The Role Of Nrf2-ARE Pathway In The Oxidative Stress Pathogenesis Of Vitiligo And Its Mechanism

Background:Vitiligo is a well-known skin depigmenting disorder characterized by the death ofmelanocytes from the lesional epidermis, and with an increasing incidence rate. Due to thepoorly understood in the pathogenesis, vitiligo is still lack of effective treatment. Recently,experimental and clinical evidences suggest that oxidative stress in the epidermis ofaffected skin lead to melanocyte degeneration. Oxidative stress can generate largeamounts of oxygen free radicals that affect the metabolism, proliferation anddifferentiation of normal cells. It may also further induce the autoimmune response toform a waterfall effect, causing irreversible damage of the epidermal melanocytes. Recentexperimental data underline that melanocytes involved in vitiligo may have inherentaberrations that make them more vulnerable to extracellular insults. However, the processand the executive disappearance mechanism lack a clear explanation. Therefore, it hasgreat significance in improving the pathogenesis and treatment of vitiligo to clarify the causes that vitiligo melanocytes is susceptible to oxidative damage and its molecularmechanism.Nrf2-ARE （Nuclear factor E2-related factor2-Antioxidant response element）pathway plays an important function in the defense against oxidative stress. Nrf2belongsto the cap’n’collar （CNC） family of basic leucine zipper proteins, and Nrf2regulates theexpression of phase II detoxifying and antioxidant genes by binding to the ARE sequence.Under unstimulated conditions, Nrf2is sequestered in the cytosol, where it is associatedwith Kelch-like ECH-associated protein1（Keap1）. When under oxidative stress, Nrf2escapes from Keap1and then translocates to the nucleus where it binds to ARE andinduces the phase II antioxidant genes. These genes encode heme oxygenase-1（HO-1）,catalase （CAT）, superoxide dismutase （SOD）, glutathione-S-transferase （GST）, glutathioneperoxidase （GPx）, NADH quinone oxidoreductase1（NQO1）, glutamate-cysteine ligasecatalytic subunit （GCLC）, and glutamyl-cystine ligase modulatory subunit （GCLM）.Recently, the functions of Nrf2and its downstream genes have been shown to beimportant for protection against oxidative stress and chemical-induced cellular damage inmany cells, tissues and organs. Therefore, we hypothesized that the Nrf2-ARE pathwaymay serve as a key mechanism of cytoprotection during H₂O₂-mediated oxidative stress inhuman melanocytes, and hypothesize that the increased sensitivity to H₂O₂-inducedoxidative insults of vitiligo melanocytes is due to the dysfunction in Nrf2-ARE redoxsignaling pathway and the activation of Nrf2can decrease the sensitivity of vitiligomelanocytes to H₂O₂-induced oxidative damage.Aims:1. To study the expression of Nrf2-ARE signaling pathway and its downstream genesin human melanocytes and to determine the role of Nrf2-ARE signaling pathway in copingwith oxidative stress in melanocytes;2. To analyze the expression and functional changes of Nrf2-ARE signaling pathwaybetween normal human melanocytes and vitiligo melanocytes;3. To explore the underlying mechanism for the aberrant expression of Nrf2-AREsignaling pathway and its downstream genes in vitiligo melanocytes. Methods:1. Firstly, we utilized H₂O₂in our studies and performed a series of dose-responseassays to determine the optimal concentration that would lead to a consistent and highdegree of cytotoxicity.2. Then, we used Nrf2siRNA and pCMV6-XL5-Nrf2to downregulate or upregulateNrf2expression in immortalized human melanocyte cell line PIG1. The melanocytes werethen analyzed under oxidative stress conditions for cell viability and apoptosis.3. After treatment with H₂O₂in PIG1cells, the expression of four main downstreamgenes （HO-1, NQO-1, GCLC and GCLM） of Nrf2pathway was determined by Real-timePCR. Then, immunocytochemistry and Real-time PCR analysis were carried out toexamine the expression of Nrf2and HO-1in the normal primary human melanocytes afterthe treatment with H₂O₂.4. Next, the cell viability, HO-1expression, the location of Nrf2, Nrf2-AREtranscriptional activity, the levels of ROS and MDA, the activities or contents of SOD,GPx, CAT and GSH were determined respectively by MTS aasay, Real-time PCR, westernblot, laser confocal scanning microscopy, dual luciferase reporter assay, flow cytometerand enzyme-linked immunosorbent assay between control and vitiligo melanocytes.5. Moreover, the expression patterns of Nrf2, p-Nrf2and HO-1in lesional skin weredetected by immunohistochemistry, and the serum levels of HO-1and interleukin （IL）-2inpatients with vitiligo were determined by ELISA.6. Finally, we used ATM siRNA or KU55933to downregulate ATM expression inPIG1cells and used Real-time PCR and western blot to determine the expression of Nrf2,HO-1and p-Nrf2. In order to observe the effect about ATM downregulation, the cellviability, the levels of ROS and MDA, melanin synthesis and tyrosinase activity weredetermined respectively by MTS aasay, flow cytometer and enzyme-linkedimmunosorbent assay. The expression patterns of ATM and p-ATM in lesional skin werealso detected by immunohistochemistry.Results:1. As determined by MTS assay, the H₂O₂-induced cell death of normal human melanocytes was concentration-dependent, and1.0mM H₂O₂caused consistentcytotoxicity but maintained high cell viability.2. MTS assay results showed that suppressed Nrf2expression led to significant celldeath under oxidative stress, and the percentage of dead cells was low in cells withupregulated Nrf2expression. Compared with the control group, the proportion ofapoptotic and necrotic cells increased after Nrf2was downregulated by Nrf2siRNA, butthere was a statistically significant induction in apoptosis48h after transfection withpCMV6-XL5-Nrf2. It suggests that modulation of Nrf2-ARE pathway can affect thecapability of melanocytes to cope with oxidative stress.3. The RT-PCR results shown that the expression levels of four main phase IIdetoxifying genes （HO-1, NQO-1, GCLC and GCLM） were upregulated after H₂O₂treatment. The most significant change was found in the HO-1gene with an approximate5-fold increase when compared to the control. ZnPP treatment exacerbated the damageinduced by H₂O₂in melanocytes, and hemin treatment enhanced cells viability comparedwith the control. The reduction of HO-1expression was associated with Nrf2knockdown,while the increase in both mRNA and protein levels of HO-1coincided with increasedNrf2levels. It suggests that HO-1can protect human melanocytes against H₂O₂-inducedoxidative damage through an Nrf2-dependent pathway.4. MTS assay results showed that no remarkable difference in cell viability was foundbetween normal human melanocytes from vitiligo melanocytes when H₂O₂was absent,while the present of1.0mM H₂O₂caused statistically significant reduction in cell viabilityof vitiligo melanocytes compared with control melanocytes. It suggests that vitiligomelanocytes showing more hypersensitive to H₂O₂-induced oxidative injury.5. Laser confocal scanning microscopy showed that nuclear Nrf2localization wasobserved mainly in control melanocytes, while vitiligo melanocytes produced primarilycytoplasmic staining. Under normal condition, the expression of nuclear Nrf2in controlmelanocytes are higher than that in vitiligo melanocytes, while the expression ofcytoplasmic Nrf2in vitiligo melanocytes are higher than that in control melanocytes.Moreover, western blot showed that in the nuclei-enriched fractions of control and vitiligo melanocytes, Nrf2content increased in response to H₂O₂. Far smaller Nrf2increases wereseen in the nuclei-enriched fractions of vitiligo melanocytes. Dual luciferase reporter assayshowed that treatment with H₂O₂increased transcriptional activity of Nrf2in terms ofARE luciferase activity in a time-dependent manner in both groups, whereas AREluciferase activity of PIG3V was much lower than that of PIG1in the presence of H₂O₂. Incontrol melanocytes, H₂O₂led to greater increases in HO-1. While in vitiligo melanocytes,the increases were far smaller compared with H₂O₂-untreated group. These data clearlyindicate that Nrf2-driven transcriptional activation of HO-1is aberrant in vitiligomelanocytes.6. Compared with normal control group, up-regulated Nrf2expression by transfectedwith pCMV6-XL5-Nrf2led to a less cell death under oxidative stress, whereas suchchanges were not observed in mock group. These data indicate that up-regulation of Nrf2restores the antioxidative ability of vitiligo melanocytes.7. Vitiligo melanocytes present a significantly higher intracellular ROS and MDAlevels. The SOD and GPx activities and low contents of total GSH and GSH in vitiligomelanocytes were comparatively lower than that in control melanocytes. Interestingly,there was no signifcant difference in activity of CAT between two groups. These dataindicate that antioxidant enzymes system is impaired in vitiligo melanocytes.8. In vitiligo lesional skin, we observed a dramatic reduction in nuclear Nrf2, p-Nrf2and HO-1in epidermal cells, which was different from the expected localizationresponding to oxidative stress.9. Serum HO-1levels were signifcantly lower in patients with vitiligo than in healthycontrols and the overall levels of serum IL-2of the patients with vitiligo were obviouslyhigher than that of healthy controls. The level of serum HO-1was inversely correlatedwith serum IL-2in vitiligo patients.10. Immunohistochemistry showed that decreased expression of ATM and increasedexpression of p-ATM were observed in epidermal cells of vitiligo lesional skin.Transfection with ATM-siRNA2lead to decreased expression of Nrf2, HO-1and p-Nrf2.Downregulation of ATM present a significantly lower cell viability and higher intracellular ROS and MDA levels, but have no effect on melanin synthesis and tyrosinase activity. Itsuggests that ATM regulates the oxidative stress level via Nrf2-ARE signaling pathway inmelanocytes, and the lack of ATM in epidermal cells of lesional skin may be the reasonthat the Nrf2activation is impaired under oxidative stress.Conclusion:In view of the above data, we demonstrate for the first time that Nrf2-ARE pathwayplays an important function in the defense against oxidative stress in human melanocytesand impaired Nrf2-ARE signaling pathway in vitiligo melanocytes shown for the first timehere contributes to the severity of oxidative stress and the failure of antioxidant inductionin response to oxidative stress. Furthermore, the underlying mechanism for the aberrantactivation of Nrf2in vitiligo melanocytes is clarified. Our work laid the foundation for thefurther understanding of the injury mechanism of vitiligo melanocytes. Meanwhile, ourstudy not only enriches and improves the oxidative stress pathogenesis in vitiligo, but alsoprovides a new target for clinical treatment.