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Human Keratinocytes (human + keratinocyte)
Kinds of Human Keratinocytes Terms modified by Human Keratinocytes 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] Functional characterization of T cells differentiated in vitro from bone marrow-derived CD34+ cells of psoriatic patients with family historyEXPERIMENTAL DERMATOLOGY, Issue 8 2010Kaiming Zhang Please cite this paper as: Functional characterization of T cells differentiated in vitro from bone marrow-derived CD34+ cells of psoriatic patients with family history. Experimental Dermatology 2010; 19: e128,e135. Abstract Background:, The strong but complex genetic background suggests that inherent and intrinsic rather than exogenous factors have a key role in immunopathogenesis of psoriasis. It is reasonable to speculate that the dysfunctional activity of psoriatic T cells may partly originate from the abnormal haematopoietic cells. Objectives:, To test if T cells originated from haematopoietic progenitor cells in psoriasis patients display functional alternations similar to previously reported abnormalities of circulating T cells. Methods:, Bone marrow CD34+ haematopoietic cells were isolated from psoriatic patients with family history and healthy subjects, and differentiated into T cells in vitro in the thymic stromal co-culture system. These cells were further subjected to functional comparisons such as in vitro proliferation, secretion of cytokines such as IL-4, IL-8 and IFN,,, and inducing the production of C-myc, Bcl-xL, and Ki67 proteins in human keratinocytes. Results:, While bone marrow-derived CD34+ cells from both patients and healthy volunteers developed into mature T cells within weeks in the thymic environment in vitro, the differentiated T cells from psoriatic patients showed higher proliferation and stronger capacity to secret TH1 cytokines in response to streptococcal superantigen. The differentiated T cells from psoriatic patients, but not from normal controls, induced overexpression of C-myc and Ki67, but not Bcl-XL, in keratinocytes. Conclusions:, T cells differentiated from CD34+ cells of psoriatic patients, but not normal controls, are functionally similar to psoriatic circulating T cells, suggesting that the dysfunctional activity of T cells in psoriatic patients can be traced back to the early development of haematopoietic cells. [source] Terrein inhibits keratinocyte proliferation via ERK inactivation and G2/Mcell cycle arrestEXPERIMENTAL DERMATOLOGY, Issue 4 2008Dong-Seok Kim Abstract:, Terrein, a fungal metabolite, has been recently shown to have a strong antiproliferative effect on skin equivalents. In the present study, we further investigated the effects of terrein on the possible signalling pathways involved in the growth inhibition of human epidermal keratinocytes by examining the regulations of extracellular signal-regulated protein kinase (ERK) and of the Akt pathway by terrein. It was observed that ERK was inactivated by terrein and that keratinocyte proliferation was inhibited, whereas Akt was unaffected. The inhibition of the ERK pathway by U0126 (a specific ERK inhibitor) also had a dose-dependent antiproliferative effect on human keratinocytes. These results indicate that ERK inhibition is involved in keratinocyte growth inhibition by terrein. Moreover, flow cytometric analysis showed that terrein inhibits DNA synthesis, as evidenced by a reduction in the S phase and an increase in the G2/M phase of the cell cycle. Thus, we next examined changes in the expressions of G2/M cell cycle-related proteins. Terrein was found to downregulate cyclin B1 and Cdc2 without Cdc2 phosphorylation, but upregulated p27KIP1 (p27), a known inhibitor of cyclin-dependent kinase. These results suggest that terrein reduces human keratinocyte proliferation by inhibiting ERK and by decreasing the expressions of cyclin B1 and Cdc2 complex. [source] The association between endothelin-1 gene polymorphisms and susceptibility to vitiligo in a Korean populationEXPERIMENTAL DERMATOLOGY, Issue 7 2007Hyun-Jin Kim Abstract:, Background:, Vitiligo is a skin disorder affected by genetic, environmental, local and endocrine factors. Endothelin-1, which is expressed by keratinocytes, has paracrine effects on melanocytes, influencing their homeostasis, proliferation and pigmentation. It is thought to play a role in the skin response to 311-nm, narrow-band ultraviolet irradiation. Objective:, To investigate the association of endothelin-1 gene (EDN1) polymorphisms with vitiligo in a Korean population. Methods:, To evaluate the expression of endothelin-1 in cultured human keratinocytes after irradiation with narrow-band ultraviolet B (NBUVB), we performed RT-PCR and ELISA. In addition, we genotyped 312 vitiligo patients and 313 matched-healthy controls, and compared the genotype, allele and haplotype frequencies of EDN1 polymorphisms (intron 4 G/A, rs2071942 and exon 5 G/T, rs5370) between the two groups, using PCR-restriction fragment length polymorphism. The effects of sex, onset age, the presence of autoimmune diseases, family history and clinical type were analysed statistically. Results:, NBUVB induced the expression of endothelin-1 in cultured human keratinocytes. The genotype distributions and allele frequencies of EDN1 polymorphisms did not differ significantly between vitiligo patients and healthy controls. Moreover, the results were not related to sex, onset age, the presence of autoimmune diseases or family history. Interestingly, the haplotype frequencies of EDN1 polymorphisms differed significantly between vitiligo patients and healthy controls. When analysed according to clinical type, the haplotype frequencies in the focal and segmental clinical types differed significantly from healthy controls. Conclusion:, This study suggests that EDN1 is related to the development of vitiligo in the Korean population. [source] Nucleofection: a new, highly efficient transfection method for primary human keratinocytes,EXPERIMENTAL DERMATOLOGY, Issue 4 2005Jörg H. W. Distler Abstract:, Transfection is an essential tool for numerous in vitro applications including studies of gene expression, promoter analysis, and intracellular signaling pathways and also for therapeutic strategies such as tissue engineering and gene therapy. However, transfection of primary cells including keratinocytes with common methods such as calcium phosphate, DEAE-dextran, liposome-mediated transfer, electroporation or viral vectors is problematic because of low transfection efficiency and the induction of terminal differentiation. Here we analyzed the use of nucleofection, a new, electroporation-based transfection method that enables the DNA to enter directly the nucleus, for the transfection of keratinocytes. Several different conditions were tested and optimized, resulting in a final transfection efficiency of 56% in primary human epidermal keratinocytes. This efficiency is superior to all non-viral transfection methods reported so far. The number of non-viable keratinocytes after nucleofection was low, varying between 14 and 16%. In contrast to other transfection protocols, nucleofection did not induce terminal differentiation in the transfected keratinocytes. In addition, nucleofection is a fast method, because the results can be analyzed within 7 h. In summary, nucleofection is a fast, easy and highly effective alternative for the transfection of primary human keratinocytes, which offers new opportunities for various research applications. [source] In vitro interactions between sensory nerves, epidermis, hair follicles and capillaries in a tissue-engineered reconstructed skinEXPERIMENTAL DERMATOLOGY, Issue 9 2004V. Gagnon Recent findings have established that cutaneous nerves modulate both skin homeostasis and various skin diseases, by influencing cell growth and differentiation, inflammation and wound healing. In order to study the influence of epidermis, hair follicles and capillaries on sensory neurons, and vice-versa, we developed a tissue-engineered model of innervated endothelialized reconstructed skin (MIERS). Mouse dorsal root ganglia neurons were seeded on a collagen sponge populated with human fibroblasts and human endothelial cells. Keratinocytes or mice newborn immature hair follicle buds were then seeded on the opposite side of the MIERS to study their influence on sensory nerves growth, and vice versa. A vigorous neurite elongation was detected inside the reconstructed dermis after 14 and 31 days of neurons culture. The presence of endothelial cells induced a significant increase of the neurite elongation after 14 days of culture. The addition of human keratinocytes totally avoided the twofold decrease in the amount of neurites observed between 14 and 31 days in controls. We have successfully developed the MIERS that allowed us to study the effects of epidermis and capillaries on nerve growth. This model will be a useful tool to study the modulation of sensory nerves on wound healing, angiogenesis, hair growth and neurogenic inflammation in the skin. [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] Expression of vanilloid receptor subtype 1 in cutaneous sensory nerve fibers, mast cells, and epithelial cells of appendage structuresEXPERIMENTAL DERMATOLOGY, Issue 3 2004Sonja Ständer Abstract:, The vanilloid receptor subtype 1 (VR1)/(TRPV1), binding capsaicin, is a non-selective cation channel that recently has been shown in human keratinocytes in vitro and in vivo. However, a description of VR1 localization in other cutaneous compartments in particular cutaneous nerve fibers is still lacking. We therefore investigated VR1 immunoreactivity as well as mRNA and protein expression in a series (n = 26) of normal (n = 7), diseased (n = 13) [prurigo nodularis (PN) (n = 10), generalized pruritus (n = 1), and mastocytosis (n = 2)], and capsaicin-treated human skin (n = 6). VR1 immunoreactivity could be observed in cutaneous sensory nerve fibers, mast cells, epidermal keratinocytes, dermal blood vessels, the inner root sheet and the infundibulum of hair follicles, differentiated sebocytes, sweat gland ducts, and the secretory portion of eccrine sweat glands. Upon reverse transcriptase-polymerase chain reaction and Western blot analysis, VR1 was detected in mast cells and keratinocytes from human skin. In pruritic skin of PN, VR1 expression was highly increased in epidermal keratinocytes and nerve fibers, which was normalized after capsaicin application. During capsaicin therapy, a reduction of neuropeptides (substance P, calcitonin gene-related peptide) was observed. After cessation of capsaicin therapy, neuropeptides re-accumulated in skin nerves. In conclusion, VR1 is widely distributed in the skin, suggesting a major role for this receptor, e.g. in nociception and neurogenic inflammation. [source] Antipsoriatic drug anthralin induces EGF receptor phosphorylation in keratinocytes: requirement for H2O2 generationEXPERIMENTAL DERMATOLOGY, Issue 2 2004Dominik Peus Abstract: Even though anthralin is a well-established topical therapeutic agent for psoriasis, little is known about its effects and biochemical mechanisms of signal transduction. In contrast to a previous report, we found that anthralin induced time- and concentration-dependent phosphorylation of epidermal growth factor receptor in primary human keratinocytes. Four lines of evidence show that this process is mediated by reactive oxygen species. First, we found that anthralin induces time-dependent generation of H2O2. Second, there is a correlation between a time-dependent increase in anthralin-induced epidermal growth factor receptor phosphorylation and H2O2 generation. Third, the structurally different antioxidants n -propyl gallate and N -acetylcysteine inhibited epidermal growth factor receptor phosphorylation induced by anthralin. Fourth, overexpression of catalase inhibited this process. The epidermal growth factor receptor-specific tyrosine kinase inhibitor PD153035 abrogated anthralin-induced epidermal growth factor receptor phosphorylation and activation of extracellular-regulated kinase 1/2. These findings establish the following sequence of events: (1) H2O2 generation, (2) epidermal growth factor receptor phosphorylation, and (3) extracellular-regulated kinase activation. Our data identify anthralin-induced reactive oxygen species and, more specifically, H2O2 as an important upstream mediator required for ligand-independent epidermal growth factor receptor phosphorylation and downstream signaling. [source] Supernatants of Pseudomonas aeruginosa induce the Pseudomonas -specific antibiotic elafin in human keratinocytesEXPERIMENTAL DERMATOLOGY, Issue 4 2003Ulf Meyer-Hoffert Abstract: Elafin is a skin-derived serine-protease inhibitor. It is thought to be important to prevent human leukocyte elastase-mediated tissue damage and might play an important role in maintaining the integrity of the human epidermis. Recent studies have provided evidence for an antimicrobial activity of elafin against P. aeruginosa. As gram-negative infections typically occur in barrier-disrupted skin we were interested to determine whether supernatants of the gram-negative bacteria P. aeruginosa and Escherichia coli were capable of inducing elafin expression. Supernatants of various P. aeruginosa strains stimulated elafin mRNA-expression and protein release, whereas supernatants of E. coli did not induce elafin expression. In non-differentiated cells the relative increase of elafin mRNA was much higher (100-fold) than in differentiated cells (sixfold), although the latter exhibited higher constitutive mRNA-expression (150-fold). However, concentrations of secreted elafin were similar in differentiated and non-differentiated cells after stimulation. We could not confirm a bactericidal effect against P. aeruginosa as described previously but observed that its growth was inhibited as demonstrated for different strains in liquid cultures. Growth of E. coli was not affected by elafin. In conclusion, the data presented in this paper suggest that elafin represents an innate immune response factor induced by secreted products of P. aeruginosa. Besides its elastase inhibitory potency elafin is an antimicrobial agent against P. aeruginosa. [source] Nickel-induced keratinocyte proliferation and up-modulation of the keratinocyte growth factor receptor expressionEXPERIMENTAL DERMATOLOGY, Issue 4 2003Cinzia Marchese Abstract: Keratinocytes play a key role in the pathogenesis of allergic contact dermatitis (ADC) induced by the sensitizing agent nickel. We analyzed here the effects of treatment with nickel and of the pretreatment with zinc on HaCaT cells and primary human keratinocytes. Cell counting, 5-bromo-2,-deoxyuridine incorporation assay and adenosine triphosphate (ATP) bioluminescence detection showed that treatment with NiSO4 induced DNA synthesis and cell proliferation and that pretreatment with ZnSO4 was able to abrogate this proliferative effect. This nickel-induced cell growth appeared enhanced when primary human keratinocytes were co-cultured with fibroblasts. Western blot analysis demonstrated that nickel ions induced up-modulation of the expression of the keratinocyte growth factor receptors (KGFR) without affecting the keratinocyte differentiation, whereas the protein levels of the epidermal growth factor receptor (EGFR) and of its ligand transforming growth factor-alpha (TGF-,) appeared unmodified by the treatment. Double immunofluorescence showed that the effect of nickel on DNA synthesis was mainly exerted on KGFR expressing cells, suggesting that KGFR up-modulation could be required for the nickel-induced cell proliferation. These results indicate that KGFR and its ligands may play a role in the mechanism of action of nickel ions and in the protective effect of zinc pretreatment. [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] Presence of immunoreactive ,-endorphin in human skinEXPERIMENTAL DERMATOLOGY, Issue 5 2001M. Wintzen Abstract: The production and its induction by ultraviolet radiation (UVR) of proopiomelanocortin (POMC)-derived peptides by keratinocytes has been reported, albeit not consistently. Recently we demonstrated that only under specific culturing conditions human keratinocytes are capable of producing a ,-endorphin (,E)-like peptide with the characteristics of ,-lipotropin (,LPH). Here the presence and UV-induction of ,E-immunoreactivity (,E-IR) in keratinocytes in human skin in vivo was investigated. ,E-IR was detectable by immunohistochemistry in keratinocytes of the follicular matrix and to some extent in cells of sweat ducts, but was absent from epidermal keratinocytes. Absence of ,E-IR was confirmed by radioimmunoassay of HPLC-fractionated extracts of normal epidermis. Repeated exposure to solar-simulated UVR had no effect. This investigation is the first to demonstrate the presence of ,E-immunoreactive material in the follicular matrix of corporal hairs and in duct cells of sweat glands. The possible meaning of these results is discussed. [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] Conversion of vitamin D3 to 1,,25-dihydroxyvitamin D3 in human skin equivalentsEXPERIMENTAL DERMATOLOGY, Issue 2 2000B. Lehmann Abstract: These results demonstrate for the first time that human keratinocytes under in vivo -like conditions have the capacity of the enzymatic hydroxylation of vitamin D3 to hormonally active calcitriol (1,,25(OH)2D3). Supplementation of the culture medium with bovine serum albumin (BSA) up to 1.5% (w/v) amplifies the conversion of vitamin D3 to 1,,25(OH)2D3. The maximum turnover rate of this reaction at 780 nM vitamin D3 in presence of 1.0% (w/v) BSA amounts to approximately 3 pmol 1,,25(OH)2D3 per 106 cells after 6 h of incubation. The hydroxylation of vitamin D3 to 1,,25(OH)2D3 is inhibited by the P-450 oxidase inhibitor ketoconazole. The generation of 1,,25(OH)2D3 from vitamin D3 has an apparent Michaelis constant (Km) of 2.3×10,6 M. The intrinsic conversion of vitamin D3 to biologically active 1,,25(OH)2D3 may be of importance for the regulation of proliferation and differentiation of keratinocytes. [source] Vanadium-induced apoptosis of HaCaT cells is mediated by c-fos and involves nuclear accumulation of clusterinFEBS JOURNAL, Issue 14 2009Soultana Markopoulou Vanadium exerts a variety of biological effects, including antiproliferative responses through activation of the respective signaling pathways and the generation of reactive oxygen species. As epidermal cells are exposed to environmental insults, human keratinocytes (HaCaT) were used to investigate the mechanism of the antiproliferative effects of vanadyl(IV) sulfate (VOSO4). Treatment of HaCaT cells with VOSO4 inhibited proliferation and induced apoptosis in a dose-dependent manner. Inhibition of proliferation was associated with downregulation of cyclins D1 and E, E2F1, and the cyclin-dependent kinase inhibitors p21Cip1/Waf1 and p27Kip1. Induction of apoptosis correlated with upregulation of the c-fos oncoprotein, changes in the expression of clusterin (CLU), an altered ratio of antiapoptotic to proapoptotic Bcl-2 protein family members, and poly(ADP-ribose) polymerase-1 cleavage. Forced overexpression of c-fos induced apoptosis in HaCaT cells that correlated with secretory CLU downregulation and upregulation of nuclear CLU (nCLU), a pro-death protein. Overexpression of Bcl-2 protected HaCaT cells from vanadium-induced apoptosis, whereas secretory CLU overexpression offered no cytoprotection. In contrast, nCLU sensitized HaCaT cells to apoptosis. Our data suggest that vanadium-mediated apoptosis was promoted by c-fos, leading to alterations in CLU isoform processing and induction of the pro-death nCLU protein. [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] 20-O-,-D-Glucopyranosyl-20 (S)-protopanaxadiol (compound K) induces expression of hyaluronan synthase 2 gene in transformed human keratinocytes and fibroblasts and increases hyaluronan in hairless mouse skinINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 6 2004S. Kim Ginsenosides, the major active ingredient of ginseng, show a variety of biomedical efficacies such as anti-aging, anti-oxidation and anti-inflammatory activities. To understand the effects of 20-O-,-D-glucopyranosyl-20 (S)-protopanaxadiol (compound K), one of the major metabolites of ginsenosides on the skin, we assessed the expression level of approximately 100 transcripts in compound K-treated HaCaT cells using cDNA microarray analysis. Compound K treatment induced differential expression of 40 genes, which have been reported to be involved in the organization of the structure of the extracellular matrix as well as defense responses in human skin cells. One of the most interesting findings is a two-fold increase in hyaluronan synthase2 (HAS2) gene expression by compound K. We found that change in expression of HAS2 gene represents a specific response of HaCaT cells to compound K because hyaluronan synthase 1,3 was not changed by treatment with compound K. We also demonstrated that the compound K effectively induced hyaluronan synthesis in human skin cells and hairless mouse skin. A human clinical study indicated that topical application of compound K containing oil-in-water emulsion showed improvement of xerosis, wrinkle and fine lines in the aged skin. We concluded that compound K has anti-aging effects by the induction of HAS2 gene expression and following hyaluronan synthase. [source] Upregulation of genes orchestrating keratinocyte differentiation, including the novel marker gene ID2, by contact sensitizers in human bulge-derived keratinocytesJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 1 2010Yoshie Yoshikawa Abstract 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 NiSO4) 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. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:10,20, 2010; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20307 [source] Cultured human keratinocytes for optical transmission measurementJOURNAL OF BIOPHOTONICS, Issue 3 2010David Schaaf Abstract The challenges of measuring optical properties of human tissues include the thickness of the sample, homogenization, or crystallization from freezing of the tissue. This investigation demonstrates a method to avoid these problems by growing optically thin samples of human keratinocytes as a substitute for ex vivo epidermis samples. Several methods of growth were investigated. Resulting samples were measured on a spectrophotometer for transmission between 300 nm and 2600 nm. The efficacy of the cell growth was confirmed with histological examination of several cultured keratinocyte samples. Limitations were the requirement to measure samples immediately after removal from the incubation environment, and the absence of the irregular structures of normal skin such as hair and glands. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Isolation and identification of 1,-hydroxy-3-epi-vitamin D3, a potent suppressor of parathyroid hormone secretionJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2005Alex J. Brown Abstract Since our original demonstration of the metabolism of 1,,25(OH)2D3 into 1,,25(OH)2 -3-epi-D3 in human keratinocytes, there have been several reports indicating that epimerization of the 3 hydroxyl group of vitamin D compounds is a common metabolic process. Recent studies reported the metabolism of 25OHD3 and 24(R),25(OH)2D3 into their respective C-3 epimers, indicating that the presence of 1, hydroxyl group is not necessary for the 3-epimerization of vitamin D compounds. To determine whether the presence of a 25 hydroxyl group is required for 3-epimerization of vitamin D compounds, we investigated the metabolism of 1,OHD3, a non-25 hydroxylated vitamin D compound, in rat osteosarcoma cells (ROS 17/2.8). We noted metabolism of 1,OHD3 into a less polar metabolite which was unequivocally identified as 1,OH-3-epi-D3 using the techniques of HPLC, GC/MS, and 1H-NMR analysis. We also identified 1,OH-3-epi-D3 as a circulating metabolite in rats treated with pharmacological concentrations of 1,OHD3. Thus, these results indicated that the presence of a 25 hydroxyl group is not required for 3-epimerization of vitamin D compounds. Furthermore, the results from the same studies also provided evidence to indicate that 1,OH-3-epi-D3, like 1,OHD3, is hydroxylated at C-25. We then evaluated the biological activities of 1,OH-3-epi-D3. Treatment of normal rats every other day for 7 days with 2.5 nmol/kg of 1,OH-3-epi-D3 did not raise serum calcium, while the same dose of 1,OHD3 increased serum calcium by 3.39,±,0.52 mg/dl. Interestingly, in the same rats which received 1,OH-3-epi-D3 we also noted a reduction in circulating PTH levels by 65,±,7%. This ability of 1,OH-3-epi-D3 to suppress PTH levels in normal rats without altering serum calcium was further tested in rats with reduced renal function. The results indicated that the ED50 of 1,OH-3-epi-D3 for suppression of PTH was only slightly higher than that of 1,,25(OH)2D3, but that the threshold dose of the development of hypercalcemia (total serum Ca >,10.5 mg/dl) was nearly 80 times higher. These findings indicate that 1,OH-3-epi-D3 is a highly selective vitamin D analog with tremendous potential for treatment of secondary hyperparathyroidism in chronic renal failure patients. © 2005 Wiley-Liss, Inc. [source] Heparin modulates the growth and adherence and augments the growth-inhibitory action of TNF-, on cultured human keratinocytesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2004Ilkka T. Harvima Abstract Previous works suggest the involvement of mast cells in the epithelialization of chronic wounds. Since heparin is a major mediator stored in the secretory granules of mast cells, the purpose of this work was to elucidate the function of heparin in epithelialization using in vitro culture models. For this, low- and high-calcium media in monolayer and epithelium cultures of keratinocytes were used. Also, an assay based on keratinocyte adherence onto plastic surface was used as well. Heparin (0.02,200 ,g/ml) inhibited keratinocyte growth in a non-cytotoxic and dose-dependent manner in low- and high-calcium media, Keratinocyte-SFM® and DMEM, in the absence of growth factors and serum. Also, heparin inhibited the growth of keratinocyte epithelium in the presence of 10% fetal calf serum and DMEM. Instead, in the presence of Keratinocyte-SFM and growth factors, heparin at 2 ,g/ml inhibited the growth by 18% but at higher heparin concentrations the inhibition was reversed to baseline. TNF-, is another preformed mediator in mast cell granules and it inhibited keratinocyte growth in monolayer and epithelium cultures. Interestingly, heparin at 2,20 ,g/ml augmented or even potentiated this growth-inhibitory effect of TNF-,. The association of TNF-, with heparin was shown by demonstrating that TNF-, bound tightly to heparin-Sepharose chromatographic material. However, heparin could not augment TNF-,-induced cell cycle arrest at G0/G1 phase or intercellular adhesion molecule-1 expression in keratinocytes. In the cell adherence assay, heparin at 2 ,g/ml inhibited significantly by 12,13% or 33% the adherence of keratinocytes onto the plastic surface coated with fibronectin or collagen, respectively, but this inhibition was reversed back to baseline at 20 or 200 ,g/ml heparin. Also, heparin affected the cell membrane rather than the protein coat on the plastic surface. In conclusion, heparin not only inhibits or modulates keratinocyte growth and adherence but it also binds and potentiates the growth-inhibitory function of TNF-,. © 2004 Wiley-Liss, Inc. [source] Expression of a releasable form of annexin II by human keratinocytesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2002Feridoun Karimi-Busheri Abstract Annexin II is a multifunctional calcium-dependent phospholipid binding protein whose presence in epidermis has previously been reported. However, like other members of annexin family, annexin II has been regarded as either an intracellular protein or associated with the cellular membrane. Here, we report the presence of a releasable annexin II and p11, two monomers of annexin II tetramer, in keratinocyte-conditioned medium (KCM). Proteins present in KCM were fractionated on a gel filtration column and following further evaluation, a releasable protein with apparent MW of 36 kDa was identified. Further characterization identified this protein as the p36 monomer of annexin II tetramer. The phospho-tyrosine antibody did not visualize this protein as the phosphorylated form of p36. Several experiments were conducted to examine whether this protein is soluble or associated with keratinocyte cell membranes in the conditioned medium. A centrifugation of conditioned medium was not able to bring this protein down into the pellet. Surprisingly, the results of Western analysis identified p36 and p11, two monomers of the annexin II tetramer, in conditioned medium derived from either keratinocytes cultured alone or keratinocytes co-cultured with fibroblasts. In contrast to the keratinocyte-conditioned medium in which annexin II was easily detectable, both monomers were barely detectable in conditioned medium collected from dermal fibroblasts. This finding was in contrast to the cell lysates in which p36 was detectable in both keratinocytes and fibroblasts. However, the amount of this protein was markedly higher in keratinocyte lysate relative to that of dermal fibroblasts. Conditioned medium derived from keratinocyte established from adult showed a higher level of annexin II compared to that of keratinocytes established from newborn babies. The expression of p11 seems to increase with differentiation of keratinocytes derived from either adult or newborn skin samples. When the site of annexin synthesis in human skin was examined by immunohistochemical staining, the antibody for p36 localized the annexin to the keratinocyte cell members in the basal and suprabasal keratinocytes. In conclusion, Western blot detection of both p36 and p11 in conditioned medium from skin cells revealed that human keratinocytes, but not fibroblasts, express a releasable monomer form of annexin II which is regulated by differentiation status of keratinocytes. This finding is consistent with the localization of annexin II detected by immunohistochemical staining. J. Cell. Biochem. 86: 737,747, 2002. © 2002 Wiley-Liss, Inc. [source] AKT and MAPK signaling in KGF-treated and UVB-exposed human epidermal cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Lavinia Vittoria Lotti Regulation of proliferation and differentiation in keratinocyte is a complex and dynamic process that involves activation of multiple signaling pathways triggered by different growth factors. Keratinocyte growth factor (KGF) is not only a potent mitogen, but differently from other growth factors, is a potent inducer of differentiation. The MAP kinase and AKT pathways are involved in proliferation and differentiation of many cell types including keratinocytes. We investigated here the role of KGF in modulating AKT and MAPK activity during differentiation of human keratinocytes. Our results show that the mechanisms of action of KGF are dose-dependent and that a sustained activation of the MAPK signaling cascade causes a negative regulation of AKT. We also demostrated increasing expression of KGFR substrates, such as PAK4 during keratinocyte differentiation parallel to the receptor upregulation. J. Cell. Physiol. 212:633,642, 2007. © 2007 Wiley-Liss, Inc. [source] Resistance to UV-induced apoptosis in human keratinocytes during accelerated senescence is associated with functional inactivation of p53JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2004V. Chaturvedi Compared to proliferating keratinocytes (KCs), growth-arrested KCs are relatively resistant to UV-light induced apoptosis. When KCs undergo confluency, or following exposure to anti-proliferative agents such as IFN-, plus a phorbol ester,12- O -tetradecanoylyphorbol-13-acetate (TPA), they convert from a proliferative to a nonproliferative state resembling senescence. Since p53 regulates UV-induced apoptosis of KCs, this report further characterizes p53 half-life, post-translational modifications, and transcriptional activity using cultured human KCs and living epidermal equivalents. The half-life of p53 in KCs was longer than fibroblasts (greater than approximately 3 h vs. 30 min). Exposure of proliferating KCs to UV-light induces post-translational modifications of p53 including acetylation of lysine-382 residues. By contrast, KCs undergoing irreversible growth arrest following confluency, or exposure to IFN-, plus TPA, were resistant to UV-induced apoptosis, and failed to undergo the acetylation modification of p53. Exposure of KCs to IFN-, plus TPA reduced total cellular p53 levels and reduced the transcriptional activity of p53. Addition of Trichostatin A (TSA), an inhibitor of de-acetylation, increased acetylation of lysine-382 in confluent KCs, thereby enhancing susceptibility of confluent cultures to UV-induced apoptosis. Pre-treatment of epidermal equivalents with IFN-, plus TPA also blocked UV-light induced increase in p53 levels, and reduced apoptosis. In conclusion, these studies demonstrate that growth arrested KCs may resist UV-light induced apoptosis by inactivating the pro-apoptotic function of p53. J. Cell. Physiol. 198: 100,109, 2004. © 2003 Wiley-Liss, Inc. [source] Calcium channel blockers inhibit galvanotaxis in human keratinocytesJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2002Donna R. Trollinger Directed migration of keratinocytes is essential for wound healing. The migration of human keratinocytes in vitro is strongly influenced by the presence of a physiological electric field and these cells migrate towards the negative pole of such a field (galvanotaxis). We have previously shown that the depletion of extracellular calcium blocks the directional migration of cultured human keratinocytes in an electric field (Fang et al., 1998; J Invest Dermatol 111:751,756). Here we further investigate the role of calcium influx on the directionality and migration speed of keratinocytes during electric field exposure with the use of Ca2+ channel blockers. A constant, physiological electric field strength of 100 mV/mm was imposed on the cultured cells for 1 h. To determine the role of calcium influx during galvanotaxis we tested the effects of the voltage-dependent cation channel blockers, verapamil and amiloride, as well as the inorganic Ca2+ channel blockers, Ni2+ and Gd3+ and the Ca2+ substitute, Sr2+, on the speed and directionality of keratinocyte migration during galvanotaxis. Neither amiloride (10 ,M) nor verapamil (10 ,M) had any effect on the galvanotaxis response. Therefore, calcium influx through amiloride-sensitive channels is not required for galvanotaxis, and membrane depolarization via K+ channel activity is also not required. In contrast, Sr2+ (5 mM), Ni2+ (1,5 mM), and Gd3+ (100 ,M) all significantly inhibit the directional migratory response to some degree. While Sr2+ strongly inhibits directed migration, the cells exhibit nearly normal migration speeds. These findings suggest that calcium influx through Ca2+ channels is required for directed migration of keratinocytes during galvanotaxis and that directional migration and migration speed are probably controlled by separate mechanisms. J. Cell. Physiol. 193: 1,9, 2002. © 2002 Wiley-Liss, Inc. [source] Stress kinase p38 mediates EGFR transactivation by hyperosmolar concentrations of sorbitolJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2002Hao Cheng Activation of the epidermal growth factor receptor (EGFR) has been shown to occur by ligand-dependent and ligand-independent mechanisms. Different molecular mechanisms have been found to be responsible for ligand-independent receptor transactivation. Here, we show that hyperosmolar concentrations of sorbitol activate the EGFR in human keratinocytes. Experiments using specific inhibitors of EGFR phosphorylation show that the increased amount of activated receptors is the result of a decreased rate of dephosphorylation. Furthermore, sorbitol treatment results in a strong activation of stress kinase p38. Treatment of the cells with SB203580, a known inhibitor of p38 , and , kinases, results in impairment of receptor activation, indicating that the stress kinase is involved in receptor activation modulation. This is further reinforced by experiments showing that addition of Toxin B, known to be an inhibitor of the small Rho GTPases rac1, cdc42, and Rho A/B, to the cells results in a strong induction of EGFR activation. Our results point, therefore, to a mechanism by which osmotic shock activates EGFR through the small Rho GTPases-p38 stress kinase pathway. © 2002 Wiley-Liss, Inc. [source] Analysis of the signal transduction pathway of nickel-induced matrix metalloproteinase-2 expression in the human keratinocytes in vitro: preliminary findingsJOURNAL OF CUTANEOUS PATHOLOGY, Issue 6 2007Brunella Perfetto Background:, Nickel can induce cellular and nuclear damages responsible for chronic diseases, like allergic contact dermatitis (ACD). We previously showed that matrix metalloproteinase-2 (MMP-2) gene expression was induced by nickel in nontumorigenic human keratinocytes cell line (HaCat). Objective:, To investigate the signal transduction pathways involved in gelatinolytic activity induced in HaCat under nickel stimulation. Methods:, We analyzed the involvement of protein kinase A (PKA), protein kinase C (PKC), tyrosine kinase (PTK), nuclear factor-kB (NF-kB) and activator protein-1 (AP-1) using specific inhibitors (H89, calphostin C, genistein, carpain and curcumin) by electrophoretic mobility shift assay, reverse transcription-polymerase chain reaction and gelatin zymography. Results:, Our results indicate that nickel-induced MMP-2 production was inhibited with PTK, PKC and AP-1 specific inhibitors. Moreover, both PKA and NF-kB were not involved in nickel pathway. Conclusions:, Using HaCat, we showed that curcumin and genistein can revert nickel-induced MMP-2 upregulation. Whether the use of PTK and AP-1 inhibitors has therapeutic ramifications in the management of ACD remains to be investigated. [source] Defective ,1 -integrins expression in arsenical keratosis and arsenic-treated cultured human keratinocytesJOURNAL OF CUTANEOUS PATHOLOGY, Issue 2 2006Chih-Hung Lee Background:, ,1 -integrins, which localize to the basolateral surface of basal keratinocytes, are important in the differentiation control and proliferation of the epidermis. Many cutaneous diseases with perturbed differentiation, including arsenical keratosis, show altered patterns of integrin distribution and expression. Arsenic may induce arsenical keratosis through the differentiation and apoptosis aberration by integrins. The purpose of this study is to investigate the role of integrin and arsenic in the pathogenesis of arsenical keratosis. Methods:, Twenty-five specimens obtained from 25 patients with arsenical keratosis disease were studied. Immunohistochemistry staining to ,1, ,2,1, or ,3,1 integrins was performed in arsenical keratosis and clinically normal perilesional skin. Western blotting was used to assess the expression of integrin ,1 and focal adhesion kinase (FAK) in arsenic-treated cultured keratinocytes. Results:, A decreased expression of ,1, ,2,1, or ,3,1 integrins was demonstrated in arsenical keratosis and clinical normal perilesional skin in a large proportion of arsenical keratosis cases studied. The expressions of integrin ,1 and FAK were both decreased in arsenic-treated keratinocytes. Conclusions:, Our results suggest that arsenic induces abnormal differentiation in arsenical keratosis via the effects of integrin expression in keratinocytes. [source] | |||||||||||||||||||||||||||||||