Hydroxyurea attenuates activated neutrophil-mediated sickle erythrocyte membrane phosphatidylserine exposure and adhesion to pulmonary vascular endothelium.

Activated neutrophils increase erythrocyte phosphatidylserine (PS) exposure. PS-exposed sickle red blood cells (SSRBCs) are more adhesive to vascular endothelium than non-PS-exposed cells. An increase in SSRBC fetal hemoglobin (HbF) concentration has been associated with improved rheology and decreased numbers of vasoocclusive episodes. This study examined the effects of HbF, PS-exposed SSRBCs, and chronic hydroxyurea (HU) treatment on activated neutrophil-mediated SSRBC retention/adherence in isolated-perfused rat lungs. Lungs were perfused with erythrocyte suspensions from 1) individuals homozygous for hemoglobin S with 0-7% HbF (SS), 2) with > or =8% HbF (SS + F), and 3) individuals homozygous for hemoglobin S treated with HU therapy for > or =1 yr (SS + HU). Retention of SSRBCs from the SS + HU group was significantly less than that seen in both the SS and SS + F groups. No difference was observed between the SS and SS + F groups. The percentage of HbF and F-cells did not differ between the SS + F and SS + HU groups. At baseline, the proportion of PS-exposed SSRBCs was not different between the SS and SS + HU groups. However, SSRBC treatment with activated neutrophil supernatant caused a twofold increase in PS-exposed SSRBCs in the SS control and no change in the SS + HU group. We conclude that 1) HU attenuates SSRBC retention/adherence in the pulmonary circulation seen in response neutrophil activation, 2) HU stabilizes SSRBC membrane PS, and 3) HU attenuation SSRBC retention/adherence in the pulmonary circulation occurs through a mechanism(s) independent of HbF.

Fetal hemoglobin induction by histone deacetylase inhibitors involves generation of reactive oxygen species.

OBJECTIVE: Several compounds, including butyrate and trichostatin A, have been shown to activate gamma-gene expression via p38 mitogen-activated protein kinase (MAPK) signaling. In eukaryotic cells, reactive oxygen species (ROS) act as signaling molecules to mediate phosphorylation of tyrosine kinases such as p38 MAPK to regulate gene expression. Therefore, we determined the role of the reactive oxygen species hydrogen peroxide (H(2)O(2)) in drug-mediated fetal hemoglobin (HbF) induction. METHODS: H(2)O(2) levels were measured using 2',7'-dichlorofluorescein-diacetate in K562 cells after drug treatments. To confirm a role for H(2)O(2) in HbF induction, studies were completed with the mitochondrial respiratory chain inhibitor myxothiazole, which prevents ROS generation. The ability of myxothiazole to block gamma-globin mRNA accumulation and HbF induction was measured in K562 cells and burst-forming unit-erythroid colonies respectively using quantitative real-time PCR and alkaline denaturation. RESULTS: Butyrate and trichostastin A stimulated p38 MAPK phosphorylation via a H(2)O(2)-dependent mechanism. Pretreatment with myxothiazole to inhibit ROS formation or SB203580 to impede p38 MAPK signaling attenuated gamma-gene activation in K562 cells and HbF induction in erythroid progenitors. However, myxothiazole had no effect on the ability of hydroxyurea to induce HbF. CONCLUSION: The findings presented herein support a ROS-p38 MAPK cell signaling mechanism for HbF induction by butyrate and trichostatin A.

Differential effects of hydroxyurea and zileuton on interleukin-13 secretion by activated murine spleen cells: implication on the expression of vascular cell adhesion molecule-1 and vasoocclusion in sickle cell anemia.

BACKGROUND: Interleukin-13 (IL-13), a TH2 cytokine, upregulates the expression of vascular cell adhesion molecule-1 on endothelial cells, a factor involved in vasoocclusion in sickle cell disease (SCD). Hydroxyurea improves clinical status of SCD patients in part by induction of fetal hemoglobin. Its effect on IL-13 secretion has not been investigated. OBJECTIVE: To determine whether hydroxyurea and zileuton, a hydroxyurea derivative with antiinflammatory properties, affect IL-13 secretion. METHODS: We measured IL-13 in the supernatant of murine spleen cells incubated without and with hydroxyurea, zileuton (10 microg/ml), concanavalin A (2.5 microg/ml), and anti-CD3 (50 ng/ml) (n=8). RESULTS: Hydroxyurea and zileuton do not affect baseline IL-13 secretion. Unexpectedly, hydroxyurea increases IL-13 levels above baseline (120%, 216.5%, [p<0.05] after 24 h and 48 h, respectively) in lymphocytes activated by anti-CD3, while zileuton reduces them by 59%-78% (p<0.005). In lymphocytes activated by concanavalin A, hydroxyurea and zileuton reduce IL-13 secretion by 24-36% and 50-87%, respectively (p<0.05). Hydroxyurea, but not zileuton, significantly inhibits spleen cell proliferative responses to mitogens (p<0.005). CONCLUSION: Data suggest that hydroxyurea up-regulates IL-13 secretion in anti-CD3-activated lymphocytes through gene activation but not by altered cell proliferation. Increased IL-13 secretion may contribute to unresponsiveness of certain SCD patients to hydroxyurea. The potential benefit of zileuton in the management of vasoocclusion is discussed.

Zileuton induces hemoglobin F synthesis in erythroid progenitors: role of the L-arginine-nitric oxide signaling pathway.

Induction of fetal hemoglobin (Hb F) is an important therapeutic tool in ameliorating complications of sickle cell disease. Nitric oxide has been implicated in the mechanism of Hb F synthesis induced by hydroxyurea (HU). This study examined whether zileuton (ZL), a structural analog of hydroxyurea, possessed Hb F-inducing properties and the potential role nitric oxide plays. ZL caused a dose-dependent increase in gamma-globin expression in K562 cells. This effect was confirmed by a dose-dependent increase in Hb F synthesis in erythroid progenitors from individuals with sickle cell anemia and normal hemoglobin genotypes. l-arginine had no effect on Hb F production; however, it dose-dependently inhibited ZL's ability to induce Hb F. The nitric oxide synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA) inhibited l-arginine's effect and restored ZL-mediated increase in Hb F synthesis. In addition, 8-PCPT-cGMP (8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate) inhibited ZL-mediated induction of Hb F synthesis. When comparing l-NMMA effects alone on ZL and HU, a partial reversal of increased Hb F synthesis was seen only with HU. Neither l-arginine alone nor l-arginine in combination with l-NMMA effected hydroxyurea-mediated induction of Hb F synthesis. This study demonstrates that ZL induces Hb F through a mechanism that involves l-arginine/nitric oxide/cGMP in a manner distinctly different from HU.

p38 MAP kinase activation mediates gamma-globin gene induction in erythroid progenitors.

OBJECTIVE: Our goal was to determine the role of p38 mitogen-activated protein kinase (MAPK) signaling in fetal hemoglobin (HbF) induction. Two histone deacetylase inhibitors (HDAIs), sodium butyrate (NB), and trichostatin (TSA) and hemin were analyzed. In addition, the effect of direct activation of p38 MAPK on gamma-globin gene activity was studied. METHOD: Primary erythroid progenitors derived from peripheral blood mononuclear cell and K562 erythroleukemia cells were analyzed. Cells were grown in NB, TSA, hemin, or anisomycin either alone or in the presence of the p38 MAPK inhibitor SB203580. The effects of the various treatments on gamma-globin RNA, HbF, and phosphorylated p38 MAPK levels were measured by RNase protection assay, alkaline denaturation, and Western blot analysis, respectively. A K562 stable line overexpressing constitutively active p38 MAPK was established using MAPK kinase kinase 3 (MKK3) and MKK6, the immediate upstream activators of p38. The direct effect of p38 MAPK overexpression on gamma-globin mRNA synthesis was analyzed. RESULTS: NB and TSA activated p38 MAPK and increased gamma-globin mRNA levels in K562 cells and primary erythroid progenitors. Pretreatment with SB203580 blocked p38 MAPK and gamma-globin gene activation. In contrast, no change in p38 activity was observed with hemin inductions. Direct activation of p38 by anisomycin or constitutive overexpression also increased gamma-globin mRNA in the absence of HbF inducers in wild-type K562 cells and in the MKK stable lines. CONCLUSION: This study supports a novel role for p38 MAPK in gamma-globin regulation in human erythroid progenitors.

Stat3 beta inhibits gamma-globin gene expression in erythroid cells.

We demonstrated previously gamma-globin gene inhibition in K562 cells and primary erythroid progenitors treated with interleukin-6. Although several cis-acting elements have been identified in the globin promoters, the precise mechanism for cytokine-mediated globin gene regulation remains to be elucidated. In this report we demonstrate inhibitors of Stat3 phosphorylation abrogate interleukin-6-mediated gamma gene silencing in erythroid cells. DNA-protein binding studies established Stat3 interaction in the 5'-untranslated gamma-globin promoter region. Furthermore, co-transfection experiments with Stat3 beta demonstrate gamma promoter inhibition in a concentration-dependent manner, which was significantly reversed when the cognate Stat3-binding site in the 5'-untranslated region was mutated. These studies establish a novel mechanism for gamma gene silencing through the STAT signal transduction pathway.