(Received for publication, May 12, 1995)
From the
Stimulatory cytokines, including granulocyte-macrophage
colony-stimulating factor (GM-CSF) and steel factor (SLF), act in a
synergistic manner to stimulate the growth of hematopoietic progenitor
cells, an effect also demonstrated for the growth factor-dependent
human hematopoietic cell line MO7e. While little is known about the
mechanisms responsible for mediating synergistic interactions of
cytokines, Raf-1, a component of the MAP kinase signaling pathway, is
thought to play a role in the stimulatory response evoked by several
cytokines, including SLF and GM-CSF. Interferon-inducible protein-10
(IP-10) and macrophage inflammatory protein-1 (MIP-1
) are
members of the chemokine family of suppressive cytokines. Prior
exposure of hematopoietic cells to chemokines, including IP-10 and
MIP-1
, inhibits the synergistic action of growth factors on
stimulating cell proliferation. We report that treatment of MO7e cells
with the combination of GM-CSF and SLF directly stimulates
statistically significant synergistic increases in the phosphorylation
and activation of Raf-1 kinase, and in cellular protein synthesis
levels. Pretreatment of MO7e cells with IP-10 or MIP-1
blocked
synergistic growth factor action, resulting in statistically
significant suppression of cell proliferation, protein synthesis, and
Raf-1 phosphorylation and activation. IP-10 and MIP-1
treatment
also evoked significant increases in intracellular cAMP levels.
Pretreatment of cells with agents which serve to raise intracellular
cAMP levels, or with cAMP analogs inhibited the synergistic actions of
GM-CSF and SLF in a manner similar to IP-10 and MIP-1
. In
addition, treatment of cells with a potent inhibitor of cAMP-dependent
protein kinase A blocked the suppressive action of MIP-1
and IP-10
on Raf-1 kinase activity and on MO7e cell proliferation. The ability of
IP-10 and MIP-1
to antagonize the synergistic action of GM-CSF and
SLF appears to involve inactivation of Raf-1 and the down-regulation of
protein synthesis. Our findings suggest that both MIP-1
and IP-10
mediate their suppressive effects in MO7e cells by stimulating
increases in cellular cAMP levels and activating protein kinase A, a
mechanism we believe to be unique to these chemokines and not one
applied to all growth suppressive members of the chemokine superfamily
(for example, interleukin 8 and platelet factor 4).
Growth of hematopoietic progenitor cells is coordinated by a
number of stimulatory and inhibitory cytokines. Several stimulatory
cytokines, including granulocyte-macrophage colony-stimulating factor
(GM-CSF) ()and steel factor (SLF), promote the growth of
hematopoietic progenitor cells in a synergistic manner when
administered in combination(1) . Upon ligand binding and
activation, cytokine receptors set into motion a cascade of
phosphorylation and dephosphorylation reactions designed to transmit
information from the cell membrane to other portions of the target cell (2) . Activation of some cytokine receptors leads to
phosphorylation and activation of receptor-associated proteins,
including mSOS and GRB-2 (3) , and the subsequent activation of
ras(4) . Activated Ras is thought to target inactive Raf-1
proteins to the cell membrane where they are phosphorylated and become
active kinases(5) . Both Ras and Raf-1 are components of the
MAP kinase signaling pathway, a major stimulatory pathway within cell
systems. While the mechanisms responsible for growth regulation within
hematopoietic cells are not completely known and even less is known
regarding synergistically induced cell proliferation, it appears likely
that cross-talk between proteins associated with and activated by
separate cytokine receptors mediate changes within target cells which
are necessary for synergistic stimulation of cell proliferation.
The
chemokine family of cytokines includes macrophage inflammatory protein
1- (MIP-1
) and interferon inducible protein-10 (IP-10) (6) . These molecules suppress the synergistic action of
combinations of stimulatory cytokines on hematopoietic progenitor cell
growth(7, 8, 9, 10) . Other
suppressive chemokines include interleukin 8 (IL-8), platelet factor 4
(PF4), MIP-2
, and macrophage chemotactic and activating factor
(MCAF; also designated MCP-1)(8, 9, 10) .
Members of the chemokine family which do not suppress progenitor cell
growth include MIP-1
and MIP-2
, GRO-
, and
RANTES(7, 8, 9) . While the suppressive
effects of chemokines have been characterized, the cellular mechanisms
through which growth inhibition is carried out have not been
elucidated. Part of the difficulty is that chemokine suppression of
growth factor action generally occurs during synergistic stimulation of
cell proliferation. Therefore, studying the mechanism of action of
specific chemokines is often limited to hematopoietic systems which not
only display synergistic growth effects in response to growth factors
but can be used readily for various biochemical analyses. Due to the
rarity of hematopoietic stem and progenitor cells and the difficulty of
isolating enough purified cells of this type for biochemical analyses,
growth factor-dependent cell lines have been used(11) . Growth
arrest resulting from serum deprivation or growth factor deprivation is
often associated with profound declines in protein synthesis rates for
many cell systems(12) . Since growth suppression mediated by
chemokines may likely trigger responses similar to those evoked by
factor deprivation, we set out to determine whether cytokine or
chemokine treatment could alter protein synthesis rates in MO7e cells.
We and others have shown previously that treatment of MO7e cells
with either GM-CSF or SLF results in increased phosphorylation and
Raf-1 kinase activity(13, 14) . More recently, we have
shown that treatment of MO7e cells with SLF results in the physical
association between Ras and Raf-1(15) , an event now considered
necessary for activation of Raf-1. Since SLF is known to synergize with
a number of cytokines in promoting cell growth, we investigated whether
Raf-1 kinase activity could be influenced by exposure of MO7e cells to
growth factors in the presence or absence of various chemokines. Given
that recent evidence has shown that Raf-1 can be inactivated through
phosphorylation of Ser, and a second serine residue in the
kinase domain, by cAMP-dependent protein kinase A(16) , we also
investigated whether chemokines or growth factors could alter cAMP
levels in MO7e cells.
Figure 1:
Effect of various
chemokines on basal levels of leucine incorporation. Eighteen h after
factor starvation, MO7e cells maintained in leucine-free RPMI medium
supplemented with [H]leucine (5 µCi/ml) were
treated with the indicated concentrations of MIP-1
, MIP-1
,
PF4, or IP-10 for 24 h. Control cells received vehicle alone. For
cycloheximide treatment, cells were exposed to cycloheximide (50
µM) for 1 h, washed with PBS, and then received control
vehicle only for the remainder of the treatment duration. Whole cell
lysates were prepared and analyzed for protein content.
[
H]Leucine incorporation was determined for
lysate aliquots (150 µg/sample) by trichloroacetic precipitating
labeled proteins onto glass fiber filters and counting the amount of
[
H]leucine present in dried filters by liquid
scintillation counting. Each point represents the mean of three
separate determinations. Incorporation levels were significantly lower
than control values (p < 0.05) for all groups treated with
MIP-1
, IP-10, or PF4 at concentrations of 20 ng/ml or greater. In
contrast, treatment with MIP-1
at any concentration did not
significantly effect basal levels of leucine incorporation. Similar
results were obtained in each of three separate
experiments.
Figure 2:
Chemokine pretreatment antagonizes the
stimulatory action of GM-CSF plus SLF on protein synthesis levels.
Factor-starved MO7e cells maintained in leucine-free RPMI supplemented
with [H]leucine (5 µCi/ml) were treated for
the indicated durations with either 100 units/ml GM-CSF plus 50 ng/ml
SLF (GM+SLF), 50 ng/ml IP-10 (IP-10), 50 ng/ml
MIP-1
(panel A), 1 µg/ml cholera toxin (C.T., panel B), 50 ng/ml IL-8 (panel C), 50
ng/ml PF4 (panel C), IP-10 for 1 h prior to GM+SLF (IP-10+GM+SLF), MIP-1
for 1 h prior to
GM+SLF (MIP-1
+GM+SLF, panel A),
cholera toxin (1 µg/ml) for 1 h prior to GM+SLF (CT+GM+SLF, panel B), IL-8 for 1 h prior to
GM+SLF (IL-8+GM+SLF, panel C),
MIp-1
for 1 h prior to GM+SLF (MIP-1
+GM+SLF, panel C), or PF4 for 1
h prior to GM+SLF (PF4+GM+SLF, panel
C). Cell lysates were analyzed for protein content and
[
H]leucine incorporation, as described in the
legend to Fig. 1. Each point represents the mean of three
separate determinations. Incorporation levels for the GM-CSF plus SLF
treatment group were significantly higher than controls at 12, 18, and
24 h for experiments shown in panels A-C (p <
0.05). Incorporation levels for MIP-1
, IP-10, or cholera toxin
pretreatment groups were significantly less than those for the GM-CSF
plus SLF group at 12, 18, and 24 h (p <
0.05).
Figure 3:
Inhibitory effectiveness of IP-10 is
related to pretreatment duration. Factor-starved MO7e cells were
treated with IP-10 at the same time as 100 units/ml GM-CSF plus 50
ng/ml SLF (IP-10(0)+G+S), or for 15 min (IP(15)+G+S), 30 min (IP(30)+G+S)
or 45 min (IP(45)+G+S) prior to treatment with
GM-CSF plus SLF in the presence of [H]leucine.
Cell lysates were analyzed for protein content and
[
H]leucine incorporation, as described in the
legend to Fig. 1. Each point represents the mean of duplicate
determinations. Similar results were obtained in each of three separate
experiments.
Figure 4:
IP-10 and MIP-1 significantly
increase cAMP levels in MO7e cells. Factor-starved MO7e cells were
treated for the indicated times with IP-10 (50 or 100 ng/ml),
MIP-1
(50 or 100 ng/ml), PF4 (100 ng/ml), IL-8 (50 ng/ml),
MIP-1
(50 ng/ml), or 1 µg/ml cholera toxin (not shown). Cell
lysates were analyzed directly for protein content and for cAMP
content, using a commercially available [
H]cAMP
assay kit (Amersham). Each sample was assayed in duplicate. Each bar represents the mean ± S.E. of separate
determinations obtained from three separate experiments. cAMP levels
evoked by treatment with either concentrations of IP-10 or MIP-1
were significantly higher than control levels at 2 and 4 h (p < 0.05). cAMP levels of cells treated with PF4, IL-8, or
MIP-1
did not differ significantly from control
levels.
Figure 5:
Effect of chemokine and cytokine treatment
on Raf-1 phosphorylation. Factor-starved MO7e cells (3 10
cells/ml) were cultured with
[
P]orthophosphate in phosphate-free RPMI, as
described under ``Experimental Procedures.'' A,
cells were treated for 10 min with 100 units/ml GM-CSF (lane
2), 50 ng/ml SLF (lane 3), or the combination of GM-CSF
plus SLF (lane 4), or were treated with 50 ng/ml IP-10 (lane 5), 50 ng/ml MIP-1
(lane 6), 1 µg/ml
cholera toxin (lane 8), or 50 µM forskolin (lane 9) 1 h prior to a 10 min treatment with GM-CSF plus SLF.
Cells were also treated with 50 ng/ml IP-10 for 15 min prior to GM-CSF
plus SLF (lane 7). B, MO7e cells were treated for 10
min with GM-CSF (100 units/ml) plus SLF (50 ng/ml) (lane 2),
for 1 h with 50 ng/ml GRO-
(lane 4), 50 ng/ml MIP-1
(lane 5), 50 ng/ml PF4 (lane 6), 50 ng/ml IL-8 (lane 7), or were treated for 1 h with 50 ng/ml GRO-1
(lane 3), 50 ng/ml MIP-1
(lane 8), or 50 ng/ml
IL-8 (lane 9) prior to a 10-min treatment with GM-CSF plus
SLF. Control cells (lane 1, panels A and B)
received vehicle only. Raf-1 proteins were immunoprecipitated from
whole cell lysates by anti-Raf-1 antibodies, separated by 12% SDS-PAGE,
transferred to PVDF membrane, and the intensity of
P-labeling visualized by autoradiography. At left is
indicated the position of the molecular weight markers. Raf-1 appears
as a single band at approximately 74 kDa, as indicated by the position
of the arrow. C, Raf-1 protein content was determined
by immunoblotting PVDF membranes used for
P analysis with
anti-Raf-1 antibodies and horseradish peroxidase-linked protein G.
Raf-1 proteins were visualized upon exposure of ECL-treated membranes
to film, as described under ``Experimental Procedures.''
Treatment groups are the same as in panel
A.
Figure 6:
Effect of cytokine and chemokine treatment
on Raf-1 kinase activity. Factor-starved MO7e cells were treated as
follows: A, 100 units/ml GM-CSF (lane 2), 50 ng/ml
SLF (lane 3), or GM-CSF plus SLF (lane 4) for 10 min,
or with 50 ng/ml IP-10 (lane 5), 50 ng/ml MIP-1 (lane
6), 1 µg/ml cholera toxin (lane 7), or 50 µM forskolin (lane 8) for 1 h prior to a 10-min treatment
with GM-CSF plus SLF. B, GM-CSF plus SLF (lane 2), or
with 50 ng/ml GRO-
(lane 3), 50 ng/ml MIP-1
(lane 4), 50 ng/ml MIP-1
(lane 5), 10-7 M 8-bromo-cAMP (lane 6), 50 ng/ml IP-10 (lane
7), 50 ng/ml PF4 (lane 8), or 50 ng/ml IL-8 (lane
9) for 1 h prior to a 10-min treatment with GM-CSF plus SLF.
Control cells (lane 1, panels A and B)
received vehicle alone. Raf-1 was immunoprecipitated from cell lysates
(150 µg/sample) with anti-Raf-1 antibodies. Immune complexes were
collected on protein G-Sepharose beads, washed, and incubated with
GST-Mek1 (1 µg) and 0.1 mM [
P]ATP
for 30 min at 30 °C. Immunocomplexes were separated by 12%
SDS-PAGE, transferred to PVDF membranes, and
P
incorporation into Mek1 was visualized by autoradiography. The position
of the molecular weight markers are depicted to the left. GST-Mek1
appears as a single band at approximately 69-70 kDa, as indicated
to the right. The band appearing above the Mek1 band represents
autophosphorylated Raf-1. C, Mek1 content was determined by
immunoblotting PVDF membranes with anti-Mek1 antibodies, horseradish
peroxidase-linked protein G, and visualized upon exposing ECL-treated
membranes to film, as described under ``Experimental
Procedures.'' Treatment groups are the same as those listed for panel A.
Figure 7:
Protein kinase A inhibitor blocks the
suppressive action of MIP-1. Factor-starved MO7e cells were
treated with control vehicle (lane 1), 100 units/ml GM-CSF
plus 50 ng/ml SLF (lane 2), 10 µg/ml PKI for 1 h prior to
10 min treatment with GM-CSF plus SLF (lane 3), 50 ng/ml
MIP-1
plus GM-CSF and SLF (lane 4), MIP-1
for 1 h
prior to 10 min treatment with GM-CSF plus SLF (lane 5), 1 h
pretreatment with 10 µg/ml PKI plus MIP-1
prior to 10 min
treatment with GM-CSF plus SLF. Raf-1 isolation and kinase activity
(Mek1 phosphorylation) assays were conducted as described under
``Experimental Procedures'' and in the legend to Fig. 6. Similar results were obtained in three separate
experiments.
In addition to examining the ability of PKI to block the
inhibitory effects of MIP-1 on Raf-1 activation, we set out to
determine whether PKI could also block the growth suppressive effects
of several chemokines on MO7e cell proliferation. The number of MO7e
CFC in S-phase were determined by the high specific activity tritiated
thymidine ([
H]Tdr) kill assay, as described under
``Experimental Procedures.'' Pulse exposure of MO7e cells for
1 h at 37 °C in vitro to 50 ng/ml IP-10, MIP-1
, PF4,
and IL-8 significantly (p < 0.01) decreased the percentage
of MO7e CFC in S-phase of the cell cycle that were responsive to
stimulation by the combination of GM-CSF (100 units/ml) and 50 ng/ml
SLF (Table 5). The suppressive activity of MIP-1
and IP-10
were blocked completely upon coincubation of cells with either
chemokine and 10 µg/ml PKI (Table 5). In contrast,
coincubation of cells with PKI and either IL-8 or PF4 failed to block
the suppressive activity of these two chemokines (Table 5). PKI
did not effect the percentage of MO7e in S-phase of the cell cycle
treated with Control medium alone (Table 5).
Raf-1 kinase plays a key role in the MAP kinase signaling pathway which links membrane-associated events with other metabolic processes occurring within target cells(35) . We present here our findings showing that the phosphorylation state and kinase activity of Raf-1 in MO7e cells can be increased synergistically in response to treatment with a combination of GM-CSF and SLF. We believe that this is the first time that direct, statistically significant synergistic activation of Raf-1 by combined cytokine treatment has been reported in a complete study. A preliminary study conducted by others and reported in abstract form observed that treatment of murine factor-dependent cells with physiological doses of SLF and either IL-3 or erythropoietin synergistically phosphorylated and activated Raf-1(36) . Our present results demonstrating GM-CSF and SLF synergistic activation of Raf-1 differ from those of our previous study (13) and that of others(14) . Differences in the immunoprecipitation buffer conditions and the amount of radionucleotide used to label target cells may account for these differences. More importantly, however, our assay system utilizes a GST-Mek1 fusion protein as a Raf-1 kinase substrate. Since Mek1 is a biological substrate for activated Raf-1, phosphorylation of GST-Mek1 provides a more sensitive and biologically relevant assay system to study Raf-1 kinase activity than previous studies which employed more broad spectrum substrates, such as histone H1(13) .
Phosphorylation of Raf-1 at serine 43 and at a
second, currently unidentified serine residue in the kinase domain, by
protein kinase A serves to inactivate Raf-1 kinase and prevent its
association with activated Ras(37) . We report here that
MIP-1 and IP-10 pretreatment can inhibit Raf-1 phosphorylation and
decrease Raf-1 kinase activity stimulated by growth factor treatment,
while simultaneously increasing cAMP levels in MO7e cells. The
suppressive action of these chemokines can be mimicked by treatment of
cells with agents which serve to raise intracellular levels of cAMP,
such as cholera toxin and forskolin, and by the cAMP analog
8-bromo-cAMP. These results are consistent with those we reported in a
recent study in which cAMP and cAMP analogs were shown to inhibit the
number of MO7e CFC in cycle, in a manner similar to the suppressive
action of ACN-treated MIP-1
(31) . Taken together with
results of Raf-1 kinase assays and MO7e CFC thymidine kill assays in
which we demonstrated that the suppressive action of MIP-1
and
IP-10 are blocked by the protein kinase A inhibitor PKI, our
observations strongly suggest that part of the inhibitory mechanism
employed by MIP-1
and IP-10 relate directly to alterations in
intracellular cAMP levels. However, our observations that rhuIL-8 and
PF4 fail to alter cAMP levels, fail to block activation of Raf-1
stimulated by growth factors, and do not appear to be sensitive to
inhibition of protein kinase A in MO7e cells indicate that inactivation
of Raf-1 by cAMP may not be applied as a general inhibitory mechanism
for all suppressive chemokines, but as one which is limited to a few
members of the chemokine superfamily.
A receptor which binds
MIP-1 with high affinity has recently been cloned(38) .
Based upon sequence information, this receptor is thought to be
comprised of seven membrane-spanning domains, a protein structure which
is consistent with membrane-bound receptors coupled through G-proteins
to adenylyl cyclase and other effector molecules(38) . While
this observation is of interest, neither direct nor indirect activation
of adenylyl cyclase has been reported for any of the chemokine
receptors. Our observation that both growth suppressive chemokines,
such as MIP-1
and IL-8, and non-suppressive chemokines, such as
MIP-1
, can bind with moderately high affinity to MO7e cells
indicates that the degree of suppressive activity may not be related
directly to the number or specificity of chemokine-binding sites, but
rather to the effector pathways activated in response to ligand
binding. However, the 2-3-fold difference between binding
affinities for MIP-1
and MIP-1
may account for the ability of
excess MIP-1
to block the suppressive action of MIP-1
, since
we have shown that greater concentrations of MIP-1
are required in
order to block MIP-1
suppressive action(18) .
We have
demonstrated that GM-CSF and SLF can stimulate increases in protein
synthesis in MO7e cells. This provides an additional model system
through which the growth promoting effects of cytokines can be
examined. By making use of protein synthesis regulation as an assay
system, we have shown that pretreatment of MO7e cells with the
chemokines IP-10 and MIP-1, and IL-8 and PF4 to a lesser extent,
can block the stimulatory effects of GM-CSF and SLF. Since declines in
protein synthesis are often associated with quiescent or
growth-arrested cells, the ability of these chemokines to alter protein
synthesis levels may be related to altering the cell cycle progression
of target cells. Our observations are consistent with results of the
thymidine kill assays in which MO7e cells were treated with various
agents and then exposed to high specific activity
[
H]thymidine. In this assay system, cells in
S-phase incorporate [
H]thymidine and are
reproductively sterilized as a result. MO7e cells pretreated with
specific chemokines, including MIP-1
and IP-10, tended to survive
the exposure to the thymidine, suggesting that chemokines protect these
cells by altering their progression into cycle and/or by slowing down
their growth rate (18) .
Down-regulation of both protein
synthesis and Raf-1 activity evoked by IP-10 and MIP-1 within the
same cell type suggests that these two events may be linked to growth
suppression. Since the ability of these chemokines to block the
stimulatory action of growth factors appears to require a minimal
pretreatment duration, it is likely that stimulatory and inhibitory
pathways utilized by cytokines and chemokines may share several key
components which, depending upon how they are modified, may ultimately
lead to growth activation or suppression. Although Raf-1 activation of
MAP kinase is thought to play a role in the growth promoting effects of
GM-CSF and SLF on hematopoietic
cells(13, 14, 15) , the connections to
factors down stream of MAP kinase which are ultimately responsible for
activation of cell growth have not been completely identified. Since
active cell growth and division are associated with periods of
increased protein synthesis, it is likely that a key component of
growth regulation by cytokines may reside in the activation of the
protein synthesis machinery within target cells. Activation of the
initiation factor eIF-4E, as an example, is thought to represent at
least one of the rate-limiting steps in the stimulation of protein
synthesis in eukaryotic systems(39) . What is of particular
interest regarding eIF-4E is that it can be phosphorylated and
activated through an unknown mechanism directly upon exposure to active
Ras and, in a manner similar to Raf-1, can be activated in response to
phosphorylation by protein kinase C(40) . Since exposure of
hematopoietic cells and MO7e cells to cAMP has been shown to be
inhibitory for cell growth(30, 31, 41) , it
may be through inactivation of eIF-4E or related proteins that protein
synthesis is shut down and cell growth is halted. Conversely,
activation of protein synthesis in some cell systems can be achieved
through inactivation of inhibitory initiation factors. Phosphorylation
of the initiation factor eIF-2
by double-stranded RNA-dependent
protein kinase has recently been shown to result in the inhibition of
protein synthesis in murine H7 cells(42) . Subsequent studies
conducted by the same group have shown that treatment with IL-3 leads
to the dephosphorylation and inactivation of both double-stranded
RNA-dependent protein kinase and eIF-2
, and the subsequent
activation of protein synthesis in IL-3 deprived murine hematopoietic
cells(43) . Similar to our present findings, results of studies
involving IL-3 and eIF-2
demonstrate a clear connection between
the stimulatory action of growth factors and regulation of protein
synthesis(42, 43) , providing evidence for one
possible mechanism by which this regulation is accomplished. By
exploring these and other links which may exist between the regulation
of protein synthesis and interactions among signaling pathways,
including Raf-1 activation and alterations in cellular cAMP levels, we
may be able to determine the sequence of events through which
hematopoietic cell growth is regulated by multiple factors.