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Normal Human Keratinocytes (normal + human_keratinocyte)
Selected AbstractsT-cadherin loss induces an invasive phenotype in human keratinocytes and squamous cell carcinoma (SCC) cells in vitro and is associated with malignant transformation of cutaneous SCC in vivoBRITISH JOURNAL OF DERMATOLOGY, Issue 2 2010D. Pfaff Summary Background, Cadherins play important roles in controlling keratinocyte growth, differentiation and survival. Atypical glycosylphosphatidylinositol-anchored T-cadherin (T-cad) is highly expressed in the basal keratinocyte layer of skin. The role of T-cad in keratinocyte biology and pathology is unclear. Objectives, To define the role of T-cad in the pathogenesis of cutaneous squamous cell carcinoma (SCC) through gain-of-function and loss-of-function studies in vitro and through examination of T-cad expression patterns in human cutaneous SCC specimens in relation to histological classification of degree of tumour differentiation. Methods,In vitro studies employed lentiviral-mediated overexpression/silencing of T-cad in normal human keratinocyte (HaCaT) and SCC (A431) cell lines, monolayer and multicellular spheroid culture models, cell morphology analyses and assays of random motility and invasion. Immunohistochemistry was performed on skin specimens from patients with actinic keratosis, Bowen disease or SCC. Results,In vitro, silencing of T-cad induced a morphologically elongated and disorganized cell phenotype, increased random motility and markedly enhanced invasive potential. Overexpression of T-cad induced a morphologically spread and compact cell phenotype and blunted invasive potential. In vivo, regional loss of T-cad expression was more frequent and prominent in SCC classified as moderately-to-poorly differentiated than in SCC classified as well differentiated. However, in both categories aberrant and/or absence of T-cad expression was associated with histological features of a potentially more malignant and invasive phenotype of cutaneous SCC. Conclusions, T-cad is a controlling determinant of SCC phenotype and invasive behaviour and its loss is associated with the process of malignant transformation from noninvasive to invasive SCC. [source] In vitro induction of matrix metalloproteinase-2 and matrix metalloproteinase-9 expression in keratinocytes by boron and manganeseEXPERIMENTAL DERMATOLOGY, Issue 8 2004Nathalie Chebassier Abstract:, Matrix metalloproteinase (MMP)-2 and MMP-9 are involved in keratinocyte migration and granulation tissue remodeling during wound healing. Thermal water cures are sometimes proposed as complementary treatment for accelerating healing of wounds resulting from burns and/or surgery, but their mechanisms of action remain unknown. Some thermal waters are rich in trace elements such as boron and manganese. Interestingly, clinical studies have shown the beneficial effects of trace elements such as boron and manganese for human wound healing. To try to specify the role of trace elements in cutaneous healing, the present study investigated the effects of these trace elements on the production of MMP-2 and MMP-9 by normal human keratinocytes cultured in vitro. Immunohistochemistry and Western blot showed that intracellular MMP-9 expression in keratinocytes was induced when incubated for 6 h with boron at 10 µg/ml or manganese at 0.2 µg/ml. Moreover, gelatin zymography on keratinocyte supernatants showed an increase of gelatinase secretion after 24 h of incubation of keratinocytes with boron or manganese, regardless of concentration. Gelatinase secretion was not associated with keratinocyte proliferation induced by trace elements. Thus, our results suggest that boron and manganese could play a role in the clinical efficiency of thermal water on wound healing. [source] Evidence for local control of gene expression in the epidermal differentiation complex,EXPERIMENTAL DERMATOLOGY, Issue 5 2002James T. Elder Abstract: The epidermal differentiation complex (EDC), located on chromosomal band 1q21, consists of at least 43 genes that are expressed during keratinocyte differentiation. Indicative of a role for chromatin structure in tissue specificity of EDC gene expression, we identified an inverse correlation between expression and DNA methylation for two EDC genes (S100A2 and S00A6) in human keratinocytes and fibroblasts. 5-azacytidine (5AC) and sodium butyrate (NaB) are two agents known to promote ,open' chromatin structure. To explore the relationship between chromatin structure and keratinocyte differentiation, we treated normal human keratinocytes (NHK) with 5AC or NaB, or with protocols known to promote their terminal differentiation. We then measured the steady-state mRNA levels for several S100 genes, small proline rich region-1, -2, and -3, loricrin, and involucrin by Northern blotting. 5AC and NaB each markedly increased expression of SPRR1/2 and involucrin in NHK. In contrast, expression of S100A2 was reduced by both agents, and by induction of keratinocyte differentiation. Moreover, while the clustered EDC genes displayed a general tendency to be expressed in epithelial cells, they displayed different patterns of cell type-specific expression. These results indicate that local, gene-specific factors play an important role in the regulation of EDC gene expression in the keratinocyte lineage and during keratinocyte terminal differentiation. [source] Zinc, copper and manganese enhanced keratinocyte migration through a functional modulation of keratinocyte integrinsEXPERIMENTAL DERMATOLOGY, Issue 6 2000I. Tenaud Abstract: The migration of keratinocytes plays an important role in the re-epithelialization of cutaneous wounds. Zinc, copper and manganese are used in vivo for their healing properties and their mechanism of action is still only partially known. Thus, they have been shown both to promote keratinocyte proliferation and to modulate integrins expression. The aim of this study was to determine if trace elements induce an increase of the migration of keratinocytes and if this effect is related to the modulation of integrins. Two independent migration assays were used to study keratinocyte migration: the scratch assay using normal human keratinocytes and the modified Boyden chamber using HaCaT cells. Inhibition studies using function-blocking antibodies directed to ,3, ,6, ,V and ,1 subunits were performed to investigate the modulator effect of trace elements on integrin function. In this way, zinc and copper gluconates increased ,3, ,V and ,1 function whereas manganese gluconate seems mainly able to modulate the function of ,3 and ,1. The stimulating effect of these trace elements on keratinocyte migration does not appear related to ,6 subunit. Thus, zinc, copper and manganese enhanced keratinocyte migration and one of the mechanisms was going through a modulation of integrin functions. [source] Escape from microenvironmental control and progression of intraepithelial neoplasiaINTERNATIONAL JOURNAL OF CANCER, Issue 6 2005Weitian Zhang Abstract We previously reported that normal human keratinocytes controlled neoplastic progression of tumor cells at an early stage of transformation in stratified squamous epithelium. We now studied if cells at a more advanced stage of transformation were also subject to such microenvironmental control. To accomplish this, 3D human tissues that mimic intraepithelial neoplasia were fabricated by mixing genetically marked (,-gal), early-stage (II-4 cells) or advanced-stage (SCC13) transformed keratinocytes with normal keratinocytes, and tumor cell fate and phenotype were monitored in organotypic culture and after surface transplantation to nude mice. In vivo, SCC13 cells evaded local growth suppression to undergo connective tissue invasion at significantly lower tumor cell volumes (12:1, 50:1 normal:tumor cells) than II-4 cells. This behavior was explained by the growth suppression of II-4 cells, while advanced-stage tumor cells escaped this control and continued to undergo clonal expansion in mixed cultures to form large, intraepithelial tumor clusters. These communities of tumor cells underwent autonomous growth that was associated with altered expression of markers of differentiation (keratin 1) and cell,cell communication (connexin-43). Furthermore, significantly greater numbers of SCC13 cells expanded into a basal position after low-calcium stripping of suprabasal cells of mixed cultures compared to II-4 cells, suggesting that expansion of these cells enabled tumor cell invasion after transplantation. These findings demonstrated that early tumor development in human stratified squamous epithelium required escape from microenvironmental growth control that was dependent on the transformation stage of intraepithelial tumor cells during the premalignant stage of cancer progression. © 2005 Wiley-Liss, Inc. [source] An optimized method for intensive screening of molecules that stimulate , -defensin 2 or 3 (hBD2 or hBD3) expression in cultured normal human keratinocytesINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 3 2005I. Pernet Synopsis Normal human skin controls the intrusion of microorganisms by the production of peptide antibiotics such as defensins. The aim of our study was to develop a culture model of normal human keratinocytes for optimal , -defensin mRNA detection which allows the screening of molecules able to stimulate hBD2 and hBD3 without inducing pro-inflammatory cytokines. A keratinocyte culture model in 96-well plates, in high calcium medium (1.7 mm) allowed to analyze hBD2 and hBD3 mRNA expression in basal condition and after cell stimulation by products from diverse vegetal extracts. The release of IL-8 and the chemokine MIP-3, was also evaluated in cell supernatants by ELISA. Among the 184 extracts tested, 75 showed a stimulatory effect on , -defensin expression: 40 on hBD2, 26 on hBD3 and nine on both defensins. Fifteen of these substances which also induced the release of pro-inflammatory cytokines were eliminated. Among the other substances, four were selected and were analyzed in a dose-dependent study (n = 4) by real-time quantitative RT-PCR and completed by a measure of MIP-3,, IL-8 and IL-1, levels. These data underline the important necessity of screening result controls by a quantitative method reproduced at least three times. This new method of intensive screening allowed us to exhibit vegetal extracts that were able to stimulate epidermal , -defensin expression without inducing an up-secretion of pro-inflammatory cytokines. Résumé La peau humaine normale exerce une fonction barrière contre l'intrusion de microorganismes par la production de peptides antibiotiques comme les défensines. Le but de cette étude a consistéà mettre au point un modèle de culture de kératinocytes humains normaux permettant une détection optimale des ARNm des défensines en général, et adapté au screening de molécules aptes à stimuler les défensines épidermiques hBD2 et hBD3 en particulier, sans induire de cytokines pro-inflammatoires. Un modèle de culture de kératinocytes en plaques 96 puits, en milieu riche en calcium (1,7 mm) permet une analyse de l'expression des ARNm de hBD2 et hBD3 en condition basale et après stimulation par divers extraits végétaux. La sécrétion d'IL-8 et de la chimiokine MIP-3, a étéévaluée dans les surnageants de culture par ELISA. Parmi les 184 extraits testés, 75 montrent un effet stimulant sur l'expression des , -défensines : 40 ont un effet sur hBD2, 26 sur hBD3 et 9 sur les 2 types de défensines. Quinze de ces actifs qui induisent aussi la sécrétion de cytokines pro-inflammatoires ont étééliminés. Parmi les autres molécules, 4 ont été sélectionnées pour faire l'objet d'une étude de leurs effets-doses (n = 4) sur l'expression des , -défensines par une technique quantitative de RT-PCR en temps réel. Cette étude est complétée par le dosage des cytokines IL-1,, IL-8 et MIP-3,. Les résultats obtenus soulignent l'importante nécessitée de contrôler au moins trois fois par une méthode quantitative les résultats d'un screening. Cette nouvelle méthode de screening intensif nous a permis de mettre en évidence des extraits végétaux capables de stimuler les défensines épidermiques sans induire de cytokines pro-inflammatoires. [source] Green Tea Phenol Extracts Reduce UVB-induced DNA Damage in Human Cells via Interleukin-12,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2008Agatha Schwarz Green tea chemoprevention has been a focus of recent research, as a polyphenolic fraction from green tea (GTP) has been suggested to prevent UV radiation-induced skin cancer. Recently, it was demonstrated that GTP reduced the risk for skin cancer in a murine photocarcinogenesis model. This was accompanied by a reduction in UV-induced DNA damage. These effects appeared to be mediated via interleukin (IL)-12, which was previously shown to induce DNA repair. Therefore, we studied whether GTP induction of IL-12 and DNA repair could also be observed in human cells. KB cells and normal human keratinocytes were exposed to GTP 5 h before and after UVB. UVB-induced apoptosis was reduced in UVB-exposed cells treated with GTP. GTP induced the secretion of IL-12 in keratinocytes. The reduction in UV-induced cell death by GTP was almost completely reversed upon addition of an anti-IL-12-antibody, indicating that the reduction of UV-induced cell death by GTP is mediated via IL-12. The ability of IL-12 to reduce DNA damage and sunburn cells was confirmed in "human living skin equivalent" models. Hence the previously reported UV-protective effects of GTP appear to be mediated in human cells via IL-12, most likely through induction of DNA repair. [source] Aquaporin-3 gene and protein expression in sun-protected human skin decreases with skin ageingAUSTRALASIAN JOURNAL OF DERMATOLOGY, Issue 2 2010Ji Li ABSTRACT Backgroud/Objectives:, Aquaporin 3 (AQP3) is a protein implicated in skin hydration. AQP3 null mice have relatively dry skin, reduced skin elasticity, and delayed recovery of barrier function after removal of the stratum corneum which is also present in skin of old people. A feature of skin aging is the change in both water content and barrier function of the skin. We investigated the expression of aquaporin 3 in non sun-exposed human skin, normal human keratinocytes and fibroblasts from different age groups to further understand the relationship between AQP3 and intrinsic skin aging. Methods:, We investigated the expression of aquaporin3 (AQP3) in normal human skin, normal human epidermal keratinocytes (NHEK) and skin fibroblast of different ages by immunohistochemistry, immunocytochemistry, the reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. The samples were derived from 60 patients of varying ages: <20 years of age, 30,45 years and >60 years of age. Twenty skin biopsies, 6 for keratinocyte/fibroblast cultures, were taken from each age group. Results:, AQP3 decreased with increasing age in both skin and NHEK samples. We demonstrated significant differences in AQP3 expression between the 3 age groups (P < 0.05). In fibroblasts, AQP3 expression levels were significantly lower in the >60 year olds compared to 30,45 year olds (P < 0.05) and <20 year olds (P < 0.05), there was no significant difference between the two younger groups (P > 0.05). Conlusions:, AQP3 may be involved in the intrinsic aging process of non sun-exposed human skin. [source] Expression of the endogenous, nicotinic acetylcholine receptor ligand, SLURP-1, in human colon cancerAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 4 2008A. Pettersson Summary 1,Secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein-1 (SLURP-1) is a recently discovered endogenous ligand at the ,7 subunit of the nicotinic acetylcholine receptors. Previous reports have shown that SLURP-1 is expressed in normal human keratinocytes seemingly with a pro-apoptotic function. Conversely, such expression was markedly attenuated in transformed cells and it was suggested that the molecule could convey protection against malignant transformation. 2,In this study, we demonstrated the mRNA expression (by RT-PCR) and protein expression (by Western blotting and immunocytochemistry) of SLURP-1 in the human colon cancer cell line, HT-29. 3,Furthermore, we demonstrated the expression of SLURP-1 (by immunohistochemistry) in tumour cells of human colon cancer tissue, and, to a greater extent, in immune and smooth muscle cells of adjacent, macroscopically tumour-free colon tissue. 4,The current findings suggest that SLURP-1 participates in the regulation of gut immune functions and motility, as well as possibly playing a role in colon carcinogenesis/cancer progression. [source] Expression of toll-like receptor 2, NOD2 and dectin-1 and stimulatory effects of their ligands and histamine in normal human keratinocytesBRITISH JOURNAL OF DERMATOLOGY, Issue 2 2009M. Kobayashi Summary Background, Epidermal keratinocytes are involved in the skin innate immunity and express toll-like receptors (TLRs) and other innate immune proteins. The epidermis is continuously exposed to pathogenic Gram-positive bacteria or fungi. However, few studies have examined the function and expression of innate immune proteins in keratinocytes. Histamine, which is well known for itch and allergy, is closely associated with innate immunity, but its influence on epidermal innate immunity is still unclear. Objectives, To clarify the expression of innate immune proteins in keratinocytes stimulated by ligand pathogen-associated molecules, and the function of histamine in this process. Methods, We investigated the effects of lipopeptide (MALP-2, 1,100 ng mL,1; ligand for TLR2), peptidoglycan (PGN, 0·02,2 ,g mL,1; ligand for NOD2) and ,-glucan (1,100 ,g mL,1; ligand for dectin-1) in the presence or absence of histamine on mRNA expression of TLR2, NOD2 and dectin-1 as well as human ,-defensin 2 by quantitative real-time polymerase chain reaction in cultured normal human epidermal keratinocytes. TLR2 expression was also examined at the cell surface and intracellularly, as determined by flow cytometry and confocal microscopy. The quantities of interleukin (IL)-1, and IL-8 produced by keratinocytes were measured using enzyme-linked immunosorbent assay. Results, At the mRNA level, TLR2 was enhanced by PGN but not by its ligand MALP-2 or by ,-glucan; NOD2 was easily induced by all three ligands; and dectin-1 was enhanced by its ligand ,-glucan. These enhanced expressions were further augmented by histamine at 1 ,g mL,1. While the surface expression of TLR2 was barely detectable by flow cytometry even after stimulation, the intracellular expression of TLR2 was apparently elevated by PGN and further promoted by histamine. A confocal microscopic analysis also revealed the enhanced expression of TLR2 in the cytoplasm. The expression of TLR2, NOD2 and dectin-1 was functional, as these pathogen-associated molecules induced the production of IL-1,, IL-8 and defensin, and again, histamine greatly enhanced this production. Conclusions, Our study demonstrated that the expression of functional innate immune receptors is augmented by the pathogen-associated molecules in a ligand-feed forward or nonrelated manner in keratinocytes, and histamine promotes their expression and the resultant production of cytokines and defensins. [source] The effect of mechanical strain on protease production by keratinocytesBRITISH JOURNAL OF DERMATOLOGY, Issue 2 2008N. Bhadal Summary Background, Intact skin is under constant tension, transmitted from the underlying dermis, but when tension is lost (i.e. upon wounding) protease activity is upregulated. Objectives, To investigate the effect of mechanical strain on protease production by both normal and transformed keratinocytes in vitro. Methods, Keratinocytes were seeded on to membranes precoated with either type I or type IV collagen. After 48 h medium was replaced with serum-free medium and mechanical strain was applied. Results, Mechanical strain resulted in decreased urokinase-type plasminogen activator (uPA) production by normal human keratinocytes (P < 0·05) but increased production by transformed keratinocytes (P < 0·05) cultured on type I and type IV collagen. Conclusions, Differential production of uPA by normal and transformed keratinocytes is relevant in the context of normal function, wound healing and tumorigenesis. [source] | ||||||||||