Hydroxyurea generates nitric oxide in human erythroid cells: mechanisms for gamma-globin gene activation.
Hydroxyurea (HU) induces fetal hemoglobin synthesis through activation of cyclic guanine monophosphate (cGMP) signaling. Studies in sickle cell patients demonstrated increased circulating nitric oxide (NO) levels after oral HU treatment. However, the direct measurement of NO in erythroid cells and its role in fetal hemoglobin induction have not been defined. Therefore, we quantified the level of nitrate and nitrite (NOx) generated by HU in human erythroid progenitors in the presence of three nitric oxide synthase inhibitors (NOS), including N(G)-monomethyl-L-arginine (L-NMMA). In addition, cGMP levels were measured in the presence or absence of the pathway inhibitor 1H-(1,2,4)ox-adiazolo(4,3-a)quinoxalin-1-one, which blocks soluble guanylyl cyclase formation. HU treatment increased NOx levels and gamma-globin transcription in K562 and primary erythroid cells, which was augmented when HU was combined with L-NMMA. Pretreatment with the cGMP pathway inhibitor reversed gamma-gene activation by HU. These data demonstrate the direct stimulation of cellular NO and cGMP signaling in erythroid progenitors by HU as a possible mechanism for gamma-globin gene activation.
Lou
Tzu-Fang
TF
University of Texas at Dallas, Department of Molecular and Cell Biology, Richardson, Texas 75080, USA.
Singh
Manisha
M
Mackie
Ashley
A
Li
Wei
W
Pace
Betty S
BS
D006152:Cyclic GMP / Q000378:metabolism; D041905:Erythroid Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D005319:Fetal Hemoglobin / Q000096:biosynthesis; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006918:Hydroxyurea / Q000031:analogs & derivatives / Q000494:pharmacology*; D020014:K562 Cells; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects*; D055545:gamma-Globins / Q000235:genetics*; D019323:omega-N-Methylarginine / Q000494:pharmacology
D009566:Nitrates; D009573:Nitrites; D055545:gamma-Globins; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D005319:Fetal Hemoglobin; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D006152:Cyclic GMP; C063449:zileuton; D006918:Hydroxyurea