Abstract
Deficiency of the interferon consensus sequence-binding protein (ICSBP) is associated with increased myeloid cell proliferation in response to hematopoietic cytokines. However, previously identified ICSBP target genes do not indicate a mechanism for this "cytokine hypersensitivity." In these studies, we identify the gene encoding neurofibromin 1 (Nf1) as an ICSBP target gene, by chromatin immunoprecipitation. Additionally, we find decreased Nf1 expression in bone marrow-derived myeloid cells from ICSBP-/- mice. Since Nf1 deficiency is also associated with cytokine hypersensitivity, our results suggested that NF1 is a functionally significant ICSBP target gene. Consistent with this, we find that the hypersensitivity of ICSBP-/- myeloid cells to granulocyte monocyte colony-stimulating factor (GM-CSF) is reversed by expression of the Nf1 GAP-related domain. We also find that treatment of ICSBP-deficient myeloid cells with monocyte colony-stimulating factor (M-CSF) results in sustained Ras activation, ERK phosphorylation, and proliferation associated with impaired Nf1 expression. These M-CSF effects are reversed by ICSBP expression in ICSBP-/- cells. Consistent with this, we find that ICSBP activates the NF1 promoter in myeloid cell line transfectants and identify an ICSBP-binding NF1 cis element. Therefore, the absence of ICSBP leads to Nf1 deficiency, impairing down-regulation of Ras activation by GM-CSF or M-CSF. These results suggest that one mechanism of increased myeloid proliferation, in ICSBP-deficient cells, is decreased NF1 gene transcription. This novel ICSBP function provides insight into regulation of myelopoiesis under normal conditions and in myeloproliferative disorders.
Highlights
Tory factor (IRF) family of transcription factors (1)
We found that expression of either interferon consensus sequencebinding protein (ICSBP) or the neurofibromin 1 (Nf1)-GAPrelated domain (GRD) decreases phosphorylated ERK in granulocyte monocyte colony-stimulating factor (GM-CSF) ϩ IL-3 in comparison with vector control-transduced ICSBPϪ/Ϫ cells
We found that GM-CSF (0.01–10.0 ng/ml) induces significantly more proliferation in control vector-transduced ICSBPϪ/Ϫ cells than in cells transduced with a vector to express ICSBP or the Nf1-GAP-related domain (Nf1-GRD) (p Ͻ 0.05, n ϭ 4) (Fig. 4A)
Summary
Tory factor (IRF) family of transcription factors (1). ICSBP is expressed exclusively in myeloid and B-cells (1) and activates genes involved in the inflammatory response. Activation of an artificial promoter construct with the PRDI consensus sequence involves interaction between ICSBP and IRF1, which requires ICSBP tyrosine phosphorylation (7). BclXL is expressed in mature myeloid cells in response to inflammatory mediators, such as lipopolysaccharide and interferon-␥ (9) In this context, BclXL expression increases survival of activated phagocytes (9). BclXL expression increases survival of activated phagocytes (9) Based upon these identified target genes, one would anticipate ICSBP deficiency to be characterized by terminal differentiation block and immune dysfunction. Mutations that inactivate Nf1 or activate Ras have been described in the myeloproliferative disorder, juvenile chronic myelomonocytic leukemia (17) This disorder is characterized by GM-CSF and stem cell factor hypersensitivity (18). In Nf1-deficient hematopoietic cells, increased Ras activity increases ERK and Akt activation, increasing proliferation (19) and decreasing apoptosis (20)
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