Keratinocytes Research Papers - Academia.edu (original) (raw)

Three in vitro methods for the prediction of the skin sensitization hazard have been validated. However, predicting sensitizer potency is a key requirement for risk assessment. Here, we report a database of 312 chemicals tested in the... more

Three in vitro methods for the prediction of the skin sensitization hazard have been validated. However, predicting sensitizer potency is a key requirement for risk assessment. Here, we report a database of 312 chemicals tested in the KeratinoSens™ assay and for kinetic peptide binding. These data were used in multiple regression analysis against potency in the local lymph node assay (LLNA). The dataset covers the majority of chemicals from the validation of the LLNA to predict human potency and this subset was analyzed for prediction of human sensitization potency by in vitro data. Global analysis yields a regression of in vitro data to LLNA pEC3 with an R(2) of 60% predicting LLNA EC3 with a mean error of 3.5-fold. The highest weight in the regression has the reaction rate with peptides, followed by Nrf2-induction and cytotoxicity in KeratinoSens™. The correlation of chemicals tested positive in vitro with human data has an R(2) of 49%, which is similar to the correlation between ...

IL-4 and IL-13 are related cytokines which induce both pro- and anti-inflammatory effects depending on the cell type they act upon and the nature of the receptors expressed. The type I receptor complex is composed of the IL-4Ralpha and... more

IL-4 and IL-13 are related cytokines which induce both pro- and anti-inflammatory effects depending on the cell type they act upon and the nature of the receptors expressed. The type I receptor complex is composed of the IL-4Ralpha and gammac and only binds IL-4, whereas, in the type II receptor, IL-4Ralpha dimerizes with IL-13Ralpha1 upon either IL-4 or IL-13 binding. Another ligand binding chain potentially implicated in the IL-4/IL-13 receptor has been described, the IL-13Ralpha2, but the regulation of its expression and its role in IL-4/IL-13 transduction is poorly understood. In this study we report that IL-4 and IL-13 upregulate IL-13Ralpha2 at both the mRNA and protein levels in the keratinocyte cell line HaCaT. In these cells, IL-4 or IL-13 were shown to activate the Janus Kinases JAK1 and JAK2, the transcription factor STAT6, and the ERK and p38 mitogen-activated protein kinases. We show that IL-4 or IL-13-induced IL-13Ralpha2 mRNA expression was inhibited by the ERK inhibitor U0126, the JAK inhibitor AG490 and, to a lesser extent, the p38 MAPK inhibitor SB203580. Moreover, expression of a constitutive active mutant of STAT6 alone did not modify IL-13Ralpha2 mRNA expression, but potentiated the effects of IL-4 or IL-13 on IL-13Ralpha2 expression. The constitutive active mutants of MEK1 or MKK6 increased the level of expression of IL-13Ralpha2 mRNA even in absence of stimulation. Our findings demonstrate, for the first time, that IL-4 and IL-13 can induce IL-13Ralpha2 expression in keratinocytes, and that the ERK and p38 MAPK together with JAK2 and STAT6 play a critical role in this process.

Skin penetrating peptides (SPPs) have garnered wide attention in recent years and emerged as a simple and effective noninvasive strategy for macromolecule delivery into the skin. Although SPPs have demonstrated their potential in... more

Skin penetrating peptides (SPPs) have garnered wide attention in recent years and emerged as a simple and effective noninvasive strategy for macromolecule delivery into the skin. Although SPPs have demonstrated their potential in enhancing skin delivery, they are still evolving as a new class of skin penetration enhancers. Detailed studies elucidating their mechanisms of action are still lacking. Using five SPPs (SPACE peptide, TD-1, polyarginine, a dermis-localizing peptide and a skin penetrating linear peptide) and a model hydrophobic macromolecule (Cyclosporine A, CsA), herein we provide a mechanistic understanding of SPPs. To evaluate the mechanism and safety of SPPs, their effects on skin lipids, proteins and keratinocyte cells were evaluated. Three SPPs (SPACE, Polyarginine and TD-1) significantly enhanced CsA penetration into the skin. SPPs did not alter the skin lipid barrier as measured by skin resistance, transepidermal water loss (TEWL) and Fourier transform infrared (FTI...

Cigarette smoke (CS) is a significant environmental source of human exposure to chemically active saturated (acetaldehyde) and α,β-unsaturated aldehydes (acrolein) inducing protein carbonylation and dysfunction. The exposure of oral... more

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

The detection of DNA-incorporated bromodeoxyuridine (BrdUrd) in mammalian cells is a well-known and important technique to study cell cycle. The use of TO-PRO-3 for detection of BrdUrd substitution of DNA by dual-laser flow cytometry has... more

The detection of DNA-incorporated bromodeoxyuridine (BrdUrd) in mammalian cells is a well-known and important technique to study cell cycle. The use of TO-PRO-3 for detection of BrdUrd substitution of DNA by dual-laser flow cytometry has been investigated. Fluorescence enhancement of TO-PRO-3 in BrdUrd-labeled cells is registered in combination with the fluorescence emission of the intercalating dye propidium iodide (PI) as a total DNA stain to give bivariate DNA/BrdUrd histograms. By the low concentration of only 0.3 mircoM TO-PRO-3, BrdUrd detection is optimized, and undisturbed total DNA content by PI can be detected as well. TO-PRO-3 is excited by a red HeNe laser and PI by an argon ion laser. In order to understand the binding of TO-PRO-3, energy transfer from PI to TO-PRO-3 has been measured as well as the influence of an external DNA binding dye such as Hoechst 33258 with Adenine-Thymine (AT) binding specificity. Cell cycle studies of human SCL-2 keratinocytes and mouse 3T3 c...

Honey has been used since ancient times for wound repair, but the subjacent mechanisms are almost unknown. We have tried to elucidate the modulatory role of honey in an in vitro model of HaCaT keratinocyte re-epithelialization by using... more

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

The expression of proliferating cell nuclear antigen (PCNA) was studied in human papillomavirus (HPV)-infected, benign and malignant lesions of the genital tract and larynx using immunocytochemical staining of formalin-fixed clinical... more

The expression of proliferating cell nuclear antigen (PCNA) was studied in human papillomavirus (HPV)-infected, benign and malignant lesions of the genital tract and larynx using immunocytochemical staining of formalin-fixed clinical specimens. We observed the induction of PCNA in squamous carcinomas and adenocarcinomas, as has been demonstrated with other malignancies. In addition, the differentiated keratinocytes of the upper spinous cells and granulocytes in condylomata acuminata and low-grade intraepithelial neoplasias showed a consistent induction of PCNA compared with the normal squamous epithelium, in which only some of the parabasal and basal cells were positive. This reactivation of PCNA synthesis correlated with the presence of high copy numbers of HPV DNA and was independent of the oncogenic risk potential of the infecting HPV genotype. We postulate that HPV gene products induce the expression of PCNA and other components of the host DNA replication machinery in different...

Cell-matrix interactions are an essential element of wound healing, while platelet derivatives are used in clinical settings for the treatment of chronic wounds. We used a platelet lysate (PL), which had been previously shown to... more

Cell-matrix interactions are an essential element of wound healing, while platelet derivatives are used in clinical settings for the treatment of chronic wounds. We used a platelet lysate (PL), which had been previously shown to accelerate in vitro the wounding of HaCaT keratinocytes and fibroblasts (J Cell Mol Med, 13, 2009, 2030; Br J Dermatol, 159, 2008, 537), to study the modulation of MMP-2 and MMP-9 collagenase expression, collagen type I and III production and syndecan-4 expression and rearrangement in these cells. Zymography and Western blot analyses showed that exposure to 20% (v/v) PL for 24 h induced an apparently ERK1/2- and p38-dependent, NF-kappaB-independent, translational upregulation of MMP-9 in HaCaT, while HaCaT MMP-2 and fibroblast collagenases were almost unaffected. The use of in-cell ELISA showed that PL induced an increase in the collagen III production of fibroblasts. In-cell ELISA and immunofluorescence microscopy revealed an increase in the expression of syndecan-4 and its rearrangement to form focal adhesions in both cell types after PL exposure. Taken together, data indicate that PL promotes keratinocyte epithelialization and regulates fibroblast matrix deposition, thus providing a molecular basis for the ability of this platelet derivative to heal severe and problematic wounds without leading to heavy scarring and keloid formation.