Abrogation of both constitutive and resveratrol-dependent EGFR and ERK phosphorylation by specific inhibitor of EGFR kinase.

<p>(<b>A</b>) Western blot analysis of whole-cell lysates. Keratinocytes were incubated with 2 µM PD168393 (PD16) for 30 minutes prior to addition of 50 µM resveratrol (Resv) for the indicated time-points. (<b>B</b>) Quantification of Western blot bands by densitometry. *<i>P</i><0.05 and ^§<i>P</i><0.01 <i>versus</i> untreated controls for each time-point. (<b>C</b>) Western blot analysis of whole-cell lysates. Keratinocytes were incubated with 2 µM PD16 prior to addition of 50 µM Rv for 1 h. Subsequently, cells were further treated for 12 h with 50 ng/ml TNFα. (<b>D</b>) Quantification of Western blot bands by densitometry. *<i>P</i><0.05 <i>versus</i> controls treated with TNFα only. Data are representative of three independent experiments.</p

Effects of resveratrol and verbascoside on the TNFα−induced phosphorylation of EGFR, ERK, and the NFκB subunit p65.

<p>(<b>A</b>) Western blot analysis performed in whole-cell lysates of human keratinocytes. Following 1 h pre-incubation with 50 µM polyphenol, cells were treated for further 12 h with TNFα (50 ng/ml). Actin was used as a loading control. (<b>B</b>) Quantification of Western blot bands by densitometry. *<i>P</i><0.05 <i>versus</i> untreated controls (0 h time-point); ^§<i>P</i><0.05 versus TNFα-treated conditions. (<b>C</b>) Binding activity of nuclear cell lysates to NFκB-specific or AP-1-specific DNA consensus sequences. *<i>P</i><0.05 <i>versus</i> untreated controls; ^§<i>P</i><0.05 versus TNFα-treated conditions. Data are representative of three independent experiments.</p

Resveratrol did not protect phosphorylated EGFR from endogenous phosphatases, led to EGFR accumulation in the keratinocyte membranes, and induced its cytosolic degradation.

<p>Western blot analysis of the plasma membrane fraction, where cadherin was used as a loading control (<b>A</b>) and of the cytosolic fraction (<b>C</b>). After 24h treatment with resveratrol (Resv), keratinocytes were stimulated with TGFα (50 ng/ml) for 10 minutes. Then, 2 µM PD168393 (PD16) were added to the medium for further 10 minutes. (<b>B</b>, <b>D</b>) Quantification of Western blot bands by densitometry. *<i>P</i><0.05 and ^§<i>P</i><0.01 <i>versus</i> untreated controls.</p

Effects of resveratrol and verbascoside on the phosphorylation status of EGFR, ERK1/2, and the NFκB subunit p65.

<p>(<b>A</b>) Western blot analysis performed in whole-cell lysates of human keratinocytes treated with 50 µM of the indicated polyphenol. Actin was used as a loading control. (<b>B, C, D</b>) Quantification of Western blot bands by densitometry. *<i>P</i><0.05 <i>versus</i> untreated controls (0 h time-point). (<b>E</b>) Western blot analysis of EGFR and ERK phosphorylation in whole-cell lysates of human keratinocytes treated with 10 and 50 µM Resv for 6 h. (<b>F</b>) Quantification of Western blot bands by densitometry. *<i>P</i><0.05 <i>versus</i> controls without Resv (0 µM). ^§<i>P</i><0.05 <i>versus</i> 10 µM Resv (0 µM). Data are representative of three independent experiments.</p

Resveratrol promoted nuclear accumulation of phosphorylated and non-phosphorylated EGFR alone and in association with TGFα.

<p>(<b>A</b>) Western blot analysis of nuclear levels of phosphorylated (nP-EGFR) and non-phosphorylated (nEGFR), and (<b>B</b>) its densitometric quantification. *<i>P</i><0.05 and ^§<i>P</i><0.01 <i>versus</i> untreated controls.</p

Effects of resveratrol on IL-8 expression are sensible to specific inhibitor of EGFR kinase.

<p>(<b>A</b>) Quantitative real-time RT-PCR measurement of <i>IL-8</i> mRNA. Cells were treated with 2 µM PD168393 (PD16) for 30 min, and then exposed to 50 µM resveratrol (Resv) for 1 h. Cells were cultivated for further 12 h, with or without TNFα (50 ng/ml). *<i>P</i><0.05 <i>versus</i> untreated controls; ^§<i>P</i><0.01 <i>versus</i> TNFα-treated conditions. (<b>B</b>) ELISA of IL-8 protein accumulation in the supernatants of human keratinocytes. Cells were treated with 2 µM PD16 for 30 min, and then, exposed to 50 µM Resv for 1 h. Cells were cultivated for further 24 h, with or without TNFα (100 ng/ml). Data are representative of three independent experiments.</p

Photo-Oxidation Products of Skin Surface Squalene Mediate Metabolic and Inflammatory Responses to Solar UV in Human Keratinocytes

<div><p>The study aimed to identify endogenous lipid mediators of metabolic and inflammatory responses of human keratinocytes to solar UV irradiation. Physiologically relevant doses of solar simulated UVA+UVB were applied to human skin surface lipids (SSL) or to primary cultures of normal human epidermal keratinocytes (NHEK). The decay of photo-sensitive lipid-soluble components, alpha-tocopherol, squalene (Sq), and cholesterol in SSL was analysed and products of squalene photo-oxidation (SqPx) were quantitatively isolated from irradiated SSL. When administered directly to NHEK, low-dose solar UVA+UVB induced time-dependent inflammatory and metabolic responses. To mimic UVA+UVB action, NHEK were exposed to intact or photo-oxidised SSL, Sq or SqPx, 4-hydroxy-2-nonenal (4-HNE), and the product of tryptophan photo-oxidation 6-formylindolo[3,2-b]carbazole (FICZ). FICZ activated exclusively metabolic responses characteristic for UV, i.e. the aryl hydrocarbon receptor (AhR) machinery and downstream <em>CYP1A1/CYP1B1</em> gene expression, while 4-HNE slightly stimulated inflammatory UV markers <em>IL-6</em>, <em>COX-2</em>, and <em>iNOS</em> genes. On contrast, SqPx induced the majority of metabolic and inflammatory responses characteristic for UVA+UVB, acting <em>via</em> AhR, EGFR, and G-protein-coupled arachidonic acid receptor (G2A).</p> <h3>Conclusions/Significance</h3><p>Our findings indicate that Sq could be a primary sensor of solar UV irradiation in human SSL, and products of its photo-oxidation mediate/induce metabolic and inflammatory responses of keratinocytes to UVA+UVB, which could be relevant for skin inflammation in the sun-exposed oily skin.</p> </div

Effects of AhR silencing (SiAhR) on metabolic system and <i>IL-8</i> expression in NHEK.

<p>A. AhR levels in NHEK treated for 24 h with 1 µM FICZ. The treatment was started 48 h after initiation of RNA silencing of AhR (SiAhR) or mock (-). The mean values of the densitometry of AhR bands (three independent determinations) are reported. Levels of <i>AhR</i> (B), <i>Cyp1A1</i> (C) and <i>IL-8</i> (D) transcripts in NHEK treated for 24 h with 1 µM FICZ. *P<0.05 and ^§P<0.01.</p

Induction of inflammatory and metabolic genes (mRNA, fold of induction) by photo-oxidized commercial squalene and squalene from photo-oxidized human skin surface lipids (SSL).

<p>Commercial squalene or SSL were exposed to UVA+UVB irradiation (dose UVA = 20 J/cm² and UVB = 2 J/cm²). Photo-exposed SSL were subjected to TLC and squalene containing spot was collected as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044472#s2" target="_blank">Materials and Methods</a>. Squalene samples or 0.1% methanol (a vehicle for squalene) were added to NHEK cultures (final concentration - 1 µg/mL) for 3 h. Then, cells were thoroughly washed and RNA was isolated. Gene expression was determined by RT-PCR and results were expressed as a fold of induction versus vehicle. Three independent experiments were carried out and the mean values ±S.D. were calculated. *P<0.05 and ^†P<0.001.</p

Time-dependent effects of UV irradiation on metabolic system in NHEK.

<p><b>A</b>. Time-dependent effects on nuclear levels of AhR and Arnt post-UV irradiation. Actin was used as a loading control; <b>B</b>. Densitometry of western blot bands of AhR and Arnt in the nucleus; *P<0.05 and ^§P<0.01 <i>versus</i> untreated controls (-). <b>C</b>. Time-course of CYP1A1 and CYP1B1 transcripts post-irradiation; *P<0.05 and ^§P<0.01 <i>versus</i> untreated controls (0 h). Significance refers to both the transcripts at each time-point. The results are representative of three independent experiments.</p