Junko Wakabayashi

Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation

Transcription factor Nrf2 (encoded by Nfe2l2) regulates a battery of detoxifying and antioxidant genes, and Keap1 represses Nrf2 function. When we ablated Keap1, Keap1-deficient mice died postnatally, probably from malnutrition resulting from hyperkeratosis in the esophagus and forestomach. Nrf2 activity affects the expression levels of several squamous epithelial genes. Biochemical data show that, without Keap1, Nrf2 constitutively accumulates in the nucleus to stimulate transcription of cytoprotective genes. Breeding to Nrf2-deficient mice reversed the phenotypic Keap1 deficiencies. These experiments show that Keap1 acts upstream of Nrf2 in the cellular response to oxidative and xenobiotic stress.

Direct Membrane Association Drives Mitochondrial Fission by the Parkinson Disease-associated Protein α-Synuclein

The protein α-synuclein has a central role in Parkinson disease, but the mechanism by which it contributes to neural degeneration remains unknown. We now show that the expression of α-synuclein in mammalian cells, including neurons in vitro and in vivo, causes the fragmentation of mitochondria. The effect is specific for synuclein, with more fragmentation by α- than β- or γ-isoforms, and it is not accompanied by changes in the morphology of other organelles or in mitochondrial membrane potential. However, mitochondrial fragmentation is eventually followed by a decline in respiration and neuronal death. The fragmentation does not require the mitochondrial fission protein Drp1 and involves a direct interaction of synuclein with mitochondrial membranes. In vitro, synuclein fragments artificial membranes containing the mitochondrial lipid cardiolipin, and this effect is specific for the small oligomeric forms of synuclein. α-Synuclein thus exerts a primary and direct effect on the morphology of an organelle long implicated in the pathogenesis of Parkinson disease.

Physiological Significance of Reactive Cysteine Residues of Keap1 in Determining Nrf2 Activity

ABSTRACT Keap1 and Cul3 constitute a unique ubiquitin E3 ligase that degrades Nrf2, a key activator of cytoprotective genes. Upon exposure to oxidants/electrophiles, the enzymatic activity of this ligase complex is inhibited and the complex fails to degrade Nrf2, resulting in the transcriptional activation of Nrf2 target genes. Keap1 possesses several reactive cysteine residues that covalently bond with electrophiles in vitro. To clarify the functional significance of each Keap1 cysteine residue under physiological conditions, we established a transgenic complementation rescue model. The transgenic expression of mutant Keap1(C273A) and/or Keap1(C288A) protein in Keap1 null mice failed to reverse constitutive Nrf2 activation, indicating that cysteine residues at positions 273 and 288 are essential for Keap1 to repress Nrf2 activity in vivo. In contrast, Keap1(C151S) retained repressor activity and mice expressing this molecule were viable. Mouse embryonic fibroblasts from Keap1(C151S) transgenic mice displayed decreased expression of Nrf2 target genes both before and after an electrophilic challenge, suggesting that Cys151 is important in facilitating Nrf2 activation. These results demonstrate critical roles of the cysteine residues in vivo in maintaining Keap1 function, such that Nrf2 is repressed under quiescent conditions and active in response to oxidants/electrophiles.

The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice

1. 1. Wakabayashi, 2. et al . 2009. J. Cell Biol. doi:10.1083/jcb.200903065 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1083%252Fjcb.200903065%26rft_id%253Dinfo%253Apmid%252F19752021%26rft.genre%253Darticle%26rft_val_fmt%

Role of Nrf2 in prevention of high-fat diet-induced obesity by synthetic triterpenoid CDDO-Imidazolide

The synthetic oleanolic triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Imidazolide or CDDO-Im) is an extremely potent activator of Nrf2 signaling. In cells undergoing adipogenesis, CDDO-Im prevents lipid accumulation in an Nrf2-dependent manner. However, in vivo evidence for effects of CDDO-Im on obesity is lacking. The goals of these studies were to determine if CDDO-Im can prevent high-fat diet-induced obesogenesis in the mouse, and to elucidate the molecular target of drug action. Wild-type and Nrf2-disrupted C57BL/6J female mice were dosed 3 times per week with 30 micromol/kg CDDO-Im or vehicle by oral gavage, during 95 days of access to a control diet or a high-fat diet. Body weights, organ weights, hepatic fat accumulation and gene expression were measured. Treatment with CDDO-Im effectively prevented high-fat diet-induced increases in body weight, adipose mass, and hepatic lipid accumulation in wild-type mice but not in Nrf2-disrupted mice. Wild-type mice on a high-fat diet and treated with CDDO-Im exhibited higher oxygen consumption and energy expenditure than vehicle-treated mice, while food intake was lower in CDDO-Im-treated than vehicle-treated mice. Levels of gene transcripts for fatty acid synthesis enzymes were downregulated after CDDO-Im treatment in the liver of wild-type mice. This inhibitory effect of CDDO-Im on lipogenic gene expression was significantly reduced in Nrf2-disrupted mice. The results indicate that CDDO-Im is an exceedingly potent agent for preventing obesity, and identify the Nrf2 pathway as a novel target for management of obesogenesis.

Regulation of Notch1 Signaling by Nrf2: Implications for Tissue Regeneration

The cell survival pathway mediated by the transcription factor Nrf2 facilitates tissue regeneration by promoting Notch1 signaling. Setting the Pace of Regeneration The ability of tissues to regenerate is critical for organismal survival, yet all of the mechanisms regulating this complex phenomenon are unknown. Nrf2 is a member of a family of transcription factors, and without the prosurvival signaling of Nrf2, mice are sensitive to oxidative and electrophilic stresses and exhibit delays in tissue regeneration. Wakabayashi et al. connect Nrf2 to the Notch signaling pathway, which is involved in determining cell proliferation and cell fate, by showing that Nrf2 transcriptionally stimulates expression of Notch1 to establish basal Notch1 signaling. The amount of Notch1 at the time of injury appears to set the pace of liver regeneration, and forced Notch signaling in hepatocytes restored liver regeneration in Nrf2-deficient mice. The Keap1-Nrf2-ARE signaling pathway elicits an adaptive response for cell survival after endogenous and exogenous stresses, such as inflammation and carcinogens, respectively. Keap1 inhibits the transcriptional activation activity of Nrf2 (p45 nuclear factor erythroid-derived 2–related factor 2) in unstressed cells by facilitating its degradation. Through transcriptional analyses in Keap1- or Nrf2-disrupted mice, we identified interactions between the Keap1-Nrf2-ARE and the Notch1 signaling pathways. We found that Nrf2 recognized a functional antioxidant response element (ARE) in the promoter of Notch1. Notch1 regulates processes such as proliferation and cell fate decisions. We report a functional role for this cross talk between the two pathways and show that disruption of Nrf2 impeded liver regeneration after partial hepatectomy and was rescued by reestablishment of Notch1 signaling.

The dynamin-related GTPase Opa1 is required for glucose-stimulated ATP production in pancreatic beta cells

The physiological function of Opa1, a dynamin-related GTPase required for mitochondrial fusion, is described in glucose-stimulated ATP production in pancreatic beta cells.

Nrf2-mediated Induction of Cytoprotective Enzymes by 15-Deoxy-Δ12,14-Prostaglandin J2 Is Attenuated by Alkenal/one Oxidoreductase

NADPH-dependent alkenal/one oxidoreductase (Aor) was discovered to be highly inducible in rat liver following treatment with the cancer chemopreventive agent 3H-1, 2-dithiole-3-thione. Aor was further characterized as an Nrf2-regulated antioxidative enzyme that reduces carbon-carbon double bonds in a variety of α, β-unsaturated aldehydes and ketones. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is a reactive membrane lipid metabolite that activates multiple pathways, including Nrf2-mediated induction of cytoprotective enzymes. Physiologically, it is postulated that 15d-PGJ2 alkylates key regulatory proteins via the electrophilic carbon centers found in two α, β-unsaturated ketone moieties. This current study addresses the metabolism of 15d-PGJ2 by rat Aor (rAor) and subsequent deactivation of the Nrf2 signaling pathway by both rat and human AOR. We demonstrate that induction of NADPH-dependent quinone oxidoreductase activity by 15d-PGJ2 is markedly attenuated in mouse embryonic fibroblasts that overexpress rAor. Luciferase reporter assay and quantitative real-time PCR confirmed these findings. Concentrations required for doubling the NADPH-dependent quinone oxidoreductase response are increased from 1.8μm in wild-type to >10μm in rat Aor transgenic fibroblasts. 15d-PGJ2 is metabolized by recombinant rAor with a Km of 9.6 μm and kcat of 18.5 min-1. The major product is 12,13-dihydro-15-deoxy-Δ12,14-prostaglandin J2 (dihydro-15d-PGJ2). The reduction of C=C by Aor yielding dihydro-15d-PGJ2 abolishes the inducibility in an antioxidant response element-driven luciferase assay. Collectively, these results demonstrate that 15d-PGJ2 can be catabolized by Aor, thereby attenuating subsequent Nrf2 signaling and possibly inflammatory and apoptotic processes also influenced by 15d-PGJ2.

GATA‐1 testis activation region is essential for Sertoli cell‐specific expression of GATA‐1 gene in transgenic mouse

Background: The erythroid transcription factor GATA‐1 is also expressed in Sertoli cells of the testis. The testicular expression of GATA‐1 is regulated in a developmental and spermatogenic stage‐specific manner. To further clarify the regulatory mechanisms of testicular GATA‐1 gene expression, we carried out transgenic reporter gene expression analyses.