Severe congenital neutropenia (CN) is a hematological disorder characterized by an early stage maturation arrest of granulopoiesis in bone marrow at promyelocytic stage. Previously we found that LEF-1 and its target gene C/EBPα are severely abrogated in promyelocytes of CN patients, which plays a decisive role in the pathomechanism of defective granulopoiesis (

Skokowa J., et al.,
Nat. Med.
2006
;
12
:
1191
). Recently it has been shown that constitutively activated STAT5a inhibits myeloid differentiation of human CD34+ cells by downregulation of C/EBPα (
Moore MA, et al.,
Exp. Hematol.
2007
;
35
:
105
). Therefore, we investigated whether the presence of constitutive activated STAT5a in CD34+ cells of healthy individuals leads to downregulation of LEF-1 and whether STAT5a is activated in myeloid progenitors of CN patients. A screen of the 10kb upstream region of LEF-1 gene revealed two putative STAT5a binding sites (−3913 bp to −3894 bp and −3728 bp to −3709 bp) and specificity of the STAT5a binding to the LEF-1 promoter in nuclear extracts of CD34+ cells was confirmed in a chromatin immunoprecipitation (ChIP) assay. Additionally, our data demonstrated that introduction of retroviral constructs expressing IRES-GFP and cDNA of constitutively activated STAT5a (mutant of STAT5a, (STAT5A[1*6]) into CD34+ cells of healthy individuals resulted in 20-fold downregulation of LEF-1 mRNA expression. To compare G-CSF- dependent phosphorylation of STAT5 in CD33+ bone marrow myeloid progenitors of CN patients and healthy individuals, we treated these cells with G-CSF in vitro and measured phosphorylation of STAT5 by intracellular staining of cells with phosphoSTAT5 (Y694) antibody. We found that G-CSF stimulation resulted in a significantly higher phosphorylation of STAT5 in myeloid progenitors of four CN patients, as compared to four healthy volunteers (mean fluorescence intensity (MFI) 436 ± 57 in CN patients vs 212 ± 30 in healthy controls). Interestingly, augmented phosphorylation of STAT5 has been observed in both groups of CN patients carrying either ELA2 or HAX1 mutations. These data confirm the hypothesis that downregulation of LEF-1 and LEF-1 target gene C/EBPα is at least in part a consequence of increased activation of STAT5a in both CD34+ cells of healthy individuals and in patients with CN.

Topics:
congenital neutropenia, down-regulation, cd34 antigens, granulocyte colony-stimulating factor, recombinant granulocyte colony stimulating factor, antibodies, cd33 antigen, colony-stimulating factors, dna, complementary, immunoprecipitation

Author notes

Disclosure: No relevant conflicts of interest to declare.

2007, The American Society of Hematology
2007
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