Hydroxyurea nitrosylates and activates soluble guanylyl cyclase in human erythroid cells

Abstract

Hydroxyurea, a drug widely used for treating myeloproliferative diseases, has also been approved for the treatment of sickle cell disease by raising fetal hemoglobin (HbF). We have shown that nitric oxide (NO) and the soluble guanylyl cyclase (sGC) pathways are involved in hydroxyurea induction of HbF levels in erythroid progenitor cells (EPCs). We demonstrate now that during erythroid differentiation, endothelial NO synthase mRNA and protein levels decline steadily, as does the production of NO derivatives and cyclic adenosine monophosphate (cAMP) levels, but guanosine 3',5'-cyclic monophosphate (cGMP) levels are stable. Hydroxyurea increased intracellular cGMP levels and cAMP levels in EPCs. The NO donor, DEANONOate, induced much higher cGMP levels, but reduced cAMP levels. Hydroxyurea (1 mM) induced production of approximately 45 pM cGMP/minute/ng of purified sGC, similar to induction by 1 muM DEANONOate. We found that hydroxyurea and ProliNONOate produced iron-nitrosyl derivatives of sGC. Thus, we confirm that hydroxyurea can directly interact with the deoxy-heme of sGC, presumably by a free-radical nitroxide pathway, and activate cGMP production. These data add to an expanding appreciation of the role of hydroxyurea as an inducer of the NO/cGMP pathway in EPCs. These mechanisms may also be involved in the cytostatic effects of hydroxyurea, as well as the induction of HbF.

Figure 1

Characterization of 2-phase liquid culture of erythroid cells. (A) NO production during erythroid differentiation (per 1 × 10⁶ cells; n = 3). (B) Total globins per total protein levels during erythroid differentiation of CD34⁺ cells. The heme group of globins gives a maximum absorbance at 415 nm, while 280 nm is the maximum absorbance for most proteins (because of tryptophan, tyrosine, and phenylalanine; n = 3). HPLC analyses of HbA and HbF during erythroid differentiation (n = 4). (C) Detection of extracellular NO levels with fluorescent indicator DAF-2 in erythroid progenitor cells at day 6, during treatment with bradykinin (n = 3). Values are means (± SEM). *P < .05 and **P < .01 compared with cells at day 4 (B) and untreated with bradykinin (C).

Figure 2

NOS levels during differentiation of erythroid cells. (A) eNOS mRNA levels during differentiation of erythroid cells. (B) iNOS mRNA levels during differentiation of erythroid cells; (C,D) eNOS protein levels during differentiation of erythroid cells: as measured by ELISA (C) and Western blotting compared with actin protein levels as a control (D). Vertical lines have been inserted to indicate a repositioned gel lane (n = 3). Values are means (± SEM). **P < .01 compared with cells at day 0.

Figure 3

Intracellular cGMP and cAMP levels in human erythroid progenitor cells. (A) Intracellular cGMP and cAMP levels in erythroid progenitor cells (left y-axis). Hydroxyurea (HU) dose-dependently inhibits erythroid cells growth after 24 and 48 hours of incubation (right y-axis). (B) Hydroxyurea (30 μM) and DEANONOate (50 μM) increased cGMP levels during 4 hours of incubation of human erythroid progenitor cells at day 4 of phase II liquid culture. (C) Hydroxyurea (30 μM) and DEANONOate (50 μM) increases and inhibits, respectively, cAMP levels during 4 hours of incubation of human erythroid progenitor cells at day 4 of phase II liquid culture (n = 3). Values are means (± SEM).

Figure 4

In vitro induction of purified sGC by hydroxyurea and NO donors.(A) Mixtures of purified sGC (0.5,1, 5, 10, and 50 ng), 3 mM MgCl₂, and 1 mM GTP were treated with DEANONOate (1 and 10 μM) during 10 minutes of incubation. (B) Mixtures of purified sGC (0.5, 1, 5, 10, and 50 ng), 3 mM MgCl₂ and 1 mM GTP were treated with hydroxyurea (0.5 and 1 mM) during 10 minutes of incubation. Background with 0 ng purified sGC, and the mixtures, was subtracted from the results (n = 3). Values are means ± SEM.

Figure 5

In vitro interaction between hydroxyurea and heme of sGC. (A) Sodium nitrite (0.001-10 mM) did not induce cGMP production of purified sGC as measured by radioimmunoassay. (B) Using tri-iodine–based reductive chemiluminescence assay, we measured the formation of the iron-nitrosyl complex during treatment of purified sGC with hydroxyurea and ProliNONOate. The numbers above columns represent percentage of conversion of deoxy-heme to the iron-nitrosyl complex (n = 3). Values are means (± SEM).