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Erythropoietin (Epo) gene expression is under the control of hypoxia-inducible factor 1 (HIF-1), and is negatively regulated by GATA. Interleukin 1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha), which increase the binding activity of GATA and inhibit Epo promoter activity, are increased in patients with anemia of chronic disease (ACD). We previously demonstrated the ability of K-7174 (a GATA-specific inhibitor), when injected intraperitoneally, to improve Epo production that had been inhibited by IL-1beta or TNF-alpha treatment. In the present study, we examined the ability of both K-11706, which inhibits GATA and enhances HIF-1 binding activity, and K-13144, which has no effect on GATA or HIF-1 binding activity, to improve Epo production following inhibition by IL-1beta or TNF-alpha in Hep3B cells in vitro and in an in vivo mouse assay. Oral administration of K-11706 reversed the decreases in hemoglobin and serum Epo concentrations, reticulocyte counts, and numbers of erythroid colony-forming units (CFU-Es) induced by IL-1beta or TNF-alpha. These results raise the possibility of using orally administered K-11706 for treating patients with ACD.

Transcription factor GATA-1 is essential for the development of the erythroid lineage. To ascertain whether strict control of GATA-1 expression level is necessary for achieving proper erythropoiesis, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of the GATA-1 gene hematopoietic regulatory domain. We examined the GATA-1 expression level by exploiting the transgenic mice and found 2 GFP-positive hematopoietic progenitor fractions in the bone marrow. One is the GFPhigh fraction containing mainly CFU-E and proerythroblasts, which coexpress transferrin receptor, while the other is the GFPlow/transferrin receptor-negative fraction containing BFU-E. Since the intensity of green fluorescence correlates well with the expression level of GATA-1, these results indicate that GATA-1 is highly expressed in erythroid colony-forming unit (CFU-E) but low in erythroid burst-forming unit (BFU-E), suggesting that the incremental expression of GATA-1 is required for the formation of erythroid progenitors. We also examined GFP-positive fractions in the transgenic mouse spleen and fetal liver and identified fractions containing BFU-E and CFU-E, respectively. This study also presents an efficient method for enriching the CFU-E and BFU-E from mouse hematopoietic tissues.

Interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), or N(G)-monomethyl-L-arginine (L-NMMA) are increased in patients with chronic disease-related anemia. They increase the binding activity of GATA and inhibit erythropoietin (Epo) promoter activity. In this study, we examined the ability of K-7174 (a GATA-specific inhibitor) to improve Epo production when inhibited by treatment with IL-1beta, TNF-alpha, or L-NMMA. Epo protein production and promoter activity were induced in Hep3B cells with 1% O2. However, 15 U/ml IL-1beta, 220 U/ml TNF-alpha, or 10(-3) M L-NMMA inhibited Epo protein production and promoter activity, respectively. Addition of 10 microM K-7174 rescued these inhibitions of Epo protein production and promoter activity induced by IL-1beta, TNF-alpha, or L-NMMA, respectively. Electrophoretic mobility shift assays revealed that addition of K-7174 decreased GATA binding activity, which was increased with the addition of IL-1beta, TNF-alpha, or L-NMMA. Furthermore, intraperitoneal injection of mice with IL-1beta or TNF-alpha decreased the hemoglobin concentrations and reticulocyte counts. However, the addition of K-7174 reversed these effects. These results raise the possibility of using K-7174 as therapy to treat anemia.

Long-term exposure of rats to cadmium (Cd) resulted in a marked suppression of erythropoietin (Epo) mRNA expression in the kidneys and the development of severe anemia. A recent report revealed that Cd inhibited hypoxia-inducible factor 1 (HIF-1) binding activity and Epo mRNA expression and protein production. However, Epo gene expression is also regulated by transcription factor GATA-2, which binds to the GATA binding site of the Epo promoter. To elucidate the mechanism of suppression of Epo by Cd, the effect of Cd on GATA-2 function was studied. Epo promoter/enhancer luciferase constructs, one with the wild-type promoter and another with a promoter with a mutant GATA site, were transfected into Hep3B cells. No significant difference in Epo promoter activity in these two types of cells was observed in the presence of Cd. The binding activity of GATA-2 was not affected by Cd. This study showed that Cd inhibited HIF-1 binding activity and Epo promoter activity, and then suppressed Epo protein production. Inhibition of Epo gene expression by Cd depends on suppression of HIF-1 binding activity, not on alteration of GATA function.

The promoter and enhancer elements of the mouse erythropoietin (mEpo) gene, which have high homology with those of the human erythropoietin (hEpo) gene, were fused with luciferase. The construct was transfected into erythropoietin-producing hepatoma cell line (Hep3B) cells by lipofectin with lacZ as an internal standard. The wild type (TGATA) showed a 39.5-fold increase in induction by hypoxia. Mouse GATA-2 inhibited the hypoxic induction of the wild-type (m3), promoterluciferase construct but not the hypoxic induction of the mutant (m4, 5) promoter-luciferase constructs. N(G)-monomethyl L-arginine (L-NMMA) inhibited the hypoxic induction of the m3 promoter-luciferase construct, but this inhibition was recovered by L-arginine. H2O2 also inhibited the hypoxic induction of the m3 promoter-luciferase construct, but this inhibition was recovered by catalase. Gel shift assays performed on nuclear extracts of 293 cells overexpressing mGATA-1, -2, and -3 revealed that mGATA-1, -2, and -3 bind to the TGATA element of the mEpo promoter. These results indicate that mGATA binds to the TGATA site of the mEpo promoter and negatively regulates mEpo gene expression. Negative regulation of mEpo gene by GATA transcriptional factors is discussed.

Background: NG-monomethyl-L-arginine (L-NMMA) decreases the expression of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) and increases the expression of GATA-2 mRNA and levels of GATA-2 binding activity, thereby inhibiting erythropoietin (Epo) promoter activity and causing a decrease in the expression of Epo protein. In the present study, we examined the effect of L-arginine on Epo gene expression in Hep3B cells and BDF1 mice., Methods: Hep3B cells were incubated with and without different concentrations of L-NMMA and/or l-arginine. Anemic mice were injected with phosphate-buffered saline (PBS) or L-NAME and L-arginine., Results: Incubation with L-NMMA under hypoxic conditions inhibited Epo expression, but this inhibition was recovered by the addition of L-arginine. Hypoxia induced the secretions of NO and cGMP, but the addition of L-NMMA inhibited these inductions, though these inhibitions of NO and cGMP by L-NMMA were recovered by the addition of L-arginine. Hep3B cells transfected with the Epo promoter/enhancer-luciferase gene had Epo promoter activity. This activity was inhibited by L-NMMA, but it could be recovered by the addition of L-arginine. L-NMMA induced the binding activity of GATA-2 under hypoxic conditions. This binding activity was inhibited by the addition of L-arginine. The addition of cGMP inhibited L-NMMA-induced GATA-2 binding activity in a dose-dependent manner. The results of an in vivo mouse assay revealed that L-NAME inhibited the expression of Epo, but this inhibition of Epo expression by L-NAME was rescued by pretreatment with L-arginine., Conclusion: L-arginine rescues decreased erythropoietin gene expression by stimulating GATA-2 with NG-monomethyl-L-arginine.

Hydrogen peroxide (H2O2) has previously been shown to inhibit the DNA binding activity of hypoxia inducible factor-1 (HIF-1), the accumulation of HIF-1alpha protein and erythropoietin (Epo) gene expression. Epo gene expression has been previously shown to be down-regulated through a GATA binding site at its promoter region. In this study, the effect of H2O2 on Epo gene expression under hypoxic conditions through a GATA transcription factor was investigated. Hypoxic induction was found to be inhibited upon the addition of H2O2, and this effect could be reversed through the addition of catalase. Hypoxic induction was found to be suppressed by co-transfection with a human GATA-2 cDNA expression plasmid. Transfection of Hep3B cells with a reporter gene bearing a mutation at the promoter GATA binding site was found to be only mildly affected by the addition of H2O2. Electrophoretic gel mobility shift assays (EMSAs), using the Epo promoter GATA site as a probe and the GATA-2 protein extracted from Hep3B cells, showed that addition of H2O2 enhanced the binding of GATA-2 while addition of catalase inhibited this binding. From these results, we conclude that H2O2 increases the binding activity of GATA-2 in a specific manner, thereby suppressing the activity of the Epo promoter and thus inhibiting Epo gene expression., (Copyright 2001 Wiley-Liss, Inc.)

N(G)-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-kappaB. 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)

We examined regulation of the human erythropoietin (Epo) gene through the GATA sequence in the Epo promoter and showed that Hep3B and HepG2 cells express human GATA-2 (hGATA-2) mRNA and protein. Nuclear extracts of QT6 cells transfected with hGATA-1, 2, or 3 transcription factors showed specific binding to the GATA element in the human Epo gene promoter by gel mobility shift assay. Transient transfection of Hep3B cells with hGATA-1, 2, or 3 showed that each of these transcription factors significantly decreased the level of expression of Epo mRNA as assessed by a competitive polymerase chain reaction. Transient transfection of Hep3B cells with hGATA-1, 2, and 3 and an Epo-reporter gene (growth hormone [GH]) construct showed significant inhibition of the Epo promoter. Antisense oligonucleotide for hGATA-2 transcription factor significantly increased the Epo protein in Hep3B cells under 1% O2 for 24 hours incubation. Furthermore, transient transfection of Hep3B cells with hGATA-1, 2, and 3 and an Epo-reporter gene (luciferase) construct also showed significant inhibition of the Epo promoter. However, transfection of the mutated GATA sequence of the Epo-luciferase gene with hGATA-1, 2, and 3 interfere with the inhibition of the Epo promoter. We conclude that the hGATA-1, 2, and 3 transcription factors specifically bind to the GATA element in the human Epo gene promoter and negatively regulate Epo gene expression.

We examined the effect of H2O2 on the regulation of the human erythropoietin (Epo) gene through the GATA sequence in the Epo promoter in Hep3B cells. The addition of exogenous H2O2 in Hep3B cells inhibited hypoxia-induced Epo production of mRNA as assessed by competitive polymerase chain reaction (PCR) and protein by EIA. Likewise, menadione, which increased production of endogenous H2O2, inhibited hypoxia-induced Epo production in Hep3B cells. Furthermore, the expression of the human GATA (hGATA) transcription factor was increased dose-dependently by the addition of H2O2 or menadione in Hep3B cells as assessed by gel mobility shift assay. We concluded that H2O2 inhibits Epo gene expression, because it enhanced the expression of the hGATA transcription factor. Our findings support the hypothesis that GATA is a transcription factor for the Epo gene acting through the oxygen sensor.

We examined regulation of the human erythropoietin (Epo) gene through the GATA sequence in the Epo promoter, and demonstrated that Hep3B and HepG2 cells express human GATA-2 (hGATA-2) mRNA and protein. Nuclear extracts of QT6 cells transfected with hGATA-1, -2 or -3 transcription factors revealed specific binding to the GATA element in the human Epo gene promoter by gel mobility shift assay. Transient transfection of Hep3B cells with hGATA-1, -2 or -3 demonstrated that each of these transcription factors significantly decreased the level of expression of Epo mRNA as assessed by a competitive polymerase chain reaction. Furthermore, transient transfection of Hep3B cells with hGATA-1, -2 and -3 and and Epo reporter gene construct showed significant inhibition of the Epo promoter. We conclude that the hGATA-1, -2 and -3 transcription factors specifically bind to the GATA element in the human Epo gene promoter and negatively regulate Epo gene expression.