Zhe Jian

Heme Oxygenase-1 Protects Human Melanocytes from H2O2-Induced Oxidative Stress via the Nrf2-ARE Pathway

Oxidative stress caused by hydrogen peroxide (H(2)O(2)) leads to cell death and has been implicated in the pathogenesis of vitiligo. The nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element (ARE), a major antioxidant pathway, regulates oxidative stress-related cytoprotective genes. We hypothesized that the Nrf2-ARE pathway protects human melanocytes from H(2)O(2)-induced oxidative damage through the induction of downstream antioxidative genes. Thus, we used Nrf2 short interfering RNA (siRNA) and pCMV6-XL5-Nrf2 to downregulate or upregulate Nrf2 expression in immortalized human melanocyte cell line PIG1. The melanocytes were then analyzed under different oxidative stress conditions for cell viability and apoptosis. Our study demonstrated that heme oxygenase-1 (HO-1) was the most induced antioxidant gene in PIG1 cells after treatment with H(2)O(2). Knockdown of Nrf2 or zinc protoporphyrin IX (ZnPP) treatment increased cell death caused by H(2)O(2) in melanocytes, but upregulation of Nrf2 or hemin treatment reduced cell death caused by H(2)O(2) in melanocytes. In addition, the H(2)O(2)-induced Nrf2-ARE/HO-1 pathway was confirmed in primary cultured human melanocytes by examining the expression and translocation of Nrf2 and HO-1. These data suggested that regulation of the Nrf2/HO-1 pathway can reduce H(2)O(2)-induced oxidative damage in human melanocytes. Our data demonstrate that HO-1 protects human melanocytes from oxidative damage via the Nrf2-ARE pathway.

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

The removal of H(2)O(2) 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 H(2)O(2)-induced apoptosis in human melanocytes. Melanocytes from the PIG1 cell line were pretreated with different concentrations of BE for 1 h, followed by exposure to 1.0 mM H(2)O(2) for 24 h. 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 H(2)O(2)-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 H(2)O(2)-induced apoptosis by inhibiting the mitochondria-dependent caspase activation and p38 MAPK pathway.

Impaired Activation of the Nrf2-ARE Signaling Pathway Undermines H2O2-Induced Oxidative Stress Response: A Possible Mechanism for Melanocyte Degeneration in Vitiligo

Vitiligo melanocytes possess higher susceptibility to oxidative insults. Consistent with this, impairment of the antioxidant defense system has been reported to be involved in the onset and progression of vitiligo. Our previous study showed that the nuclear factor E2-related factor 2-antioxidant response element (Nrf2-ARE) pathway and its downstream antioxidant enzyme heme oxygenase-1 (HO-1) are crucial for melanocytes to cope with H2O2-induced oxidative damage. Here, we sought to determine whether the diminished Nrf2-ARE activity that contributes to reduced downstream antioxidant enzymes and increased oxidative stress could be the reason why melanocytes are more vulnerable to vitiligo. We found that vitiligo melanocytes exhibited hypersensitivity to H2O2-induced oxidative injury because of reduced Nrf2 nuclear translocation and transcriptional activity, which led to decreased HO-1 expression and aberrant redox balance. Moreover, we also found that the level of serum HO-1 was significantly decreased and that of IL-2 was markedly increased in 113 vitiligo patients when compared with healthy controls. These data demonstrate that impaired activation of Nrf2 under oxidative stress could result in decreased expression of antioxidant enzymes and increased death of vitiligo melanocytes.

Association between FOXP3 polymorphisms and vitiligo in a Han Chinese population

Vitiligo is an autoimmune chronic depigmentation disorder caused by melanocyte loss. Previous studies found that CD4+CD25+ regulatory T‐cell (Treg) dysfunction was involved in the pathogenesis of vitiligo and that gene polymorphisms in forkhead box P3 (FOXP3) – a master regulator of Treg development and function – were associated with susceptibility to some autoimmune disorders. Therefore, we hypothesized that functional polymorphisms of the FOXP3 gene might be associated with vitiligo via dysregulation of Treg cells.

Foxp3 expression in melanoma cells as a possible mechanism of resistance to immune destruction.

The forkhead transcription factor Foxp3 is the only definitive marker of CD4+CD25+ regulatory T cells (Tregs) and has been identified as a key regulator in the development and function of Tregs. Foxp3 expression has been reported in a variety of solid tumors, including melanoma. In this study, we validated Foxp3 expression in both tumor-infiltrating Tregs and melanoma cells by performing immunohistochemical analysis of human melanoma tissue sections. Further, we assessed Foxp3 expression in melanoma cell lines by performing flow cytometry, confocal microscopic analysis, reverse transcription-polymerase chain reaction (RT–PCR), and Western blotting. Inhibition of Foxp3 expression in melanoma cells using small interfering RNA (siRNA) resulted in downregulation of B7-H1 and transforming growth factor (TGF)-β expression; in contrast, Foxp3 overexpression resulted in the upregulation of the expression of these proteins. Coculture of Foxp3-expressing melanoma cells with naive CD4+CD25− T cells resulted in strong inhibition of T-cell proliferation. This antiproliferative effect was partially abrogated by specific inhibition of Foxp3 expression and was effectively enhanced by overexpression of Foxp3. We observed an attenuated antiproliferative effect even when melanoma cells and T cells in the coculture were separated using Transwell inserts. These findings indicated that melanoma cells could have Foxp3-dependent Treg-like suppressive effects on T cells and suggested that the mimicking of Treg function by melanoma cells may represent a possible mechanism of tumor resistance to immune destruction in the melanoma tumor microenvironment.

Oxidative stress drives CD8+ T-cell skin trafficking in patients with vitiligo through CXCL16 upregulation by activating the unfolded protein response in keratinocytes.

Background In patients with vitiligo, an increased reactive oxygen species (ROS) level has been proved to be a key player during disease initiation and progression in melanocytes. Nevertheless, little is known about the effects of ROS on other cells involved in the aberrant microenvironment, such as keratinocytes and the following immune events. CXCL16 is constitutively expressed in keratinocytes and was recently found to mediate homing of CD8+ T cells in human skin. Objective We sought to explicate the effect of oxidative stress on human keratinocytes and its capacity to drive CD8+ T‐cell trafficking through CXCL16 regulation. Methods We first detected putative T‐cell skin‐homing chemokines and ROS in serum and lesions of patients with vitiligo. The production of candidate chemokines was detected by using quantitative real‐time PCR and ELISA in keratinocytes exposed to H2O2. Furthermore, the involved mediators were analyzed by using quantitative real‐time PCR, Western blotting, ELISA, and immunofluorescence. Next, we tested the chemotactic migration of CD8+ T cells from patients with vitiligo mediated by the CXCL16‐CXCR6 pair using the transwell assay. Results CXCL16 expression increased and showed a positive correlation with oxidative stress levels in serum and lesions of patients with vitiligo. The H2O2‐induced CXCL16 expression was due to the activation of 2 unfolded protein response pathways: kinase RNA (PKR)–like ER kinase–eukaryotic initiation factor 2&agr; and inositol‐requiring enzyme 1&agr;–X‐box binding protein 1. CXCL16 produced by stressed keratinocytes induced migration of CXCR6+CD8+ T cells derived from patients with vitiligo. CXCR6+CD8+ T‐cell skin infiltration is accompanied by melanocyte loss in lesions of patients with vitiligo. Conclusion Our study demonstrated that CXCL16‐CXCR6 mediates CD8+ T‐cell skin trafficking under oxidative stress in patients with vitiligo. The CXCL16 expression in human keratinocytes induced by ROS is, at least in part, caused by unfolded protein response activation.

The association of functional polymorphisms in the aryl hydrocarbon receptor (AHR) gene with the risk of vitiligo in Han Chinese populations.

Background  Vitiligo is an acquired depigmentation disorder resulting from selective destruction of melanocytes. The aryl hydrocarbon receptor (AHR) is vital to the regulation of melanogenesis and melanocyte proliferation and differentiation through modulating the expressions of melanogenesis‐related genes. AHR mutations may negatively affect AHR proteins and its target genes. Therefore, we hypothesized that AHR polymorphisms might be involved in vitiligo by impacting the transcriptional activities of related genes as mentioned above.

AHR promoter variant modulates its transcription and downstream effectors by allele-specific AHR-SP1 interaction functioning as a genetic marker for vitiligo

Vitiligo is an acquired depigmentation disorder largely caused by defective melanocyte- or autoimmunity-induced melanocyte destruction. The aryl hydrocarbon receptor (AHR) is essential for melanocyte homeostasis and immune process, and abnormal AHR was observed in vitiligo. We previously identified the T allele of AHR −129C > T variant as a protective factor against vitiligo. However, biological characterization underlying such effects is not fully certain, further validation by mechanistic research is warranted and was conducted in the present study. We showed that −129T allele promoted AHR transcriptional activity through facilitating its interaction with SP1 transcription factor (SP1) compared with −129C allele. We subsequently found reduced peripheral AHR and SP1 transcript expressions in vitiligo and a negative correlation of AHR level with disease duration. We also investigated AHR-related cytokines and observed increased serum TNF-α concentration and diminished serum levels of IL-10 and TGF-β1 in vitiligo. Further genetic analysis showed that -129T carriers possessed higher levels of AHR and IL-10 than −129C carriers. Therefore, our study indicates that the modulation of AHR transcription by a promoter variant has a profound influence on vitiligo, not only advancing our understanding on AHR function but also providing novel insight into the pathogenesis of degenerative or autoimmune diseases including vitiligo.

Simvastatin Protects Human Melanocytes from H2O2-Induced Oxidative Stress by Activating Nrf2

The prevention of hydrogen peroxide (H2O2)-induced oxidative stress has proved to be beneficial to vitiligo patients. Simvastatin possesses antioxidative capacity and has shown protective effect in various oxidative stress-related diseases. However, whether simvastatin can protect human melanocytes against oxidative stress has not been investigated. In this study, we initially found that pretreatment with 0.1 μmol/L to 1.0 μmol/L simvastatin led to increased cell viability and decreased cell apoptosis of melanocytes in response to H2O2. In addition, simvastatin was able to potentiate the activity of antioxidant enzymes and lessen intracellular reactive oxygen species accumulation. Furthermore, we found that simvastatin promoted the activation of nuclear erythroid 2-related factor (Nrf2) and that knockdown of Nrf2 abolished the protective effect of simvastatin against H2O2-induced oxidative damage. More importantly, the mutual enhancement between mitogen-activated protein kinase pathways and p62 contributed to simvastatin-induced Nrf2 activation in melanocytes. Finally, simvastatin showed more antioxidative capacity and better protective effect than aspirin in H2O2-treated melanocytes. Taken together, our results show that simvastatin protects human melanocytes from H2O2-induced oxidative stress by activating Nrf2, thus supporting simvastatin as a potential therapeutic agent for vitiligo.

Aspirin induces Nrf2-mediated transcriptional activation of haem oxygenase-1 in protection of human melanocytes from H2O2-induced oxidative stress

The removal of hydrogen peroxide (H2O2) by antioxidants has been proven to be beneficial to patients with vitiligo. Aspirin (acetylsalicylic acid, ASA) has antioxidant activity and has great preventive and therapeutical effect in many oxidative stress‐relevant diseases. Whether ASA can protect human melanocytes against oxidative stress needs to be further studied. Here, we investigated the potential protective effect and mechanisms of ASA against H2O2‐induced oxidative injury in human melanocytes. Human melanocytes were pre‐treated with different concentrations of ASA, followed by exposure to 1.0 mM H2O2. Cell apoptosis, intracellular reactive oxygen species (ROS) levels were evaluated by flow cytometry, and cell viability was determined by an Cell Counting Kit‐8 assay. Total and phosphorylated NRF2 expression, NRF2 nuclear translocation and antioxidant response element (ARE) transcriptional activity were assayed with or without Nrf2‐siRNA transfection to investigate the possible molecular mechanisms. Concomitant with an increase in viability, pre‐treatment of 10‐90 μmol/l ASA resulted in decreased rate of apoptotic cells, lactate dehydrogenase release and intracellular ROS levels in primary human melanocytes. Furthermore, we found ASA dramatically induced NRF2 nuclear translocation, enhanced ARE‐luciferase activity, increased both p‐ NRF2 and total NRF2 levels, and induced the expression of haem oxygenase‐1 (HO‐1) in human melanocytes. In addition, knockdown of Nrf2 expression or pharmacological inhibition of HO‐1 abrogated the protective action of ASA on melanocytes against H2O2‐induced cytotoxicity and apoptosis. These results suggest that ASA protects human melanocytes against H2O2‐induced oxidative stress via Nrf2‐driven transcriptional activation of HO‐1.