Matthew Dodson

Role of Nrf2 and Autophagy in Acute Lung Injury

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are the clinical manifestations of severe lung damage and respiratory failure. Characterized by severe inflammation and compromised lung function, ALI/ARDS result in very high mortality of affected individuals. Currently, there are no effective treatments for ALI/ARDS, and ironically, therapies intended to aid patients (specifically mechanical ventilation, MV) may aggravate the symptoms. Key events contributing to the development of ALI/ARDS are increased oxidative and proteotoxic stresses, unresolved inflammation, and compromised alveolar-capillary barrier function. Since the airways and lung tissues are constantly exposed to gaseous oxygen and airborne toxicants, the bronchial and alveolar epithelial cells are under higher oxidative stress than other tissues. Cellular protection against oxidative stress and xenobiotics is mainly conferred by Nrf2, a transcription factor that promotes the expression of genes that regulate oxidative stress, xenobiotic metabolism and excretion, inflammation, apoptosis, autophagy, and cellular bioenergetics. Numerous studies have demonstrated the importance of Nrf2 activation in the protection against ALI/ARDS, as pharmacological activation of Nrf2 prevents the occurrence or mitigates the severity of ALI/ARDS. Another promising new therapeutic strategy in the prevention and treatment of ALI/ARDS is the activation of autophagy, a bulk protein and organelle degradation pathway. In this review, we will discuss the strategy of concerted activation of Nrf2 and autophagy as a preventive and therapeutic intervention to ameliorate ALI/ARDS.

ABCF2, an Nrf2 target gene, contributes to cisplatin resistance in ovarian cancer cells

Previously, we have demonstrated that NRF2 plays a key role in mediating cisplatin resistance in ovarian cancer. To further explore the mechanism underlying NRF2‐dependent cisplatin resistance, we stably overexpressed or knocked down NRF2 in parental and cisplatin‐resistant human ovarian cancer cells, respectively. These two pairs of stable cell lines were then subjected to microarray analysis, where we identified 18 putative NRF2 target genes. Among these genes, ABCF2, a cytosolic member of the ABC superfamily of transporters, has previously been reported to contribute to chemoresistance in clear cell ovarian cancer. A detailed analysis on ABCF2 revealed a functional antioxidant response element (ARE) in its promoter region, establishing ABCF2 as an NRF2 target gene. Next, we investigated the contribution of ABCF2 in NRF2‐mediated cisplatin resistance using our stable ovarian cancer cell lines. The NRF2‐overexpressing cell line, containing high levels of ABCF2, was more resistant to cisplatin‐induced apoptosis compared to its control cell line; whereas the NRF2 knockdown cell line with low levels of ABCF2, was more sensitive to cisplatin treatment than its control cell line. Furthermore, transient overexpression of ABCF2 in the parental cells decreased apoptosis and increased cell viability following cisplatin treatment. Conversely, knockdown of ABCF2 using specific siRNA notably increased apoptosis and decreased cell viability in cisplatin‐resistant cells treated with cisplatin. This data indicate that the novel NRF2 target gene, ABCF2, plays a critical role in cisplatin resistance in ovarian cancer, and that targeting ABCF2 may be a new strategy to improve chemotherapeutic efficiency.

Non-Canonical Activation of NRF2: New Insights and Its Relevance to Disease

Purpose of ReviewThe goal of this review is to summarize the current knowledge in the field regarding the non-canonical activation of the NRF2 pathway. Specifically, we address what role p62 plays in mediating this pathway, which pathologies have been linked to the p62-dependent activation of NRF2, as well as what therapeutic strategies could be used to treat diseases associated with the non-canonical pathway.Recent FindingsIt has recently been shown that autophagic dysfunction leads to the aggregation or autophagosomal accumulation of p62, which sequesters KEAP1, resulting in prolonged activation of NRF2. The ability of p62 to outcompete NRF2 for KEAP1 binding depends on its abundance, or post-translational modifications to its key domains. Furthermore, the relevance of the p62-dependent activation of NRF2 in disease has been demonstrated in human hepatocellular carcinomas, as well as neurodegenerative diseases.SummaryThese findings indicate that targeting p62, or the enzymes that modify it, could prove to be an advantageous strategy for treating diseases associated with autophagy dysregulation and prolonged activation of NRF2. Other therapeutic possibilities include restoring proper autophagic function, or directly inhibiting NRF2 or its targets, to restore redox and metabolic homeostasis. Future studies will help further clarify the mechanisms, regulation, and relevance of the non-canonical pathway in driving disease pathogenesis.

Multifunctional p62 Effects Underlie Diverse Metabolic Diseases

p62, a protein capable of binding both ubiquitin and autophagy substrates, is well established as a key regulator in cancer and neurodegenerative diseases. Recently, there has been accumulating evidence that p62 is also a pivotal regulator in metabolic diseases, such as obesity, T2DM, NAFLD, metabolic bone disease, gout and thyroid disease. This review summarizes the emerging role of p62 on these diseases by considering its functional domains, phenotypes in genetically modified animals, clinically observed alterations, and its effects on downstream metabolic signaling pathways. At the same time, we highlight the need to explore the roles played by p62 in the gastrointestinal environment and immune system, and the extent to which its elevated expression may confer protection against metabolic disorders.

Low-level arsenic causes proteotoxic stress and not oxidative stress

&NA; Prolonged exposure to arsenic has been shown to increase the risk of developing a number of diseases, including cancer and type II diabetes. Arsenic is present throughout the environment in its inorganic forms, and the level of exposure varies greatly by geographical location. The current recommended maximum level of arsenic exposure by the EPA is 10 &mgr;g/L, but levels > 50–1000 &mgr;g/L have been detected in some parts of Asia, the Middle East, and the Southwestern United States. One of the most important steps in developing treatment options for arsenic‐linked pathologies is to understand the cellular pathways affected by low levels of arsenic. Here, we show that acute exposure to non‐lethal, low‐level arsenite, an environmentally relevant arsenical, inhibits the autophagy pathway. Furthermore, arsenite‐induced autophagy inhibition initiates a transient, but moderate ER stress response. Significantly, low‐level arsenite exposure does not exhibit an increase in oxidative stress. These findings indicate that compromised autophagy, and not enhanced oxidative stress occurs early during arsenite exposure, and that restoring the autophagy pathway and proper proteostasis could be a viable option for treating arsenic‐linked diseases. As such, our study challenges the existing paradigm that oxidative stress is the main underlying cause of pathologies associated with environmental arsenic exposure. HighlightsAcute, low‐level arsenite does not induce oxidative stress.Low‐level arsenite inhibits autophagy.Restoring autophagy may be a viable therapy to treat arsenic‐linked disease.

Modulating NRF2 in Disease: Timing Is Everything

The transcription factor nuclear factor erythroid 2 (NF-E2)-related factor 2 (NRF2) is a central regulator of redox, metabolic, and protein homeostasis that intersects with many other signaling cascades. Although the understanding of the complex nature of NRF2 signaling continues to grow, there is only one therapeutic targeting NRF2 for clinical use, dimethyl fumarate, used for the treatment of multiple sclerosis. The discovery of new therapies is confounded by the fact that NRF2 levels vary significantly depending on physiological and pathological context. Thus, properly timed and targeted manipulation of the NRF2 pathway is critical in creating effective therapeutic regimens. In this review, we summarize the regulation and downstream targets of NRF2. Furthermore, we discuss the role of NRF2 in cancer, neurodegeneration, and diabetes as well as cardiovascular, kidney, and liver disease, with a special emphasis on NRF2-based therapeutics, including those that have made it into clinical trials.

RPA1 binding to NRF2 switches ARE-dependent transcriptional activation to ARE-NRE-dependent repression.

Significance Our findings shift the paradigm of NRF2 as a transcriptional activator to one in which NRF2 can also act as a transcriptional repressor, which we believe will stimulate new research areas and interests among scientists from other fields. While the majority of the data provided in this paper center on suppression of MYLK expression and the resulting pathological significance, the more far-reaching findings are the in silico and RNA-seq datasets indicating that the NRF2-replication protein A1 (RPA1)-ARE-NRE complex transcriptionally represses other genes as well, again highlighting the broad scope and significance of NRF2 repression of target genes. NRF2 regulates cellular redox homeostasis, metabolic balance, and proteostasis by forming a dimer with small musculoaponeurotic fibrosarcoma proteins (sMAFs) and binding to antioxidant response elements (AREs) to activate target gene transcription. In contrast, NRF2-ARE–dependent transcriptional repression is unreported. Here, we describe NRF2-mediated gene repression via a specific seven-nucleotide sequence flanking the ARE, which we term the NRF2-replication protein A1 (RPA1) element (NRE). Mechanistically, RPA1 competes with sMAF for NRF2 binding, followed by interaction of NRF2-RPA1 with the ARE-NRE and eduction of promoter activity. Genome-wide in silico and RNA-seq analyses revealed this NRF2-RPA1-ARE-NRE complex mediates negative regulation of many genes with diverse functions, indicating that this mechanism is a fundamental cellular process. Notably, repression of MYLK, which encodes the nonmuscle myosin light chain kinase, by the NRF2-RPA1-ARE-NRE complex disrupts vascular integrity in preclinical inflammatory lung injury models, illustrating the translational significance of NRF2-mediated transcriptional repression. Our findings reveal a gene-suppressive function of NRF2 and a subset of negatively regulated NRF2 target genes, underscoring the broad impact of NRF2 in physiological and pathological settings.

Increased O-GlcNAcylation of SNAP29 Drives Arsenic-Induced Autophagic Dysfunction

ABSTRACT Environmental exposure to arsenic is linked to adverse health effects, including cancer and diabetes. Pleiotropic cellular effects are observed with arsenic exposure. Previously, we demonstrated that arsenic dysregulated the autophagy pathway at low, environmentally relevant concentrations. Here we show that arsenic blocks autophagy by preventing autophagosome-lysosome fusion. Specifically, arsenic disrupts formation of the STX17-SNAP29-VAMP8 SNARE complex, where SNAP29 mediates vesicle fusion through bridging STX17-containing autophagosomes to VAMP8-bearing lysosomes. Mechanistically, arsenic inhibits SNARE complex formation, at least in part, by enhancing O-GlcNAcylation of SNAP29. Transfection of O-GlcNAcylation-defective, but not wild-type, SNAP29 into clustered regularly interspaced short palindromic repeat (CRISPR)-mediated SNAP29 knockout cells abolishes arsenic-mediated autophagy inhibition. These findings reveal a mechanism by which low levels of arsenic perturb proteostasis through inhibition of SNARE complex formation, providing a possible therapeutic target for disease intervention in the more than 200 million people exposed to unsafe levels of arsenic.

Sensing Oxidative Stress: The NRF2 Signaling Pathway

Abstract Cells are constantly exposed to a variety of both endogenous and exogenous oxidative and electrophilic insults that affect their biomolecules and compromise their function. Sensing the stress and mounting adaptive cytoprotective stress responses, particularly in a timely and controlled manner to remove the source of stress and repair the damaged components, are extremely important in maintaining cellular homeostasis and an organism’s wellbeing. The transcription factor nuclear factor erythroid-2 (NF-E2)-related factor 2 (NRF2) controls the expression of a set of genes that counteract oxidative and electrophilic stresses. Under physiological conditions, the protein levels of NRF2 are tightly controlled by Kelch-ECH associated protein 1 (KEAP1), a redox sensor and substrate adaptor for a Cullin 3-containing E3 ubiquitin ligase that ubiquitylates NRF2, targeting it for proteasomal degradation. During increased oxidative or electrophilic stress, KEAP1 cysteines are modified, resulting in a conformational change that prevents the ubiquitylation and degradation of NRF2. Newly synthesized NRF2 can then accumulate, translocate to the nucleus, and activate the transcription of antioxidant response element (ARE)-containing genes. This canonical activation of the NRF2 pathway involves the oxidative/electrophilic modification of KEAP1. However, NRF2 can also be activated through dysregulation of the autophagy pathway, namely the non-canonical mechanism of NRF2 activation. Non-canonical activation of NRF2 occurs independently of oxidative stress, and instead is a result of the direct interaction of KEAP1 and p62, an autophagy adaptor protein, and their aggregation in autophagosomes. This non-canonical mechanism of NRF2 activation results in prolonged activation of NRF2. Discovery of the non-canonical activation of NRF2, and the realization that certain cancer types have constitutively high levels of NRF2, have revealed that controlled, canonical activation of NRF2 is cytoprotective, whereas uncontrolled or prolonged activation is deleterious (“dark side” of NRF2). This chapter describes our current knowledge of the molecular mechanisms of NRF2 regulation, and the potential of NRF2-based strategies for disease prevention and therapeutic intervention.