Toru Yanagawa

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.

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.

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.

Induction of osteoblast differentiation indices by statins in MC3T3‐E1 cells

Statins inhibit 3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) reductase, which catalyzes conversion of HMG‐CoA to mevalonate, a rate‐limiting step in cholesterol synthesis. The present study was undertaken to understand the events of osteoblast differentiation induced by statins. Simvastatin at 10−7 M markedly increased mRNA expression for bone morphogenetic protein‐2 (BMP‐2), vascular endothelial growth factor (VEGF), alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin (OCN) in nontransformed osteoblastic cells (MC3T3‐E1), while suppressing gene expression for collagenase‐1, and collagenase‐3. Extracellular accumulation of proteins such as VEGF, OCN, collagenase‐digestive proteins, and noncollagenous proteins was increased in the cells treated with 10−7 M simvastatin, or 10−8 M cerivastatin. In the culture of MC3T3‐E1 cells, statins stimulated mineralization; pretreating MC3T3‐E1 cells with mevalonate, or geranylgeranyl pyrophosphate (a mevalonate metabolite) abolished statin‐induced mineralization. Statins stimulate osteoblast differentiation in vitro, and may hold promise drugs for the treatment of osteoporosis in the future.

Peroxiredoxin I expression in oral cancer: a potential new tumor marker

This study investigates the applicability of the novel antioxidant protein, peroxiredoxin (Prx) I as a marker for tumor status in oral squamous cell carcinoma (SCC). Samples from 53 patients with SCC in the oral cavity were examined by immunohistochemistry. Correlations between the expression level of Prx I and proliferating cell nuclear antigen (PCNA), the clinical features of tumors, and their histopathological classifications were statistically analyzed. Cases exhibiting low Prx I expression level included significantly more with larger tumor mass cases (T-category, P=0.004), positive lymph node metastasis (N-category, P=0.015), advanced stage (P=0.002), and poorly differentiated cells (P=0.020). There was no significant difference between Prx I expression and the other indices.

Factors associated with diagnostic delay of oral squamous cell carcinoma

Shortening the diagnostic delay from the onset of symptoms to the final diagnosis leads to early cancer detection and a reduced incidence of advanced cases. To analyze factors contributing to delays in the diagnosis of oral cancer, information was collected from the medical charts of 152 consecutive patients with oral squamous cell carcinoma, and factors associated with diagnostic delay were examined retrospectively. No characteristic was significantly associated with delay caused by patients. Referral by a non-initial professional, initial visit to a dentist, T1 cancer, and the presence of an ulcerative lesion were significantly associated with delay caused by the initial professionals. Patients with N0 were significantly associated with diagnostic delay caused by the final professional. These results re-emphasize the important role of the initial professional, particularly the dentist, and the diagnostic difficulty posed by ulcerative lesions and small-sized or early-stage oral cancer.

Deletion of nuclear factor-E2-related factor-2 leads to rapid onset and progression of nutritional steatohepatitis in mice.

Oxidative stress is a critical mediator in liver injury of steatohepatitis. The transcription factor Nrf2 serves as a cellular stress sensor and is a key regulator for induction of hepatic detoxification and antioxidative stress systems. The involvement of Nrf2 in defense against the development of steatohepatitis remains unknown. We aimed to investigate the protective roles of Nrf2 in nutritional steatohepatitis using wild-type (WT) and Nrf2 gene-null (Nrf2-null) mice. WT and Nrf2-null mice were fed a methionine- and choline-deficient (MCD) diet for 3 and 6 wk, and the liver tissues were analyzed for pathology and for expression levels of detoxifying enzymes and antioxidative stress genes via the Nrf2 transcriptional pathway. In WT mice fed an MCD diet, Nrf2 was potently activated in the livers, and steatohepatitis did not develop over the observation periods. However, in Nrf2-null mice fed an MCD diet, the pathological state of the steatohepatitis was aggravated in terms of fatty changes, inflammation, fibrosis, and iron accumulation. In the livers of the Nrf2-null mice, oxidative stress was significantly increased compared with that of WT mice based on the increased levels of 4-hydroxy-2-nonenal and malondialdehyde. This change was associated with the decreased levels of glutathione, detoxifying enzymes, catalase, and superoxide dismutase activity. Correlating well with the liver pathology, the mRNA levels of factors involved in fatty acid metabolism, inflammatory cytokines, and fibrogenesis-related genes were significantly increased in the livers of the Nrf2-null mice. These findings demonstrate that Nrf2 deletion in mice leads to rapid onset and progression of nutritional steatohepatitis induced by an MCD diet. Activation of Nrf2 could be a promising target toward developing new options for prevention and treatment of steatohepatitis.

Transcriptional activation of p62/A170/ZIP during the formation of the aggregates: possible mechanisms and the role in Lewy body formation in Parkinson's disease.

Formation of intracellular inclusion bodies due to defects in the protein degradation machinery is associated with the pathogenesis of neurodegenerative diseases. Sequestosomal protein p62/A170/ZIP, which is an oxidative stress-related protein and a ubiquitin-binding protein, is a component protein of Lewy bodies that are observed in patients with Parkinsons disease. The association of p62 with poly-ubiquitinated proteins may be an important step in the formation of intracellular protein aggregates like Lewy bodies. To study the role of p62 in the formation of protein aggregates in PC12 cells, we monitored the intracellular localizations of p62 and ubiquitinated proteins and the levels of both components during treatment with MG132, a proteasome inhibitor. In the early stage of aggregate formation, p62 did not always co-localize with ubiquitin. In contrast, these proteins were always co-localized in later stages. After the treatment of the cells with MG132, we found that the expression level of p62 increased due to the transcriptional activation of the gene and that higher molecular sizes of p62, corresponding to mono- and di-ubiquitinated formes, were also formed. Both the transcriptional inhibitor actinomycin D and an antisense oligonucleotide of p62 inhibited the MG132-mediated increase of p62, the sequestration of ubiquitinated proteins, and the enlargement of the aggregates. Furthermore, p62-positive aggregates were observed primarily in surviving cells. Together, these results suggest that p62 plays an important role in the protection of cells from the toxicity of misfolded proteins by enhancing aggregate formation especially in the later stages.

Novel roles of peroxiredoxins in inflammation, cancer and innate immunity.

Peroxiredoxins possess thioredoxin or glutathione peroxidase and chaperone-like activities and thereby protect cells from oxidative insults. Recent studies, however, reveal additional functions of peroxiredoxins in gene expression and inflammation-related biological reactions such as tissue repair, parasite infection and tumor progression. Notably, peroxiredoxin 1, the major mammalian peroxiredoxin family protein, directly interacts with transcription factors such as c-Myc and NF-κB in the nucleus. Additionally, peroxiredoxin 1 is secreted from some cells following stimulation with TGF-β and other cytokines and is thus present in plasma and body fluids. Peroxiredoxin 1 is now recognized as one of the pro-inflammatory factors interacting with toll-like receptor 4, which triggers NF-κB activation and other signaling pathways to evoke inflammatory reactions. Some cancer cells release peroxiredoxin 1 to stimulate toll-like receptor 4-mediated signaling for their progression. Interestingly, peroxiredoxins expressed in protozoa and helminth may modulate host immune responses partly through toll-like receptor 4 for their survival and progression in host. Extracellular peroxiredoxin 1 and peroxiredoxin 2 are known to enhance natural killer cell activity and suppress virus-replication in cells. Peroxiredoxin 1-deficient mice show reduced antioxidant activities but also exhibit restrained tissue inflammatory reactions under some patho-physiological conditions. Novel functions of peroxiredoxins in inflammation, cancer and innate immunity are the focus of this review.

Activation of Nrf2 and accumulation of ubiquitinated A170 by arsenic in osteoblasts

Sub-lethal levels of arsenic induce upregulation of stress proteins. We here report for the first time that inorganic arsenic activates the transcription factor Nrf2, which controls the expression of oxidative stress-induced proteins. Treatment of cultured MC3T3-E1 osteoblasts with arsenite or arsenate induced increase of Nrf2, followed by transcriptional activation of target genes encoding HO-1, Prx I, and A170. We found that arsenate (200-800 micro M) only slightly increased the normal 60kDa A170 protein but markedly increased higher molecular mass forms of A170 (HMM-A170) that appeared as smeared bands. Arsenate also markedly increased ubiquitin-conjugated cellular proteins, suggesting that HMM-A170 was one of the poly-ubiquitinated proteins. Arsenite (50-100 micro M) also induced accumulation of HMM-A170 and ubiquitin-conjugated proteins. These results provide the first direct evidence that toxic arsenics impair the normal function of A170. Our findings provide a potential diagnostic tool for monitoring biotoxicity in cells and tissues in response to arsenic compounds.