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All mice harboring the X-linked Gata1 low mutation in a predominantly CD1 background are born anemic and thrombocytopenic. They recover from anemia at 1 month of age but remain thrombocytopenic all their life and develop myelofibrosis, a syndrome similar to human idiopathic myelofibrosis, at 12 months. The effects of the genetic background on the myelofibrosis developed by Gata1 low mice was assessed by introducing the mutation, by standard genetic approaches, in the C57BL/6 and DBA/2 backgrounds and by analyzing the phenotype of the different mutants at 12 to 13 (by histology) and 16 to 20 (by cytofluorimetry) months of age. Although all the Gata1 low mice developed fibrosis at 12 to 13 months, variegations were observed in the severity of the phenotype expressed by mutants of different backgrounds. In C57BL/6 mice, the mutation was no longer inherited in a Mendelian fashion, and fibrosis was associated with massive osteosclerosis. Instead, DBA/2 mutants, although severely anemic, expressed limited fibrosis and osteosclerosis and did not present tear-drop poikilocytes in blood or extramedullary hemopoiesis in liver up to 20 months of age. We propose that the variegation in myelofibrosis expressed by

Glucocorticoid receptor (GR) agonists increase erythropoiesis in vivo and in vitro.To clarify the effect of the dominant negative GR␤ isoform (unable to bind STAT-5) on erythropoiesis, erythroblast (EB) expansion cultures of mononuclear cells from 18 healthy (nondiseased) donors (NDs) and 16 patients with polycythemia vera (PV) were studied. GR␤ was expressed in all PV EBs but only in EBs from 1 ND. The A3669G polymorphism, which stabilizes GR␤ mRNA, had greater frequency in PV (55%; n ‫؍‬ 22; P ‫؍‬ .0028) and myelofibrosis (35%; n ‫؍‬ 20) patients than in NDs (9%; n ‫؍‬ 22) or patients with essential thrombocythemia (6%; n ‫؍‬ 15). Dexamethasone stimulation of ND cultures increased the number of immature EBs characterized by low GATA1 and ␤-globin expression, but PV cultures generated great numbers of immature EBs with low levels of GATA1 and ␤-globin irrespective of dexamethasone stimulation. In ND EBs, STAT-5 was not phosphorylated after dexamethasone and erythropoietin treatment and did not form transcriptionally active complexes with GR␣, whereas in PV EBs, STAT-5 was constitutively phosphorylated, but the formation of GR/STAT-5 complexes was prevented by expression of GR␤. These data indicate that GR␤ expression and the presence of A3669G likely contribute to development of erythrocytosis in PV and provide a potential target for identification of novel therapeutic agents. (Blood. 2011;118(2):425-436) IntroductionPolycythemia vera (PV) is a Philadelphia chromosome-negative myeloproliferative neoplasm (MPN) characterized by increased production of erythroid cells. 1 As in other MPNs, PV is associated with a gain-of-function mutation (JAK2V617F) of the JAK2 gene. [2][3][4] JAK2 is the first transduction element of many hematopoietic growth factor receptors, including the receptor for erythropoietin, the primary growth factor that controls erythroid cell production. 5 The observation that inhibition of JAK2V617F abrogates erythropoietin-independent growth of erythroid progenitors suggested the hypothesis that in PV, increased erythroid production is caused by constitutive activation of erythropoietin receptor signaling (STAT-5 and/or PI3K/Akt). [6][7][8] Because low and high levels of STAT-5 activation favor maturation and proliferation, respectively, in normal hematopoietic cells, 9,10 the presence of the JAK2V617F mutation in PV may increase the intrinsic proliferative potential of erythroid cells by increasing This concept is indirectly supported by the observation that hematopoietic progenitor cells from PV patients generate greater numbers of erythroblasts (EBs) in liquid culture than cells from healthy (nondiseased) donors (NDs). 7,12 In addition to erythropoietin, erythroid proliferation is also controlled by nuclear receptors such as the glucocorticoid receptor (GR). Evidence for this regulatory role is provided by clinical observations and in vitro studies of cultured EBs. Patients may develop erythrocytosis as the first manifestation of Cushing syndrome. 13 In addition, 50% of patients with Diamond-...

We have identified two new histone deacetylase (HDAC) inhibitors (9 and 24) capable of inducing the expression of ␥-globin and/or ␤-globin promoter-driven reporter genes in a synthetic model of Hb switch. Both compounds also increased, with different mechanisms, the ␥/(␥ϩ␤) ratio expressed in vitro by normal human erythroblasts. Compound 9 increased the levels of ␥-globin mRNA and the ␥/(␥ϩ␤) ratio (both by 2-fold). Compound 24 increased by 3-fold the level of ␥-globin and decreased by 2-fold that of ␤-globin mRNA, increasing the ␥/(␥ϩ␤) ratio by 6-fold, and raising (by 50%) the cell HbF content. Both compounds raised the acetylation state of histone H4 in primary cells, an indication that their activity was mediated through HDAC inhibition. Compounds 9 and 24 were also tested as ␥/(␥ϩ␤) mRNA inducers in erythroblasts obtained from patients with ␤ 0 thalassemia. Progenitor cells from patients with ␤ 0 thalassemia generated in vitro morphologically normal proerythroblasts that, unlike normal cells, failed to mature in the presence of EPO and expressed low ␤-globin levels but 10 times higher-than-normal levels of the ␣ hemoglobin-stabilizing protein (AHSP) mRNA. Both compounds ameliorated the impaired in vitro maturation in ␤ 0 thalassemic erythroblasts, decreasing AHSP expression to normal levels. In the case of two patients (of five analyzed), the improved erythroblast maturation was associated with detectable increases in the ␥/(␥ϩ␤) mRNA ratio. The low toxicity exerted by compounds 9 and 24 in all of the assays investigated suggests that these new HDAC inhibitors should be considered for personalized therapy of selected patients with ␤ 0 thalassemia.

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The high number (>10(8-10)) of primary human pro-erythroblasts (CD36high/CD235alow) obtainable in HEMA culture (Migliaccio et al., 2002) is exploited here to analyse the expression of proteins implicated in erythropoietin (EPO)-signalling (STATs, PI-3K, and PLCs) during the process of erythroid maturation. Human pro-erythroblasts progressed in 4 days of culture with EPO into basophilic- (CD36high/CD235amedium, 24 h), polychromatic-(CD36high/CD235ahigh, 48 h), and, finally, orthochromatic-(CD36low/CD235ahigh, 72-96 h) erythroblasts. During this maturation, STAT-1 was expressed up to the orthochromatic stage, expression of STAT-5, as well as of its target proteins BclxL and IRF1, remained constant up to 48 h (polychromatic-erythroblasts) but decreased by 96 h (orthochromatic-erythroblasts), while that of STAT-3 decreased constantly from 24 h on and became undetectable by 96 h. Expression of PI-3K rapidly decreased with differentiation since only 50% of original protein levels were detected by 48 h. On the other hand, among the members of PLC families investigated, PLC beta4 was not expressed, PLC beta2, delta1, and gamma2 were expressed at constant levels throughout the maturation process, while expression of PLC beta3 and of PLC gamma1 decreased, as PI-3K, by 24 h and that of PLC beta1 was induced by 6 h and became undetectable by 24 h. In conclusion, these data depict the dynamic signalling scenario associated with the maturation of erythroid cells and provide the first indication that members of PLC families (PLC beta1, beta3, and gamma1) might be involved in the control of erythroid differentiation in humans.

Ex vivo expanded erythroblasts from normal donors express modestly imbalanced levels of alpha-globin and gamma + beta-globin fully compensated by AHSP expression, likely ensuring normal function and survival.