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

Coordinated regulation of Mitf expression and CREB activation.

<p>(A) One representative western blot anti-Mitf used to quantify RNA interference efficiency after 24 h of nucleofection. (B) Analysis of Mitf siRNA interference on CREB level of phosphorylation was analyzed measuring CREB level of phosphorylation by measuring PE-CREB-Ser133 median fluorescence intensity (MFI) in duplicates. Histogram represents means±SD of MFI of three independent experimets. (C) Dot plot analysis of one representative experiments of Mitf-siRNA showing higher MFI of PE-CREB-Ser133 in samples presenting low levels of Mitf expression (region R2) in comparison with the cell fraction presenting high levels of Mitf (region R3).</p

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

The effect of forced β-catenin expression on pyridinyl imidazoles-dependent melanogenesis inhibition.

<p>Western blot analysis of β-catenin in whole-cell lysates prepared from B16-F0 cells transfected with pCS2-β-cat-wt, pCS2-β-cat-mut plasmids or pCS2 empty vector. 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) Twenty-four hours after transfection cells were treated 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, and then the extracellular and intracellular levels of melanin were determined as described above. The data show the mean±SD of three independent experiments performed in duplicate. (C) B16-F0 cells were cotrasfected with lucerase reporter plasmids TopFlash and pCS2-β-cat-wt, pCS2-β-cat-mut plasmids. The pTK-Renilla was inserted as an internal control for each experimental sample as a control for transfection efficiency. Firefly luciferase activity, normalized to the renilla luciferase activity was expressed as fold decrease compared with control cells. Values represent mean±SD of three representative experiments performed in duplicate.</p

Effect of pyridinyl imidazoles compounds on melanin synthesis in B16-F0 melanoma cells.

<p>(A) Following incubation with α-MSH (0.1 µM) and increasing concentrations (1, 2.5, 5, 10, 20 µM) of pyridinyl imidazoles for 72 h, the extracellular and intracellular levels of melanin were determined separately by measuring the absorbance at 405 nm. Standard curves of synthetic melanin were used to extrapolate the absolute values of melanin content. The total amount of melanin was calculated for each experimental point by adding the extracellular and intracellular melanin values after normalization for protein content. Total melanin produced at the end-point by control (DMSO-treated cells) and hormone-stimulated cells (α-MSH plus DMSO-treated cells) is reported for comparison. (B) B16-F0 cells were also treated with pyridinyl imidazoles compounds for 96 h in absence of α-MSH. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033021#s2" target="_blank">Results</a> are expressed as percentage of untreated control samples. The data show the mean±SD of three experiments performed in duplicate. *P≤0.05; #P≤0.01 versus control.</p

The effect of pyridinyl imidazoles on melanogenic gene expression.

<p>(A) Semi-qunatitative real-time PCR was used to measure Mitf, tyrosinase, TRP1 and TRP2 mRNAs expression in B16-F0 cells after 6 h and (B) 24 h of tratments. The graphs show fold differences in transcript abundance in untreated cells, and α-MSH-treated cells (0.1 µM) in presence or not of PI compounds (SB202474, SB202190, SB203580, SB220025, PD169316 20 µM: MAPK Inh III 10 µM). The results shown in (A) and (B) were normalized by the β-actin mRNA levels. The data show the mean±SD of four experiments performed in triplicate. *P≤0.05; #P≤0.01 versus control.</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

The effect of p38 silencing on Wnt/β-catenin pathway signal transduction.

<p>(A) One representative western blot used to quantify p38-siRNA interference efficiency and β-catenin 24 h of transfection. Total cellular proteins (20 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) Luciferase activity of TopFlash reporter plasmid in cells transfected with p38-siRNA and NS-siRNA. Luciferase activity was assayed after 24 h of transfection. 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. (C) 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 24 h of transfection with p38-siRNA. The graphs show fold differences in transcript abundance in comparison with non-specific siRNA-treated 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 versus control.</p

Vitiligo: A Possible Model of Degenerative Diseases

<div><p>Vitiligo is characterized by the progressive disappearance of pigment cells from skin and hair follicle. Several <i>in vitro</i> and <i>in vivo</i> studies show evidence of an altered redox status, suggesting that loss of cellular redox equilibrium might be the pathogenic mechanism in vitiligo. However, despite the numerous data supporting a pathogenic role of oxidative stress, there is still no consensus explanation underlying the oxidative stress-driven disappear of melanocytes from the epidermis. In this study, <i>in vitro</i> characterization of melanocytes cultures from non-lesional vitiligo skin revealed at the cellular level aberrant function of signal transduction pathways common with neurodegenerative diseases including modification of lipid metabolism, hyperactivation of mitogen-activated protein kinase (MAPK) and cAMP response element-binding protein (CREB), constitutive p53-dependent stress signal transduction cascades, and enhanced sensibility to pro-apoptotic stimuli. Notably, these long-term effects of subcytotoxic oxidative stress are also biomarkers of pre-senescent cellular phenotype. Consistent with this, vitiligo cells showed a significant increase in p16 that did not correlate with the chronological age of the donor. Moreover, vitiligo melanocytes produced many biologically active proteins among the senescence-associated secretory phenotype (SAPS), such as interleukin-6 (IL-6), matrix metallo proteinase-3 (MMP3), cyclooxygenase-2 (Cox-2), insulin-like growth factor-binding protein-3 and 7 (IGFBP3, IGFBP7). Together, these data argue for a complicated pathophysiologic puzzle underlying melanocytes degeneration resembling, from the biological point of view, neurodegenerative diseases. Our results suggest new possible targets for intervention that in combination with current therapies could correct melanocytes intrinsic defects.</p> </div

p53 and PML expression co-localize with melanocyte specific markers.

<p>Double immunofluoresce staining was used to co-localize the expression of the melanocyte-specific marker tyrosinase and the expression of p53 and PML. Nuclei were labelled with bisbenzidine (DAPI). Original magnification 40×.</p