N(G)-monomethyl-L-arginine inhibits erythropoietin gene expression by stimulating GATA-2.
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)
Tarumoto
T
T
Department of Hematology, Jichi Medical School, Minamikawachi-machi, Tochigi-ken, Japan.
Imagawa
S
S
Ohmine
K
K
Nagai
T
T
Higuchi
M
M
Imai
N
N
Suzuki
N
N
Yamamoto
M
M
Ozawa
K
K
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D006152:Cyclic GMP / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004921:Erythropoietin / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D050989:GATA2 Transcription Factor; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D000860:Hypoxia; D008156:Luciferases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000187:drug effects* / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019323:omega-N-Methylarginine / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D050989:GATA2 Transcription Factor; C494711:GATA2 protein, human; C494712:Gata2 protein, mouse; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004921:Erythropoietin; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D004247:DNA; D008156:Luciferases; D006152:Cyclic GMP; D019331:NG-Nitroarginine Methyl Ester