Regulation of Nrf2—an update

Nrf2:INrf2 (Keap1) are cellular sensors of oxidative and electrophilic stress. Nrf2 is a nuclear factor that controls the expression and coordinated induction of a battery of genes that encode detoxifying enzymes, drug transporters, antiapoptotic proteins, and proteasomes. In the basal state, Nrf2 is constantly degraded in the cytoplasm by its inhibitor, INrf2. INrf2 functions as an adapter for Cul3/Rbx1 E3 ubiquitin ligase-mediated degradation of Nrf2. Chemicals, including antioxidants, tocopherols including α-tocopherol (vitamin E), and phytochemicals, and radiation antagonize the Nrf2:INrf2 interaction and lead to the stabilization and activation of Nrf2. The signaling events involve preinduction, induction, and postinduction responses that tightly control Nrf2 activation and repression back to the basal state. Oxidative/electrophilic signals activate unknown tyrosine kinases in a preinduction response that phosphorylates specific residues on Nrf2 negative regulators, INrf2, Fyn, and Bach1, leading to their nuclear export, ubiquitination, and degradation. This prepares nuclei for unhindered import of Nrf2. Oxidative/electrophilic modification of INrf2 cysteine 151 followed by PKC phosphorylation of Nrf2 serine 40 in the induction response results in the escape or release of Nrf2 from INrf2. Nrf2 is thus stabilized and translocates to the nucleus, resulting in a coordinated activation of gene expression. This is followed by a postinduction response that controls the “switching off” of Nrf2-activated gene expression. GSK3β, under the control of AKT and PI3K, phosphorylates Fyn, leading to Fyn nuclear localization. Fyn phosphorylates Nrf2 Y568, resulting in nuclear export and degradation of Nrf2. The activation and repression of Nrf2 provide protection against oxidative/electrophilic stress and associated diseases, including cancer. However, deregulation of INrf2 and Nrf2 due to mutations may lead to nuclear accumulation of Nrf2 that reduces apoptosis and promotes oncogenesis and drug resistance.     

Nrf2 prevents diabetic cardiomyopathy via antioxidant effect and normalization of glucose and lipid metabolism in the heart

Metabolic disorders and oxidative stress are the main causes of diabetic cardiomyopathy. Activation of nuclear factor erythroid 2-related factor 2 (Nrf2) exerts a powerful antioxidant effect and prevents the progression of diabetic cardiomyopathy. However, the mechanism of its cardiac protection and direct action on cardiomyocytes are not well understood. Here, we investigated in a cardiomyocyte-restricted Nrf2 transgenic mice (Nrf2-TG) the direct effect of Nrf2 on cardiomyocytes in DCM and its mechanism. In this study, cardiomyocyte-restricted Nrf2 transgenic mice (Nrf2-TG) were used to directly observe whether cardiomyocyte-specific overexpression of Nrf2 can prevent diabetic cardiomyopathy and correct glucose and lipid metabolism disorders in the heart. Compared to wild-type mice, Nrf2-TG mice showed resistance to diabetic cardiomyopathy in a streptozotocin-induced type 1 diabetes mouse model. This was primarily manifested as improved echocardiography results as well as reduced myocardial fibrosis, cardiac inflammation, and oxidative stress. These results showed that Nrf2 can directly act on cardiomyocytes to exert a cardioprotective role. Mechanistically, the cardioprotective effects of Nrf2 depend on its antioxidation activity, partially through improving glucose and lipid metabolism by directly targeting lipid metabolic pathway of AMPK/Sirt1/PGC-1α activation via upstream genes of sestrin2 and LKB1, and indirectly enabling AKT/GSK-3β/HK-Ⅱ activity via AMPK mediated p70S6K inhibition.     

Structural and functional characterization of Nrf2 degradation by glycogen synthase kinase 3/β-TrCP

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of cellular homeostasis that controls the expression of more than 1% of human genes related to biotransformation reactions, redox homeostasis, energetic metabolism, DNA repair, and proteostasis. Its activity has a tremendous impact on physiology and pathology and therefore it is very tightly regulated, mainly at the level of protein stability. In addition to the very well established regulation by the ubiquitin E3 ligase adapter Keap1, recent advances have identified a novel mechanism based on signaling pathways that regulate glycogen synthase kinse-3 (GSK-3). This kinase phosphorylates specific serine residues in the Neh6 domain of Nrf2 to create a degradation domain that is then recognized by the ubiquitin ligase adapter β-TrCP and tagged for proteasome degradation by a Cullin1/Rbx1 complex. Here we review the mechanistic elements and the signaling pathways that participate in this regulation by GSK-3/β-TrCP. These pathways include those activated by ligands of tyrosine kinase, G protein-coupled, metabotropic, and ionotropic receptors that activate phosphatidyl inositol 3-kinase (PI3K)/ATK and by the canonical WNT signaling pathway, where a fraction of Nrf2 interacts with Axin1/GSK-3. Considering that free Nrf2 protein is localized in the nucleus, we propose a model termed “double flux controller” to explain how Keap1 and β-TrCP coordinate the stability of Nrf2 in several scenarios. The GSK-3/β-TrCP axis provides a novel therapeutic strategy to modulate Nrf2 activity.     

Metallothionein plays a prominent role in the prevention of diabetic nephropathy by sulforaphane via up-regulation of Nrf2

Sulforaphane (SFN) prevents diabetic nephropathy (DN) in type 1 diabetes via up-regulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, it has not been addressed whether SFN also prevents DN from type 2 diabetes or which Nrf2 downstream gene(s) play(s) the key role in SFN renal protection. Here we investigated whether Nrf2 is required for SFN protection against type 2 diabetes-induced DN and whether metallothionein (MT) is an Nrf2 downstream antioxidant using Nrf2 knockout (Nrf2-null) mice. In addition, MT knockout mice were used to further verify if MT is indispensable for SFN protection against DN. Diabetes-increased albuminuria, renal fibrosis, and inflammation were significantly prevented by SFN, and Nrf2 and MT expression was increased. However, SFN renal protection was completely lost in Nrf2-null diabetic mice, confirming the pivotal role of Nrf2 in SFN protection from type 2 diabetes-induced DN. Moreover, SFN failed to up-regulate MT in the absence of Nrf2, suggesting that MT is an Nrf2 downstream antioxidant. MT deletion resulted in a partial, but significant attenuation of SFN renal protection from type 2 diabetes, demonstrating a partial requirement for MT for SFN renal protection. Therefore, the present study demonstrates for the first time that as an Nrf2 downstream antioxidant, MT plays an important, though partial, role in mediating SFN renal protection from type 2 diabetes.     

Rg1 protects H9C2 cells from high glucose‐/palmitate‐induced injury via activation of AKT/GSK‐3β/Nrf2 pathway

Our previous studies have assessed ginsenoside Rg1 (Rg1)‐mediated protection in a type 1 diabetes rat model. To uncover the mechanism through which Rg1 protects against cardiac injury induced by diabetes, we mimicked diabetic conditions by culturing H9C2 cells in high glucose/palmitate. Rg1 had no toxic effect, and it alleviated the high glucose/palmitate damage in a dose‐dependent manner, as indicated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay and lactate dehydrogenase release to the culture medium. Rg1 prevented high glucose/palmitate‐induced cell apoptosis, assessed using cleaved caspase‐3 and terminal deoxynucleotidyl transferase dUTP nick end labelling staining. Rg1 also reduced high glucose‐/palmitate‐induced reactive oxygen species formation and increased intracellular antioxidant enzyme activity. We found that Rg1 activates protein kinase B (AKT)/glycogen synthase kinase‐3 (GSK‐3β) pathway and antioxidant nuclear factor erythroid 2‐related factor 2 (Nrf2) pathway, indicated by increased phosphorylation of AKT and GSK‐3β, and nuclear translocation of Nrf2. We used phosphatidylinositol‐3‐kinase inhibitor Ly294002 to block the activation of the AKT/GSK‐3β pathway and found that it partially reversed the protection by Rg1 and decreased Nrf2 pathway activation. The results suggest that Rg1 exerts a protective effect against high glucose and palmitate damage that is partially AKT/GSK‐3β/Nrf2‐mediated. Further studies are required to validate these findings using primary cardiomyocytes and animal models of diabetes.