Tetsuro Ishii

Transcription Factor Nrf2 Coordinately Regulates a Group of Oxidative Stress-inducible Genes in Macrophages

Electrophiles and reactive oxygen species have been implicated in the pathogenesis of many diseases. Transcription factor Nrf2 was recently identified as a general regulator of one defense mechanism against such havoc. Nrf2 regulates the inducible expression of a group of detoxication enzymes, such as glutathione S-transferase and NAD(P)H:quinone oxidoreductase, via antioxidant response elements. Using peritoneal macrophages from Nrf2-deficient mice, we show here that Nrf2 also controls the expression of a group of electrophile- and oxidative stress-inducible proteins and activities, which includes heme oxygenase-1, A170, peroxiredoxin MSP23, and cystine membrane transport (system xc −) activity. The response to electrophilic and reactive oxygen species-producing agents was profoundly impaired in Nrf2-deficient cells. The lack of induction of system xc − activity resulted in the minimum level of intracellular glutathione, and Nrf2-deficient cells were more sensitive to toxic electrophiles. Several stress agents induced the DNA binding activity of Nrf2 in the nucleus without increasing its mRNA level. Thus Nrf2 regulates a wide-ranging metabolic response to oxidative stress.

A Role for NBR1 in Autophagosomal Degradation of Ubiquitinated Substrates

Autophagy is a catabolic process where cytosolic cellular components are delivered to the lysosome for degradation. Recent studies have indicated the existence of specific receptors, such as p62, which link ubiquitinated targets to autophagosomal degradation pathways. Here we show that NBR1 (neighbor of BRCA1 gene 1) is an autophagy receptor containing LC3- and ubiquitin (Ub)-binding domains. NBR1 is recruited to Ub-positive protein aggregates and degraded by autophagy depending on an LC3-interacting region (LIR) and LC3 family modifiers. Although NBR1 and p62 interact and form oligomers, they can function independently, as shown by autophagosomal clearance of NBR1 in p62-deficient cells. NBR1 was localized to Ub-positive inclusions in patients with liver dysfunction, and depletion of NBR1 abolished the formation of Ub-positive p62 bodies upon puromycin treatment of cells. We propose that NBR1 and p62 act as receptors for selective autophagosomal degradation of ubiquitinated targets.

Cloning and Expression of a Plasma Membrane Cystine/Glutamate Exchange Transporter Composed of Two Distinct Proteins

Transport system xc − found in plasma membrane of cultured mammalian cells is an exchange agency for anionic amino acids with high specificity for anionic form of cystine and glutamate. We have isolated cDNA encoding the transporter for system xc − from mouse activated macrophages by expression in Xenopus oocytes. The expression of system xc − activity in oocytes required two cDNA transcripts, and the sequence analysis revealed that one is identical with the heavy chain of 4F2 cell surface antigen (4F2hc) and the other is a novel protein of 502 amino acids with 12 putative transmembrane domains. The latter protein, named xCT, showed a significant homology with those recently reported to mediate cationic or zwitterionic amino acid transport when co-expressed with 4F2hc. Thus xCT is a new member of a family of amino acid transporters that form heteromultimeric complex with 4F2hc, with a striking difference in substrate specificity. The expression of system xc − was highly regulated, and Northern blot analysis demonstrated that the expression of both 4F2hc and xCT was enhanced in macrophages stimulated by lipopolysaccharide or an electrophilic agent. However, the expression of xCT was more directly correlated with the system xc − activity.

Keap1 regulates both cytoplasmic‐nuclear shuttling and degradation of Nrf2 in response to electrophiles

Background: Transcription factor Nrf2 regulates the expression of a set of detoxifying and anti‐oxidant enzyme genes. Several lines of evidence suggest that electrophiles and reactive oxygen species liberate Nrf2 from its cytoplasmic repressor Keap1 and provoke the accumulation of Nrf2 in the nucleus. To elucidate the molecular mechanisms as to how Nrf2 is activated by inducers, we examined the cytoplasmic‐nuclear shuttling and turnover of Nrf2.

Role of Nrf2 in the Regulation of CD36 and Stress Protein Expression in Murine Macrophages: Activation by Oxidatively Modified LDL and 4-Hydroxynonenal

Abstract— CD36 is an important scavenger receptor mediating uptake of oxidized low-density lipoproteins (oxLDLs) and plays a key role in foam cell formation and the pathogenesis of atherosclerosis. We report the first evidence that the transcription factor Nrf2 is expressed in vascular smooth muscle cells, and demonstrate that oxLDLs cause nuclear accumulation of Nrf2 in murine macrophages, resulting in the activation of genes encoding CD36 and the stress proteins A170, heme oxygenase-1 (HO-1), and peroxiredoxin I (Prx I). 4-Hydroxy-2-nonenal (HNE), derived from lipid peroxidation, was one of the most effective activators of Nrf2. Using Nrf2-deficient macrophages, we established that Nrf2 partially regulates CD36 expression in response to oxLDLs, HNE, or the electrophilic agent diethylmaleate. In murine aortic smooth muscle cells, expressing negligible levels of CD36, both moderately and highly oxidized LDL caused only limited Nrf2 translocation and negligible increases in A170, HO-1, and Prx I expression. However, treatment of smooth muscle cells with HNE significantly enhanced nuclear accumulation of Nrf2 and increased A170, HO-1, and Prx I protein levels. Because PPAR-&ggr; can be activated by oxLDLs and controls expression of CD36 in macrophages, our results implicate Nrf2 as a second important transcription factor involved in the induction of the scavenger receptor CD36 and antioxidant stress genes in atherosclerosis.

Transcription Factor Nrf2 Regulates Inflammation by Mediating the Effect of 15-Deoxy-Δ12,14-Prostaglandin J2

ABSTRACT Activated macrophages express high levels of Nrf2, a transcription factor that positively regulates the gene expression of antioxidant and detoxication enzymes. In this study, we examined how Nrf2 contributes to the anti-inflammatory process. As a model system of acute inflammation, we administered carrageenan to induce pleurisy and found that in Nrf2-deficient mice, tissue invasion by neutrophils persisted during inflammation and the recruitment of macrophages was delayed. Using an antibody against 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), it was observed that macrophages from pleural lavage accumulate 15d-PGJ2. We show that in mouse peritoneal macrophages 15d-PGJ2 can activate Nrf2 by forming adducts with Keap1, resulting in an Nrf2-dependent induction of heme oxygenase 1 and peroxiredoxin I (PrxI) gene expression. Administration of the cyclooxygenase 2 inhibitor NS-398 to mice with carrageenan-induced pleurisy caused persistence of neutrophil recruitment and, in macrophages, attenuated the 15d-PGJ2 accumulation and PrxI expression. Administration of 15d-PGJ2 into the pleural space of NS-398-treated wild-type mice largely counteracted both the decrease in PrxI and persistence of neutrophil recruitment. In contrast, these changes did not occur in the Nrf2-deficient mice. These results demonstrate that Nrf2 regulates the inflammation process downstream of 15d-PGJ2 by orchestrating the recruitment of inflammatory cells and regulating the gene expression within those cells.

Listeria monocytogenes ActA-mediated escape from autophagic recognition

Autophagy degrades unnecessary organelles and misfolded protein aggregates, as well as cytoplasm-invading bacteria. Nevertheless, the bacteria Listeria monocytogenes efficiently escapes autophagy. We show here that recruitment of the Arp2/3 complex and Ena/VASP, via the bacterial ActA protein, to the bacterial surface disguises the bacteria from autophagic recognition, an activity that is independent of the ability to mediate bacterial motility. L. monocytogenes expressing ActA mutants that lack the ability to recruit the host proteins initially underwent ubiquitylation, followed by recruitment of p62 (also known as SQSTM1) and LC3, before finally undergoing autophagy. The ability of ActA to mediate protection from ubiquitylation was further demonstrated by generating aggregate-prone GFP–ActA–Q79C and GFP–ActA–170* chimaeras, consisting of GFP (green fluorescent protein), the ActA protein and segments of polyQ or Golgi membrane protein GCP170 (ref. 6). GFP–ActA–Q79C and GFP–ActA–170* formed aggregates in the host cell cytoplasm, however, these ActA-containing aggregates were not targeted for association with ubiquitin and p62. Our findings indicate that ActA-mediated host protein recruitment is a unique bacterial disguise tactic to escape from autophagy.

Regulatory mechanisms of cellular response to oxidative stress

An antioxidant responsive element (ARE) or electrophile responsive element (EpRE) mediates the transcriptional activation of genes encoding phase II drug metabolizing enzymes. The ARE consensus sequence shows high similarity to an erythroid gene regulatory element, and based on the observation, we have recently found that transcription factor Nrf2 is essential for the coordinate induction of phase II detoxifying enzymes. The expression of anti-oxidative stress enzyme genes is also regulated by Nrf2. Detailed analysis of the regulatory mechanisms of Nrf2 activity has ultimately led us to the identification of a new protein, which we have named Keap1, that suppresses Nrf2 activity by specific binding to its evolutionarily-conserved N-terminal Neh2 regulatory domain.

Peroxiredoxin family proteins are key initiators of post-ischemic inflammation in the brain

Post-ischemic inflammation is an essential step in the progression of brain ischemia-reperfusion injury. However, the mechanism that activates infiltrating macrophages in the ischemic brain remains to be clarified. Here we demonstrate that peroxiredoxin (Prx) family proteins released extracellularly from necrotic brain cells induce expression of inflammatory cytokines including interleukin-23 in macrophages through activation of Toll-like receptor 2 (TLR2) and TLR4, thereby promoting neural cell death, even though intracellular Prxs have been shown to be neuroprotective. The extracellular release of Prxs in the ischemic core occurred 12 h after stroke onset, and neutralization of extracellular Prxs with antibodies suppressed inflammatory cytokine expression and infarct volume growth. In contrast, high mobility group box 1 (HMGB1), a well-known damage-associated molecular pattern molecule, was released before Prx and had a limited role in post-ischemic macrophage activation. We thus propose that extracellular Prxs are previously unknown danger signals in the ischemic brain and that its blocking agents are potent neuroprotective tools.

Keap1 degradation by autophagy for the maintenance of redox homeostasis

The Kelch-like ECH-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) system is essential for cytoprotection against oxidative and electrophilic insults. Under unstressed conditions, Keap1 serves as an adaptor for ubiquitin E3 ligase and promotes proteasomal degradation of Nrf2, but Nrf2 is stabilized when Keap1 is inactivated under oxidative/electrophilic stress conditions. Autophagy-deficient mice show aberrant accumulation of p62, a multifunctional scaffold protein, and develop severe liver damage. The p62 accumulation disrupts the Keap1-Nrf2 association and provokes Nrf2 stabilization and accumulation. However, individual contributions of p62 and Nrf2 to the autophagy-deficiency–driven liver pathogenesis have not been clarified. To examine whether Nrf2 caused the liver injury independent of p62, we crossed liver-specific Atg7::Keap1-Alb double-mutant mice into p62- and Nrf2-null backgrounds. Although Atg7::Keap1-Alb::p62−/− triple-mutant mice displayed defective autophagy accompanied by the robust accumulation of Nrf2 and severe liver injury, Atg7::Keap1-Alb::Nrf2−/− triple-mutant mice did not show any signs of such hepatocellular damage. Importantly, in this study we noticed that Keap1 accumulated in the Atg7- or p62-deficient mouse livers and the Keap1 level did not change by a proteasome inhibitor, indicating that the Keap1 protein is constitutively degraded through the autophagy pathway. This finding is in clear contrast to the Nrf2 degradation through the proteasome pathway. We also found that treatment of cells with tert-butylhydroquinone accelerated the Keap1 degradation. These results thus indicate that Nrf2 accumulation is the dominant cause to provoke the liver damage in the autophagy-deficient mice. The autophagy pathway maintains the integrity of the Keap1-Nrf2 system for the normal liver function by governing the Keap1 turnover.