Cytoprotective Transcription Factor Nrf2 Research Articles

ML385, an Nrf2 Inhibitor, Synergically Enhanced Celastrol Triggered Endoplasmic Reticulum Stress in Lung Cancer Cells.

Lung cancer is one of the leading causes of death. Celastrol is a natural product that has shown anticancer activity but has not yet been applied in clinical settings due to its narrow therapeutic window. In this study, we discovered that celastrol stimulates an abnormal rise in the reactive oxygen species (ROS) level in lung cancer cells and that the ROS scavenger N-acetylcysteine (NAC) could counteract the cell death caused by celastrol. At the same time, celastrol upregulated the expression of cytoprotective transcription factor Nrf2 and its downstream proteins, which are effective in preventing the oxidative damage caused by ROS accumulation. Notably, we found that the overexpression of Nrf2 enhances the tolerance of lung cancer cells to celastrol and that lung cancer cells H460 with a Keap1 mutation are insensitive to celastrol. This indicates that the increase in Nrf2 contributes to the survival of lung cancer cells. Thus, we brought in an Nrf2 inhibitor ML385 to suppress the activation of Nrf2. We found that when ML385 and celastrol were added together the survival rates of lung cancer cells decreased more and the detected ROS level became much higher compared to treatment with celastrol alone. We also discovered that ML385 suppressed the expression of HO-1 and GCLC, which amplified celastrol-induced ATF4/CHOP-dependent endoplasmic reticulum stress (ER stress). Above all, our study found that ML385 enhanced celastrol-induced increases in ROS and ER stress, leading to lung cancer cell death. This research provides a potential strategy for the preclinical investigation of celastrol.

Hepatic WDR23 proteostasis mediates insulin homeostasis by regulating insulin-degrading enzyme capacity

Maintaining insulin homeostasis is critical for cellular and organismal metabolism. In the liver, insulin is degraded by the activity of the insulin-degrading enzyme (IDE). Here, we establish a hepatic regulatory axis for IDE through WDR23-proteostasis. Wdr23KO mice have increased IDE expression, reduced circulating insulin, and defective insulin responses. Genetically engineered human cell models lacking WDR23 also increase IDE expression and display dysregulated phosphorylation of insulin signaling cascade proteins, IRS-1, AKT2, MAPK, FoxO, and mTOR, similar to cells treated with insulin, which can be mitigated by chemical inhibition of IDE. Mechanistically, the cytoprotective transcription factor NRF2, a direct target of WDR23-Cul4 proteostasis, mediates the enhanced transcriptional expression of IDE when WDR23 is ablated. Moreover, an analysis of human genetic variation in WDR23 across a large naturally aging human cohort in the US Health and Retirement Study reveals a significant association of WDR23 with altered hemoglobin A1C (HbA1c) levels in older adults, supporting the use of WDR23 as a new molecular determinant of metabolic health in humans.

Acidosis Activates the Nrf2 Pathway in Renal Proximal Tubule-Derived Cells through a Crosstalk with Renal Fibroblasts

Crosstalk of renal epithelial cells with interstitial fibroblasts plays an important role in kidney pathophysiology. A previous study showed that crosstalk between renal epithelial cells and renal fibroblasts protects against acidosis-induced damage. In order to gain further mechanistic insight into this crosstalk, we investigated the effect of acidosis on the transcriptome of renal epithelial cells (NRK-52E) and renal fibroblasts (NRK-49F) in co-culture by RNASeq, bioinformatics analysis and experimental validation. Cells were exposed to acidic media or control media for 48 h. RNA and protein from whole cell lysate were isolated. In addition, cells were fractionated into cytosol, nucleus and chromatin. RNASeq data were analyzed for differential expression and pathway enrichment (ingenuity pathway analysis, IPA, QIAGEN). Total and phosphorylated protein expression was assessed by Western blot (WB). Transcription factor activity was assessed by luciferase reporter assay. Bioinformatic analysis using differentially expressed genes according to RNASeq (7834 for NRK-52E and 3197 for NRK-49F) predicted the antioxidant and cell-protective Nrf2 pathway as acidosis-induced in NRK-52E and NRK-49F cells. Activation of Nrf2 comprises enhanced Nrf2 phosphorylation, nuclear translocation, DNA binding and initiation of a cell protective transcriptional program. Our data show that acidosis enhances chromatin-associated Nrf2 expression and the abundance of phosphorylated Nrf2 in the chromatin fraction of NRK-52E cells in co-culture but not in monoculture. Furthermore, acidosis enhances the activity of a reporter for Nrf2 (ARE-luciferase). Despite the bioinformatics prediction, NRK-49F cells did not respond with Nrf2 activation. Transketolase (TKT) is an important regulator of antioxidant and homeostatic responses in the kidney and a canonical Nrf2 target gene. We show that protein and mRNA expression of TKT is increased in NRK-52E cells under co-culture but not under monoculture conditions. In conclusion, our data show that extracellular acidosis activates the cytoprotective transcription factor Nrf2 in renal epithelial cells co-cultivated with renal fibroblasts, thereby enhancing the expression of cytoprotective TKT. This protective response is not observed in monoculture. Activation of the Nrf2 pathway represents a co-operative cellular strategy of protection against acidosis.

Manjari Medika Grape Seed Extract Protects Methotrexate-Induced Hepatic Inflammation: Involvement of NF-κB/NLRP3 and Nrf2/HO-1 Signaling System.

Grape Seed Extract is a natural source of various polyphenols, which have been shown to possess potent antioxidant and free radical-scavenging activities. The earlier studies have reported that grape seed extract exhibits broad-spectrum pharmacological activities. Therefore, studying the hepatoprotective effects and elucidation of mechanisms of action of the Indian Variety, Manjari Medika grape seed extract (GSE), may give an insight into therapeutic benefits. Methotrexate (MTX) is the first-line pharmacological therapy for different rheumatic diseases. The major adverse events such as hepatotoxicity are evident even in the low doses used for the treatment. The present study investigated the role of MTX on hepatic damage in murine liver and the plausible protective effects of the Indian grape variety, Manjari Medika grape seed extract, in ameliorating it. To assess the hepatological modulation, mice were divided into eight groups to investigate the ameliorative potential of this GSE (75 and 125 mg/kg) and correlate the experimental findings. The active components of the extract were assessed through UPLC-(ESI)-QToF-MS analysis. On the other hand, various biochemical and immunological indices were carried out to correlate the experimental data. The result demonstrated that the prophylactic administration of GSE reduced MTX-induced hepatic toxicity indices, which subsequently restored the hepatic morphological architecture. Moreover, the application of GSE in a dual dosage (75 and 125 mg/kg) suppressed MTX-induced reactive oxygen species generation, followed by lipid peroxidation and cellular nitrite formation. MTX-induced inflammasome activation through the redox-assisted cascade of TLR4/NF-κB signaling was further reduced by applying the GSE. The results showed that the activation of cytoprotective transcription factor Nrf2 enhanced the level of endogenous antioxidants. Furthermore, through the regulation of TLR4/NF-κB and Nrf2/HO-1 axis, this extract could reduce the MTX-mediated hepatic damage. Our findings suggest that Manjari Medika seed extract could be used as a therapeutic agent to relieve the side effects of MTX and other hepatic disorders.

Hinokitiol functions as a ferroptosis inhibitor to confer neuroprotection.

The intrinsic link of ferroptosis to neurodegeneration, such as Parkinson's disease and Alzheimer's disease, has set promises to apply ferroptosis inhibitors for treatment of neurodegenerative disorders. Herein, we report that the natural small molecule hinokitiol (Hino) functions as a potent ferroptosis inhibitor to rescue neuronal damages in vitro and in vivo. The action mechanisms of Hino involve chelating irons and activating cytoprotective transcription factor Nrf2 to upregulate the antioxidant genes including solute carrier family 7 member 11, glutathione peroxidase 4 and Heme oxygenase-1. In vivo studies demonstrate that Hino rescues the deficits of locomotor activity and neurodevelopment in zebrafishes. In addition, Hino shows the efficient blood-brain barrier permeability in mice, supporting the application of Hino for brain disorders. Paclitaxel is one of the most widely used broad-spectrum antineoplastic agents. However, its neurotoxic side effect is a severe concern. We demonstrate that the neurotoxicity of paclitaxel is ferroptosis-related and Hino also alleviates the paclitaxel-induced neurotoxicity without compromising its cytotoxicity to cancer cells. Hino also salvages the neurobehavioral impairment by paclitaxel in zebrafishes. Collectively, the discovery of Hino as a novel ferroptosis inhibitor and disclosure of its action mechanisms establish a foundation for the further development of Hino as a neuroprotective agent.

Diosmin and Trolox Have Anti-Arthritic, Anti-Inflammatory and Antioxidant Potencies in Complete Freund's Adjuvant-Induced Arthritic Male Wistar Rats: Roles of NF-κB, iNOS, Nrf2 and MMPs.

Rheumatoid arthritis (RA) is a chronic, progressive, autoimmune disease caused by a malfunction of the immune system. The aim of this study was to examine the anti-arthritic effects and suggest the mechanisms of actions of diosmin and trolox in male Wistar rats. Complete Freund’s adjuvant (CFA) was used to establish RA in the animals by subcutaneous injection of 100 µL CFA/rat into plantar region of right hind leg in two consecutive days. Diosmin and/or trolox were administered orally at a dosage of 20 mg/kg/day to CFA-induced arthritic rats for 2 weeks. The normal and arthritic control groups were orally given the same equivalent volume of a vehicle (1% carboxymethyl cellulose) in which treatment agents were dissolved. At the end of the experiment, blood samples were collected from the jugular vein for the detection of the total leukocyte count (TLC) and differential leukocyte count (DLC) in blood and the detection of rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPA), tumor necrosis factor-α (TNF-α), interleukin-13 (IL-13), and interleukin-17 (IL-17) levels by enzyme-linked immunosorbent assay (ELISA), as well as markers of oxidative stress and the antioxidant defense system in serum. The right hind ankle regions of three rats from each group were dissected out and fixed in 10% neutral-buffered formalin for histological examination and the other three were kept at −30 °C for Western blot analysis of nuclear factor-kappa B (NF-κB) protein 50 (NF-κB p50), NF-κB p65, inducible nitric oxide synthase (iNOS), nuclear factor erythroid-2-related factor 2 (Nrf2), and matrix metalloproteinase (MMP)-1 (MMP-1), MMP-3, and MMP-9. The CFA injection was deleterious to the ankle joint’s histological architecture, manifesting as infiltration of inflammatory cells into the articular cartilage, hyperplasia of the synovium, and erosion of the cartilage. All these effects were ameliorated by diosmin and/or trolox, with the combined dose being the most effective. The two compounds significantly lowered the elevated serum levels of RF, ACPA, TNF-α, and IL-17, as well as other pro-inflammatory mediators, such as NF-κB p50, NF-κB p65, iNOS, MMP-1, MMP-3 and MMP-9. They also increased the levels of the anti-inflammatory cytokine, IL-13, and the cytoprotective transcription factor Nrf2. The compounds stimulated higher activities of antioxidants, such as glutathione, glutathione-S-transferase, catalase, and superoxide dismutase, and reduced lipid peroxidation in the serum of arthritic rats. In conclusion, diosmin, trolox, and their combination, which was the most potent, exerted anti-arthritic, anti-inflammatory and antioxidant effects by suppressing NF-κB signaling, inhibiting matrix metalloproteinases, and activating Nrf2.

Interaction of the NRF2 and p63 transcription factors promotes keratinocyte proliferation in the epidermis.

Epigenetic regulation of cell and tissue function requires the coordinated action of transcription factors. However, their combinatorial activities during regeneration remain largely unexplored. Here, we discover an unexpected interaction between the cytoprotective transcription factor NRF2 and p63- a key player in epithelial morphogenesis. Chromatin immunoprecipitation combined with sequencing and reporter assays identifies enhancers and promoters that are simultaneously activated by NRF2 and p63 in human keratinocytes. Modeling of p63 and NRF2 binding to nucleosomal DNA suggests their chromatin-assisted interaction. Pharmacological and genetic activation of NRF2 increases NRF2–p63 binding to enhancers and promotes keratinocyte proliferation, which involves the common NRF2–p63 target cyclin-dependent kinase 12. These results unravel a collaborative function of NRF2 and p63 in the control of epidermal renewal and suggest their combined activation as a strategy to promote repair of human skin and other stratified epithelia.

The transcription factor NRF2 shapes the identity of radio-resistant tumor cells

Tumor recurrence and resistance to therapy remain challenging issues in oncology. Various theories have been tested, including the stem cell hypothesis, but conflicting results were obtained, and are not applicable to all tumors types. We have addressed an overarching theory focused on cell “survivology” in cancer, and analyzed the existing information on the involvement of the cytoprotective transcription factor NRF2 in tumor progression, tumorigenicity and radio-resistance, with special emphasis on cancer stem cells (CSCs). The review is focused on NRF2-mediated responses of tumor cells under the pressure of selective intracellular and microenvironmental forces that shape their genotype and phenotype, deciding which cells survive and which ones die. The cytoprotection provided by NRF2 distinctively to normal and tumor cells, the common and unique mechanisms accounting for the survival and evolution of tumor cells and CSCs in the harsh tumor environment, including the particular NRF2-driven hallmark features of CSCs in the context of radiotherapy, are emphasized. Finally, existing natural compounds and small molecules showing at preclinical and clinical level an inhibitory action on NRF2 are revised. Altogether, the review highlights that NRF2 might be considered as an oncogenic marker with clinical prognostic value for disease evolution and response to radiotherapy-based treatment strategies.

IGF-1 Signalling Regulates Mitochondria Dynamics and Turnover through a Conserved GSK-3β-Nrf2-BNIP3 Pathway.

The Insulin-like Growth Factor I (IGF-1) signalling pathway is essential for cell growth and facilitates tumourogenic processes. We recently reported that IGF-1 induces a transcriptional programme for mitochondrial biogenesis, while also inducing expression of the mitophagy receptor BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), suggesting that IGF-1 has a key mitochondria-protective role in cancer cells. Here, we investigated this further and delineated the signaling pathway for BNIP3 induction. We established that IGF-1 induced BNIP3 expression through a known AKT serine/threonine kinase 1 (AKT)-mediated inhibitory phosphorylation on Glycogen Synthase Kinase-3β (GSK-3β), leading to activation of Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2/Nrf2) and acting through the downstream transcriptional regulators Nuclear Respiratory Factor-1 (NRF1) and Hypoxia-inducible Factor 1 subunit α (HIF-1α). Suppression of IGF-1 signaling, Nrf2 or BNIP3 caused the accumulation of elongated mitochondria and altered the mitochondrial dynamics. IGF-1R null Mouse Embryonic Fibroblasts (MEFs) were impaired in the BNIP3 expression and in the capacity to mount a cell survival response in response to serum deprivation or mitochondrial stress. IGF-1 signalling enhanced the cellular capacity to induce autophagosomal turnover in response to activation of either general autophagy or mitophagy. Overall, we conclude that IGF-1 mediated a mitochondria-protective signal that was coordinated through the cytoprotective transcription factor Nrf2. This pathway coupled mitochondrial biogenesis with BNIP3 induction, and increased the cellular capacity for autophagosome turnover, whilst enhancing survival under conditions of metabolic or mitochondrial stress.

Tussilagonone Ameliorates Psoriatic Features in Keratinocytes and Imiquimod-Induced Psoriasis-Like Lesions in Mice via NRF2 Activation

Psoriasis is a common inflammatory skin disorder that is characterized by keratinocyte hyperproliferation and abnormal differentiation, resulting in the thickening of the epidermis and stratum corneum. In this study, we investigated invitro and invivo pharmacological effects of tussilagonone (TGN), a sesquiterpenoid isolated from Tussilago farfara, on transcription factors relevant for the pathogenesis of psoriasis. TGN inhibited activation of NF-κB and STAT3, leading to the attenuated expression of psoriasis-related inflammatory genes and suppression of keratinocyte hyperproliferation. Mechanistically, we show that the inhibition of NF-κB and STAT3 by TGN is mediated through activation of the cytoprotective transcription factor NRF2. Evaluation of invivo antipsoriatic effects of topical TGN in the imiquimod-induced psoriasis-like dermatitis mouse model demonstrated amelioration of imiquimod-induced phenotypical changes, lesion severity score, epidermal thickening, and reduction in dermal cellularity. The spleen index also diminished in TGN-treated mice, suggesting anti-inflammatory properties of TGN. Moreover, TGN significantly attenuated the imiquimod-induced mRNA levels of psoriasis-associated inflammatory cytokines and antimicrobial peptides and reduced epidermal hyperproliferation. Taken together, TGN, as a potent NRF2 activator, is a promising therapeutic candidate for the development of antipsoriatic agents derived from medicinal plants.

The expression profile of redox genes in human monocytes exposed in vitro to γ radiation

BackgroundIt is known that γ radiation is rapidly triggering in cells and in the surrounding medium a hydrogen peroxide burst through water radiolysis, which may have early and late consequence on the viability and functions of irradiated cells. Investigations on monocytes and macrophages are of utmost importance for assessing radiation risks, considering that they are highly responsive to danger/damage signals, are master regulators of the immune response and actively contribute to repair processes in tissues damaged by radiation. AimIn the present study we investigated the expression changes of redox genes in human SC monocytes exposed in vitro to γ rays, aiming to identify delayed disturbances of the redox gene network in irradiated cells. MethodsSC monocytes were exposed to γ radiation in the dose range of 1–5 Gy, at various dose rates (1.6 Gy/h, 4 Gy/h and 10 Gy/h). Cells were further cultivated and were analyzed at various time points after irradiation (24 h and 48 h) regarding cell viability and the expression profile of 84 redox genes critically involved in oxidative stress and antioxidant responses, addressing ROS metabolism, oxidative stress responsive genes, antioxidant genes and pathway activity signature genes. ResultsOver-expression of particular genes encoding various members of the NADPH oxidase family or myeloperoxidase evidenced an increased potential of irradiated cells to generate reactive oxygen or nitrogen species, especially superoxide anion. Alternatively, up-regulation of genes encoding antioxidant molecules indirectly proved that an increased oxidative activity occurred in irradiated monocytes. Redox-mediated cellular deregulation was evidenced by the over-expression of the BNIP3 gene which is involved in cell death by apoptosis or autophagy, and was complemented by the up-regulation of the DUSP1 gene involved in cell cycle arrest and functional silencing of monocytes. At lower doses and dose rates, an enhanced molecular fingerprint of antioxidant responses mediated by the cytoprotective transcription factor NRF2 was found, albeit not being able to avoid the observed reduction of viable cells in irradiated cultures. Moreover, only a low transcriptional activity of NRF2 was registered at higher doses which probably inflicted profound oxidative damages that compromised critical cellular defense mechanisms. ConclusionPathway-focused analysis of redox gene expression allowed us to highlight delayed oxidative alterations in monocytes exposed in vitro to γ rays, indicating a persistent oxidative activity after irradiation. Moreover, we emphasized the necessity to boost the endogenous antioxidant system in normal cells exposed to higher doses of γ rays, in order to protect them against the deleterious effects of radiation. A therapeutic option might be the pharmacological activation of the transcription factor NRF2 which can rapidly react to even small increases in intracellular oxidants, and regulates the expression of many antioxidant and cytoprotective genes.

SUN-048 Maternal Caloric Restriction during Pregnancy Activates the Nrf2 Antioxidant Program in Embryonic Mouse Liver

Obesity and type 2 diabetes in adult life are influenced by the intrauterine environment. The fetus adapts to the environment to which it was exposed, leading potentially to resistance to similar exposures experienced after birth. Studies in both mice and humans suggest that exposure of pregnant females to nutrient deprivation may lead their offspring to develop obesity, insulin resistance and ultimately cardiovascular disease in their adulthood. As the cytoprotective transcription factor Nrf2 (Nfe2l2) is known to be activated upon oxidative and electrophilic stress and has an emerging role in metabolism and specifically in hepatic gluconeogenesis and lipogenesis, we hypothesized that it should be important in the adaptation of the embryos to the metabolic stress induced by maternal caloric restriction. To this end, pregnant C57BL6 mice underwent calorie restriction (CR) by providing them with 50% of the necessary calories per body weight (assessed in pilot experiments) from gestation day 10 to gestation day 16. This CR scheme guarantees the delivery of viable pups that weigh 50% less than the pups whose mothers were fed ad libitum standard chow diet. This CR experiment was performed using either wild-type (WT) or Nrf2 knockout (Nrf2KO) parents. Pregnant females of both genotypes fed ad libitum were used as control. The pregnant females were sacrificed on gestational day 16 and embryos were extracted. Embryonic liver was used for DNA extraction for sex determination by PCR and for RNA preparation to be used for quantitative real-time PCR (qPCR). Statistical significance was set at p<0.05. 10 male embryonic livers were used in qPCR per each condition (CR or control) per each genotype (WT or Nrf2KO). The prototypical Nrf2 antioxidant target gene Nqo1 was increased 2-fold in CR mice. Nrf2-regulated glutathione transferases Gsta1 and Gsta4 were also induced 7.7-fold and 2-fold respectively after CR. Deletion of Nrf2 abrogated completely Nqo1 induction after CR and reduced Gsta1 induction to just 1.4-fold while Gsta1 induction remained unchanged. De novo lipogenesis genes Fasn and Acc2 expression was ~50% and ~90% lower after CR and this change was not significantly affected by Nrf2 deletion. The expression of G6Pase, one of the key gluconeogenic enzymes was also repressed 5-fold in CR mice while expression of Pepck remained unaffected. Lack of Nrf2 impaired the repression of G6Pase to 2-fold. Our study shows for the first time induction of Nrf2 pathway in embryonic liver exposed to CR. Molecular mechanisms and physiological significance of this effect in adult life of the CR WT and Nrf2KO embryos are currently investigated.

Strigolactone GR24 upregulates target genes of the cytoprotective transcription factor Nrf2 in skeletal muscle.

GR24 is a synthetic strigolactone analog, demonstrated to regulate the development of plants and arbuscular mycorrhizal fungi. GR24 possesses anti-cancer and anti-apoptotic properties, enhances insulin sensitivity and mitochondrial biogenesis in skeletal myotubes, inhibits adipogenesis, decreases inflammation in adipocytes and macrophages and downregulates the expression of hepatic gluconeogenic enzymes. Transcription factor Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) is a master regulator of antioxidant response, regulating a multitude of genes involved in cellular stress responses and anti-inflammatory pathways, thus maintaining cellular redox homeostasis. Nrf2 activation reduces the deleterious effects of mitochondrial toxins and has multiple roles in promoting mitochondrial function and dynamics. We studied the role of GR24 on gene expression in rat L6 skeletal muscle cells which were differentiated into myotubes. The myotubes were treated with GR24 and analyzed by microarray gene expression profiling. GR24 upregulated the cytoprotective transcription factor Nrf2 and its target genes, activating antioxidant defences, suggesting that GR24 may protect skeletal muscle from the toxic effects of oxidative stress.

Strigolactone GR24 upregulates target genes of the cytoprotective transcription factor Nrf2 in skeletal muscle

GR24 is a synthetic strigolactone analog, demonstrated to regulate the development of plants and arbuscular mycorrhizal fungi. GR24 possesses anti-cancer and anti-apoptotic properties, enhances insulin sensitivity and mitochondrial biogenesis in skeletal myotubes, inhibits adipogenesis, decreases inflammation in adipocytes and macrophages and downregulates the expression of hepatic gluconeogenic enzymes. Transcription factor Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) is a master regulator of antioxidant response, regulating a multitude of genes involved in cellular stress responses and anti-inflammatory pathways, thus maintaining cellular redox homeostasis. Nrf2 activation reduces the deleterious effects of mitochondrial toxins and has multiple roles in promoting mitochondrial function and dynamics. We studied the role of GR24 on gene expression in rat L6 skeletal muscle cells which were differentiated into myotubes. The myotubes were treated with GR24 and analyzed by microarray gene expression profiling. GR24 upregulated the cytoprotective transcription factor Nrf2 and its target genes, activating antioxidant defences, suggesting that GR24 may protect skeletal muscle from the toxic effects of oxidative stress.

Opsonic Phagocytosis in Chronic Obstructive Pulmonary Disease Is Enhanced by Nrf2 Agonists.

Previous studies have identified defects in bacterial phagocytosis by alveolar macrophages (AMs) in patients with chronic obstructive pulmonary disease (COPD), but the mechanisms and clinical consequences remain incompletely defined. To examine the effect of COPD on AM phagocytic responses and identify the mechanisms, clinical consequences, and potential for therapeutic manipulation of these defects. We isolated AMs and monocyte-derived macrophages (MDMs) from a cohort of patients with COPD and control subjects within the Medical Research Council COPDMAP consortium and measured phagocytosis of bacteria in relation to opsonic conditions and clinical features. COPD AMs and MDMs have impaired phagocytosis of Streptococcus pneumoniae. COPD AMs have a selective defect in uptake of opsonized bacteria, despite the presence of antipneumococcal antibodies in BAL, not observed in MDMs or healthy donor AMs. AM defects in phagocytosis in COPD are significantly associated with exacerbation frequency, isolation of pathogenic bacteria, and health-related quality-of-life scores. Bacterial binding and initial intracellular killing of opsonized bacteria in COPD AMs was not reduced. COPD AMs have reduced transcriptional responses to opsonized bacteria, such as cellular stress responses that include transcriptional modules involving antioxidant defenses and Nrf2 (nuclear factor erythroid 2-related factor 2)-regulated genes. Agonists of the cytoprotective transcription factor Nrf2 (sulforaphane and compound 7) reverse defects in phagocytosis of S. pneumoniae and nontypeable Haemophilus influenzae by COPD AMs. Patients with COPD have clinically relevant defects in opsonic phagocytosis by AMs, associated with impaired transcriptional responses to cellular stress, which are reversed by therapeutic targeting with Nrf2 agonists.

Strigolactone GR24 upregulates Nrf2 target genes and may protect against oxidative stress in skeletal muscle

GR24 is a synthetic strigolactone analog, demonstrated to regulate the development of plants and arbuscular mycorrhizal fungi. GR24 possesses anti-cancer and anti-apoptotic properties, enhances insulin sensitivity and mitochondrial biogenesis in skeletal myotubes, inhibits adipogenesis, decreases inflammation in adipocytes and macrophages and downregulates the expression of hepatic gluconeogenic enzymes. Transcription factor Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) is a master regulator of antioxidant response, regulating a multitude of genes involved in cellular stress responses and anti-inflammatory pathways, thus maintaining cellular redox homeostasis. Nrf2 activation reduces the deleterious effects of mitochondrial toxins and has multiple roles in promoting mitochondrial function and dynamics. We studied the role of GR24 on gene expression in rat L6 skeletal muscle cells which were differentiated into myotubes. The myotubes were treated with GR24 and analyzed by microarray gene expression profiling. GR24 upregulated the cytoprotective transcription factor Nrf2 and its target genes, activating antioxidant defences, suggesting that GR24 may protect skeletal muscle from the toxic effects of oxidative stress.

Crosstalk between NRF2 and HIPK2 shapes cytoprotective responses

Homeodomain interacting protein kinase-2 (HIPK2) is a member of the HIPK family of stress-responsive kinases that modulates cell growth, apoptosis, proliferation and development. HIPK2 has several well-characterised tumour suppressor roles, but recent studies suggest it can also contribute to tumour progression, although the underlying mechanisms are unknown. Herein, we have identified novel crosstalk between HIPK2 and the cytoprotective transcription factor NRF2. We show that HIPK2 is a direct transcriptional target of NRF2, identifying a functional NRF2 binding site in the HIPK2 gene locus and demonstrating for the first time a transcriptional mode of regulation for this kinase. In addition, HIPK2 is required for robust NRF2 responsiveness in cells and in vivo. By using both gain-of-function and loss-of-function approaches, we demonstrate that HIPK2 can elicit a cytoprotective response in cancer cells via NRF2. Our results have uncovered a new downstream effector of HIPK2, NRF2, which is frequently activated in human tumours correlating with chemoresistance and poor prognosis. Furthermore, our results suggest that modulation of either HIPK2 levels or activity could be exploited to impair NRF2-mediated signalling in cancer cells, and thus sensitise them to chemotherapeutic drugs.

A Glutathione-Nrf2-Thioredoxin Cross-Talk Ensures Keratinocyte Survival and Efficient Wound Repair.

The tripeptide glutathione is the most abundant cellular antioxidant with high medical relevance, and it is also required as a co-factor for various enzymes involved in the detoxification of reactive oxygen species and toxic compounds. However, its cell-type specific functions and its interaction with other cytoprotective molecules are largely unknown. Using a combination of mouse genetics, functional cell biology and pharmacology, we unraveled the function of glutathione in keratinocytes and its cross-talk with other antioxidant defense systems. Mice with keratinocyte-specific deficiency in glutamate cysteine ligase, which catalyzes the rate-limiting step in glutathione biosynthesis, showed a strong reduction in keratinocyte viability in vitro and in the skin in vivo. The cells died predominantly by apoptosis, but also showed features of ferroptosis and necroptosis. The increased cell death was associated with increased levels of reactive oxygen and nitrogen species, which caused DNA and mitochondrial damage. However, epidermal architecture, and even healing of excisional skin wounds were only mildly affected in the mutant mice. The cytoprotective transcription factor Nrf2 was strongly activated in glutathione-deficient keratinocytes, but additional loss of Nrf2 did not aggravate the phenotype, demonstrating that the cytoprotective effect of Nrf2 is glutathione dependent. However, we show that deficiency in glutathione biosynthesis is efficiently compensated in keratinocytes by the cysteine/cystine and thioredoxin systems. Therefore, our study highlights a remarkable antioxidant capacity of the epidermis that ensures skin integrity and efficient wound healing.

CD11d integrin blockade reduces the systemic inflammatory response syndrome after traumatic brain injury in rats

Traumatic CNS injury triggers a systemic inflammatory response syndrome (SIRS), in which circulating inflammatory cells invade body organs causing local inflammation and tissue damage. We have shown that the SIRS caused by spinal cord injury is greatly reduced by acute intravenous treatment with an antibody against the CD11d subunit of the CD11d/CD18 integrin expressed by neutrophils and monocyte/macrophages, a treatment that reduces their efflux from the circulation. Traumatic brain injury (TBI) is a frequently occurring injury after motor vehicle accidents, sporting and military injuries, and falls. Our studies have shown that the anti-CD11d treatment diminishes brain inflammation and oxidative injury after moderate or mild TBI, improving neurological outcomes. Accordingly, we examined the impact of this treatment on the SIRS triggered by TBI.The anti-CD11d treatment was given at 2h after a single moderate (2.5–3.0atm) or 2 and 24h after each of three consecutive mild (1.0–1.5atm) fluid percussion TBIs. Sham-injured, saline-treated rats served as controls. At 24h, 72h, and 4 or 8weeks after the single TBI and after the third of three TBIs, lungs of rats were examined histochemically, immunocytochemically and biochemically for downstream effects of SIRS including inflammation, tissue damage and expression of oxidative enzymes.Lung sections revealed that both the single moderate and repeated mild TBI caused alveolar disruption, thickening of inter-alveolar tissue, hemorrhage into the parenchyma and increased density of intra-and peri-alveolar macrophages. The anti-CD11d treatment decreased the intrapulmonary influx of neutrophils and the density of activated macrophages and the activity of myeloperoxidase after these TBIs. Moreover, Western blotting studies showed that the treatment decreased lung protein levels of oxidative enzymes gp91phox, inducible nitric oxide synthase and cyclooxygenase-2, as well as the apoptotic pathway enzyme caspase-3 and levels of 4-hydroxynonenal-bound proteins (an indicator of lipid peroxidation). Decreased expression of the cytoprotective transcription factor Nrf2 reflected decreased lung oxidative stress. Anti-CD11d treatment also diminished the lung concentration of free radicals and tissue aldehydes.In conclusion, the substantial lung component of the SIRS after single or repeated TBIs is significantly decreased by a simple, minimally invasive and short-lasting anti-inflammatory treatment.

Intracellular accumulation of subviral HBsAg particles and diminished Nrf2 activation in HBV genotype G expressing cells lead to an increased ROI level

Hepatitis B virus genotype G (HBV/G) is characterized by a lack of HBeAg secretion and very low HBsAg secretion. This study aimed at (1) comparing HBV genotype G and A2 with respect to morphogenesis and release of HBV-derived particles, (2) characterizing factors contributing to HBV/G-associated pathogenesis. HBV/G- and HBV/A-expressing hepatoma cells and infected HepaRG cells were analyzed by confocal laser scanning microscopy, Western blot, real-time PCR, density gradient centrifugation, and electron microscopy. Modulation of the transcription factors Nrf2 and AP-1 was analyzed. While the release of viral particles is not affected in HBV/G replicating cells, the secretion of subviral particles is impaired, although they are produced in high amounts. These subviral particles, which display an increased density and a predominantly filamentous morphology, accumulate at the endoplasmic reticulum. The PreS1PreS2 domain of genotype G, which forms aggregates, causes the block of HBsAg-secretion at the ER and leads to decreased transcriptional activator function of LHBs. Intracellular accumulation of HBsAg and impaired induction of the cytoprotective transcription factor Nrf2 lead to an elevated level of ROIs. This results in activation of JNK and as a consequence in Ser-phosphorylation of IRS-1, which is known to impair insulin signaling, a key factor for liver regeneration. Although competent for release of viral particles, secretion of subviral particles is impaired in HBV/G expressing cells leading to ER-stress. In parallel, HBV-induced Nrf2 activation diminishes, which causes a decrease of the capacity to inactivate ROIs. This might be related to genotype-specific pathogenesis.