Effect of PPARγ modulation on PUVA-induced expression of senescence-like phenotype in HDFs.

<p>After PUVA treatment, HDFs were cultured in the absence or in the presence of 2µM Octa. The medium was changed every 3 days to ensure efficient antioxidant capacity. (A) To evaluate fibroblast morphology, 2 weeks after PUVA in the absence or presence of Octa treatment, cells were fixed and stained with Comassie Brilliant Blue. Scale bar 50 µm. (B) SA-β-gal expression was detected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104045#s2" target="_blank"><b>Materials and Methods</b></a>. The <i>inset</i> represents fibroblasts after PUVA-treatment revealing a senescent phenotype with enlarged cytoplasmic morphology and SA-β-gal expression. The number of SA-β-gal positive fibroblasts is shown as mean ± SD of three independent experiments. **p<0.001 as compared with mock treated controls; ^##p<0.001 as compared with PUVA-treated fibroblasts. (C) Supernatants were collected from mock-treated fibroblasts, at 24 h, 48 h and 1 week post PUVA-treatment. MMP-1 release was assessed by ELISA-kit. Three independent experiments in each donor (n = 3) were performed to determine specific MMP-1 protein concentrations in the supernatants. **p<0.001 as compared with mock-treated fibroblasts; ^#p<0.05; ^##p<0.001 as compared with PUVA-treated fibroblasts. (D) Total cellular proteins (30µg/lane) were subject to 10% SDS-PAGE. Variation of protein loading was determined by reblotting membrane with an anti-β-tubulin antibody. Western Blot assays are representative of at least three experiments. Increase of p53 and p21 proteins expression is remarkable 24 h after irradiation as well as until 7 days. Octa treatment decreased PUVA-induced expression of p53 protein (at 24 and 48 h) and of its target gene p21 (at 1 week).</p

Evaluation of PUVA induced effects on PPARγ expression and activity.

<p>(A) Real-time RT-PCR was performed to measure the expression of PPAR-γ mRNA 6 h and 24 h after PUVA exposure. The level of PPAR-γ mRNA was normalized to the expression of GAPDH and is expressed relative to untreated control cells (*p<0.05 respect to Ctr). (B) Luciferase activity analysis of cells transfected with pGL3-(Jwt)3TKLuc reporter construct. After 24 h of transfection, cells were treated with PUVA. The measurement of luciferase activity was carried out 24 h and 48 h after treatment (*p<0.05 respect to Ctr). (C) Real-time RT-PCR was performed to measure the effect of Octa post-treatment on the expression of PPAR-γ mRNA 6 h and 24 h after PUVA exposure. The level of PPAR-γ mRNA was normalized to the expression of GAPDH and is expressed relative to untreated control cells (*p<0.05 respect to Ctr; ^#p<0.05 respect to PUVA). (D) Luciferase activity analysis of cells transfected with pGL3-(Jwt) 3TKLuc reporter construct. After 24 h of transfection, cells were treated with PUVA and post-incubated with Octa. The measurement of luciferase activity was carried out 24 h and 48 h after treatment (*p<0.05 respect to Ctr; ^#p<0.05 respect to PUVA).</p

Protective action of PPARγ modulation on PUVA-induced imbalance of cell antioxidant system.

<p>HDFs (1×10⁶) were lysed in PBS and protease inhibitor cocktail. Cell lysates were used for analytical determinations. (A) Total antioxidant capacity (TAC) was assessed by BAP-test as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104045#s2" target="_blank"><b>Materials and Methods</b></a> section. (B) Cat enzyme activity was determined by spectrophotometry as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104045#s2" target="_blank"><b>Materials and Methods</b></a>. (C) GSH concentrations were determined by HPLC-MS as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104045#s2" target="_blank"><b>Materials and Methods</b></a>. (D) α-Toc is measured by GC-MS as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104045#s2" target="_blank"><b>Materials and Methods</b></a>. *p<0.05; **p<0.001 respect to control fibroblasts; ^#p<0.05; ^##p<0.001 compared with PUVA-treated fibroblasts.</p

Modulation of PPARγ Provides New Insights in a Stress Induced Premature Senescence Model

<div><p>Peroxisome proliferator-activated receptor gamma (PPARγ) may be involved in a key mechanism of the skin aging process, influencing several aspects related to the age-related degeneration of skin cells, including antioxidant unbalance. Therefore, we investigated whether the up-modulation of this nuclear receptor exerts a protective effect in a stress-induced premature senescence (SIPS) model based on a single exposure of human dermal fibroblasts to 8-methoxypsoralen plus + ultraviolet-A-irradiation (PUVA). Among possible PPARγ modulators, we selected 2,4,6-octatrienoic acid (Octa), a member of the parrodiene family, previously reported to promote melanogenesis and antioxidant defense in normal human melanocytes through a mechanism involving PPARγ activation. Exposure to PUVA induced an early and significant decrease in PPARγ expression and activity. PPARγ up-modulation counteracted the antioxidant imbalance induced by PUVA and reduced the expression of stress response genes with a synergistic increase of different components of the cell antioxidant network, such as catalase and reduced glutathione. PUVA-treated fibroblasts grown in the presence of Octa are partially but significantly rescued from the features of the cellular senescence-like phenotype, such as cytoplasmic enlargement, the expression of senescence-associated-β-galactosidase, matrix-metalloproteinase-1, and cell cycle proteins. Moreover, the alterations in the cell membrane lipids, such as the decrease in the polyunsaturated fatty acid content of phospholipids and the increase in cholesterol levels, which are typical features of cell aging, were prevented. Our data suggest that PPARγ is one of the targets of PUVA-SIPS and that its pharmacological up-modulation may represent a novel therapeutic approach for the photooxidative skin damage.</p></div

The effect of pyridinyl imidazoles on cAMP/PKA/CREB signal transduction.

<p>(A) Expression of Mitf in B16-F0 cells after 6 h of treatment with α-MSH (0.1 µM) in presence or not of PI compounds (SB202474, SB202190, SB203580, SB220025, PD169316 20 µM: MAPK Inh III 10 µM). Total cellular proteins (30 µg/lane) were subject to 10% SDS-PAGE. Variation of loading was determined by blotting with anti-β-tubulin antibody. Western blot assays are representative of at least three experiments. (B) Concentration of cAMP of control and treated cells were determined using the cAMP bioluminescent assay. Following incubation with α-MSH (0.1 µM) in presence or not of PI compounds, the cAMP levels were measured and compared to the untreated control samples. The results are the mean±SD of three experiments performed in duplicates. (C) Analysis of the time-dependent effect α-MSH treatment on CREB level of phosphorylation. Cells were stained with anti-phospho-CREB-PE (Ser133), and then analyzed measuring median fluorescence intensity (MFI) in duplicates. Histogram represents means ± SD of MFI of three independent experimets. (D) Comparative analysis of CREB-Ser133 level of phosphorylation in untreated cells, α-MSH-treated cells and α-MSH plus PI coumpond-treated cells.</p

Evidence for PPARγ-induced promotion of cell antioxidant defence.

<p>(A) Octa treatment for 24 h, 48 h and 1 week determined a significant increase of antioxidant cell response. TAC was assessed by BAP-test and Cat enzyme activity was determined by spectrophotometry as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104045#s2" target="_blank"><b>Materials and Methods</b></a> section. (B) HDFs were transfected with siRNA specific for PPARγ (siPPARγ) or non-specific siRNA (siCtr). PPARγ level was evaluated by real-time RT-PCR (C) The activity of Cat was assessed in HDFs transfected with siPPARγ or siCtr and exposed to 2µM Octa for 6 h. In parallel Cat activity was measured in HDFs transfected with siPPARγ or siCtr and exposed to PUVA w/o post-incubation with 2µM Octa. *p<0.05; **p<0.001 respect to control fibroblasts; ^#p<0.05 compared with PUVA-treated fibroblasts.</p

Possible interference of PPARγ against PUVA induced modulation of the cellular stress response system.

<p>(A) RT-PCR was performed to measure the expression of NRF2 mRNA 6 and 24 h after PUVA exposure, w/o Octa post-incubation. The level of NRF2 mRNA was normalized to the expression of GAPDH and is expressed relative to untreated control cells (**p<0.001 respect to Ctr; ^##p<0.001 compared with PUVA-treated fibroblasts). (B) RT-PCR was performed to measure the expression of HO-1 mRNA 6 and 24 h after PUVA exposure, w/o Octa post-incubation. The level of HO-1 mRNA was normalized to the expression of GAPDH and is expressed relative to untreated control cells (**p<0.001 respect to Ctr; ^##p<0.001 compared with PUVA-treated fibroblasts). (C) GSH concentrations were determined by HPLC-MS) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104045#s2" target="_blank"><b>Materials and Methods</b></a> (*p<0.05 respect to control fibroblasts). (D) RT-PCR was performed to measure the expression of FoxO1 mRNA 6 and 24 h after PUVA exposure, w/o Octa post-incubation. The level of FoxO1 mRNA was normalized to the expression of GAPDH and is expressed relative to untreated control cells. *p<0.05 respect to control fibroblasts; ^#p<0.05 compared with PUVA-treated fibroblasts.</p

Analysis of the role of Mitf expression in pyridinyl imidazoles-dependent melanogenesis inhibition.

<p>(A) To B16-F0 melanoma cells were transiently transfected with a plasmid encoding for Mitf cDNA (pCAAG-mi-S) or a control construct carrying Mitf cDNA in antisense orientation (pCAAG-mi-AS). Following incubation with α-MSH (0.1 µM) in presence of pyridinyl imidazoles (SB202474, SB202190, SB203580, SB220025, PD169316 20 µM: MAPK Inh III 10 µM) for 72 h, or not, the extracellular and intracellular levels of melanin were determined as described above. The data show the mean±SD of three experiments performed in duplicate. (B–C) Analysis of luciferase activity of the Mitf melanocyte-specific promoter (M promoter) in the presence of pyridinyl imidazoles in B16-F0 and HeLa cells. Twenty-four hours after transient transfection cells were treated with PI compounds in presence (C) or not (B) of α-MSH (0.1 µM) (B16-F0) or forskolin (1 µM) (HeLa). Luciferase activity was assayed after 6 h of treatment. Firefly luciferase activity, normalized to the corrisponding renilla luciferase activity was expressed as fold change compared with control cells. Values represent mean ± SD of three representative experiments performed in duplicate.</p

Regulation of Wnt/β-catenin signaling by pyridinyl imidazoles.

<p>(A) Inhibition of the β-catenin/Tcf/Lef1-responsive luciferase reporter gene by PI compounds. The pTK-Renilla was inserted as an internal control. Twenty-four hours after transfection, cells were treated with PI compounds (SB202474, SB202190, SB203580, SB220025, PD169316 20 µM: MAPK Inh III 10 µM) for 6 h. Firefly luciferase activity, normalized to the corresponding renilla luciferase activity was expressed as fold decrease compared with control cells. Values represent mean ± SD of three representative experiments performed in duplicate. (B) Semi-qunatitative real-time PCR was used to measure Wnt/β-catenin-target genes Axin2, Lef1 and Wisp1 mRNAs expression in B16-F0 cells after 6 h of treatments with PI compounds. The graphs show fold differences in transcript abundance in comparison with untreated cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033021#s2" target="_blank">Results</a> shown were normalized by the β-actin mRNA levels. The data show the mean±SD of three experiments performed in triplicate. *P≤0.05; #P≤0.01 versus control. (C) Expression of β-catenin in B16-F0 cells after 6 h of treatment with PI compounds (SB202474, SB202190, SB203580, SB220025, PD169316 20 µM: MAPK Inh III 10 µM). Total cellular proteins (30 µg/lane) were subject to 10% SDS-PAGE. Variation of loading was determined by blotting with anti-β-tubulin antibody. Western blot assays are representative of at least three experiments. (D) Immunofluorescence analysis of β-catenin. B16-F0 cells were grown on glass coverslips and then treated with SB202474, PD169316 (20 µM) or DMSO respectively. Six hours later, cells were fixed and analyzed by immunofluorescence labelling with a mouse monoclonal anti-β-catenin followed by Alexa-Fluor-546-conjugated goat anti-mouse IgG antibody. Nuclei were labelled with bisbenzidine (DAPI). Original magnification 20×.</p

Evidence for Octa-mediated activation of PPARγ-linked signal transduction.

<p>(A) Activation of RARE. Cells (2×10⁴cells/well) were plated in a 24-well plate and after 24 h they were transfected with RARE. After 24 h, cells were treated with 5µM ReOH for 6 h, 5µM AtRA for 6–48 h, and 2µM Octa for 6–48 h. Measurement of luciferase activity was assessed as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104045#s2" target="_blank"><b>Materials and Methods</b></a>. (B) Quantitative real-time RT-PCR was performed to measure the expression of CRABPII and CYP26A1 mRNA at various time points after treatment with 2µM Octa or 5 µM AtRA. The values were normalized to GAPDH mRNA levels. (C) Quantitative real-time RT-PCR was performed to measure the expression of FABP5 and PPARγ mRNA at various time points after treatment with 2µM Octa or 5µM AtRA. The values were normalized to GAPDH mRNA levels. (D) Luciferase activity analysis of cells transfected with pGL3-(Jwt)3TKLuc reporter construct. After 24 h of transfection, cells were treated with 2µM Octa. The measurement of luciferase activity was carried out 24 h and 48 h after treatment. *p<0.05; **p<0.001 respect to untreated control cells.</p