Oxidative Stress and Vitiligo: The Nrf2-ARE Signaling Connection

Abstract

Hypersensitivity of epidermal melanocytes to oxidative stress is known to contribute to vitiligo pathogenesis. Molecular mechanisms that connect melanocyte redox homeostasis to the complex disease phenotype are not fully understood. Jian et al. show that vitiligo melanocytes have impaired Nrf2-ARE signaling and decreased activation of the antioxidant enzyme system. In patients with vitiligo, higher serum levels of IL-2 correlate with lower levels of HO-1, a product of the Nrf2 target gene.

Degeneration of melanocytes, the cellular source of melanin pigment, is the pathologic hallmark of depigmented macules in patients with vitiligo. Vitiligo is now recognized as a disorder of multifactorial etiology culminating in melanocyte loss, although its underlying pathogenic mechanisms are not fully understood. Both intrinsic (gene polymorphisms/mutations, autoimmunity, auto-toxicity of melanocytes, oxidative stress) and extrinsic factors (xenobioticsand infections) are known to contribute to vitiligo, and it is recognized that hypersensitivity to oxidative stress plays an important role in ultimate destruction of epidermal melanocytes (Taieb, 2000). Aberrant accumulation of reactive oxygen species (ROS) induced by oxidative insults disrupts the redox homeostasis and eventually contributes to its pathogenesis. In this issue, Jian et al. (2014), using exposure to hydrogen peroxide (H₂O₂) to simulate oxidative stress, report on the role of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway in melanocyte homeostasis.

High levels of oxidative stress are damaging to skin architecture, and impaired cellular redox control is implicated in many skin disorders. Recent studies have demonstrated a crucial function for the Nrf2-ARE signaling pathway in regulating skin homeostasis under oxidative stress (Marrotet al., 2008). The Nrf2-ARE pathway is an early sensor of oxidative stress, and it acts to dampen oxidative damage by upregulating the expression of phase II detoxifying enzymes.

Nrf2, a basic leucine zipper transcription factor with a Cap’n’ Collar (CNC) structure, is ubiquitously expressed in human tissues. It regulates cellular defense against oxidative stress by inducing expression of genes that code many phase II detoxification enzymes (Kensler et al., 2007). Phase II detoxification enzymes, which convert metabolites to species less reactive against cell components, are critical components in responses to oxidative stress. The ARE sequence in the 5’-flanking region of many phase II detoxifying and antioxidant genes is a promoter element to which Nrf2 binds. In non-stressed cells, Nrf2 is localized primarily in the cytoplasm due to its binding to kelch-like ECH associating protein 1 (Keap1), which negatively regulates Nrf2 function by initiating its proteasomal degradation (Itoh et al., 1999). Under oxidative stress, dissociation of Nrf2 from and Keap1 results in Nrf2 activation, its translocation and accumulation in the nucleus. Binding of Nrf2 to AREs activates expression of phase II enzymes such as hemeoxygenase-1 (HO-1), catalase (CAT), and superoxide dismutase (SOD). To assess the role of the Nrf2-ARE pathway in vitiligo in a previous study, Jian and coworkers measured serum levels of antioxidant enzymes in vitiligo patients and compared them with age- and sex-matched healthy controls.

In the present study, Jian et al. (2014) demonstrate that vitiligo melanocytes exhibit a diminished H₂O₂-induced oxidative response as a result of impaired Nrf2-ARE signaling. Using melanocytes derived from control and vitiligo patients, they first confirmed the hypersensitivity of vitiligo melanocytes to H₂O₂-induced oxidative insults in a cell viability assay. When exposed to H₂O₂, vitiligo melanocytes showed a less prominent increase in HO-1 expression (both mRNA and protein) than did control melanocytes. Nuclear translocation of Nrf2 from cytosol was also impaired in the vitiligo melanocytes. Impaired activation of antioxidant enzymes and higher levels of cellular oxidative stress were also detected, confirming the correlation between Nrf2-ARE signaling and oxidative stress and its role in the destruction of melanocytes in vitiligo.

Employing normal human epidermal melanocytes (PIG1), a vitiligo melanocyte cell line (PIG3V) and promoter-reporter assays, Jian et al examined Nrf2 transcription factor activity upon induction of oxidative stress. As expected, transcriptional activity of Nrf2 was significantly lower in the PIG3V cell line than in PIG1 cells. The most significant observation of this study, however, was that overexpression of Nrf2 (by transfection with an Nrf2 expression plasmid) restored antioxidative activity in PIG3V cells. This observation supports an important role for Nrf2 in regulating intracellular antioxidative capacity of melanocyte.

To extend these in vitro observations to patients and to search for an immunological link to the oxidative stress, the authors measured serum HO-1 and interleukin-2 (IL-2) levels in 113 non-segmental vitiligo patients and 113 healthy controls. They found that, in patients with both stable and progressive vitiligo, serum levels of HO-1 were significantly lower, while IL-2 levels were higher compared to normal individuals, i. e., serum IL-2 levels were inversely proportional to HO-1, an indicator of an oxidative stress response. This was interesting in view of previous studies that have shown HO-1, through production of carbon monoxide, to suppress T cell proliferation via inhibition of IL-2 production (Paeet al., 2004). Thus, the dampened Nrf2-ARE signaling pathway may also account for IL-2 activation in patients with vitiligo. This will be an interesting avenue for investigators to explore in looking for a mechanistic link between oxidative stress and immune activation in vitiligo.

In summary, by confirming the antioxidant function of the Nrf2-ARE pathway in human melanocytes, Jian et al provide mechanistic insight into the response of melanocytes to oxidative stress and its role in the pathogenesis of vitiligo. This study opens the possibility that the Nrf2-ARE pathway could be exploited as a preventive and/or therapeutic strategy to counter oxidative damage in melanocytes as well as to dampen IL-2 production.

There are severap limitations to this study, specifically as it relates to melanocytes in vivo and the potential for clinical translation. First, the melanocytes used in this study are immortalized cell lines, which may exhibit altered oxidative stress responses compared to primary melanocytes. Immunohistochemical evaluation of the levels/localization of Nrf2 and/or antioxidant enzymes in normal and lesional skin will be of considerable value. This will also address the role of keratinocyte antioxidant responses in vitiligo pathogenesis, since keratinocytes are thought to be ROS donors to melanocytes. Second, although H₂O₂ is a widely used and widely accepted oxidative stress inducer, additional stressors may need to be tested to confirm the role of the Nrf2-ARE pathway. Third, although provocative, the observations on the inverse relationship between serum HO-1 and IL-2 levels do not identify the source of HO-1 (melanocyte intracellular vs. systemically derived) or the correlations among serum HO-1, IL-2 levels, and lesional T lymphocyte accumulation. These limitations notwithstanding, this study by Jian et al. (2014) shine a spotlight on antioxdative mechanisms in vitiligo and highlight the need to explore strategies to restore a balanced redox system, thereby protectingmelanocytes from destruction.

Clinical Highlights.

• The study by Jian et al in this issue highlight the role of the Nrf2-ARE signaling pathway in protecting melanocytes from oxidative stress, and it implicates an impaired Nrf2-ARE signaling response in melanocyte death in patients with vitiligo.

• Inverse correlations between serum HO-1 and IL-2 levels found in a large cohort of vitiligo patients suggests a link between impaired oxidative damage and a heightened immune response.

• Evaluating and monitoring exposure to oxidative stress and developing strategies to reduce oxidative stress may ultimately be included in the preventive and therapeutic armamentarium for vitiligo.