Alexandria Lau

A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62

ABSTRACT In response to stress, cells can utilize several cellular processes, such as autophagy, which is a bulk-lysosomal degradation pathway, to mitigate damages and increase the chances of cell survival. Deregulation of autophagy causes upregulation of p62 and the formation of p62-containing aggregates, which are associated with neurodegenerative diseases and cancer. The Nrf2-Keap1 pathway functions as a critical regulator of the cells defense mechanism against oxidative stress by controlling the expression of many cellular protective proteins. Under basal conditions, Nrf2 is ubiquitinated by the Keap1-Cul3-E3 ubiquitin ligase complex and targeted to the 26S proteasome for degradation. Upon induction, the activity of the E3 ubiquitin ligase is inhibited through the modification of cysteine residues in Keap1, resulting in the stabilization and activation of Nrf2. In this current study, we identified the direct interaction between p62 and Keap1 and the residues required for the interaction have been mapped to 349-DPSTGE-354 in p62 and three arginines in the Kelch domain of Keap1. Accumulation of endogenous p62 or ectopic expression of p62 sequesters Keap1 into aggregates, resulting in the inhibition of Keap1-mediated Nrf2 ubiquitination and its subsequent degradation by the proteasome. In contrast, overexpression of mutated p62, which loses its ability to interact with Keap1, had no effect on Nrf2 stability, demonstrating that p62-mediated Nrf2 upregulation is Keap1 dependent. These findings demonstrate that autophagy deficiency activates the Nrf2 pathway in a noncanonical cysteine-independent mechanism.

Brusatol enhances the efficacy of chemotherapy by inhibiting the Nrf2-mediated defense mechanism

The major obstacle in cancer treatment is the resistance of cancer cells to therapies. Nrf2 is a transcription factor that regulates a cellular defense response and is ubiquitously expressed at low basal levels in normal tissues due to Keap1-dependent ubiquitination and proteasomal degradation. Recently, Nrf2 has emerged as an important contributor to chemoresistance. High constitutive expression of Nrf2 was found in many types of cancers, creating an environment conducive for cancer cell survival. Here, we report the identification of brusatol as a unique inhibitor of the Nrf2 pathway that sensitizes a broad spectrum of cancer cells and A549 xenografts to cisplatin and other chemotherapeutic drugs. Mechanistically, brusatol selectively reduces the protein level of Nrf2 through enhanced ubiquitination and degradation of Nrf2. Consequently, expression of Nrf2-downstream genes is reduced and the Nrf2-dependent protective response is suppressed. In A549 xenografts, brusatol and cisplatin cotreatment induced apoptosis, reduced cell proliferation, and inhibited tumor growth more substantially when compared with cisplatin treatment alone. Additionally, A549-K xenografts, in which Nrf2 is expressed at very low levels due to ectopic expression of Keap1, do not respond to brusatol treatment, demonstrating that brusatol-mediated sensitization to cisplatin is Nrf2 dependent. Moreover, a decrease in drug detoxification and impairment in drug removal may be the primary mechanisms by which brusatol enhances the efficacy of chemotherapeutic drugs. Taken together, these results clearly demonstrate the effectiveness of using brusatol to combat chemoresistance and suggest that brusatol can be developed into an adjuvant chemotherapeutic drug.

Regulation of the Nrf2–Keap1 Antioxidant Response by the Ubiquitin Proteasome System: An Insight into Cullin-Ring Ubiquitin Ligases

Nrf2 is a transcription factor that has emerged as the cells main defense mechanism against many harmful environmental toxicants and carcinogens. Nrf2 is negatively regulated by Keap1, a substrate adaptor protein for the Cullin3 (Cul3)-containing E3-ligase complex, which targets Nrf2 for ubiquitination and degradation by the ubiquitin proteasome system (UPS). Recent evidence suggests that constitutive activation of Nrf2, due to mutations in Keap1 or Nrf2, is prominent in many cancer types and contributes to chemoresistance. Regulation of Nrf2 by the Cul3-Keap1-E3 ligase provides strong evidence that tight regulation of Cullin-ring ligases (CRLs) is imperative to maintain cellular homeostasis. There are seven known Cullin proteins that form various CRL complexes. They are regulated by neddylation/deneddylation, ubiquitination/deubiquitination, CAND1-assisted complex assembly/disassembly, and subunit dimerization. In this review, we will discuss the regulation of each CRL using the Cul3-Keap1-E3 ligase complex as the primary focus. The substrates of CRLs are involved in many signaling pathways. Therefore, deregulation of CRLs affects several cellular processes, including cell cycle arrest, DNA repair, cell proliferation, senescence, and death, which may lead to many human diseases, including cancer. This makes CRLs a promising target for novel cancer drug therapies.

Arsenic inhibits autophagic flux, activating the Nrf2-Keap1 pathway in a p62-dependent manner.

ABSTRACT The Nrf2-Keap1 signaling pathway is a protective mechanism promoting cell survival. Activation of the Nrf2 pathway by natural compounds has been proven to be an effective strategy for chemoprevention. Interestingly, a cancer-promoting function of Nrf2 has recently been observed in many types of tumors due to deregulation of the Nrf2-Keap1 axis, which leads to constitutive activation of Nrf2. Here, we report a novel mechanism of Nrf2 activation by arsenic that is distinct from that of chemopreventive compounds. Arsenic deregulates the autophagic pathway through blockage of autophagic flux, resulting in accumulation of autophagosomes and sequestration of p62, Keap1, and LC3. Thus, arsenic activates Nrf2 through a noncanonical mechanism (p62 dependent), leading to a chronic, sustained activation of Nrf2. In contrast, activation of Nrf2 by sulforaphane (SF) and tert-butylhydroquinone (tBHQ) depends upon Keap1-C151 and not p62 (the canonical mechanism). More importantly, SF and tBHQ do not have any effect on autophagy. In fact, SF and tBHQ alleviate arsenic-mediated deregulation of autophagy. Collectively, these findings provide evidence that arsenic causes prolonged activation of Nrf2 through autophagy dysfunction, possibly providing a scenario similar to that of constitutive activation of Nrf2 found in certain human cancers. This may represent a previously unrecognized mechanism underlying arsenic toxicity and carcinogenicity in humans.

Autophagy Suppresses RIP Kinase-Dependent Necrosis Enabling Survival to mTOR Inhibition

mTOR inhibitors are used clinically to treat renal cancer but are not curative. Here we show that autophagy is a resistance mechanism of human renal cell carcinoma (RCC) cell lines to mTOR inhibitors. RCC cell lines have high basal autophagy that is required for survival to mTOR inhibition. In RCC4 cells, inhibition of mTOR with CCI-779 stimulates autophagy and eliminates RIP kinases (RIPKs) and this is blocked by autophagy inhibition, which induces RIPK- and ROS-dependent necroptosis in vitro and suppresses xenograft growth. Autophagy of mitochondria is required for cell survival since mTOR inhibition turns off Nrf2 antioxidant defense. Thus, coordinate mTOR and autophagy inhibition leads to an imbalance between ROS production and defense, causing necroptosis that may enhance cancer treatment efficacy.

The Predicted Molecular Weight of Nrf2: It Is What It Is Not

Dear Editor: The transcription factor NF-E2-related factor 2 (Nrf2) is rapidly being recognized as a critical regulator of the cellular stress response. Explosions of publications in the field are investigating the role of Nrf2 in disease prevention and progression; however, this rapid expansion is coming at a cost. As new investigators break into the emerging field of Nrf2 research, confusion regarding the correct migratory pattern of Nrf2 is causing doubts about the accuracy and reproducibility of published results. This letter provides solid evidence that the biologically relevant molecular weight of Nrf2 is ∼95–110 kilodalton (kDa) and not the predicted ∼55–65 kDa based on its 2-kb open reading frame. The data discussed and presented here will hopefully lead to a uniform acceptance that future experiments and publications should be designed around detecting Nrf2 at the apparent molecular weight of ∼95–110 kDa. Since its discovery over a decade ago, Nrf2 has emerged as one of the cardinal transcription factors for the adaptive stress response. The Nrf2 pathway reaches broadly across many systems of biology and is involved in the prevention and pathogenesis of multiple complex human diseases such as cancer, diabetes, and cardiovascular and neurodegenerative diseases. The field of Nrf2 biology has exponentially grown over the past decade. Subsequently, the accurate reporting of Nrf2 molecular weight is a major issue that has arisen with the influx of researchers entering the Nrf2 field from other disciplines. The common misconception relating to Nrf2 is that it migrates at a predicted molecular weight of ∼55–65 kDa by sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS-PAGE). This prediction is made according to Nrf2s open reading frame size of ∼2.2-kb. In this letter, we provide evidence through chemical activation, vector driven mammalian expression, and recombinant protein expression that in fact, the biologically relevant species of Nrf2 migrates between ∼95 and 110 kDa. As a reviewer for journals and grants, it is worrisome to see that many researchers are reporting an incorrect migratory species of Nrf2. Furthermore, I receive weekly inquiries from investigators both nationally and internationally regarding the “abnormal” migration of Nrf2 from its predicted size. To remove the current confusion in the field, we want to make this information publically available in a highly credible, broadly impactful journal. This report is crucial to allow investigators to report accurate and reliable data and to progress the field. Nrf2 is a member of the cap ‘n’ collar subfamily of basic-region leucine zipper transcription factors that was first identified, cloned, and characterized in 1994 (5). Nrf2 knockout mice appear to be normal and fertile, indicating that Nrf2 is not essential for the normal development of mice (1). Over the past decade, much of the molecular mechanisms regulating the Nrf2 pathway have been broadly elucidated. The substrate adaptor protein Kelch-like ECH-associated protein 1 (Keap1) forms an E3 ubiquitin ligase complex with Cullin 3 (Cul3) and RING-box protein 1 (Rbx1) to negatively regulate Nrf2 protein levels. Under unstressed conditions, the Keap1-Cul3-Rbx1 E3 ubiquitin ligase complex conjugates ubiquitin onto Nrf2, leading to its degradation by the 26S proteasome to maintain low basal levels of Nrf2. Upon exposure to electrophiles or oxidative stress, essential cysteine residues in Keap1 act as sensors and are modified, resulting in stabilization of Nrf2. Subsequently, Nrf2 enters the nucleus and heterodimerizes with a small Maf protein to activate transcription of targets bearing an antioxidant response element in the promoter. These cytoprotective genes encode for phase II detoxifying enzymes, intracellular redox-balancing proteins, and transporters (4). Nrf2 is known to be the master regulator of a major cellular defense mechanism due to its ability to eliminate toxicants or carcinogens and reinstate cellular homeostasis. Therefore, activation of Nrf2 by small molecules and natural products has been shown to protect against a variety of human diseases. The beneficial role of Nrf2 inducers in preventing or alleviating pathological alterations by harmful substances has been demonstrated using various murine disease models. More importantly, several clinical trials have yielded promising results using broccoli sprouts to activate Nrf2 to prevent aflatoxin-induced liver cancer (3). It was not until 2008, when the “dark” side of Nrf2 was revealed (7). Somatic mutations in Nrf2 or Keap1, and loss of Keap1 expression, were found in different types of tumors that allow Nrf2 to escape Keap1-mediated degradation, thus causing constitutive activation of Nrf2. Furthermore, high basal levels of Nrf2 have been proven to contribute to both intrinsic and acquired chemoresistance in cancer. Nrf2 is widely expressed at low basal levels in all tissues. Its transcript is 2.2-kb, which is predicted to be a ∼66-kDa protein. However, the very first in vitro transcribed and translated protein from full-length Nrf2 cDNA showed not only a band at ∼66 kDa, but also at ∼96 kDa (5). In the following year, Yamamotos group made an antibody against the basic region leucine-zipper domain of Nrf2 (ECH) (2). This antibody detected bands at ∼63 and ∼97 kDa when the cDNA for the ECH domain was expressed in vitro (2). The first use of a commercial antibody from Santa Cruz Biotechnology detected two bands at 66 and 110 kDa (6). Now, mounting evidence supports the biologically relevant size Nrf2 to be ∼95–110 kDa under reduced and denatured conditions. The nature of the aberrant migration pattern of Nrf2 is still uncertain. However, the abundance of acidic residues in Nrf2 may offer an explanation for the unpredicted migration of Nrf2. The dual role of Nrf2 in cancer and other diseases has piqued the interest of an interdisciplinary group of researchers causing the field to exponentially grow over the last few years. However, this aberrant migration of Nrf2 has caused major controversy and confusion in the field. Most commercial antibody sources indicate Nrf2 protein to be at its predicted molecular weight of ∼55–65 kDa, which has caused investigators to litter the literature with somewhat misleading data and snowballing this molecular weight mystery. Therefore, the intention of this commentary is to make clear that the molecular weight of Nrf2 ranges from ∼95 to 110 kDa depending upon composition of the SDS-PAGE gel used. Here, we provide strong evidence that Nrf2 indeed migrates at ∼95–110 kDa. Immunoblot analysis with overexpressed hemagglutinin-tagged Nrf2 shows a prominent protein band at ∼110 kDa and 95 kDa on a 7.5% SDS-PAGE and a commercial 4%–12% gradient gel, respectively (Fig. 1a). The well-known Nrf2 activator, tert-butylhydroquinone (tBHQ), increased these ∼110 and 95 kDa band intensities, further confirming that the ∼95–110 kDa signal is indeed Nrf2 (Fig. 1a). A band of similar size was also detected when Nrf2 protein was produced and purified from Escherichia coli. Purified GST-Nrf2 or GST-cleaved Nrf2 was detected at ∼80–130 kDa, by either Coomassie blue staining or immunoblot analysis using an Nrf2 antibody (Santa Cruz [SC]-H300) (Fig. 1b). Endogenous Nrf2 in a variety of cell lines was also detected at ∼95–110 kDa (Fig. 1c). Moreover, endogenous Nrf2 displays induction by well-established activators, sulforaphane or tBHQ. This induction only occurs at the ∼95–110 kDa range with no alterations in levels of other nonspecific bands or any migratory species of ∼55–65 kDa (Fig. 1c). Mouse Nrf2 also migrates at ∼110 kDa since only this band significantly increased by SF treatment in Nrf2+/+ and Nrf2+/− mouse embryonic fibroblast (MEF) cells, whereas no ∼110 kDa band was detected in Nrf2−/− MEFs regardless of treatment (Fig. 1d). More importantly, no prominent bands were detected at the predicted molecular weight of 55–65 kDa in any of the experiments. Notably, human Nrf2 frequently appears as a doublet in low percentage SDS-PAGE gels (<7.5%), however, the reasons for the apparent doublet have not yet been scientifically postulated. Lastly, we compared commercially available antibodies from three different sources in addition to SC-H300 and were able to only detect a band that could be induced by sulforaphane at 110 kDa (Fig. 1e). FIG. 1. NF-E2-related factor 2 (Nrf2) migrates at ∼95–110 kDa. (a) Vector alone (V) or hemagglutinin (HA)-tagged Nrf2 was transfected into human kidney epithelial (HEK293) cells. Cells were either not treated (N) or treated with 25 μ ... Due to the misconception that Nrf2 runs at ∼55–65 kDa, companies that commercialize antibodies may actually discard more specific Nrf2 antibodies that only detect the relevant species of ∼95–110 kDa and instead, commercialize antibodies that show inherently nonspecific bands. Quite often the ∼95–110 kDa band is labeled as “nonspecific” on the antibody data sheet due to a lack of knowledge of an apparent Nrf2 molecular weight of ∼95–110 kDa. The reality that Nrf2 in fact migrates at ∼95–110 kDa has created a huge negative impact on Nrf2 research. A vast amount of the published literature refers to a change in the ∼55–65 kDa band as modulation of Nrf2 protein expression by certain compounds or physiological conditions. As the field begins to move into the translational phase of research by targeting Nrf2 for disease prevention and intervention, proper recognition of the apparent Nrf2 molecular weight and generation of specific Nrf2 antibodies are essential to avoid misinterpretation of results.

Arsenic-Mediated Activation of the Nrf2-Keap1 Antioxidant Pathway

Arsenic is present in the environment and has become a worldwide health concern due to its toxicity and carcinogenicity. However, the specific mechanism(s) by which arsenic elicits its toxic effects has yet to be fully elucidated. The transcription factor nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) has been recognized as the master regulator of a cellular defense mechanism against toxic insults. This review highlights studies demonstrating that arsenic activates the Nrf2‐Keap1 antioxidant pathway by a distinct mechanism from that of natural compounds such as sulforaphane (SF) found in broccoli sprouts or tert‐butylhyrdoquinone (tBHQ), a natural antioxidant commonly used as a food preservative. Evidence also suggests that arsenic prolongs Nrf2 activation and may mimic constitutive activation of Nrf2, which has been found in several human cancers due to disruption of the Nrf2‐Keap1 axis. The current literature strongly suggests that activation of Nrf2 by arsenic potentially contributes to, rather than protects against, arsenic toxicity and carcinogenicity. The mechanism(s) by which known Nrf2 activators, such as the natural chemopreventive compounds SF and lipoic acid, protect against the deleterious effects caused by arsenic will also be discussed. These findings will provide insight to further understand how arsenic promotes a prolonged Nrf2 response, which will lead to the identification of novel molecular markers and development of rational therapies for the prevention or intervention of arsenic‐induced diseases.

Nrf2 promotes neuronal cell differentiation

The transcription factor Nrf2 has emerged as a master regulator of the endogenous antioxidant response, which is critical in defending cells against environmental insults and in maintaining intracellular redox balance. However, whether Nrf2 has any role in neuronal cell differentiation is largely unknown. In this report, we have examined the effects of Nrf2 on cell differentiation using a neuroblastoma cell line, SH-SY5Y. Retinoic acid (RA) and 12-O-tetradecanoylphorbol 13-acetate, two well-studied inducers of neuronal differentiation, are able to induce Nrf2 and its target gene NAD(P)H quinone oxidoreductase 1 in a dose- and time-dependent manner. RA-induced Nrf2 up-regulation is accompanied by neurite outgrowth and an induction of two neuronal differentiation markers, neurofilament-M and microtubule-associated protein 2. Overexpression of Nrf2 in SH-SY5Y cells promotes neuronal differentiation, whereas inhibition of endogenous Nrf2 expression inhibited neuronal differentiation. More remarkably, the positive role of Nrf2 in neuronal differentiation was verified ex vivo in primary neuron culture. Primary neurons isolated from Nrf2-null mice showed a retarded progress in differentiation, compared to those from wild-type mice. Collectively, our data demonstrate a novel role for Nrf2 in promoting neuronal cell differentiation, which will open new perspectives for therapeutic uses of Nrf2 activators in patients with neurodegenerative diseases.

Tanshinone I Activates the Nrf2-Dependent Antioxidant Response and Protects Against As(III)-Induced Lung Inflammation In Vitro and In Vivo

AIMS The NF-E2 p45-related factor 2 (Nrf2) signaling pathway regulates the cellular antioxidant response and activation of Nrf2 has recently been shown to limit tissue damage from exposure to environmental toxicants, including As(III). In an attempt to identify improved molecular agents for systemic protection against environmental insults, we have focused on the identification of novel medicinal plant-derived Nrf2 activators. RESULTS Tanshinones [tanshinone I (T-I), tanshinone IIA, dihydrotanshinone, cryptotanshinone], phenanthrenequinone-based redox therapeutics derived from the medicinal herb Salvia miltiorrhiza, have been tested as experimental therapeutics for Nrf2-dependent cytoprotection. Using a dual luciferase reporter assay overexpressing wild-type or mutant Kelch-like ECH-associated protein-1 (Keap1), we demonstrate that T-I is a potent Keap1-C151-dependent Nrf2 activator that stabilizes Nrf2 by hindering its ubiquitination. In human bronchial epithelial cells exposed to As(III), T-I displays pronounced cytoprotective activity with upregulation of Nrf2-orchestrated gene expression. In Nrf2 wild-type mice, systemic administration of T-I attenuates As(III) induced inflammatory lung damage, a protective effect not observed in Nrf2 knockout mice. INNOVATION Tanshinones have been identified as a novel class of Nrf2-inducers for antioxidant tissue protection in an in vivo As(III) inhalation model, that is relevant to low doses of environmental exposure. CONCLUSION T-I represents a prototype Nrf2-activator that displays cytoprotective activity upon systemic administration targeting lung damage originating from environmental insults. T-I based Nrf2-directed systemic intervention may provide therapeutic benefit in protecting other organs against environmental insults.

KPNA6 (Importin α7)-mediated nuclear import of Keap1 represses the Nrf2-dependent antioxidant response

ABSTRACT The transcription factor Nrf2 has emerged as a master regulator of cellular redox homeostasis. As an adaptive response to oxidative stress, Nrf2 activates the transcription of a battery of genes encoding antioxidants, detoxification enzymes, and xenobiotic transporters by binding the cis-antioxidant response element in the promoter regions of genes. The magnitude and duration of inducible Nrf2 signaling is delicately controlled at multiple levels by Keap1, which targets Nrf2 for redox-sensitive ubiquitin-mediated degradation in the cytoplasm and exports Nrf2 from the nucleus. However, it is not clear how Keap1 gains access to the nucleus. In this study, we show that Keap1 is constantly shuttling between the nucleus and the cytoplasm under physiological conditions. The nuclear import of Keap1 requires its C-terminal Kelch domain and is independent of Nrf1 and Nrf2. We have determined that importin α7, also known as karyopherin α6 (KPNA6), directly interacts with the Kelch domain of Keap1. Overexpression of KPNA6 facilitates Keap1 nuclear import and attenuates Nrf2 signaling, whereas knockdown of KPNA6 slows down Keap1 nuclear import and enhances the Nrf2-mediated adaptive response induced by oxidative stress. Furthermore, KPNA6 accelerates the clearance of Nrf2 protein from the nucleus during the postinduction phase, therefore promoting restoration of the Nrf2 protein to basal levels. These findings demonstrate that KPNA6-mediated Keap1 nuclear import plays an essential role in modulating the Nrf2-dependent antioxidant response and maintaining cellular redox homeostasis.