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RED CELLS
From the Department of Laboratory Medicine, University
of California, San Francisco; and Children's Hospital Oakland Research
Institute, Oakland, CA.
p45NF-E2 is a member of the cap `n' collar (CNC)-basic leucine
zipper family of transcriptional activators that is expressed at high
levels in various types of blood cells. Mice deficient in p45NF-E2 that
were generated by gene targeting have high mortality from bleeding
resulting from severe thrombocytopenia. Surviving p45nf-e2 Eukaryotic cells have a battery of mechanisms
to defend against the harmful effects of reactive oxygen
molecules,1,2 including antioxidant molecules that
function by directly inactivating reactive oxygen molecules and enzymes
that metabolically convert toxic compounds to forms that are readily
excreted by cells. In many cases, transcriptional activation of genes
that play a role in detoxification of xenobiotics and defense against
oxidative stress is mediated partly by the antioxidant response element (ARE). For example, AREs were found in promoter sequences of genes including nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase, heme oxygenase, glutathione-S- transferases
(GST), and glutamylcysteine synthetase.3-7 Several
different transcription factors, including basic leucine zipper (bZIP)
proteins, activator protein 1 (AP-1), and other novel factors, were
shown to bind the ARE.4,8
The ARE consensus sequence is very similar to the NF-E2/AP1-like
sequence of the Expression of p45NF-E2 is restricted to cells of the hematopoietic
lineage Nrf1 and Nrf2 have been implicated in the regulation of antioxidant
gene expression. The knockout of nrf1 results in embryonic death,22,23 and fibroblasts derived from nrf1
null embryos have enhanced sensitivity to the toxic effects of various
oxidant compounds.24 Although nrf2
knockout mice are viable and outwardly normal, mutant animals have
diminished expression of phase 2 enzyme genes and are sensitive to
redox cycling agents.25,26
Does p45NF-E2 have a role in the oxidative-stress response? Because CNC
factors may represent an important class of regulators of antioxidant
gene expression by means of the ARE, one possibility is that p45NF-E2
is involved in regulating oxidative-stress response genes in the RBCs,
while ubiquitous-expressing Nrf1 and Nrf2 assume this role in all other
tissues. It is possible that a compensated hemolytic state contributes
to the erythroid abnormalities observed in p45NF-E2 knockout mice.
Because of their function as oxygen carriers, RBCs are constantly
exposed to oxidative stress. Yet, despite the limited biosynthetic
repertoire available to mature RBCs, they are resilient to
oxidant-induced damage. Clearly, antioxidants in the form of scavengers
and detoxifying enzymes provide an important protective system in
RBCs,27 but our knowledge of the molecular mechanisms that
regulate expression of antioxidants in RBCs is limited.
In this study, we found that RBCs from p45NF-E2-deficient mice are
sensitive to oxidative stress. RBCs from p45NF-E2-deficient mice
accumulated high levels of free radicals when exposed to oxidants, and
this correlated with increased formation of methemoglobin and loss of
membrane deformability. In addition, severe anemia developed in
p45NF-E2-deficient mice treated with oxidative-stress-inducing drugs,
and mutant RBCs had decreased survival. One likely cause of increased
sensitivity to oxidative stress is reduced catalase activity in mutant
RBCs. Our finding that messenger RNA (mRNA) and protein levels for
catalase are reduced in mutant RBCs suggests that the mouse catalase
gene is regulated by p45NF-E2. These findings indicate that anemia in
p45NF-E2 knockout mice has another cause aside from bleeding. Our
findings should provide important clues to the role of p45NF-E2 in RBC
function and have possible relevance in hemolytic diseases.
Measurement of intracellular reactive oxygen intermediates
Methemoglobin formation assay
Osmotic gradient ektacytometry Ektacytometry measurements were done as described previously by using a Technicon ektacytometer (Technicon Instruments, Tarrytown, NY).30 Oxidant damage was induced by incubating fresh RBCs (~2.5%-5% hematocrit) in normal saline containing 4 mM sodium azide and various concentrations of H2O2. After a 10-minute incubation at room temperature, cells were pelleted by centrifugation at 5000g and resuspended in a buffered polyvinyl pyrrolidone (PVP) solution (36 g/L PVP, 20 mM sodium phosphate [pH 7.4], and sodium chloride [NaCl] to a final osmolality of 300 mOsm/kg) with a final viscosity of 20 cP. The osmotic deformability of untreated RBCs and RBCs treated with H2O2 was analyzed by continuous measurements of a deformability index as a function of increasing osmolality (80-500 mOsm/kg) at a constant shear stress of 160 dyne/cm2.Analysis of mice and RBCs Mice used in the studies had a mixed 129/Sv and C57BL/6 background. Dapsone (4,4'-diaminodiphenyl sulfone), H2O2, and phenylhydrazine hydrochloride were purchased from Sigma (St Louis, MO). Phenylhydrazine was prepared in 1 × PBS and adjusted to a pH of 7.4 with sodium hydroxide. Working solutions of dapsone were made in 1 × PBS by diluting a concentrated solution of dapsone prepared in dimethyl sulfoxide. Anemia was induced in adult mice (8-10 weeks old) by an intraperitoneal injection of dapsone (50 µmol/kg) or phenylhydrazine (50 mg/kg) solution. Blood was collected 24 to 48 hours after the injection from either the retro-orbital venous plexus or the tail vein into heparin-treated capillary tubes (Kimble, Vineland, NJ). For retro-orbital collections, mice were anesthetized with methoxyflurane before the procedure. RBC counts, hemoglobin concentrations, hematocrit levels, and other RBC variables were assessed and analyzed by using a Hemavet 850 machine (CDC Technologies, Oxford, CT) and software designed for murine blood studies. Blood smears were prepared and stained with Wright-Giemsa stain by using an automated stainer (Hematek, Branchburg, NJ). Reticulocytes were enumerated on thin films after staining of cells with new methylene blue dye. The percentage of reticulocytes was determined by counting approximately 1000 RBCs from random fields in blood films under 1000× magnification.RBC survival studies The turnover of RBCs was measured in 2 ways. RBCs were labeled with biotin by injection of approximately 100 µL of a 60-mg/mL solution of EZ-Link-N-hydroxy succinimide (NHS)-biotin (Pierce, Rockford, IL) in PBS (pH 7.4). More than 95% of the murine RBCs were biotinylated after labeling. Alternatively, heparin-treated whole blood was washed in HBSS (pH 7.4, 330 mOsm). A solution of 4.43 mg/mL EZ-Link-NHS-biotin (Pierce) in PBS (pH 7.4) was prepared. To 800 µL washed RBCs (hematocrit ~ 0.5), 10 µL of the biotinylation mixture was added to achieve a final concentration of 100 µM. After a 1-hour incubation at room temperature, the cells were washed in buffer and resuspended to achieve a 0.5 hematocrit in 330 mOsm NaCl, and 100 µL was injected into the tail vein. This resulted in the presence of approximately 7% to 10% biotinylated cells in the cell population.The first blood sample for determining the quantity of biotinylated cells was obtained 30 minutes after either injection of biotin or transfusion of biotinylated cells. Thereafter, blood samples were obtained from the tail vein at various intervals for 4 weeks for quantitation of biotin-labeled cells remaining in the circulation. Samples were washed with HBSS (pH 7.4, 330 mOsm) and incubated with fluorescein-labeled avidin solution (0.2 mg/mL in the same buffer) for 30 minutes at room temperature in the dark. Unbound avidin was removed by washing with buffer, and this was followed by flow cytometric detection of fluorescein-labeled cells. The percentage of biotinylated cells was calculated as a ratio of positive (fluorescein-labeled) cells to all RBCs. Ter-119 sorting and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis Ter-119-positive (Ter-119+) cells from the spleen and bone marrow of mice were isolated by using magnetically activated cell-sorter (MACS) Ter-119 microbeads according to the manufacturer's instructions (Miltenyi Biotec, Germany). Briefly, bone marrow and spleens from 3 or 4 mice were pooled and processed to obtain a single-cell suspension by using standard procedures. Contaminating RBCs were then lysed in a solution of 0.8% ammonium chloride and 10 mM EDTA, and cell aggregates and debris were removed by passage over a 70-µM filter (Falcon; Becton Dickinson, Franklin Lakes, NY). Lymphocytes and granulocytes were labeled by using a combination of biotinylated anti-CD2, 19, and Gr-1 antibodies (Pharmigen, San Diego, CA). Depletion was achieved by incubating labeled cells with streptavidin microbeads and passage over a magnetic column (MidiMACS; Miltenyi Biotec). Erythrocytes in the flow-through fraction were then magnetically isolated from the depleted fraction by using Ter-119 microbeads. An aliquot of the resulting cells was stained with Wright-Giemsa stain to determine the purity of isolated cells. RNA from Ter-119-sorted cells was isolated by using Ultraspec RNA extraction solution (Biotecx, Houston, TX). For RT-PCR, first-strand complementary DNA was synthesized by using random hexamer primers according to the manufacturer's protocol (Pharmacia, Piscataway, NJ). PCRs were carried out in 10 mM Tris-hydrochloric acid (pH 8.3), 50 mM potassium chloride, 1.5 mM magnesium chloride, 0.2 mM deoxynucleoside triphosphate, 0.0037 MBq (0.1 µCi) -phosphorous
32-deoxycytidine triphosphate (1.11 MBq8/mM [3000
Ci/mM]; Amersham, Arlington Heights, IL), 10 pmol of each of the
primers, and 2.5 U Amplitaq polymerase (Perkin Elmer, Foster City, CA).
Standard procedures were used for gels, and band intensities were
quantitated by means of phosphoimaging. PCR primer sequences were as
follows: catalase, 5'-GGCACACTTTGACAGAGAGCGGAT and
3'-AGTTTTTGATGCCCTGGTCGGTCT; GST-Yc, 5'-GAGATCGACGGGATGAAACTGGTG and
3'-GCGCTTTCAGGAGAGGGAAGTTGT; erythropoietin receptor,
5'-AGATGATGAGGGGCCCTTACTGGA and 3'-AAGGCTGTTCTCATAGGGGTGGGA; and
glyceraldehyde-3-phosphate dehydrogenase, 5'-ACCACAGTCCATGCCATCAC and
3'-TCCACCACCCTGTTGCTGTA.
Cellular catalase assays and immunoblot analysis Catalase activity in RBCs was measured as described by Beutler.53 For immunoblot experiments, RBCs were lysed in T-Per Extraction Buffer (Pierce). Lysates (50 µg) were electrophoresed in 10% denaturing polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was blocked with 1×triethanolamine-buffered saline containing 5% (wt/vol) nonfat dry milk and 0.2% Tween 20. Catalase was detected with a rabbit polyclonal antibody against human catalase (Oxis, Portland, OR) and peroxidase-labeled goat antirabbit antibody.Statistical analyses Statistical analyses were done with Statview software (SAS, Cupertino, CA). Data from the same number of +/+ and / mice were
used to determine the mean (± SEM). The significance of difference between mean values was analyzed by using the Student t
test. Differences were considered significant if the P value
was less than .05.
RBCs deficient in p45NF-E2 accumulate elevated levels of reactive oxygen intermediates To determine whether p45nf-e2 / RBCs are
susceptible to oxidative stress, we measured oxidation of the DCFHDA
fluorescent dye as a marker for reactive oxygen species (ROS) inside
RBCs. We found that p45nf-e2/ RBCs had a
3-fold elevation in basal levels of fluorescent DCFHDA formation
compared with normal control cells (Figure
1A). This finding indicates that
p45nf-e2/ RBCs have an increased level of
ROS compared with wild-type cells under steady-state conditions.
Challenging the cells with H2O2 treatment led
to a further increase in fluorescent DCFHDA levels in both wild-type
and mutant cells (Figure 1B and 1C). However, the increase in
fluorescent level was consistently greater in p45NF-E2-deficient RBCs.
Mutant RBCs treated with 0.05 mM H2O2 had an
approximately 5-fold increase in fluorescence, whereas wild-type cells
had a 3-fold increase (Figure 1B). Increasing the concentration of
H2O2 to 0.5 mM further increased fluorescence in wild-type cells, to values about 7-fold above baseline. In contrast,
mutant RBCs had a significantly greater increase (20-fold; Figure 1C).
Similar results were obtained when cells were incubated with
phenylhydrazine (data not shown).
To further assess the consequences of oxidative treatment on mutant
RBCs, we measured oxidation of hemoglobin in the form of methemoglobin.
In H2O2-treated cells, increased levels of
methemoglobin were observed in p45NF-E2-deficient RBCs compared with
normal cells. Methemoglobin was readily induced in
p45nf-e2
Increased osmotic fragility in p45NF-E2-deficient RBCs Because the deformability of erythrocytes was previously shown to be affected by oxidative stress,30,31 we assessed the effects of H2O2 on p45NF-E2 mutant cells by using ektacytometry. As shown in Figure 3, the osmotic deformability profile of p45NF-E2 mutant RBCs was considerably different than that of normal RBCs. A decrease in the maximal deformability index was readily apparent even in untreated p45NF-E2 mutant cells compared with normal controls (Figure 3A). Challenge with as little as 0.05 mM H2O2 led to a further decrease in the deformability index in mutant RBCs. Virtually no change was detected in normal cells that were treated similarly (Figure 3B). Comparison studies (Figure 3B and 3C) showed that the alteration in the deformability profile depended on the H2O2 concentration. At a concentration of 0.5 mM H2O2, the deformability of p45nfe2/ RBCs in comparison to normal RBCs
was completely abolished (Figure 3C). Whether the abnormal membrane
deformability at baseline was caused only by increased ROS in the
p45NF-E2-deficient erythrocytes is not known, but it is clear that
loss of deformability was further exacerbated by oxidative stress.
Similar changes in the osmotic deformability profile and a decrease in
the deformability index due to oxidation-induced changes in RBC
membranes were previously observed in hemoglobinopathies such as
thalassemia.32-34 The oxidant-induced decreased
deformability in the hypertonic arm of the profile indicates changes in
the mechanical properties of the membrane35 and suggests that mutant cells may be more rigid than normal cells. This may lead to
their premature removal as they move through the reticuloendothelial system.36,37
Phenylhydrazine induces severe anemia in p45NF-E2-deficient mice We examined whether p45NF-E2-deficient mice also have an increased susceptibility to oxidative agents in vivo. Thus, both normal control and p45nf-e2/ mice were given
intraperitoneal injections of phenylhydrazine. Forty-eight hours later,
there was an approximately 5-point drop in hematocrit level in normal
control mice (Figure 4). In
p45nf-e2/ mice, however, the decrease in
hematocrit was significantly greater (~15 points;
P < .01), and it was accompanied by a dramatic increase in reticulocytes (30%) above the normally elevated levels in mutant mice (Figure 4). In contrast, no significant increase in reticulocytes was observed in normal control mice. Additionally, oxidative
treatment of p45nf-e2/ mice significantly
altered the morphologic features of their RBCs compared with those in
normal control mice. Echinocytes were readily apparent after
phenylhydrazine or dapsone treatment in p45nf-e2/ RBCs (data not shown). Others have
also observed formation of echinocytes when erythrocytes are treated
with various oxidants, including H2O2 and
dapsone.31,38 The morphologic alterations have been
attributed to peroxidation of membrane proteins as a result of
oxidative stress. These findings suggest that
p45nf-e2/ mice have an increased
susceptibility to oxidative-stress-induced hemolysis as a result of
drug treatment.
RBCs from p45NF-E2-deficient mice have decreased survival Because the findings described above indicated that mutant RBCs are prone to oxidative damage, we conducted RBC survival studies to test the idea that p45nf-e2/ RBCs have an
accelerated turnover rate. Labeling of RBCs by tail-vein injection of
biotin provided RBC survival data in normal mice (Figure
5A). The labeling resulted in
biotinylation of more than 95% of the RBC population. The data were
normalized to express the percentage of biotinylated cells relative to
time zero. Our data indicated a linear replacement of murine RBCs in
the control mice (k = 0; R = 0.99), with an potential lifespan (T)
of 33 days, or a turnover of approximately 3% per day with a half-life
of 16.5 days (Figure 5A). This corresponds well with a
reticulocyte count in these mice of 2.5%. In contrast, random RBC
removal was observed in the p45nf-e2/ mice.
The extinction time (T) was still about 30 days, but extra destruction
was also observed (k = 0.11; R = 0.98), and it resulted in a
half-life of 5 days. These data showed that another RBC
destruction factor, independent of RBC age, was present in
p45nf-e2/ mice.
To evaluate whether the premature removal of RBCs was the result of RBC
characteristics only or depended on other factors in the mice, we
labeled RBCs ex vivo and monitored their survival in normal mice
(Figure 5B). Infusion of biotinylated RBCs in the tail vein resulted in
7% to 10% biotinylated RBCs in the population. The data were
normalized to express the percentage of biotinylated cells relative to
time zero. The survival of ex vivo-labeled normal RBCs in control mice
(Figure 5B) was virtually identical to the results shown in Figure 5A
for in vivo biotinylated cells. Our data again indicated a linear
replacement (k = 0; R = 0.99), with an potential lifespan (T) of
34.5 days, or a turnover of approximately 2.9% per day resulting in a
half-life of 17 days. In contrast, p45nf-e2 Expression of catalase gene is reduced in p45NF-E2-deficient RBCs To begin to define the molecular basis of the increased sensitivity to oxidants in mutant RBCs, we measured mRNA levels of various genes involved in protecting RBCs from oxidative stress. Total RNA from sorted Ter-119+ cells of wild-type and p45nf-e2/ mice was prepared and analyzed by
RT-PCR. In mutant Ter-119+ cells, the transcripts encoding
the GST-Yc and NQO1 genes were reduced in comparison to wild-type cells
(Figure 6A and data not shown). RT-PCR
also revealed reduced catalase levels in mutant Ter-119+
cells. The reduction in the catalase mRNA was accompanied by a
significant decrease in catalase protein and activity in RBCs from
p45nf-e2/ mice (Figure 6). In contrast, we
did not detect significant differences in transcripts encoding the
glutathione synthesis genes (data not shown). The reduction in catalase
is a possible mechanism for the diminished capacity of the RBCs to deal
with oxidants.
Mature RBCs are particularly prone to oxidative damage because they are constantly exposed to high levels of oxygen. In addition, iron released from hemoglobin can be deleterious because it acts as a catalyst for generation of hydroxyl radicals by means of Fenton-type reactions.39,40 Moreover, mature RBCs have a limited capacity to replace damaged proteins by de novo synthesis, and they must circulate for an extended period in the body. Under normal circumstances, however, RBCs are protected against oxidative damage with its complement of antioxidant enzymes and molecules. The importance of the protective mechanisms of RBCs is evident from a consideration of human hemolytic disorders due to a variety of enzyme deficiencies involving pathways that maintain intracellular reductive molecules.27,41-44 Deficiencies compromising the capacity to detoxify oxidant molecules such as H2O2 and oxygen radicals result in oxidant-induced denaturation of intracellular molecules and premature destruction of RBCs. Thus, patients with deficiencies in glutathione synthesis also have hemolytic anemia, as well as other disorders.45,46 Presumably, the failure to maintain glutathione levels compromises antioxidant defenses and ultimately results in oxidative destruction of RBCs. Other enzymopathies that might compromise intracellular reductive capacity have also been described; they include abnormalities involving glutathione peroxidase and glutathione reductase activity.41,44,47,48 However, the cause and effect relations in these abnormalities remain unknown. Although it is clear that antioxidants play an important role in
protecting cells, knowledge of the molecular mechanisms that regulate
their expression in RBCs is limited. Expression of a variety of genes
encoding antioxidant enzymes is mediated partly by a
cis-active DNA element designated the ARE.4
Although several proteins bind the ARE, factors mediating expression of
genes controlled by the ARE have not been identified. Among possible
factors, Nrf1 and Nrf2 were implicated as playing a part in
ARE-mediated regulation of antioxidant gene expression. In transfection
studies, Nrf1 and Nrf2 activated expression of a human NQO1 promoter
plasmid that contained an ARE.49 In our study
investigating the role of Nrf1 in the oxidative-stress response, we
demonstrated that nrf1 This study found an additional phenotype in p45NF-E2-deficient mice.
We identified a role for p45NF-E2 in protecting RBCs from oxidative
stress. Our in vitro analysis made it clear that RBCs from
p45nf-e2 It is possible that the inability to maintain reducing power observed
in this study was caused by abnormalities in p45NF-E2-deficient RBCs
that have not yet been characterized. Alternatively, the reduced
capacity to deal with oxidative stress might involve diminished activity in various antioxidant enzymes or reflect a diminished reserve
in the reductive capacity in mutant RBCs. Because of findings from
analyses of other CNC knockouts, we believe that the increase in
sensitivity results from decreased expression of oxidative-stress response genes. Considering that Nrf1 was found to play a role in
regulating glutathione synthesis, it seems likely that p45-NFE2 assumes
this role in RBCs. However, we were unable to consistently detect
differences in the expression of glutathione synthase and glutamylcysteine synthetase genes in this study. The finding that GST
and catalase genes are down-regulated in mutant RBCs provides a
possible explanation for a molecular basis for the observed sensitivity
to oxidant compounds. The promoters for NQO1 and various GSTs are
regulated by AREs; however, a role for AREs in the regulation of
catalase and GST-Yc has not been reported. We believe our findings suggest that p45NF-E2 has an important role in maintaining redox status
in RBCs through regulating expression of various antioxidant genes
under the control of the ARE. We previously found that catalase has an
essential role in the detoxification of
H2O2-derived radical species in the
RBC.52 A reduction in catalase activity would therefore be
one important factor in the sensitivity of the RBCs of
p45nf-e2
We thank members of the laboratory and Y. W. Kan for reading the manuscript and Paul Dazin for technical assistance with flow cytometry.
Submitted June 27, 2000; accepted November 27, 2000.
Supported by grants DK50267 and DK02603 from the National Institutes of Health.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: J. Chan, 533 Parnassus Ave, Rm U442, University of California, San Francisco, San Francisco, CA 94143-0793; e-mail: jchan{at}socrates.ucsf.edu.
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Top
Abstract
Introduction
-globin locus control region, which was found to be
essential for globin gene expression. Multiple proteins can interact
with the NF-E2 consensus sequence. The cap `n' collar (CNC)-bZIP
factor family of proteins was identified from searches for proteins
that bind and activate the NF-E2/AP1 site of the 
megakaryocytes, neutrophils, mast cells, and developing erythrocytes.
-gcs(l) and gss expression in mouse fibroblasts.
J Biol Chem.
1999;274:37491-37498