of gene expression and coregulation during skin cancer progression. Detailed analysis of differential expressed genes according to their functional annotation confirmed the
FBS is chelex-treated in batch with Chelex-100 resin (BioRad, cat # 1422832) to remove free Ca++. Use 100 g chelex resin per 500 ml FBS. Swell 100 g chelex resin in 400-500 ml distilled water, then titrate to pH 7.4 with HCl while stirring (pH will take a while to stabilize during titration). Filter through Whatman #1 paper. Scrape resin slurry into 500 ml FBS and stir at room temp for 3 hr or at 4°C overnight. Filter the chelated FBS through Whatman #1 paper and discard the resin slurry. Filter the chelated FBS through a 0.2μm bottle filter to sterilize it. Aliquot sterile, chelated FBS at 20 ml/tube and store at 20°C. Add 20 ml chelated FBS per 500 ml bottle of EMEM (final concn = 4%). ...
UV radiation-mediated photodamage to the skin has been implicated in premature aging and photoaging-related skin cancer and melanoma. Little is known about the cellular events that underlie premature senescence, or how to impede these events. In the present study we demonstrate that PPARδ (peroxisome-proliferator-activated receptor δ) regulates UVB-induced premature senescence of normal keratinocytes. Activation of PPARδ by GW501516, a specific ligand of PPARδ, significantly attenuated UVB-mediated generation of ROS (reactive oxygen species) and suppressed senescence of human keratinocytes. Ligand-activated PPARδ up-regulated the expression of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and suppressed the PI3K (phosphatidylinositol 3-kinase)/Akt pathway. Concomitantly, translocation of Rac1 to the plasma membrane, which leads to the activation of NADPH oxidases and generation of ROS, was significantly attenuated. siRNA (small interfering RNA)-mediated knockdown of PTEN ...
Abstract: Infection by human papillomavirus (HPV) alters the microenvironment of keratinocytes as a mechanism to evade the immune system. A-to-I editing by ADAR1 has been reported to regulate innate immunity in response to viral infections. Here, we evaluated the role of ADAR1 in HPV infection in vitro and in vivo. Innate immune activation was characterized in human keratinocyte cell lines constitutively infected or not with HPV. ADAR1 knockdown induced an innate immune response through enhanced expression of RIG-I-like receptors (RLR) signaling cascade, over-production of type-I IFNs and pro-inflammatory cytokines. ADAR1 knockdown enhanced expression of HPV proteins, a process dependent on innate immune function as no A-to-I editing could be identified in HPV transcripts. A genetic association study was performed in a cohort of HPV/HIV infected individuals followed for a median of 6 years (range 0.1-24). We identified the low frequency haplotype AACCAT significantly associated with recurrent ...
Previous studies in our laboratory demonstrated that 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) treatment induced apoptosis and mitochondrial translocation of the tumor suppressor p53 in a mouse skin carcinogenesis model, suggesting that oncogenic versus cell death signaling involve a common mediator. Mutational activation of oncogenic Ras is an early event and has been demonstrated to play a critical role in skin carcinogenesis. A malignant skin keratinocyte cell line (308), which carries a H-ras mutation at codon 61, showed elevated p53 levels, increased caspase 3 activity and enhanced apoptosis after TPA treatment. In contrast, the non-malignant counterpart (C50) showed undetectable levels of p53 and less apoptosis than 308 cells similarly treated. Inhibition of NADPH-oxidase (NOX) by diphenyleneiodonium suppressed p53 activation and apoptosis in 308 cells, linking Ras mutation to NOX-induced p53 activation, which was further supported by the finding that ...
Although the expression of VR1 receptors has been well established in sensory neurons (Szallasi, 1995; Mezey et al., 2000), the existence of VR1 in keratinocytes is controversial. Previous studies indicated that VR1 expression in skin was localized on the terminals of afferent neurons at the dermal/epidermal junction (Guo et al., 1999). Several lines of evidence indicate other cell types, including keratinocytes, express a functional VR1. First, VR1 has recently been identified in cardiomyocytes (Dvorakova and Kummer, 2001), bronchial epithelial cells (Veronesi et al., 1999b), and urinary bladder epithelial cells (Birder et al., 2001) independent of sensory neurons, thus establishing that non-neuronal cells can express VR1. Keratinocytes express receptors that were previously thought to be confined to neuronal cells, including nicotinic (Grando et al., 1995), muscarinic (Ndoye et al., 1998), and μ-opiate (Bigliardi-Qi et al., 1999), although the functional role for many of these receptors has ...
ATCC ® Normal Adult Human Primary Epidermal Keratinocytes, when grown in Dermal Cell Basal Media supplemented with Keratinocyte Growth Kit components, provide an ideal cell system to propagate keratinocytes in serum-free (not animal free) conditions. The cells are cryopreserved in the first passage to ensure the highest viability and plating efficiency. ATCC ® Primary Cell Solutions™ cells, media, supplements and reagents are quality tested together to guarantee optimum performance and reliability.
The transcription factor p63 plays a pivotal role in keratinocyte proliferation and differentiation in the epidermis. However, how p63 regulates epidermal genes during differentiation is not yet clear. Using epigenome profiling of differentiating human primary epidermal keratinocytes, we characterized a catalog of dynamically regulated genes and p63-bound regulatory elements that are relevant for epithelial development and related diseases. p63-bound regulatory elements occur as single or clustered enhancers, and remarkably, only a subset is active as defined by the co-presence of the active enhancer mark histone modification H3K27ac in epidermal keratinocytes. We show that the dynamics of gene expression correlates with the activity of p63-bound enhancers rather than with p63 binding itself. The activity of p63-bound enhancers is likely determined by other transcription factors that cooperate with p63. Our data show that inactive p63-bound enhancers in epidermal keratinocytes may be active ...
Cell biology is part of the R&D activity in dermocosmetology. At HCS Pharma, we are developing new democosmetology assays on human primary keratinocytes using our automation plateform and high content analysis suite.. Assessment of compounds activity/safety can be performed on several parameters (wound healing, hydration, inflammation, toxicity, genotoxicity). Further parameters could be analyzed on demand using probes and immunocytochemistry.. Thought the use of high content analysis technologies, we can provide a wide range of results from phenotypes quantification cell by cell to live cells imaging movies.. View and download on Slideshare (low quality) : http://fr.slideshare.net/hcspharma/in-vitro-dermocosmetology-high-content-analysis-approach-using-human-primary-keratinocytes. Ask for high quality link by putting your email below ...
Integrin-linked kinase (ILK) is a ubiquitous scaffold protein that mediates cellular responses to integrin stimulation by extracellular matrix proteins. Mice with inactivation of the Ilk gene in squamous epithelia display defects in skin regeneration after injury, failure to thrive, and perinatal death. ILK-deficient epidermis exhibits reduced adhesion to the basement membrane and impaired hair follicle morphogenesis. In culture, ILK-deficient keratinocytes fail to attach and spread efficiently, and demonstrate decreased survival. We now show that ILK-deficient keratinocytes exhibit lower proliferative capacity and increased apoptosis in the absence or presence of growth factors. This reduced viability appears to be independent of the AKT pathway, as ILK-deficient cells exhibit normal levels of active, phosphorylated AKT. They do, however, display higher levels of cleaved Caspase-3 and PARP, both associated with caspase-dependent programmed cell death. We have also observed an increase in γH2A.X, a
Chronic wounds are a substantial problem in todays health care and place significant strains on the patient. Successful modelling of the wound healing process is pivotal for the advancement of wound treatment research. Wound healing is a dynamic and multifactorial process involving all constituents of the skin. The progression from haemostasis and inflammation to proliferation of epidermal keratinocytes and dermal fibroblasts, and final scar maturation can be halted and result in a chronic wound that fails to re-epithelialise. The wound healing process constitutes an example of dynamic reciprocity in tissue where cellular changes take place on cues from the extracellular matrix and vice versa when tissue homeostasis is disturbed. The extracellular matrix provides a structural context for the resident cells and the epidermal keratinocytes, and a functioning interplay between the two tissue compartments is crucial for successful wound healing to take place. Work included in this thesis has ...
BALB/MK keratinocyte cell line cultured in a defined medium. b Overexpressed. c FRSK cell line. d PAM212 cell line. c HPK1A cell line.. duced. Approximately 106-107 cells are injected in 0.1 ml PBS into the interscapular region of the host.19 Most malignant keratinocytes can grow subcutaneously, whereas papilloma or benign tumor cells do not form tumors94 (Table III).. 91 D. A. Greenhalgh and S. H. Yuspa, Mol Carcinog. 1, 134 (1988).. 92 C. M. Kim, J. Vogel, G. Jay, and J. S. Rhim, Oncogene 7, 1525 (1992).. 93 E. Finzi, A. Kilkenny, J. E. Strickland, M. Balaschak, T. Bringman, R. Derynck, S. Aaron-son, and S. H. Yuspa, Mot. Carcinog. 1, 7 (1988).. 94 J. E. Strickland, D. A. Greenhalgh, A. Koceva-Chyla, H. Hennings, C. Restrepo, M. Balaschak, and S. H. Yuspa, Cancer Res. 48, 165 (1988).. Was this article helpful?. ...
Bioalternatives present his test NHEK-0009 : NHEK (Normal Human Epidermal Keratinocytes) : Elafin release|beta-defensin 2 release|S100A7 release
HSV-1 infected keratinocytes release IL-1αPrimary human keratinocytes were treated with medium only, 25 μg ml-1 poly(I:C), or HSV-1 as indicated. After one ho
The most prevalent cultivation method for maintenance of mouse and human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells is co-cultivation on primary mouse embryonic fibroblast (mEF) feeder cell layers. Feeder cell layers of NIH3T3 fibroblasts or stromal derived OP9 cells are also frequently used in co-culture systems: NIH3T3 fibroblasts support primary keratinocyte cultures, OP9 cells are used for hematopoietic induction of ES cells. - USA
Glucocorticoids (GCs) have a long history of use as therapeutic agents for numerous skin diseases. Surprisingly, their specific molecular effects are largely unknown. To characterize GC action in epidermis, we compared the transcriptional profiles of primary human keratinocytes untreated and treated with dexamethasone (DEX) for 1, 4, 24, 48, and 72 h using large scale microarray analyses. The majority of genes were found to be regulated only after 24 h and remained regulated throughout treatment. In addition to regulation of the expected pro-inflammatory genes, we found that GCs regulate cell fate, tissue remodeling, cell motility, differentiation, and metabolism. GCs suppress the expression of essentially all IFNgamma-regulated genes, including IFNgamma receptor and STAT-1, an effect that was previously unknown. GCs also block STAT-1 activation and nuclear translocation. Unexpectedly, GCs induce the expression of anti-apoptotic genes and repress pro-apoptotic ones, preventing UV-induced ...
Ultraviolet (UV) B is the most prominent physical carcinogen in the environment leading to the development of various skin cancers. We have previously demonstrated that the human ortholog of the Arabidopsis thaliana constitutive photomorphogenesis 1 (COP1) protein, huCOP1, is expressed in keratinocytes in a UVB-regulated manner and is a negative regulator of p53 as a posttranslational modifier. However, it was not known whether huCOP1 plays a role in mediating the UVB-induced early transcriptional responses of human keratinocytes. In this study, we report that stable siRNA-mediated silencing of huCOP1 affects the UVB response of several genes within 2 h of irradiation, indicating that altered huCOP1 expression sensitizes the cells toward UVB. Pathway analysis identified a molecular network in which 13 of the 30 examined UVB-regulated genes were organized around three central proteins. Since the expression of the investigated genes was upregulated by UVB in the siCOP1 cell line, we hypothesize ...
U1 RNA stimulates skin barrier genes in a TLR3-dependent mannerTLR3 was silenced in normal human epidermal keratinocytes (NHEKs) for 48 hours before treatment w
Our results show that cannabinoids inhibit keratinocyte proliferation, and therefore support a potential role for cannabinoids in the treatment of psoriasis.
Long-term exposure of immortalized keratinocytes to arsenic induces EMT, impairs differentiation in organotypic skin models and mimics aspects of human skin der
A generic research platform with 2-dimensional (2D) cell culture technology, a 3-dimensional (3D) in vitro tissue model, and a scaled-down cell culture and imaging system in between, was utilized to address the problematic issues associated with the use of serum in skin tissue engineering. Human dermal fibroblasts (HDFs) and immortalized keratinocytes (HaCat cells) mono- or co-cultured in serum or serum-free medium were compared and analyzed via the platform. It was demonstrated that serum depletion had significant influence on the attachment of HaCat cells onto tissue culture plastic (TCP), porous substrates and cellulosic scaffolds, which was further enhanced by the pre-seeded HDFs. The complex structures formed by the HDFs colonized within the porous substrates and scaffolds not only prevented the seeded HaCat cells from filtering through the open pores, but also acted as cellular substrates for HaCat cells to attach onto. When mono-cultured on TCP, both HDFs and HaCat cells were less proliferative
IFN-γ is a well-characterized macrophage differentiation signal. Macrophages pretreated with IFN-γ achieve a higher level of activation, e.g., IFN-γ-primed macrophages become more sensitive to other stimuli, such as bacterial products and cytokines, as evidenced by the synergistic induction of many inflammatory gene products, e.g., iNOS (12)(35)(36) and IL-12 p40 (13)(37)(42)(43). In this paper, we show that activation with IFN-γ, as well as its priming effect, can be extended to Cox-2, another gene with a central role in inflammatory responses. Although IFN-γ regulation of Cox-2 has been described previously (26)(44)(45), little is known of the molecular mechanisms that underlie activation of this gene by IFN-γ. For example, in bronchial epithelial cells (44) and in normal human epidermal keratinocytes (26), Cox-2 induction is controlled through autocrine loops via the epidermal growth factor receptor and its ligands, such as TGF-α, resulting in a delayed activation. However, in ...
Cytoskeletal modifications are thought to require the activation of Rho GTPases. Bacterial pathogens can modulate or mimic Rho GTPase signaling, leading to modifications of the actin cytoskeleton that help the bacteria to invade a host cell and/or gain motility in the cell (4). There is first evidence that viruses also take advantage of Rac1/Cdc42 signaling; the most prominent example is vaccinia virus (16). During herpesvirus infection, activation of Rho GTPases is often observed; however, the functional significance is not yet fully understood. We addressed the impact of Rac1 and Cdc42 during initiation of HSV-1 infection in keratinocytes and performed overexpression and RNA interference studies. In addition, we used a mouse model to study the effects in the epidermis, which serves as an entry portal for HSV-1 in vivo. Our findings provide the first evidence that Rac1/Cdc42 signaling pathways are not required for efficient initiation of HSV-1 infection in keratinocytes.. Initially we ...
BioAssay record AID 683120 submitted by ChEMBL: Inhibition of human ALK5 activity in human HaCaT cells expressing p3TP-luciferase reporter construct assessed as TGFbeta-induced p3TP-luciferase reporter activity at 30 nM relative to control.
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