Oxidative stress and inflammation are two critical factors that drive the formation of plaques in atherosclerosis. Nrf2 is a redox-sensitive transcription factor that upregulates a battery of antioxidative genes and cytoprotective enzymes that constitute the cellular response to oxidative stress. Our previous studies have shown that disruption of Nrf2 in mice (Nrf2−/−) causes increased susceptibility to pulmonary emphysema, asthma and sepsis due to increased oxidative stress and inflammation. Here we have tested the hypothesis that disruption of Nrf2 in mice causes increased atherosclerosis.
To investigate the role of Nrf2 in the development of atherosclerosis, we crossed Nrf2−/− mice with apoliporotein E-deficient (ApoE−/−) mice. ApoE−/− and ApoE−/− Nrf2−/− mice were fed an atherogenic diet for 20 weeks, and plaque area was assessed in the aortas. Surprisingly, ApoE−/− Nrf2−/− mice exhibited significantly smaller plaque area than ApoE−/− controls (11.5% vs 29.5%). This decrease in plaque area observed in ApoE−/− Nrf2−/− mice was associated with a significant decrease in uptake of modified low density lipoproteins (AcLDL) by isolated macrophages from ApoE−/− Nrf2−/− mice. Furthermore, atherosclerotic plaques and isolated macrophages from ApoE−/− Nrf2−/− mice exhibited decreased expression of the scavenger receptor CD36.
Nrf2 is pro-atherogenic in mice, despite its antioxidative function. The net pro-atherogenic effect of Nrf2 may be mediated via positive regulation of CD36. Our data demonstrates that the potential effects of Nrf2-targeted therapies on cardiovascular disease need to be investigated.
The apolipoprotein E (APOE) genotype is an important risk factor for ageing and age-related diseases. The APOE4 genotype (in contrast to APOE3) has been shown to be associated with oxidative stress and chronic inflammation. Metallothioneins (MT) exhibit antioxidant and anti-inflammatory activity, and MT overexpression has been shown to increase lifespan in mice. Interactions between APOE and MT, however, are largely unknown. Hence, we determined the effect of the APOE4 versus APOE3 genotype on MT levels in targeted gene replacement mice. APOE4 versus APOE3 mice exhibited significantly lower hepatic MT1 and MT2 mRNA as well as lower MT protein levels. The decrease in hepatic MT protein levels in APOE4 as compared to APOE3 mice was accompanied by lower nuclear Nrf1, a protein partly controlling MT gene expression. Cell culture experiments using hepatocytes identified allyl-isothiocyanate (AITC) as a potent MT inductor in vitro. Therefore, we supplemented APOE3 and APOE4 mice with AITC. However, AITC (15 mg/kg b.w.) could only partly correct for decreased MT1 and MT2 gene expression in APOE4 mice in vivo. Furthermore, cholesterol significantly decreased both Nrf1 and MT mRNA levels in Huh7 cells indicating that differences in MT gene expression between the two genotypes could be related to differences in hepatic cholesterol concentrations. Overall, present data suggest that the APOE genotype is an important determinant of tissue MT levels in mice and that MT gene expression may be impaired by the APOE4 genotype.
APOE; Metallothionein; Nrf1; Ageing; Mice
apoE deficiency causes hyperlipidemia and premature atherosclerosis. To determine if macrophage-specific expression of apoE would decrease the extent of atherosclerosis, we expressed human apoE in macrophages of apoE-null mice (apoE-/-) and assessed the effect on lipid accumulation in cells of the arterial wall. Macrophage-specific expression of human apoE in normal mice was obtained by use of the visna virus LTR. These animals were bred with apoE-/- mice to produce animals hemizygous for expression of human apoE in macrophages in the absence of murine apoE (apoE-/-,hTgE+/0). Low levels of human apoE mRNA were present in liver and spleen and high levels in lung and peritoneal macrophages. Human apoE was secreted by peritoneal macrophages and was detected in Kupffer cells of the liver. Human apoE in the plasma of apoE-/-,hTgE+/0 mice (n = 30) was inversely correlated (P < 0.005) with the plasma cholesterol concentration. After 15 wk on a normal chow diet, atherosclerosis was assessed in apoE-/-,hTgE+/0 animals and in apoE-/-,hTgE0/0 littermates matched for plasma cholesterol level (approximately 450 mg/dl) and lipoprotein profile. There was significantly less atherosclerosis in both the aortic sinus and in the proximal aorta (P < 0.0001) in the animals expressing the human apoE transgene. In apo-E-/-,hTgE+/0 animals, which had detectable atherosclerotic lesions, human apoE was detected in the secretory apparatus of macrophage-derived foam cells in the arterial wall. The data demonstrate that expression of apoE by macrophages is antiatherogenic even in the presence of high levels of atherogenic lipoproteins. The data suggest that apoE prevents atherosclerosis by promoting cholesterol efflux from cells of the arterial wall.
Oxidative stress-mediated destruction of normal parenchymal cells during hepatic inflammatory responses contributes to the pathogenesis of immune-mediated hepatitis and is implicated in the progression of acute inflammatory liver injury to chronic inflammatory liver disease. The transcription factor NF-E2-related factor 2 (Nrf2) regulates the expression of a battery of antioxidative enzymes and Nrf2 signaling can be activated by small-molecule drugs that disrupt Keap1-mediated repression of Nrf2 signaling. Therefore, genetic and pharmacologic approaches were used to activate Nrf2 signaling to assess protection against inflammatory liver injury. Profound increases in ind of cell death were observed in both Nrf2 wild-type (Nrf2-WT) mice and Nrf2-disrupted (Nrf2-KO) mice 24-hr following intravenous injection of concanavalin A (12.5 mg/kg, ConA), a model for T cell-mediated acute inflammatory liver injury. However, hepatocyte-specific conditional Keap1 null (Alb-Cre:Keap1flox/−, cKeap1-KO) mice with constitutively enhanced expression of Nrf2-regulated antioxidative genes as well as Nrf2-WT mice but not Nrf2-KO mice pretreated with three daily doses of a triterpenoid that potently activates Nrf2 (30 µmole/kg, CDDO-Im) were highly resistant to ConA-mediated inflammatory liver injury. CDDO-Im pretreatment of both Nrf2-WT and Nrf2-KO mice resulted in equivalent suppression of serum pro-inflammatory soluble proteins suggesting that the hepatoprotection afforded by CDDO-Im pretreatment of Nrf2-WT mice but not Nrf2-KO mice was not due to suppression of systemic pro-inflammatory signaling, but instead was due to activation of Nrf2 signaling in the liver. Enhanced hepatic expression of Nrf2-regulated antioxidative genes inhibited inflammation-mediated oxidative stress, thereby preventing hepatocyte necrosis. Attenuation of hepatocyte death in cKeap1-KO mice and CDDO-Im pretreated Nrf2-WT mice resulted in decreased late-phase pro-inflammatory gene expression in the liver thereby diminishing the sustained influx of inflammatory cells initially stimulated by the ConA challenge. Taken together, these results clearly illustrate that targeted cytoprotection of hepatocytes through Nrf2 signaling during inflammation prevents the amplification of inflammatory responses in the liver.
Liver inflammation; Nrf2; Keap1; antioxidative enzymes; cytoprotection; triterpenoid
The transcription factor NFE2-related factor 2 (Nrf2) mediates detoxification and antioxidant gene transcription following electrophile exposure and oxidative stress. Mice deficient in Nrf2 (Nrf2-null) are highly susceptible to acetaminophen (APAP) hepatotoxicity, and exhibit lower basal and inducible expression of cytoprotective genes, including NADPH quinone oxidoreductase 1 (Nqo1) and glutamate cysteine ligase (catalytic subunit, or Gclc). Administration of toxic APAP doses to C57BL/6J mice generates electrophilic stress and subsequently increases levels of hepatic Nqo1, Gclc and the efflux multidrug resistance-associated protein transporters 1–4 (Mrp1-4). It was hypothesized that induction of hepatic Mrp1-4 expression following APAP is Nrf2-dependent. Plasma and livers from wild-type (WT) and Nrf2-null mice were collected 4, 24 and 48 hrs after APAP. As expected, hepatotoxicity was greater in Nrf2-null compared to WT mice. Gene and protein expression of Mrp1-4 and the Nrf2 targets, Nqo1 and Gclc, was measured. Induction of Nqo1 and Gclc mRNA and protein after APAP was dependent on Nrf2 expression. Similarly, APAP treatment increased hepatic Mrp3 and Mrp4 mRNA and protein in WT, but not Nrf2-null mice. Mrp1 was induced in both genotypes after APAP, suggesting that elevated expression of this transporter was independent of Nrf2. Mrp2 was not induced in either genotype at the mRNA or protein levels. These results show that Nrf2 mediates induction of Mrp3 and Mrp4 after APAP, but does not affect Mrp1 or Mrp2. Thus coordinated regulation of detoxification enzymes and transporters by Nrf2 during APAP hepatotoxicity is a mechanism by which hepatocytes may limit intracellular accumulation of potentially toxic chemicals.
Nuclear factor-E2-related factor 2; Nrf2; acetaminophen; APAP; hepatotoxicity; multidrug resistance-associated proteins; Mrp3; Mrp4
Apolipoprotein (apo) E is best known for its ability to lower plasma cholesterol and protect against atherosclerosis. Although the liver is the major source of plasma apoE, extra-hepatic sources of apoE, including from macrophages, account for up to 10% of plasma apoE levels. This study examined the contribution of macrophage-derived apoE expression levels in diet-induced hyperlipidemia and atherosclerosis.
Hypomorphic apoE (Apoeh/h) mice expressing wildtype mouse apoE at ∼2–5% of physiological levels in all tissues were derived by gene targeting in embryonic stem cells. Cre-mediated gene repair of the Apoeh/h allele in Apoeh/hLysM-Cre mice raised apoE expression levels by 26 fold in freshly isolated peritoneal macrophages, restoring it to 37% of levels seen in wildtype mice. Chow-fed Apoeh/hLysM-Cre and Apoeh/h mice displayed similar plasma apoE and cholesterol levels (55.53±2.90 mg/dl versus 62.70±2.77 mg/dl, n = 12). When fed a high-cholesterol diet (HCD) for 16 weeks, Apoeh/hLysM-Cre mice displayed a 3-fold increase in plasma apoE and a concomitant 32% decrease in plasma cholesterol when compared to Apoeh/h mice (602.20±22.30 mg/dl versus 888.80±24.99 mg/dl, n = 7). On HCD, Apoeh/hLysM-Cre mice showed increased apoE immunoreactivity in lesional macrophages and liver-associated Kupffer cells but not hepatocytes. In addition, Apoeh/hLysM-Cre mice developed 35% less atherosclerotic lesions in the aortic root than Apoeh/h mice (167×103±16×103 µm2 versus 259×103±56×103 µm2, n = 7). This difference in atherosclerosis lesions size was proportional to the observed reduction in plasma cholesterol.
Macrophage-derived apoE raises plasma apoE levels in response to diet-induced hyperlipidemia and by such reduces atherosclerosis proportionally to the extent to which it lowers plasma cholesterol levels.
Alpha-naphthylisothiocyanate (ANIT) causes intrahepatic cholestasis by injuring biliary epithelial cells. Adaptive regulation of hepatobiliary transporter expression has been proposed to reduce liver injury during cholestasis. Recently, the oxidative stress transcription factor Nrf2 (nf-e2–related factor 2) was shown to regulate expression of hepatobiliary transporters. The purpose of this study was to determine whether ANIT-induced hepatotoxicity and regulation of hepatobiliary transporters are altered in the absence of Nrf2. For this purpose, wild-type and Nrf2-null mice were administered ANIT (75 mg/kg po). Surprisingly, ANIT-induced hepatotoxicity was similar in both genotypes at 48 h. Accumulation of bile acids in serum and liver was lower in Nrf2-null mice compared with wild-types treated with ANIT. Transporter mRNA profiles differed between wild-type and Nrf2-null mice after ANIT. Bsep (bile salt export pump), Mdr2 (multidrug resistance gene), and Mrp3 (multidrug resistance–associated protein) efflux transporters were increased by ANIT in wild-type, but not in Nrf2-null mice. In contrast, mRNA expression of two hepatic uptake transporters, Ntcp (sodium-taurocholate cotransporting polypeptide) and Oatp1b2 (organic anion transporting peptide), were decreased in both genotypes after ANIT, with larger declines in Nrf2-null mice. mRNA expression of the transcriptional repressor of Ntcp, small heterodimeric partner (SHP), was increased in Nrf2-null mice after ANIT. Furthermore, hepatocyte nuclear factor 1α (HNF1α), which regulates Oatp1b2, was downregulated in ANIT-treated Nrf2-null mice. Preferential upregulation of SHP and downregulation of HNF1α and uptake transporters likely explains why Nrf2-null mice exhibited similar injury to wild-types after ANIT. A subsequent study revealed that treatment of mice with the Nrf2 activator oltipraz protects against ANIT-induced histological injury. Despite compensatory changes in Nrf2-null mice to limit ANIT toxicity, pharmacological activation of Nrf2 may represent a therapeutic option for intrahepatic cholestasis.
Nrf2; ANIT; Nqo1; oxidative stress; Mrps
Mice lacking the transcription factor NF-E2 p45-related factor 2 (Nrf2) develop more severe nonalcoholic steatohepatitis (NASH), with cirrhosis, than wild-type (Nrf2+/+) mice when fed a high-fat (HF) diet for 24 weeks. Although NASH is usually associated with insulin resistance, HF-fed Nrf2−/− mice exhibited better insulin sensitivity than HF-fed Nrf2+/+ mice. In livers of HF-fed mice, loss of Nrf2 resulted in greater induction of lipogenic genes, lower expression of β-oxidation genes, greater reduction in AMP-activated protein kinase (AMPK) levels, and diminished acetyl coenzyme A (CoA) carboxylase phosphorylation than in the wild-type livers, which is consistent with greater fatty acid (FA) synthesis in Nrf2−/− livers. Moreover, primary Nrf2−/− hepatocytes displayed lower glucose and FA oxidation than Nrf2+/+ hepatocytes, with FA oxidation partially rescued by treatment with AMPK activators. The unfolded protein response (UPR) was perturbed in control regular-chow (RC)-fed Nrf2−/− mouse livers, and this was associated with constitutive activation of NF-κB and JNK, along with upregulation of inflammatory genes. The HF diet elicited an antioxidant response in Nrf2+/+ livers, and as this was compromised in Nrf2−/− livers, they suffered oxidative stress. Therefore, Nrf2 protects against NASH by suppressing lipogenesis, supporting mitochondrial function, increasing the threshold for the UPR and inflammation, and enabling adaptation to HF-diet-induced oxidative stress.
Although much is known about the pathophysiological processes contributing to diabetic retinopathy (DR), the role of protective pathways has received less attention. The transcription factor nuclear factor erythroid-2-related factor 2 (also known as NFE2L2 or NRF2) is an important regulator of oxidative stress and also has anti-inflammatory effects. The objective of this study was to explore the potential role of NRF2 as a protective mechanism in DR.
Retinal expression of NRF2 was investigated in human donor and mouse eyes by immunohistochemistry. The effect of NRF2 modulation on oxidative stress was studied in the human Müller cell line MIO-M1. Non-diabetic and streptozotocin-induced diabetic wild-type and Nrf2 knockout mice were evaluated for multiple DR endpoints.
NRF2 was expressed prominently in Müller glial cells and astrocytes in both human and mouse retinas. In cultured MIO-M1 cells, NRF2 inhibition significantly decreased antioxidant gene expression and exacerbated tert-butyl hydroperoxide- and hydrogen peroxide-induced oxidative stress. NRF2 activation strongly increased NRF2 target gene expression and suppressed oxidant-induced reactive oxygen species. Diabetic mice exhibited retinal NRF2 activation, indicated by nuclear translocation. Superoxide levels were significantly increased by diabetes in Nrf2 knockout mice as compared with wild-type mice. Diabetic Nrf2 knockout mice exhibited a reduction in retinal glutathione and an increase in TNF-α protein compared with wild-type mice. Nrf2 knockout mice exhibited early onset of blood–retina barrier dysfunction and exacerbation of neuronal dysfunction in diabetes.
These results indicate that NRF2 is an important protective mechanism regulating the progression of DR and suggest enhancement of the NRF2 pathway as a potential therapeutic strategy.
Diabetic retinopathy; Inflammation; Müller glial cells; Neuronal dysfunction; NF-E2-related factor-2; Reactive oxygen species; Transcription factor; Vascular permeability
High mobility group box protein 1 (HMGB1) and receptor for the advanced glycation end product (RAGE) play pivotal roles in vascular inflammation and atherosclerosis. The aim of this study was to determine whether the HMGB1-RAGE axis was involved in the actions of simvastatin on vascular inflammation and atherosclerosis in ApoE−/− mice.
Five-week old ApoE−/− mice and wild-type C57BL/6 mice were fed a Western diet. At 8 weeks of age, ApoE−/− mice were administered simvastatin (50 mg·kg−1·d−1) or vehicle by gavage, and the wild-type mice were treated with vehicle. The mice were sacrificed at 11 weeks of age, and the atherosclerotic lesions in aortic sinus were assessed with Oil Red O staining. Macrophage migration was determined with scanning EM and immunohistochemistry. Human umbilical vein endothelial cells (HUVECs) were used for in vitro study. Western blots were used to quantify the protein expression of HMGB1, RAGE, vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1).
Vehicle-treated ApoE−/− mice exhibited significant increases in aortic inflammation and atherosclerosis as well as enhanced expression of HMGB1, RAGE, VCAM-1, and MCP-1 in aortic tissues as compared to the wild-type mice. Furthermore, serum total cholesterol, triglyceride and LDL levels were markedly increased, while serum HDL level was decreased in vehicle-treated ApoE−/− mice. Administration with simvastatin in ApoE−/− mice markedly attenuated the vascular inflammation and atherosclerotic lesion area, and decreased the aortic expression of HMGB1, RAGE, VCAM-1, and MCP-1. However, simvastatin did not affect the abnormal levels of serum total cholesterol, triglyceride, LDL and HDL in ApoE−/− mice. Exposure of HUVECs to HMGB1 (100 ng/mL) markedly increased the expression of HMGB1, RAGE and VCAM-1, whereas pretreatment of the cells with simvastatin (10 μmol/L) blocked the HMGB1-caused changes.
Simvastatin inhibits vascular inflammation and atherosclerosis in ApoE−/− mice, which may be mediated through downregulation of the HMGB1-RAGE axis.
atherosclerosis; inflammation; simvastatin; ApoE−/− mice; human umbilical vein endothelial cells (HUVECs); aortic sinus; HMGB1; RAGE; VCAM-1; MCP-1; macrophages
Apolipoprotein E (apoE) plays a key role in lipoprotein metabolism and may have other important biological functions. In humans, there are three common, naturally occurring isoforms of apoE that are associated with differences in lipid levels and atherosclerosis. However, the direct in vivo effects of the apoE isoforms on lipoprotein metabolism and atherosclerosis are not yet fully understood. To investigate the effect of the apoE isoforms in vivo, we constructed second-generation recombinant adenoviruses encoding each of the apoE isoforms. These recombinant adenoviruses were injected intravenously into apoE-deficient mice fed a Western diet (mean baseline cholesterol level 1401 mg/dl) in order to study their effects in the absence of endogenous mouse apoE. Hepatic expression of apoE3 and apoE4 completely normalized the lipoprotein profile; 3 d after injection, mean plasma cholesterol levels were 194 and 217 mg/ dl, respectively, and this effect was maintained for at least 6 wk. Expression of apoE2 had much less effect on lipoprotein levels (mean cholesterol level 752 mg/dl 3 d after injection), despite much higher plasma levels of apoE2 compared with apoE3 and apoE4; by 6 wk after injection the cholesterol levels had returned to baseline levels in the apoE2-expressing mice. Expression of all three isoforms significantly increased HDL cholesterol levels by approximately threefold and was independent of the cholesterol-lowering effect. ApoE transgene expression was substantially prolonged compared with that achieved using a first generation adenovirus and apoE was readily detected in plasma 3 mo after virus injection. These studies demonstrate: (a) prolonged in vivo expression of human apoE isoforms in apoE deficient mice after second-generation recombinant adenovirus-mediated somatic gene transfer; and (b) significantly impaired ability of apoE2 in vivo to mediate clearance of remnant lipoproteins in apoE-deficient mice fed a Western diet compared with apoE3 and apoE4.
Aim: A growing body of evidence has shown that increased formation of oxidized molecules and reactive oxygen species within the vasculature (i.e., the extracellular space) plays a crucial role in the initiation and progression of atherosclerosis and in the formation of unstable plaques. Peroxiredoxin 4 (PRDX4) is the only known secretory member of the antioxidant PRDX family. However, the relationship between PRDX4 and susceptibility to atherosclerosis has remained unclear. Results: To define the role of PRDX4 in hyperlipidemia-induced atherosclerosis, we generated hPRDX4 transgenic (Tg) and apolipoprotein E (apoE) knockout mice (hPRDX4+/+/apoE−/−). After feeding the mice a high-cholesterol diet, they showed fewer atheromatous plaques, less T-lymphocyte infiltration, lower levels of oxidative stress markers, less necrosis, a larger number of smooth muscle cells, and a larger amount of collagen, resulting in thickened fibrous cap formation and possible stable plaque phenotype as compared with apoE−/− mice. We also detected greater suppression of apoptosis and decreased Bax expression in hPRDX4+/+/apoE−/− mice than in apoE−/− mice. Bone marrow transplantation from hPRDX4+/+ donors to apoE−/− mice confirmed the antiatherogenic aspects of PRDX4, revealing significantly suppressed atherosclerotic progression. Innovation: In this study, we demonstrated for the first time that PRDX4 suppressed the development of atherosclerosis in apoE−/− mice fed a high-cholesterol diet. Conclusion: These data indicate that PRDX4 is an antiatherogenic factor and, by suppressing oxidative damage and apoptosis, that it may protect against the formation of vulnerable (unstable) plaques. Antioxid. Redox Signal. 17, 1362–1375.
To evaluate the effects of a genetic reduction of Lias gene expression on atherosclerosis development.
Methods and Results
Heterozygous knockout mice for the lipoid acid synthase gene (Lias+/−) were crossed with apolipoprotein E–deficient (ApoE−/−) mice, and the plaque size in aortic sinuses of Lias+/− ApoE−/− mice was evaluated at 6 months of age. Lesions at the aortic sinus in Lias+/− ApoE−/− males were significantly larger (1.5X) than those in Lias+/+ ApoE−/− littermate males. The lesion size was inversely correlated with an increased erythrocyte reduced glutathione/ oxidized glutathione (GSH/GSSH) ratio, an systemic index of body redox balance. Lias+/− ApoE−/− males also had significantly increased plasma cholesterol and reduced pyruvate dehydrogenase complex activity in the liver. Significant reductions in the expression of genes for antioxidant enzymes, including superoxide dismutase 1 (SOD1) and SOD2, were observed in aortas of Lias+/− ApoE−/− males. Female Lias+/− ApoE−/− also exhibited changes in these parameters, parallel to those observed in males. However, the Lias gene effects for the majority of these factors, including atherosclerotic lesion size, were not significant in females.
Our data provide evidence that Lias deficiency enhances atherosclerosis in male mice, at least in part due to reduce antioxidant capacity. The notable absence of such effects in females leaves open the possibility of a gender-specific protection mechanism.
antioxidant; lipoic acid; Lias mouse model; atherosclerosis; apolipoprotein E null mice
BACKGROUND: The induction of phase 2 enzymes by dithiolethiones such as oltipraz is an effective means for achieving protection against environmental carcinogens in animals and humans. Transcriptional control of the expression of at least some of these protective enzymes is mediated through the antioxidant response element (ARE) found in the upstream regulatory region of many phase 2 genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of proto-typical phase 2 enzymes such as glutathione S-transferase (GST) Ya, Yp, and NAD(P)H: quinone reductase (NQO1) in vivo. MATERIALS AND METHODS: In the present study, 3H-1,2-dithiole-3-thione (D3T) was used as a potent model inducer whose effects on gene expression and chemopreventive efficacy have been extensively characterized in the rat. Over a dozen putative D3T-inducible genes were examined in wild-type and nrf2-disrupted mice by Northern blot hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to elucidate whether loss of Nrf2 function also affects the induction of a broader representation of phase 2 and antioxidative enzymes. The effects of D3T on hepatic Nrf2 expression and localization were also examined in vivo by Northern blot hybridization, electromobility shift assay, and Western blot analysis. RESULTS: Specific activities of hepatic GST and NQO1 were increased by D3T in wild-type mice and were largely blunted in the nrf2-deficient mice. However, changes in levels of RNA transcripts following D3T treatment of nrf2-disrupted mice were multidirectional, dependent upon the particular gene examined. Although elevation of mRNAs for GST Ya, NQO1, microsomal epoxide hydrolase and gamma-glutamylcysteine synthetase regulatory chain were blocked in the mutant mice, elevation of GST Yp mRNA was largely unimpeded. Increases in levels of mRNA for the heavy and light chains of ferritin were only seen in the nrf2-disrupted mice. Transcript levels of UDP-glucuronyl-transferase 1A6, heme oxygenase-1, maganese superoxide dismutase, which were inducible in the wild-type mice, actually decreased in the mutant mice, whereas levels of mRNA for GST Yc, aflatoxin B1 aldehyde reductase and catalase decreased following D3T treatment in the mutant mice in the absence of any inductive effect by D3T in the wild-type mice. In wild-type mice, treatment with D3T lead to 3-fold increases in hepatic Nrf2 mRNA levels within several hours following dosing as assessed by Northern blot and RT-PCR analyses. Gel shift analyses with oligonucleotide probes for human NQO1 ARE, murine GST Ya ARE, and erythroid transcription factor (NF-E2) binding site showed increased intensity of binding with nuclear extracts prepared from livers of D3T-treated mice compared to vehicle-treated controls. Antibody to Nrf2 supershifted the DNA binding bands of these nuclear extracts. Moreover, immunoblot analysis indicated accumulation of Nrf2 in extracts prepared from hepatic nuclei of D3T-treated mice at the same time points. CONCLUSIONS: Nrf2 plays a central role in the regulation of constitutive and inducible expression of multiple phase 2 and antioxidative enzymes by chemoprotective dithiolethiones in vivo, although patterns of response vary among different genes. Knowledge of the factors controlling the specificity of actions of enzyme inducers will be exceedingly helpful in the design and isolation of more efficient and selective chemoprotective agents.
Acetaminophen (AA) is a widely used antipyretic drug that causes hepatotoxicity at high doses. Nuclear factor erythroid 2–related factor 2 (Nrf2) is a transcription factor that mitigates electrophilic stress from AA by inducing genes, such as NAD(P)H:quinone oxidoreductase 1 (Nqo1), multidrug resistance–associated proteins (Mrps), and glutathione (GSH) synthesis enzymes. To determine whether Nrf2 activation alters the biotransformation and excretion of AA, male wild-type, Nrf2-null, and Keap1 (Kelch-like ECH-associated protein 1)-knockdown (Keap1-kd) mice (which have increased activation of Nrf2) were administered a single subtoxic dose of AA (50 mg/kg, iv), after which, AA and its metabolites (AA-glucuronide [AA-GLUC]; AA-sulfate [AA-SULF]; AA-glutathione [AA-GSH]) were quantified in plasma, bile, and liver. AA-GLUC concentrations were reduced in plasma and elevated in livers of Nrf2-null mice due to decreased glucuronidation activity and lower expression of the basolateral efflux transporter Mrp3. In contrast, Keap1-kd mice had higher plasma and lower hepatic AA-GLUC concentrations, due to higher Mrp3 expression. Lower glucuronidation activity of Nrf2-null mice increased the proportion of AA available for sulfation, resulting in elevated AA-SULF concentrations in plasma, bile, and liver. Decreased AA-sulfation activity in Keap1-kd mice resulted in lower AA-SULF concentrations. AA-GSH conjugates were increased in Nrf2-null mice and tended to be lower in Keap1-kd mice. Furthermore, Nqo1, an enzyme capable of detoxifying the reactive intermediate of AA metabolism, N-acetyl-p-benzoquinone imine (NAPQI), had 85% lower activity in Nrf2-null mice and 415% higher activity in Keap1-kd mice relative to wild-type. In conclusion, lack of Nrf2 results in decreased AA glucuronidation, leading to increased AA available for NAPQI formation and decreased efflux of AA-GLUC via Mrp3; however, activation of Nrf2, as in Keap1-kd mice, results in decreased sulfotransferase activity, decreased AA-SULF formation, and enhanced elimination of AA-GLUC due to increased expression of Mrp3.
Nrf2; acetaminophen; transporters; pharmacokinetics
Atherosclerosis is a chronic inflammatory disease that evolves from the interaction of activated endothelial cells, macrophages, lymphocytes and modified lipoproteins (LDLs). In the last years many molecules with crucial metabolic functions have been shown to prevent important steps in the progression of atherogenesis, including peroxisome proliferator activated receptors (PPARs) and the class III histone deacetylase (HDAC) SIRT1. The PPARγ coactivator 1 alpha (Ppargc1a or PGC-1α) was identified as an important transcriptional cofactor of PPARγ and is activated by SIRT1. The aim of this study was to analyze total PGC-1α deficiency in an atherosclerotic mouse model.
To investigate if total PGC-1α deficiency affects atherosclerosis, we compared ApoE−/− PGC-1α−/− and ApoE−/− PGC-1α+/+ mice kept on a high cholesterol diet. Despite having more macrophages and a higher ICAM-1 expression in plaques, ApoE−/− PGC-1α−/− did not display more or larger atherosclerotic plaques than their ApoE−/− PGC-1α+/+ littermates. In line with the previously published phenotype of PGC-1α−/− mice, ApoE−/− PGC-1α−/− mice had marked reduced body, liver and epididymal white adipose tissue (WAT) weight. VLDL/LDL-cholesterol and triglyceride contents were also reduced. Aortic expression of PPARα and PPARγ, two crucial regulators for adipocyte differentitation and glucose and lipid metabolism, as well as the expression of some PPAR target genes was significantly reduced in ApoE−/− PGC-1α−/− mice. Importantly, the epididymal WAT and aortic expression of IL-18 and IL-18 plasma levels, a pro-atherosclerotic cytokine, was markedly reduced in ApoE−/− PGC-1α−/− mice.
ApoE−/− PGC-1α−/− mice, similar as PGC-1α−/− mice exhibit markedly reduced total body and visceral fat weight. Since inflammation of visceral fat is a crucial trigger of atherogenesis, decreased visceral fat in PGC-1α-deficient mice may explain why these mice do not develop enhanced atherosclerosis.
Radiation therapy is an integral part of treatment for cancer patients; however, major side effects of this modality include aberrant bone remodeling and bone loss. Ionizing radiation (IR) is a major external factor that contributes to a significant increase in oxidative stress such as reactive oxygen species (ROS), has been implicated in osteoporotic phenotypes, and has been implicated in osteoporotic phenotypes, bone loss, and fracture risk. One of the major cellular defenses against heightened oxidative stress is mediated by nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a master transcription factor that regulates induction of antioxidant gene expression and phase II antioxidant enzymes. Our objective was to test the hypothesis that loss of functional Nrf2 increases radiation-induced bone loss. We irradiated (single dose, 20 Gy) the hindlegs of age- and sex-matched Nrf2+/+ and Nrf2−/− mice. After 1 month, microCT analysis and histology revealed a drastic overall decrease in the bone volume after irradiation of mice lacking Nrf2. Although radiation exposure led to bone loss in mice with intact Nrf2, it was dramatically enhanced by loss of Nrf2. Furthermore, in the absence of Nrf2, a decrease in osteoblast mineralization was noted in calvarial osteoblasts compared with wild-type controls, and treatment with a common antioxidant, N-acetyl-l-cysteine (NAC), was able to rescue the mineralization. As expected, we observed a higher number of osteoclasts in Nrf2−/− mice compared to Nrf2+/+ mice, and after irradiation, the trend remained the same. RT-PCR analysis of calvarial osteoblasts revealed that in the absence of Nrf2, the expression of RANKL was increased after irradiation. Interestingly, RANKL expression was suppressed when the calvarial osteoblasts were treated with NAC before IR exposure. Taken together, our data suggest that loss of Nrf2 leads to heightened oxidative stress and increased susceptibility to radiation-induced bone loss.
Ionizing radiation; Bone loss; Oxidative stress; N-acetylcysteine; Osteoblast differentiation; RANKL; Nrf2; Reactive oxygen species; Free radicals
Transcription factor Nrf2 is considered a master regulator of antioxidant defense in mammals. However, it is unclear whether this concept is applicable to nonmammalian vertebrates, because no animal model other than Nrf2 knockout mice has been generated to examine the effects of Nrf2 deficiency. Here, we characterized a recessive loss-of-function mutant of Nrf2 (nrf2fh318) in a lower vertebrate, the zebrafish (Danio rerio). In keeping with the findings in the mouse model, nrf2fh318 mutants exhibited reduced induction of the Nrf2 target genes in response to oxidative stress and electrophiles but were viable and fertile, and their embryos developed normally. The nrf2fh318 larvae displayed enhanced sensitivity to oxidative stress and electrophiles, especially peroxides, and pretreatment with an Nrf2-activating compound, sulforaphane, decreased peroxide-induced lethality in the wild type but not nrf2fh318 mutants, indicating that resistance to oxidative stress is highly dependent on Nrf2 functions. These results reveal an evolutionarily conserved role of vertebrate Nrf2 in protection against oxidative stress. Interestingly, there were no significant differences between wild-type and nrf2fh318 larvae with regard to their sensitivity to superoxide and singlet oxygen generators, suggesting that the importance of Nrf2 in oxidative stress protection varies based on the type of reactive oxygen species (ROS).
Nrf2, a master regulator of intracellular redox homeostasis, is indicated to participate in fatty acid metabolism in liver. However, its role in diet-induced obesity remains controversial. In the current study, genetically engineered Nrf2-null, wild-type (WT), and Nrf2-activated, Keap1-knockdown (K1-KD) mice were fed either a control or a high-fat western diet (HFD) for 12 weeks. The results indicate that the absence or enhancement of Nrf2 activity did not prevent diet-induced obesity, had limited effects on lipid metabolism, but affected blood glucose homeostasis. Whereas the Nrf2-null mice were resistant to HFD-induced glucose intolerance, the Nrf2-activated K1-KD mice exhibited prolonged elevation of circulating glucose during a glucose tolerance test even on the control diet. Feeding a HFD did not activate the Nrf2 signaling pathway in mouse livers. Fibroblast growth factor 21 (Fgf21) is a liver-derived anti-diabetic hormone that exerts glucose- and lipid-lowering effects. Fgf21 mRNA and protein were both elevated in livers of Nrf2-null mice, and Fgf21 protein was lower in K1-KD mice than WT mice. The inverse correlation between Nrf2 activity and hepatic expression of Fgf21 might explain the improved glucose tolerance in Nrf2-null mice. Furthermore, a more oxidative cellular environment in Nrf2-null mice could affect insulin signaling in liver. For example, mRNA of insulin-like growth factor binding protein 1, a gene repressed by insulin in hepatocytes, was markedly elevated in livers of Nrf2-null mice. In conclusion, genetic alteration of Nrf2 does not prevent diet-induced obesity in mice, but deficiency of Nrf2 improves glucose homeostasis, possibly through its effects on Fgf21 and/or insulin signaling.
Nrf2; high-fat diet; obesity; Fgf21; glucose intolerance; redox signaling
Nuclear factor erythroid 2–related factor 2 (Nrf2) is a transcription factor critical for protection against electrophilic and oxidative stress. In a recently engineered mouse with knockdown of kelch-like ECH associated protein 1 (Keap1-kd mice), the cytosolic repressor of Nrf2, there is a 55% decrease in Keap1 mRNA and a 200% increase in Nrf2 protein in liver. Experiments with Nrf2-null mice have demonstrated the effects of a lack of Nrf2. However, little is known about the biological effects of more Nrf2 activation. Accordingly, the hepatic phenotype of Keap1-kd mice, as well as the hepatic mRNA expression of cytoprotective genes were compared among wild-type, Nrf2-null, and Keap1-kd mice. Three distinct patterns of hepatic gene expression were identified among wild-type, Nrf2-null, and Keap1-kd mice. The first pattern encompassed genes that were lower in Nrf2-null mice and considerably higher in Keap1-kd mice than wild-type mice, which included genes mainly responsible for the detoxification and elimination of electrophiles, such as NAD(P)H:quinone oxidoreductase 1 and glutathione-S-transferases (Gst), and multidrug resistance–associated proteins. The second pattern encompassed genes that were lower in Nrf2-null mice but not increased in Keap1-kd mice, and included genes, such as epoxide hydrolase-1, UDP-glucuronosyltransferases, aldehyde dehydrogenases, as well as genes important in the detoxification of reactive oxygen species, such as superoxide dismutase 1 and 2, catalase, and peroxiredoxin 1. The third pattern encompassed genes that were not different among wild-type, Nrf2-null, and Keap1-kd mice and included genes such as glutathione peroxidase, microsomal Gsts, and uptake transporters. In conclusion, the present study suggests that increased activation of hepatic Nrf2 is more important for the detoxification and elimination of electrophiles than reactive oxygen species.
Nrf2; electrophilic stress; oxidative stress; cytoprotection
The transcription factor Nrf2 is a master regulator of cellular defence: Nrf2 null mice (Nrf2(−/−)) are highly susceptible to chemically induced toxicities. We report a comparative iTRAQ-based study in Nrf2(−/−) mice treated with a potent inducer, methyl-2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate (CDDO-me; bardoxolone -methyl), to define both the Nrf2-dependent basal and inducible hepatoproteomes. One thousand five hundred twenty-one proteins were fully quantified (FDR < 1%). One hundred sixty-one were significantly different (P < 0.05) between WT and Nrf2(−/−) mice, confirming extensive constitutive regulation by Nrf2. Treatment with CDDO-me (3 mg/kg; i.p.) resulted in significantly altered expression of 43 proteins at 24 h in WT animals. Six proteins were regulated at both basal and inducible levels exhibiting the largest dynamic range of Nrf2 regulation: cytochrome P4502A5 (CYP2A5; 17.2-fold), glutathione-S-transferase-Mu 3 (GSTM3; 6.4-fold), glutathione-S-transferase Mu 1 (GSTM1; 5.9-fold), ectonucleoside-triphosphate diphosphohydrolase (ENTPD5; 4.6-fold), UDP-glucose-6-dehydrogenase (UDPGDH; 4.1-fold) and epoxide hydrolase (EPHX1; 3.0-fold). These proteins, or their products, thus provide a potential source of biomarkers for Nrf2 activity. ENTPD5 is of interest due to its emerging role in AKT signalling and, to our knowledge, this protein has not been previously shown to be Nrf2-dependent. Only two proteins altered by CDDO-me in WT animals were similarly affected in Nrf2(−/−) mice, demonstrating the high degree of selectivity of CDDO-me for the Nrf2:Keap1 signalling pathway.
The Nrf2:Keap1 signalling pathway is attracting considerable interest as a therapeutic target for different disease conditions. For example, CDDO-me (bardoxolone methyl) was investigated in clinical trials for the treatment of acute kidney disease, and dimethyl fumarate, recently approved for reducing relapse rate in multiple sclerosis, is a potent Nrf2 inducer. Such compounds have been suggested to act through multiple mechanisms; therefore, it is important to define the selectivity of Nrf2 inducers to assess the potential for off-target effects that may lead to adverse drug reactions, and to provide biomarkers with which to assess therapeutic efficacy. Whilst there is considerable information on the global action of such inducers at the mRNA level, this is the first study to catalogue the hepatic protein expression profile following acute exposure to CDDO-me in mice. At a dose shown to evoke maximal Nrf2 induction in the liver, CDDO-me appeared highly selective for known Nrf2-regulated proteins. Using the transgenic Nrf2(−/−) mouse model, it could be shown that 97% of proteins induced in wild type mice were associated with a functioning Nrf2 signalling pathway. This analysis allowed us to identify a panel of proteins that were regulated both basally and following Nrf2 induction. Identification of these proteins, which display a large magnitude of variation in their expression, provides a rich source of potential biomarkers for Nrf2 activity for use in experimental animals, and which may be translatable to man to define individual susceptibility to chemical stress, including that associated with drugs, and also to monitor the pharmacological response to Nrf2 inducers.
•Liver proteomes from WT, Nrf2-null and Nrf2-induced mice were compared by iTRAQ•Of 1521 proteins quantified, 161 were regulated basally and 43 following induction•Six proteins were both basally and inducibly regulated, with high dynamic ranges•In order of fold change, these proteins were CYP2A5, GSTM3, GSTM1, ENTPD5, G6PD, EPHX1•These proteins may yield translatable biomarkers for clinical development
Nrf2; iTRAQ; ENTPD5; CYP2A5; Hepatoproteome; CDDO
Rationale: Nuclear factor erythroid 2–related factor 2 (Nrf2), an important regulator of lung antioxidant defenses, declines in chronic obstructive pulmonary disease (COPD). However, Nrf2 also regulates the proteasome system that degrades damaged and misfolded proteins. Because accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and ER stress-induced apoptosis, Nrf2 may potentially prevent ER stress-mediated apoptosis in COPD.
Objectives: To determine whether Nrf2-regulated proteasome function affects ER stress-mediated apoptosis in COPD.
Methods: We assessed the expression of Nrf2, Nrf2-dependent proteasomal subunits, proteasomal activity, markers of ER stress, and apoptosis in emphysematous lungs of mice exposed to cigarette smoke (CS) as well as peripheral lung tissues from normal control subjects and patients with COPD.
Measurements and Main Results: Compared with wild-type mice, emphysematous lungs of CS-exposed Nrf2-deficient mice exhibited markedly lower proteasomal activity and elevated markers of ER stress and apoptosis. Furthermore, compared with normal control subjects, lungs of patients with mild and advanced COPD showed a marked decrease in the expression of Nrf2-regulated proteasomal subunits and total proteasomal activity. However, they were associated with greater levels of ER stress and apoptosis markers. In vitro studies have demonstrated that enhancing proteasomal activity in Beas2B cells either by sulforaphane, an activator of Nrf2, or overexpression of Nrf2-regulated proteasomal subunit PSMB6, significantly inhibited cigarette smoke condensate (CSC)-induced ER stress and cell death.
Conclusions: Impaired Nrf2 signaling causes significant decline in proteasomal activity and heightens ER stress response in lungs of patients with COPD and CS-exposed mice. Accordingly, pharmacological approaches that augment Nrf2 activity may protect against COPD progression by both up-regulating antioxidant defenses and relieving ER stress.
Nrf2; proteasome system; endoplasmic reticulum stress; unfolded protein response; chronic obstructive pulmonary disease lungs
Interruption of the enterohepatic circulation of bile acids induces hepatic bile acid synthesis, increases hepatic cholesterol demand, and increases clearance of apoB-containing lipoproteins in plasma. Based on these effects, bile acid sequestrants have been used for many years to treat hypercholesterolemia and the associated atherosclerosis. The objective of this study was to determine the effect of blocking ileal apical versus basolateral membrane bile acid transport on the development of hypercholesterolemia and atherosclerosis in mouse models.
Methods and Results
ApoE−/− and Ldlr−/− mice deficient in the apical sodium-dependent bile acid transporter (Asbt) or apoE−/− mice deficient in the basolateral bile acid transporter (Ostα) were fed an atherogenic diet for 16 weeks. Bile acid metabolism, cholesterol metabolism, gene expression, and development of atherosclerosis were examined. Mice deficient in Asbt exhibited the classic response to interruption of the enterohepatic circulation of bile acids, including significant reductions in hepatic and plasma cholesterol levels, and reduced aortic cholesteryl ester content. Ileal Fibroblast Growth Factor-15 (FGF15) expression was significantly reduced in Asbt−/−apoE−/− mice and was inversely correlated with expression of hepatic cholesterol 7α-hydroxylase (Cyp7a1). Ileal FGF15 expression was directly correlated with plasma cholesterol levels and aortic cholesterol content. In contrast, plasma and hepatic cholesterol levels and atherosclerosis development were not reduced in apoE−/− mice deficient in Ostα.
Decreases in ileal FGF15, with subsequent increases in hepatic Cyp7a1 expression and bile acid synthesis appear to be necessary for the plasma cholesterol-lowering and atheroprotective effects associated with blocking intestinal bile acid absorption.
Bile acids; cholesterol; apoE knockout mouse; atherosclerosis; Fibroblast growth factor 15; ileum; transporters
Chronic alcohol ingestion induces the expression of transforming growth factor beta-1(TGFβ1), inhibits nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated activation of the antioxidant response element (ARE), depletes alveolar glutathione pools, and potentiates acute lung injury. In this study, we examined the mechanistic relationship between TGFβ1 and Nrf2-ARE signaling in the experimental alcoholic lung.
Wild-type mice were treated ± alcohol in drinking water for 8 weeks and their lungs were assessed for Nrf2 expression. In parallel, mouse lung fibroblasts were cultured ± alcohol and treated ± sulforaphane (SFP; an activator of Nrf2), ±TGFβ1, ±TGFβ1 neutralizing antibody, and/or ±activin receptor-like kinase 5 inhibitors (to block TGβ1 receptor signaling) and then analyzed for the expression of Nrf2, Kelch-like ECH-associated protein 1 (Keap1) and TGFβ1, Nrf2-ARE activity, and the expression of the Nrf2-ARE-dependent antioxidants glutathione s-transferase theta 2 (GSTT2) and glutamate-cysteine ligase catalytic subunit (GCLC). Finally, silencing RNA (siRNA) of Nrf2 was then performed prior to alcohol exposure and subsequent analysis of TGFβ1 expression.
Alcohol treatment in vivo or in vitro decreased Nrf2 expression in murine whole lung and lung fibroblasts, respectively. In parallel, alcohol exposure in vitro decreased Keap1 gene and protein expression in lung fibroblasts. Furthermore, alcohol exposure increased TGFβ1 expression but decreased Nrf2-ARE activity and expression of the ARE-dependent genes for GSTT2 and GCLC. These effects of alcohol were prevented by treatment with SFP; in contrast, Nrf2 SiRNA expression exacerbated alcohol-induced TGFβ1 expression. Finally, TGFβ1 treatment directly suppressed Nrf2-ARE activity whereas blocking TGFβ1 signaling attenuated alcohol-induced suppression of Nrf2-ARE activity.
Alcohol-induced oxidative stress is mediated by TGFβ1, which suppresses Nrf2-ARE-dependent expression of antioxidant defenses and creates a vicious cycle that feeds back to further increase TGFβ1 expression. These effects of alcohol can be mitigated by activation of Nrf2, suggesting a potential therapy in individuals at risk for lung injury due to alcohol abuse.
Nrf2; Antioxidant Response Element; TGFβ1; Glutathione; Lung Fibroblasts; Alcohol
Oxidative stress has been proposed as an important promoter of the progression of fatty liver diseases. The current study investigates the potential functions of the Nrf2–Keap1 signaling pathway, an important hepatic oxidative stress sensor, in a rodent fatty liver model. Mice with no (Nrf2-null), normal (wild type, WT), and enhanced (Keap1 knockdown, K1-kd) expression of Nrf2 were fed a methionine- and choline-deficient (MCD) diet or a control diet for 5 days. Compared to WT mice, the MCD diet-caused hepatosteatosis was more severe in the Nrf2-null mice and less in the K1-kd mice. The Nrf2-null mice had lower hepatic glutathione and exhibited more lipid peroxidation, whereas the K1-kd mice had the highest amount of glutathione in the liver and developed the least lipid peroxidation among the three genotypes fed the MCD diet. The Nrf2 signaling pathway was activated by the MCD diet, and the Nrf2-targeted cytoprotective genes Nqo1 and Gstα1/2 were induced in WT and even more in K1-kd mice. In addition, Nrf2-null mice on both control and MCD diets exhibited altered expression profiles of fatty acid metabolism genes, indicating Nrf2 may influence lipid metabolism in liver. For example, mRNA levels of long chain fatty acid translocase CD36 and the endocrine hormone Fgf21 were higher in livers of Nrf2-null mice and lower in the K1-kd mice than WT mice fed the MCD diet. Taken together, these observations indicate that Nrf2 could decelerate the onset of fatty livers caused by the MCD diet by increasing hepatic antioxidant and detoxification capabilities.
Nrf2; Fatty liver disease; MCD diet; Mouse