Redox-regulated Transcription Factors Research Articles

Carrier-free delivery of nucleic acid and photosensitizer nanoparticles for enhanced photodynamic and gene antitumour therapy

Activated anti-oxidation reactions in cells partially diminish the anticancer effect of photodynamic therapy (PDT), significantly hindering efforts to increase the efficacy of PDT. The expression of transcription factor E2 related factor 2 (Nrf2), an important redox-regulated transcription factor, can be downregulated by Nrf2 siRNA, leading to greatly enhanced PDT effects. However, the efficient co-delivery of photosensitizers and siRNAs remains a key problem because these agents are complex to synthesize, exhibit poor biocompatibility and load drugs with a low efficiency. Herein, we designed a carrier–free and extremely simple strategy to co-deliver a photosensitizer and Nrf2 siRNA to cancer cells. In this nanoplatform, an indocyanine green photosensitizer, siRNA and FeⅡ were self-assembled to form a spherical hybrid structure with a uniform size, high loading ratio and adjustable component ratio. The platform can effectively transfer photosensitizers and siRNAs into cells and effectively inhibit tumour growth in vivo. Overall, the self-assembly approach shows great potential for clinical application and provides a simple method to achieve photodynamic therapy and enhanced photothermal therapy.

Nephroprotective potential of syringic acid in experimental diabetic nephropathy: Focus on oxidative stress and autophagy.

Diabetic nephropathy (DN) is a chronic hyperglycemic manifestation of microvascular damage in the kidneys. Widespread research in this area suggests the involvement of perturbed redox homeostasis and autophagy in renal cells phrase- promote the progression of DN. Reframed sentences-The present study investigates the pharmacological effect of Syringic acid (SYA), in streptozotocin (STZ, 55 mg/kg, i.p) induced diabetic nephropathy model and in high glucose (30 mM) challenged rat renal epithelial cells (NRK 52E) cells with a focus on oxidative stress and autophagy mechanisms. Both in vivo and in vitro experimental data revealed elevated oxidative stress markers along with compromised levels of nuclear factor erythroid 2-related factor 2 (Nrf2), a pivotal cellular redox-regulated transcription factor in renal cells upon glycemic stress. Elevated blood glucose also reduced the autophagy process as indicated by low expression of light chain (LC) 3-IIB in diabetic kidney and in NRK 52E cells subjected to excess glucose. SYA (25 and 50 mg/kg, p.o.) administration for 4 weeks to diabetic rats, Reframed sentence-preserved the renal function as evidenced by reduced serum creatinine levels as well as improved urine creatinine and urea levles as compared to non treated diabetic animals. At the molecular level, SYA improved renal expression of Nrf2 and autophagy-related proteins (Atg5, Atg3, and Atg7) in diabetic rats. Similarly, SYA (10 and 20 μM) co-treatment in high glucose-treated NRK 52E cells displayed increased levels of Nrf2 and autophagy induction. Results from this study signify the renoprotective effect of SYA and highlight the modulation of oxidative stress and autophagy mechanisms to mitigate diabetic kidney disease.

Drug Inhibition of Redox Factor-1 Restores Hypoxia-Driven Changes in Tuberous Sclerosis Complex 2 Deficient Cells.

Therapies with the mechanistic target of rapamycin complex 1 (mTORC1) inhibitors are not fully curative for tuberous sclerosis complex (TSC) patients. Here, we propose that some mTORC1-independent disease facets of TSC involve signaling through redox factor-1 (Ref-1). Ref-1 possesses a redox signaling activity that stimulates the transcriptional activity of STAT3, NF-kB, and HIF-1α, which are involved in inflammation, proliferation, angiogenesis, and hypoxia, respectively. Here, we demonstrate that redox signaling through Ref-1 contributes to metabolic transformation and tumor growth in TSC cell model systems. In TSC2-deficient cells, the clinically viable Ref-1 inhibitor APX3330 was effective at blocking the hyperactivity of STAT3, NF-kB, and HIF-1α. While Ref-1 inhibitors do not inhibit mTORC1, they potently block cell invasion and vasculature mimicry. Of interest, we show that cell invasion and vasculature mimicry linked to Ref-1 redox signaling are not blocked by mTORC1 inhibitors. Metabolic profiling revealed that Ref-1 inhibitors alter metabolites associated with the glutathione antioxidant pathway as well as metabolites that are heavily dysregulated in TSC2-deficient cells involved in redox homeostasis. Therefore, this work presents Ref-1 and associated redox-regulated transcription factors such as STAT3, NF-kB, and HIF-1α as potential therapeutic targets to treat TSC, where targeting these components would likely have additional benefits compared to using mTORC1 inhibitors alone.

Antioxidant Micronutrients and Oxidative Stress Biomarkers.

Chronic inflammatory diseases are the major causes of mortality in humans and recent research has improved our understanding of the major impact of lifestyle factors upon inflammatory diseases and conditions. One of the most influential of these is nutrition, which may drive both pro-inflammatory as well asanti-inflammatory cascades at molecular and cellular levels. There are a variety of model systems that may be employed to investigate the impact of micronutrients and macronutrients upon inflammatory pathways, many of which operate through oxidative stress, either at the level of controlling the redox state of the cell and downstream redox-regulated gene transcription factors, and other acting as free radical generating or scavenging agents. This chapter focuses upon biological sample preparation prior to assay and details methods for analyzing certain antioxidant micronutrients and biomarkers of oxidative stress.

ClNOX1/ClNOXR-mediated MAPK and cAMP-PKA signalling pathways and ROS metabolism are involved in Curvularia lunata sexual reproduction and host infection.

NADPH oxidases (NOXs) and hydrogen peroxide (H2 O2 ) are involved in physiological and pathological processes, and cell fate decisions in organisms. However, regulatory mechanism of NOXs and the role of H2 O2 on fungal sexual reproduction and host infection remain largely unexplored. Here, we identified ROS metabolic genes and key signalling genes of MAPK and cAMP-PKA pathways in Curvularia lunata, which were NOX ClNOX1 and ClNOXR, superoxide dismutase ClSOD1 and catalase ClCAT4, redox-regulated transcription factor ClAP1, Ras small GTPases Clg2P, pheromone-response MAPK ClK1 and cAMP-PKA ClSCHA, and characterized the functions of these genes. The results showed that ClNOX1 localized to the plasma membrane. ClNOX1 and ClNOXR were involved in sexual reproduction and host infection via ClNOX1/ClNOXR-derived H2 O2 as well as MAPK and cAMP-PKA signalling pathways. H2 O2 acted as a signalling molecule to regulate sexual reproduction and host infection in C. lunata.

Flavones 7,8-DHF, Quercetin, and Apigenin Against Tau Toxicity via Activation of TRKB Signaling in ΔK280 TauRD-DsRed SH-SY5Y Cells

Alzheimer’s disease (AD) is a progressive neurodegenerative disease with memory loss and cognitive decline. Neurofibrillary tangles (NFTs) formed by hyperphosphorylated Tau protein are one of the pathological hallmarks of several neurodegenerative diseases including AD. Heat shock protein family B (small) member 1 (HSPB1) is a molecular chaperone that promotes the correct folding of other proteins in response to environmental stress. Nuclear factor erythroid 2-like 2 (NRF2), a redox-regulated transcription factor, is the master regulator of the cellular response to excess reactive oxygen species. Tropomyosin-related kinase B (TRKB) is a membrane-bound receptor that, upon binding brain-derived neurotrophic factor (BDNF), phosphorylates itself to initiate downstream signaling for neuronal survival and axonal growth. In this study, four natural flavones such as 7,8-dihydroxyflavone (7,8-DHF), wogonin, quercetin, and apigenin were evaluated for Tau aggregation inhibitory activity and neuroprotection in SH-SY5Y neuroblastoma. Among the tested flavones, 7,8-DHF, quercetin, and apigenin reduced Tau aggregation, oxidative stress, and caspase-1 activity as well as improved neurite outgrowth in SH-SY5Y cells expressing ΔK280 TauRD-DsRed folding reporter. Treatments with 7,8-DHF, quercetin, and apigenin rescued the reduced HSPB1 and NRF2 and activated TRKB-mediated extracellular signal-regulated kinase (ERK) signaling to upregulate cAMP-response element binding protein (CREB) and its downstream antiapoptotic BCL2 apoptosis regulator (BCL2). Knockdown of TRKB attenuated the neuroprotective effects of these three flavones. Our results suggest 7,8-DHF, quercetin, and apigenin targeting HSPB1, NRF2, and TRKB to reduce Tau aggregation and protect cells against Tau neurotoxicity and may provide new treatment strategies for AD.

Role of NRF2 in immune modulator expression in developing lung.

After birth, the alveolar epithelium is exposed to environmental pathogens and high O2 tensions. The alveolar type II cells may protect this epithelium through surfactant production. Surfactant protein, SP-A, an immune modulator, is developmentally upregulated in fetal lung with surfactant phospholipid synthesis. Herein, we observed that the redox-regulated transcription factor, NRF2, and co-regulated C/EBPβ and PPARγ, were markedly induced during cAMP-mediated differentiation of cultured human fetal lung (HFL) epithelial cells. This occurred with enhanced expression of immune modulators, SP-A, TDO2, AhR, and NQO1. Like SP-A, cAMP induction of NRF2 was prevented when cells were exposed to hypoxia. NRF2 knockdown inhibited induction of C/EBPβ, PPARγ, and immune modulators. Binding of endogenous NRF2 to promoters of SP-A and other immune modulator genes increased during HFL cell differentiation. In mouse fetal lung (MFL), a developmental increase in Nrf2, SP-A, Tdo2, Ahr, and Nqo1 and decrease in Keap1 occurred from 14.5 to 18.5dpc. Developmental induction of Nrf2 in MFL was associated with increased nuclear localization of NF-κB p65, a decline in p38 MAPK phosphorylation, increase in the MAPK phosphatase, DUSP1, induction of the histone acetylase, CBP, and decline in the histone deacetylase, HDAC4. Thus, together with surfactant production, type II cells protect the alveolar epithelium through increased expression of NRF2 and immune modulators to prevent inflammation and oxidative stress. Our findings further suggest that lung cancer cells have usurped this developmental pathway to promote immune tolerance and enhance survival.

Redox Homeostasis in Pancreatic β-Cells: From Development to Failure.

Redox status is a key determinant in the fate of β-cell. These cells are not primarily detoxifying and thus do not possess extensive antioxidant defense machinery. However, they show a wide range of redox regulating proteins, such as peroxiredoxins, thioredoxins or thioredoxin reductases, etc., being functionally compartmentalized within the cells. They keep fragile redox homeostasis and serve as messengers and amplifiers of redox signaling. β-cells require proper redox signaling already in cell ontogenesis during the development of mature β-cells from their progenitors. We bring details about redox-regulated signaling pathways and transcription factors being essential for proper differentiation and maturation of functional β-cells and their proliferation and insulin expression/maturation. We briefly highlight the targets of redox signaling in the insulin secretory pathway and focus more on possible targets of extracellular redox signaling through secreted thioredoxin1 and thioredoxin reductase1. Tuned redox homeostasis can switch upon chronic pathological insults towards the dysfunction of β-cells and to glucose intolerance. These are characteristics of type 2 diabetes, which is often linked to chronic nutritional overload being nowadays a pandemic feature of lifestyle. Overcharged β-cell metabolism causes pressure on proteostasis in the endoplasmic reticulum, mainly due to increased demand on insulin synthesis, which establishes unfolded protein response and insulin misfolding along with excessive hydrogen peroxide production. This together with redox dysbalance in cytoplasm and mitochondria due to enhanced nutritional pressure impact β-cell redox homeostasis and establish prooxidative metabolism. This can further affect β-cell communication in pancreatic islets through gap junctions. In parallel, peripheral tissues losing insulin sensitivity and overall impairment of glucose tolerance and gut microbiota establish local proinflammatory signaling and later systemic metainflammation, i.e., low chronic inflammation prooxidative properties, which target β-cells leading to their dedifferentiation, dysfunction and eventually cell death.

Context-Dependent Regulation of Nrf2/ARE Axis on Vascular Cell Function during Hyperglycemic Condition.

Diabetes mellitus is associated with an increased risk of micro and macrovascular complications. During hyperglycemic conditions, endothelial cells and vascular smooth muscle cells are exquisitely sensitive to high glucose. This high glucose-induced sustained reactive oxygen species production leads to redox imbalance, which is associated with endothelial dysfunction and vascular wall remodeling. Nrf2, a redox-regulated transcription factor plays a key role in the antioxidant response element (ARE)-mediated expression of antioxidant genes. Although accumulating data indicate the molecular mechanisms underpinning the Nrf2 regulated redox balance, understanding the influence of the Nrf2/ARE axis during hyperglycemic condition on vascular cells is paramount. This review focuses on the context-dependent role of Nrf2/ARE signaling on vascular endothelial and smooth muscle cell function during hyperglycemic conditions. This review also highlights improving the Nrf2 system in vascular tissues, which could be a potential therapeutic strategy for vascular dysfunction.

Oxidative Stress and New Pathogenetic Mechanisms in Endothelial Dysfunction: Potential Diagnostic Biomarkers and Therapeutic Targets.

Cardiovascular diseases (CVD), including heart and pathological circulatory conditions, are the world’s leading cause of mortality and morbidity. Endothelial dysfunction involved in CVD pathogenesis is a trigger, or consequence, of oxidative stress and inflammation. Endothelial dysfunction is defined as a diminished production/availability of nitric oxide, with or without an imbalance between endothelium-derived contracting, and relaxing factors associated with a pro-inflammatory and prothrombotic status. Endothelial dysfunction-induced phenotypic changes include up-regulated expression of adhesion molecules and increased chemokine secretion, leukocyte adherence, cell permeability, low-density lipoprotein oxidation, platelet activation, and vascular smooth muscle cell proliferation and migration. Inflammation-induced oxidative stress results in an increased accumulation of reactive oxygen species (ROS), mainly derived from mitochondria. Excessive ROS production causes oxidation of macromolecules inducing cell apoptosis mediated by cytochrome-c release. Oxidation of mitochondrial cardiolipin loosens cytochrome-c binding, thus, favoring its cytosolic release and activation of the apoptotic cascade. Oxidative stress increases vascular permeability, promotes leukocyte adhesion, and induces alterations in endothelial signal transduction and redox-regulated transcription factors. Identification of new endothelial dysfunction-related oxidative stress markers represents a research goal for better prevention and therapy of CVD. New-generation therapeutic approaches based on carriers, gene therapy, cardiolipin stabilizer, and enzyme inhibitors have proved useful in clinical practice to counteract endothelial dysfunction. Experimental studies are in continuous development to discover new personalized treatments. Gene regulatory mechanisms, implicated in endothelial dysfunction, represent potential new targets for developing drugs able to prevent and counteract CVD-related endothelial dysfunction. Nevertheless, many challenges remain to overcome before these technologies and personalized therapeutic strategies can be used in CVD management.

Homologous-targeting biomimetic nanoparticles for photothermal therapy and Nrf2-siRNA amplified photodynamic therapy against oral tongue squamous cell carcinoma

Photothermal therapy (PTT) and photodynamic therapy (PDT) have some advantages such as site selectivity, non- or little-invasive property and negligible drug resistance for treatment of oral tongue squamous cell carcinoma (OTSCC). However, the activation of intracellular antioxidant responses will partly counteract anticancer effects of PDT. As a well-known redox-regulated transcription factor, Nrf2 has been found to participate in the resistance of tumor cells against PDT. Hereon, a biomimetic nanoparticle system was designed for co-loading and targeted delivery of photosensitizer indocyanine green (ICG) and Nrf2-siRNA, thus hope to achieve synergistic effects of PTT and amplified PDT in OTSCC. Poly(β-amino ester) (PBAE)/poly lactic-co-glycolic acid (PLGA) blended nanoparticles co-loaded with ICG and Nrf2-siRNA were prepared and then encapsulated within the vesicles of cancer cell membrane (CCM) specifically derived from homologous OTSCC, thus obtained biomimetic nanoparticles named as M@PPI-siRNA. Both in vitro and in vivo, M@PPI-siRNA nanoparticles exhibited strong OTSCC-targeting ability due to tumor-homing effect of homologous CCM. Upon laser irradiation at 808 nm, M@PPI-siRNA nanoparticles significantly down-regulated the expressions of Nrf2 and its regulated genes, glutamate-cysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM) that play important roles in ROS detoxification, and subsequently indirectly enhanced PDT anticancer effects by maintaining the accumulation of intracellular ROS. Through cooperation of PTT and Nrf2-siRNA amplified PDT, M@PPI-siRNA nanoparticles significantly inhibited the proliferation and induced the apoptosis of OTSCC cells, and also notably suppressed tumor growth and angiogenesis in OTSCC tumor-bearing mice. In summary, this study provides a promising OTSCC-targeted delivery system for both photosensitizer and gene, and also confirms that Nrf2-siRNA is an efficient synergist for amplifying PDT.

Redox Balance-DDR-miRNA Triangle: Relevance in Genome Stability and Stress Responses in Plants.

Plants are continuously faced with complex environmental conditions which can affect the oxidative metabolism and photosynthetic efficiency, thus leading to the over-production of reactive oxygen species (ROS). Over a certain threshold, ROS can damage DNA. DNA damage, unless repaired, can affect genome stability, thus interfering with cell survival and severely reducing crop productivity. A complex network of pathways involved in DNA damage response (DDR) needs to be activated in order to maintain genome integrity. The expression of specific genes belonging to these pathways can be used as indicators of oxidative DNA damage and effective DNA repair in plants subjected to stress conditions. Managing ROS levels by modulating their production and scavenging systems shifts the role of these compounds from toxic molecules to key messengers involved in plant tolerance acquisition. Oxidative and anti-oxidative signals normally move among the different cell compartments, including the nucleus, cytosol, and organelles. Nuclei are dynamically equipped with different redox systems, such as glutathione (GSH), thiol reductases, and redox regulated transcription factors (TFs). The nuclear redox network participates in the regulation of the DNA metabolism, in terms of transcriptional events, replication, and repair mechanisms. This mainly occurs through redox-dependent regulatory mechanisms comprising redox buffering and post-translational modifications, such as the thiol-disulphide switch, glutathionylation, and S-nitrosylation. The regulatory role of microRNAs (miRNAs) is also emerging for the maintenance of genome stability and the modulation of antioxidative machinery under adverse environmental conditions. In fact, redox systems and DDR pathways can be controlled at a post-transcriptional level by miRNAs. This review reports on the interconnections between the DDR pathways and redox balancing systems. It presents a new dynamic picture by taking into account the shared regulatory mechanism mediated by miRNAs in plant defense responses to stress.

Costunolide-A Bioactive Sesquiterpene Lactone with Diverse Therapeutic Potential.

Sesquiterpene lactones constitute a major class of bioactive natural products. One of the naturally occurring sesquiterpene lactones is costunolide, which has been extensively investigated for a wide range of biological activities. Multiple lines of preclinical studies have reported that the compound possesses antioxidative, anti-inflammatory, antiallergic, bone remodeling, neuroprotective, hair growth promoting, anticancer, and antidiabetic properties. Many of these bioactivities are supported by mechanistic details, such as the modulation of various intracellular signaling pathways involved in precipitating tissue inflammation, tumor growth and progression, bone loss, and neurodegeneration. The key molecular targets of costunolide include, but are not limited to, intracellular kinases, such as mitogen-activated protein kinases, Akt kinase, telomerase, cyclins and cyclin-dependent kinases, and redox-regulated transcription factors, such as nuclear factor-kappaB, signal transducer and activator of transcription, activator protein-1. The compound also diminished the production and/expression of proinflammatory mediators, such as cyclooxygenase-2, inducible nitric oxide synthase, nitric oxide, prostaglandins, and cytokines. This review provides an overview of the therapeutic potential of costunolide in the management of various diseases and their underlying mechanisms.

Oxidative Stress Induced by the Deubiquitinase Inhibitor b-AP15 Is Associated with Mitochondrial Impairment.

Inhibitors of the 20S proteasome such as bortezomib are cytotoxic to tumor cells and have been proven to be valuable for the clinical management of multiple myeloma. The therapeutic efficacy of bortezomib is, however, hampered by the emergence of acquired resistance. Available data suggest that blocking proteasome activity at the level of proteasome-associated deubiquitinases (DUBs) provides a mechanism to overcome resistance to bortezomib and also to other cancer therapies. The small molecule b-AP15 is an inhibitor of proteasome-associated DUB activity that induces both proteotoxic stress and increases in the levels of reactive oxygen species (ROS) in tumor cells. Antioxidants have been shown to decrease apoptosis induction by b-AP15 and we here addressed the question of the mechanism of redox perturbation by this compound. We show that oxidative stress induction by b-AP15 is abrogated in cells deprived of mitochondrial DNA (ρ 0 cells). We also show associations between the level of proteotoxic stress, the degree of mitochondrial dysfunction, and the extent of induction of hemeoxygenase-1 (HO-1), a target of the redox-regulated Nrf-2 transcription factor. Decreased expression of COX5b (cytochrome c oxidase subunit 5b) and TOMM34 (translocase of outer mitochondrial membrane 34) was observed in b-AP15-treated cells. These findings suggest a mitochondrial origin of the increased levels of ROS observed in cells exposed to the DUB inhibitor b-AP15.

249 - Roles of Ohr (a Cys based peroxidase) and OhrR (a redox regulated transcription factor) from Pseudomonas aeruginosa in a mouse infection model

Pseudomonas aeruginosa is a human pathogen that represents a serious threat worldwide because they present resistance to several clinical relevant antibiotics. Pathogenic bacteria have evolved several defense strategies, including antioxidant mechanisms to overcome host responses. Organic hydroperoxide resistance protein (Ohr, a Cys-based peroxidase) and its transcriptional repressor (OhrR, a redox regulated transcription factor) comprise a system that is central in the defense of bacteria to organic hydroperoxides. To understand the role of Ohr/OhrR system in the virulence of P. aeruginosa, an acute pneumonia model in mice was used. Initially, the ∆ohr, ∆ohrR and ∆ohr/∆ohrR mutants of P. aeruginosa PA14 (WT) were constructed by homologous recombination. Female BALB/c mice (8–12 weeks old) were intratracheally inoculated with 60 µL of bacterial suspension (2×106 cells/ml) or PBS in the control group. After a 24 hour-infection, the lungs, spleen, liver and blood were harvested, and bacterial burden, pro-inflammatory cytokines and neutrophil recruitment were analyzed. No CFU difference was observed between the mice infected with the WT strain and mutants strains in the organs. Although, higher levels of TNF-α and IL-1β was observed in the lungs of mice infected with the ∆ohr, ∆ohrR and ∆ohr/∆ohrR mutants as compared to the wild-type infected ones, suggesting a pro-inflammatory processes. Notably, less neutrophil recruitment was observed in the lungs of animals infected with ∆ohrR, suggesting that overexpression of Ohr might promote anti-inflammatory responses in this area. Corroborating this data, an increased survival trend was observed in the ∆ohrR infected mice, suggesting a less virulent phenotype. Since hydroperoxides of arachidonic acid are modulators of inflammatory processes and are reduced by Ohr to alcohols at extremely high rates, it is tempting to speculate that Ohr/OhrR system may subvert host immune responses.

NF-\u03baB-related decrease of glioma angiogenic potential by graphite nanoparticles and graphene oxide nanoplatelets

Gliomas develop an expanded vessel network and a microenvironment characterized by an altered redox environment, which produces high levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that fuel its growth and malignancy. ROS and RNS can influence tumor cell malignancy via the redox-regulated transcription factor NF-κB, whose activation is further regulated by the mutation status of p53. The objective of this study was to assess the influence of graphite nanoparticles (NG) and graphene oxide nanoplatelets (nGO) on the angiogenic potential of glioma cell lines with different p53 statuses. Nanoparticle treatment of glioma cells decreased the angiogenesis of human umbilical vein endothelial cells (HUVEC) cocultured with U87 (p53 wild type) and was not effective for U118 (p53 mutant) cells. Nanoparticle activity was related to the decreased level of intracellular ROS and RNS, which downregulated NF-κB signaling depending on the p53 status of the cell line. Activation of NF-κB signaling affected downstream protein levels of interleukin 6, interleukin 8, growth-regulated oncogene α, and monocyte chemotactic protein 1. These results indicate that the activity of NG and nGO can be regulated by the mutation status of glioma cells and therefore give new insights into the use of nanoparticles in personalized biomedical applications regarding glioma angiogenesis and its microenvironment.

A mathematical analysis of Prx2-STAT3 disulfide exchange rate constants for a bimolecular reaction mechanism.

Appreciation of peroxiredoxins as the major regulators of H2O2 concentrations in human cells has led to a new understanding of redox signaling. In addition to their status as the primary reducers of H2O2 to water, the oxidized peroxiredoxin byproduct of this reaction has recently been shown capable of participation in H2O2-mediated signaling pathways through disulfide exchange reactions with the transcription factor STAT3. The dynamics of peroxidase-transcription factor disulfide exchange reactions have not yet been considered in detail with respect to how these reactions fit into the larger network of competing reactions in human cells. In this study, we used a kinetic model of oxidation and reduction reactions related to H2O2 metabolism in the cytosol of human cells to study the dynamics of peroxiredoxin-2 mediated oxidation of the redox-regulated transcription factor STAT3. In combination with previously reported experimental data, the model was used to estimate the rate coefficient of a biomolecular reaction between Prx2 and STAT3 for two sets of assumptions that constitute lower and upper bound cases. Using these estimates, we calculated the relative rates of the reaction of oxidized peroxiredoxin-2 and STAT3 and other competing reactions in the cytosol. These calculations revealed that peroxiredoxin-2-mediated oxidation of STAT3 likely occurs at a much slower rate than competing reactions in the cytosol. This analysis suggests the existence of more complex mechanisms, potentially involving currently unknown protein-protein recognition partners, which facilitate disulfide exchange reactions between peroxiredoxin-2 and STAT3.

Redox-Sensitive Transcription Factor NRF2 Enhances Trophoblast Differentiation via Induction of miR-1246 and Aromatase.

Dysregulation of human trophoblast invasion and differentiation with placental hypoxia can result in preeclampsia, a hypertensive disorder of pregnancy. Herein, we characterized the role and regulation of miR-1246, which is markedly induced during human syncytiotrophoblast differentiation. miR-1246 targets GSK3β and AXIN2, inhibitors of WNT/β-catenin signaling, which is crucial for placental development, and is predicted to target JARID2, which promotes silencing of developmentally regulated genes. Human cytotrophoblasts cultured in 20% O2 spontaneously differentiate to syncytiotrophoblast with induction of hCYP191A/aromatase, a marker of differentiation. miR-1246 was induced >150-fold during syncytiotrophoblast differentiation in 20% O2, whereas targets-GSK3β, AXIN2, and JARID2-were significantly decreased. However, when cytotrophoblasts were cultured in 2% O2, miR-1246 and aromatase induction were prevented. miR-1246 was significantly decreased in placentas of women with severe preeclampsia, whereas AXIN2, GSK3β, and JARID2 were increased, compared with normotensive subjects. To identify factors that regulate miR-1246, we investigated the redox-regulated transcription factor NRF2, which has predicted binding sites in the miR-1246 promoter. Intriguingly, NRF2 messenger RNA was upregulated during syncytiotrophoblast differentiation and significantly reduced by hypoxia and in preeclamptic placentas. Moreover, NRF2 knockdown in cytotrophoblasts inhibited induction of miR-1246 and hCYP19A1, as well as transcription factors C/EBPβ and PPARγ, which are implicated in placental differentiation. Using chromatin immunoprecipitation-quantitative polymerase chain reaction, we found that binding of endogenous NRF2 to the miR-1246 and hCYP191A promoters increased during syncytiotrophoblast differentiation. Thus, NRF2 promotes syncytiotrophoblast differentiation by inducing C/EBPβ, PPARγ, hCYP19A1, and miR-1246, which targets WNT inhibitors and JARID2 and is dysregulated in preeclampsia.

Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis.

Nrf2, a redox regulated transcription factor, has recently been shown to play a role in cartilage integrity but the mechanism remains largely unknown. Osteoarthritis (OA) is a multifactorial disease in which focal degradation of cartilage occurs. Here, we studied whether Nrf2 exerts chondroprotective effects by suppressing the oxidative stress and apoptosis in IL-1β stimulated human OA chondrocytes. Expression of Nrf2 and its target genes HO-1, NQO1 and SOD2 was significantly high in OA cartilage compared to normal cartilage and was also higher in damaged area compared to smooth area of OA cartilage of the same patient. Human chondrocytes treated with IL-1β resulted in robust Nrf2/ARE reporter activity, which was inhibited by pretreatment with antioxidants indicating that Nrf2 activity was due to IL-1β-induced ROS generation. Ectopic expression of Nrf2 significantly suppressed the IL-1β-induced generation of ROS while Nrf2 knockdown significantly increased the basal as well as IL-1β-induced ROS levels in OA chondrocytes. Further, Nrf2 activation significantly inhibited the IL-1β-induced activation of extrinsic and intrinsic apoptotic pathways as determined by inhibition of DNA fragmentation, activation of Caspase-3,-8,-9, cleavage of PARP, release of cytochrome-c, suppression of mitochondrial dysfunction and mitochondrial ROS production in OA chondrocytes. Nrf2 over-expression in OA chondrocytes increased the expression of anti-apoptotic proteins while pro-apoptotic proteins were suppressed. Importantly, Nrf2 over-expression activated ERK1/2 and its downstream targets-ELK1, P70S6K and P90RSK and suppressed the IL-1β-induced apoptosis whereas inhibition of ERK1/2 activation abrogated the protective effects of Nrf2 in OA chondrocytes. Taken together, our data demonstrate that Nrf2 is a stress response protein in OA chondrocytes with anti-oxidative and anti-apoptotic function and acts via activation of ERK1/2/ELK1-P70S6K-P90RSK signaling axis. These activities of Nrf2 make it a promising candidate for the development of novel therapies for the management of OA.

Pleiotropic Roles of Nrf2 as Regulators of Chondrocyte Apoptosis, Oxidative Stress, Inflammatory Response and Catabolic and Anabolic Pathways in Osteoarthritis

Nrf2, a redox regulated transcription factor, has recently been shown to play a role in cartilage integrity but the mechanism remains largely unknown. Osteoarthritis (OA) is a multifactorial disease in which focal degradation of cartilage occurs. Here, we investigated the regulation of oxidative stress, inflammatory response, apoptosis, catabolic and anabolic factors by Nrf2 in IL-1β-stimulated human OA chondrocytes. Ectopic expression of Nrf2 in OA chondrocytes suppressed the generation of ROS while Nrf2 knockdown significantly increased the ROS levels in IL-1β-stimulated OA chondrocytes. Further, Nrf2 activation significantly inhibited the IL-1β-induced activation of extrinsic and intrinsic apoptotic pathways as determined by inhibition of DNA fragmentation, activation of Caspase-3,-8,-9, cleavage of PARP, release of cytochrome-c, suppression of mitochondrial dysfunction, suppression of pro-apoptotic proteins Bax, Bad and Bid expression and enhanced expression of anti-apoptotic proteins Bcl2, Bcl-xl and Mcl-1 in OA chondrocytes. To examine the role of Nrf2 in IL-1β-induced inflammatory response, we determined the mRNA expression and protein levels of several cytokines and chemokines using PCR array and multiplex immunoassay. Our data demonstrate that Nrf2 activation inhibited the IL-1β-mediated expression of several inflammatory mediators and Gene Ontology analysis revealed significant enrichment of MAPK signaling, TNF signaling, NF-κβ signaling and cytokine receptor signaling pathways. Additionally, Nrf2 activation inhibited the expression matrix degrading proteases-MMP-13, 3, 9, and ADAMTS-4 and enhanced the expression of OA related anabolic factors-COL2A1, ACAN, FRZB and GDF5 and SOX-5, 8 and 9 in IL-1β-stimulated OA chondrocytes. Importantly, we also discovered that Nrf2 mediated suppression of catabolic factors was dependent on ERK1/2 activation by Nrf2. Our data demonstrate that Nrf2 regulates multiple pathways involved in chondrocyte apoptosis, inflammation and matrix degradation via activation of ERK1/2/ELK1-P70S6K-P90RSK axis. Our findings collectively support the development of therapies targeting Nrf2 for the management of OA.