Samantha A. Whitman

Therapeutic Potential of Nrf2 Activators in Streptozotocin-Induced Diabetic Nephropathy

OBJECTIVE To determine whether dietary compounds targeting NFE2-related factor 2 (Nrf2) activation can be used to attenuate renal damage and preserve renal function during the course of streptozotocin (STZ)-induced diabetic nephropathy. RESEARCH DESIGN AND METHODS Diabetes was induced in Nrf2+/+ and Nrf2−/− mice by STZ injection. Sulforaphane (SF) or cinnamic aldehyde (CA) was administered 2 weeks after STZ injection and metabolic indices and renal structure and function were assessed (18 weeks). Markers of diabetes including blood glucose, insulin, polydipsia, polyuria, and weight loss were measured. Pathological alterations and oxidative damage in glomeruli were also determined. Changes in protein expression of the Nrf2 pathway, as well as transforming growth factor-β1 (TGF-β1), fibronectin (FN), collagen IV, and p21/WAF1Cip1 (p21) were analyzed. The molecular mechanisms of Nrf2-mediated protection were investigated in an in vitro model using human renal mesangial cells (HRMCs). RESULTS SF or CA significantly attenuated common metabolic disorder symptoms associated with diabetes in Nrf2+/+ but not in Nrf2−/− mice, indicating SF and CA function through specific activation of the Nrf2 pathway. Furthermore, SF or CA improved renal performance and minimized pathological alterations in the glomerulus of STZ-Nrf2+/+ mice. Nrf2 activation reduced oxidative damage and suppressed the expression of TGF-β1, extracellular matrix proteins and p21 both in vivo and in HRMCs. In addition, Nrf2 activation reverted p21-mediated growth inhibition and hypertrophy of HRMCs under hyperglycemic conditions. CONCLUSIONS We provide experimental evidence indicating that dietary compounds targeting Nrf2 activation can be used therapeutically to improve metabolic disorder and relieve renal damage induced by diabetes.

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.

Nrf2 suppresses lupus nephritis through inhibition of oxidative injury and the NF-κB-mediated inflammatory response

The generation of reactive oxygen species plays a pivotal role in both acute and chronic glomerular injuries in patients with lupus nephritis. Since the transcription factor Nrf2 is a major regulator of the antioxidant response and is a primary cellular defense mechanism we sought to determine a role of Nrf2 in the progression of lupus nephritis. Pathological analyses of renal biopsies from patients with different types of lupus nephritis showed oxidative damage in the glomeruli, accompanied by an active Nrf2 antioxidant response. A murine lupus nephritis model using Nrf2+/+ and Nrf2−/− mice was established using pristine injection. In this model, Nrf2−/− mice suffered from greater renal damage and had more severe pathological alterations in the kidney. In addition, Nrf2+/+ mice showed ameliorative renal function when treated with sulforaphane, an Nrf2 inducer. Nrf2−/− mice had higher expression of TGFβ1, fibronectin and iNOS. In primary mouse mesangial cells, the nephritogenic monoclonal antibody R4A activated the NF-κB pathway and increased the level of reactive oxygen species, iNOS, TGFβ1 and fibronectin. Knockdown of Nrf2 expression aggravated all aforementioned responses induced by R4A. Thus, these results suggest that Nrf2 improves lupus nephritis by neutralizing reactive oxygen species and by negatively regulating the NF-κB and TGFβ1 signaling pathways.

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.

Basic ResearchNrf2 suppresses lupus nephritis through inhibition of oxidative injury and the NF-κB-mediated inflammatory response

The generation of reactive oxygen species plays a pivotal role in both acute and chronic glomerular injuries in patients with lupus nephritis. Since the transcription factor Nrf2 is a major regulator of the antioxidant response and is a primary cellular defense mechanism we sought to determine a role of Nrf2 in the progression of lupus nephritis. Pathological analyses of renal biopsies from patients with different types of lupus nephritis showed oxidative damage in the glomeruli, accompanied by an active Nrf2 antioxidant response. A murine lupus nephritis model using Nrf2+/+ and Nrf2−/− mice was established using pristine injection. In this model, Nrf2−/− mice suffered from greater renal damage and had more severe pathological alterations in the kidney. In addition, Nrf2+/+ mice showed ameliorative renal function when treated with sulforaphane, an Nrf2 inducer. Nrf2−/− mice had higher expression of TGFβ1, fibronectin and iNOS. In primary mouse mesangial cells, the nephritogenic monoclonal antibody R4A activated the NF-κB pathway and increased the level of reactive oxygen species, iNOS, TGFβ1 and fibronectin. Knockdown of Nrf2 expression aggravated all aforementioned responses induced by R4A. Thus, these results suggest that Nrf2 improves lupus nephritis by neutralizing reactive oxygen species and by negatively regulating the NF-κB and TGFβ1 signaling pathways.

Desmoplakin and Talin2 Are Novel mRNA Targets of Fragile X–Related Protein-1 in Cardiac Muscle

Rationale: The proper function of cardiac muscle requires the precise assembly and interactions of numerous cytoskeletal and regulatory proteins into specialized structures that orchestrate contraction and force transmission. Evidence suggests that posttranscriptional regulation is critical for muscle function, but the mechanisms involved remain understudied. Objective: To investigate the molecular mechanisms and targets of the muscle-specific fragile X mental retardation, autosomal homolog 1 (FXR1), an RNA binding protein whose loss leads to perinatal lethality in mice and cardiomyopathy in zebrafish. Methods and Results: Using RNA immunoprecipitation approaches we found that desmoplakin and talin2 mRNAs associate with FXR1 in a complex. In vitro assays indicate that FXR1 binds these mRNA targets directly and represses their translation. Fxr1 KO hearts exhibit an up-regulation of desmoplakin and talin2 proteins, which is accompanied by severe disruption of desmosome as well as costamere architecture and composition in the heart, as determined by electron microscopy and deconvolution immunofluorescence analysis. Conclusions: Our findings reveal the first direct mRNA targets of FXR1 in striated muscle and support translational repression as a novel mechanism for regulating heart muscle development and function, in particular the assembly of specialized cytoskeletal structures.

Desmoplakin and Talin2 Are Novel mRNA Targets of Fragile X–Related Protein-1 in Cardiac Muscle Whitman; Dsp and Tln2 Are mRNA Targets of FXR1 in the Heart

Rationale: The proper function of cardiac muscle requires the precise assembly and interactions of numerous cytoskeletal and regulatory proteins into specialized structures that orchestrate contraction and force transmission. Evidence suggests that posttranscriptional regulation is critical for muscle function, but the mechanisms involved remain understudied. Objective: To investigate the molecular mechanisms and targets of the muscle-specific fragile X mental retardation, autosomal homolog 1 (FXR1), an RNA binding protein whose loss leads to perinatal lethality in mice and cardiomyopathy in zebrafish. Methods and Results: Using RNA immunoprecipitation approaches we found that desmoplakin and talin2 mRNAs associate with FXR1 in a complex. In vitro assays indicate that FXR1 binds these mRNA targets directly and represses their translation. Fxr1 KO hearts exhibit an up-regulation of desmoplakin and talin2 proteins, which is accompanied by severe disruption of desmosome as well as costamere architecture and composition in the heart, as determined by electron microscopy and deconvolution immunofluorescence analysis. Conclusions: Our findings reveal the first direct mRNA targets of FXR1 in striated muscle and support translational repression as a novel mechanism for regulating heart muscle development and function, in particular the assembly of specialized cytoskeletal structures.

Cellular self-organization by autocatalytic alignment feedback

Myoblasts aggregate, differentiate and fuse to form skeletal muscle during both embryogenesis and tissue regeneration. For proper muscle function, long-range self-organization of myoblasts is required to create organized muscle architecture globally aligned to neighboring tissue. However, how the cells process geometric information over distances considerably longer than individual cells to self-organize into well-ordered, aligned and multinucleated myofibers remains a central question in developmental biology and regenerative medicine. Using plasma lithography micropatterning to create spatial cues for cell guidance, we show a physical mechanism by which orientation information can propagate for a long distance from a geometric boundary to guide development of muscle tissue. This long-range alignment occurs only in differentiating myoblasts, but not in non-fusing myoblasts perturbed by microfluidic disturbances or other non-fusing cell types. Computational cellular automata analysis of the spatiotemporal evolution of the self-organization process reveals that myogenic fusion in conjunction with rotational inertia functions in a self-reinforcing manner to enhance long-range propagation of alignment information. With this autocatalytic alignment feedback, well-ordered alignment of muscle could reinforce existing orientations and help promote proper arrangement with neighboring tissue and overall organization. Such physical self-enhancement might represent a fundamental mechanism for long-range pattern formation during tissue morphogenesis.

Nrf2 modulates contractile and metabolic properties of skeletal muscle in streptozotocin-induced diabetic atrophy

The role of Nrf2 in disease prevention and treatment is well documented; however the specific role of Nrf2 in skeletal muscle is not well described. The current study investigated whether Nrf2 plays a protective role in an STZ-induced model of skeletal muscle atrophy. Modulation of Nrf2 through siRNA resulted in a more robust differentiation of C2C12s, whereas increasing Nrf2 with sulforaphane treatment inhibited differentiation. Diabetic muscle atrophy was not dramatically influenced by Nrf2 genotype, since no differences were observed in total atrophy (all fiber types combined) between WT+STZ and KO+STZ animals. Nrf2-KO animals however illustrated alterations in muscle size of Fast, Type II myosin expressing fibers. KO+STZ animals show significant alterations in myosin isoform expression in the GAST. Similarly, KO controls mimic both WT+STZ and KO+STZ muscle alterations in mitochondrial subunit expression. PGC-1α, a well-established player in mitochondrial biogenesis and myosin isoform expression, was decreased in KO control, WT+STZ and KO+STZ SOL muscle. Similarly, PGC-1α protein levels are correlated with Nrf2 levels in C2C12s after modulation by Nrf2 siRNA or sulforaphane treatment. We provide experimental evidence indicating Nrf2 plays a role in myocyte differentiation and governs molecular alterations in contractile and metabolic properties in an STZ-induced model of muscle atrophy.