Notch1 and Notch2 Inhibit Myeloid Differentiation in Response to Different Cytokines

We have compared the ability of two mammalian Notch homologs, mouse Notch1 and Notch2, to inhibit the granulocytic differentiation of 32D myeloid progenitor cells. 32D cells undergo granulocytic differentiation when stimulated with either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of the activated intracellular domain of Notch1 inhibits the differentiation induced by G-CSF but not by GM-CSF; conversely, the corresponding domain of Notch2 inhibits differentiation in response to GM-CSF but not to G-CSF. The region immediately C-terminal to the cdc10 domain of Notch confers cytokine specificity on the cdc10 domain. The cytokine response patterns of Notch1 and Notch2 are transferred with this region, which we have termed the Notch cytokine response (NCR) region. The NCR region is also associated with differences in posttranslational modification and subcellular localization of the different Notch molecules. These findings suggest that the multiple forms of Notch found in mammals have structural differences that allow their function to be modulated by specific differentiation signals.     

Notch signaling enhances survival and alters differentiation of 32D myeloblasts

The Notch transmembrane receptors play important roles in precursor survival and cell fate specification during hematopoiesis. To investigate the function of Notch and the signaling events activated by Notch in myeloid development, we expressed truncated forms of Notch1 or Notch2 proteins that either can or cannot activate the core binding factor 1 (CBF1) in 32D (clone 3) myeloblasts. 32D cells proliferate as blasts in the presence of the cytokines, GM-CSF or IL-3, but they initiate differentiation and undergo granulopoiesis in the presence of granulocyte CSF (G-CSF). 32D cells expressing constitutively active forms of Notch1 or Notch2 proteins that signal through the CBF1 pathway maintained significantly higher numbers of viable cells and exhibited less cell death during G-CSF induction compared with controls. They also displayed enhanced entry into granulopoiesis, and inhibited postmitotic terminal differentiation. In contrast, Notch1 constructs that either lacked sequences necessary for CBF1 binding or that failed to localize to the nucleus had little effect. Elevated numbers of viable cells during G-CSF treatment were also observed in 32D cells overexpressing the basic helix-loop-helix protein (bHLH), HES1, consistent with activation of the CBF1 pathway. Taken together, our data suggest that Notch signaling enhances 32D cell survival, promotes entry into granulopoiesis, and inhibits postmitotic differentiation through a CBF1-dependent pathway.     

Selection of lineage-restricted cell lines immortalized at different stages of hematopoietic differentiation from the murine cell line 32D

Erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor- (G-CSF) dependent cell lines have been derived from the murine hematopoietic cell line 32D with a selection strategy involving the culture of the cells in FBS-deprived medium supplemented only with pure recombinant Epo, GM-CSF, or G-CSF. The cells retain the diploid karyotype of the original 32D clone, do not grow in the absence of exogenous growth factor, and do not induce tumors when injected into syngeneic recipients. The morphology of the Epo-dependent cell lines (32D Epo1, -2, and -3) was heterogeneous and evolved with passage. The percent of differentiated cells also was a function of the cell line investigated. Benzidine-positive cells ranged from 1-2% (32D Epo3) to 50-60% (32D Epo1). These erythroid cells expressed carbonic anhydrase I and/or globin mRNA but not carbonic anhydrase II. The GM-CSF- and G-CSF-dependent cell lines had predominantly the morphology of undifferentiated myeloblasts or metamyelocytes, respectively. The GM-CSF-dependent cell lines were sensitive to either GM-CSF or interleukin-3 (IL-3) but did not respond to G-CSF. The G-CSF-dependent cell lines grew to a limited extent in IL-3 but did not respond to GM-CSF. These results indicate that the cell line 32D, originally described as predominantly a basophil/mast cell line, has retained the capacity to give rise to cells which proliferate and differentiate in response to Epo, GM-CSF, and/or G-CSF. These cells represent the first nontransformed cell lines which can be maintained in growth factors other than IL-3 and which differentiate in the presence of physiologic signals. As such, they may represent a model to study the molecular mechanisms underlying the process of hematopoietic differentiation, as well as sensitive targets for bioassays of specific growth factors.     

Acceleration of granulocyte colony-stimulating factor-induced neutrophilic nuclear lobulation by overexpression of Lyn tyrosine kinase.

Stimulation with granulocyte colony-stimulating factor (G-CSF) induces myeloid precursor cells to differentiate into neutrophils, and tyrosine phosphorylation of certain cellular proteins is crucial to this process. However, the signaling pathways for neutrophil differentiation are still obscure. As the Src-like tyrosine kinase, Lyn, has been reported to play a role in G-CSF-induced proliferation in avian lymphoid cells, we examined its involvement in G-CSF-induced signal transduction in mammalian cells. Expression plasmids for wild-type Lyn (Lyn) and kinase-negative Lyn (LynKN) were introduced into a murine granulocyte precursor cell line, GM-I62M, that can respond to G-CSF with neutrophil differentiation, and cell lines that overexpressed these molecules (GM-Lyn, GM-LynKN) were established. Upon G-CSF stimulation, both the GM-Lyn and GM-LynKN cells began to differentiate into neutrophils, showing early morphological changes within a few days, much more rapidly than did the parental cells, which started to exhibit nuclear lobulation about 10 days after the cells were transferred to G-CSF-containing medium. However, the time course of expression of the myeloperoxidase gene, another neutrophil differentiation marker, was not affected by the overexpression of Lyn or LynKN. Therefore, in normal cells, protein interactions with Lyn, but not its kinase activity, are important for the induction of G-CSF-induced neutrophilic nuclear lobulation in mammalian granulopoiesis.     

Granulocyte colony-stimulating factor negatively regulates Toll-like receptor agonist-induced cytokine production in human neutrophils

We studied the effect of G-CSF on TLR agonist-induced cytokine production in human neutrophils. Human neutrophils produced IL-8 and TNF-α in response to stimulation with TLR agonists such as LPS and N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine. This response was dependent on activation of ERK, p38, and PI3K, but not JNK. TLR agonist-induced cytokine production in neutrophils was inhibited by G-CSF, whereas it was enhanced by GM-CSF, and GM-CSF-mediated enhancement was attenuated by G-CSF. G-CSF and GM-CSF did not affect TLR agonist-induced phosphorylation of ERK, p38, JNK, Akt, and IκBα. STAT3 activation was much greater in G-CSF-stimulated neutrophils than that in GM-CSF-stimulated cells. G-CSF-mediated STAT3 phosphorylation and inhibition of TLR agonist-induced cytokine production were prevented by pretreatment of cells with AG-490 (JAK2 inhibitor). These findings suggest that G-CSF and GM-CSF exert the opposite effects on TLR agonist-induced cytokine production, and G-CSF negatively regulates TLR agonist-induced cytokine production in neutrophils via activation of STAT3.     

Involvement of protein kinase Cepsilon in the negative regulation of Akt activation stimulated by granulocyte colony-stimulating factor

Stimulation of cells with G-CSF activates multiple signaling cascades, including the serine/threonine kinase Akt pathway. We show in this study that G-CSF-induced activation of Akt in myeloid 32D was specifically inhibited by treatment with PMA, a protein kinase C (PKC) activator. PMA treatment also rapidly attenuated sustained Akt activation mediated by a carboxy truncated G-CSF receptor, expressed in patients with acute myeloid leukemia evolving from severe congenital neutropenia. The inhibitory effect of PMA was abolished by pretreatment of cells with specific PKC inhibitor GF109203X, suggesting that the PKC pathway negatively regulates Akt activation. Ro31-8820, a PKCepsilon inhibitor, also abrogated PMA-mediated inhibition of Akt activation, whereas rottlerin and Go6976, inhibitors of PKCdelta and PKCalphabetaI, respectively, exhibited no significant effects. Furthermore, overexpression of the wild-type and a constitutively active, but not a kinase-dead, forms of PKCepsilon markedly attenuated Akt activation, and inhibited the proliferation and survival of cells in response to G-CSF. The expression of PKCepsilon was down-regulated with G-CSF-induced terminal granulocytic differentiation. Together, these results implicate PKCepsilon as a negative regulator of Akt activation stimulated by G-CSF and indicate that PKCepsilon plays a negative role in cell proliferation and survival in response to G-CSF.     

Resolution and purification of three distinct factors produced by Krebs ascites cells which have differentiation-inducing activity on murine myeloid leukemic cell lines

The use of different myeloid leukemic cell lines (WEHI-3B D+ and M1) and different sources of factors has led to discrepancies concerning the identity of factors capable of inducing differentiation in leukemic cells. We have biochemically fractionated medium conditioned by one such source (Krebs II ascites cells) and assayed fractions for their bone marrow colony-stimulating activity as well as their differentiation-inducing activity for WEHI-3B D+ and M1 cells. This resulted in the resolution of four distinct molecular species with differentiation-inducing activity. One activity was purified to homogeneity and shown by a variety of biochemical, biological, and receptor-binding criteria to be authentic granulocyte colony-stimulating factor (G-CSF). A second activity was identified as granulocyte-macrophage colony-stimulating factor (GM-CSF). Two other activities termed LIF-A and LIF-B (leukemia inhibitory factor) were shown to probably be different glycosylation variants of the same protein and one of these (LIF-A) was purified 12,000-fold to homogeneity. G-CSF induced differentiation in both WEHI-3B D+ and at higher concentrations M1 cells while GM-CSF weakly induced differentiation in WEHI-3B D+ cells. LIF-A had no colony-stimulating activity and induced differentiation in and inhibited the proliferation of only M1 cells. Each factor bound to a unique cell surface receptor with no evidence of direct cross-reactivity.     

G-CSF stimulates Jak2-dependent Gab2 phosphorylation leading to Erk1/2 activation and cell proliferation

Granulocyte colony-stimulating factor (G-CSF), the major cytokine regulator of neutrophilic granulopoiesis, stimulates both the proliferation and differentiation of myeloid precursors. A variety of signaling proteins have been identified as mediators of G-CSF signaling, but understanding of their specific interactions and organization into signaling pathways for particular cellular effects is incomplete. The present study examined the role of the scaffolding protein Grb2-associated binding protein-2 (Gab2) in G-CSF signaling. We found that a chemical inhibitor of Janus kinases inhibited G-CSF-stimulated Gab2 phosphorylation. Transfection with Jak2 antisense and dominant negative constructs also inhibited Gab2 phosphorylation in response to G-CSF. In addition, G-CSF enhanced the association of Jak2 with Gab2. In vitro, activated Jak2 directly phosphorylated specific Gab2 tyrosine residues. Mutagenesis studies revealed that Gab2 tyrosine 643 (Y643) was a major target of Jak2 in vitro, and a key residue for Jak2-dependent phosphorylation in intact cells. Mutation of Gab2 Y643 inhibited G-CSF-stimulated Erk1/2 activation and Shp2 binding to Gab2. Loss of Y643 also inhibited Gab2-mediated G-CSF-stimulated cell proliferation. Together, these results identify a novel signaling pathway involving Jak2-dependent Gab2 phosphorylation leading to Erk1/2 activation and cell proliferation in response to G-CSF.     

Tyrosine 763 of the murine granulocyte colony-stimulating factor receptor mediates Ras-dependent activation of the JNK/SAPK mitogen-activated protein kinase pathway.

The receptor for granulocyte colony-stimulating factor (G-CSF) can mediate differentiation and proliferation of hemopoietic cells. A proliferative signal is associated with activation of the ERK mitogen-activated protein kinase (MAPK) pathway. To determine whether other MAPK pathways are activated by G-CSF signalling, we have investigated activation of JNK/SAPK in cells proliferating in response to G-CSF. Here we show that G-CSF and interleukin-3 activate JNK/SAPK in two hemopoietic cell lines. The region of the G-CSF receptor required for G-CSF-induced JNK/SAPK activation is located within the C-terminal 68 amino acids of the cytoplasmic domain, which contains Tyr 763. Mutation of Tyr 763 to Phe completely blocks JNK/SAPK activation. However, the C-terminal 68 amino acids are not required for ERK2 activation. We show that activation of JNK/SAPK, like that of ERK2, is dependent on Ras but that higher levels of Ras-GTP are associated with activation of JNK/SAPK than with activation of ERK2. Two separate functional regions of the G-CSF receptor contribute to activation of Ras. The Y763F mutation reduces G-CSF-induced Ras activation from 30 to 35% Ras-GTP to 10 to 13% Ras-GTP. Low levels of Ras activation (10 to 13% Ras-GTP), which are sufficient for ERK2 activation, require only the 100 membrane-proximal amino acids. High levels of Ras-GTP provided by expression of oncogenic Ras are not sufficient to activate JNK/SAPK. An additional signal, also mediated by Tyr 763, is required for activation of JNK/SAPK.     

IL-1α and TNFα act synergistically to stimulate production of myeloid colony-stimulating factors by cultured human bone marrow stromal cells and cloned stromal cell strains

Human bone marrow stromal cells repond to stimulation by the monokines IL-1 and TNF by producing colony-stimulating factors such as GM-CSF and G-CSF. In this study we show that IL-1α and TNFα act synergistically to stimulate GM-CSF and G-CSF production by cultured marrow stromal cells. We further show that IL-1α and TNFα synergistically stimulate production of GM-CSF and G-CSF by a clonal stroma-derived cell strain. Although IL-1 and TNF share many of the same biological activities, we show that IL-1α and TNFα have an unequal ability to induce myeloid-CSF production by both cultures, with IL-1α being the more potent inducer. We found that induction by IL-1α and TNFα was independent of cell proliferation. The effect of IL-1α and TNFα on production of the two myeloid-CSFs by the clonal cells was significantly greater than the unfractionated passaged stromal cultures, having the greater effect on G-CSF production. The clonally derived stromal cells constitutively produced colony-stimulating activity, in particular GM-CSF, at levels easily detected by ELISA. These findings show that, in addition to the overlapping and additive activities of IL-1α and TNFα, they can interact synergistically. Our findings further suggest that a small subpopulation of stroma cells may be the major producer of G-CSF in the marrow microenvironment during immune response. © 1994 wiley-Liss, Inc.     

Interleukin 1 stimulates human endothelial cells to produce granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor

Endothelial cells are a potent source of hematopoietic growth factors when stimulated by soluble products of monocytes. Interleukin 1 (IL 1) is released by activated monocytes and is a mediator of the inflammatory response. We determined whether purified recombinant human IL 1 could stimulate cultured human umbilical vein endothelial cells to release hematopoietic growth factors. As little as 1 U/ml of IL 1 stimulated growth factor production by the endothelial cells, and increasing amounts of IL 1 enhanced growth factor production in a dose-dependent manner. Growth factor production increased within 2 to 4 hr and remained elevated for more than 48 hr. To investigate the molecular basis for these findings, oligonucleotide probes for granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and multi-CSF were hybridized to poly(A)-containing RNA prepared from unstimulated and IL 1-stimulated endothelial cells. Significant levels of GM-CSF and G-CSF, but not M-CSF or multi-CSF, mRNA were detected in the IL 1-stimulated endothelial cells. Biological assays performed on the IL 1-stimulated endothelial cell-conditioned medium confirmed the presence of both GM- and G-CSF. These results demonstrate that human recombinant IL 1 can stimulate endothelial cells to release GM-CSF and G-CSF, and provide a mechanism by which IL 1 could modulate both granulocyte production and function during the course of an inflammatory response.     

Tyrosine phosphorylation of receptor beta subunits and common substrates in response to interleukin-3 and granulocyte-macrophage colony-stimulating factor.

The receptors for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) consist of two polypeptides each belonging to a new class of molecules referred to as the hemopoietin receptor family. When expressed alone, receptor polypeptides of this family often bind their respective factors with lower affinity than the receptors identified in whole cells. Despite the lack of structural evidence for any enzymatic activity of the receptor polypeptides, both IL-3 and GM-CSF stimulate tyrosine phosphorylation of multiple intracellular substrates. We investigated IL-3 and GM-CSF receptor structure and signaling in a myeloid cell line, FDC-P1, which is dependent on either IL-3 or GM-CSF for growth. Antiphosphotyrosine antibodies were used to immunoprecipitate tyrosine-phosphorylated proteins from 32P-labeled cells or to probe immunoblots. Both IL-3 and GM-CSF stimulated the phosphorylation of a similar pattern of polypeptides on tyrosine. One tyrosine phosphorylated polypeptide migrated with M(r) = 135,000 and increased to 150,000 over a period of 10 min following stimulation of cells with IL-3 or GM-CSF. The M(r) = 135,000-150,000 polypeptide phosphorylated in response to IL-3 was shown to be primarily the Aic-2A polypeptide, the low affinity IL-3 receptor. Phosphatase treatment showed that the dramatic IL-3-induced shift in apparent molecular weight from M(r) = 125,000 in unstimulated cells was entirely due to phosphorylation. The closely related receptor, Aic-2B, was also tyrosine phosphorylated in response to IL-3, although to a lesser extent than Aic-2A. Treatment with GM-CSF resulted in tyrosine phosphorylation of the Aic-2B polypeptide exclusively. It was intriguing that GM-CSF treatment did affect the mobility of the Aic-2A polypeptide on polyacrylamide gels. Together, these results suggest that the Aic-2A polypeptide is part of the IL-3 receptor complex, but not the GM-CSF receptor. In contrast, the Aic-2B polypeptide is a component of the GM-CSF receptor, but it can also be utilized in an IL-3 receptor.