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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 816-825
Prolonged Expression of c-fos Suppresses Cell Cycle Entry of
Dormant Hematopoietic Stem Cells
By
Seiji Okada,
Tetsuya Fukuda,
Kunimasa Inada, and
Takeshi Tokuhisa
From the Department of Developmental Genetics, Chiba University
Graduate School of Medicine, Chiba, Japan.
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ABSTRACT |
The proto-oncogene c-fos was transiently upregulated in
primitive hematopoietic stem (Lin Sca-1+)
cells stimulated with stem cell factor, interleukin-3 (IL-3), and IL-6.
To investigate a role of the c-fos in hematopoietic stem cells,
we used bone marrow (BM) cells from transgenic mice carrying the
c-fos gene under the control of the
interferon- / -inducible Mx-promoter (Mx-c-fos), and fetal liver
cells from c-fos-deficient mice. Prolonged expression of the
c-fos in LinSca-1+ BM cells
inhibited factor-dependent colony formation and hematopoiesis on a
stromal cell layer by keeping them at G0/G1 phase of the cell cycle.
These LinSca-1+ BM cells on a stromal
layer entered into the cell cycle whenever exogenous c-fos was
downregulated. However, ectopic c-fos did not perturb colony
formation by LinSca-1+ BM cells after they
entered the cell cycle. Furthermore, endogenous c-fos is not
essential to cell cycle progression of hematopoietic stem cells because
the factor-dependent and the stroma-dependent hematopoiesis by
LinSca-1+ fetal liver cells from
c-fos-deficient mice was not impaired. These results suggest
that the c-fos induced in primitive hematopoietic stem cells
negatively controls cell cycle progression and maintains them in a
dormant state.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
IT IS WIDELY CONSIDERED that pluripotent
hematopoietic stem cells (PHSC) are highly quiescent in
normal steady state bone marrow (BM), and that dormancy plays an
important role in their preservation.1 However, mechanisms
involved in retaining PHSC at a dormant state are not well understood.
Several cytokines are involved in initiation of cell cycle progression
of the dormant PHSC.1 Those cytokines activate signal
transduction pathways via their own receptors in the PHSC to induce
expression of immediate early genes. Those early gene products function
as transcription factors that regulate expression of target genes. Many
transcription factors control proliferation and differentiation of
PHSC.2,3 Although lineage-restricted transcription factors
such as tal-1/SCL and rtbn2/LMO2 may be candidates as key regulator for
differentiation of PHSC,4-9 widely or even ubiquitously
expressed transcription factors may have a special role in maintaining
PHSC in a dormant state.2,3
The c-fos proto-oncogene, one of the immediate early genes, is
transiently expressed on stimulation by external stimuli leading to
cell cycle progression.10 Its product (c-Fos) forms a
complex with the product of another proto-oncogene c-jun (AP-1)
that regulates expression of AP-1-binding genes at their
transcriptional level.10-12 Thus, c-Fos may play a key role
in the transduction of signals induced by external
stimuli.12-14 c-Fos is known to be critical for the G0/G1
transition and cell cycle progression in fibroblasts.13,14 The overexpression of c-fos in transgenic mice leads to a
deregulated bone growth and results in sarcomas,15,16 and
the overexpression in several cell lines leads to acceleration of cell
cycle progression.17,18 On the contrary, overexpression of
c-Fos negatively regulates cell cycle progression in some cell
types.19 Thus, functions of c-Fos in cell cycle progression
have remained open to question.
c-Fos is thought to be important in various differentiation processes
as well as in development.2,10-12 Although the
developmental capacity of PHSC lacking the c-fos gene appears
to be fairly normal,20-23 functions of c-Fos in controlling
proliferation and differentiation of PHSC are unknown. Inducible type
transgenic mice are powerful tools to investigate the gene function at
certain stages of development.24 Using the transgenic mice
carrying the c-fos gene under the control of the interferon
(IFN)- /-inducible Mx-promoter (Mx-c-fos mice),25 we
have shown that c-Fos interferes cell cycle progression of mature B
cells at the G1/S transition of the cell cycle.26
Furthermore, c-Fos induces apoptosis in pro-B cells27 and
germinal-center B cells28 from Mx-c-fos mice. Thus,
functions of c-Fos in regulating proliferation and differentiation of
PHSC can be investigated using PHSC from Mx-c-fos mice.
Recent progress in the stem-cell biology revealed that
PHSC and progenitors can be identified in BM cells, based on their surface marker profile.29 They lack lineage-specific
antigens (Lin) and express c-kit, H-2K, low level of
Thy-1, and a high affinity to WGA.30-32 PHSC can be further
isolated from committed progenitors by cell-surface staining with
monoclonal antibody against Sca-1 or CD34, or by nuclear staining with
supravital staining dye (rhodamine-123, Hoechst-33342).29,33-36 This method has made feasible
studies on the nature of PHSC at the clonal level. We investigated the role of c-Fos in cell cycle progression of PHSC using primitive hematopoietic stem (LinSca-1+) cells
isolated from Mx-c-fos mice and also c-fos-deficient mice. We
show here that the c-fos gene is transiently expressed in
LinSca-1+ BM cells stimulated with stem
cell factor (SCF), interleukin-3 (IL-3), and IL-6. The prolonged
expression of c-fos inhibits
LinSca-1+ BM cells from entering the
cell cycle. The role of c-Fos in maintenance of PHSC in a dormant state
is discussed.
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MATERIALS AND METHODS |
Mice.
C57BL/6CrSlc mice were purchased from Japan SLC Co, Ltd (Hamamatsu,
Japan). Transgenic mice carrying the mouse c-fos gene under the
control of the Mx gene promoter (Mx-c-fos)25 and
c-fos-deficient mice,20 provided by Dr E.F. Wagner
(IMP, Vienna, Austria), were maintained by heterozygous mating in our
animal facility.
Reverse-transcribed polymerase chain reaction (RT-PCR) analysis.
Total RNA was extracted from 1 × 104 of
LinSca-1+ BM cells using an ISOGEN total
RNA isolating kit (Waco, Tokyo, Japan). RNAs were reverse-transcribed
using Superscript (Life Technologies, Grand Island, NY) and oligo(dT)
(Pharmacia, Piscataway, NJ), in a final volume of 20 µL, and 1 µL
of cDNA was used for PCR. After an initial 7-minute incubation at
95°C, 22 cycles of PCR were performed using the following
conditions: c-fos cDNA, denaturation at 95°C for 1.5 minutes, annealing at 55°C for 1.5 minutes, and polymerization at
72°C for 1.5 minutes; G3PDH cDNA, denaturation at 95°C for 1 minute, annealing at 60°C for 1 minute, and polymerization at
72°C for 1 minute. PCR primers for the cDNA amplification were as
follows: the c-fos primers,37
5'-TTCTCGGGTTTCAACGCC-3' and 5'-GGCGTTGAAACCCGAGAA-3'; and the G3PDH
primers,38 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3' and
5'-CATGTAGGCCATGAGGTCCACCAC-3'.
PCR products were separated on a 1.5% agarose gel, transferred onto a
nylon membrane (Boehringer Mannheim, Mannheim, Germany) and fixed by
cross-linking with ultraviolet irradiation and by baking at 80°C
for 3 hours. The filter was hybridized overnight with the digoxigenin
(DIG)-labeled probe at 42°C. Following hybridization, the filter
was washed twice with 0.1× standard saline citrate/0.1% sodium
dodecyl sulfate at 68°C for 15 minutes. The probe on the filter was
detected by sheep anti-DIG antibody conjugated with alkaline
phosphatase. The antibody detection reaction was performed using the
enhanced chemiluminescent detection system (Boehringer Mannheim GmbH,
Mannheim, Germany). The probes were the full-length murine
c-fos cDNA and murine G3PDH cDNA that were subcloned into pGEM
vectors and labeled by DIG using PCR with T7 and SP6 primers.
Hematopoietic growth factors.
Purified recombinant human IL-6, purified recombinant murine IL-3, and
conditioned medium (CM) of Chinese hamster ovary (COS) cells that had
been transfected with an expression plasmid containing the murine SCF
cDNA, were provided by Dr Tetsuo Sudo (Toray Industries, Kamakura,
Japan). Concentration of SCF CM was assessed as 3 µg/mL in the
proliferation assay by BaF cells transfected with mouse c-kit receptor.
Recombinant murine SCF was purchased from Pepro Tech (Rocky Hill, NJ).
Unless specified otherwise, SCF CM was used as a source of SCF.
Recombinant human erythropoietin (Epo) was provided by Kirin Brewery
(Tokyo, Japan). Concentration of the cytokines used here was as
follows: IL-3, 200 U/mL; IL-6, 20 ng/mL; SCF CM, 5%; SCF, 100 ng/mL;
Epo, 2 U/mL.
Antibodies.
Biotinylated monoclonal antibodies against B220 (RA3-6B2), Mac-1
(M1/70), Gr-1 (RB6-8C5), CD4 (GK1.5), CD8 (53-6.72), and TER119 were
purchased from PharMingen (San Diego, CA) and used to detect lineage
markers. Fluorescein isothiocyanate (FITC)-conjugated anti-Sca-1
(Ly6A/E) antibody was purchased from PharMingen. Biotinylated antibodies were visualized using Streptavidin-phycoerythrin (PE; PharMingen).
Isolation of LinSca-1+ cells from BM
cells or fetal liver (FL) cells.
Total BM cells from Mx-c-fos mice and their littermates were stained
with a cocktail of biotinylated monoclonal antibodies against lineage
markers for 20 minutes at 4°C. FL cells isolated from
c-fos-deficient embryos and their littermates on day 14.5 postcoitus were stained with a cocktail of biotinylated monoclonal antibodies against lineage markers except for anti-Mac-1 antibody, as
described.39 After washing the cells 3 times with staining medium (phosphate-buffered saline with 3% fetal calf serum [FCS] and
0.1% sodium azide), the cells were treated with
streptavidin-conjugated immunomagnetic beads (BioMag; Perceptive
Diagnostics, Cambridge, MA) for 30 minutes to remove lineage marker
highly positive cells. The remaining cells were collected and stained
with FITC-anti-Sca-1 antibody and Streptavidin-PE at 4°C for 20 minutes. After washing, the cells were resuspended in staining medium
supplemented with propidium iodide (PI; 1 µg/mL). Stained cells were
analyzed by FACS Vantage (Becton Dickinson, San Jose, CA), and the
LinSca-1+ cells were sorted and used as
a primitive hematopoietic stem-cell fraction.40
In vitro colony assay.
Methylcellulose culture was performed using the modified
method32 described by Iscove.41 Briefly, 1 mL
of culture medium contained an adequate number of total BM cells or
sorted LinSca-1+ cells, 1.2%
methylcellulose (Shin-etsu Chemical Co, Tokyo, Japan), alpha-medium
(Flow Laboratories, North Ryde, Australia), 30% FCS (Flow
Laboratories), 1% deionized bovine serum albumin (Sigma Chemical, St
Louis, MO), 0.1 mmol/L -2-mercaptoethanol (Eastman Organic Chemical,
Rochester, NY), and appropriate concentrations of growth factors in the
presence or absence of IFN-/ (Sigma Chemical). The cultures were
prepared in 35-mm nontissue culture dishes (Beckton Dickinson Labware,
Lincoln Park, NJ) and incubated at 37°C in a humidified atmosphere
of 5% CO2. The number of colonies was counted using an
inverted microscope. When IFN-/ was added in the culture on day
4, 100 µL of alpha-medium with 200 U of IFN-/ was added to the
methylcellulose culture dish.
Short-term liquid culture.
Total BM cells or sorted LinSca-1+ cells
were cultured in a 24-well plate (Beckton Dickinson Labware) with 1 mL
alpha-medium containing 20% FCS and appropriate concentrations of
growth factors in the presence (200 U/mL) or absence of IFN-/
under a humidified 5% CO2 atmosphere at 37°C. After 7 days of culture, cells were obtained and nucleated cells were counted
using Trypan blue (Life Technologies).
Spleen colony assay.
The spleen colony assay of Till and McCulloch42 was used.
Freshly isolated or cultured LinSca-1+
cells were injected into lethally irradiated mice (9.0 Gy total body
irradiation). The spleens were removed on day 8 or day 12 after
transplantation, fixed in Bouin's solution, and macroscopically visible colonies were counted and scored as colony forming unit in
spleen (CFU-S).
Coculture of LinSca-1+ cells with
stromal cells.
PA-6 stromal cells support myelopoiesis.43 PA-6 cells (3 × 105/well) were seeded in a 6-well plate (PRIMARIA,
Beckton Dickinson Labware) 1 day before coculture. Five hundred sorted
LinSca-1+ cells were cultured on the
PA-6 stromal layer with 3 mL of alpha-medium containing 10% FCS in the
presence (200 U/mL) or absence of IFN-/.
Cell cycle analysis.
Cell cycle analysis was performed as described by
Nicoletti.44 Briefly, sorted
LinSca-1+ cells were cultured for 24 hours with SCF, IL-3, and IL-6 in the presence or absence of
IFN-/. Those cells were incubated in hypotonic lysing buffer
(0.1% sodium citrate, 0.01% Triton X, 0.1 mg/mL RNase, and 0.1 mg/mL
PI). DNA content in each nuclei was analyzed on FACScalibur (Becton
Dickinson, Mountain View, CA) using Cell Quest software
(Becton Dickinson) for Macintosh (Apple Computet Inc, Cupertino, CA).
5-bromo-2'-deoxyuridine (BrdU) incorporation assay.
Sorted LinSca-1+ cells were cultured
with SCF, IL-3, and IL-6 in the presence or absence of IFN-/ for
36 hours and pulsed with 10 µmol/L BrdU (Sigma) for 3 hours. Those
cells were spread on a slide glass by Cytospin (Shandon Southern
Instruments Inc, Sewickley, PA) and fixed with cold acetone for 10 minutes. The cells were then incubated with 2N HCl for 1 hour, followed
by reaction with mouse monoclonal antibody to BrdU (Boehringer
Mannheim, Indianapolis, IN). These cells were further incubated with
F(ab')2 fragment of anti-mouse Ig labeled with
horseradish peroxidase (Nycomed Amersham plc, Buckinghamshire, UK). The
DAB kit (Nichirei, Tokyo, Japan) was used to visualize peroxidase, and
counter staining was done using hematoxylin.45
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RESULTS |
Expression of the c-fos gene in primitive hematopoietic stem
cells stimulated with SCF, IL-3, and IL-6.
To examine expression of the c-fos gene in primitive
hematopoietic stem cells after stimulation,
LinSca-1+ BM cells from Mx-c-fos
mice and their control littermates were stimulated with SCF, IL-3, and
IL-6 in the presence or absence of IFN-/. Expression of
c-fos was analyzed by RT-PCR with Southern blotting
(Fig 1A). c-fos RNA was detected in
the LinSca-1+ cells from both Mx-c-fos
and control mice at 1 hour but not at 7 hours after stimulation in the
absence of IFN-/, thereby indicating transient expression induced
in these hematopoietic stem cells. When the cells were stimulated with
SCF, IL-3, and IL-6 in the presence of IFN-/ (200 U/mL), a large
amount of c-fos RNA was evident in the Mx-c-fos but not in
control stem cells, even 7 hours after stimulation. When IFN-/
was added to those cultures on day 0 and day 4, c-fos mRNA was
continuously detected until day 7 of culture (Fig 1B). Because
IFN-/ receptor is present in almost every cell type, including
hematopoietic stem cells,46 we concluded that all of the
cells derived from Mx-c-fos LinSca-1+
cells in the culture express the exogenous c-fos in the
presence of IFN-/.
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| Fig 1.
Prolonged expression of c-fos mRNA in primitive
hematopoietic stem cells from Mx-c-fos mice.
LinSca-1+ BM cells were cultured with SCF,
IL-3, and IL-6 in the presence (200 U/mL) or absence of IFN-/ for
7 hours (A), or by the addition of IFN-/ (200 U/mL) on day 0 and
day 4 (B). Levels of c-fos mRNA were measured by RT-PCR
analysis followed by Southern blotting, as described in Materials and
Methods. G3PDH mRNA served as an internal control for the amount of
RNA.
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Effect of the prolonged expression of c-fos on colony
formation by primitive hematopoietic stem cells stimulated with SCF,
IL-3, and IL-6.
Total BM cells or LinSca-1+ BM cells
from Mx-c-fos mice and their control littermates were cultured with
SCF, IL-3, and IL-6 in the presence of various concentrations of
IFN-/, and the number of colonies was counted in those cultures
on day 8. As shown in Fig 2A, colony
formation by BM cells from Mx-c-fos mice was suppressed in the
presence of IFN-/ at more than 200 U/mL. However, the suppression
was not so significant in control cultures even at 2,000 U/mL of
IFN-/, although size of each colony was reduced at more than
1,000 U/mL of IFN-/ (data not shown). The suppression was more
evident in the LinSca-1+ cell cultures
from Mx-c-fos mice in the presence of IFN-/ (Fig 2B),
which means that c-fos suppressed on onset of colony formation from primitive hematopoietic stem cells but not from progenitors.
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| Fig 2.
Inhibitory effect of c-fos on colony formation by
primitive hematopoietic stem cells. Total BM cells or
LinSca-1+ BM cells from Mx-c-fos mice or
control littermates were cultured with SCF, IL-3, and IL-6 with various
concentrations of IFN-/. On day 8 of culture, the number of
colonies was scored. Results represent mean and SD of four dishes. The
data presented are representative of two independent experiments.
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Kinetics of colony formation by
LinSca-1+ cells was further examined by
adding IFN-/ to the cultures on day 0 and day 4 (Fig 3). The number of colonies increased
and reached a plateau in both Mx-c-fos and control cultures on day 8 of culture in the absence of IFN-/. When IFN-/ (200 U/mL)
was added to the culture on day 0, colony formation was delayed and the
plateau level was slightly reduced in the Mx-c-fos but not in the
control cultures. Furthermore, the addition of IFN-/ to those
cultures on day 0 and day 4 slowed down the colony formation and
lowered the plateau level in the Mx-c-fos cultures but not in the
control cultures. These observations suggest that prolonged expression
of c-fos suppresses the onset of colony formation by primitive
hematopoietic stem cells stimulated with SCF, IL-3, and IL-6.
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| Fig 3.
Kinetics of colony formation by primitive hematopoietic
stem cells. LinSca-1+ BM cells (3 × 102) from Mx-c-fos mice or control littermates were
cultured with SCF, IL-3, and IL-6 ( ). IFN-/ (200 U/mL) was
added on day 0 ( ), or on day 0 and day 4 ( ). The number of
colonies was scored every 4 days. Results represent mean and SD of four
dishes. The data presented are representative of two independent
experiments.
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To further analyze the inhibitory effect of c-fos on growth of
the colony, we serially plotted growth rate of each colony through
colony mapping study, as described by Ikebuchi.47 One hundred and fifty of LinSca-1+ cells per
dish were cultured with SCF, IL-3, IL-6, and Epo, and emergence of a
new colony later showed the CFU-Mix and its subsequent rate of
proliferation were analyzed (Fig 4).
Although the number of CFU-Mix was about 13 to 16 per dish in both
Mx-c-fos and control cultures in the presence (200 U/mL) or
absence of IFN-/, the onset of each colony was delayed in
Mx-c-fos cultures but not in control cultures when IFN-/ was
added on day 0 of culture. When IFN-/ was added to those cultures
on day 0 and day 4, the onset of each colony was further delayed in the
Mx-c-fos cultures. However, the proliferation rate of each colony
developed in the Mx-c-fos cultures did not differ
significantly from that in the control cultures in the presence of
IFN-/. Becuase 200 U/mL of IFN-/ can induce expression of
the exogenous c-fos gene in BM cells from Mx-c-fos mice for
more than 2 days,26,28 these results suggest that after
entering the cell cycle c-fos does not inhibit growth of
colonies derived from primitive hematopoietic stem cells.
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| Fig 4.
Mapping study of colony formation by primitive
hematopoietic stem cells. LinSca-1+ BM
cells (1 × 102) from Mx-c-fos mice or control
littermates were cultured with SCF, IL-3, IL-6, and Epo. IFN-/
(200 U/mL) was added on day 0, or on day 0 and day 4. Graphic
presentation indicates cell number changes in individual colonies that
later became a mixed colony (CFU-Mix). The data represent colonies
identified in two plates. The data presented are representative of two
independent experiments.
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Effect of c-fos on cell growth of primitive hematopoietic
stem cells stimulated with various combinations of cytokines in the
liquid culture.
The combination of SCF, IL-3, and IL-6 provides one of the strongest
signals for differentiation of stem cells and
progenitors.1,36 Stimulation of SCF and IL-6 is required
for expansion of immature cells48 and that of IL-3 alone
supports differentiation of stem cells and progenitors in a cycling
state.1,47 To examine which cytokine signals were inhibited
by c-fos, we did short-term liquid cultures of stem cells from
Mx-c-fos mice, using various combinations of cytokines by
adding IFN-/ (200 U/mL) on day 0 and day 4 of culture, and the
number of nucleated cells was counted on day 7 of culture
(Fig 5). When total BM cells were cultured
with combinations of cytokines, cell growth in the Mx-c-fos cultures
with SCF+IL-6 stimulation was suppressed in the presence of
IFN-/. However, cell growth in Mx-c-fos cultures with
SCF+IL-3+IL-6 or IL-3 alone was not suppressed, suggesting that the
prolonged expression of c-fos inhibits cell growth of primitive
hematopoietic stem cells. Indeed, cell growth in cultures of
LinSca-1+ BM cells from Mx-c-fos
mice but not from control littermates with all of the combinations
of cytokines used was inhibited in the presence of IFN-/.
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| Fig 5.
Inhibitory effect of c-fos on cell proliferation
by primitive hematopoietic stem cells.
LinSca-1+ BM cells from Mx-c-fos or
control littermates were cultured with SCF+IL-3+IL-6, SCF+IL-6,
or IL-3. IFN-/ (200 U/mL) was added to the culture on day 0 and
day 4. On day 7 of culture, the number of viable cells was counted by
Trypan blue dye exclusion. Results represent mean and SD of four
dishes. The data presented are representative of three independent
experiments. Purified recombinant SCF was used in this experiment.
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To confirm the suppressive effect of c-fos on cell growth of
primitive hematopoietic stem cells, cell cycle analysis of
LinSca-1+ BM cells stimulated with SCF,
IL-3, and IL-6 in the liquid culture was done using
fluorescence-activated cell sorter (FACS). As shown in
Fig 6A, the percentage of cells in the
S/G2/M phase was approximately 15% in Mx-c-fos and control cultures
in the absence of IFN-/ and in control cultures in the presence
of IFN-/ (200 U/mL) 24 hours after stimulation. In contrast, only
3.3% of the LinSca-1+ cells from
Mx-c-fos mice were in the S/G2/M phase in the presence of IFN-/.
Similar results were obtained in the case of BrdU incorporation assay
(Fig 6B). Approximately 30% of cells were BrdU positive in cultures
from Mx-c-fos and from control mice in the absence of
IFN-/ and in control cultures in the presence of IFN-/ 36 hours after stimulation. However, only 5% of cells were positive for
BrdU in Mx-c-fos cultures in the presence of IFN-/.
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| Fig 6.
Cell cycle analysis of primitive hematopoietic stem cells
after stimulation. LinSca-1+ BM cells from
Mx-c-fos or control littermates were cultured with SCF, IL-3,
and IL-6 in the presence (200 U/mL) or absence of IFN-/. (A)
After 24 hours, the cells were lysed and the nuclei were stained with
propidium iodide. DNA content in the nuclei was determined by FACS.
Percentage of PI-labeled nuclei in S/G2/M phase of the cell cycle is
indicated. (B) After 36 hours, cells were pulsed with BrdU for 3 hours.
Incorporated BrdU was detected by anti-BrdU monoclonal antibody and
positive cells were counted. Results represent mean and SD of four
dishes. The data presented are representative of three independent
experiments.
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To examine the suppressive effect of c-fos on differentiation
of hematopoietic stem cells, we performed the spleen colony assay.42 Five hundred
LinSca-1+ BM cells were cultured with
SCF+IL-3+IL-6 in the presence (200 U/mL) or absence of IFN-/. On
day 4 of culture, cells were collected and injected into lethally
irradiated mice. As shown in Table 1,
generation of day-8 CFU-S but not day-12 CFU-S was suppressed in mice
injected with these cultured cells from Mx-c-fos mice in the presence
of IFN-/. The number of day-8 CFU-S and that of day-12 CFU-S
reflect the number of hematopoietic progenitor cells and that of more
primitive hematopoietic stem cells,42,49 respectively.
Therefore, these results indicate that prolonged expression of
c-fos suppresses differentiation of hematopoietic stem cells.
Effect of c-fos on hematopoiesis by primitive hematopoietic
stem cells cultured on a stromal cell layer.
To investigate the suppressive effect of c-fos on hematopoiesis
by primitive hematopoietic stem cells on a stromal cell layer, LinSca-1+ BM cells were cultured
on a layer of PA-6 stromal cells. Medium in the cultures was changed
twice a week, and the number of nonadherent cells was counted by Trypan
blue exclusion. As shown in Fig 7, the
number of cells in both Mx-c-fos and control cultures exponentially increased and reached a plateau after day 12 of culture. When IFN-/ (200 U/mL) was added to medium from the start of culture, the number of cells in Mx-c-fos cultures did not clearly increase. When addition of IFN-/ in the medium was ceased after day 12 of
culture, the number of cells increased and caught up to the control
level in the Mx-c-fos cultures within 1 week. These data suggest that
prolonged expression of c-fos to primitive hematopoietic stem cells also inhibits stroma-dependent hematopoiesis.
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| Fig 7.
Inhibitory effect of c-fos on the
stroma-dependent hematopoiesis by primitive hematopoietic stem cells.
LinSca-1+ BM cells (3 × 102 cells/well in 6-well tissue culture plate) were
cultured on a PA-6 stromal-cell layer in the presence (closed square;
200 U/mL) or absence () of IFN-/. On day 12 of culture, the
cells from Mx-c-fos culture with IFN-/ were obtained and washed
with medium, and continued to culture on the PA-6 stromal layer with
( ) or without () IFN-/. Nonadherent cells obtained from at
least four dishes by gentle pipetting at every medium change were
rinsed once with medium, pooled, and counted. The data presented are
representative of three independent experiments.
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Factor-dependent and stroma-dependent hematopoiesis by primitive
hematopoietic stem cells from c-fos-deficient mice.
FL cells from c-fos-deficient mice was used as the source of
primitive hematopoietic stem cells because of the osteopetrosis in
c-fos-deficient mice.20,21 Kinetics of colony
formation by LinSca-1+ cells was
examined in the cultures with SCF+IL-3+IL-6 or IL-3 alone. As shown in
Fig 8, kinetics of colony formation did not significantly differ between c-fos-deficient and control
littermates. In addition, stromal-dependent hematopoiesis by
LinSca-1+ cells from
c-fos-deficient mice was normal because kinetics of cell
growth on a PA-6 stromal layer by the
LinSca-1+ cells from
c-fos-deficient mice showed similar features to those seen in
case of control littermates (Fig
9). These results suggest that endogenous c-fos is not
required for cell cycle progression of primitive hematopoietic stem
cells.
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| Fig 8.
Kinetics of colony formation by primitive hematopoietic
stem cells from c-fos-deficient mice.
LinSca-1+ cells (3 × 102)
from FL of c-fos-deficient mice or control littermates were
cultured with SCF, IL-3, and IL-6 (), or IL-3 alone (). The
number of colonies was scored and plotted. Results represent mean and
SD of four dishes. The data presented are representative of three
independent experiments.
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| Fig 9.
Kinetics of the stroma-dependent hematopoiesis by
primitive hematopoietic stem cells from c-fos-deficient mice.
LinSca-1+ cells (3 × 102
cells/well in 6-well tissue culture plate) from FL of
c-fos-deficient mice () or control littermates
(c-fos+/+, ; c-fos+/, ) were
cultured on a PA-6 stromal cell layer. The number of nonadherent cells
was counted (see Fig 7 legend). The data presented are representative
of three independent experiments.
|
|
| |
DISCUSSION |
In the steady state, the majority of hematopoietic stem cells reside in
BM at a dormant state,1,49,50 and only a few stem cells
supply all of the hematopoietic cells at a given time.1,51 Because hematopoietic stem cells in BM are always exposed to various forms of stimuli, mechanisms to maintain the stem cells in a dormant state no doubt exist. We found that c-fos negatively controls cell cycle progression of primitive hematopoietic stem cells. Initiation of cell cycling and subsequent proliferation of stem cells
appear to require collaboration of early acting cytokines. Ogawa
proposed that cytokines regulating proliferation of primitive hematopoietic progenitors may be separated into three arbitrary groups.1 The combination among these three groups such as
SCF, IL-3, and IL-6 is one of the most effective stimuli supporting the
early process of hematopoiesis.1,36 These stimulations transiently induce expression of c-fos in primitive
hematopoietic stem cells (Fig 1). Because prolonged expression of
c-fos inhibits G0/G1 transition of dormant hematopoietic stem
cells in both cytokine-dependent (Figs 2 to 5) and stroma-dependent
(Fig 8) hematopoiesis, downregulation of the c-fos may initiate
G0/G1 transition. Indeed, cell proliferation began in the stem cell
culture from Mx-c-fos mice whenever addition of IFN-/ to the
culture was stopped (Fig 8). Therefore, c-Fos may be a gate keeper for
cell cycle entry of dormant hematopoietic stem cells.
c-Fos is an important positive regulator of cell growth and notably of
the G0/G1 transition.2,10-12,14 Expression of c-fos antisense RNA or injection of anti-Fos antibodies inhibit
serum-stimulated cells to enter into S phase of the cell cycle and to
reduce the growth rate of asynchronously growing cells.52
In addition, overexpression of c-fos in fibroblasts and myeloid
cell lines accelerates growth rate.17,18 On the other hand,
Balsalobre and Jolicoeur19 showed that G0-S progression of
rat-1 fibroblasts was delayed by overexpression of c-fos. We
reported that prolonged expression of c-fos also perturbs cell
cycle progression of mature B cells by surface Ig
cross-linking.26 The c-fos gene is transiently induced in B cells, and prolonged expression inhibits B cells from
passing into S phase of the cell cycle. This perturbation of cell cycle
progression is due to poor degradation of the cyclin kinase inhibitor
p27kip1 in the G1 phase. However, prolonged expression of
c-fos accelerates cell cycle progression of mature B cells
stimulated with LPS.53 Taken together, c-Fos may act as a
negative and as a positive regulator of cell growth that is dependent
on cell types, stimulation signals, differentiation stages, and cell
cycle states.
The inhibitory effect of c-fos on cell cycle progression of
dormant hematopoietic stem cells was also shown in a mapping study (Fig
4). However, growth rate was not significantly affected by the
expression after colony formation began. This is supported by the
finding that colony formation by total BM cells was affected by
c-fos much less than that by purified
LinSca-1+ BM cells because the majority
of colony-forming cells in total BM are committed progenitors. These
results suggest that endogenous c-fos negatively regulates cell
cycle progression of dormant hematopoietic stem cells.
Embryonal stem cells and 3T3-type fibroblasts lacking the c-fos
gene divide at a normal rate.54,55 Furthermore,
c-fos-deficient mice are viable but do have osteopetrosis, as
a primary pathology.20,21 Although they have extramedullary
hematopoiesis in the spleen, B lymphopenia, and thymic
atrophy,20,21 we have found that hematopoietic stem cells
can appear normal,22 except for failure differentiation
into functional osteoclasts.56 In the present study,
we found that both factor-dependent and stroma-dependent hematopoiesis
by primitive hematopoietic stem cells from c-fos-deficient mice are normal (Fig 8 and 9). Colony formation by stem cells stimulated with IL-3+IL-6+SCF or IL-3 alone was almost the same as that seen in control littermates (Fig 8). These results indicate that c-fos is not essential for cell proliferation in several cell types and that redundancy with other members of the fos
gene family may exist.11,12 These results also support the
notion that c-Fos is a gate keeper for cell cycle entry of primitive hematopoietic stem cells.
c-Fos is also implicated in apoptosis.11,57 Treatment of
cells with antisense oligonucleotide against c-fos increased
survival of growth factor-derived lymphoid cells,37
suggesting that expression of c-fos may represent an early
event in activating programmed cell death. High levels of c-fos
expression were also observed in mouse tissues in which apoptosis is
part of normal development.57 We also reported that
overexpression of c-fos induced apoptosis of CD43+
pro-B cells.27 On the other hand, c-fos plays a
protective function from apoptosis of fibroblasts in response to stress
such as ultraviolet irradiation.58 Mapping study (Fig 4)
and spleen colony assay (Table 1) showed that significant number of
primitive hematopoietic stem cells could survive despite the prolonged
expression of c-fos. Furthermore, hematopoietic stem cells with
long-term repopulating ability remain after the liquid culture for 7 days with SCF, IL-3, and IL-6 in the presence of IFN-/ (data not shown). These results indicate that c-fos is not related to
apoptosis in the dormant-state hematopoietic stem cells. Alternatively, as SCF, IL-3, and IL-6 are survival factors for primitive hematopoietic stem cells,59,60 these factors may contribute to survival
of hematopoietic stem cells with overexpression of c-fos.
In summary, the c-fos was transiently induced in primitive
hematopoietic stem cells stimulated with SCF, IL-3, and IL-6. The prolonged expression of c-fos inhibited cell-cycle entry of
primitive hematopoietic stem cells stimulated with SCF, IL-3, and
IL-6 as well as in case of cultures on a stromal cell layer.
Hematopoietic stem cells with the c-fos expression in culture
survived in a dormant state and entered the cell cycle after
c-fos was downregulated. We propose that c-Fos plays the role
of gate keeper in cell cycle progression of dormant
hematopoietic stem cells.
| |
ACKNOWLEDGMENT |
We thank Drs T. Suda and M. Hatano for helpful discussions, Dr E.F.
Wagner for c-fos-deficint mice, Dr T. Sudo for reagents, E. Furusawa and N. Fujita for secretarial services, and M. Ohara for
comments on the manuscript.
| |
FOOTNOTES |
Submitted June 17, 1998; accepted September 28, 1998.
Supported in part by Grants-in-Aid from the Ministry of Education,
Science, Sports and Culture of Japan and a Research Grant from the
Inohana Foundation.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Takeshi Tokuhisa, MD, Department of
Developmental Genetics, Chiba University Graduate School of Medicine,
Chiba 260-8670, Japan; e-mail: tokuhisa{at}med.m.chiba-u.ac.jp.
| |
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Top
Abstract
Introduction