By
§
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
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Abstract |
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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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Introduction |
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Ras proteins regulate the growth and differentiation of many cell types by acting as molecular switches that transduce signals from the extracellular environment to the nucleus (1). The biochemical output of Ras proteins is tightly regulated by their ability to cycle between an active guanosine triphosphate (GTP)-bound state (Ras-GTP) and an inactive guanosine diphosphate (GDP)-bound state (Ras-GDP) (1). Ras activation is an essential component of proliferative responses induced after receptor binding by a variety of growth factors including IL-3, GM-CSF, CSF-1, and stem cell factor (SCF)1 (5). Stimulation by each of these cytokines induces an increase in the percentage of Ras-GTP in the target cell (5, 6, 8, 9). Ras-GTP recruits Raf-1 to the plasma membrane and Raf-1, in turn, activates a series of downstream effectors such as mitogen-activated protein (MAP) kinase (7, 10, 11). GTPase-activating proteins (GAPs) regulate Ras output by stimulating the slow intrinsic Ras GTPase (4, 12, 13). Because it is Ras-GTP that transduces signals, GAPs act (at least in part) as negative regulators of Ras function (1, 12). Two GAPs, p120 GAP (also known as Ras-GAP) and neurofibromin (the protein encoded by the NF1 gene) regulate Ras output in mammalian cells (4, 12) by promoting the conversion of Ras-GTP to Ras-GDP (4).
Mutations of NF1 cause neurofibromatosis type 1 (NF1),
an autosomal dominant disorder with an incidence of 1 in
4,000 (14). Affected individuals are predisposed to the
development of benign and malignant neoplasms that arise
primarily in cells derived from the embryonic neural crest
(14). In addition, children (but not adults) with NF1
have a markedly increased risk of developing malignant
myeloid disorders and comprise as many as 10% of de novo
cases of preleukemia in the pediatric age group (19). Juvenile chronic myelogenous leukemia (JCML), a myeloproliferative syndrome characterized by leukocytosis, thrombocytopenia, and hepatosplenomegaly with infiltrates of myeloid
cells, is the most common leukemia seen in children with
NF1 (20). Genetic and biochemical data from studies of
human leukemias have shown that NF1 functions as a tumor suppressor gene in hematopoietic cells by negatively regulating Ras signaling (20, 22). Mice that are heterozygous for a targeted disruption of Nf1 are predisposed
to a number of cancers including myeloid leukemia (26).
Leukemic cells from these animals show loss of the normal
Nf1 allele (26). Although mice homozygous for disruption
of Nf1 die during embryonic development, Nf1/
fetal
liver cells efficiently reconstitute hematopoiesis and consistently induce a JCML-like myeloproliferative syndrome in irradiated recipients (27).
A hallmark of low density blood and bone marrow cells
from children with JCML is an abnormal pattern of in vitro
progenitor growth that is characterized by the appearance
of factor-independent myeloid colonies in methylcellulose
cultures (28, 29). A second consistent finding is that JCML
cells form higher numbers of colonies derived from lineage-restricted colony-forming unit granulocyte-macrophage progenitors (CFU-GM) in the presence of low concentrations of GM-CSF (21, 28, 29). Hypersensitive growth has
not been observed in cultures in which IL-3 or GM-CSF was
added, and these results have led to the hypothesis that a
specific defect within the GM-CSF signal transduction
pathway plays a central role in the pathogenesis of JCML
(28, 29). Nf1/
fetal liver cells also demonstrate a similar
pattern of selective GM-CSF hypersensitivity in myeloid
progenitor assays (27, 30) and provide a model system to
investigate both the role of neurofibromin in regulating
Ras signaling in hematopoietic cells and also the pathogenesis of JCML.
We have used Nf1 mice to address two questions related
to the role of neurofibromin in regulating hematopoietic
cell growth through the Ras-MAP kinase pathway. First,
since the hematopoietic system is hierarchial and has multiple compartments, it is possible that neurofibromin is only
required to regulate growth of specific subpopulations of
cells in response to a restricted number of hematopoietic
growth factors. Previous studies of human and murine cells
examined only the responsiveness of relatively differentiated, lineage-restricted progenitors from mixed populations
of hematopoietic cells, and in most instances cultures were
established using single growth factors (21, 27, 29). We
isolated highly pure hematopoietic progenitors and examined their growth in response to GM-CSF, IL-3, and stem
cell factor (SCF) singly and in combination with other factors. SCF promotes the survival and proliferation of immature, multilineage hematopoietic progenitors (34) and
acts synergistically with growth factors, such as GM-CSF,
that primarily affect differentiated, lineage-restricted cells
(38). Second, the selective hypersensitivity of Nf1/
and JCML cells to GM-CSF in colony-forming assays is
perplexing because GM-CSF and IL-3 both increase Ras-GTP levels in cell lines (8, 9) and the cell surface receptors
for these ligands share a common
chain that transduces
signals (for review see references 42). To address these
questions, we measured constitutive and cytokine-stimulated MAP kinase activation in well-defined populations of
primary Nf1
/
and Nf1+/+ hematopoietic cells and correlated these data with in vitro colony-forming assays that assessed the size and growth factor responsiveness of these
populations. Our results implicate neurofibromin as regulating the size and growth factor responsiveness of both
immature and lineage-restricted hematopoietic progenitor
populations by modulating Ras output in response to multiple cytokines.
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Materials and Methods |
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Isolation of Fetal Hematopoietic Cells.
Heterozygous Nf1+/Genotyping Fetal Tissues.
Once the liver was removed, the remainder of the embryonic tissues were used for genotype analysis. Genomic DNA from fetal tissues were isolated as previously described (30). An assay based on the PCR was used to perform genotype analysis. The targeting vector used to disrupt the murine Nf1 gene truncates exon 31 and inserts a neomycin resistance gene (neo). The 3' (reverse) primer is complimentary to a sequence in the 3' region of exon 31 that is present in both wild-type and targeted Nf1 genes. Two 5' primers were designed to distinguish disrupted and wild-type genes. The first is complimentary to neo and the second is complimentary to a wild-type exon 31 sequence. Amplification with the neo and 3' Nf1 primer pair yields a 350-bp product, whereas amplification with the 5' Nf1 and 3' Nf1 pair gives a 230-bp fragment. The sequences for the PCR primers used in these studies are: 5' Nf1 exon 31: CACCTTTGTTTGGAATAT, 3' Nf1 exon 31: TTCAATACCTGCCCAAGG, and neo: ATTCGCCAATGACAAGAC.Transplantation into Irradiation-conditioned Congenic Mice.
Unfractionated fetal liver cells (1-2 × 106) from Nf1Purification of Hematopoietic Progenitors.
Low density cells were incubated with a PE-conjugated c-kit monoclonal antibody or a PE-conjugated Sca1+ monoclonal antibody whose corresponding Ly6A/E antigen is highly expressed on all primitive hematopoietic progenitors and cells in the stem cell compartment in C57Bl6 strain (45). Cells were incubated with 1 µg of c-kit or Sca1 antibody/106 cells, (PharMingen, San Diego, CA), and the cells were then incubated on ice for 20 min, pelleted, washed, repelleted, and then suspended in FACS buffer. Cells were also simultaneously stained with antibodies to the following lineage markers including: anti-B220, anti-CD3Hematopoietic Progenitor Colony Growth.
Recombinant GM-CSF, IL-3, and SCF were obtained from Peprotech (Rocky Hill, NJ); recombinant mIL-1Kinase Assays.
The incorporation of phosphate into myelin basic protein (MBP) (Sigma Chemical Co., St. Louis MO) was used as a measure of MAP kinase activity as previously described (48). Cell pellets were lysed in lysis buffer (10 mM K2HPO4, 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 1 mM Na3 V04, 50 mMLiquid Cultures.
Purified populations of c-kit+ cells isolated from the bone marrows of mice transplanted with Nf1 ![]() |
Results |
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Previous studies in JCML bone marrow samples have
implicated cytokine production by accessory cells as contributing to the hypersensitive pattern of myeloid progenitor growth (31, 32). We therefore isolated highly pure (95-
99%) Sca1+lin/dim cells derived from day 13.5 fetal livers
by flow cytometric cell sorting and assayed the growth of
CFU-GM in methylcellulose medium supplemented with
varying concentrations of either GM-CSF or IL-3. A representative FACS® analysis of cells before and after cell
sorting is shown in Fig. 1. The results of four independent
experiments indicate that CFU-GM derived from Nf1
/
fetal liver Sca1+lin
/dim cells retain a hypersensitive pattern
of colony formation in response to GM-CSF but not IL-3
(Fig. 2, a and b). These data suggest that the alteration of
growth responsiveness to GM-CSF seen in Nf1
/
cells is
intrinsic to the progenitor compartment.
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We next examined the effects of adding a low concentration of murine SCF (10 ng/ml) in combination with either GM-CSF or IL-3 on Sca1+lin/dim cells on colony formation. CFU-GM colony growth under these conditions is shown in Fig. 2, c and d, and the absolute numbers of
progenitor colonies enumerated at maximal concentrations
of GM-CSF or IL-3 singly and in combination with SCF
are shown in Table 1. As with GM-CSF alone, the dose-
response curve for CFU-GM colony formation from Nf1
/
cells was significantly left-shifted relative to Nf1+/+ cells in
cultures stimulated with GM-CSF + SCF (Fig. 2 c). The hypersensitive pattern of growth seen in Nf1
/
cells was
more pronounced in parallel cultures stimulated with GM-CSF and SCF than in plates containing GM-CSF alone,
and there was an increase in the absolute number of progenitors enumerated (Table 1). Surprisingly, the addition of
SCF to cultures stimulated with IL-3 induced a hypersensitive pattern of growth in Nf1
/
cells (Fig. 2 d) that was not
seen with IL-3 alone (Fig. 2 b). Although SCF is relatively
inefficient at inducing colony formation in the absence of
other growth factors (Table 1), we also detected a significant left shift in the dose-response curve of Nf1
/
versus
Nf1+/+ cells cultured in the presence of SCF alone (Fig. 2 e).
Taken together, these data suggest that loss of Nf1 deregulates the growth of hematopoietic progenitors in response
to SCF, which acts on more immature populations of progenitors than do IL-3 or GM-CSF, and demonstrate that
SCF can synergize with either factor to induce a hypersensitive pattern of in vitro growth in Nf1
/
progenitors.
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One potential explanation for the hypersensitive growth
seen in response to SCF alone or in combination with IL-3
is that SCF stimulation induces autocrine production of
GM-CSF by Nf1/
progenitor cells. To test this hypothesis, an anti-GM-CSF neutralizing antibody was added to
the methylcellulose cultures stimulated with a combination
of IL-3 and SCF. In pilot experiments, a concentration of
anti-GM-CSF antibody that completely blocked CFU-GM
colony formation in response to 10 U/ml of GM-CSF
(maximum stimulating activity) was established and used
in subsequent experiments (data not shown). Fig. 3 summarizes the results of three independent experiments examining the number of CFU-GM colonies seen when either
anti-GM-CSF or an isotype control antibody was added
to cultures containing IL-3 + SCF. These data indicate
that the abnormal pattern of CFU-GM colony formation
observed in Nf1
/
Sca1+lin
/dim cells is maintained in the
presence of anti-GM-CSF antibody, and therefore we conclude that hypersensitive growth in the presence of IL-3 + SCF is not due to autocrine GM-CSF production by Nf1
/
cells.
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The experiments described above assayed the growth of
colonies derived from CFU-GM, a relatively differentiated,
lineage-restricted population of myeloid progenitors. However, data from cultures stimulated with SCF suggested
that neurofibromin might also regulate the growth of more
primitive hematopoietic compartments. To investigate this
possibility, two-layer agar cultures were established to assay
the growth of primitive progenitors with significant replating potential indicative of a primitive stem/progenitor
cell (46) and referred to as HPP-CFC. Fetal liver cells from
Nf1/
, Nf1+/
, and Nf1+/+ embryos were cultured in two
different combinations of four growth factors at saturating activities to enumerate HPP-CFC. The frequency of
HPP-CFC observed in cultures were then multiplied by
the number of low density cells per organ to calculate the
absolute number of HPP-CFC per fetal liver. The results
shown in Fig. 4 demonstrate that significantly higher numbers
of HPP-CFC are present from the Nf1
/
fetal liver cells.
The data suggest that the loss of Nf1 results in an expansion
of HPP-CFC in vivo in the fetal liver and that neurofibromin is important in modulating the growth of primitive myeloid progenitor compartments as well as CFU-GM
progenitors.
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Stimulation with a variety of growth factors including
SCF, GM-CSF, or IL-3 activates the Ras-Raf-MAP kinase
cascade in cultured cell lines (5). We hypothesized that if
neurofibromin is critical to regulate Ras signaling in response to these cytokines in primary hematopoietic progenitors, then loss of Nf1 function might result in an augmented MAP kinase activity after stimulation. To test this
possibility, c-kit+ cells were isolated from the bone marrows of mice previously irradiated and then reconstituted
with fetal liver cells from day 13.5 gestation Nf1/
and
Nf1+/+ littermates. In pilot experiments, we determined
that the c-kit+ cells isolated from transplant recipients had a
similar hyperresponsiveness to GM-CSF in clonogenic assays as did Nf1
/
fetal liver cells (data not shown). After
isolation using fluorescence-activated cell sorting, c-kit+
cells were maintained in liquid cultures overnight without
exogenous growth factors. The cells were then stimulated
with either SCF, IL-3, or GM-CSF alone or with a combination of SCF + IL-3. MAP kinase activity was measured
in unstimulated cells as well as at 5 and 60 min after the addition of growth factor(s). Three independent experiments
yielded similar results; data from one of these is shown in
Fig. 5. A threefold elevation in MAP kinase activity was
observed in unstimulated c-kit+ cells isolated from the
Nf1
/
recipients. Kinase activity increased significantly in
Nf1
/
cells after exposure to IL-3, SCF, or SCF + IL-3,
and persisted above baseline at 60 min. There was also an
increase in MAP kinase activity at 60 min after stimulation
with GM-CSF that approached statistical significance. In
contrast, c-kit+ cells from the Nf1+/+ recipients had a much
less pronounced increase in MAP kinase activity at 5 min
after stimulation, and the kinase returned to baseline by 60 min. We conclude that absence of neurofibromin in c-kit+
hematopoietic progenitors results in a constitutive activation of Ras-MAP kinase signaling that can be further stimulated in response to SCF, IL-3, and GM-CSF.
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To confirm an association between the clonogenic and
biochemical data presented above, c-kit+ cells were again
isolated from transplant recipients. MAP kinase activity was
determined on aliquots of Nf1+/+ and Nf1/
cells after
overnight incubation in the presence or absence of an inhibitor of MEK. The remaining cells were either immediately cultured in methylcellulose to score the growth of
myeloid progenitor colonies or were placed into liquid culture containing SCF and IL-3 together with MEK or the
vehicle control for 48 h before plating. Biochemical data
from one of three representative experiments are shown in
Fig. 6 and the colony numbers are presented in Fig. 7. Addition of the MEK inhibitor reduced the MAP kinase activity of unstimulated Nf1
/
cells to wild-type levels and
blocked kinase activation in response to SCF and IL-3.
Similarly, addition of the MEK inhibitor to liquid cultures
of c-kit+ cells for 48 h before plating in methylcellulose
markedly decreased the growth of differentiated cells, differentiated progenitor cells (LPP-CFC), and primitive hematopoietic progenitors (HPP-CFC) as compared with
cultures containing the vehicle only. A reduced biochemical and cellular response to the MEK inhibitor was noted in
the Nf1+/+ hematopoietic cells. Taken together, these data
implicate deregulated signaling through the Ras-MEK-MAP kinase in the aberrant pattern of cytokine-induced
hematopoietic progenitor colony growth seen in Nf1
/
cells.
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Discussion |
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Ras signaling plays a central role in normal myelopoiesis
and is deregulated by at least three distinct genetic mechanisms in myeloid leukemia (for review see references 20,
53, 54). Ligand-induced activation of many hematopoietic
growth factor receptors transiently increases the Ras-GTP
levels, which in turn influences the survival, proliferation,
and differentiation of myeloid-lineage cells. In leukemic
cells, acquired mutations of NRAS or KRAS, formation of
a chimeric protein as a result of BCR-ABL translocations,
and inactivation of NF1 all deregulate Ras signaling. Lines
of knockout mice provide novel model systems for ascertaining how loss of function of specific genes influences
complex biologic processes such as hematopoiesis and leukemogenesis. Although inactivation of the murine homologues of many human tumor suppressor genes results in a
spectrum of neoplasms that differs significantly from the respective human cancers, Nf1 mice spontaneously develop a
type of leukemia that closely resembles JCML and Nf1/
cells display a similar pattern of GM-CSF hypersensitivity
in myeloid colony-forming assays.
A distinct advantage of this murine model is the ability to reconstitute irradiated mice only lacking Nf1 and isolate highly purified populations of phenotypically defined cells to characterize their in vitro colony growth and intracellular signaling.
This study addressed two questions: (a) what is the role of neurofibromin in modulating the cytokine responsiveness and growth of both primitive myeloid progenitors with multilineage and replating potential, and of more differentiated lineage-restricted myeloid progenitors?, and (b) how does inactivation of Nf1 affect constitutive and cytokine-stimulated intracellular signaling through the Ras-Raf-MAP kinase pathway in these primary hematopoietic cells? We used antibodies to purify immature hematopoietic cells and focused on the SCF/c-kit signaling pathway because the binding of SCF to c-kit increases Ras-GTP levels and activates Raf-1 and MAP kinase activity in myeloid cell lines (7). In addition, the presence of multiple hematopoietic defects in lines of mice with mutations affecting either c-kit (dominant white spotting, W) or SCF (Steel) (34, 39, 55, 56), as well as numerous in vitro studies showing a synergistic effect between SCF and other cytokines in promoting the survival and proliferation of primitive hematopoietic progenitor cells, indicate that the SCF/c-kit signaling plays an important role in early hematopoiesis (38).
An intriguing finding demonstrated here was that SCF
alone or in combination with IL-3 induced a hypersensitive pattern of CFU-GM colony growth in Nf1/
cells.
Though some reports using JCML cells have observed hypersensitive colony growth in response to only GM-CSF,
others have inferred the involvement of other cytokines in
the pathogenesis of the myeloproliferative disease (31,
57). Our results address an interesting paradox: since the
GM-CSF and IL-3 receptors share a common
signaling
chain that has been implicated in Ras activation, it has been
perplexing why cultured Nf1
/
cells are hypersensitive to
GM-CSF but not IL-3 in progenitor colony-forming assays. One explanation that has been suggested previously is
that p120 GAP, which is abundant and active in Nf1
/
hematopoietic cells (27, 30), is able to downregulate Ras-GTP when it associates in a complex with the IL-3 receptor and other proteins, but not when Ras associates with
the GM-CSF receptor (27, 30). Biochemical evidence that
MAP kinase is hyperactivated in c-kit+ Nf1
/
cells in response to IL-3, or SCF (Fig. 5), and cell culture data showing that SCF alone and in combination with IL-3 or GM-CSF induce a hypersensitive pattern of in vitro growth in
Nf1
/
cells (Fig. 2), suggest that aberrant intracellular signaling occurs in response to IL-3, GM-CSF, and SCF.
However, the biochemical abnormality is not translated to
hypersensitive colony formation in response to IL-3 only.
It is possible that SCF and GM-CSF activate downstream
signaling molecules important for terminal differentiation that are not induced by IL-3. For example, focal adhesion
kinase is a recently identified kinase involved in the terminal differentiation of myeloid cells that is activated in response to GM-CSF (58) and SCF (59) but not IL-3. Nf1-deficient hematopoietic cells and cell lines should prove
useful for further characterizing signaling pathways that are
differentially activated and regulated in response to GM-CSF and IL-3. Our studies of purified Sca1+lin
/dim cells
suggest that the hypersensitive pattern of colony formation is intrinsic to the progenitors and is not dependent on accessory cells as suggested by some (31), but not all (21),
previous studies in JCML cells.
Cultures established from primary d13.5 Nf1/
fetal
liver cells also demonstrated an increase in the numbers of
primitive progenitor (HPP-CFC) cells in the fetal liver. In
fact the expansion in this compartment was greater than
was noted for more differentiated progenitors (LPP-CFC)
scored in simultaneous experiments (our unpublished results). As the HPP-CFC and LPP-CFC assays were performed in the presence of saturating concentrations of hematopoietic growth factors, our data indicate that inactivation
of Nf1 is not only associated with a leftward shift in the
dose-response curves for colony formation in response to
exogenous growth factors but also with an absolute increase in the numbers of clonogenic progenitors in vivo.
Preliminary data indicate that this increase in progenitors is
associated with a relative resistance of Nf1
/
cells to apoptosis upon withdrawal of hematopoietic growth factors (Zhang, Y.-Y., and D.W. Clapp, unpublished results).
Biochemical data from c-kit positive hematopoietic cells
demonstrating constitutive activation of MAP kinase signaling and a prolonged hyperactivation of MAP kinase activity in response to stimulation with cytokines suggests
that deregulation of the Ras/MAP kinase pathway accounts for the perturbations of in vitro colony growth. Indeed, reducing the MAP kinase activity with an inhibitor
of MEK was associated with a profound, specific reduction in colony formation from Nf1/
c-kit+ cells. The elevated
and sustained levels of MAP kinase that we measured in response to SCF, IL-3, or GM-CSF in highly progenitor-enriched primary cells are consistent with the previous observation of a sustained elevation of Ras-GTP levels after
GM-CSF stimulation in Myb immortalized Nf1
/
fetal hematopoietic cell lines (27). Similarly, primary bone marrow
cells from children with NF1 and leukemia showed an elevated percentage of Ras-GTP (30). Although the biochemical data presented here, the predilection to myeloid
leukemia seen in children with NF1 and in Nf1+/
mice,
and the myeloid disorder that invariably develops in the recipients of Nf1
/
fetal liver cells provide compelling evidence that neurofibromin is essential to regulate Ras output in myeloid lineage cells, future studies are indicated to
further delineate how neurofibromin modulates Ras in response to cytokines such as SCF. In particular, p120 GAP
accounts for most of the in vivo and in vitro GTPase activating protein activity in bone marrow cells (30, 60, 61). It
is possible that p120 GAP is sequestered away from growth factor receptor-Ras complexes in activated myeloid cells or
is inactivated by a more direct mechanism. In this respect,
the recent cloning of the gene encoding p62Dok (Dok) is of
interest as this protein is phosphorylated in leukemic cells
that carry the BCR-ABL translocation and phosphorylated Dok selectively associates with p120 GAP through Src homology 2 (SH2) domains. Dok is rapidly phosphorylated in
hematopoietic cells in response to SCF (62). If the binding
of phosphorylated p62Dok inhibits the function of p120
GAP, cells that have been stimulated with SCF may require
neurofibromin to properly regulate Ras-GTP (neurofibromin does not contain SH2 domains [for review see references 4, 63, 64], and is unlikely to interact with Dok). Additional biochemical data are required to examine if SCF-induced
phosphorylation of p62Dok affects the function of p120
GAP in normal and Nf1-deficient myeloid lineage cells.
In summary, we have shown that the loss of Nf1 results
in constitutive activation of Ras signaling in primitive and
lineage-restricted hematopoietic progenitors. This is associated with hyperresponsiveness to multiple growth factors in
biochemical and progenitor colony-forming assays, as well
as in expansion of myeloid compartments in Nf1/
embryos
and in adult recipients reconstituted with Nf1
/
fetal liver
cells. Taken together, these data strongly suggest that the
size and growth factor sensitivity of cells within these multiple hematopoietic compartments are regulated by neurofibromin through its ability to effect the activation state
of the Ras/MAP kinase cascade. These data have implications for the use of GM-CSF antagonists to treat JCML.
On the one hand, our data showing that inactivation of
Nf1 results in aberrant signaling in response to other
growth factors provide evidence that GM-CSF-specific inhibitors might not show efficacy. However, mice that
overexpress GM-CSF develop a JCML-like disorder (65),
and in one recent study xenograft mice transplanted with
JCML cells and treated with antagonists to GM-CSF had a
significant reduction in JCML cells as compared with control mice treated with an isoimmune nonspecific antibody
(66). Thus, it may be that the loss of Nf1 results in aberrant
Ras activation in multiple myeloid compartments, the importance of GM-CSF on terminal differentiation may be
pivotal to acquisition of the clinical disease, and it remains
possible that the clinical disorder will be abrogated by
blocking the GM-CSF receptor. Nf1 mice provide a valuable model system to test basic aspects of myeloid growth
control and to evaluate novel therapeutics directed against
hyperactive Ras.
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Footnotes |
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Address correspondence to Wade Clapp, Riley Hospital for Children, Cancer Research Institute, 1044 West Walnut, Rm. 402, Indianapolis, IN 46202-5254. Phone: 317-274-4719; Fax: 317-274-8679.
Received for publication 4 February 1998 and in revised form 19 March 1998.
We thank Patricia Fox for secretarial support.
This work was supported by Public Health Service grant P5ODK49218 R29 CA-74177-01 (to D.W. Clapp) and American Cancer Society grant 1874SC (to D.W. Clapp and K. Shannon). Tyler Jacks is an investigator of the Howard Hughes Medical Institute.
1Abbreviations used in this paper CFC, colony-forming cells; GAP, GTPase-activating protein; HPP, high proliferating potential; JCML, juvenile chronic myelogenous leukemia; LPP, low proliferating potential; MAP, mitogen-activated protein; MBP, myelin basic protein; MEK, MAP kinase/extracellular signal-regulated kinase; NF1, neurofibromatosis type 1; SCF, stem cell factor.
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References |
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1. | Bourne, H.R., D.A. Sanders, and F. McCormick. 1990. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 348: 125-132 [Medline]. |
2. | Bourne, H.R., D.A. Sanders, and F. McCormick. 1991. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 349: 117-127 [Medline]. |
3. | Hall, A.. 1990. The cellular functions of small GTP-binding proteins. Science. 249: 635-640 [Medline]. |
4. |
Hall, A..
1992.
Signal transduction through small GTPases![]() |
5. | Hill, C.S., and R. Treisman. 1995. Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell. 80: 199-211 [Medline]. |
6. | Marshall, C.J.. 1995. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 80: 179-185 [Medline]. |
7. | Miyazawa, K., P.C. Hendrie, C. Mantel, K. Wood, L.K. Ashman, and H.E. Broxmeyer. 1991. Comparative analysis of signaling pathways between mast cell growth factor (c-kit ligand) and granulocyte-macrophage colony-stimulating factor in a human factor-dependent myeloid cell line involves phosphorylation of Raf-1, GTPase-activating protein and mitogen-activated protein kinase. Exp. Hematol. 19: 1110-1123 [Medline]. |
8. | Satoh, T., M. Nakafuku, A. Miyajima, and Y. Kaziro. 1991. Involvement of ras p21 protein in signal-transduction pathways from interleukin 2, interleukin 3, and granulocyte/macrophage colony-stimulating factor, but not from interleukin 4. Proc. Natl. Acad. Sci. USA. 88: 3314-3318 [Abstract]. |
9. |
Satoh, T.,
Y. Uehara, and
Y. Kaziro.
1992.
Inhibition of interleukin 3 and GM-CSF stimulated increase in Ras-GTP by
herbamycin A, a specific inhibitor of tyrosine kinases.
J. Biol.
Chem.
267:
2537-2541
|
10. | Leevers, S., H. Peterson, and C. Marshall. 1994. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature. 369: 411-414 [Medline]. |
11. | Stokoe, D., S. MacDonald, K. Cadwallader, M. Symons, and J. Hancock. 1994. Activation of Raf as a result of recruitment to the plasma membrane. Science. 264: 1463-1467 [Medline]. |
12. | Boguski, M., and F. McCormick. 1993. Proteins regulating Ras and its relatives. Nature. 366: 643-654 [Medline]. |
13. |
Clark, G.J.,
J.K. Drugan,
R.S. Terrel,
C. Bradham,
J.D. Channing,
R.M. Bell, and
S. Campbell.
1996.
Peptides containing a consensus Ras binding sequence from Raf-1 and the
GTPase activating protein NF1 inhibit Ras function.
Proc.
Natl. Acad. Sci. USA.
93:
1577-1581
|
14. | DeClue, J.E., A.G. Papageorge, J.A. Fletcher, S.R. Diehl, N. Ratner, W.C. Vass, and D.R. Lowy. 1992. Abnormal regulation of mammalian p21ras contributes to malignant tumor growth in von Recklinghausen (type 1) neurofibromatosis. Cell. 69: 265-273 [Medline]. |
15. | Xu, G., P. O'Connell, D. Viskochil, R. Cawthon, M. Robertson, M. Culver, D. Dunn, J. Stevens, R. Gesteland, R. White, and R. Weiss. 1990. The neurofibromatosis type 1 gene encodes a protein related to GAP. Cell. 62: 599-608 [Medline]. |
16. | Legius, E., D.A. Marchuk, F.S. Collins, and T.W. Glover. 1993. Somatic deletion of the neurofibromatosis type 1 gene in a neurofibrosarcoma supports a tumor suppressor gene hypothesis. Nat. Genet. 3: 122-126 [Medline]. |
17. | Menon, A.G., K.M. Anderson, V.M. Riccardi, R.Y. Chung, J.M. Whaley, D.W. Yandell, G.E. Farmer, R.N. Freiman, J.K. Lee, F.P. Li, et al . 1990. Chromosome 17p deletions and p53 gene mutations associated with the formation of malignant neurofibrosarcomas in von Recklinghausen neurofibromatosis. Proc. Natl. Acad. Sci. USA. 87: 5435-5439 [Abstract]. |
18. | Skuse, G.R., B.A. Kosciolek, and P.T. Rowley. 1989. Molecular genetic analysis of tumors in von Recklinghausen neurofibromatosis: loss of heterozygosity for chromosome 17. Genes Chromosomes Cancer. 1: 36-41 [Medline]. |
19. | Johnson, M.R., A.T. Look, J.E. DeClue, M.B. Valentine, and D.R. Lowy. 1993. Inactivation of the NF1 gene in human melanoma and neuroblastoma cell lines without impaired regulation of GTP-Ras. Proc. Natl. Acad. Sci. USA. 90: 5539-5543 [Abstract]. |
20. |
Brodeur, G.M..
1994.
The NF1 gene in myelopoiesis and
childhood myelodysplastic syndromes.
N. Engl. J. Med.
330:
637-639
|
21. | Emanuel, P.D., L.J. Bates, S.-W. Zhu, R.P. Castleberry, R.J. Gualtieri, and K.S. Zuckerman. 1991. The role of monocyte-derived hemopoietic growth factors in the regulation of myeloproliferation in juvenile chronic myelogenous leukemia. Exp. Hematol 19: 1017-1024 [Medline]. |
22. |
Side, L.,
B. Taylor,
M. Cayouette,
E. Conner,
P. Thompson,
M. Luce, and
K. Shannon.
1997.
Homozygous inactivation
of the NF1 gene in bone marrow cells from children with
neurofibromatosis type 1 and malignant myeloid disorders.
N. Engl. J. Med
336:
1713-1720
|
23. | Neubauer, A., K.M. Shannon, and E. Liu. 1991. Mutations of the ras proto-oncogenes in childhood monosomy 7. Blood. 77: 594-598 [Abstract]. |
24. | Shannon, K.M., J. Watterson, P. Johnson, P. O'Connell, B. Lange, N. Shah, P. Steinherz, Y.W. Kan, and J.R. Priest. 1992. Monosomy 7 myeloproliferative disease in children with neurofibromatosis, type 1: epidemiology and molecular analysis. Blood. 79: 1311-1318 [Abstract]. |
25. |
Shannon, K.M.,
P. O'Connell,
G.A. Martin,
D. Paderanga,
K. Olson,
P. Dinndorf, and
F. McCormick.
1994.
Loss of the
normal NF1 allele from the bone marrow of children with
type 1 neurofibromatosis and malignant myeloid disorders.
N. Engl. J. Med
330:
597-601
|
26. | Jacks, T., S. Shih, E.M. Schmitt, R.T. Bronson, A. Bernards, and R.A. Weinberg. 1994. Tumor predisposition in mice heterozygous for a targeted mutation in Nf1. Nat. Genet. 7: 353-361 [Medline]. |
27. | Largaespada, D.A., C.I. Brannan, N.A. Jenkins, and N.G. Copeland. 1996. Nf1 deficiency causes Ras-mediated granulocyte/macrophage colony stimulating factor hypersensitivity and chronic myeloid leukaemia. Nat. Genet. 12: 137-143 [Medline]. |
28. | Emanuel, P.D., L.J. Bates, R.P. Castleberry, R.J. Gualtieri, and K.S. Zuckerman. 1991. Selective hypersensitivity to granulocyte-macrophage colony stimulating factor by juvenile chronic myeloid leukemia hematopoietic progenitors. Blood. 77: 925-929 [Abstract]. |
29. | Gualtieri, R.J., P.D. Emanuel, K.S. Zuckerman, G. Martin, S.C. Clark, R.K. Shadduck, R.A. Draker, J. Akabutu, R. Nitschke, M.L. Hetherington, et al . 1989. Granulocyte-macrophage colony-stimulating factor is an endogenous regulator of cell proliferation in juvenile chronic myelogenous leukemia. Blood 74: 2360-2367 [Abstract]. |
30. | Bollag, G., D.W. Clapp, S. Shih, F. Adler, Y.Y. Zhang, P. Thompson, B.J. Lange, M.H. Freedman, F. McCormick, T. Jacks, and K. Shannon. 1996. Loss of NF1 results in activation of the Ras signaling pathway and leads to aberrant growth in haematopoietic cells. Nat. Genet. 12: 144-148 [Medline]. |
31. | Bagby, G.C.J., C.A. Dinarello, R.C. Neerhout, D. Ridgway, and E. McCall. 1988. Interleukin 1-dependent paracrine granulopoiesis in chronic granulocytic leukemia of the juvenile type. J. Clin. Invest. 82: 1430-1436 [Medline]. |
32. | Freedman, M.H., A. Cohen, T. Grunberger, N. Bunin, R.E. Luddy, E.F. Saunders, N. Shahidi, A. Lau, and Z. Estrov. 1992. Central role of tumor necrosis factor, GM-CSF, and interleukin 1 in the pathogenesis of juvenile chronic myelogenous leukemia. Br. J. Hematol. 80: 40-48 [Medline]. |
33. |
Schiro, R.,
D. Longoni,
V. Rossi,
O. Maglia,
A. Doni,
M. Arsura,
G. Carrara,
G. Masera,
E. Vannier,
C.A. Dinarello, et al
.
1994.
Suppression of juvenile chronic myelogenous leukemia colony growth by interleukin-1 receptor antagonist.
Blood.
83:
460-465
|
34. | Martin, F., S. Suggs, K. Langley, H. Lu, J. Ting, K. Okino, C. Morris, I. McNiece, F. Jacobsen, E. Mendiaz, et al . 1990. Primary structure and functional expression of rat and human stem cell factor DNAs. Cell. 63: 203-211 [Medline]. |
35. | Nocka, K., J. Buck, E. Levi, and P. Besmer. 1990. Candidate ligand for the c-kit transmembrane dinase receptor: KL, a fibroblast derived growth factor stimulates mast cells and erythroid progenitors. EMBO (Eur. Mol. Biol. Organ.) J. 9: 3287-3294 [Abstract]. |
36. | Rottapel, R., M. Reeduk, D. Williams, S. Lyman, D. Anderson, T. Pawson, and A. Bernstein. 1991. The Steel/W transduction pathway: kit autophosphorylation and its association with a unique subset of cytoplasmic signaling proteins is induced by the steel factor. Mol. Cell. Biol. 11: 3043-3051 [Medline]. |
37. | Zsebo, K., D. Williams, E. Geissier, V. Broudy, F. Martin, H. Atkins, R.-Y. Hasu, N. Birkett, K. Okino, D. Murdock, et al . 1990. Stem cell factor is encoded at the SE locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell. 63: 213-224 [Medline]. |
38. |
Brandt, J.,
K. Bhalla, and
R. Hoffman.
1994.
Effects of interleukin-3 and c-kit ligand on the survival of various classes of
human hematopoietic progenitor cells.
Blood.
83:
1507-1514
|
39. |
Lowry, P.,
K. Zsebo,
D. Deacon,
C. Eichman, and
P. Quesenberry.
1991.
Effects of rrSCF on multiple cytokine responsive HPP-CFC generated from SCA+Lin![]() |
40. | McNiece, I., K. Langley, and K. Zsebo. 1991. Recombinant human stem cell factor synergises with GM-CSF, G-CSF, IL-3 and Epo to stimulate human progenitor cells of the myeloid and erythroid lineages. Exp. Hematol. 19: 226-231 [Medline]. |
41. | Molineaux, G., A. Migdalska, M. Szmitkowski, K. Zsebo, and T. Dexter. 1991. The effects on hematopoiesis of recombinant stem cell factor (ligand for c-kit) administered in vivo to mice either alone or in combination with granulocyte colony-stimulating factor. Blood. 78: 961-966 [Abstract]. |
42. |
Kitamura, T.,
N. Sato,
K. Arai, and
A. Miyajima.
1991.
Expression cloning of the human IL-3 receptor cDNA reveals a
shared ![]() |
43. | Mui, A.L., and A. Miyajima. 1994. Interleukin-3 and granulocyte-macrophage colony-stimulating factor receptor signal transduction. Proc. Soc. Exp. Biol. Med. 206: 284-288 [Abstract]. |
44. | Sakamoto, K.M., R.C. Mignacca, and J.C. Gasson. 1994. Signal transduction by granulocyte-macrophage colony-stimulating factor and interleukin-3 receptors. Receptors Channels. 2: 175-181 [Medline]. |
45. | Spangrude, G.J., and D.W. Brooks. 1993. Mouse strain variability in the expression of the hematopoietic stem cell antigen Ly-6A by bone marrow cells. Blood. 82: 3327-3332 [Abstract]. |
46. | Bradley, T.R., and G.S. Hodgson. 1979. Detection of primitive macrophage progenitor cells in mouse bone marrow. Blood. 54: 1446-1450 [Abstract]. |
47. |
Clapp, D.W.,
B. Freie,
E. Srour,
M.C. Yoder,
K. Fortney, and
S.L. Gerson.
1995.
Myeloproliferative sarcoma virus directed expression of ![]() |
48. |
Ahn, N.G.,
R. Seger,
R.L. Bratlien,
C.D. Diltz,
N.K. Tonks, and
E.G. Krebs.
1991.
Multiple components in an epidermal
growth factor-stimulated protein kinase cascade. In vitro activation of a myelin basic protein/microtubule-associated
protein 2 kinase.
J. Biol. Chem.
266:
4220-4227
|
49. | Calvo, V., B.E. Bierer, and T.A. Vik. 1991. T cell receptor activation of a ribosomal S6 kinase activity. Eur. J. Immunol. 22: 457-462 . |
50. | Sturgill, T.W., L.B. Ray, N.G. Anderson, and A.K. Erickson. 1991. Purification of mitogen-activated protein kinase from epidermal growth factor-treated 3T3-L1 fibroblasts. Methods Enzymol. 200: 342-351 [Medline]. |
51. | Vik, T.A., L.J. Sweet, and R.L. Erikson. 1990. Coinfection of insect cells with recombinant baculovirus expressing pp60v-src results in the activation of a serine-specific protein kinase pp90rsk. Proc. Natl. Acad. Sci. USA. 87: 2685-2689 [Abstract]. |
52. | Dudley, D.T., L. Pang, S.J. Decker, A.J. Bridges, and A.R. Saltiel. 1995. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. USA. 92: 7686-7689 [Abstract]. |
53. | Bos, J.L.. 1989. ras oncogenes in human cancer: a review. Cancer Res. 49: 4682-4689 [Abstract]. |
54. | Rodenhuis, S.. 1992. ras and human tumors. Semin. Cancer Biol. 3: 241-247 [Medline]. |
55. | Copeland, N., D. Gilbert, B. Cho, P. Donovan, N. Jenkins, D. Cosman, D. Anderson, S. Lyman, and D. Williams. 1990. Mast cell growth factor maps near the steel locus on mouse chromosome 10 and is deleted in a number of steel alleles. Cell. 63: 175-183 [Medline]. |
56. | Paulson, R.F., S. Vesely, K.A. Siminovitch, and A. Bernstein. 1996. Signalling by the W/Kit receptor tyrosine kinase is negatively regulated in vivo by the protein tyrosine phosphatase Shp 1. Nat. Genet. 13: 309-315 [Medline]. |
57. | Sawai, N., K. Koike, S. Ito, N. Okumura, T. Kamijo, M. Shiohara, Y. Amano, K. Tsuji, T. Nakahata, M. Oda, et al . 1996. Aberrant growth of granulocyte-macrophage progenitors in juvenile chronic myelogenous leukemia in serum-free culture. Exp. Hematol. 24: 116-122 [Medline]. |
58. |
Kume, A.,
H. Nishiura,
J. Suda, and
T. Suda.
1997.
Focal adhesion kinase upregulated by granulocyte-macrophage colony-stimulating factor but not by interleukin-3 in differentiating myeloid cells.
Blood.
89:
3434-3442
|
59. |
Takahira, H.,
A. Gotoh,
A. Ritchie, and
H.E. Broxmeyer.
1997.
Steel factor enhances integrin-mediated tyrosine phosphorylation of focal adhesion kinase (pp 125FAK) and paxillin.
Blood.
89:
1574-1584
|
60. | Bollag, G., and F. McCormick. 1991. Differential regulation of rasGAP and neurofibromatosis gene product activities. Nature. 351: 576-579 [Medline]. |
61. | Bollag, G., and F. McCormick. 1992. NF is enough of GAP. Nature. 356: 663-664 [Medline]. |
62. | Carpino, N., D. Wisniewski, A. Strife, D. Marshak, R. Kobayashi, B. Stillman, and B. Clarkson. 1997. p62dok: a constitutively tyrosine-phosphorylated, GAP-associated protein in chronic myelogenous leukemia progenitor cells. Cell. 88: 197-204 [Medline]. |
63. | Koch, C.A., D. Anderson, M.F. Moran, C. Ellis, and T. Pawson. 1991. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 252: 668-674 [Medline]. |
64. | Marchuk, D.A., A.M. Saulino, R. Tavakkol, M. Swaroop, M.R. Wallace, L.B. Andersen, A.L. Mitchell, D.H. Gutmann, M. Boguski, and F.S. Collins. 1991. cDNA cloning of the type 1 neurofibromatosis gene: complete sequence of the NF1 gene product. Genomics. 11: 931-940 [Medline]. |
65. | Lang, R., D. Metcalf, R.A. Cuthbertson, I. Lyons, E. Stanley, A. Kelso, G. Kannourakis, D.J. Williamson, G.K. Klintworth, T.J. Gonda, and A.R. Dunn. 1987. Transgenic mice expressing a hemopoietic growth factor gene (GM-CSF) develop accumulations of macrophages, blindness, and fatal syndrome of tissue damage. Cell. 51: 675-686 [Medline]. |
66. |
Iversen, P.O.,
I.D. Lewis,
S. Turczynowicz,
H. Hasle,
C. Niemeyer,
K. Schmiegelow,
S. Bastiras,
A. Biondi,
T.P. Hughes, and
A.F. Lopez.
1997.
Inhibition of granulocyte-macrophage
colony-stimulating factor prevents dissemination and induces
remission of juvenile myelomonocytic leukemia in engrafted
immunodeficient mice.
Blood.
90:
4910-4917
|