|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
HEMATOPOIESIS
From the Department of Hematology, Jichi Medical
School, Minamikawachi-machi, Tochigi-ken, Japan; Division of
Hematology, Institute of Clinical Medicine, University of Tsukuba,
Tennoudai, Tsukuba, Ibaraki, Japan; Center for Tsukuba Advanced
Research Alliance and Institute of Basic Medical Sciences, University
of Tsukuba, Tennoudai, Tsukuba, Ibaraki, Japan; Chugai Pharmaceutical
Co, Ltd, Tokyo, Japan.
NG-monomethyl-L-arginine (L-NMMA) has been
reported to be elevated in uremic patients. Based on the hypothesis
that the pathogenesis of the anemia of renal disease might be due to
the perturbation of transcription factors of the erythropoietin
(Epo) gene by L-NMMA, the present study was designed to
investigate the effect of L-NMMA on Epo gene expression
through the GATA transcription factor. L-NMMA caused decreased levels
of NO, cyclic guanosine monophosphate (cGMP), and Epo protein in Hep3B
cells. L-NAME (analogue of L-NMMA) also inhibited Epo production in
anemic mice. Transfection of the Epo promoter-luciferase gene into
Hep3B cells revealed that L-NMMA inhibited the Epo promoter activity.
However, L-NMMA did not inhibit the Epo promoter activity when mutated
Epo promoter (GATA to TATA) was transfected, and L-NMMA did not affect
the enhancer activity. Electrophoretic mobility shift assays
demonstrated the stimulation of GATA binding activity by L-NMMA.
However, L-NMMA had no effect on the binding activity of hepatic
nuclear factor-4, COUP-TF1, hypoxia-inducing factor-1, or NF- In humans and mammals, erythropoiesis is regulated
by the 30.4-kd glycoprotein hormone erythropoietin (Epo).1
Epo gene expression is regulated by hypoxia through an
oxygen sensor.1 The major sites of Epo production are the
liver in the fetus2 and the kidney in the
adult.3 Peritubular capillary interstitial cells are
thought to be the major site of production of Epo in the
kidney.4 The cause of the anemia of renal disease is
believed to be damage to this site of the Epo production by renal
failure.5 In this regard, however, it is interesting to
note that some patients with the anemia of renal disease still have the
ability to produce Epo in response to acute blood loss and
hypoxia.6 On the other hand, other patients with renal
failure do not have anemia.7 These observations suggest
that chronic perturbation of oxygen sensing or signal transduction or
both underlie the pathogenesis of the anemia of renal disease rather
than damage at the site of Epo production. Recently,
NG-monomethyl-L-arginine (L-NMMA) was reported
to be undetectable in nonuremic subjects, whereas the concentration of
L-NMMA was markedly elevated in uremic patients.8 Based on
this observation, we hypothesized that this substance may be a
candidate uremic toxin responsible for renal anemia. However, the
precise function of L-NMMA in mediating expression of the
Epo gene remains to be elucidated. Because L-NMMA functions
as an inhibitor of nitric oxide synthase (NOS),9 it is
expected to suppress the production of nitric oxide (NO) and cyclic
guanosine 3', 5'-monophosphate (cGMP). We have found that GATA
transcription factors bind to a GATA site in the Epo gene
promoter and negatively regulate the gene expression in Hep3B cells
(Figure 1A).10 In this
study, we demonstrate that L-NMMA suppresses Epo gene
expression by up-regulation of the GATA transcription factor.
Cell culture and RNA preparation
Transfection
DNA binding assay Nuclear extracts were prepared as previously described.13 Protein concentrations were determined by assay (Bio-Rad) using bovine serum albumin (BSA) as a standard. Sense-strand oligonucleotide (wild-type: CATGCAGATAA CAGCCCCGAC) was end-labeled with T4 polynucleotide kinase (Toyobo, Tokyo, Japan) and annealed to a 4-fold excess of the unlabeled antisense oligonucleotide. Two nanograms of labeled probe was used in each binding reaction. The binding buffer consisted of 10 mmol/L Tris HCl (pH 7.5), 1 mmol/L EDTA, 4% Ficoll, 1 mmol/L dithiothreitol, and 75 mmol/L KCl. An equimolar mixture of poly[d(I-C)] and poly[d(A-T)] (25 ng; Sigma, St Louis, MO) was used as a nonspecific competitor. The reaction mixtures (25 µL) were incubated for 15 minutes at 4°C and then electrophoresed on 5% nondenaturing polyacrylamide gels in 0.25 × TBE buffer (22 mmol/L Tris borate, 22 mmol/L boric acid, 0.5 mmol/L EDTA) at room temperature at 150 V for 1.5 hours as previously described.13 Gels were vacuum dried and then autoradiography was performed using intensifying screens at 80°C
for 24 hours. Monoclonal antibodies to hGATA-1, -2, and -3 were
prepared as previously described.14
Plasmid vectors We used the reporter plasmid pEPLuc described by Blanchard and coworkers15 as a basic plasmid construct, in which both the 126-bp 3' Epo enhancer (120 to 245-bp 3' of the poly(A) addition site) and the 144-bp minimal Epo promoter (from118 to +26 relative to the
transcription initiation site) were placed upstream of the firefly
luciferase (Luc) gene in pXP2,16 resulting in
Pwt17 or V2-Ewt-Pwt-pXP217 (Figure 1B). This
enhancer contained hypoxia-inducible factor 1 (HIF-1) binding site and
steroid receptor response element (SRRE). In the mutant construct, the
GATA sequence in the Epo promoter was mutated to TATA (AGATAACAG to
ATATAAAAG). This mutant construct is called
Pm717 or V3-Ewt-Pm7-pXP217 (Figure 1B). The
144-bp minimal Epo promoter (from 118 to +26 relative to the
transcription initiation site) was placed upstream of the Luc gene in pXP2, resulting in  18pXP2 (Figure 1C). In the
mutant construct, the GATA sequence in the Epo promoter was mutated to TATA (AGATAACAG to ATATAAAAG). This mutant
construct is called 18m7pXP2 (Figure 1C). Construction of the
hGATA-2 expression plasmid has been previously
described.14
Northern blot analysis Probes were labeled with [ -32P]deoxycytidine
triphosphate by random priming and used in RNA blot
hybridization.18 Formaldehyde gels for RNA electrophoresis
were prepared as described.18 RNA blot hybridization was
performed using 25 µg of total RNA from Hep3B cells. The filter was
hybridized to probe of hGATA-2 complementary DNA (cDNA). The same
filter was stripped and rehybridized to probe consisting of the Epo
cDNA, and a probe for ribosomal RNA to determine the level of RNA in
each lane. Autoradiography was performed at 80°C and quantitated by
densitometric scanning.
Anemic mice The BDF1 mice were injected intraperitoneally with 0.2 mL of 10 mg NG-nitro-L-arginine methyl ester (L-NAME)/mL phosphate-buffered saline (PBS) or 0.2 mL of PBS. Blood samples (0.3 mL) were obtained from the orbital vein at 0, 12, and 24 hours after injection of L-NAME. Epo levels in the serum were determined by radioimmunosorbent assay (RIA).Other assays Transfected Hep3B cells were washed with PBS and lysed in 10-cm dishes with 800 µL of cell lysis buffer (PicaGene, Toyo Ink, Tokyo). Luc activity in 20 µL of the cell extract was determined by Autolumat luminometer (Berthorude, Tokyo, Japan) for 10 seconds. Each measurement of relative light units was corrected by subtraction of the background and standardized to the RSVCAT or -galactosidase internal
transfection control activity. Hypoxic inducibility was defined as the
ratio of the corrected relative light units of the hypoxic (1%
O2) dish to those of the normoxic (21% O2)
dish. CAT activity was determined as described by Neumann and
colleagues.19 NO was detected by the
2,3-diaminonaphthalene method,20 cGMP was measured by
enzyme immunosorbent assay (EIA),21 and -fetoprotein (AFP) was measured by RIA.
Inhibition of Epo protein by L-NMMA We first confirmed that L-NMMA was not cytotoxic at concentrations of up to 10 2 mol/L for Hep3B cells by the trypan blue dye
exclusion method and the methyl-thiazol-diphenyl-tetrazolium
(MTT) method (data not shown). Similarly, Fisher and coworkers
reported that L-NMMA concentrations of up to 103 mol/L
were not cytotoxic for Hep3B cells.22 We then examined the
effect of L-NMMA on the production of Epo protein in Hep3B cells.
Incubation for 24 hours with 102 mol/L L-NMMA under
hypoxic conditions showed an 80% inhibition of Epo, whereas
105 mol/L,104 mol/L, and 103
mol/L L-NMMA each showed a 60% inhibition of Epo (Figure
2). To make sure that this inhibition of
Epo protein by L-NMMA was specific, AFP was measured by RIA. Up to
102 mol/L L-NMMA did not inhibit the level of AFP (data
not shown). These results suggest that L-NMMA specifically inhibited
the production of Epo protein in Hep3B cells.
Inhibition of NO and cGMP by L-NMMA L-NMMA is known to be an NOS inhibitor, and, therefore, a decrease in NO from cells incubated with L-NMMA was expected. To this end, Hep3B cells were incubated with different concentrations of L-NMMA. Hypoxia induced the secretion of NO, but the addition of L-NMMA inhibited this induction (Figure 3). Because NO stimulates guanylate cyclase (GC) to produce cGMP,23 a decrease in cGMP from cells incubated with L-NMMA was also expected. Hypoxic Hep3B cells were incubated with different concentrations of L-NMMA. As shown in Figure 4, L-NMMA inhibited the secretion of cGMP from the cells.
Inhibition of serum Epo by L-NAME from L-NAME-injected mice L-NAME was examined using an in vivo mouse assay, because L-NMMA is reported to be catabolyzed by NG-dimethylarginine dimethylaminohydrolase (DDHA) in the intact kidney24,25; however, L-NAME is not catabolyzed by this enzyme.26 Hecker and associates observed that L-NMMA was rapidly hydrolyzed to L-citrulline and lost the inhibitory effect in endothelial cells.27 Furthermore, L-NMMA is continuously released into body fluids during the in vivo breakdown of the proteins and is assumed to be readily excreted in urine without reincorporation into proteins or further degradation in intact animals.24 To identify the effect of L-NAME on Epo production in vivo, BDF1 mice were injected intraperitoneally with 0.2 mL of 10 mg L-NAME/mL PBS or 0.2 mL of PBS as a control. Blood samples (0.3 mL) were obtained from the orbital vein immediately after (0 hours) and at 12 and 24 hours after the injection of L-NAME (Figure 5). The serum Epo from the control (dashed line) increased to 66.4 mU/mL at 12 hours and to 276.8 mU/mL at 24 hours after the injection. The serum Epo from the mice injected with L-NAME (straight line) was 28.5 mU/mL at 12 hours and 98.8 mU/mL at 24 hours after the injection. These values were significantly lower than those of the control (Figure 5). These in vivo results are comparable with those obtained from the in vitro incubation of Hep3B cells (Figure 2).
Inhibition of Epo promoter activity by L-NMMA Expression of the Epo gene has been shown to be induced by hypoxia.1 To elucidate the molecular mechanisms underlying the hypoxic induction of the Epo gene, plasmids containing both the promoter and enhancer of the Epo gene were used in a transient transfection assay into Hep3B cells.10 One GATA and 2 CACCC motifs are in the promoter region, and one HIF-1 and one hepatic nuclear factor 4 (HNF-4) binding site are in the enhancer (Figure 1A). Both the promoter and enhancer were inserted upstream of the Luc gene to give rise to Pwt and Pm7 plasmids (Figure 1B). The latter contains a mutation in the promoter GATA element. We transfected Pwt and Pm7 into Hep3B cells and incubated the cells in the presence or absence of L-NMMA under 21% (normoxia) or 1% (hypoxia) oxygen for 24 hours. The hypoxic induction of Luc gene expression is represented as a hypoxia/normoxia ratio, as previously described.10 Hypoxic induction from Pwt was 55.8 ± 5.5 fold higher than that from normoxic Pwt (mean ± 1 SD, n = 3) (Table 1 and Figure 6). Interestingly, the addition of L-NMMA inhibited the hypoxic induction of the Luc reporter gene expression from Pwt with hypoxia/normoxia ratio of only 33.0 ± 9.2 fold, 59.1% of that from Pwt incubated without L-NMMA (Table 1 and Figure 6). These results indicate that the hypoxic induction of the Epo gene expression is suppressed by L-NMMA through the Epo gene regulatory regions.
We previously found that the GATA element in the promoter plays an important role in limiting the hypoxic induction of the Epo gene.10 We therefore examined the contribution of the GATA site to the L-NMMA suppression of hypoxic Epo gene induction. To this end, Hep3B cells were co-transfected with Pwt and an hGATA-2 expression vector. The expression of hGATA-2 resulted in the inhibition of the hypoxic induction of the Epo gene. The Luc reporter gene was induced in the presence of hGATA-2 by 21.5 ± 3.8 fold, 43.2% of that from Pwt incubated without hGATA-2 (Table 1 and Figure 6). Furthermore, the exposure of Hep3B cells co-transfected with Pwt and hGATA-2 expression vector to L-NMMA resulted in further suppression with hypoxic induction of the reporter gene of only 14.2 ± 3.4 fold, 25.4% of that from the cells incubated without L-NMMA and hGATA-2 (Table 1 and Figure 6). These results suggest that hGATA-2 acts as a repressor of the hypoxic induction of the Epo gene and that the GATA sequence in the promoter mediates L-NMMA suppression. The GATA element in the promoter only contributes to L-NMMA suppression The contribution of the GATA element was further tested by using a reporter plasmid, Pm7, which contains GATA site mutations. We previously found that alone this GATA mutation affects the basal level expression of Luc reporter activity.10 As was the case for Pwt, Luc expression was also strongly induced following the exposure of the transfected cells to hypoxia, 96.9 ± 15.9 fold (Table 1 and Figure 6). However, L-NMMA failed to affect the GATA mutant Pm7 Luc activity with hypoxic induction of 103.7 ± 20.6 fold, 107.0% of that from Pm7 only (Table 1 and Figure 6). Transfection of Pm7 into Hep3B cells, which express hGATA-2, resulted in hypoxic induction of 147.3 ± 17.1 fold, 152.0% of that from Pm7 only (Table 1 and Figure 6). Furthermore, the exposure of the cells co-transfected with Pm7 and hGATA-2 expression vector to L-NMMA showed 123.5 ± 10.6 fold, and 127.5% of that from Pm7 only (Table 1 and Figure 6). These results suggest that L-NMMA inhibits Epo gene expression through the GATA site in the Epo promoter rather than through the enhancer activity. To clearly identify whether this inhibitory effect of L-NMMA on Epo gene expression was due to a GATA-2 or HIF-1 binding site or both, use of the construct that contained the Epo promoter only was advantageous. To this end, we then transfected18pXP2 (wild
type) or 18m7pXP2 (GATA site mutant) plasmids into Hep3B cells and incubated the cells both with or without additional L-NMMA under 21%
or 1% oxygen for 24 hours (Figure 7).
Hypoxic induction from 18pXP2 was 7.6 ± 1.0 fold higher than that
from normoxic 18pXP2 (mean ± 1 SD, n = 4) (Figure 7). The
addition of L-NMMA significantly inhibited the hypoxic induction of the
Luc reporter gene expression from 18pXP2 with
hypoxia/normoxia ratio of only 4.0 ± 0.6 fold, 52.6% of that from
18pXP2 incubated without L-NMMA (Figure 7). Hypoxic induction from
18m7pXP2 was 7.3 ± 0.2 fold higher that from normoxic
18m7pXP2. L-NMMA failed to affect the 18m7pXP2 Luc activity with
hypoxic induction of 8.2 ± 0.5 fold, 112.3% of that from
18m7pXP2 only (Figure 7). These results clearly indicate that the
inhibitory effect of L-NMMA on Epo gene expression was due
to GATA-2, not HIF-1.
Enhancement of GATA-2 binding activity by L-NMMA To examine whether L-NMMA treatment affects the binding activity of hGATA-2, nuclear extracts were prepared from the cells stimulated by L-NMMA for 1 hour under normoxic or hypoxic conditions, and electrophoretic mobility shift assays (EMSA) were performed with an oligonucleotide containing the wild-type GATA element (AGATAA) (Figure 8). L-NMMA induced the binding activity of GATA-2 (indicated by the circle) under normoxic and hypoxic conditions (lanes 3-6, 9-11). This binding activity was abolished by self-competitor (lanes 14-17, 20-22). To confirm that the band indicated by the circle was GATA-2, nuclear extracts were prepared from Hep3B cells under hypoxia with 104 mol/L L-NMMA for 1 hour, and incubated with 0.5 or 1.0 µL monoclonal antibodies of
hGATA-1, 2, and 3 and then EMSA was performed under the same conditions
as described in Figure 8. The control revealed a band of increased
intensity (Figure 9, lane 2), and the
addition of FCS further increased the intensity of this band, though
the mechanism of this increase is unknown (Figure 9, lane 3). The addition of monoclonal antibodies of hGATA-1 and -3 resulted in bands
of similar intensities (lanes 4, 5 and 8, 9); however, the band
indicated by the circle disappeared with the addition of monoclonal
hGATA-2 antibody (lanes 6 and 7). These results strongly suggest that
the band indicated by the circle was a specific GATA-2 band.
To clarify the effect of cGMP on GATA-2 binding activity, nuclear
extracts were prepared from Hep3B cells under hypoxia with the addition
of 10 GATA-2 messenger RNA expression was induced by L-NMMA To examine whether GATA-2 and Epo messenger RNA (mRNA) expression levels were affected by the addition of L-NMMA, Northern blot analysis was performed. Northern blot analysis showed hypoxia-induced Epo mRNA expression; however, the addition of L-NMMA inhibited this induction of Epo mRNA (Figure 10A, middle panel). In contrast, hypoxia reduced GATA-2 mRNA expression, whereas L-NMMA induced the expression of GATA-2 mRNA (Figure 10A, upper panel, and B). The 28S used as a control revealed a constant level of mRNA expression from the cells incubated under normoxia or hypoxia and with or without L-NMMA (Figure 10A, lower panel).
It was reported that L-NAME, also an NOS inhibitor, significantly
blunted interferon-
The predialysis concentration of L-NMMA in patients with chronic renal
failure is approximately 10 In the present study, the effect of L-NMMA on GATA was investigated in
detail. The effects of L-NMMA on HIF-1, HNF-4, COUP-TF1, NF- The effect of NO on VEGF expression via HIF-1 is controversial. Some recent reports show an inhibitory effect of NO on VEGF expression. Huang and colleagues36 and Sogawa and associates37 have demonstrated that sodium nitroprusside (SNP, NO donor) suppresses hypoxia-induced VEGF gene activation and HIF-1 binding activity. SNP inhibits the hypoxic induction of the VEGF gene in a dose-dependent manner, in contrast to the effects S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and 3-(2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazino)-1-propanamine (NOC5) (another NO donor) as shown by Kimura and colleagues.35 This concentration is clearly due to the specific nature of SNP. As to the effect of NO on Epo, Fisher and coworkers reported that serum levels of Epo in hypoxic polycythemic mice were significantly increased after injections of 200 µg/kg SNP.22 Furthermore, cGMP levels in hypoxic Hep3B cells were also elevated. SNP (10 and 100 µmol/L) and NO (2 µmol/L) increased cGMP levels in Hep3B cells.22 These results are compatible with our data and strongly suggest that L-NMMA inhibits Epo production via the GATA transcription factor. Further analysis of the GATA-2 expression level and NO and cGMP from patients with renal anemia is necessary to clarify the details of the pathogenesis of renal anemia.
We thank D. L. Galson for providing the Pwt, Pm7,
Submitted November 22, 1999; accepted April 25, 2000.
Supported by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan, and the Chugai Foundation, Tokyo, Japan.
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: Shigehiko Imagawa, Division of Hematology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan; e-mail: simagawa{at}md.tsukuba.ac.jp.
1.
Bunn HF, Poyton RO.
Oxygen sensing and molecular adaptation to hypoxia.
Physiol Rev.
1996;76:839-885 2. Zanjani ED, Poster J, Burlington H, Mann LI, Wasserman LR. Liver as the primary site of erythropoietin formation in the fetus. J Lab Clin Med. 1977;89:640-644[Medline] [Order article via Infotrieve]. 3. Jacobson LO, Goldwasser E, Fried W, Plzak L. Role of the kidney in erythropoiesis. Nature. 1957;179:633-634[Medline] [Order article via Infotrieve]. 4. Lacombe C, Da Silva J-L, Bruneval P, et al. Peritubular cells are the site of erythropoietin synthesis in the murine hypoxic kidney. J Clin Invest. 1988;81:620-623. 5. McGonigle RJS, Wallin JD, Shadduck RK, Fisher JW. Erythropoietin deficiency and erythropoiesis in renal insufficiency. Kidney Int. 1984;25:437-444[Medline] [Order article via Infotrieve]. 6. Esbach JW, Adamson JW. Anemia of end-stage renal disease. Kidney Int. 1985;28:1-5[Medline] [Order article via Infotrieve]. 7. Walle AJ, Wong GY, Clemons GK, Garcia JF, Niedermayer W. Erythropoietin-hematocrit feedback circuit in the anemia of end-stage renal disease. Kidney Int. 1987;31:1205-1209[Medline] [Order article via Infotrieve]. 8. Ribeiro ACM, Roberts NB, Lane C, Yaqoob M, Ellory JC. Accumulation of the endogenous L-arginine analogue NG-monomethyl-L arginine in human end-stage renal failure patients on regular haemodialysis. Exp Physiol. 1996;81:475-481[Abstract]. 9. Gray GA, Schott C, Julou-Schaeffer G, Fleming I, Paratt JR, Stoclet J. The effect of inhibitors of the L-arginine/nitric oxide pathway on endotoxin-induced loss of vascular responsiveness in anesthetized rats. Br J Pharmacol. 1991;103:1218-1224[Medline] [Order article via Infotrieve].
10.
Imagawa S, Yamamoto M, Miura Y.
Negative regulation of the erythropoietin gene expression by the GATA transcription factors.
Blood.
1997;89:1430-1439
11.
Imagawa S, Goldberg MA, Doweiko J, Bunn HF.
Regulatory elements of the erythropoieitin gene.
Blood.
1991;77:278-285 12. Gilman M. Current Protocols in Molecular Biology Vol 1. New York: John Wiley & Sons; 1988:4.1.4.
13.
Yamamoto M, Ko LJ, Leonard MW, Beug H, Orkin SH, Engel JD.
Activity and tissue-specific expression of the transcription factor NF-E1 multi gene family.
Genes Dev.
1990;4:1650-1662
14.
Nagai T, Harigae H, Ishihara H, et al.
Transcription factor GATA-2 is expressed in erythroid, early myeloid, and CD34+ human leukemia-derived cell lines.
Blood.
1994;84:1074-1084
15.
Blanchard KL, Acquaviva AM, Galson DL, Bunn H.
Hypoxic induction of the human erythropoietin gene: cooperation between the promoter and enhancer, each of which contains steroid receptor response elements.
Mol Cell Biol.
1992;12:5373-5385 16. Nordeen SK. Luciferase reporter gene vectors for analysis of promoters and enhancers. Biotechniques. 1988;6:454-457[Medline] [Order article via Infotrieve]. 17. Galson DL, Tsuchiya T, Tendler DS, et al. The orphan receptor hepatic nuclear factor 4 functions as a transcriptional activator for tissue-specific and hypoxia-specific erythropoietin gene expression and is antagonized by EAR3/COUP-TF1. Mol Cell Biol. 1995;15:2135-2144[Abstract].
18.
Riddle RD, Yamamoto M, Engel JD.
Expression of 19. Neumann JR, Morency CA, Russian KO. A novel rapid assay for chloramphenicol acetyltransferase gene expression. Biotechniques. 1987;5:444. 20. Misco TP, Schilling RJ, Salvemini D, Moore WM, Currie MG. A fluorometric assay for the measurement of nitrite in biological samples. Anal Biochem. 1993;214:11-16[Medline] [Order article via Infotrieve].
21.
Ueno M, Rondon I, Beckman B, et al.
Increased secretion of erythropoietin in human renal carcinoma cells in response to hypoxia.
Am J Physiol.
1990;259 (Cell Physiol. 28):C427-C431 22. Ohigashi T, Brookins J, Fisher JW. Interaction of nitric oxide and cyclic guanosine 3', 5'-monophosphate in erythropoietin production. J Clin Invest. 1993;92:1587-1591. 23. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathology, and pharmacology. Pharmacol Rev. 1991;43:109-142[Medline] [Order article via Infotrieve].
24.
Kimoto M, Whitley G, St J, Tsuji H, Ogawa T.
Detection of NG, NG-dimethylarginine dimethylaminohydrolase in human tissues using a monoclonal antibody.
J Biochem.
1995;117:237-238 25. Kimoto M, Tsuji H, Ogawa T, Sasaoka K. Detection of NG, NG-dimethylarginine dimethylaminohydrolase in the nitric oxide-generating systems of rats using monoclonal antibody. Arch Biochem Biophys. 1993;300:657-662[Medline] [Order article via Infotrieve].
26.
Ogawa T, Kimoto M, Sasaoka K.
Purification and properties of a new enzyme, NG, NG-dimethylarginine dimethylaminohydrolase, from rat kidney.
J Biol Chem.
1989;264:10205-10209 27. Hecker M, Mitchell JA, Harris HJ, Katsura M, Thiemermann C, Vane JR. Endothelial cells metabolize NG-monomethyl-L-arginine to L-citrulline and subsequently to L-arginine. Biochem Biophys Res Commun. 1990;167:1037-1043[Medline] [Order article via Infotrieve]. 28. Ignarro LJ. Endothelium-derived nitric oxide: actions and properties. FASEB J. 1989;3:31-36[Abstract]. 29. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature Lond. 1987;327:524-526[Medline] [Order article via Infotrieve]. 30. Forstermann U, Schmidt HHHW, Pollock JS, et al. Isoform of nitric oxide: characterization and purification from different cell types. Biochem Pharmacol. 1991;42:1849-1857[Medline] [Order article via Infotrieve]. 31. Marletta MA, Yoon PS, Iyengar R, Leaf CD, Wishnok JS. Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry. 1988;27:8706-8711[Medline] [Order article via Infotrieve]. 32. Ito S, Ren Y. Evidence for the role of nitric oxide in macula densa control of glomerular hemodynamics. J Clin Invest. 1993;92:1093-1098. 33. Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet. 1992;339:572-575[Medline] [Order article via Infotrieve]. 34. Vallance P, Leone A, Calver A, Collier J, Moncada S. Endogenous dimethylarginine as an inhibitor of nitric oxide synthesis. J Cardiovasc Pharmacol. 1992;20(suppl 12):S60-S62.
35.
Kimura H, Weisz A, Kurashima Y, et al.
Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxia-inducible factor-1 activity by nitric oxide.
Blood.
2000;95:189-197
36.
Huang LE, Gu J, Schau M, Bunn HF.
Regulation of hypoxia inducible factor 1
37.
Sogawa K, Numayama-Tsuruta K, Ema M, Abe M, Abe H, Fujii-Kuriyama Y.
Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia.
Proc Natl Acad Sci U S A.
1998;95:7368-7373
© 2000 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  N. Obara, N. Suzuki, K. Kim, T. Nagasawa, S. Imagawa, and M. Yamamoto Repression via the GATA box is essential for tissue-specific erythropoietin gene expression Blood, May 15, 2008; 111(10): 5223 - 5232. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Zumberg and C. Leissinger Acquired underproduction anemias ASH Self-Assessment Program, January 1, 2007; 2007(1): 78 - 101. [Full Text] [PDF]
|
|
|
|
|
|
Y. Nakano, S. Imagawa, K. Matsumoto, C. Stockmann, N. Obara, N. Suzuki, T. Doi, T. Kodama, S. Takahashi, T. Nagasawa, et al. Oral administration of K-11706 inhibits GATA binding activity, enhances hypoxia-inducible factor 1 binding activity, and restores indicators in an in vivo mouse model of anemia of chronic disease Blood, December 15, 2004; 104(13): 4300 - 4307. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Lasbury, X. Tang, P. J. Durant, and C.-H. Lee Effect of Transcription Factor GATA-2 on Phagocytic Activity of Alveolar Macrophages from Pneumocystis carinii-Infected Hosts Infect. Immun., September 1, 2003; 71(9): 4943 - 4952. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ezoe, I. Matsumura, S. Nakata, K. Gale, K. Ishihara, N. Minegishi, T. Machii, T. Kitamura, M. Yamamoto, T. Enver, et al. GATA-2/estrogen receptor chimera regulates cytokine-dependent growth of hematopoietic cells through accumulation of p21WAF1 and p27Kip1 proteins Blood, November 15, 2002; 100(10): 3512 - 3520. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Minami and W. C. Aird Thrombin Stimulation of the Vascular Cell Adhesion Molecule-1 Promoter in Endothelial Cells Is Mediated by Tandem Nuclear Factor-kappa B and GATA Motifs J. Biol. Chem., December 7, 2001; 276(50): 47632 - 47641. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. T. Furuta, J. R. Turner, C. T. Taylor, R. M. Hershberg, K. Comerford, S. Narravula, D. K. Podolsky, and S. P. Colgan Hypoxia-inducible Factor 1-dependent Induction of Intestinal Trefoil Factor Protects Barrier Function during Hypoxia J. Exp. Med., April 30, 2001; 193(9): 1027 - 1034. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
B.
Furthermore, cGMP inhibited the L-NMMA-induced GATA binding activity.
L-NMMA also increased GATA-2 messenger RNA expression. These results
demonstrate that L-NMMA suppresses Epo gene expression by
up-regulation of the GATA transcription factor and support the
hypothesis that L-NMMA is one of the candidate substances that underlie
the pathogenesis of renal anemia.
(Blood. 2000;96:1716-1722)
) or
hypoxic (1% O2, 
) conditions for 4 hours in the
presence or absence of L-NMMA. NO was measured by the
2,3-diaminonaphthalene method. Three separate experiments were
performed (n = 3). Error bars represent 1 SD.
*P < .01.
(IFN-
-aminolevulinate syntase in avian cells: separate genes encode erythroid specific and nonspecific isozymes.
Proc Natl Acad Sci U S A.
1989;86:792-796