Sekhar P. Reddy

Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis

Host genetic factors that regulate innate immunity determine susceptibility to sepsis. Disruption of nuclear factor-erythroid 2-related factor 2 (Nrf2), a basic leucine zipper transcription factor that regulates redox balance and stress response, dramatically increased the mortality of mice in response to endotoxin- and cecal ligation and puncture-induced septic shock. LPS as well as TNF-alpha stimulus resulted in greater lung inflammation in Nrf2-deficient mice. Temporal analysis of pulmonary global gene expression after LPS challenge revealed augmented expression of large numbers of proinflammatory genes associated with the innate immune response at as early as 30 minutes in lungs of Nrf2-deficient mice, indicating severe immune dysregulation. The expression profile indicated that Nrf2 has a global influence on both MyD88-dependent and -independent signaling. Nrf2-deficient mouse embryonic fibroblasts showed greater activation of NF-kappaB and interferon regulatory factor 3 in response to LPS and polyinosinic-polycytidylic acid [poly(I:C)] stimulus, corroborating the effect of Nrf2 on MyD88-dependent and -independent signaling. Nrf2s regulation of cellular glutathione and other antioxidants is critical for optimal NF-kappaB activation in response to LPS and TNF-alpha. Our study reveals Nrf2 as a novel modifier gene of sepsis that determines survival by mounting an appropriate innate immune response.

The transcription factor NRF2 protects against pulmonary fibrosis

The molecular mechanisms of pulmonary fibrosis are poorly understood, although reactive oxygen species are thought to have an important role. NRF2 is a transcription factor that protects cells and tissues from oxidative stress by activating protective antioxidant and detoxifying enzymes. We hypothesized that NRF2 protects lungs from injury and fibrosis induced by bleomycin, an anti‐neoplastic agent that causes pulmonary fibrosis in susceptible patients. To test this hypothesis, mice with targeted deletion of Nrf2 (Nrf2‒/‒) and wild‐type (Nrf2+/+) mice were treated with bleomycin or vehicle, and pulmonary injury and fibrotic responses were compared. Bleomycin‐induced increases in lung weight, epithelial cell death, and inflammation were significantly greater in Nrf2‒/‒ mice than in Nrf2+/+ mice. Indices of lung fibrosis (hydroxyproline content, collagen accumulation, fibrotic score, cell proliferation) were significantly greater in bleomycin‐treated Nrf2‒/‒ mice, compared with Nrf2+/+ mice. NRF2 expression and activity were elevated in Nrf2+/+ mice by bleomycin. Bleomycin caused greater up‐regulation of several NRF2‐inducible antioxidant enzyme genes and protein products in Nrf2+/+ mice compared with Nrf2‒/‒ mice. Further, bleomycin‐induced transcripts and protein levels of lung injury and fibrosis markers were significantly attenuated in Nrf2+/+ mice compared with Nrf2‒/‒ mice. Results demonstrated that NRF2 has a critical role in protection against pulmonary fibrosis, presumably through enhancement of cellular antioxidant capacity. This study has important implications for the development of intervention strategies against fibrosis.

Functional polymorphisms in the transcription factor NRF2 in humans increase the risk of acute lung injury

We recently used positional cloning to identify the transcription factor Nrf2 (NF‐E2 related factor 2) as a susceptibility gene in a murine model of oxidant‐induced acute lung injury (ALI). NRF2 binds to antioxidant response elements (ARE) and up‐regulates protective detoxifying enzymes in response to oxidative stress. This led us to investigate NRF2 as a candidate susceptibility gene for risk of development of ALI in humans. We identified multiple single nucleotide polymorphisms (SNPs) by resequencing NRF2 in ethnically diverse subjects, and one (—617 C/A) significantly (P< 0.001) diminished luciferase activity of promoter constructs containing the SNP and significantly decreased the binding affinity (P<0.001) relative to the wild type at this locus (—617 CC). In a nested case‐control study, patients with the —617 A SNP had a significantly higher risk for developing ALI after major trauma (OR 6.44; 95% CI 1.34, 30.8;P=0.021) relative to patients with the wild type (—617 CC). This translational investigation provides novel insight into the molecular mechanisms of susceptibility to ALI and may help to identify patients who are predisposed to develop ALI under at risk conditions, such as trauma and sepsis. Furthermore, these findings may have important implications in other oxidative stress related illnesses.–Marzec J. M., Christie, J. D., Reddy, S. P., Jedlicka, A. E., Vuong, H., Lanken, P. N., Aplenc, R., Yamamoto, T., Yamamoto, M., Cho, H.‐Y., Klee‐berger S. R. Functional polymorphisms in the transcription factor NRF2 in humans increase the risk of acute lung injury. FASEB J. 21, 2237–2246 (2007)

ERK1 and ERK2 Activate CCAAAT/Enhancer-binding Protein-β-dependent Gene Transcription in Response to Interferon-γ

Interferons (IFNs) regulate the expression of a number of cellular genes by activating the JAK-STAT pathway. We have recently discovered that CCAAAT/enhancer-binding protein-β (C/EBP-β) induces gene transcription through a novel IFN response element called the γ-IFN-activated transcriptional element (Roy, S. K., Wachira, S. J., Weihua, X., Hu, J., and Kalvakolanu, D. V. (2000) J. Biol. Chem. 275, 12626–12632. Here, we describe a new IFN-γ-stimulated pathway that operates C/EBP-β-regulated gene expression independent of JAK1. We show that ERKs are activated by IFN-γ to stimulate C/EBP-β-dependent expression. Sustained ERK activation directly correlated with C/EBP-βdependent gene expression in response to IFN-γ. Mutant MKK1, its inhibitors, and mutant ERK suppressed IFN-γ-stimulated gene induction through the γ-IFN-activated transcriptional element. Ras and Raf activation was not required for this process. Furthermore, Raf-1 phosphorylation negatively correlated with its activity. Interestingly, C/EBP-β-induced gene expression required STAT1, but not JAK1. A C/EBP-β mutant lacking the ERK phosphorylation site failed to promote IFN-stimulated gene expression. Thus, our data link C/EBP-β to IFN-γ signaling through ERKs.

Transcription factor Nrf2 is protective during ischemic and nephrotoxic acute kidney injury in mice

Oxidative stress is involved in acute kidney injury due to ischemia-reperfusion and chemotherapy-induced nephrotoxicity. To investigate their basic mechanisms we studied the role of nuclear factor-erythroid 2-p45-related factor 2 (Nrf2), a redox-sensitive transcription factor that regulates expression of several antioxidant and cytoprotective genes. We compared the responses of Nrf2-knockout mice and their wild-type littermates in established mouse models of ischemia-reperfusion injury and cisplatin-induced nephrotoxicity. Several Nrf2-regulated genes encoding antioxidant enzymes/proteins were significantly upregulated in the kidneys of wild type but not Nrf2-knockout mice following renal ischemia. Renal function, histology, vascular permeability, and survival were each significantly worse in the Nrf2 knockout mice. Further, proinflammatory cytokine and chemokine expression tended to increase after ischemia in the knockout compared to the wild-type mice. Treatment of the knockout mice with the antioxidants N-acetyl-cysteine or glutathione improved renal function. The knockout mice were more susceptible to cisplatin-induced nephrotoxicity, and this was blunted by N-acetyl-cysteine pretreatment. Our study demonstrates that Nrf2-deficiency enhances susceptibility to both ischemic and nephrotoxic acute kidney injury, and identifies this transcription factor as a potential therapeutic target in these injuries.

Disruption of Nrf2 impairs the resolution of hyperoxia-induced acute lung injury and inflammation in mice

Aberrant tissue repair and persistent inflammation following oxidant-mediated acute lung injury (ALI) can lead to the development and progression of various pulmonary diseases, but the mechanisms underlying these processes remain unclear. Hyperoxia is widely used in the treatment of pulmonary diseases, but the effects of this oxidant exposure in patients undergoing recovery from ALI are not clearly understood. Nrf2 has emerged as a crucial transcription factor that regulates oxidant stress through the induction of several detoxifying enzymes and other proteins. Using an experimental model of hyperoxia-induced ALI, we have examined the role of oxidant stress in resolving lung injury and inflammation. We found that when exposed to sublethal (72 h) hyperoxia, Nrf2-deficient, but not wild-type mice, succumbed to death during recovery. When both genotypes were exposed to a shorter period of hyperoxia-induced ALI (48 h), the lungs of Nrf2-deficient mice during recovery exhibited persistent cellular injury, impaired alveolar and endothelial cell regeneration, and persistent cellular infiltration by macrophages and lymphocytes. Glutathione (GSH) supplementation in Nrf2-deficient mice immediately after hyperoxia remarkably restored their ability to recover from hyperoxia-induced damage in a manner similar to that of wild-type mice. Thus, the results of the present study indicate that the Nrf2-regulated transcriptional response and, particularly GSH synthesis, is critical for lung tissue repair and the resolution of inflammation in vivo and suggests that a dysfunctional Nrf2-GSH pathway may compromise these processes in vivo.

Genetic disruption of the Nrf2 compromises cell-cycle progression by impairing GSH-induced redox signaling.

Genetic disruption of Nrf2 greatly enhances susceptibility to prooxidant- and carcinogen-induced experimental models of various human disorders; but the mechanisms by which this transcription factor confers protection are unclear. Using Nrf2-proficient (Nrf2+/+) and Nrf2-deficient (Nrf2−/−) primary epithelial cultures as a model, we now show that Nrf2 deficiency leads to oxidative stress and DNA lesions, accompanied by impairment of cell-cycle progression, mainly G2/M-phase arrest. Both N-acetylcysteine and glutathione (GSH) supplementation ablated the DNA lesions and DNA damage–response pathways in Nrf2−/− cells; however only GSH could rescue the impaired colocalization of mitosis-promoting factors and the growth arrest. Akt activation was deregulated in Nrf2−/− cells, but GSH supplementation restored it. Inhibition of Akt signaling greatly diminished the GSH-induced Nrf2−/− cell proliferation and wild-type cell proliferation. GSH depletion impaired Akt signaling and mitosis-promoting factor colocalization in Nrf2+/+ cells. Collectively, our findings uncover novel functions for Nrf2 in regulating oxidative stress-induced cell-cycle arrest, especially G2/M-checkpoint arrest, and proliferation, and GSH-regulated redox signaling and Akt are required for this process.

CD36 participates in a signaling pathway that regulates ROS formation in murine VSMCs

CD36 is a membrane glycoprotein expressed on platelets, monocytes, macrophages, and several other cell types that was recently demonstrated to be involved in platelet activation in response to oxidized phospholipids, including oxidized LDL. Although the role of CD36 in other vascular cells has not been well defined, previous studies have demonstrated that cd36-knockout (cd36-/-) mice have prolonged thrombosis times after vascular injury, which can be protective in the state of hyperlipidemia. Here, we found significantly less ROS in the vessel walls of cd36-/- mice compared with WT after chemically induced arterial injury, suggesting that CD36 may contribute to ROS generation in the VSMCs themselves. Gene expression analysis revealed that the antioxidant enzymes peroxiredoxin-2 (Prdx2) and heme oxygenase-1 were upregulated in cd36-/- VSMCs. Molecular dissection of the pathway in isolated mouse VSMCs revealed CD36 ligand-dependent induction of Fyn phosphorylation, with subsequent phosphorylation and degradation of the redox-sensitive transcription factor Nrf2. Chromatin immunoprecipitation experiments further showed that Nrf2 directly occupied the Prdx2 promoter. The importance of this pathway was evidenced by increased ROS generation in prdx2-/- mice and decreased thrombosis times in both prdx2-/- and nrf2-/- mice after vascular injury. These data suggest that CD36-mediated downregulation of antioxidant systems in VSMCs may contribute to its prothrombotic, proinflammatory, and atherogenic effects.

Enhancing Nrf2 Pathway by Disruption of Keap1 in Myeloid Leukocytes Protects against Sepsis

RATIONALE Sepsis syndrome is characterized by inappropriate amplified systemic inflammatory response and bacteremia that promote multiorgan failure and mortality. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulates a pleiotropic cytoprotective defense program including antioxidants and protects against several inflammatory disorders by inhibiting oxidative tissue injuries. However, the role of enhanced Nrf2 activity in modulating innate immune responses to microbial infection and pathogenesis of sepsis is unclear. OBJECTIVES To determine whether Nrf2 in myeloid leukocytes alters inflammatory response and protects against sepsis. METHODS Mice with deletion of Nrf2 or kelch-like ECH-associated protein (Keap1) in myeloid leukocyte cells and respective floxed controls were subjected to cecal ligation and puncture-induced sepsis and were assessed for survival, organ injury, systemic inflammation, and bacteremia. Using LPS-stimulated peritoneal macrophages, Toll-like receptor (TLR) 4 surface trafficking and downstream signaling events were analyzed. MEASUREMENTS AND MAIN RESULTS Mortality, organ injury, circulating levels of inflammatory mediators, and bacteremia were markedly reduced in LysM-Keap1(-/-) compared with respective floxed controls (Keap1(f/f) or Nrf2(f/f)) and significantly elevated in LysM-Nrf2(-/-) mice after cecal ligation and puncture. Peritoneal macrophages from septic LysM-Keap1(-/-) mice showed a greater bacterial phagocytic activity compared with LysM-Nrf2(-/-) and floxed controls. LPS stimulation resulted in greater reactive oxygen species-induced cell surface transport of TLR4 from trans-Golgi network and subsequent TLR4 downstream signaling (recruitment of MYD88 and TRIF, phosphorylation of IkB and IRF3, and cytokine expression) in macrophages of LysM-Nrf2(-/-) compared with LysM-Keap1(-/-) mice and floxed controls. CONCLUSIONS Our study shows that Nrf2 acts as a critical immunomodulator in leukocytes, controls host inflammatory response to bacterial infection, and protects against sepsis.

MEKK1 plays a critical role in activating the transcription factor C/EBP-β-dependent gene expression in response to IFN-γ

IFN-γ induces a number of genes to up-regulate cellular responses by using specific transcription factors and the cognate elements. We recently discovered that CCAAT/enhancer-binding protein-β (C/EBP-β) induces gene transcription through an IFN-response element called γ-IFN-activated transcriptional element (GATE). Using mutant cells, chemical inhibitors, and specific dominant negative inhibitors, we show that induction of GATE-driven gene expression depends on MEK1 (mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase kinase) and ERKs (extracellular signal-regulated protein kinases) but is independent of Raf-1. Interestingly in cells lacking the MEKK1 gene or expressing the dominant negative MEKK1, ERK activation, and GATE dependent gene expression is inhibited. A dominant negative MEKK1 blocks C/EBP-β-driven gene expression stimulated by IFN-γ. These studies describe an IFN-γ-stimulated pathway that involves MEKK1-MEK1-ERK1/2 kinases to regulate C/EBP-β-dependent gene expression.