Theresa Zibello

C/EBP epsilon mediates myeloid differentiation and is regulated by the CCAAT displacement protein (CDP/cut).

Neutrophils from CCAAT enhancer binding protein epsilon (C/EBPɛ) knockout mice have morphological and biochemical features similar to those observed in patients with an extremely rare congenital disorder called neutrophil-specific secondary granule deficiency (SGD). SGD is characterized by frequent bacterial infections attributed, in part, to the lack of neutrophil secondary granule proteins (SGP). A mutation that results in loss of functional C/EBPɛ activity has recently been described in an SGD patient, and has been postulated to be the cause of the disease in this patient. We have previously demonstrated that overexpression of CCAAT displacement protein (CDP/cut), a highly conserved transcriptional repressor of developmentally regulated genes, suppresses expression of SGP genes in 32Dcl3 cells. This phenotype resembles that observed in both C/EBPɛ−/− mice and in SGD patients. Based on these observations we investigated potential interactions between C/EBPɛ and CDP/cut during neutrophil maturation. In this study, we demonstrate that inducible expression of C/EBPɛ in 32Dcl3/tet cells results in granulocytic differentiation. Furthermore, Northern blot analysis of G-CSF-induced CDP/cut overexpressing 32Dcl3 cells revealed absence of C/EBPɛ mRNA. We therefore hypothesize that C/EBPɛ positively regulates SGP gene expression, and that C/EBPɛ is itself negatively regulated by CDP/cut during neutrophil maturation. We further demonstrate that the C/EBPɛ promoter is regulated by CDP/cut during myeloid differentiation.

C/EBPε directs granulocytic-vs-monocytic lineage determination and confers chemotactic function via Hlx

OBJECTIVE Mutations in the CCAAT enhancer binding protein epsilon (C/EBPepsilon) gene have been identified in the cells of patients with neutrophil specific granule deficiency, a rare congenital disorder marked by recurrent bacterial infections. Their neutrophils, in addition to lacking specific granules required for normal respiratory burst activity, also lack normal phagocytosis and chemotaxis. Although the specific granule deficiency phenotype has been replicated in C/EBPepsilon(-/-) (knockout [KO]) mice, the mechanisms by which C/EBPepsilon mutations act to decrease neutrophil function are not entirely clear. MATERIALS AND METHODS In order to determine the role of C/EBPepsilon in neutrophil differentiation and migration, we generated immortalized progenitor cell lines from C/EBPepsilon KO and wild-type mice and performed expression and flow cytometric analysis and functional studies. RESULTS Expression of lineage-specific cell surface antigens on our in vitro differentiated cell lines revealed persistent expression of monocytic markers on KO granulocytes. We verified this in primary murine peripheral blood and bone marrow cells. In addition, KO bone marrow had an increase in immature myeloid precursors at the common myeloid progenitor and granulocyte/monocyte progenitor levels, suggesting a critical role for C/EBPepsilon not only in granulocyte maturation beyond the promyelocyte stage, but also in the monocyte/granulocyte lineage decision. We found that restoration of Hlx (H2.0-like homeo box 1) expression, which was decreased in C/EBPepsilon KO cells, rescued chemotaxis, but not the other defects of C/EBPepsilon KO neutrophils. CONCLUSIONS We show two new regulatory functions of C/EBPepsilon in myelopoiesis: in the absence of C/EBPepsilon, there is not only incomplete differentiation of granulocytes, but myelopoiesis is disrupted with the appearance of an intermediate cell type with monocyte and granulocyte features, and the neutrophils have abnormal chemotaxis. Restoration of expression of Hlx provides partial recovery of function; it has no effect on neutrophil maturation, but can completely ameliorate the chemotaxis defect in C/EBPepsilon KO cells.

Modulation of a calcium/calmodulin-dependent protein kinase cascade by retinoic acid during neutrophil maturation.

Retinoic acid is a lipophilic derivative of vitamin A that can cause differentiation in a variety of cell types. A large body of evidence has shown that normal retinoid signaling is required for proper neutrophil maturation in vitro and in vivo. In this study, we have found that calcium/calmodulin dependent (CaM) protein kinase kinase alpha (CaMKKalpha) is upregulated in an immediate early fashion during retinoic acid induced neutrophil maturation. Furthermore, we describe the expression and modulation of various components of the CaM kinase cascade during neutrophil maturation. We have confirmed upregulation of CaMKKalpha protein by Western analysis and further show that CaMKKbeta is expressed, although its protein levels are constant throughout induction. We also find that neutrophil progenitor cells express both CaMKI and CaMKIV transcripts. RNase protection and Western analysis show that CaMKIV is downregulated during neutrophil maturation. In contrast, CaMKI transcript and protein is expressed in uninduced cells and is induced by all-trans retinoic acid. These data represent the first report of a CaM kinase cascade in myeloid cells and suggests that this cascade may mediate some of the well-characterized effects of calcium on neutrophil function. These observations also support the idea that the retinoic acid receptors play a major role in mediating neutrophil specific gene expression and differentiation.

Coordinate Regulation of Neutrophil Secondary Granule Protein Gene Expression

Evidence from study of both normal and leukemic cells suggests that a crucial step in neutrophil maturation occurs in the transition from the promyelocyte to the myelocyte stage. The transition from the promyelocyte to the myelocyte in normal marrow cells is accompanied both by the loss of proliferative capacity associated with terminal maturation [1], and by the loss of the capacity for alternative maturation [2]. Normal promyelocytes respond to stimuli of both granulocyte and monocyte differentiation, but myelocytes are restricted to terminal granulocyte maturation [2]. In this regard, it is striking that acute myeloid leukemias invariably involve proliferation of cells arrested in development at or before the promyelocyte stage. Consequently, an understanding of the transition from the promyelocyte to the myelocyte stage should provide crucial insights into both the control mechanisms governing normal hematopoietic cell differentiation and the ways in which disruption of the control mechanisms can contribute to leukemic transformation.

p120 nucleolar-proliferating antigen is a direct target of G-CSF signaling during myeloid differentiation.

Granulocyte‐colony stimulating factor (G‐CSF) is an essential cytokine, which contributes to proliferation and differentiation of granulocyte precursor cells in the bone marrow. Despite recent progress in understanding G‐CSF signaling events, the mechanisms that underlie the distinct spectrum of biological functions attributed to G‐CSF‐mediated gene expression remain unclear. Previous studies have identified a number of genes, which are up‐regulated in G‐CSF‐stimulated myeloid precursor cells. In this study, we sought to identify additional target genes of G‐CSF‐mediated proliferation and/or differentiation. cDNA representational difference analysis was used with the 32Dcl3 cell line as a model system to isolate genes, which are up‐regulated in an immediate‐early manner upon G‐CSF stimualtion. We isolated p120 nucleolar‐proliferation antigen (NOL1), a highly conserved, nucleolar‐specific, RNA‐binding protein of unknown function, and confirmed its expression by Northern blot analysis in 4‐h, G‐CSF‐induced 32Dcl3 cells. Isolation of a mouse p120 genomic clone revealed the presence of a signal tranducer and activator of transcription (STAT)‐binding site in the first intron of the gene. We demonstrate the importance of STAT3 and STAT5 in mediating the G‐CSF response with respect to p120 expression by transient transfection analysis, oligonucleotide pull‐down assays, and the loss of p120 expression in the bone marrow of mice lacking normal STAT3 signaling. In addition, overexpression of p120 in G‐CSF‐induced 32D cells revealed normal, morphologic maturation and growth characteristics but loss of lactoferrin expression, a marker of normal neutrophil maturation, suggesting that inappropriate expression of the p120 gene can result in aberrant neutrophil maturation.