Gene Expression In Human Keratinocytes Research Articles

The Natural Janus Kinase Inhibitor Agerarin Downregulates Interleukin-4-Induced PER2 Expression in HaCaT Keratinocytes.

The circadian clock system is closely associated with inflammatory responses. Dysregulation of the circadian clock genes in the skin impairs the skin barrier function and affects the pathophysiology of atopic dermatitis. Interleukin 4 (IL-4) is a proinflammatory cytokine derived from T-helper type 2 cells; it plays a critical role in the pathogenesis of atopic dermatitis. Agerarin (6,7-dimethoxy-2,2-dimethyl-2H-chromene) is a natural JAK1/2/3 inhibitor isolated from Ageratum houstonianum that has a protective effect on the epidermal skin barrier. However, it remains unclear whether agerarin affects the circadian clock system. The aim of this study is to investigate the effect of agerarin on IL-4-induced PER2 gene expression in human keratinocytes through reverse transcription (RT)-PCR, quantitative real-time PCR (qPCR), immunoblotting, immunofluorescence microscopic analysis, and real-time bioluminescence analysis. We found that agerarin reduced IL-4-induced PER2 mRNA expression by suppressing the JAK-STAT3 pathway. In addition, real-time bioluminescence analysis in PER2:luc2p promoter-reporter cells revealed that agerarin restored the oscillatory rhythmicity of PER2 promoter activity altered by IL-4. These findings suggest that agerarin may be useful as a cosmeceutical agent against inflammatory skin conditions associated with disrupted circadian rhythms, such as atopic dermatitis.

Zinc Chloride: Time-Dependent Cytotoxicity, Proliferation and Promotion of Glycoprotein Synthesis and Antioxidant Gene Expression in Human Keratinocytes.

Simple SummaryZinc ions are involved in the biology of cell growth, proliferation, differentiation or apoptosis by regulating many biological molecules, such as transcription factors, enzymes and growth factors. In this study, the time-dependent cytotoxicity, cell proliferation and gene expression in human keratinocytes HaCaT cells were evaluated when exposed to ZnCl2. The results of this study showed non-cytotoxic effects up to 10 µg/mL after 24 h, no significant effect on cell proliferation when exposed to 5 or 1 µg/mL ZnCl2 at 72 h and upregulation of eight genes, with great potential in the biomedical field, particularly for regenerative-medicine applications and wound healing.The use of ionic metals such as zinc (Zn2+) is providing promising results in regenerative medicine. In this study, human keratinocytes (HaCaT cells) were treated with different concentrations of zinc chloride (ZnCl2), ranging from 1 to 800 µg/mL, for 3, 12 and 24 h. The results showed a time–concentration dependence with three non-cytotoxic concentrations (10, 5 and 1 µg/mL) and a median effective concentration value of 13.5 µg/mL at a cell exposure to ZnCl2 of 24 h. However, the zinc treatment with 5 or 1 µg/mL had no effect on cell proliferation in HaCaT cells in relation to the control sample at 72 h. The effects of the Zn2+ treatment on the expression of several genes related to glycoprotein synthesis, oxidative stress, proliferation and differentiation were assessed at the two lowest non-cytotoxic concentrations after 24 h of treatment. Out of 13 analyzed genes (superoxide dismutase 1 (SOD1), catalase (CAT), matrix metallopeptidase 1 (MMP1), transforming growth factor beta 1 (TGFB1), glutathione peroxidase 1 (GPX1), fibronectin 1 (FN1), hyaluronan synthase 2 (HAS2), laminin subunit beta 1 (LAMB1), lumican (LUM), cadherin 1 (CDH1), collagen type IV alpha (COL4A1), fibrillin (FBN) and versican (VCAN)), Zn2+ was able to upregulate SOD1, CAT, TGFB1, GPX1, LUM, CDH1, FBN and VCAN, with relative expression levels of at least 1.9-fold with respect to controls. We found that ZnCl2 promoted glycoprotein synthesis and antioxidant gene expression, thus confirming its great potential in biomedicine.

AhR Regulates Peptidoglycan-Induced Inflammatory Gene Expression in Human Keratinocytes

Bacterial peptidoglycan (PGN) stimulates toll-like receptor 2 (TLR2) on the surface of keratinocytes (KCs), triggering signaling pathways that promote an innate immune response. However, excessive TLR2 activation can lead to inappropriate inflammation, which contributes to skin conditions such as rosacea. To better treat these conditions, there is a need to understand the molecular mechanisms that regulate the cellular response to TLR2 activation in the skin. Aryl hydrocarbon receptor (AhR) is a transcription factor that modulates the immune response in KCs and is a promising therapeutic target for inflammatory skin diseases. Here, we investigated the role of the AhR in regulating the transcriptional response of human KCs to PGN. We performed whole-transcriptome sequencing in wild-type and AhR-depleted KCs after PGN stimulation. AhR depletion altered the expression of 72 genes in response to PGN, leading to increased expression of 48 genes and repression of 24 genes, including interleukin (IL)-1β. Chromatin immunoprecipitation showed that PGN stimulation resulted in AhR binding the promoters of IL-1β and IL-6 to activate them. More broadly, AhR promoted inflammatory gene expression by increasing JNK/mitogen-activated protein kinase signaling and FosB expression. Finally, we observed that AhR depletion increased TLR2 expression itself, raising the hypothesis that AhR may serve to restrain TLR2-mediated inflammation in KCs through negative feedback. Viewed together, our findings demonstrate a significant and complex role for AhR in modulating the expression of inflammatory genes in KCs in response to PGN.

Effect of extracts and isolated compounds derived from Retama monosperma (L.) Boiss. on anti-aging gene expression in human keratinocytes and antioxidant activity

Ethnopharmacological relevanceMoroccan folk medicine treats skin cicatrization with Retama monosperma (L.) Boiss. locally named "Rtem", but the mechanism involved is still not well known. Traditional healers use the plant in small doses as an anthelmintic, disinfectant and an effective abortive. In addition, the cladodes powder mixed with honey is employed as purgative and vermifuge.Equally, the SIRT1 and SIRT3 genes activation and sirtuin proteins expression, which delay cellular senescence, participate in wound healing and skin regeneration especially, SIRT1 the most studied gene, leads to fast skin restoration and cicatrization. Aim of the studyIn this study, we evaluated the ability of the Retama monosperma (L.)Boiss. flowers and seeds extracts and the isolated compounds in augmenting the SIRT1 and SIRT3 gene expression in HaCaT cells and expressing the antioxidant activity. Materials and methodsWe examined for quantitative expression levels of SIRT1 and SIRT3 in HaCaT cell by qRT-PCR and the antioxidant activity by four tests (conjugated diene, TBARS assay, DPPH scavenging activity and H2O2 radical scavenging assay) of diethyl ether extract of flowers (DEF extract) and ethyl acetate extract of seeds (EAS extract) of R. monosperma(L.) Boiss. and the isolated compounds (quercetin, 6-methoxykaempferol, kaempferol and genistein). ResultsThe screening system by EGFP fluorescence revealed that all samples and resveratrol significantly increase SIRT1 and SIRT3 promoters activities in HaCaT cells with p< 0.05. Furthermore, EAS, quercetin, 6-methoxykaempferol and kaempferol increase significantly (p< 0.05) SIRT1 (3.43, 1.18, 2.62, and 1.72 expression quantity, respectively) and SIRT3 (16.27, 5.01, 3.01, and 6.18 expression quantity, respectively) in HaCaT cells. On the other hand, genistein has a moderate activity on SIRT1 and SIRT3 with 1.43 and 2.04 expression levels. For the antioxidant activity, the EAS and the pure compounds exhibited stronger antioxidant activity than BHT. While DEF and genistein have a moderate antioxidant activity when compared with BHT. ConclusionsIn this study, the expression levels of SIRT1 and SIRT3 in HaCaT cells increase in the presence of extracts of R. monosperma (L.) Boiss. and the pure compounds.

3′-Hydroxypterostilbene Inhibits 7,12-Dimethylbenz[a]anthracene (DMBA)/12-O-Tetradecanoylphorbol-13-Acetate (TPA)-Induced Mouse Skin Carcinogenesis

BackgroundA natural pterostilbene analogue isolated from the herb Sphaerophysa salsula, 3′-hydroxypterostilbene (HPSB), exhibits antiproliferative activity in several cancer cell lines; however, the inhibitory effects of HPSB on skin carcinogenesis remains unclear. PurposeThe aim of this study was to evaluate the inhibitory effects of HPSB on two-stage skin carcinogenesis in mice and its potential mechanism. Study Design and MethodsThis study investigated the anti-inflammatory and anti-tumor effects of HPSB in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated acute skin inflammation and 7,12-dimethylbenz[a]anthracene (DMBA)/TPA-induced two-stage skin carcinogenesis model. In addition, the effects of HPSB on the modulation of the phase I and phase II metabolizing enzymes in the DMBA-induced HaCaT cell model were investigated. ResultsThe results provide evidence that topical treatment with HPSB significantly inhibits TPA-induced epidermal hyperplasia and leukocyte infiltration through the down-regulation of cyclooxygenase-2 (COX-2), matrix metalloprotein-9 (MMP-9), and ornithine decarboxylase (ODC) protein expression in mouse skin. Furthermore, HPSB suppresses DMBA/TPA-induced skin tumor incidence and multiplicity via the inhibition of proliferating cell nuclear antigen (PCNA), Cyclin B1 and cyclin-dependent kinase 1 (CDK1) expression in the two-stage skin carcinogenesis model. In addition, pretreatment with HPSB markedly reduces DMBA-induced cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1B1 (CYP1B1) gene expression in human keratinocytes; however, HPSB does not significantly affect the gene expression of the phase II enzymes. ConclusionThis is the first study to show that topical treatment with HPSB prevents mouse skin tumorigenesis. Overall, our study suggests that natural HPSB may serve as a novel chemopreventive agent capable of preventing carcinogen activation and inflammation-associated tumorigenesis.

종대황과 선복화 에탄올 추출물의 인간 피부 세포주인 HaCaT 세포에서 NRF2/ARE에 의존적인 유전자 발현의 유도를 통한 항산화 효과

Mammalian cells control cellular homeostasis using a variety of defensive enzymes in order to combat against environmental oxidants and electrophiles. NF-E2-related factor-2 (NRF2) is a transcription factor that, in response to an exposure to oxidative stress, translocates into the nucleus and modulates the inducible expression of various phase II cytoprotective enzymes by binding to the antioxidant response element (ARE). In the present study, we have acquired 400 ethanol extracts of traditional medicinal plants and attempted to find out possible extract(s) that can increase the NRF2/ARE-dependent gene expression in human keratinocytes. As a result, we have identified that ethanol extracts of Rheum undulatum and Inula japonica strongly activated the ARE-dependent luciferase activity in HaCaT- ARE-luciferase cells. Exposure of ethanol extracts of Rheum undulatum and Inula japonica increased the viability and activated transcription and translation of NRF2-dependent phase II cytoprotective enzymes in HaCaT cells, such as heme oxygenase-1 (HO-1) and NAD[P]H:quinone oxidorecutase- 1 (NQO1). In addition, ethanol extracts of Rheum undulatum and Inula japonica suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced generation of intracellular reactive oxygen species (ROS), thereby inhibiting the formation of 8-hydroxyguanosine (8-OHG) and 4-hydroxynonenal (4-HNE) in HaCaT cells. Together, our results demonstrate that ethanol extracts of Rheum undulatum and Inula japonica exert anti-oxidant effects via the induction of NRF2/ARE-dependent gene expression in human keratinocytes.

Transcriptome Analysis Identifies the Dysregulation of Ultraviolet Target Genes in Human Skin Cancers

Exposure to ultraviolet radiation (UVR) is a major risk factor for both melanoma and non-melanoma skin cancers. In addition to its mutagenic effect, UVR can also induce substantial transcriptional instability in skin cells affecting thousands of genes, including many cancer genes, suggesting that transcriptional instability may be another important etiological factor in skin photocarcinogenesis. In this study, we performed detailed transcriptomic profiling studies to characterize the kinetic changes in global gene expression in human keratinocytes exposed to different UVR conditions. We identified a subset of UV-responsive genes as UV signature genes (UVSGs) based on 1) conserved UV-responsiveness of this subset of genes among different keratinocyte lines; and 2) UV-induced persistent changes in their mRNA levels long after exposure. Interestingly, 11 of the UVSGs were shown to be critical to skin cancer cell proliferation and survival. Through computational Gene Set Enrichment Analysis, we demonstrated that a significant portion of the UVSGs were dysregulated in human skin squamous cell carcinomas, but not in other human malignancies. This highlights the potential and specificity of the UVSGs in clinical diagnosis of UV damage and stratification of skin cancer risk.

Characterization of TNF-α– and IL-17A–Mediated Synergistic Induction of DEFB4 Gene Expression in Human Keratinocytes through IκBζ

Human β-defensin 2 (hBD2), encoded by the DEFB4 gene, is an antimicrobial peptide playing an essential role in inflammatory processes in the skin. hBD2 expression is regulated synergistically by tumor necrosis factor-α (TNF-α) and IL-17A; however, the underlying regulatory mechanisms are unknown. The purpose of this study was to characterize the molecular mechanism by which TNF-α and IL-17A synergistically induce hBD2 expression. In cultured human keratinocytes we show that a constitutive noninducible binding of the transcription factor organic cation transporter 1 (OCT1) to the DEFB4 promoter is crucial for IL-17A/TNF-α-mediated synergistic induction of hBD2 but not the synergistic induction of CCL20, IL8, IL17C and LCN2. Interestingly, stimulation with IL-17A results in a p38 mitogen-activated protein kinase-dependent accumulation of inhibitor of nuclear factor κB ζ (IκBζ), which is a necessity for synergistic induction of hBD2. Finally, co-stimulation with TNF-α induces DNA binding of NF-κB and activator protein 1 (AP-1) to two specific sites in the DEFB4 promoter region. Hence, our study shows how two inflammatory stimuli are integrated by three different signaling pathways into the regulation of one specific target gene involving the three specific transcription factors OCT1, NF-κB, and AP-1 as well as the transcriptional cofactor IκBζ. These findings may be important in psoriasis, where TNF-α and IL-17A have been identified as key pathogenic cytokines.

Bioinformatics approach for choosing the correct reference genes when studying gene expression in human keratinocytes.

Reverse transcription polymerase chain reaction (qRT-PCR) has become a mainstay in many areas of skin research. To enable quantitative analysis, it is necessary to analyse expression of reference genes (RGs) for normalization of target gene expression. The selection of reliable RGs therefore has an important impact on the experimental outcome. In this study, we aimed to identify and validate the best suited RGs for qRT-PCR in human primary keratinocytes (KCs) over a broad range of experimental conditions using the novel bioinformatics tool 'RefGenes', which is based on a manually curated database of published microarray data. Expression of 6 RGs identified by RefGenes software and 12 commonly used RGs were validated by qRT-PCR. We assessed whether these 18 markers fulfilled the requirements for a valid RG by the comprehensive ranking of four bioinformatics tools and the coefficient of variation (CV). In an overall ranking, we found GUSB to be the most stably expressed RG, whereas the expression values of the commonly used RGs, GAPDH and B2M were significantly affected by varying experimental conditions. Our results identify RefGenes as a powerful tool for the identification of valid RGs and suggest GUSB as the most reliable RG for KCs.

521: Role of hypoxia and Hypoxia Inducible Factor 1a (HIF1a) in regulation of the HSPA2 gene expression in human keratinocytes

521: Role of hypoxia and Hypoxia Inducible Factor 1a (HIF1a) in regulation of the HSPA2 gene expression in human keratinocytes

Limited dependence of IL-4-regulated keratinocyte differentiation and gene expression on STAT6 (HYP6P.261)

Abstract Epidermal Differentiation Complex (EDC) genes are critical for barrier function in the skin, and Th2 cytokines that promote allergic inflammation inhibit EDC gene expression in keratinocytes. To further define the effects of the atopic immune response on keratinocyte differentiation, we investigated IL-4-mediated changes in gene expression in human keratinocytes via RNA-seq. Human keratinocytes were differentiated for 2 days with calcium chloride and then stimulated with IL-4 for 24 hours, or keratinocytes were differentiated with calcium chloride and chronically stimulated with IL-4 for 5 days. At day 2, IL-4 increased expression of genes associated with cell cycle, and decreased expression of genes involved with cell adhesion. At day 5 of differentiation, IL-4-stimulated keratinocytes showed increased expression of genes involved with cell projection and adhesion, and decreased expression of genes that participate in wound repair, epidermis development and host defense. STAT6 inhibition by siRNA significantly altered the expression of less than 20% of genes induced by IL-4. Thus, although IL-4 dramatically alters keratinocyte gene expression and differentiation, only a subset of this response is dependent upon STAT6, and eliminating STAT6 expression has IL-4-independent changes in gene expression. This suggests that keratinocyte gene regulation by IL-4 requires additional molecular pathways.

The Retinoid-Related Orphan Receptor RORα Promotes Keratinocyte Differentiation via FOXN1

RORα is a retinoid-related orphan nuclear receptor that regulates inflammation, lipid metabolism, and cellular differentiation of several non-epithelial tissues. In spite of its high expression in skin epithelium, its functions in this tissue remain unclear. Using gain- and loss-of-function approaches to alter RORα gene expression in human keratinocytes (HKCs), we have found that this transcription factor functions as a regulator of epidermal differentiation. Among the 4 RORα isoforms, RORα4 is prominently expressed by keratinocytes in a manner that increases with differentiation. In contrast, RORα levels are significantly lower in skin squamous cell carcinoma tumors (SCCs) and cell lines. Increasing the levels of RORα4 in HKCs enhanced the expression of structural proteins associated with early and late differentiation, as well as genes involved in lipid barrier formation. Gene silencing of RORα impaired the ability of keratinocytes to differentiate in an in vivo epidermal cyst model. The pro-differentiation function of RORα is mediated at least in part by FOXN1, a well-known pro-differentiation transcription factor that we establish as a novel direct target of RORα in keratinocytes. Our results point to RORα as a novel node in the keratinocyte differentiation network and further suggest that the identification of RORα ligands may prove useful for treating skin disorders that are associated with abnormal keratinocyte differentiation, including cancer.

P38α and p38δ MAPK are key regulators of p21Cip1 gene expression in human keratinocytes

PKCδ increases keratinocyte differentiation via a mechanism that involves activation of p38δ MAPK. We recently showed that PKCδ also suppresses cell proliferation by increasing expression of the p21Cip1 cyclin‐dependent kinase inhibitor. However, the downstream effectors that mediate this PKCδ‐dependent regulation are not known. Our present studies suggest that PKCδ activates p38δ which regulates p21Cip1 promoter activity, as exogenously expressed p38δ increases p21Cip1 mRNA and protein level in human keratinocytes. Treatment with SB202190, a p38α/β inhibitor, or dominant negative p38α, leads to a decrease in PKCδ‐dependent p21Cip1 promoter activity, implicating p38α as a second mediator of this regulation. p21Cip1 promoter truncation experiments indicate that p38δ increases the activity via a putative p53 response element. This suggests that p38α and δ may act in conjunction with or through p53 to regulate p21Cip1 gene expression. In addition, we show that p38α and p38δ overexpression increases p53 promoter activity, suggesting that p38α and δ regulate p53 gene expression. We propose that PKCδ activates p38α and p38δ which in turn activates p53 to increase p21Cip1 expression leading to cessation of keratinocyte proliferation.

Activation of endogenous opioid gene expression in human keratinocytes and fibroblasts by pulsed radiofrequency energy fields

BackgroundPulsed radiofrequency energy (PRFE) fields are being used increasingly for the treatment of pain arising from dermal trauma. However, despite their increased use, little is known about the biological and molecular mechanism(s) responsible for PRFE-mediated analgesia. In general, current therapeutics used for analgesia target either endogenous factors involved in inflammation, or act on endogenous opioid pathways.Methods and ResultsUsing cultured human dermal fibroblasts (HDF) and human epidermal keratinocytes (HEK), we investigated the effect of PRFE treatment on factors, which are involved in modulating peripheral analgesia in vivo. We found that PRFE treatment did not inhibit cyclooxygenase enzyme activity, but instead had a positive effect on levels of endogenous opioid precursor mRNA (proenkephalin, pro-opiomelanocortin, prodynorphin) and corresponding opioid peptide. In HEK cells, increases in opioid mRNA were dependent, at least in part, on endothelin-1. In HDF cells, additional pathways also appear to be involved. PRFE treatment was also followed by changes in endogenous expression of several cytokines, including increased levels of interleukin-10 mRNA and decreased levels of interleukin-1β mRNA in both cell types.ConclusionThese findings provide a new insight into the molecular mechanism underlying PRFE-mediated analgesia reported in the clinical setting.

Microarray profiling of gene expression in human keratinocytes suggests a new protective activity against UV-induced DNA damage for a compound previously known to interact with SCF-KIT signalling pathway

The stem cell factor (SCF) and its protein-tyrosine kinase receptor KIT are together implicated in the regulation of diverse biological processes and particularly in melanogenesis. Indeed, this signalling pathway controls melanoblast migration from the neural crest during embryogenesis and allows the communication between keratinocytes and melanocytes in the adult. In melanocytes, the binding of SCF to its transmembrane receptor leads to the activation of signalling pathways implicating protein kinases which finally control the expression of pigmentation-related genes. We have developed a biological compound called IV09.007, which we previously described as a modulator of the SCF/KIT signalling pathway with a pro-pigmenting effect. In the present work, we have studied the expression and localization of both SCF and KIT mRNAs and proteins in the skin or skin-derived cell lines. Then, we explored with a microarray approach the ability of IV09.007 to modulate the expression of genes in human keratinocytes and melanocytes in culture. Thereby, we observed the regulation of genes implicated in DNA repair, mainly related to base/nucleotides excision pathways. A modulated transcriptional response was also observed for some genes implicated in the response against oxidative stress, in apoptosis inhibition and in lowering inflammatory immune response. These microarray results predicted a conferred protective effect of IV09.007 and we verified this hypothesis by performing comet assays on UVB-irradiated keratinocytes or melanocytes, to demonstrate the efficacy of IV09.007 on preventing DNA damage.

EGFR and IL-1 Signaling Synergistically Promote Keratinocyte Antimicrobial Defenses in a Differentiation-Dependent Manner

Ligands of the EGF family regulate autocrine keratinocyte proliferation, and IL-1 family cytokines orchestrate epithelial defense responses. Although members of both families are overexpressed in wound healing and psoriasis, their roles in regulating the innate immune functions of keratinocytes remain incompletely explored. Using sensitive assays, we found significant increases of heparin-binding EGF-like growth factor, transforming growth factor-α, and amphiregulin mRNA and protein in lesional psoriasis compared with uninvolved or control skin. In normal human keratinocyte (NHK) monolayers, EGFR ligands were ineffective in inducing DEFB4, S100A7, and CCL20 mRNAs and human β-defensin (hBD)-2 peptide. Combined with IL-1α, however, EGFR ligands provoked 250 × more DEFB4 and CCL20 and a 9-fold rise in S100A7 mRNA relative to the EGFR ligand alone. This synergy was also reflected in secreted hBD-2 protein, both from NHK and reconstituted human epidermis. Keratinocyte differentiation was critical for these responses, as postconfluent NHK yielded mRNA and protein levels an order of magnitude greater than subconfluent cells. Differentiation also influenced signal transduction, with subconfluent cells using NF-κB and postconfluent cells using EGFR, MEK1/2, and p38. We propose that EGFR ligands are important modifiers of IL-1 activity, synergizing with IL-1 to stimulate epidermal production of hBD-2, S100A7, and CCL20, three of the most upregulated transcripts in psoriatic plaques.

Sulforaphane but not ascorbigen, indole‐3‐carbinole and ascorbic acid activates the transcription factor Nrf2 and induces phase‐2 and antioxidant enzymes in human keratinocytes in culture

Nrf2 is a basic leucine zipper transcriptional activator essential for the coordinated transcriptional induction of phase-2 and antioxidant enzymes. Brassica vegetables contain phytochemicals including glucoraphanin, the precursor of sulforaphane (SFN) and glucobrassicin, the precursor of indole-3-carbinole (I3C) and ascorbigen (ABG). The degradation products SFN, I3C and ABG may be capable of inducing cytoprotective genes in skin. In this study, we tested the potency of SFN, ABG and I3C in affecting Nrf2-dependent gene expression in human keratinocytes in culture. SFN but not ABG and its precursors I3C and ascorbic acid induced Nrf2 dependent gene expression at a relatively low concentration (5 micromol/l). Induction of Nrf2 due to SFN was accompanied by an increase in mRNA and protein levels of NADPH quinone oxidoreductase 1, heme oxygenase 1 and gamma-glutamylcysteine-synthetase. Furthermore, SFN elevated cellular glutathione levels and antagonized tumor necrosis factor-alpha-induced NFkappaB transactivation. Therefore, SFN treatment may present a strategy for enhancing the cellular defense mechanisms in skin.

A central role for transcription factor C/EBP-β in regulating CD1d gene expression in human keratinocytes

A central role for transcription factor C/EBP-β in regulating CD1d gene expression in human keratinocytes

A Central Role for Transcription Factor C/EBP-β in Regulating CD1d Gene Expression in Human Keratinocytes

CD1d is a nonclassical Ag-presenting molecule that presents glycolipid Ags to NKT cells that are involved in immune defense and tumor rejection. It also plays a role in immunoregulatory functions in the epidermis. The mechanisms controlling the expression of CD1d are not well understood. Therefore, we cloned the CD1d gene promoter and characterized its activities in primary human keratinocytes and other cell lines of epithelial origin. We found that a CCAAT box in the CD1d promoter is required for its expression in keratinocytes. We show here that transcription factor C/EBP-beta binds to the CCAAT box in the CD1d promoter in vitro and in vivo. Consistent with these observations, deletion of the gene encoding for C/EBP-beta caused a loss of CD1d expression. The in vivo regulation of CD1d has significant implications for the pathologic mechanisms of certain immunologic skin diseases in which NKT cells play a role, such as allergic contact dermatitis and psoriasis. Together, these data show a central role for C/EBP-beta in regulating CD1d transcription.

Ultraviolet irradiation‐induces epidermal growth factor receptor (EGFR) nuclear translocation in human keratinocytes

Epidermal growth factor receptor (EGFR) plays a critical role in mediating ultraviolet (UV) irradiation-induced signal transduction and gene expression in human keratinocytes. EGFR activation results from increased phosphorylation on specific tyrosine residues in the C-terminal intracellular domain. It has recently been reported that following growth factor stimulation EGFR translocates from the surface membrane to the nucleus, where it may directly regulate gene transcription. We have investigated the ability of UV irradiation to induce EGFR nuclear translocation in human primary and HaCaT keratinocytes. UV irradiation caused rapid nuclear translocation of EGFR. Significant accumulation of EGFR in the nucleus was observed within 15 min after UV irradiation exposure. Maximal translocation occurred at 30 min post-UV irradiation, and resulted in a 10-fold increase in EGFR in the nucleus, as determined by Western blot analysis of nuclear extracts and confirmed by immunofluorescence. Inhibition of nuclear export by Leptomycin B did not alter UV irradiation-induced nuclear accumulation. EGFR tyrosine kinase inhibitor (PD169540) reduced UV irradiation-induced EGFR nuclear translocation 50%. Mutation of either tyrosine 1148 or tyrosine 1173 reduced nuclear translocation 70%, while mutation of tyrosine 1068 was without effect. In addition, over-expression of receptor type protein tyrosine phosphatase-kappa (RPTP-kappa), which specifically dephosphorylates EGFR tyrosines, decreased UV irradiation-induced EGFR nuclear translocation in human keratinocytes. These data demonstrate that UV irradiation stimulates rapid EGFR nuclear translocation, which is dependent on phosphorylation of specific EGFR tyrosine residues. EGFR nuclear translocation may act in concert with conventional signaling pathways to mediate UV irradiation-induced responses in human keratinocytes.