L-arginine rescues decreased erythropoietin gene expression by stimulating GATA-2 with L-NMMA.
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.


CONCLUSIONS
L-arginine rescues decreased erythropoietin gene expression by stimulating GATA-2 with NG-monomethyl-L-arginine.
Imagawa
Shigehiko
S
Division of Hematology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan. simagawa@md.tsukuba.ac.jp
Tarumoto
Takahisa
T
Suzuki
Norio
N
Mukai
Harumi Y
HY
Hasegawa
Yuichi
Y
Higuchi
Masato
M
Neichi
Tomohiro
T
Ozawa
Keiya
K
Yamamoto
Masayuki
M
Nagasawa
Toshiro
T
D000740:Anemia / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000494:pharmacology*; D015687:Cell Hypoxia / Q000502:physiology; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004921:Erythropoietin / Q000037:antagonists & inhibitors / Q000097:blood / Q000235:genetics*; D050989:GATA2 Transcription Factor; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D051379:Mice; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000032:analysis; D014157:Transcription Factors / Q000378:metabolism*; D014511:Uremia / 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; D012333:RNA, Messenger; D014157:Transcription Factors; D004921:Erythropoietin; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D001120:Arginine; D006152:Cyclic GMP; D010100:Oxygen; D019331:NG-Nitroarginine Methyl Ester