Microphthalmia-associated transcription factor regulates RAB27A gene expression and controls melanosome transport.

Melanosomes are lysosome-related organelles specialized in melanin synthesis and transport. In this study, we show that microphthalmia-associated transcription factor (MITF) silencing induces melanosome gathering around the nucleus and causes the relocalization of Rab27A, Slac2a-Mlph, and Myo5a that control the transport of melanosomes on the actin network. In an attempt to elucidate the mechanism by which MITF controls melanosome distribution, we identify RAB27A as a new MITF target gene. Indeed, MITF silencing leads to a dramatic decrease in Rab27A expression and blocks the stimulation of Rab27A expression evoked by cAMP. Further, forced expression of MITF increases Rab27A expression, indicating that MITF is required and sufficient for Rab27A expression in melanoma cells. MITF binds to two E-boxes in the proximal region of the Rab27A promoter and stimulates its transcriptional activity. Finally, re-expression of Rab27A, in MITF-depleted cells, restores the transport of melanosomes to the cell periphery. These results show that RAB27A is a new direct transcriptional target of MITF and link MITF to melanosome transport, another key parameter of melanocyte differentiation and skin pigmentation. Interestingly, Rab27A is involved in other fundamental physiological functions, such as the transport of lytic granules and insulin secretion. Thus our results, deciphering the mechanism of Rab27A transcriptional regulation, have an interest that goes beyond the skin pigmentation field

GSK3β inhibition promotes melanogenesis in Mouse B16 Melanoma cells and human normal melanocytes

Glycogen synthase kinase 3beta (GSK3beta) is implicated in many biological events, including embryonic development, cell differentiation, apoptosis, and the insulin response. GSK3beta also plays a key role in the Wnt/beta-catenin pathway. The master regulator of the pigmentation microphthalmia-associated transcription factor (MITF) is a target for the Wnt pathway, however, to date, the regulatory role of GSK3beta in the control of melanogenesis has not been elucidated. In this study, we evaluated the effect of inhibiting GSK3beta activity on the regulation of melanocyte differentiation. Exposure of the murine melanoma cell line B16 and normal human melanocytes to GSK3beta specific inhibitors (SB216763, SB415286, BIO, and LiCl) resulted in a dose-dependent accumulation of beta-catenin. This is associated with the induction of melanocyte differentiation-associated markers such as melanin synthesis, tyrosinase activity, and expression of tyrosinase and the microphthalmia-associated transcription factor. Attenuation of GSK3beta activity has an inhibitory effect on cell growth, and this was accompanied by morphological changes. Moreover, treatment of B16 cells with a siRNA targeted against beta-catenin completely abolished the promelanogenic effect of GSK3beta inhibition, however, the overexpression of a constitutively active mutant form of beta-catenin (pCS2beta-cat-mut) only slightly increased the degree of pigmentation. These results demonstrated that GSK3beta is implicated in the regulation of melanogenesis and that pharmacological inhibition of its activity could increase melanin synthesis through mechanisms probably not restricted to Wnt/beta-catenin pathway activation

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

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

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

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

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

Summary scheme of possible role of PPARγ modulation in counteracting PUVA-SIPS of HDFs.

<p>PUVA exposure induced intracellular generation of ROS, alteration of mitochondria function, activation of antioxidant stress response and MAPK phosphorylation pathway, dysregulation of membrane lipid metabolism, DNA-oxidative damage and altered expression of cell cycle regulators. PPARγ modulation by Octa may counteract PUVA-induced senescence-like phenotype. Moreover, Octa ability to reduce phospholipid oxidation and oxysterol generation contributes to the reduction of PUVA-induced inflammatory response and redox imbalance.</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

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