Z‐ligustilide ameliorated ultraviolet B‐induced oxidative stress and inflammatory cytokine production in human keratinocytes through upregulation of Nrf2/HO‐1 and suppression of NF‐κB pathway

Ultraviolet B (UVB), a harmful environmental factor, is responsible for a variety of skin disorders including skin inflammation through reactive oxygen species (ROS) and inflammatory mediator production. Here, we investigated the effect of Z‐ligustilide (Z‐lig), an active ingredient isolated from the medicinal plants Cnidium officinale and Angelica acutiloba, on UVB‐induced ROS generation and inflammatory mediator production in normal human epidermal keratinocytes (NHEKs) as well as its underlying mechanisms. Z‐lig significantly rescued UVB‐induced NHEKs damage in a dosage‐dependent manner. Pretreatment of NHEKs with Z‐lig inhibited UVB‐induced ROS production in NHEKs. Both silencing the nuclear factor E2‐related factor 2 (Nrf2) and the supplement of tin protoporphyrin IX (SnPP), a haeme oxygenase‐1 (HO‐1) inhibitor, cancelled the inhibitory effect of Z‐lig on UVB‐induced ROS upregulation in NHEKs. Moreover, pretreatment of NHEKs with Z‐lig reduced UVB‐induced nuclear factor kappa B (NF‐κB)‐dependent inflammatory mediators (IL‐6, IL‐8 and MCP‐1) production at both mRNA and protein level. In the presence of Z‐lig, UVB‐induced NF‐κB subunit p65 nuclear translocation was abolished, and the IκBα degradation was suppressed. Taken together, these findings suggest that Z‐lig can suppress UVB‐induced ROS generation through Nrf2/HO‐1 upregulation and inflammation by suppressing the NF‐κB pathway, suggesting that Z‐lig may be beneficial in protecting skin from UVB exposure.     

Identification of ketoconazole as an AhR-Nrf2 activator in cultured human keratinocytes: the basis of its anti-inflammatory effect

Ketoconazole (KCZ) has been shown to exhibit anti-inflammatory effects in addition to its inhibitory effects against fungi; however, the underlying molecular mechanism remains poorly understood. Aryl hydrocarbon receptor (AhR), a receptor that is activated by polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons such as dioxin, is a sensor of the redox system against oxidative stress and regulates nuclear factor-erythroid 2-related factor-2 (Nrf2), a master switch of the redox machinery. To clarify whether KCZ modulates AhR-Nrf2 function leading to redox system activation, cultured human keratinocytes were treated with KCZ. Confocal microscopic analysis revealed that KCZ induced AhR nuclear translocation, resulting in the upregulation of CYP1A1 mRNA and protein expression. Furthermore, KCZ actively switched on Nrf2 nuclear translocation and quinone oxidoreductase 1 expression. Tumor necrosis factor-α- and benzo(a)pyrene (BaP)-induced reactive oxidative species (ROS) and IL-8 production were effectively inhibited by KCZ. Knockdown of either AhR or Nrf2 abolished the inhibitory capacity of KCZ on ROS and IL-8 production. In addition, KCZ-induced Nrf2 activation was canceled by AhR knockdown. Moreover, KCZ inhibited BaP-induced 8-hydroxydeoxyguanosine and IL-8 production. In conclusion, the engagement of AhR by KCZ exhibits the cytoprotective effect mediated by the Nrf2 redox system, which potently downregulates either cytokine-induced (AhR-independent) or PAH-induced (AhR-dependent) oxidative stress.     

An environmental contaminant, benzo(a)pyrene, induces oxidative stress-mediated interleukin-8 production in human keratinocytes via the aryl hydrocarbon receptor signaling pathway

Background

Benzo(a)pyrene (BaP) is an environmental contaminant found in cigarette smoke. It is well known that cigarette smoking exacerbates interleukin-8 (IL-8)-related inflammatory skin diseases such as psoriasis, palmoplantar pustulosis and acne. Although BaP has been shown to exert its biological effects via the aryl hydrocarbon receptor (AhR) signaling pathway, the mechanism of its inflammatory effects on skin remains unanswered.

Objective

To elucidate whether or not BaP cause AhR activation and subsequent oxidative stress leading to IL-8 production in normal human epidermal keratinocytes (NHEKs).

Methods

NHEKs exposed to BaP were analyzed. Immunofluorescence, real-time PCR, Western blotting, ELISA, reactive oxygen species (ROS) detection using H2DCFDA and RNA interference using si (small interfering) RNA were employed.

Results

Immunofluorescence analysis clearly demonstrated that BaP induced nuclear translocation of AhR from cytoplasm. The AhR activation subsequently induced CYP1A1 mRNA and protein expression in a dose-dependent manner. In addition, ROS and IL-8 production were coordinately augmented by BaP, whereas this was not the case in IL-1α, IL-6, TNF-α or GM-CSF production. Knockdown of AhR expression using siRNA transfection inhibited BaP-induced-ROS and IL-8 production, suggesting that these responses are strongly dependent on the AhR signaling pathway. Furthermore, the addition of N-acetyl cystein or catalase cancelled the IL-8 production by BaP, indicating that ROS production is essential for IL-8 production.

Results

This data highlights AhR-ROS-dependent regulation of IL-8 in NHEKs by BaP, providing a plausible explanation, at least in part, for why cigarette smoking exacerbates IL-8-related skin diseases such as psoriasis, palmoplantar pustulosis and acne.     

Antioxidant soybean tar Glyteer rescues T‐helper‐mediated downregulation of filaggrin expression via aryl hydrocarbon receptor

Soybean tar Glyteer (Gly) has been widely used for the treatment of various inflammatory skin diseases in Japan since 1924 as an alternative to coal tar remedy. Recently, coal tar has been shown to induce barrier repair in atopic dermatitis via aryl hydrocarbon receptor (AhR). In this study, we demonstrated that Gly activated AhR by inducing its cytoplasmic to nuclear translocation in keratinocytes. The AhR ligation by Gly was biologically active, with significant and dose‐dependent upregulation of CYP1A1 expression, which is a specific marker for AhR activation. Gly upregulated the expression of filaggrin in an AhR‐dependent manner because its enhancing effect was completely abrogated in AhR‐knockdown keratinocytes. T‐helper (Th)2 cytokines inhibited the expression of filaggrin; however, Gly completely restored the Th2‐mediated inhibition of filaggrin expression. Furthermore, Gly coordinately upregulated a series of epidermal differentiation complex genes, including involucrin, loricrin and hornerin. In addition, Gly exhibited potent antioxidant activity through the activation of nuclear factor‐erythroid 2‐related factor‐2 (Nrf2) and downstream antioxidant enzymes such as NAD(P)H:quinone oxidoreductase 1 (Nqo1), which actually inhibited the generation of reactive oxygen species in keratinocytes treated with tumor necrosis factor‐α or benzo[α]pyrene. In conclusion, antioxidant Gly rescues the downregulated expression of filaggrin (and plausibly other barrier proteins) in a Th2‐skewed milieu via AhR activation, which may partly explain its empirical anti‐inflammatory therapeutic effects.     

Induction of a chloracne phenotype in an epidermal equivalent model by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent on aryl hydrocarbon receptor activation and is not reproduced by aryl hydrocarbon receptor knock down

Background2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent activator of the aryl hydrocarbon receptor (AhR) and causes chloracne in humans. The pathogenesis and role of AhR in chloracne remains incompletely understood.ObjectiveTo elucidate the mechanisms contributing to the development of the chloracne-like phenotype in a human epidermal equivalent model and identify potential biomarkers.MethodsUsing primary normal human epidermal keratinocytes (NHEK), we studied AhR activation by XRE-luciferase, AhR degradation and CYP1A1 induction. We treated epidermal equivalents with high affinity TCDD or two non-chloracnegens: β-naphthoflavone (β-NF) and 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Using Western blotting and immunochemistry for filaggrin (FLG), involucrin (INV) and transglutaminase-1 (TGM-1), we compared the effects of the ligands on keratinocyte differentiation and development of the chloracne-like phenotype by H&E.ResultsIn NHEKs, activation of an XRE-luciferase and CYP1A1 protein induction correlated with ligand binding affinity: TCDD > β-NF > ITE. AhR degradation was induced by all ligands. In epidermal equivalents, TCDD induced a chloracne-like phenotype, whereas β-NF or ITE did not. All three ligands induced involucrin and TGM-1 protein expression in epidermal equivalents whereas FLG protein expression decreased following treatment with TCDD and β-NF. Inhibition of AhR by α-NF blocked TCDD-induced AhR activation in NHEKs and blocked phenotypic changes in epidermal equivalents; however, AhR knock down did not reproduce the phenotype.ConclusionLigand-induced CYP1A1 and AhR degradation did not correlate with their chloracnegenic potential, indicating that neither CYP1A1 nor AhR are suitable biomarkers. Mechanistic studies showed that the TCDD-induced chloracne-like phenotype depends on AhR activation whereas AhR knock down did not appear sufficient to induce the phenotype.     

Malassezia‐derived aryl hydrocarbon receptor ligands enhance the CCL20/Th17/soluble CD163 pathogenic axis in extra‐mammary Paget's disease

Malassezia yeast play a role in the pathogenesis of chronic dermatitis, especially in apocrine areas, by polarizing the local immunologic background to a Th2/Th17 state through aryl hydrocarbon receptor (AhR)‐dependent pathways. Extra‐mammary Paget's disease (EMPD) is an adenocarcinoma of apocrine origin, and except for cases associated with Malassezia yeast and their metabolites, the lesions typically develop in areas not exposed to environmental material. The purpose of this study was to investigate (a) the immunomodulatory effects of Malassezia metabolites on normal human keratinocytes (NHKCs), focusing on interleukin (IL)‐17 and related cytokines/chemokines (IL‐23, IL‐36γ, CCL20), (b) the expression of these factors in lesion‐affected skin in EMPD and (c) the activation of tumor‐associated macrophages (TAMs) by these factors. Malassezia metabolites augmented the expression of cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), CCL20 and IL‐36γ mRNA in NHKCs in vitro. In lesion‐affected skin of patients with EMPD, epidermal keratinocytes expressed CYP1A1 and CCL20. In addition, Paget cells expressed CCL20 and IL‐23. IL‐17–producing cells were distributed adjacent to Paget cells. Compared to healthy donors, patients with EMPD exhibited significantly increased serum levels of soluble (s)CD163, CXCL5, CXCL10 and CCL20. In addition, serum levels of sCD163 decreased significantly following tumor resection. Our study demonstrates a possible mechanism for the development of EMPD involving AhR‐mediated signalling by epidermal keratinocytes and RANKL‐induced recruitment of Th17 cells and TAMs.     

Melanocyte‐protective effect of afzelin is mediated by the Nrf2‐ARE signalling pathway via GSK‐3β inactivation

Vitiligo is an acquired condition characterized by depigmented, cutaneous lesions that result from the death of pigment‐producing cells, melanocytes. The occurrence of oxidative stress has been proposed as a pathogenetic mechanism for melanocyte degeneration in vitiligo. Therefore, in this study, we investigated the cytoprotective effects of afzelin against oxidative stress and its mechanism of action in human epidermal melanocytes. We found that afzelin significantly inhibited hydrogen peroxide‐induced cell death, cellular reactive oxygen species production and lipid peroxidation in melanocytes. In an antioxidant response element (ARE)‐luciferase reporter assay, afzelin increased ARE‐luciferase reporter activity in a concentration‐dependent manner. Consistently, the expression of antioxidant genes, including NF‐E2‐related factor 2 (Nrf2), haem oxygenase‐1 (HO‐1) and catalase, was enhanced by afzelin treatment. Nuclear translocation of Nrf2 also increased in response to afzelin treatment. In addition, the phosphorylation of glycogen synthase kinase‐3β (GSK‐3β) was induced by afzelin treatment. The enhancement of HO‐1 gene expression by afzelin treatment was reduced by Nrf2‐siRNA expression. Furthermore, we found that the expression of Nrf2‐siRNA significantly attenuated the cytoprotective effect of afzelin against hydrogen peroxide. These data suggest that the cytoprotective effects of afzelin against hydrogen peroxide may be mediated by Nrf2‐ARE signalling via GSK‐3β inactivation. Our data suggest the novel use of afzelin for the prevention of oxidative stress‐induced damage in melanocytes and its potential as a therapeutic agent for vitiligo.     

Baicalein inhibits matrix metalloproteinase 1 expression via activation of TRPV1‐Ca‐ERK pathway in ultraviolet B–irradiated human dermal fibroblasts

Increased matrix metalloproteinase 1 (MMP‐1) expression is a feature of photo‐aged skin. We investigated the effects of baicalein and sulphoraphane on ultraviolet B (UVB) irradiation–induced MMP‐1 expression and apoptosis using human dermal fibroblasts. UVB irradiation not only increased MMP‐1 expression, but also caused apoptosis. Both baicalein and sulphoraphane protected cells from UVB irradiation–induced apoptosis, but only baicalein inhibited MMP‐1 expression. UVB irradiation activated 12‐lipoxygenase, and its product, 12‐hydroxyeicosatetraenoic acid, activated TRPV1 channels. The resulting UVB irradiation–induced Ca²⁺ increase was blocked by the 12‐lipoxygenase inhibitor baicalein and the TRPV1 blocker capsazepine, but not by the Nrf2 inducer sulphoraphane. UVB irradiation also increased ROS generation and decreased Nrf2 protein levels. UVB irradiation–induced MMP‐1 expression was blocked by the Ca²⁺ chelator BAPTA, by capsazepine and by TRPV1 silencing. However, induction was unaffected by the antioxidant N‐acetylcysteine. ERK phosphorylation and JNK phosphorylation were induced by UVB irradiation, but only ERK phosphorylation was Ca²⁺ sensitive. Increased MMP‐1 expression was blocked by PD98059, but not by SP600125. Thus, increased MMP‐1 expression is mediated by increased cytosolic Ca²⁺ and ERK phosphorylation. UVB irradiation–induced ROS generation is also Ca²⁺ sensitive, and UVB irradiation–induced apoptosis is caused by increased ROS. Thus, baicalein, by blocking the UVB irradiation–induced cytosolic Ca²⁺ increase, protects cells from UVB irradiation–induced MMP‐1 expression and apoptosis. In contrast, sulphoraphane, by decreasing cellular ROS, protects cells from only UVB‐induced apoptosis. Thus, targeting 12‐lipoxygenase may provide a therapeutic approach to improving the health of photo‐aged human skin.     

Differential response of normal human epidermal keratinocytes and HaCaT cells to hydrogen peroxide-induced oxidative stress

Background. Normal human epidermal keratinocytes (NHEKs) and HaCaT cells are the most common models used to study the effects of various factors on skin cells. These cell lines share some common characteristics, but little is known about their differences in handling hydrogen peroxide (H₂O₂)‐induced oxidative stress. Aim. To investigate the differential response of NHEKs and HaCaT cells to H₂O₂‐induced oxidative stress. Methods. We examined differences in NHEKs and HaCaT cells after H₂O₂ treatment, assessing changes in cell viability; levels of intracellular reactive oxygen species (ROS), superoxide dismutase (SOD) and caspase‐3/7; percentage of cells arrested in G1 phase; number of senescence‐associated β‐galactosidase (SA‐β‐Gal)‐positive cells and; expression of senescence‐related protein Klotho. Results. The viability of NHEKs and HaCaT cells decreased in a concentration‐dependent and time‐dependent manner after exposure to H₂O₂. The inhibitory effect of 150 μmol/L H₂O₂ on cell viability was greater in HaCaT cells than in NHEKs (P < 0.05). Levels of ROS and caspase‐3/7, and the percentage of cells arrested in G1 phase, were higher in HaCaT cells than in NHEKs, whereas intracellular SOD was higher in NHEKs than in HaCaT cells after exposure to 150 μmol/L H₂O₂ (P < 0.05). SA‐β‐Gal positive cells increased significantly in NHEKs after treatment with H₂O₂ (P < 0.05). Klotho was significantly downregulated in both NHEKs and HaCaT cells after H₂O₂ treatment, but no SA‐β‐Gal‐positive HaCaT cells were seen, even after treatment with H₂O₂. Conclusions. Normal human epidermal keratinocytes are more resistant than HaCaT cells to H₂O₂‐induced oxidative stress. HaCaT cells have senescence phenotypes, but do not express β‐Gal.     

Differentiation-dependent expression of NADP(H):quinone oxidoreductase-1 via NF-E2 related factor-2 activation in human epidermal keratinocytes

Background

NADP(H):quinone oxidoreductase-1 (NQO-1) is known for its protective role in skin carcinogenesis, but the expression of NQO-1 during keratinocyte (KC) differentiation has not been studied.

Objective

The purpose of the current study was to evaluate modulation of NQO-1 and NF-E2-related factor-2 (Nrf2) during KC differentiation.

Methods

Normal human epidermal keratinocytes (NHEKs) were induced to differentiation by prolonged culture after confluency (postconfluence).

Results

NQO-1 was induced at the late stage of differentiation of NHEKs (7th day of postconfluence). The expression of postconfluence-induced NQO-1 was stimulated by 0.1 mM H₂O₂, but attenuated by 5 mM N-acetylcysteine, implying that reactive oxygen species (ROS) are implicated in the expression of NQO-1 in differentiated KCs. Nrf2 was up-regulated at the earlier than NQO-1 induction (3rd day of postconfluence). The Nrf2-dependent expression of NQO-1 was further supported by Nrf2-siRNA experiments. A confocal study confirmed the differentiation-dependent induction and activation of NOQ-1 and Nrf-2 in NHEKs. Immunohistochemistry showed that NQO-1 was accentuated in the upper epidermal layers, supporting the notion that differentiation-dependent NQO-1 expression is functional in human skin in vivo.

Conclusion

These results demonstrate that NQO-1 is modulated during KC differentiation via Nrf2 pathway, suggesting the active role of NQO-1 in the differentiating epidermis.     

Licochalcone A activates Nrf2 in vitro and contributes to licorice extract‐induced lowered cutaneous oxidative stress in vivo

The retrochalcone licochalcone A (LicA) has previously been shown to possess antimicrobial and anti‐inflammatory properties. In this study, we focused on pathways responsible for the antioxidative properties of LicA. In vitro, LicA protected from oxidative stress mediated by reactive oxygen species (ROS) by activating the expression of cytoprotective phase II enzymes. LicA induced nuclear translocation of NF‐E2‐related factor 2 (Nrf2) in primary human fibroblasts and elevated the expression of the cytoprotective and anti‐inflammatory enzymes heme oxygenase 1 and glutamate–cysteine ligase modifier subunit. LicA‐treated cells displayed a higher ratio of reduced to oxidized glutathione and decreased concentrations of ROS in UVA‐irradiated human dermal fibroblasts, as well as in activated neutrophils. In vivo, ultraweak photon emission analysis of skin treated with LicA‐rich licorice extract revealed a significantly lowered UVA‐induced luminescence, indicative for a decrease in oxidative processes. We conclude from these data that topical application of licorice extract is a promising approach to induce Nrf2‐dependent cytoprotection in human skin.     

High calcium enhances the expression of double‐stranded RNA sensors and antiviral activity in epidermal keratinocytes

Double‐stranded RNA (dsRNA) sensors including TLR3, MDA5 and RIG‐I are expressed in epidermal keratinocytes and play an important immunological role by enhancing various innate and adaptive immune responses. Although the role of elevated extracellular calcium concentration in keratinocyte differentiation is well understood, the effect of high calcium on dsRNA sensors is not well studied. We investigated alterations in dsRNA sensor expression and antiviral activity induced by a high extracellular concentration of calcium in epidermal keratinocytes. Normal human epidermal keratinocytes (NHEKs) were stimulated with high calcium and/or synthetic dsRNA, poly (I:C). TLR3, IFIH1 (MDA5) and DDX58 (RIG‐I) expression were measured via qPCR, and IFN‐β and human beta‐defensin 2 (HBD2) levels were measured using ELISA. TLR3 localization was evaluated with immunocytofluorescence. Antiviral activity was quantified with virus plaque assays using herpes simplex virus type 1 (HSV‐1). High calcium significantly upregulated mRNA expression of TLR3, IFIH1 and DDX58 in NHEKs. In addition, high calcium significantly enhanced poly (I:C)‐induced anti‐HSV‐1 activity in NHEKs. The antiviral molecule HBD2 but not IFN‐β induction by poly (I:C) was enhanced by high calcium. Our findings indicate that high levels of extracellular calcium enhance the expression of dsRNA sensors and augment antiviral activity in epidermal keratinocytes.     

IL‐24 is expressed during wound repair and inhibits TGFα‐induced migration and proliferation of keratinocytes

Please cite this paper as: IL‐24 is expressed during wound repair and inhibits TGFα ‐induced migration and proliferation of keratinocytes. Experimental Dermatology 2010; 19 : 714–722. Abstract: Interleukin (IL)‐24 is the protein product of melanoma differentiation‐associated gene 7 (MDA‐7). Originally identified as a tumor suppressor molecule, MDA‐7 was renamed IL‐24 and classified as a cytokine because of its chromosomal location in the IL‐10 locus, its mRNA expression in leukocytes, and its secretory sequence elements. We previously reported that IL‐24 is expressed by cytokine‐activated monocytes and T lymphocytes. Here, we show that IL‐24 is expressed in keratinocytes during wound repair. Paraffin‐embedded tissues prepared from human skin sampled at days 2, 6, and 10 after wounding were examined by immunohistochemistry for the expression of IL‐24. Protein expression was detected in the keratinocyte population with maximum expression at days 2 and 6, and no expression by day 10 (four of four subjects). In vitro studies showed that cytokines involved in wound repair, most notably transforming growth factor α (TGFα), TGFβ, IFNγ, and IFNβ, upregulated IL‐24 protein expression in normal human epidermal keratinocytes (NHEKs). Examination of the function of IL‐24 in both in vitro wound repair and migration assays demonstrated that IL‐24 inhibits TGFα‐induced proliferation and migration of NHEKs. These data support the hypothesis that IL‐24 functions during an inflammatory response in the skin by inhibiting the proliferation and migration of keratinocytes.