Pathway In Human Keratinocytes Research Articles

Rosmarinic acid inhibits poly(I:C)-induced inflammatory reaction of epidermal keratinocytes

AimsKeratinocytes are the predominant cells in the epidermis, exerting their primary role of physical barrier through sophisticated differentiation process. In addition, keratinocytes contribute to the activation of innate immunity, providing the surveillant role against external pathogens. It has been known that chronic skin inflammatory disease such as psoriasis can be provoked by viral pathogens including double-stranded RNA. In this study, we demonstrated that rosmarinic acid (RA) has an inhibitory potential on inflammatory reaction induced by double-stranded RNA mimic poly(I:C) in epidermal keratinocytes. Main methodsWe cultured human epidermal keratinocytes and induced inflammatory reaction by poly(I:C) treatment. The effect of RA on inflammatory reaction of keratinocytes was determined by RT-PCR and Western blot. Key findingsRA significantly inhibited poly(I:C)-induced expression of inflammatory cytokines including IL-1β, IL-6, IL-8, CCL20, and TNF-α, and downregulated NF-κB signaling pathway in human keratinocytes. In addition, RA significantly inhibited poly(I:C)-induced inflammasome activation, in terms of secretion of active form of IL-1β and caspase-1. Furthermore, RA markedly inhibited poly(I:C)-induced NLRP3 and ASC expression. SignificanceThese results indicate that RA can inhibit poly(I:C)-induced inflammatory reaction of keratinocytes, and suggest that it may be a potential candidate for the treatment of psoriasis.

Icariin inhibits TNF-α/IFN-γ induced inflammatory response via inhibition of the substance P and p38-MAPK signaling pathway in human keratinocytes

Pro-inflammatory cytokines play a crucial role in the etiology of atopic dermatitis. We demonstrated that Herba Epimedii has anti-inflammatory potential in an atopic dermatitis mouse model; however, limited research has been conducted on the anti-inflammatory effects and mechanism of icariin, the major active ingredient in Herba Epimedii, in human keratinocytes. In this study, we evaluated the anti-inflammatory potential and mechanisms of icariin in the tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ)-induced inflammatory response in human keratinocytes (HaCaT cells) by observing these cells in the presence or absence of icariin. We measured IL-6, IL-8, IL-1β, MCP-1 and GRO-α production by ELISA; IL-6, IL-8, IL-1β, intercellular adhesion molecule-1 (ICAM-1) and tachykinin receptor 1 (TACR1) mRNA expression by real-time PCR; and P38-MAPK, P-ERK and P-JNK signaling expression by western blot in TNF-α/IFN-γ-stimulated HaCaT cells before and after icariin treatment. The expression of TNF-α-R1 and IFN-γ-R1 during the stimulation of the cell models was also evaluated before and after icariin treatment. We investigated the effect of icariin on these pro-inflammatory cytokines and detected whether this effect occurred via the mitogen-activated protein kinase (MAPK) signal transduction pathways. We further specifically inhibited the activity of two kinases with 20μM SB203580 (a p38 kinase inhibitor) and 50μM PD98059 (an ERK1/2 kinase inhibitor) to determine the roles of the two signal pathways involved in the inflammatory response. We found that icariin inhibited TNF-α/IFN-γ-induced IL-6, IL-8, IL-1β, and MCP-1 production in a dose-dependent manner; meanwhile, the icariin treatment inhibited the gene expression of IL-8, IL-1β, ICAM-1 and TACR1 in HaCaT cells in a time- and dose-dependent manner. Icariin treatment resulted in a reduced expression of p-P38 and p-ERK signal activation induced by TNF-α/IFN-γ; however, only SB203580, the p38 alpha/beta inhibitor, inhibited the secretion of inflammatory cytokines induced by TNF-α/IFN-γ in cultured HaCaT cells. The differential expression of TNF-α-R1 and IFN-γ-R1 was also observed after the stimulation of TNF-α/IFN-γ, which was significantly normalized after the icariin treatment. Collectively, we illustrated the anti-inflammatory property of icariin in human keratinocytes. These effects were mediated, at least partially, via the inhibition of substance P and the p38-MAPK signaling pathway, as well as by the regulation of the TNF-α-R1 and IFN-γ-R1 signals.

Inhibitory effect of cucurbitacin B on imiquimod-induced skin inflammation

Psoriasis is a common skin disease, of which pathogenesis involves the increase of inflammatory reaction in epidermal cells. In an attempt to find therapeutics for psoriasis, we found that cucurbitacin B has an inhibitory potential on imiquimod-induced inflammation of keratinocytes. Cucurbitacin B significantly inhibited imiquimod-induced expression of crucial psoriatic cytokines, such as IL-8 and CCL20, via down-regulation of NF-κB and STAT3 signaling pathway in human keratinocytes. In addition, keratinocyte proliferation was markedly inhibited by cucurbitacin B. The potential beneficial effect of cucurbitacin B on psoriasis was further validated in imiquimod-induced psoriasiform dermatitis of experimental animal. Topical application of cucurbitacin B resulted in significant reduction of epidermal hyperplasia and inflammatory cytokines production, and ameliorated the psoriatic symptom. Taken together, these results suggest that cucurbitacin B may be a potential candidate for the treatment of psoriasis.

ZnO nanoparticle induces apoptosis by ROS triggered mitochondrial pathway in human keratinocytes

Zinc Oxide nanoparticles (ZnO NPs) in its small size with large reactive surfaces can lead to toxicological injury by generating reactive oxygen species (ROS) and oxidative stress. Recently, ZnO NPs were shown to play a role in acute or chronic toxicities with mammalian cells, where its mechanism of toxicity is not fully characterized yet. In this study, the potential mechanisms of ZnO NPs in inducing and increasing oxidative stress for causing cellular apoptosis were investigated with human keratinocytes. Indeed, ZnO NPs induced significant intracellular ROS and mitochondrial ROS productions. And it seemed that cellular ROS levels induced by ZnO NPs led to the dissipation of the mitochondrial membrane potentials and elicited the cellular apoptosis. The induced ROS production by ZnO NPs was blocked with chelator treatment, which inhibited the ZnO ion. The present study demonstrates that increased levels of ROS by ZnO NPs cause mitochondrial dysfunction and cellular apoptosis.

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.

Can nitroxides evoke the Keap1–Nrf2–ARE pathway in skin?

Nitroxides are stable cyclic radicals of diverse size, charge, and lipophilicity. They are cell-permeative, which effectively protects cells, tissues, isolated organs, and laboratory animals from radical-induced damage. The mechanisms of activity through which nitroxides operate are diverse, including superoxide dismutase-mimetic activity, oxidation of semiquinone radicals, oxidation of reduced metal ions, procatalase-mimetic activity, interruption of radical chain reactions, and indirect modulation of NO levels. Nitroxides possess both a nucleophilic (reducing properties) and an electrophilic (oxidizing properties) nature and, therefore, they may affect different cellular pathways. In the current study, a novel mechanism of action by which nitroxides provide skin protection based on their electrophilic nature is suggested. This study shows that nitroxides may act as electrophiles, directly or indirectly, capable of activating the Keap1–Nrf2–ARE pathway in human keratinocytes (HaCaT) and in human skin (human organ culture model). The high potency of oxoammonium cations versus hydroxylamines in activating the system is demonstrated. The mechanism of action by which nitroxides activate the Keap1–Nrf2–ARE pathway is discussed. Understanding the mechanism of activity may expand the usage of nitroxides as a skin protection strategy against oxidative stress-related conditions.

DNAX-activating Protein 10 (DAP10) Membrane Adaptor Associates with Receptor for Advanced Glycation End Products (RAGE) and Modulates the RAGE-triggered Signaling Pathway in Human Keratinocytes

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.

Fucoxanthin enhances the level of reduced glutathione via the Nrf2-mediated pathway in human keratinocytes.

Fucoxanthin, a natural carotenoid, is abundant in seaweed with antioxidant properties. This study investigated the role of fucoxanthin in the induction of antioxidant enzymes involved in the synthesis of reduced glutathione (GSH), synthesized by glutamate-cysteine ligase catalytic subunit (GCLC) and glutathione synthetase (GSS), via Akt/nuclear factor-erythroid 2-related (Nrf2) pathway in human keratinocytes (HaCaT) and elucidated the underlying mechanism. Fucoxanthin treatment increased the mRNA and protein levels of GCLC and GSS in HaCaT cells. In addition, fucoxanthin treatment promoted the nuclear translocation and phosphorylation of Nrf2, a transcription factor for the genes encoding GCLC and GSS. Chromatin immune-precipitation and luciferase reporter gene assays revealed that fucoxanthin treatment increased the binding of Nrf2 to the antioxidant response element (ARE) sequence and transcriptional activity of Nrf2. Fucoxanthin treatment increased phosphorylation of Akt (active form), an up-regulator of Nrf2 and exposure to LY294002, a phosphoinositide 3-kinase (PI3K)/Akt inhibitor, suppressed the fucoxanthin-induced activation of Akt, Nrf2, resulting in decreased GCLC and GSS expression. In accordance with the effects on GCLC and GSS expression, fucoxanthin induced the level of GSH. In addition, fucoxanthin treatment recovered the level of GSH reduced by ultraviolet B irradiation. Taken together, these findings suggest that fucoxanthin treatment augments cellular antioxidant defense by inducing Nrf2-driven expression of enzymes involved in GSH synthesis via PI3K/Akt signaling.

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

BackgroundThe 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. ObjectiveThe 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. MethodsHaCaT 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. ResultsZnO-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. ConclusionsThe present study demonstrated that ZnO-NPs might induce inflammatory response via ROS-ERK-Egr-1 pathway in human keratinocytes.

Evidence for a novel anti-apoptotic pathway in human keratinocytes involving the aryl hydrocarbon receptor, E2F1, and checkpoint kinase 1

Exposure of keratinocytes (KC) to ultraviolet (UV) radiation results in the initiation of apoptosis, a protective mechanism that eliminates cells harboring irreparable DNA damage. Hence, a manipulation of UV-induced apoptosis may significantly influence photocarcinogenesis. We have discovered that the aryl hydrocarbon receptor (AHR), a key regulator of drug metabolism and an UVB-sensitive transcription factor, serves an anti-apoptotic function in UVB-irradiated human KC. Chemical and shRNA-mediated inhibition of AHR signaling sensitized KC to UVB-induced apoptosis by decreasing the expression of E2F1 and its target gene checkpoint kinase 1 (CHK1). The decreased expression of these cell-cycle regulators was due to an enhanced expression of p27(KIP1) and an associated decrease in phosphorylation of both cyclin-dependent kinase 2 and its substrate molecule retinoblastoma protein. The subsequent inhibition of E2F1 autoregulation and downstream CHK1 expression resulted in an enhanced susceptibility of damaged cells to undergo apoptosis. Accordingly, ectopic overexpression of either E2F1 or CHK1 in AHR-knockdown KC attenuated the observed sensitization to UVB-induced apoptosis. Using an AHR-knockout SKH-1 hairless mouse model, we next demonstrated the physiological relevance of the anti-apoptotic function of AHR. In contrast to their AHR-proficient littermates, the constitutive expression of E2F1 and CHK1 was significantly reduced in the skin of AHR-knockout mice. Accordingly, a single exposure of the animals to UVB resulted in an enhanced cleavage of caspase-3 in the skin of AHR-knockout mice. These results identify for the first time the AHR-E2F1-CHK1 axis as a novel anti-apoptotic pathway in KC, which may represent a suitable target for chemoprevention of non-melanoma skin cancer.

Water extract of edible bird’s nest attenuated the oxidative stress-induced matrix metalloproteinase-1 by regulating the mitogen-activated protein kinase and activator protein-1 pathway in human keratinocytes

To better understand the molecular mechanisms underlying the anti-aging properties of water extract of edible bird’s nest (WEBN), the effect of WEBN on oxidative stress-induced matrix metalloproteinase-1 (MMP-1) was assessed in human HaCaT keratinocyte. Upon exposure to H2O2, WEBN inhibited H2O2-induced cytotoxicity and scavenged intracellular reactive oxygen species, down-regulated H2O2-induced MMP-1 mRNA expression, protein expression and activity, and lowered the activation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinases (JNK), which are the upstream of MMP-1 expression. Furthermore, WEBN inhibited the expression of c-Fos and phospho c-Jun, which are components of the transcription factor activator protein-1 (AP-1), AP-1 transcriptional activity, and AP-1 binding to the MMP-1 promoter. These data indicate that the anti-aging properties of WEBN involve the inhibition of MMP-1 expression via down-regulation of the ERK /JNK and AP-1 pathway.

Baicalein Attenuates Oxidative Stress-Induced Expression of Matrix Metalloproteinase-1 by Regulating the ERK/JNK/AP-1 Pathway in Human Keratinocytes

The matrix metalloproteinase (MMP) family is involved in the breakdown of the extracellular matrix during normal physiological processes such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes such as pathological aging, arthritis, and metastasis. Oxidative conditions generate reactive oxygen species (ROS) (e.g., hydrogen peroxide [H2O2]) in cells, which subsequently induce the synthesis of matrix metalloproteinase-1 (MMP-1). MMP-1, an interstitial collagenase, in turn stimulates an aging phenomenon. In this study, baicalein (5,6,7-trihydroxyflavone) was investigated for its in vitro activity against H2O2-induced damage using a human skin keratinocyte model. Baicalein pretreatment significantly inhibited H2O2-induced up-regulation of MMP-1 mRNA, MMP-1 protein expression and MMP-1 activity in cultured HaCaT keratinocytes. In addition, baicalein decreased the transcriptional activity of activator protein-1 (AP-1) and the expression of c-Fos and c-Jun, both components of the heterodimeric AP-1 transcription factor. Furthermore, baicalein reduced phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK), which are upstream of the AP-1 transcription factor. The results of this study suggest that baicalein is involved in the inhibition of oxidative stress-induced expression of MMP-1 via inactivation of the ERK/JNK/AP-1 signaling pathway.

Extracellular Double-Stranded RNA Induces TSLP via an Endosomal Acidification- and NF-κB-Dependent Pathway in Human Keratinocytes

Double-stranded RNA (dsRNA) causes keratinocytes to release thymic stromal lymphopoietin (TSLP), which plays a key role in allergic diseases. Endosomal Toll-like receptor 3 (TLR3) and cytosolic RIG-like receptors (RLRs) and PKR have been reported to recognize dsRNA. Here, we demonstrate that dsRNA induces TSLP in keratinocytes via an endosomal acidification-dependent and NF-κB-mediated pathway. After treatment with pharmacologic inhibitors or transfection with small interfering RNAs (siRNAs), primary human keratinocytes were stimulated. Bafilomycin A1, which inhibits endosomal acidification to block the TLR3 pathway, blocked the dsRNA-induced expression of TSLP, IL-8, IFN-β, and other molecules including the dsRNA sensors, whereas it did not inhibit diacyllipopeptide-induced expression of TSLP and IL-8. The dsRNA-induced gene expression of TSLP depended on RelA, a component of NF-κB, but not IRF3, similar to IL-8 but different from IFN-β, which depended on both IRF3 and RelA. The results indicate that endosomal acidification and the subsequent activation of NF-κB are necessary to sense extracellular dsRNA, suggesting the importance of the TLR3-NF-κB axis to trigger production of TSLP against the self dsRNA released from damaged cells or viral dsRNA, in the epidermis, relating to skin inflammation including atopic dermatitis (AD).

Up‐regulation of TNF‐alpha secretion by cigarette smoke is mediated by Egr‐1 in HaCaT human keratinocytes

Many epidemiologic studies have pointed to a significant association between cigarette smoking and inflammatory skin disease such as psoriasis. Cigarette smoke induces expression of regulators of inflammation such as interleukin (IL)-1, IL-6, IL-8 and tumor necrosis factor (TNF)-alpha. It was recently demonstrated that early growth response-1 (Egr-1) transcription factor is significantly up-regulated in the skin lesions of patients with psoriasis. The mechanism by which cigarette smoke extract (CSE) regulates inflammatory cytokine expression in keratinocyte was still unknown. The aim of this study was to investigate the signalling of CSE-induced Egr-1 expression and the role for Egr-1 in CSE-induced TNF-alpha expression. Cytotoxicity of CSE in HaCaT cells was measured by thiazolyl blue tetrazolium bromide (MTT) assay. CSE-induced Egr-1 expression was investigated by western blot, luciferase reporter assay and confocal microscopy. TNF-alpha expression was measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Involvement of Egr-1 in CSE-induced TNF-alpha secretion was determined by using Egr-1 specific siRNA. CSE increases the Egr-1 expression, promoter activity and its nuclear translocation in human HaCaT keratinocytes. CSE activates mitogen-activated protein kinase (MAPK) pathways including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). Up-regulation of Egr-1 expression by CSE stimulation was found to be inhibited by an ERK and JNK but not p38 inhibitor. CSE increases TNF-alpha expression and secretion. This increase is mediated by CSE-induced Egr-1 expression. Our results showed that CSE induces Egr-1 expression via MAPK pathway in human keratinocytes and TNF-alpha expression by Egr-1. This pathway may contribute to the development of inflammatory disease such as psoriasis.

Cytosolic RNA Recognition Pathway Activates 14-3-3 Protein Mediated Signaling and Caspase-Dependent Disruption of Cytokeratin Network in Human Keratinocytes

The skin is the primary boundary between the body and the environment. In addition to its properties as a physical barrier, skin keratinocytes actively participate in many defense mechanisms. Viral double-stranded RNA (dsRNA) is the most important viral structure involved in activation of immune response. Intracellular detection of dsRNA by cytoplasmic receptors activates well-characterized antiviral response, as well as pro-inflammatory response and apoptosis of virus-infected cells. Here, we have used quantitative subcellular proteomics to characterize the signaling pathways activated by cytosolic dsRNA recognition pathway in human keratinocytes. Cytoplasmic and mitochondrial proteomes were analyzed using 2-DE in combination with MS, immunoblotting and confocal microscopy. We have identified 239 reproducibly differentially expressed proteins upon dsRNA stimulation. The identified proteins include several key proteins involved in cytoskeletal dynamics, cell signaling, cell death, and stress response. Our analysis provides novel information how the cytokeratin network is disrupted in a caspase-dependent manner upon dsRNA stimulation as well as Encephalomyocarditis virus or Vesicular stomatitis virus infection. We show that this caspase-dependent disruption of cytokeratin is activated by cytoplasmic RNA recognition pathway. In addition, we show that viral infection activates 14-3-3 protein mediated signaling pathways in human keratinocytes which suggest an important role of 14-3-3 proteins in antiviral innate immune response.

Inflammatory Cytokine Tumor Necrosis Factor-α Enhances Nerve Growth Factor Production in Human Keratinocytes, HaCaT Cells

The skin lesions of inflammatory skin diseases (e.g., atopic dermatitis or psoriasis) accompany infiltration of inflammatory cells like macrophages, where abnormal sensory innervations and elevation of nerve growth factor (NGF) level are observed. It is thought that increased NGF mediates the abnormal innervations and this may cause the hypersensitivity of the skin. However, the mechanism of this increased NGF production in the skin is still unknown. Here, we show that tumor necrosis factor (TNF)-α, but not interferon-γ or interleukin-6, enhanced the NGF production in human keratinocytes. The enhanced NGF production was abolished by both Raf-1 kinase and MEK inhibitors, whereas specific inhibitors of p38 mitogen–activated protein kinase and c-Jun N-terminal kinase did not. The extracellular signal–regulated kinase (ERK) phosphorylation and expression of NGF mRNA were accelerated by TNF-α treatment. Furthermore, serum was necessary for the NGF production and epidermal growth factor could substitute for serum in the effect on NGF secretion. These results indicate that TNF-α enhances NGF production via the Raf-1 / MEK / ERK pathway in human keratinocytes, suggesting that regulating TNF-α is a therapeutic target to control NGF production and subsequent sensory innervations.

Leukotriene B4 enhances tumour necrosis factor‐α‐induced CCL27 production in human keratinocytes

A chemokine CCL27 recruits skin-homing T cells. CCL27 production by epidermal keratinocytes is dependent on nuclear factor-kappaB (NF-kappaB) activity and is enhanced in lesions with atopic dermatitis or allergic contact dermatitis. A lipid mediator leukotriene B(4) (LTB(4)) may be involved in the development of these allergic dermatoses. LTB(4) acts on cell surface G-protein-coupled receptors, BLT1 and BLT2. The aim of this study was to investigate the in vitro effects of LTB(4) on CCL27 production in human keratinocytes. Keratinocytes were incubated with TNF-alpha and LTB(4). CCL27 secretion and mRNA levels were analysed by ELISA and RT-PCR, respectively. NF-kappaB activities were analysed by luciferase assays. Protein levels or phosphorylation status were analysed by cell-based ELISA. LTB(4) alone did not enhance CCL27 production and modestly enhanced NF-kappaB activity in human keratinocytes. However, LTB(4) potently enhanced TNF-alpha-induced CCL27 secretion and mRNA expression and NF-kappaB activity. LTB(4) alone or together with TNF-alpha, induced phosphorylation and degradation of inhibitory NF-kappaB alpha (IkappaBalpha) and phosphorylation of NF-kappaB p65. These effects of LTB(4) were suppressed by BLT1 antagonist U75302, pertussis toxin, phosphoinositide-3 kinase (PI3K) inhibitor LY294002 and extracellular signal-regulated kinase (ERK) kinase inhibitor U0126, but not by BLT2 antagonist LY255283. LTB(4) induced phosphorylation of ERK and Akt, downstream kinase of PI3K; LY294002 suppressed phosphorylation of both kinases while U0126 suppressed only the former. These results suggest that LTB(4) may enhance TNF-alpha-induced CCL27 production by activating NF-kappaB via the BLT1/G(i/o)/PI3K/ERK pathway in human keratinocytes. LTB(4) may contribute to the enhanced CCL27 production of keratinocytes in lesions with atopic dermatitis or allergic contact dermatitis.

Corticotropin-Releasing Hormone (CRH) Downregulates Interleukin-18 Expression in Human HaCaT Keratinocytes by Activation of p38 Mitogen-Activated Protein Kinase (MAPK) Pathway

It is generally accepted that corticotropin-releasing hormone (CRH) acts as the main coordinator of the central response to stress. Stress or an abnormal response to stressors has been found to modify the evolution of skin disorders, including psoriasis and atopic dermatitis. Nevertheless, the specific pathogenic role of stress remains unknown in skin diseases. Interleukin (IL)-18, a member of the IL-1 family, is a key mediator of peripheral inflammation and host defense responses, and is secreted by human keratinocytes. Here, we investigated the regulatory effect of CRH on expression of IL-18 in skin keratinocytes. Exposure of HaCaT cells to CRH resulted in a reduction of IL-18 mRNA transcripts and its production was in a concentration-dependent manner. In order to investigate whether the mitogen-activated protein kinase (MAPK) signaling pathway is involved in the downregulation of IL-18 production, cells were pre-treated with SB203580, an inhibitor of p38 MAPK, prior to the addition of CRH. This pre-treatment blocked the decrease in IL-18 production. In addition, CRH treatment induced rapid phosphorylation of p38 MAPK. SB203580 were able to inhibit CRH-induced p38 MAPK phosphorylation. CRH also inhibited production of IL-18 in human primary keratinocytes. These results suggest that CRH regulates IL-18 production through the MAPK signaling pathway in human keratinocytes.

AKT status controls susceptibility of malignant keratinocytes to the early-activated and UVB-induced apoptotic pathway.

In previous work, we have described an early-activated and ultraviolet B (UVB)-induced apoptotic pathway in human keratinocytes, which can be completely inhibited by AKT activation. We now compared this response of primary human keratinocytes with the response of two p53-mutated squamous cell carcinoma (SCC)-derived cell lines (A431 and A253) to an apoptotic UVB dose. In these cell lines, both the basal AKT phosphorylation status and the apoptotic response to UVB diverged strongly from the response of healthy primary keratinocytes. Even more, a remarkable correlation was found between the two. Although a constitutive dual phosphorylation of AKT rendered the A253 SCC cell line completely resistant to the early-activated and UVB-induced apoptotic pathway, deficient T308 phosphorylation of AKT in the SCC cell line A431 led to a greatly augmented sensitivity to the early-activated, UVB-induced apoptotic pathway. These results indicate that the preservation of a healthy AKT pathway is essential for a wild-type UVB-induced apoptotic response in skin, and suggest that AKT-mediated dysregulation of the early-activated apoptotic response to UVB is an important event in the oncogenic transformation of keratinocytes.

Differential and bi-directional regulation between TR2/TR4 orphan nuclear receptors and a specific ligand mediated-peroxisome proliferator-activated receptor α in human HaCaT keratinocytes

Background: We have reported that human TR2 orphan nuclear receptor (TR2) can modulate the transcriptional activity of the reporter gene containing an AGGTCA direct repeat-hormone response element. Objective: The aim of this study is to investigate the potential role and regulation of TR2 in human HaCaT keratinocytes. Methods: We performed mainly chloramphenicol acetyltransferase reporter gene assays (CAT assays), and Western blot analysis. Results: From CAT assays, TR2 can suppress retinoic acid (RA)-induced transactivation by 44.7% in HaCaT keratinocytes. This suppression is similar to our previous report showing TR4 orphan nuclear receptor (TR4) can suppress RA-induced transactivation. However, TR4 but not TR2 can significantly repress Wy-14643-mediated peroxisome proliferator-activated receptor α (PPARα) transactivation by 95%. Western blot analysis suggested that Wy-14643 can differentially regulate the expression of TR2 and TR4 (by increasing the expression of TR4 protein and decreasing that of TR2) in HaCaT keratinocytes. Conclusion: Our data not only provides the first evidence to demonstrate that close members of orphan nuclear receptors group, such as TR2 and TR4, can have distinct functions, but also suggests the existence of differential and bi-directional regulation between PPARα and TR2/TR4, that may play some important roles in the PPARα signaling pathway in human keratinocytes.