Reactive oxygen species (ROS) can induce premature cellular senescence, which is believed to contribute to aging and age-related diseases. The nuclear erythroid 2 p45-related factor-2 (Nrf2) is a transcription factor that mediates cytoprotective responses against stress. We demonstrate that caveolin-1 is a direct binding partner of Nrf2, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82-101) to the caveolin-binding domain of Nrf2 (amino acids 281-289). Biochemical studies show that Nrf2 is concentrated into caveolar membranes in human and mouse fibroblasts, where it colocalizes with caveolin-1, under resting conditions. After oxidative stress, caveolin-1 limits the movement of Nrf2 from caveolar membranes to the nucleus. In contrast, Nrf2 is constitutively localized to the nucleus before and after oxidative stress in caveolin-1-null mouse embryonic fibroblasts (MEFs), which do not express caveolin-1. Functional studies demonstrate that caveolin-1 acts as an endogenous inhibitor of Nrf2, as shown by the enhanced up-regulation of NQO1, an Nrf2 target gene, in caveolin-1-null MEFs and the activation or inhibition of a luciferase construct carrying an antioxidant responsive element (ARE) after down-regulation of caveolin-1 by small interfering RNA or overexpression of caveolin-1, respectively. Expression of a mutant form of Nrf2 that cannot bind to caveolin-1 (Φ→A-Nrf2) hyperactivates ARE and inhibits oxidative stress-induced activation of the p53/p21(Waf1/Cip1) pathway and induction of premature senescence in fibroblasts. Finally, we show that overexpression of caveolin-1 in colon cancer cells inhibits oxidant-induced activation of Nrf2-dependent signaling, promotes premature senescence, and inhibits their transformed phenotype. Thus, by inhibiting Nrf2-mediated signaling, caveolin-1 links free radicals to the activation of the p53/senescence pathway.

Previous studies using in vitro cell culture systems have shown the role of the dynamin-related GTPase Opa1 in apoptosis prevention and mitochondrial DNA (mtDNA) maintenance. However, it remains to be tested whether these functions of Opa1 are physiologically important in vivo in mammals. Here, using the Cre-loxP system, we deleted mouse Opa1 in pancreatic beta cells, in which glucose-stimulated ATP production in mitochondria plays a key role in insulin secretion. Beta cells lacking Opa1 maintained normal copy numbers of mtDNA; however, the amount and activity of electron transport chain complex IV were significantly decreased, leading to impaired glucose-stimulated ATP production and insulin secretion. In addition, in Opa1-null beta cells, cell proliferation was impaired, whereas apoptosis was not promoted. Consequently, mice lacking Opa1 in beta cells develop hyperglycemia. The data suggest that the function of Opa1 in the maintenance of the electron transport chain is physiologically relevant in beta cells.

The Nrf2 (nuclear factor E2 p45-related factor 2) transcription factor responds to diverse oxidative and electrophilic environmental stresses by circumventing repression by Keap1, translocating to the nucleus, and activating cytoprotective genes. Nrf2 responses provide protection against chemical carcinogenesis, chronic inflammation, neurodegeneration, emphysema, asthma and sepsis in murine models. Nrf2 regulates the expression of a plethora of genes that detoxify oxidants and electrophiles and repair or remove damaged macromolecules, such as through proteasomal processing. However, many direct targets of Nrf2 remain undefined. Here, mouse embryonic fibroblasts (MEF) with either constitutive nuclear accumulation (Keap1(-/-)) or depletion (Nrf2(-/-)) of Nrf2 were utilized to perform chromatin-immunoprecipitation with parallel sequencing (ChIP-Seq) and global transcription profiling. This unique Nrf2 ChIP-Seq dataset is highly enriched for Nrf2-binding motifs. Integrating ChIP-Seq and microarray analyses, we identified 645 basal and 654 inducible direct targets of Nrf2, with 244 genes at the intersection. Modulated pathways in stress response and cell proliferation distinguish the inducible and basal programs. Results were confirmed in an in vivo stress model of cigarette smoke-exposed mice. This study reveals global circuitry of the Nrf2 stress response emphasizing Nrf2 as a central node in cell survival response.

Mitochondria divide and fuse continuously, and the balance between these two processes regulates mitochondrial shape. Alterations in mitochondrial dynamics are associated with neurodegenerative diseases. Here we investigate the physiological and cellular functions of mitochondrial division in postmitotic neurons using in vivo and in vitro gene knockout for the mitochondrial division protein Drp1. When mouse Drp1 was deleted in postmitotic Purkinje cells in the cerebellum, mitochondrial tubules elongated due to excess fusion, became large spheres due to oxidative damage, accumulated ubiquitin and mitophagy markers, and lost respiratory function, leading to neurodegeneration. Ubiquitination of mitochondria was independent of the E3 ubiquitin ligase parkin in Purkinje cells lacking Drp1. Treatment with antioxidants rescued mitochondrial swelling and cell death in Drp1KO Purkinje cells. Moreover, hydrogen peroxide converted elongated tubules into large spheres in Drp1KO fibroblasts. Our findings suggest that mitochondrial division serves as a quality control mechanism to suppress oxidative damage and thus promote neuronal survival.

Notch signaling coordinates cell differentiation processes in the intestinal epithelium. The transcription factor Nrf2 orchestrates defense mechanisms by regulating cellular redox homeostasis, which, as shown previously in murine liver, can be amplified through signaling crosstalk with the Notch pathway. However, interplay between these 2 signaling pathways in the gut is unknown.

The dynamin-related guanosine triphosphatase Drp1 mediates the division of mitochondria and peroxisomes. To understand the in vivo function of Drp1, complete and tissue-specific mouse knockouts of Drp1 were generated. Drp1-null mice die by embryonic day 11.5. This embryonic lethality is not likely caused by gross energy deprivation, as Drp1-null cells showed normal intracellular adenosine triphosphate levels. In support of the role of Drp1 in organelle division, mitochondria formed extensive networks, and peroxisomes were elongated in Drp1-null embryonic fibroblasts. Brain-specific Drp1 ablation caused developmental defects of the cerebellum in which Purkinje cells contained few giant mitochondria instead of the many short tubular mitochondria observed in control cells. In addition, Drp1-null embryos failed to undergo developmentally regulated apoptosis during neural tube formation in vivo. However, Drp1-null embryonic fibroblasts have normal responses to apoptotic stimuli in vitro, suggesting that the apoptotic function of Drp1 depends on physiological cues. These findings clearly demonstrate the physiological importance of Drp1-mediated organelle division in mice.

Lipodystrophy is a disorder featuring loss of normal adipose tissue depots due to impaired production of normal adipocytes. It leads to a gain of fat deposition in ectopic tissues such as liver and skeletal muscle that results in steatosis, dyslipidemia, and insulin resistance. Previously, we established a Rosa NIC/NIC::AdiCre lipodystrophy model mouse. The lipodystrophic phenotype that included hepatomegaly accompanied with hepatic damage due to higher lipid accumulation was attenuated substantially by amplified systemic NRF2 signaling in mice with hypomorphic expression of Keap1; whole-body Nrf2 deletion abrogated this protection. To determine whether hepatic-specific NRF2 signaling would be sufficient for protection against hepatomegaly and fatty liver development, direct, powerful, transient expression of Nrf2 or its target gene Nqo1 was achieved by administration through hydrodynamic tail vein injection of pCAG expression vectors of dominant-active Nrf2 and Nqo1 in Rosa NIC/NIC::AdiCre mice fed a 9% fat diet. Both vectors enabled protection from hepatic damage, with the pCAG-Nqo1 vector being the more effective as seen with a ~50% decrease in hepatic triglyceride levels. Therefore, activating NRF2 signaling or direct elevation of NQO1 in the liver provides new possibilities to partially reduce steatosis that accompanies lipodystrophy.

The Notch family of intermembrane receptors is highly conserved across species and is involved in cell fate and lineage control. Previous in vitro studies have shown that Notch may inhibit adipogenesis. Here we describe the role of Notch in adipose tissue by employing an in vivo murine model which overexpresses Notch in adipose tissue.

Small molecules of plant origin offer presumptively safe opportunities to prevent carcinogenesis, mutagenesis and other forms of toxicity in humans. However, the mechanisms of action of such plant-based agents remain largely unknown. In recent years the stress responsive transcription factor Nrf2 has been validated as a target for disease chemoprevention. Withania somnifera (WS) is a herb used in Ayurveda (an ancient form of medicine in South Asia). In the recent past, withanolides isolated from WS, such as Withaferin A (WA) have been demonstrated to be preventive and therapeutic against multiple diseases in experimental models. The goals of this study are to evaluate withanolides such as WA as well as Withania somnifera root extract as inducers of Nrf2 signaling, to probe the underlying signaling mechanism of WA and to determine whether prevention of acetaminophen (APAP)-induced hepatic toxicity in mice by WA occurs in an Nrf2-dependent manner. We observed that WA profoundly protects wild-type mice but not Nrf2-disrupted mice against APAP hepatotoxicity. WA is a potent inducer of Nrf2-dependent cytoprotective enzyme expression both in vivo and in vitro. Unexpectedly, WA induces Nrf2 signaling at least in part, in a Keap1-independent, Pten/Pi3k/Akt-dependent manner in comparison to prototypical Nrf2 inducers, sulforaphane and CDDO-Im. The identification of WA as an Nrf2 inducer that can signal through a non-canonical, Keap1-independent pathway provides an opportunity to evaluate the role of other regulatory partners of Nrf2 in the dietary and pharmacological induction of Nrf2-mediated cytoprotection.

Bardoxolone methyl (CDDO-Me) is an oleanane triterpenoid in late-stage clinical development for the treatment of patients with diabetic kidney disease. Preclinical studies in rodents demonstrate the efficacy of triterpenoids against carcinogenesis and other diseases, including renal ischemia-reperfusion injury, hyperoxia-induced acute lung injury, and immune hepatitis. Genetic disruption of Nrf2 abrogates protection by triterpenoids, suggesting that induction of the NRF2 pathway may drive this protection. Herein, we examined the effect of a point mutation (C151S) in KEAP1, a repressor of NRF2 signaling, at cysteine 151 in mouse embryo fibroblasts and mouse liver. Induction of target gene transcripts and enzyme activity by CDDO-Me was lost in C151S mutant fibroblasts compared with wild-type. Protection against menadione toxicity was also nullified in the mutant fibroblasts. In mouse liver, CDDO-Me evoked the nuclear translocation of NRF2, followed by increased transcript and activity levels of a prototypic target gene, Nqo1, in wild-type, but not C151S mutant, mice. To test the role of KEAP1 Cys151 in governing the broader pharmacodynamic action of CDDO-Me, wild-type and C151S mutant mice were challenged with concanavalin A to induce immune hepatitis. Strong protection was seen in wild-type but not C151S mutant mice. RNA-seq analysis of mouse liver from wild-type, C151S mutant, and Nrf2-knockout mice revealed a vigorous response of the NRF2 transcriptome in wild-type, but in neither C151S mutant nor Nrf2-knockout, mice. Activation of "off-target" pathways by CDDO were not observed. These data highlight the singular importance of the KEAP1 cysteine 151 sensor for activation of NRF2 signaling by CDDO-Me. SIGNIFICANCE STATEMENT: KEAP1 serves as a key sensor for induction of the cytoprotective signaling pathway driven by the transcription factor NRF2. Mutation of a single cysteine (C151) in KEAP1 abrogates the induction of NRF2 signaling and its downstream cytoprotective actions in vitro and in vivo by bardoxolone methyl (CDDO-Me), a drug in late-stage clinical development. Further, at these bioeffective concentrations/doses, activation of "off-target" pathways by CDDO-Me are not observed, highlighting the singular importance of NRF2 in its mode of action.