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Cited by 150Neurofibromatosis and Schwannoma CasesProtein Tyrosine PhosphatasesAcute Myeloid Leukemia Research

The DOI matches its Crossref record; 68 of the 70 references it cites were found in a registry or an index.

Nf1 Regulates Hematopoietic Progenitor Cell Growth and Ras Signaling in Response to Multiple Cytokines

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Zhang, You-Yan, Vik, Terry A., Ryder, John W., Srour, Edward F., Jacks, Tyler, Shannon, Kevin, Wade Clapp, D.·1998·The Journal of Experimental Medicine
DOI 10.1084/jem.187.11.1893PMC PMC2212307
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Abstract

Neurofibromin, the protein encoded by the NF1 tumor-suppressor gene, negatively regulates the output of p21ras (Ras) proteins by accelerating the hydrolysis of active Ras-guanosine triphosphate to inactive Ras-guanosine diphosphate. Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile chronic myelogenous leukemia (JCML) and other malignant myeloid disorders, and heterozygous Nf1 knockout mice spontaneously develop a myeloid disorder that resembles JCML. Both human and murine leukemias show loss of the normal allele. JCML cells and Nf1−/− hematopoietic cells isolated from fetal livers selectively form abnormally high numbers of colonies derived from granulocyte-macrophage progenitors in cultures supplemented with low concentrations of granulocyte-macrophage colony stimulating factor (GM-CSF). Taken together, these data suggest that neurofibromin is required to downregulate Ras activation in myeloid cells exposed to GM-CSF. We have investigated the growth and proliferation of purified populations of hematopoietic progenitor cells isolated from Nf1 knockout mice in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF), as well as to GM-CSF. We found abnormal proliferation of both immature and lineage-restricted progenitor populations, and we observed increased synergy between SCF and either IL-3 or GM-CSF in Nf1−/− progenitors. Nf1−/− fetal livers also showed an absolute increase in the numbers of immature progenitors. We further demonstrate constitutive activation of the Ras-Raf-MAP (mitogen-activated protein) kinase signaling pathway in primary c-kit+ Nf1−/− progenitors and hyperactivation of MAP kinase after growth factor stimulation. The results of these experiments in primary hematopoietic cells implicate Nf1 as playing a central role in regulating the proliferation and survival of primitive and lineage-restricted myeloid progenitors in response to multiple cytokines by modulating Ras output.

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Figures

Figure 1 Isolation of Sca1+lin−/dim hematopoietic progenitors from fetal liver cells. Pooled day 13.5 gestation fetal liver cells from each respective genotype were prepared for culture by isolation of low density cells using a Ficoll-Hypaque gradient. Low density cells were simultaneously stained with isotype controls or antibodies to Sca1 (PE) and lineage markers including anti-B220 (pan–B lymphocytes), anti-CD3ε (pan–T cells), anti–Gr-1 (granulocytes), and anti–Mac 1 (monocyte-macrophages) all conjugated to FITC. Sca1+lin−/dim cells were identified relative to isotype control antibodies in the upper left corner of the dual parameter dot plot in A. These cells were isolated by fluorescence-activated cell sorting and a sample of sorted cells was reanalyzed to confirm the purity of isolated cells (B).
Figure 1 Isolation of Sca1+lin−/dim hematopoietic progenitors from fetal liver cells. Pooled day 13.5 gestation fetal liver cells from each respective genotype were prepared for culture by isolation of low density cells using a Ficoll-Hypaque gradient. Low density cells were simultaneously stained with isotype controls or antibodies to Sca1 (PE) and lineage markers including anti-B220 (pan–B lymphocytes), anti-CD3ε (pan–T cells), anti–Gr-1 (granulocytes), and anti–Mac 1 (monocyte-macrophages) all conjugated to FITC. Sca1+lin−/dim cells were identified relative to isotype control antibodies in the upper left corner of the dual parameter dot plot in A. These cells were isolated by fluorescence-activated cell sorting and a sample of sorted cells was reanalyzed to confirm the purity of isolated cells (B).CC BY-NC-SA · PubMed Central
Figure 2 Dose–response curves of progenitor colony growth in response to cytokines. Growth of Sca1+lin−/dim cells from wild-type (Nf1 +/+), heterozygous (Nf1 +/−), and homozygous (Nf1 −/−) mice is shown at the indicated concentrations of mGM-CSF (a), mIL-3 (b), mGM-CSF and 10 ng/ml SCF (c), mIL-3 and 10 ng/ml mSCF (d), or mSCF (e). The data are expressed as percentage of maximal numbers of CFU-GM colonies.
Figure 2 Dose–response curves of progenitor colony growth in response to cytokines. Growth of Sca1+lin−/dim cells from wild-type (Nf1 +/+), heterozygous (Nf1 +/−), and homozygous (Nf1 −/−) mice is shown at the indicated concentrations of mGM-CSF (a), mIL-3 (b), mGM-CSF and 10 ng/ml SCF (c), mIL-3 and 10 ng/ml mSCF (d), or mSCF (e). The data are expressed as percentage of maximal numbers of CFU-GM colonies.CC BY-NC-SA · PubMed Central
Figure 3 Progenitor colony growth in response to varying concentrations of IL-3 and SCF in the presence of neutralizing anti–GM-CSF antibodies. Growth of Sca1+lin−/dim from wild-type (Nf1 +/+) and homozygous (Nf1 −/−) mice is shown at the indicated concentrations of mIL-3 and 10 ng SCF in the presence of anti– GM-CSF neutralizing antibodies or isotype control antibodies.
Figure 3 Progenitor colony growth in response to varying concentrations of IL-3 and SCF in the presence of neutralizing anti–GM-CSF antibodies. Growth of Sca1+lin−/dim from wild-type (Nf1 +/+) and homozygous (Nf1 −/−) mice is shown at the indicated concentrations of mIL-3 and 10 ng SCF in the presence of anti– GM-CSF neutralizing antibodies or isotype control antibodies.CC BY-NC-SA · PubMed Central
Figure 4 Growth of colonies derived from primitive progenitors in fetal livers. Total numbers of HPP-CFC from wild-type (Nf1 +/+), heterozygous (Nf1 +/−), and homozygous (Nf1 −/−) fetal livers are shown after culture in saturating concentrations of SCF, M-CSF, IL-1α, and either GM-CSF (A) or IL-3 (B).
Figure 4 Growth of colonies derived from primitive progenitors in fetal livers. Total numbers of HPP-CFC from wild-type (Nf1 +/+), heterozygous (Nf1 +/−), and homozygous (Nf1 −/−) fetal livers are shown after culture in saturating concentrations of SCF, M-CSF, IL-1α, and either GM-CSF (A) or IL-3 (B).CC BY-NC-SA · PubMed Central
Figure 5 MAP kinase activity in c-kit+ hematopoietic cells. c-kit+ bone marrow cells from mice previously reconstituted with Nf1 −/− or Nf1 +/+ fetal liver cells were isolated, stimulated with cytokines for specified times, and harvested, and cell lysates were prepared. MAP kinase activity was measured in whole cell extracts. Activity was calculated by determining the rate of incorporation of phosphate in MBP per min/μg of lysate and is indicated in the graph for each genotype, cytokine, and length of stimulation. The genotype and length of stimulation are indicated.
Figure 5 MAP kinase activity in c-kit+ hematopoietic cells. c-kit+ bone marrow cells from mice previously reconstituted with Nf1 −/− or Nf1 +/+ fetal liver cells were isolated, stimulated with cytokines for specified times, and harvested, and cell lysates were prepared. MAP kinase activity was measured in whole cell extracts. Activity was calculated by determining the rate of incorporation of phosphate in MBP per min/μg of lysate and is indicated in the graph for each genotype, cytokine, and length of stimulation. The genotype and length of stimulation are indicated.CC BY-NC-SA · PubMed Central
Figure 6 Inhibition of MAP kinase activity in Nf1 −/− c-kit+ cells exposed to MEK inhibitor before stimulation with cytokines. c-kit+ bone marrow cells from mice reconstituted with Nf1 −/− fetal liver cells (left) or Nf1 +/+ (right) were exposed to a MEK inhibitor or vehicle control before stimulation with a maximum stimulating capacity of IL-3 and SCF. Cells were lysed 5 min after stimulation with cytokines. The autoradiographs and activities of one of three independent experiments is shown. The cytokine and MEK inhibitor treatment of cells is indicated by + or −.
Figure 6 Inhibition of MAP kinase activity in Nf1 −/− c-kit+ cells exposed to MEK inhibitor before stimulation with cytokines. c-kit+ bone marrow cells from mice reconstituted with Nf1 −/− fetal liver cells (left) or Nf1 +/+ (right) were exposed to a MEK inhibitor or vehicle control before stimulation with a maximum stimulating capacity of IL-3 and SCF. Cells were lysed 5 min after stimulation with cytokines. The autoradiographs and activities of one of three independent experiments is shown. The cytokine and MEK inhibitor treatment of cells is indicated by + or −.CC BY-NC-SA · PubMed Central
Figure 7 Effect of preincubation with an MEK inhibitor on progenitor colony formation from Nf1 −/− cells. c-kit+ bone marrow cells (4 × 104) were cultured with SCF and IL-3 for 48 h in the presence of the MEK inhibitor (MEKI) or the vehicle control (DMSO). After liquid culture, progenitor assays for growth of HPP-CFC and LPP-CFC were established. The total number of nucleated cells (A), LPP-CFC (B), and HPP-CFC (C) after culture for each genotype and condition are indicated. Based on cultures established in agar before treatment with the MEK inhibitor, 1,850 LPP-CFCs ± 10 SEM and 260 HPP-CFC ± 40 SEM per well were present in the input Nf1 −/− cells, and 825 LPP-CFC ± 205 SEM and 100 HPP-CFC ± 40 SEM were present in input Nf1 +/+ cells.
Figure 7 Effect of preincubation with an MEK inhibitor on progenitor colony formation from Nf1 −/− cells. c-kit+ bone marrow cells (4 × 104) were cultured with SCF and IL-3 for 48 h in the presence of the MEK inhibitor (MEKI) or the vehicle control (DMSO). After liquid culture, progenitor assays for growth of HPP-CFC and LPP-CFC were established. The total number of nucleated cells (A), LPP-CFC (B), and HPP-CFC (C) after culture for each genotype and condition are indicated. Based on cultures established in agar before treatment with the MEK inhibitor, 1,850 LPP-CFCs ± 10 SEM and 260 HPP-CFC ± 40 SEM per well were present in the input Nf1 −/− cells, and 825 LPP-CFC ± 205 SEM and 100 HPP-CFC ± 40 SEM were present in input Nf1 +/+ cells.CC BY-NC-SA · PubMed Central

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Research works printed but not linked: 2

  1. Bos, 1989, rasoncogenes in human cancer: a review, Cancer Res, 49, 4682
  2. Rodenhuis, 1992, rasand human tumors, Semin Cancer Biol, 3, 241

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Metadata

Authors
Zhang, Y.-Y., Vik, T. A., Ryder, J. W., Srour, E. F., Jacks, T., Shannon, K., & Wade Clapp, D.
Affiliations
  • From the *Department of Pediatrics, the ‡Department of Microbiology and Immunology, the §Department of Medicine, and ‖The Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana 46202; the ¶Howard Hughes Medical Institute and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; and the **Department of Pediatrics, University of California San Francisco, San Francisco, California 94143
  • Howard Hughes Medical Institute (US)
ORCID
Venue
The Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
1998-06-01
Volume
187
Issue
11
Pages
1893-1902
ISSN
  • 0022-1007 (ISSN-L)
  • 1540-9538
DOI
10.1084/jem.187.11.1893
PMC
PMC2212307
OpenAlex
W2156597055
Cited by
150
References
70
Citations by year
2026: 3 · 2025: 1 · 2024: 1 · 2023: 3 · 2022: 2 · 2021: 1 · 2020: 2 · 2019: 3 · 2017: 2 · 2016: 4 · 2015: 2 · 2014: 2 · 2013: 3 · 2012: 6
FWCI
6.35
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • The Journal of Experimental Medicine (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
  • IUScholarWorks (Indiana University) (submittedVersion, cc-by-nc-sa)
Funders
  • National Cancer Institute (R29 CA74177-01, R29CA074177)
  • National Institute of Diabetes and Digestive and Kidney Diseases (P50DK049218)
Topics
  • Health Sciences › Medicine › Clinical Neurology › Neurofibromatosis and Schwannoma Cases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Tyrosine Phosphatases
  • Health Sciences › Medicine › Hematology › Acute Myeloid Leukemia Research
Keywords
neurofibromin, Ras signaling, hematopoietic progenitor cells, juvenile myelomonocytic leukemia, neurofibromatosis type 1, GM-CSF, interleukin-3, stem cell factor, cytokine synergy
MeSH terms
Animals, Cell Division, Female, Hematopoietic Stem Cells, Interleukin-3, Macrophages, Male, Mice, Inbred C57BL, Proteins, Signal Transduction, Granulocyte-Macrophage Colony-Stimulating Factor, Proto-Oncogene Proteins p21(ras), Calcium-Calmodulin-Dependent Protein Kinases, Stem Cell Factor, Neurofibromin 1, Mice

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