Woo In Ryu

Effect of the size and surface charge of silica nanoparticles on cutaneous toxicity

Silica nanoparticles (NPs) are widely applied in many fields, such as chemical industry, medicine, cosmetics, and agriculture. However, the hazardous effects of silica NPs exposure are not completely understood. In this study, the two different sizes (20 nm and 100 nm) and different charges (negatively charged [NC] and weakly negatively charged [WNC]) of silica NPs were used. The present study investigated the cytotoxicity and reactive oxygen species (ROS) generation of silica NPs on keratinocytes. The phototoxicity test of silica NPs was performed on skin fibroblast cells. In addition, skin irritation and skin sensitization of silica NPs were studied on HSEM and mouse skin, respectively. The cell viability of NC 20 nm silica NPs was decreased. However, there are no cytotoxicity for NC 100 nm silica NPs and WNC silica NPs (20 and 100 nm). The results for silica NPs-induced ROS generation are consistent with the cytotoxicity test by silica NPs. Further, NC and WNC silica NPs induced no phototoxicity, acute cutaneous irritation, or skin sensitization. These results suggested that silica NPs-induced ROS generation was the determinant of cytotoxicity. This study showed that the smaller size (20 nm) of silica NPs had more toxicity than the larger size (100 nm) of silica NPs for NC silica NPs. Moreover, we observed an effect of surface charge in cytotoxicity and ROS generation, by showing that the NC silica NPs (20 nm) had more toxic than the WNC silica NPs (20 nm). These findings suggested that the surface charge of silica NPs might be the important parameter for silica NPs-induced toxicity. Further study is needed to assess the effect of surface modification of nanotoxicity.

ZnO nanoparticles induce TNF-α expression via ROS-ERK-Egr-1 pathway in human keratinocytes

BACKGROUND The area of nanotechnology continues to expand rapidly and zinc oxide (ZnO) nanoparticles (NPs) are widely being used in cosmetics and sunscreens. Although ZnO-NPs are considered materials that can potentially cause skin inflammation, the underlying mechanisms remain elusive. OBJECTIVE The aim of this study was to investigate the signaling pathways of a cutaneous inflammatory response induced by ZnO-NPs. ZnO-NPs increased the early growth response-1 (Egr-1) expression, promoter activity and its nuclear translocation in HaCaT cells. METHODS HaCaT cells and primary keratinocytes were exposed to ZnO NPs over a range of doses and time course. Protein levels and mRNA levels of Egr-1 and mitogen-activated protein kinase (MAPK) were measured by Western blot and ELISA, respectively. As an in vivo study, ZnO-NPs were applicated on mouse skin, and immunohistochemical stain with TNF-α and Egr-1 was done. RESULTS ZnO-NPs activated extracellular signal-regulated kinase (ERK) of MAPK pathways. The up-regulation of Egr-1 expression by ZnO-NPs stimulation was found to be inhibited by an ERK inhibitor, but by neither c-Jun-N-terminal kinase (JNK) nor p38 inhibitor. Antioxidative N-acetyl-cysteine (NAC) strongly inhibited the level of Egr-1 and phosphorylated ERK expression in ZnO-NPs treated cells. ZnO NPs also increased tumor necrosis factor (TNF)-α expression and secretion, which were inhibited by the blockade of Egr-1 expression. CONCLUSIONS The present study demonstrated that ZnO-NPs might induce inflammatory response via ROS-ERK-Egr-1 pathway in human keratinocytes.

Thymic stromal lymphopoietin downregulates filaggrin expression by signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) phosphorylation in keratinocytes

IL-33–activated mast cells and ILC2s, and may be involved in severe asthma via the recruitment of neutrophils and smooth muscle activation. Furthermore, the presence of other immune cells in the airway in an IL-33 milieu may result in an abundance of non–type 2 mediators, which could affect differential gene expression not otherwise seen in the coculture system. Thus, further studies need to be conducted in vivo to examine the effects of anti–IL-13 on airway inflammation, mucus expression, and hyperreactivity in response to IL-33. In conclusion, we have shown that IL-33–activated mast cells and ILC2s drive predominantly IL-13–regulated gene expression when cocultured with NHBE cells, which initiates a transcriptional program that can subsequently exacerbate airway pathology and contribute to fibrosis, eosinophilia, and mucous metaplasia. Although mast cells and ILC2s produce a diverse set of cytokines and other mediators of intercellular communication in response to IL-33, the predominant effect on airway epithelium in this in vitro coculture system is mediated by IL-13.

UVB Induces HIF-1α-Dependent TSLP Expression via the JNK and ERK Pathways

Thymic stromal lymphopoietin (TSLP) may have a key role in the initiation and maintenance of allergic inflammatory diseases, including atopic dermatitis. The present study revealed that UVB radiation exposure could induce TSLP expression in human keratinocytes and a human skin equivalent model. In addition, we investigated the regulatory mechanism of UVB-induced TSLP expression in keratinocytes. TSLP expression was upregulated by transfection with pcDNA3-hypoxia-inducible factor (HIF)-1α (P402A and P564A), which stably expresses HIF-1α protein. UVB-induced TSLP induction in keratinocytes was suppressed in the treatment of mitogen-activated protein kinase inhibitors or small interfering RNAs against HIF-1α. The results of chromatin immunoprecipitation assays indicate the direct involvement of HIF-1α in UVB-mediated TSLP induction. Taken together, these findings indicate that UVB exposure may increase TSLP expression through a HIF-1α-dependent mechanism via the c-JUN N-terminal kinase and extracellular signal-regulated kinase pathways in human keratinocytes. Our data showed that UVB-induced TSLP might increase secretion of the T-helper type 2-attracting chemokine (c-c motif) ligand 17 by human dendritic cells. The present study suggests an important role of HIF-1α in UVB-mediated immune response in keratinocytes.

IL‐33 induces Egr‐1‐dependent TSLP expression via the MAPK pathways in human keratinocytes

Atopic dermatitis (AD) is a chronic inflammatory skin disease in which T‐helper type 2 (Th2)‐type immune responses are dominant. Th2 cytokine, interleukin (IL)‐33 and thymic stromal lymphopoietin (TSLP) have been suggested to have an important role in AD. IL‐33 is highly expressed in AD, but its role in AD has not yet been fully understood. To further identify the role of IL‐33 in AD, we investigated the expression of TSLP induced by IL‐33 in keratinocytes. This study revealed that IL‐33 induced TSLP expression in human keratinocytes. Early growth response protein 1 (Egr)‐1, which is an inflammatory transcriptional factor, is induced by IL‐33. IL‐33‐mediated TSLP induction in keratinocytes was suppressed by treatment with mitogen‐activated protein kinase (MAPK) inhibitors or small interfering RNA against Egr‐1. Chromatin immunoprecipitation (ChIP) assay indicated the direct involvement of Egr‐1 in IL‐33‐mediated TSLP induction. Taken together, these findings indicate that IL‐33 may increase TSLP expression through an Egr‐1‐dependent mechanism via ERK1/2, JNK and p38 activation in keratinocytes. These data suggest that the IL‐33‐ERK/JNK/p38/Egr‐1/TSLP axis is involved in allergic skin Th2 inflammation, and it may be a novel therapeutic target.

TSLP Down-Regulates S100A7 and ß-Defensin 2 Via the JAK2/STAT3-Dependent Mechanism

Elevated T-helper type 2 cytokines in atopic skin, such as IL-4 and IL-13, were thought to be responsible for an impaired expression of antimicrobial proteins, which may contribute to the increased susceptibility to skin infections in patients with atopic dermatitis. In this study, the relationship between thymic stromal lymphopoietin and antimicrobial proteins and the involved molecular pathway was defined in normal human epidermal keratinocytes and human skin equivalent model. Stimulation of normal human epidermal keratinocytes with thymic stromal lymphopoietin decreased both mRNA and levels of S100A7 and human β-defensin 2 in a dose-dependent manner, and the regulation was JAK2/STAT3-dependent. Thymic stromal lymphopoietin decreased the antimicrobial protein expression, even in the presence of IL-17, which is their strong inducer. STAT3 directly regulated the S100A7 and human β-defensin 2 promoters in normal human epidermal keratinocytes. Immunohistochemically, lesional atopic skin stained more intensely with phospho-STAT3 compared with healthy control. Our results show that up-regulated thymic stromal lymphopoietin may contribute to the deficiency of antimicrobial proteins in atopic dermatitis, including S100A7 and human β-defensin 2, by a JAK2/STAT3-dependent mechanism and that STAT3/Sin3a might directly control the transcriptional activity of the antimicrobial protein promoters in normal human epidermal keratinocytes. Taken together, a key role of the JAK2/STAT3/Sin3a signaling pathway in thymic stromal lymphopoietin-mediated immune response in normal human epidermal keratinocytes might give us clues to understanding the pathological signal transductions in atopic dermatitis.

Egr-1 is a key regulator of IL-17A-induced psoriasin upregulation in psoriasis

The early growth response (Egr)‐1 is a transcriptional factor which plays an important role in the regulation of cell growth, differentiation, cell survival and immune responses. Emerging evidences including our data demonstrate that the Egr‐1 expression is up‐regulated in the psoriatic skin lesions. The purpose of this study was to investigate the significance and regulatory mechanism of Egr‐1 in the pathogenesis of psoriasis. Through microarray analysis, we found out that psoriasin (S100A7) expression was increased in the Egr‐1 overexpressed cells. Our results showed that IL‐17A increased Egr‐1 expression in the skin of psoriatic patients and cultured human keratinocytes. We then investigated activation of mitogen‐activated protein kinase as an upstream signal regulator of Egr‐1 expression. IL‐17A‐induced Egr‐1 expression was suppressed by ERK inhibitor. In addition, IL‐17A induced psoriasin expression in cultured keratinocytes and the skin of IL‐17A intradermally injected mouse. IL‐17A‐mediated psoriasin upregulation was reduced after treatment of small interfering RNAs against Egr‐1. Furthermore, the results of chromatin immunoprecipitation assays demonstrated that Egr‐1 directly binds the psoriasin promoter. Our findings present a novel signalling mechanism by which IL‐17A can induce the Egr‐1‐dependent psoriasin expression via the ERK pathway in human keratinocytes. This study suggests that Egr‐1 may be a novel and important modulator in IL‐17A‐mediated immune response in psoriasis.

Toluene induces early growth response-1 dependent thymic stromal lymphopoietin expression in human keratinocytes

Volatile organic compounds (VOCs) can be an important environmental risk factor in the development and/or aggravation of atopic dermatitis (AD). In a several studies to evaluate the clinical effects of air pollution including VOCs on skin symptoms in AD patients. Although these accumulated data shows an association between exposure to VOCs and aggravation of AD, the molecular effects of VOCs on the aggravation of AD is still not clear. The present study demonstrates increases of thymic stromal lymphopoietin (TSLP) in keratinocytes following exposure to toluene. We further investigated the regulatory mechanisms of toluene-induced TSLP expression in human keratinocytes. Toluene induces early growth response-1 (Egr-1) protein expression in human keratinocytes. Moreover, treatment of Egr-1 siRNA abolished toluene-induced TSLP protein expression. Taken together, these findings suggest that in human keratinocytes, the up-regulation of TSLP by toluene is induced through an Egr-1 dependent mechanism.

Chloroform induces cystein-rich 61, a mediator of collagen homeostasis via early growth response-1 dependent pathway in human skin dermal fibroblasts

Volatile organic compounds (VOCs) are major air pollutants known to have adverse effects on skin. Many reports have shown an association between air pollutants and skin aging. However, there is no report on VOCs association with skin aging. The aim of this study is to identify the biologic effects of VOCs on fibroblasts. We investigated the effects of chloroform on cysteine-rich 61 (CYR61) expression in human skin fibroblasts using western blot and reverse transcriptionpolymerase chain reaction. We also observed the change of type I procollagen and matrix metalloproteinase after exposure to chloroform using western blot. We explored the involvement of early growth response-1 (Egr-1) using siRNA s in the fibroblasts. Chloroform exposure increased the expression of CYR61 protein and mRNA level. Elevated CYR61 downregulates type I procollagen and upregulates MMP-1. Egr-1 overexpression upregulates CYR61 promoter activity. CYR61 promoter activity was lower in Egr-1 siRNA-transfected fibroblasts. The present study demonstrates that CYR61 is transcriptionally regulated by chloroform exposure through transcription factor Egr-1, and alters collagen homeostasis in human skin fibroblasts.

Intracellular ROS levels determine the apoptotic potential of keratinocyte by Quantum Dot via blockade of AKT Phosphorylation

Quantum dots (QDs) have shown great potential for biomedical use in a broad range including diagnostic agents. However, the regulatory mechanism of dermal toxicity is poorly understood. In this study, we investigated how QDs‐induced apoptosis is regulated in human keratinocytes. We also examined the effect of carboxylic acid‐coated QDs (QD 565 and QD 655) on reactive oxygen species (ROS) production and apoptosis‐related cellular signalling. The viability of keratinocyte was inhibited by two types of QDs in a concentration‐dependent manner. QDs induce ROS production and blockade of AKT phosphorylation. Moreover, the cleavage of AKT‐dependent pro‐apoptotic proteins such as poly (ADP‐ribose) polymerase, caspases‐3 and caspases‐9 was significantly increased. We also found that a decrease in cellular ROS level by ROS scavenger, N‐acetylcysteine (NAC), resulting in the abolishment of QDs‐induced AKT de‐phosphorylation and cellular apoptosis. Interestingly, QD 655 had a more cytotoxic effect including oxidative stress and AKT‐dependent apoptosis than QD 565. In addition, QD 655 had the cytotoxic potential in the human skin equivalent model (HSEM). These data show that QD‐induced intracellular ROS levels may be an important parameter in QD‐induced apoptosis. These findings from this study indicate that intracellular ROS levels might determine the apoptotic potential of keratinocyte by QD via blockade of AKT phosphorylation.