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Background & Aims 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. Methods Mice modified genetically to amplify Nrf2 in the intestinal epithelium (Keap1f/f::VilCre) were generated as well as pharmacological activation of Nrf2 and subjected to phenotypic and cell lineage analyses. Cell lines were used for reporter gene assays together with Nrf2 overexpression to study transcriptional regulation of the Notch downstream effector. Results Constitutive activation of Nrf2 signaling caused increased intestinal length along with expanded cell number and thickness of enterocytes without any alterations of secretory lineage, outcomes abrogated by concomitant disruption of Nrf2. The Nrf2 and Notch pathways in epithelium showed inverse spatial profiles, where Nrf2 activity in crypts was lower than villi. In progenitor cells of Keap1f/f::VilCre mice, Notch downstream effector Math1, which regulates a differentiation balance of cell lineage through lateral inhibition, showed suppressed expression. In vitro results demonstrated Nrf2 negatively regulated Math1, where 6 antioxidant response elements located in the regulatory regions contributed to this repression. Conclusions Activation of Nrf2 perturbed the dialog of the Notch cascade though negative regulation of Math1 in progenitor cells, leading to enhanced enterogenesis. The crosstalk between the Nrf2 and Notch pathways could be critical for fine-tuning intestinal homeostasis and point to new approaches for the pharmacological management of absorptive deficiencies., Graphical abstract

Vascular dysfunctions such as vascular hyporeactivity following ischemic/hypoxic injury are a major cause of death in injured patients. In this study, we showed that treatment with mitochondrial division inhibitor 1 (Mdivi-1), a selective inhibitor of dynamin-related protein 1 (Drp1), significantly improved vascular reactivity in ischemic rats by attenuating oxidative stress. The antioxidative effects of Mdivi-1 were relatively Drp1-independent, and possibly due to an increase in the levels of the antioxidant enzymes, SOD1 and catalase, as well as to enhanced Nrf2 expression. In addition, we found that while Mdivi-1 had little effect on Drp1 GTPase activity in vascular smooth muscle cells, it inhibited hypoxia-induced Drp1 phosphorylation at Ser-616, reducing excessive mitochondrial fission and slightly enhancing mitochondrial fusion. These effects possibly contributed to vascular protection at an early stage of ischemic/hypoxic injury. Finally, Mdivi-1 stabilized hemodynamics, increased vital organ perfusion, and improved rat survival after ischemic/hypoxic injury, proving a promising therapeutic agent for ischemic/hypoxic injury., Highlights • Mdivi-1 improved vascular contractility in ischemic rats. • Mdivi-1 attenuated hypoxia-induced oxidative stress and mitochondrial changes. • Drp1 recruitment to mitochondria, not GTPase activity, involved in Mdivi-1 effects. • Mdivi-1 has therapeutic potential against ischemic injury.

Enzymatic and non-enzymatic oxidation of unsaturated fatty acids gives rise to reactive species that covalently modify nucleophilic residues within redox sensitive protein sensors in a process called lipoxidation. This triggers adaptive signaling pathways that ultimately lead to increased resistance to stress. In this graphical review, we will provide an overview of pathways affected by protein lipoxidation and the key signaling proteins being altered, focusing on the KEAP1-NRF2 and heat shock response pathways. We review the mechanisms by which lipid peroxidation products can serve as second messengers and evoke cellular responses via covalent modification of key sensors of altered cellular environment, ultimately leading to adaptation to stress., Graphical abstract fx1

Nrf2 pathway has been known to be protective against cancer progression however recent studies have revealed that the antioxidant activity of Nrf2 contributes to chemotherapy resistance. For many years, hyperthermia has been used as an additional therapy to increase the efficiency of chemotherapy and radiotherapy. Besides the positive effects of hyperthermia during treatment procedure, thermotolerance has been found to develop against heat treatment. Although the involved molecular mechanisms have not been fully clarified, heat shock proteins (HSP) and proteasome activity are known to be involved in the acquisition of thermotolerance. The aim of this study was to investigate the potential beneficial effects of combining hyperthermia with Nrf2 silencing to inhibit molecular mechanisms leading to induction of defense mechanisms in transcription level. Following heat treatment of HT22 cells, HSP70 and the proteasome levels and as well as proteasome activity were found to be elevated in the nucleus. Our results demonstrated that Nrf2 silencing reduced defense mechanisms against heat treatment both in antioxidant and proteolytic manner and Nrf2 may be a potential target for therapeutic approach in order to improve the beneficial effects of hyperthermia in cancer therapy., Graphical abstract fx1, Highlights • Hyperthermia increases HSP70, β5 levels and Proteasome activity in the nucleus. • Nrf2 pathway contributes to thermotolerance by HO-1 and GSTα. • Nrf2 contributes to thermotolerance via proteasome and HSP related degradation. • Nrf2 Inhibition may be a useful approach to overcome the thermotolerance.

Reactive oxygen species (ROS) are integral components of multiple cellular pathways even though excessive or inappropriately localized ROS damage cells. ROS function as anti-microbial effector molecules and as signaling molecules that regulate such processes as NF-kB transcriptional activity, the production of DNA-based neutrophil extracellular traps (NETs), and autophagy. The main sources of cellular ROS are mitochondria and NADPH oxidases (NOXs). In contrast to NOX-generated ROS, ROS produced in the mitochondria (mtROS) were initially considered to be unwanted by-products of oxidative metabolism. Increasing evidence indicates that mtROS have been incorporated into signaling pathways including those regulating immune responses and autophagy. As metabolic hubs, mitochondria facilitate crosstalk between the metabolic state of the cell with these pathways. Mitochondria and ROS are thus a nexus of multiple pathways that determine the response of cells to disruptions in cellular homeostasis such as infection, sterile damage, and metabolic imbalance. In this review, we discuss the roles of mitochondria in the generation of ROS-derived anti-microbial effectors, the interplay of mitochondria and ROS with autophagy and the formation of DNA extracellular traps, and activation of the NLRP3 inflammasome by ROS and mitochondria., Graphical abstract fx1, Highlights • Mitochondrial ROS production is regulated by and incorporated into immunity pathways. • ROS regulate mitophagy and xenophagy and neutrophil extracellular trap formation. • Mitochondria and mitochondrial ROS regulate NLRP3 inflammasome activation.

Neonatal hypoxic-ischemic (HI) injury is a major cause of neonatal death and neurological dysfunction. H2S has been shown to protect against hypoxia-induced injury and apoptosis of neurons. L-Cysteine is catalyzed by cystathionine-β-synthase (CBS) in the brain and sequentially produces endogenous H2S. The present study was designed to investigate whether L-Cysteine could attenuate the acute brain injury and improve neurobehavioral outcomes following HI brain injury in neonatal mice by releasing endogenous H2S. L-Cysteine treatment significantly attenuated brain edema and decreased infarct volume and neuronal cell death, as shown by a decrease in the Bax/Bcl-2 ratio, suppression of caspase-3 activation, and reduced phosphorylation of Akt and ERK at 72 h after HI. Additionally, L-Cysteine substantially up-regulated NF-E2-related factor 2 and heme oxygenase-1 expression. L-Cysteine also decreased endoplasmic reticulum (ER) stress-associated pro-apoptotic protein expression. Furthermore, L-Cysteine had long-term effects by protecting against the loss of ipsilateral brain tissue and improving neurobehavioral outcomes. Importantly, pre-treatment with a CBS inhibitor significantly attenuated the neuroprotection of L-Cysteine on HI insult. Thus, L-Cysteine exerts neuroprotection against HI-induced injury in neonates via the CBS/H2S pathway, mediated in part by anti-apoptotic effects and reduced oxidative stress and ER stress. Thus, L-Cysteine may be a promising treatment for HI., Graphical abstract fx1, Highlights • L-Cysteine administration at 24 h after HI insult has neuroprotective effect. • L-Cysteine administration attenuated HI-induced oxidative stress and ER stress. • L-Cysteine administration had long-term effects in improving neurobehavioral function at 14 and 28 days after HI insult. • Pre-treatment with a CBS inhibitor significantly attenuated the neuroprotection of L-Cysteine on HI in neonatal mice.

Free radicals originate from both exogenous environmental sources and as by-products of the respiratory chain and cellular oxygen metabolism. Sustained accumulation of free radicals, beyond a physiological level, induces oxidative stress that is harmful for the cellular homeodynamics as it promotes the oxidative damage and stochastic modification of all cellular biomolecules including proteins. In relation to proteome stability and maintenance, the increased concentration of oxidants disrupts the functionality of cellular protein machines resulting eventually in proteotoxic stress and the deregulation of the proteostasis (homeostasis of the proteome) network (PN). PN curates the proteome in the various cellular compartments and the extracellular milieu by modulating protein synthesis and protein machines assembly, protein recycling and stress responses, as well as refolding or degradation of damaged proteins. Molecular chaperones are key players of the PN since they facilitate folding of nascent polypeptides, as well as holding, folding, and/or degradation of unfolded, misfolded, or non-native proteins. Therefore, the expression and the activity of the molecular chaperones are tightly regulated at both the transcriptional and post-translational level at organismal states of increased oxidative and, consequently, proteotoxic stress, including ageing and various age-related diseases (e.g. degenerative diseases and cancer). In the current review we present a synopsis of the various classes of intra- and extracellular chaperones, the effects of oxidants on cellular homeodynamics and diseases and the redox regulation of chaperones., Graphical abstract Differential regulation of chaperones activity, under physiological conditions or during oxidative stress-mediated proteome instability., Highlights • Free radicals originate from various sources and at physiological concentrations are essential for the modulation of cell signalling pathways. • Abnormally high levels of free radicals induce oxidative stress and damage all cellular biomolecules, including proteins. • Molecular chaperones facilitate folding of nascent polypeptides, as well as holding, folding, and/or degradation of damaged proteins. • The expression and the activity of chaperones during oxidative stress are regulated at both the transcriptional and post-translational level.

Hepcidin is a liver-derived antimicrobial peptide that regulates iron absorption and is also an integral part of the acute phase response. In a previous report, we found evidence that this peptide could also be induced by toxic heavy metals and xenobiotics, thus broadening its teleological role as a defensin. However it remained unclear how its sensing of disparate biotic and abiotic stressors might be integrated at the transcriptional level. We hypothesized that its function in cytoprotection may be regulated by NFE2-related factor 2 (Nrf2), the master transcriptional controller of cellular stress defenses. In this report, we show that hepcidin regulation is inextricably linked to the acute stress response through Nrf2 signaling. Nrf2 regulates hepcidin expression from a prototypical antioxidant response element in its promoter, and by synergizing with other basic leucine-zipper transcription factors. We also show that polyphenolic small molecules or phytoestrogens commonly found in fruits and vegetables including the red wine constituent resveratrol can induce hepcidin expression in vitro and post-prandially, with concomitant reductions in circulating iron levels and transferrin saturation by one such polyphenol quercetin. Furthermore, these molecules derepress hepcidin promoter activity when its transcription by Nrf2 is repressed by Keap1. Taken together, the data show that hepcidin is a prototypical antioxidant response or cytoprotective gene within the Nrf2 transcriptional circuitry. The ability of phytoestrogens to modulate hepcidin expression in vivo suggests a novel mechanism by which diet may impact iron homeostasis., Graphical abstract fx1, Highlights • Nrf2 regulates HAMP transcription. • Phytoestrogens differentially induce HAMP transcription by Nrf2. • Post-prandial hepcidin induction reduces serum iron and transferrin saturation. • Nrf2 knockout results in iron-overload in mouse embryonic fibroblasts.

NADPH oxidase-4 (Nox4) is an important modulator of redox signaling that is inducible at the level of transcriptional expression in multiple cell types. By contrast to other Nox enzymes, Nox4 is continuously active without requiring stimulation. We reported recently that expression of Nox4 is induced in the adult heart as an adaptive stress response to pathophysiological insult. To elucidate the potential downstream target(s) regulated by Nox4, we performed a microarray screen to assess the transcriptomes of transgenic (tg) mouse hearts in which Nox4 was overexpressed. The screen revealed a significant increase in the expression of many antioxidant and detoxifying genes regulated by Nrf2 in tg compared to wild-type (wt) mouse hearts, and this finding was subsequently confirmed by Q-PCR. Expression of glutathione biosynthetic and recycling enzymes was increased in tg hearts and associated with higher levels of both GSH and the ratio of reduced:oxidised GSH, compared to wt hearts. The increases in expression of the antioxidant genes and the changes in glutathione redox effected by Nox4 were ablated in an Nrf2-null genetic background. These data therefore demonstrate that Nox4 can activate the Nrf2-regulated pathway, and suggest a potential role for Nox4 in the regulation of GSH redox in cardiomyocytes.