The Role of Nrf2 in Cardiovascular Function and Disease

Abstract

Free radicals, reactive oxygen/nitrogen species (ROS/RNS), hydrogen sulphide, and hydrogen peroxide play an important role in both intracellular and intercellular signaling; however, their production and quenching need to be closely regulated to prevent cellular damage. An imbalance, due to exogenous sources of free radicals and chronic upregulation of endogenous production, contributes to many pathological conditions including cardiovascular disease and also more general processes involved in aging. Nuclear factor erythroid 2-like 2 (NFE2L2; commonly known as Nrf2) is a transcription factor that plays a major role in the dynamic regulation of a network of antioxidant and cytoprotective genes, through binding to and activating expression of promoters containing the antioxidant response element (ARE). Nrf2 activity is regulated by many mechanisms, suggesting that tight control is necessary for normal cell function and both hypoactivation and hyperactivation of Nrf2 are indicated in playing a role in different aspects of cardiovascular disease. Targeted activation of Nrf2 or downstream genes may prove to be a useful avenue in developing therapeutics to reduce the impact of cardiovascular disease. We will review the current status of Nrf2 and related signaling in cardiovascular disease and its relevance to current and potential treatment strategies.

Figure 1

Nrf2 and KEAP1 structure. Nrf2 is a cap‘n'collar-basic region leucine zipper (CNC-bZIP), and its human sequence contains 605 amino acids, divided into seven domains: Neh1 to Neh7. Neh1 contains a CNC-bZIP motif, allowing heterodimerization with Maf proteins and DNA binding [54]. The Neh2 domain contains the Keap1 binding site (DLG and ETGE motifs), necessary for its cytoplasmic retention and degradation [55]. The Neh3 domain is fundamental for Nrf2 transcriptional activation through binding with chromo-ATPase/helicase DNA-binding protein 6 (CHD6) [56]. Neh4 and Neh5 provide an interaction site for nuclear cofactor RAC3/AIB1/SRC-3 [57] and CREB-binding protein (CBP) [58] which enhances the Nrf2/ARE activation pathways, partially by promoting acetylation of Nrf2 [59]. Additionally, Nrf2 possesses a redox-sensitive nuclear exporting signal within the Neh5 transactivation domain able to regulate its cellular localization [60]. The serine-rich Neh6 domain contains two motifs (DSGIS and DSAPGS) involved in the negative regulation of Nrf2. Glycogen synthase kinase 3 (GSK-3) phosphorylates serine residues within Neh6 enabling the interaction with the β-transducin repeat-containing protein (β-TrCP) which acts as a substrate receptor for Skp1–Cul1–Rbx1/Roc1 ubiquitin ligase complex, leading to KEAP1-independent degradation [41]. Neh7 domain interacts with retinoid X receptor alpha (RXRα), responsible for Nrf2/ARE signaling inhibition [61]. Human Kelch-like ECH-associated protein 1 (KEAP1) is a 69 kD protein, containing 27 cysteine residues. It is a substrate adaptor for cullin (Cul3) which contains E3 ubiquitin ligase (E3). KEAP1 is composed of five domains starting from the N-terminal region, a BTB dimerization domain (Broad-Complex, Tramtrack, and Bric-à-brac) which contains the Cys151 residue, a cysteine-rich intervening region (IVR) domain with two cysteine domain residues Cys273 and Cys288, critical for stress sensing. A Kelch domain/double glycine repeat (DGR) domain possessing 6 Kelch repeats and ending with a C-terminal region [62]. KEAP1 needs a domain capable to homodimerize and interact with Cul3, forming the Nrf2 inhibitor complex (iNrf2), and this is the BTB domain [63]. The Cys151 in the same domain plays an important role on Nrf2 activation in response to oxidative stress [64]. Furthermore, the IVR domain is highly sensitive to oxidation and contains three cysteines, 273, 288, and 297 which regulate Nrf2 activation and repression [16, 65]. The DGR domain acts as an Nrf2 repressor; it contains six repetitive Kelch structures that specifically bind to the Neh2 domain on Nrf2 [15].

Figure 2

ROS-induced uncoupling of eNOS and the generation of O₂•. Excess ROS induce the conversion of BH4 to BH2 with subsequent eNOS uncoupling and synthesis of O₂• instead of NO. eNOS: endothelial nitric oxide synthase; ROS: reactive oxygen species; NO: nitric oxide; O₂•: superoxide; BH4: tetrahydrobiopterin; BH2: dihydrobiopterin.

Figure 3

Hyperglycemia-induced ROS generation in the heart. A schematic model showing the potential pathways involved in cardiomyopathy and how Nrf2 could be targeted to reduce ROS and prevent the development of this pathology. AGEs: advanced glycation end products; NADPH: nicotinamide adenine dinucleotide phosphate; PKC: protein kinase C; eNOS: endothelial nitric oxide synthase; ETC: electron transport chain; MPTP: mitochondrial permeability transition pore.