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

4-hydroxy-2-nonenal (HNE) effects on signal transduction and expression of pro-inflammatory mediators in NHEK.

<p>Time-dependent effects of 25 µM HNE on ERK1/2 and Akt1 phosphorylation: <b>A</b>. Western blot and <b>B</b>. Densitometry of total cell extracts. Dynamics of HNE effects on COX-2 and iNOS (<b>C</b>) and pro-inflammatory cytokine (<b>D</b>) gene expression. Cytokine levels in the culture medium after 24 h treatment with 25 µM HNE (<b>E</b>). *P<0.05 and ^§P<0.01 <i>versus</i> untreated controls.</p

Effects of intact and UV-exposed skin surface lipids on signal transduction and AhR-CYP1 axis in NHEK.

<p>NHEK cultures were exposed to skin surface lipids normalised by the content of squalene (Sq): intact freshly prepared skin surface lipids (SSL, with final Sq content 1 µg/mL), Sq isolated from the SSL (final content 1 µg/mL), SSL exposed to UVA+UVB for 20 min (oxSSL with final content of oxidised Sq (PxSq) 1 µg/mL), or SqPx isolated from oxSSL (final content of SqPx 1 µg/mL), or SqPx-<i>in vivo</i> isolated from the in vivo photo-exposed skin surface lipids (final content of SqPx-<i>in vivo</i> 1 µg/mL). After 20 min of co-incubation, differential lysis was carried out, cytoplasmic and nuclear proteins extracted, and subjected to electrophoresis (<b>A</b>). Western blots were quantified by densitometry: (<b>B</b>) nuclear levels of AhR and (<b>C</b>) cytoplasmic levels of phosphorylated EGFR (P-EGFR), ERK (P-ERK), and the NFκB constituent p65 (P-p65). Transcripts of <i>CYP1A1</i> and <i>CYP1B1</i> were determined by RT-PCR at 3 h co-incubation (<b>D</b>). *P<0.05 and ^§P<0.01 <i>versus</i> control treated with the vehicle for the lipids (0.1% methanol).</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

Inflammatory responses to UVA+UVB in normal human epidermal keratinocytes (NHEK).

<p><b>A</b>. Time course of post-UV cytokine transcripts; <b>B</b>. TNFα, IL-6 and IL-8 proteins in the conditioned medium of sham-irradiated NHEK and 24 h post UVA+UVB irradiation (1.0+0.1 mJ/cm²); <b>C</b>. Time course of post-UV <i>COX-2</i> transcript; <b>D</b>. Time course of post-UV <i>IL-1R1</i> and <i>TLR4</i> transcripts. *P<0.05 and ^§P<0.01<i>versus</i> sham-irradiated control.</p

Effects of photo-oxidized linoleic acid and <i>tert</i>-buthyl hydroperoxide (tBOOH) on the expression of solar UV-sensitive genes in cultivated primary human keratinocytes.

<p>NHEK were incubated with photo-oxidized (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044472#s2" target="_blank">Materials and Methods</a>) linoleic acid or with <i>tert</i>- butyl hydroperoxide (tBOOH) or with their vehicle methyl alcohol (0.02%, control) for 3 hours. Gene expression was determined by real-time PCR and results (fold of gene expression) are espressed as mean values ±S.D. of three independent experiments. *P<0.05, ^&P<0.01, and ^#P<0.001 versus control.</p

Effects of FICZ on metabolic system in NHEK.

<p><b>A</b>. Western blots of nuclear AhR and Arnt after 30 min exposure to 0.1 or 1.0 µM FICZ. <b>B</b>. Densitometry of western blot bands. <b>C</b>. Western blots of total whole cell AhR after 30 min exposure to 0.1 or 1.0 µM FICZ. <b>D</b>. Densitometry of western blot bands. *P<0.05 and §P<0.01 <i>versus</i> untreated controls (-). Time-course of AhR, CypA1 and Cyp1B1 levels following treatment with 0.1 µM FICZ (<b>E</b>) and 1 µM FICZ (<b>F</b>). *P<0.05 and §P<0.01 <i>versus</i> untreated controls (0 h). The results are representative of three independent experiments.</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

Effects of UV and FICZ on the EGFR signalling in NHEK.

<p><b>A</b>. Western blot detection of the phosphorylated forms of EGFR (P-EGFR) and ERK (P-ERK) in total lysates of untreated NHEK (-) and post-UV irradiation; <b>B</b>. Densitometry of the bands. *P<0.05 and ^§P<0.01 <i>versus</i> untreated controls (-). <b>C</b>. Western blots of nuclear non-phosphorylated (nEGFR) and phosphorylated EGFR (nP-EGFR) before and 20 min after exposure to UV. Histone 4 (H4) was used as a loading control; <b>D</b>. Densitometry of nEGFR and nP-EGFR bands. *P<0.05 <i>versus</i> untreated control (-). <b>E</b>. Western blot detection of the phosphorylated forms of EGFR (P-EGFR) and ERK (P-ERK) in total lysates of untreated NHEK (-) and after 30 min exposure to 0.1 or 1.0 µM FICZ. <b>F</b>. Densitometry of the bands. *P<0.05 <i>versus</i> untreated control (-). <b>G</b>. Western blots of nuclear non-phosphorylated (nEGFR) and phosphorylated EGFR (nP-EGFR) after 30 min exposure to 0.1 or 1.0 µM FICZ; <b>H</b>. Densitometry of nEGFR and nP-EGFR bands. *P<0.05 <i>versus</i> untreated controls.</p