NRF2 and Key Transcriptional Targets in Melanoma Redox Manipulation

Abstract

Melanocytes are dendritic, pigment-producing cells located in the skin and are responsible for its protection against the deleterious effects of solar ultraviolet radiation (UVR), which include DNA damage and elevated reactive oxygen species (ROS). They do so by synthesizing photoprotective melanin pigments and distributing them to adjacent skin cells (e.g., keratinocytes). However, melanocytes encounter a large burden of oxidative stress during this process, due to both exogenous and endogenous sources. Therefore, melanocytes employ numerous antioxidant defenses to protect themselves; these are largely regulated by the master stress response transcription factor, nuclear factor erythroid 2-related factor 2 (NRF2). Key effector transcriptional targets of NRF2 include the components of the glutathione and thioredoxin antioxidant systems. Despite these defenses, melanocyte DNA often is subject to mutations that result in the dysregulation of the proliferative mitogen-activated protein kinase (MAPK) pathway and the cell cycle. Following tumor initiation, endogenous antioxidant systems are co-opted, a consequence of elevated oxidative stress caused by metabolic reprogramming, to establish an altered redox homeostasis. This altered redox homeostasis contributes to tumor progression and metastasis, while also complicating the application of exogenous antioxidant treatments. Further understanding of melanocyte redox homeostasis, in the presence or absence of disease, would contribute to the development of novel therapies to aid in the prevention and treatment of melanomas and other skin diseases.

Keywords: HO-1; HSP70; NQO1; NRF2; SOD; antioxidant; glutathione; melanoma; peroxiredoxin; thioredoxin.

Figure 1

Scheme of the domains present in (A) NRF2 and (B) KEAP1 and associated functions. Above each protein domain are referenced interactions and below are associated functions. CBP, CREB binding protein; CHD6, chromodomain-helicase-DNA binding 6; CUL3, E3 ubiquitin ligase cullin-3; KEAP1, kelch-like ECH-associated protein 1; Neh, NRF2-ECH homology; NRF2, nuclear factor erythroid 2-like 2; RXRα, retinoid x receptor α; and sMAF, small musculoaponeurotic fibrosarcoma.

Figure 2

Sources of oxidative stress in melanocytes and subsequent activation of the NRF2/KEAP1/ARE-signaling pathway. Endogenous sources of oxidative stress include ROS produced by mitochondrial respiration, melanosomal melanogenesis, and the activity of NOX enzymes as well as RNS produced by NOS enzymes. It should be noted that NOX enzymes are expressed in several subcellular compartments in addition to the plasma membrane, as depicted here. Exogenous sources of oxidative stress include environmental stressors such as UVR and exposure of the skin to pro-oxidant chemical substances. Under normal conditions, there is a consistent low level of NRF2 present that is sequestered in the cytosol by KEAP1. CUL3 can then bind to the NRF2/KEAP1 complex leading to the ubiquitination and subsequent degradation of NRF2. However, under conditions of oxidative stress, there are redox-sensitive cysteine residues on KEAP1 that become modified leading to the release of NRF2. Released NRF2 can then enter the nucleus, bind to AREs, and promote the expression of antioxidant genes. ARE, antioxidant response element; CUL3, E3 ubiquitin ligase cullin-3; GSH, glutathione; KEAP1, kelch-like ECH-associated protein 1; NOS, nitric oxide synthase; NOX, NADPH oxidase; NRF2, nuclear factor erythroid 2-like 2; RNS, reactive nitrogen species; ROS, reactive oxygen species; TRX, thioredoxin; and UVR, ultraviolet radiation.

Figure 3

Schematic overview of the cysteine-dependent glutathione (top) and thioredoxin (bottom) antioxidant systems. The glutathione antioxidant system is comprised of the enzymes necessary for the synthesis, utilization, and recycling of the tripeptide glutathione. The amino acid cysteine is essential to both the glutathione and thioredoxin antioxidant systems and is taken into the cell in the form of the disulfide dimer cystine by the cystine/glutamate antiporter xCT (system x_c⁻), the protein subunits of which are encoded by NRF2 transcriptional targets SLC7A11 and SLC3A2. Cystine is rapidly reduced into cysteine in cytosol to be further utilized by the cell. The thioredoxin antioxidant system is comprised of the enzymes necessary for the utilization and recycling of the thioredoxin enzyme. Proteins in grey shapes are encoded by genes directly regulated by NRF2 through AREs. The blue text in the figure indicates the oxidized form of a molecule/protein, and the red text indicates the reduced form. ARE, antioxidant response element; G6PD, glucose-6-phosphate dehydrogenase; GCL, glutamate cysteine ligase; GPX, glutathione peroxidase; GSH, glutathione GSR, glutathione reductase; GSS, glutathione synthetase; GST, glutathione-S-transferase; PRDX, peroxiredoxin; TR, thioredoxin reductase; and TRX, thioredoxin.