Apoptosis In Human Melanocytes Research Articles

NO-induced apoptosis in human melanocytes from lightly and darkly pigmented skin.

Nitric oxide (NO) is a potent modulator of programmed cell death, with the ability to both induce and prevent apoptosis. Some of the factors that are capable of triggering apoptosis of skin cells also cause NO overproduction in the epidermis. Unlike keratinocytes, melanin-producing melanocytes are highly resistant to apoptotic death. To investigate whether NO can induce apoptosis in normal human epidermal melanocytes and whether the pigmentation phenotype of the cells can affect their response to NO. Human epidermal melanocytes, derived from lightly and darkly pigmented neonatal foreskins, were cultured in the presence of various concentrations of SPER/NO. The effect of NO released from its donor on the cell morphology, viability, and proliferation was assessed. The ability of NO to induce cell apoptosis was evaluated by Hoechst 33342 staining, DNA fragmentation assay, flow cytometry with annexin V and propidium iodide staining, determination of caspase 3/7, 8, and 9 activities, and assessment of changes in the cell expression levels of BAX and BCL-2. We have shown that NO is capable of inducing apoptosis in normal human epidermal melanocytes in vitro, with preferential activation of the intrinsic (mitochondrial) pathway. Melanocytes from darkly pigmented skin showed a strong increase in BCL-2 expression in response to NO and were significantly more resistant to apoptosis than those from lightly pigmented skin. The pigmentation phenotype may be an important factor modulating the response of human epidermal melanocytes to proapoptotic activity of extracellular NO.

MAPK and Notch-Mediated Effects of Meso-Xanthin F199 Compounds on Proliferative Activity and Apoptosis of Human Melanocytes in Three-Dimensional Culture.

Meso-Xanthin (Meso-Xanthin F199™) is a highly active antiaging injection drug of the latest generation. The main acting compound is fucoxanthin, supplemented with several growth factors, vitamins, and hyaluronic acid. Previous examination of fucoxanthin on melanocytes showed its ability to inhibit skin pigmentation through different signaling pathways focused on suppression of melanogenic-stimulating receptors. In turn, the anticancer property of fucoxanthin is realized through MAPK and PI3K pathways. We aimed to evaluate the effect of fucoxanthin and supplemented growth factors on melanocyte growth and transformation at a proteomic level. The effect of fucoxanthin on melanocytes cultivated in three-dimensional (3D) condition was examined using high-throughput proteomic and system biology approaches to disclose key molecular events of the targeted action. Our results demonstrated significant inhibition of cell differentiation and ubiquitination processes. We found that the negative regulation of PSME1 and PTGIS largely determines the inhibition of NF-κB and MAPK2. Besides, fucoxanthin selectively inhibits cell differentiation via negative regulation of Raf signaling and the upstream activation of IL-1 signaling. It is assumed that inhibition of Raf influences the Notch-4 signaling and switches off the MAPK/MAPK2 cascade. Blockage of MAPK/MAPK2 is feasible due to suppression of Ras and NF-κB by the addressed action of IKKB, IKK2, and TRAF6. Suggestively, Meso-Xanthin F199™ can manage processes of proliferative activity and inhibition of apoptosis due to composition of fucoxanthin and growth-stimulating factors, which may increase the risk of skin cancer development under certain condition.

IFN‑γ induces apoptosis in human melanocytes by activating the JAK1/STAT1 signaling pathway.

The present study aimed to investigate the role of janus kinase (JAK)1/STAT1 in interferon (IFN)-γ-induced apoptosis in human melanocytes. Following IFN-γ treatment, the viability of human melanocytes were analyzed using a Cell Counting Kit-8 assay and the apoptotic rate was determined using flow cytometry. Western blotting was also performed to analyze the phosphorylation levels of JAK1, JAK2 and the transcriptional factor STAT1, as well as the expression levels of Bcl-2, Bax, Bcl-2 homologous antagonist killer (Bak) and cleaved caspase-3. Finally, following the pretreatment with the STAT1 inhibitor fludarabine, human melanocytes were treated with IFN-γ and flow cytometry was used to detect the apoptotic rate. The results revealed that IFN-γ reduced the proliferation and induced the apoptosis of human melanocytes. In addition, IFN-γ treatment led to decreased expression levels of Bcl-2 and increased expression levels of Bax, Bak and cleaved caspase-3, alongside the activation of the JAK1/STAT1 signaling pathway. Conversely, the pretreatment with the STAT1 inhibitor fludarabine decreased the apoptotic rate of human melanocytes following IFN-γ induction. In conclusion, the findings of the present study suggested that IFN-γ may induce the apoptosis of human melanocytes by activating the JAK1/STAT1 signaling pathway, alongside increasing the expression levels of Bax, Bak and cleaved caspase-3, and decreasing the expression levels of Bcl-2.

Mesenchymal stem cells promote human melanocytes proliferation and resistance to apoptosis through PTEN pathway in vitiligo

BackgroundVitiligo is an acquired chronic and recurrent skin disease that causes a depigmentation disorder, resulting in selective destruction of melanocytes (MC). However, the mechanism that leads to melanocyte dysfunction and death remains unclear.MethodsWe performed RNA sequencing, immunohistochemistry, and immunoblotting to characterize the patterns of phosphatase and tensin homolog (PTEN)/phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway activation in vitiligo. We also cocultured primary melanocytes with mesenchymal stem cells (MSCs) in a Transwell system to explore how MSCs inhibit the PTEN/PI3K/AKT pathway in melanocytes.ResultsWe identified that vitiligo normal-lesional junction skin presented with high expression of PTEN, which led to the inhibition of AKT phosphorylation (p-AKT) at S-473. Furthermore, PTEN overexpression led to oxidative stress-induced apoptosis in melanocytes. Coculturing with MSCs enhanced the cell proliferation of human melanocytes and repressed PTEN expression, which inhibited oxidative stress-induced apoptosis.ConclusionWe report that vitiligo patients present with high PTEN expression, which may play a role in the impairment of melanocytes. Furthermore, our study provides evidence that MSCs target the PTEN/PI3K/AKT pathway to regulate cell proliferation and apoptosis in human melanocytes, indicating that MSCs may serve as a promising therapy for vitiligo.

Fas-FasL interaction in cytotoxic T cell-mediated vitiligo: The role of lesional expression of tumor necrosis factor-α and interferon-γ in Fas-mediated melanocyte apoptosis.

Vitiligo is an acquired skin depigmentation disorder resulting from the selective loss of epidermal melanocytes, and previous studies have suggested that a T lymphocyte-mediated mechanism has a role in melanocyte loss. Although Fas-Fas ligand (FasL) interactions are important for T lymphocytes to mediate cytotoxicity, there are only few reports examining the involvement of the Fas-FasL pathway in vitiligo using in vivo mouse models. In addition, there have been no reports concerning Fas-mediated apoptosis in human melanocytes in vitro. In this study, we found that the Fas-mediated pathway is involved in cytotoxic T lymphocyte (CTL)-dependent vitiligo in a mouse model and FasL-induced apoptosis of human melanocytes. Tumor necrosis factor (TNF)-α and interferon (IFN)-γ, the expression levels of which have been reported to be elevated in lesional skin of patients with vitiligo, synergistically upregulated Fas expression on human melanocytes but inhibited the Fas-mediated apoptosis. Treatment with TNF-α and IFN-γ synergistically upregulated the expression of the anti-apoptotic genes, c-IAP2, c-FLIP and MCL1. A siRNA knock-down study showed that c-FLIP and MCL1, but not c-IAP2, were involved in inducing synergistic inhibitory effects on Fas-mediated apoptosis. Furthermore, we found that FasL and TNF-related apoptosis-inducing ligand (TRAIL) synergistically induced apoptosis on human melanocytes. In conclusion, our results suggest that the Fas-FasL pathway is involved in CTL-dependent vitiligo and the elevated expression levels of TNF-α and IFN-γ in lesional skin may act synergistically on melanocytes to suppress Fas-mediated apoptosis.

Baicalein protects human vitiligo melanocytes from oxidative stress through activation of NF-E2-related factor2 (Nrf2) signaling pathway

Vitiligo is a complex disorder characterized by patchy loss of skin pigmentation due to abnormal melanocyte function. Overwhelming evidences have suggested that oxidative stress plays a major role in the loss of melanocytes thereby mediating the onset and progression of vitiligo. The nuclear factor erythroid 2-like factor 2 (Nrf2) is a master regulator of cellular redox homeostasis and the activation of Nrf2 signaling pathway is impaired in the vitiligo melanocytes. Baicalein, as flavonoid extracted from the Scutellaria baicalensis, has been proved to possess the ability to activate Nrf2 signaling pathway in other cell types and mouse model. Our previous data found that baicalein exerts a cytoprotective role in H2O2-induced apoptosis in human melanocytes cell line (PIG1). Based on these founding, we hypothesized that baicalein activates Nrf2 signaling pathway, alleviates H2O2-induced mitochondrial dysfunction and cellular damage, thereby protecting human vitiligo melanocytes from oxidative stress. In the present study, we found that baicalein effectively inhibited H2O2-induced cytotoxicity and apoptosis in human vitiligo melanocytes (PIG3V). Further results demonstrated that baicalein promoted Nrf2 nucleus translocation as well as up-regulated the expression of Nrf2 and its target gene, heme oxygenase-1 (HO-1). Moreover, the protective effects of baicalein against H2O2-induced cellular damage and apoptosis as well as mitochondrial dysfunction were abolished by Nrf2 knockdown. Additionally, we observed that Nrf2 knockdown suppressed proliferation and increased the sensitivity of PIG3V cells to H2O2 treatment. Finally, we explored the mechanism of baicalein associated with Nrf2 activation and found that the phosphorylation of Nrf2 as well as ERK1/2and PI3K/AKT signaling were not involved in the baicalein-induced activation of Nrf2. Taken together, these data clearly suggest that baicalein enhances cellular antioxidant defense capacity of human vitiligo melanocytes through the activation of the Nrf2 signaling pathway, providing beneficial evidence for the application of baicalein in the vitiligo treatment.

HMGB1/RAGE Mediates UVB-Induced Secretory Inflammatory Response and Resistance to Apoptosis in Human Melanocytes

The major modifiable risk factor in melanomagenesis is UV exposure and mutagenesis of melanocytes. Other UV-induced events that contribute to early tumorigenesis are poorly understood. Herein we show that therepeated exposure of human primary melanocytes to UVB results in a sustained senescence response, increases in expression of signal transducer and activator of transcription 1, MX1, OAS2, and IRF7 proteins of up to 75-fold, and resistance to subsequent UVB-induced apoptosis. In the setting of UVB-induced DNA damage, we detected time-dependent increases in the release of damage-associated molecular patterns such as high-mobility group box 1 (HMGB1). After intermittent UVB exposure, melanocytes treated with the JAK inhibitor ruxolitinib reduced expression of HMGB1 and MX1 as well as activation of JAK1 (pJAK1) and signal transducer and activator of transcription 1 (pSTAT1). In addition, melanocytes expressing small hairpin RNA selective for the HMGB1 receptor, receptor for advanced glycosylation end product (RAGE), exhibited decreased expression of both HMGB1 and MX1 after UVB exposure. The response of small hairpin RAGE-infected cells to human recombinant HMGB1 was blunted with decreased MX1 expression and JAK activation. Finally, depletion of receptor for advanced glycosylation end product decreased UVB-induced resistance to apoptosis (P < 0.05). These findings highlight a cell autonomous response to UV damage, contribute to their resistance to apoptosis and cell death, and may have implications for early stages of melanoma development.

1215 RAGE mediates UVB-induced persistent DNA Damage Response (DDR) and resistance to apoptosis in human melanocytes

1215 RAGE mediates UVB-induced persistent DNA Damage Response (DDR) and resistance to apoptosis in human melanocytes

A single‐nucleotide polymorphism of miR‐196a‐2 and vitiligo: an association study and functional analysis in a Han Chinese population

Recent evidence indicates that oxidative stress and genetic factors play an important role in the pathogenesis of vitiligo. SNPs in miRNAs involved in oxidative stress could potentially influence the development of vitiligo. In this case-control study, we investigated the association of a functional SNP of rs11614913 in miR-196a-2 with risk of vitiligo. A significantly lower risk of vitiligo was associated with the rs11614913 miR-196a-2 CC genotype (adjusted OR, 0.77; CI, 0.60-0.98). In addition, TYRP1 gene expression was considerably down-regulated by the rs11614913 C allele in miR-196a-2, which lowered the levels of intracellular reactive oxygen species (ROS) and reduced the proportion of early apoptosis in human melanocytes in response to H2 O2 treatment. Our data suggest that the rs11614913 C allele in miR-196a-2 confers potential protection against oxidative effects on human melanocytes through the modulation of the target gene, TYRP1, which may account for the decreased risk of vitiligo in this study population.

Baicalein protects Human melanocytes from H2O2-induced apoptosis via inhibiting mitochondria-dependent caspase activation and the p38 MAPK pathway

The removal of H2O2 by antioxidants has been proven to be beneficial to patients with vitiligo. Baicalein (5,6,7-trihydroxyflavone; BE) has antioxidant activity and has been used in vitiligo therapy in Chinese traditional medicine. In this study, we investigated the potential protective effect and mechanisms of BE against H2O2-induced apoptosis in human melanocytes. Melanocytes from the PIG1 cell line were pretreated with different concentrations of BE for 1h, followed by exposure to 1.0 mM H2O2 for 24h. Cell apoptosis, reactive oxygen species levels, and mitochondrial membrane potentials were evaluated by flow cytometry, and cell viability was determined by an MTT assay. The expressions of Bax, Bcl-2, caspase-3, total and phosphorylated ERKs, and p38 MAPK were assayed by Western blot to investigate the possible molecular mechanisms. Our results showed that BE significantly inhibited H2O2-induced apoptosis, intracellular reactive oxygen species generation, and changes in the mitochondrial membrane potential. It also reduced the Bax/Bcl-2 ratio, the release of cytochrome c, the activation of caspase-3, and the phosphorylation of p38 MAPK in a concentration-dependent manner. The results demonstrate for the first time that BE exerts a cytoprotective role in H2O2-induced apoptosis by inhibiting the mitochondria-dependent caspase activation and p38 MAPK pathway.

Redox-Dependent Translocation of p53 to Mitochondria or Nucleus in Human Melanocytes after UVA- and UVB-Induced Apoptosis

The p53 protein is an important transcription factor and tumor suppressor that is induced in response to many forms of cellular stress. UVA irradiation of human melanocytes caused generation of reactive oxygen species, which altered the intracellular redox balance and was accompanied by translocation of p53 to mitochondria. In contrast, UVB did not affect the redox status and p53 was translocated to the nucleus. Although different intracellular location of p53, UVA/B induced apoptosis through the intrinsic pathway detected as translocation of Bax to mitochondria, release of cytochrome c, and activation of caspases. These events were all prevented by inhibition of p53 with pifithrin-alpha. Furthermore, inhibition of p53 prevented lysosomal membrane permeabilization, detected as translocation of cathepsins to the cytosol, after UVB exposure, whereas UVA-induced lysosomal release was unaffected by inhibition of p53. In control cells, p53 coimmunoprecipitated with the antiapoptotic proteins Bcl-2 and Bcl-x(L) and upon UVA exposure the interaction was replaced by binding to the proapoptotic proteins Bax, Noxa, and Puma. Our findings suggest that UVA-induced apoptosis is caused by extensive oxidative damage leading to p53-regulated mitochondrial release, whereas UVB induces DNA damage and apoptosis signaling upstream of lysosomal membrane permeabilization.

JNK mediates UVB-induced apoptosis upstream lysosomal membrane permeabilization and Bcl-2 family proteins

UVB irradiation induced phosphorylation of JNK and subsequent apoptosis in human melanocytes. Depletion of both JNK1 and JNK2 expression using siRNA transfection, protected against apoptosis, as detected by decreased nuclear fragmentation and caspase-3 activity, as well as reduced translocation of Bax to mitochondria. Moreover, release of cathepsin B and D from lysosomes to the cytosol was reduced when JNK expression was suppressed by siRNA, demonstrating a JNK dependent regulation of lysosomal membrane permeabilization. In unirradiated control melanocytes, coimmunoprecipitation showed that Bim was sequestered by Mcl-1, which had a pro-survival function. After UVB irradiation, a significant decrease in Mcl-1 protein level was found, which was prevented by addition of a proteasome inhibitor. The interaction between Bim and Mcl-1 was reduced in response to UVB irradiation and Bim was phosphorylated in a JNK dependent manner. In conclusion, these findings suggest JNK to have an important pro-apoptotic function following UVB irradiation in human melanocytes, by acting upstream of lysosomal membrane permeabilization and Bim phosphorylation.

Ultraviolet (UV) A- and UVB-induced redox alterations and activation of nuclear factor-κB in human melanocytes-protective effects ofα-tocopherol

Despite compelling evidence that ultraviolet (UV) irradiation causes melanoma the knowledge concerning reaction pathways and signalling transduction in melanocytes is still limited. To evaluate the protective capacity of alpha-tocopherol and beta-carotene during UVA and UVB irradiation of human melanocytes in vitro. Primary cultures of normal human melanocytes were irradiated by different wavelengths within the UV spectrum (UVA 6 J cm(-2), UVB 60 mJ cm(-2)). Redox alterations and apoptosis were studied and the protective potential of alpha-tocopherol and beta-carotene was evaluated. UVA and UVB irradiation decreased the intracellular concentration of reduced glutathione and activated the transcription factor nuclear factor (NF)-kappaB, detected as the increased level of the p65 subunit and translocation to the nucleus. This coincided with a rise in the level of gamma-glutamyl-cysteine-synthetase, the rate-limiting enzyme of the glutathione synthesis. UVA and UVB caused apoptotic cell death as detected by nuclear fragmentation and caspase activation 24 h postirradiation. Pretreatment with alpha-tocopherol prevented UVA- and UVB-induced glutathione loss, NF-kappaB translocation and diminished apoptosis, but beta-carotene did not show a similar protective capacity. Further, exposure to alpha-tocopherol by itself reduced cell proliferation rate. UVA and UVB irradiation affected the intracellular redox state and increased the frequency of apoptosis in human melanocytes in vitro. alpha-Tocopherol might be a useful substance in protecting melanocytes from UV-induced damage.

UVA/B-Induced Apoptosis in Human Melanocytes Involves Translocation of Cathepsins and Bcl-2 Family Members

We demonstrate UVA/B to induce apoptosis in human melanocytes through the mitochondrial pathway, displaying cytochrome c release, caspase-3 activation, and fragmentation of nuclei. The outcome of a death signal depends on the balance between positive and negative apoptotic regulators, such as members of the Bcl-2 protein family. Apoptotic melanocytes, containing fragmented nucleus, show translocation of the proapoptotic proteins Bax and Bid from the cytosol to punctate mitochondrial-like structures. Bcl-2, generally thought to be attached only to membranes, was in melanocytes localized in the cytosol as well. In the fraction of surviving melanocytes, that is, cells with morphologically unchanged nucleus, the antiapoptotic proteins Bcl-2 and Bcl-X(L) were translocated to mitochondria following UVA/B. The lysosomal proteases, cathepsin B and D, which may act as proapoptotic mediators, were released from lysosomes to the cytosol after UVA/B exposure. Proapoptotic action of the cytosolic cathepsins was confirmed by microinjection of cathepsin B, which induced nuclear fragmentation. Bax translocation and apoptosis were markedly reduced in melanocytes after pretreatment with either cysteine or aspartic cathepsin inhibitors. No initial caspase-8 activity was detected, excluding involvement of the death receptor pathway. Altogether, our results emphasize translocation of Bcl-2 family proteins to have central regulatory functions of UV-induced apoptosis in melanocytes and suggest cathepsins to be proapoptotic mediators operating upstream of Bax.

Dopamine-induced apoptosis in human melanocytes involves generation of reactive oxygen species

Previous studies have shown that significant increases in urinary and plasma levels of several monoamines and their metabolites characterize the onset of vitiligo and its progression. Recently, both epidermal keratinocytes and melanocytes were found to have the capacity for the biosynthesis of several catecholamines and serotonin. Some monoamines and their metabolites can induce apoptosis and cytotoxicity in neural cells. However, no previous report has investigated the potential role of these monoamines in inducing apoptosis or cytotoxicity in melanocytes. To study the effects of dopamine (DA), norepinephrine (NE), epinephrine (EP), and serotonin (5-HT) on melanocyte cytotoxicity and apoptosis. Primary cultures of normal human melanocytes established from the foreskins of normal individuals were treated with different concentrations of DA, NE, EP and 5-HT for 5 and 7 days. Cell viability was measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Melanocyte apoptosis was evaluated by morphological examination and flow cytometric analysis. We also measured the generation of reactive oxygen species (ROS) after DA treatment. Among the four monoamines used in this study, only DA had an effect, dose-dependently decreasing the melanocyte viability at concentrations ranging from 0.01 to 100 micromol L(-1) (0.1 and 1 micromol L(-1), P < 0.05; 10 micromol L(-1), P < 0.01). In addition, DA-induced melanocyte apoptosis was evidenced by the increased percentage of sub-G1 cells from 7.71 +/- 0.28% (control) to 12.22 +/- 1.05% (0.1 micromol L(-1) DA) (P < 0.005), and treatment with the antioxidant N-acetylcysteine (NAC) reversed this apoptotic effect. DA treatment led to the generation of ROS, which could be prevented by pretreatment with NAC. DA can induce melanocyte apoptosis, which might be related to the generation of ROS. This novel effect might play an important role in the development or progression of vitiligo, which is currently viewed as a disease process closely related to melanocyte apoptosis.

Sphingosine-1-phosphate-induced ERK activation protects human melanocytes from UVB-induced apoptosis.

Ultraviolet B (UVB) is known to induce apoptosis in human melanocytes. Here we show the cytoprotective effect of sphingosine-1-phosphate (S1P) against UVB-induced apoptosis. We also show that UVB-induced apoptosis of melanocytes is mediated by caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage, and that S1P prevents apoptosis by inhibiting this apoptotic pathway. We further investigated three major mitogen-activated protein (MAP) kinases after UVB irradiation. UVB gradually activated c-Jun N-terminal kinase (JNK) and p38 MAP kinase, while extracellular signal-regulated protein kinase (ERK) was inactivated transiently. Blocking of the p38 MAP kinase pathway using SB203580 promoted cell survival and inhibited the activation of caspase-3 and PARP cleavage. These results suggest that p38 MAP kinase activation may play an important role in the UVB-induced apoptosis of human melanocytes. To explain this cytoprotective effect, we next examined whether S1P could inhibit UVB-induced JNK and p38 MAP kinase activation. However, S1P was not found to have any influence on UVB-induced JNK or p38 MAP kinase activation. In contrast, S1P clearly stimulated the phosphorylation of ERK, and the specific inhibition of the ERK pathway using PD98059 abolished the cytoprotective effect of S1P. Based on these results, we conclude that the activation of p38 MAP kinase plays an important role in UVB-induced apoptosis, and that S1P may show its cytoprotective effect through ERK activation in human melanocytes.