|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 813-822
Protein Kinase C Mediates the Mitogenic Action of Thrombopoietin
in c-Mpl-Expressing UT-7 Cells
By
Ying Hong,
Dominique Dumènil,
Bernd van der Loo,
Frédérique Goncalves,
William Vainchenker, and
Jorge D. Erusalimsky
From the Cruciform Project and Department of Medicine, University
College London, London, UK; and INSERM U362, Institut Gustave Roussy,
Villejuif Cedex, France.
 |
ABSTRACT |
Protein kinase C (PKC) has been implicated in signal transduction
events elicited by several hematopoietic growth factors. Thrombopoietin
(TPO) is the major regulator of megakaryocytic lineage development, and
its receptor, c-Mpl, transduces signals for the proliferation and
differentiation of hematopoietic progenitors. In this study we have
examined the effect of TPO on the subcellular distribution of PKC (a
measure of enzyme activation) in a growth factor-dependent pluripotent
hematopoietic cell line that was engineered to express the c-Mpl
receptor (UT-7/mpl). In addition, we have assessed the significance of
this activation for the induction of both mitogenesis and
differentiation. Using a PKC translocation assay, TPO was found to
stimulate a time- and dose-dependent increase in the total content of
PKC activity present in the membrane fraction of UT-7/mpl cells
(maximum increase = 2.3-fold above basal level after 15 minutes
with 40 ng/mL TPO, EC50 = 7 ng/mL). Accordingly, a
decrease of PKC content in the cytosolic fraction was observed. Immunoblot analysis using PKC isotype-specific antibodies showed that
TPO treatment led to a marked increase of the
Ca2+/diacylglycerol-sensitive PKC isoforms  and  found in the membrane fraction. In contrast, the subcellular
distribution of these isoforms did not change after treatment with
granulocyte-macrophage colony-stimulating factor (GM-CSF). Exposure of
UT-7/mpl cells to the selective PKC inhibitor GF109203X completely
inhibited the PKC activity associated to the membrane fraction after
TPO treatment, and blocked the mitogenic effect of TPO. In contrast,
GF109203X had no effect on the TPO-induced expression of GpIIb, a
megakaryocytic differentiation antigen. Downregulation of PKC isoforms
and to less than 25% of their initial level by treatment with
phorbol 12,13-dibutyrate also abolished the TPO-induced mitogenic
response, but had no significant effect when this response was induced
by GM-CSF. Taken together, these findings suggest that (1) TPO
stimulates the activation of PKC, (2) PKC activation mediates the
mitogenic action of TPO, and (3) PKC activation is not required for
TPO-induced expression of megakaryocytic surface markers.
| |
INTRODUCTION |
THROMBOPOIETIN (TPO) is the major
regulator of platelet production.1,2 This hematopoietic
growth factor stimulates the proliferation of megakaryocyte progenitor
cells, promotes megakaryocyte terminal differentiation, and is
essential for the production and maintenance of normal levels of
thrombopoiesis.3-8 In addition, TPO stimulates the
proliferation of erythroid progenitors,9,10 plays a role in
the production of progenitor cells for other myeloid lineages,11-13 and may suppress apoptosis.14,15
TPO is produced by the liver3 and exhibits significant
homology to erythropoietin (EPO),3 the lineage-specific
growth factor that regulates erythropoiesis. The receptor for TPO, a
member of the cytokine-receptor superfamily, is encoded by the
proto-oncogene c-mpl.16,17 c-Mpl is expressed on
primitive hematopoietic progenitor cells and cells of the
megakaryocytic lineage.18 Binding of TPO to c-Mpl induces
tyrosine phosphorylation of various signaling
proteins,19-26 including Shc and Jak2, which in turn
respectively engage the RAS/MAP kinase and Jak/STAT signaling
cascades.27
Ligand binding to various members of the cytokine receptor superfamily,
including those for interleukin-3,28
erythropoietin,29 and prolactin,30 has been
shown to induce the activation of protein kinase C (PKC). PKC is a
family of phospholipid-dependent ser/thr kinases activated by second
messenger action,31,32 and thought to participate in the
transmission of signals for growth and differentiation in many cell
types, including hematopoietic cells.28,33-36
Tumor-promoting phorbol esters, which mimic the action of the second
messenger diacylglycerol, bind and activate PKC directly,37
and stimulate concomitantly the translocation of cytosolic PKC isoforms
to membrane sites.33 These agents have been shown to induce
megakaryocytic differentiation in bone marrow cultures38
and in hematopoietic cell lines,39 suggesting that PKC
mediates signals for megakaryocyte development. Based on the above
knowledge, we hypothesized that in addition to stimulating tyrosine
phosphorylation, TPO could also modulate the activity of PKC. To test
this hypothesis, we used a growth factor-dependent hematopoietic cell
line that was engineered to express the c-Mpl receptor (UT-7/mpl). Like
parental UT-7 cells,40,41 UT-7/mpl cells depend on
granulocyte-macrophage colony-stimulating factor (GM-CSF) or EPO for
growth and survival, but in addition, they proliferate and
differentiate in the presence of TPO. Here we report that in UT-7/mpl
cells, TPO stimulates the translocation of individual PKC isoforms,
from the cytosol to a membrane compartment. We also show that PKC is
required by TPO for the induction of a mitogenic response but not for
the induction of differentiation antigens.
| |
MATERIALS AND METHODS |
Materials.
Phorbol esters were purchased from Sigma Chemical Co (Poole Dorset,
UK). The bisindolylmaleimide GF109203X
(2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) was purchased from Calbiochem-Novabiochem (Nottingham,
UK). Stock solutions (2 mmol/L) of phorbol esters were
made in ethanol and stored at  20°C. GF109203X (10 mmol/L) was
dissolved in dimethylsulfoxide and stored at 4°C.
[methyl-3H]Thymidine (80 Ci/mmol) and
[ -32P]adenosine triphosphate (ATP) (>5,000 Ci/mmol)
were purchased from Amersham International (Amersham, UK). Tissue
culture media and additives were from Gibco Life Technologies (Paisley,
UK). Human recombinants TPO and GM-CSF were from R&D Systems (Oxon, UK). Other reagents were from standard suppliers or as listed in the
text.
Retroviral infection of UT-7 cells.
Stable cell clones expressing the c-Mpl receptor were obtained by
retroviral infection of the human pluripotent cell line UT-7 as
previously described.42 Briefly, the retroviral vector pBTZen-c-mpl-SVNeo, containing a 1,500-bp fragment
of the human c-mpl cDNA (Sal 1-Not 1 fragment),
was transfected into the amphotropic packaging cell line
Gp+envAm12.43 Individual geneticin-resistant clones were
derived in the presence of 1 mg/mL G418, and then supernatants were
tested for retroviral production on NIH3T3 cells. Cell clones with the
highest retroviral production (4 × 105 c-mpl
infectious retroviral particles per milliliter of supernatant) were
selected. UT-7 cells were then infected by coculturing for 48 hours on
subconfluent irradiated (10 Gy) Gp+envAm12-c-mpl cells in the
presence of GM-CSF. Nonadherent cells were then plated in a semisolid
medium consisting of 1% methylcellulose (Fluka, Saint-Quentin
Fallavier, France) in -minimum essential medium without
deoxyribonucleotides (-MEM) supplemented with 10% fetal calf serum
(FCS), 2.5 ng/mL GM-CSF, and 1 mg/mL G418. Individual colonies were
picked up 10 days later and expanded in liquid medium in the presence
of 2.5 ng/mL GM-CSF and 1 mg/mL G418. c-Mpl expression was
assessed by flow cytometry using the anti-human Mpl mouse monoclonal
antibody (MoAb) M1, and by Northern blot analysis using a
PvuII-PvuII fragment of c-mpl c-DNA, as
previously described.18 Three independent UT-7/mpl clones
(5.3, 5.1, and 1101C) expressing relatively low, intermediate, and high
levels of c-Mpl, respectively, were examined in the present study.
However, for most experiments, UT-7/mpl 5.1 was chosen as the
representative clone.
Cell culture.
UT-7/mpl cells were maintained in exponential growth (between 1 and
5 × 105 cell/mL) in -MEM supplemented with 10% FCS,
2 ng/mL GM-CSF, and 0.5 mg/mL G418 (referred to below as culture
medium), under 5% CO2/95% air in a humidified incubator.
Parental UT-7 cells were grown under the same conditions except for the
omission of G418 in the culture medium. For experiments involving
measurements of PKC, before stimulation, cells were rendered quiescent
by washing twice in culture medium lacking GM-CSF, followed by
incubation for 18 hours in this medium, as described by Pallard et
al.23 CMK cells were maintained in RPMI
supplemented with 10% FCS. CMK cells were rendered quiescent by
incubation for 18 hours in RPMI containing 0.5% FCS. Cells were judged
to be quiescent based on [3H]thymidine incorporation
measurements. These measurements showed a greater than 25-fold
reduction in the rate of DNA synthesis compared with cells growing in
complete media (data not shown). Quiescent cells were stimulated at
37°C as described in the respective figure legends, and then
harvested as follows.
Preparation of cytosolic and membrane fractions.
Aliquots of 5 × 106 cells were washed with ice-cold
Dulbecco's phosphate-buffered saline (PBS), resuspended in 0.5 mL
ice-cold homogenization buffer (5 mmol/L EDTA, 10 mmol/L EGTA, 0.3%
-mercaptoethanol, 1 mmol/L phenylmethylsulfonyl fluoride, 10 mmol/L
benzamidine and 20 mmol/L Tris-HCl, pH 7.5), and disrupted by
sonication (3 cycles of 5 seconds at 22 Hz with intervals of 15 seconds) using a Soniprep 150 Ultrasonic Disintegrator (MSE Scientific
Instruments, Nottingham, UK). The resulting lysates were
centrifuged at 100,000g for 45 minutes at 4°C and
supernatants were collected (cytosolic fraction). Pellets were
resuspended in 0.5 mL homogenization buffer (as above) containing 0.5%
Brij 58 to solubilize membrane-associated proteins. After standing on
ice for 30 minutes with occasional vortexing, detergent-insoluble
proteins were removed by centrifugation as above, and supernatants were
collected (membrane fraction). The cytosolic and membrane fractions
were placed on ice until assayed for PKC activity.
Assay of PKC activity.
Total PKC activity in the membrane and cytosolic fractions was
determined using a commercially available assay system (Biotrak, Amersham, UK), as described by the manufacturer. This assay system is
based upon the PKC-catalyzed transfer of the
[32P]phosphate group from [-32P]ATP into
a PKC-specific peptide substrate (amino acids 651-658 of the epidermal
growth factor receptor with the phosphorylation site on Thr-654), in
the presence of Ca2+, phosphatidylserine, and phorbol
12-myristate 13-acetate (PMA). Reactions were carried out at 37°C for
15 minutes, using dilutions of each fraction equivalent to 1 × 105 cells. Reactions were performed in duplicate and the
results averaged. Radioactivity values resulting from the
phosphorylation of endogenous substrates were subtracted from all
determinations. One U of PKC was defined as the amount of enzyme that
catalyzes the transfer of 1 pmol 32P from
[-32P]ATP into the PKC substrate per minute at 37°C.
Immunoblot analysis of protein kinase C isoforms.
The distribution of individual PKC isoforms in the cytosolic and
membrane fractions was assessed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis followed by immunoblotting
using PKC isotype-specific mouse MoAbs (Transduction Laboratories,
Lexington, KY). Briefly, after boiling in sodium dodecyl sulfate
(SDS)-sample buffer (2.5% SDS, 1 mmol/L EDTA, 2% -mercaptoethanol,
5% glycerol, 10 mmol/L Tris/HCl, pH 6.8), solubilized proteins were
resolved on SDS-polyacrylamide gels (7.5% acrylamide) and transferred
to Immobilon PVDF membranes (Millipore, UK). Prestained molecular
weight markers and individual PKC isoforms (Transduction Laboratories),
used as positive controls, were run in parallel. Membranes were blocked
for 1 hour at room temperature with 5% nonfat dried milk in PBS/0.1%
Tween 20, and then incubated for 2 hours at room temperature with the
indicated anti-PKC MoAbs diluted (1:1,200 to 1:2,500) in PBS/0.1%
Tween 20. Immunoreactive bands were detected using horseradish
peroxidase-labeled rabbit anti-mouse IgG (Amersham) and enhanced
chemiluminescence (ECL, Amersham). The relative intensity of the bands
was quantified by scanning densitometry using a UVP Gel Documentation
System (Ultra-Violet Products, Ltd, Cambridge, UK). For
assessment of PKC isoform downregulation, PBS-washed cell pellets
(1.5 × 106 cells) were solubilized in 0.2 mL 2× SDS
sample buffer without prior fractionation, and total cellular proteins
were electrophoresed and immunoblotted as described above.
Cell proliferation assay.
For measurement of [3H]thymidine incorporation into DNA,
UT-7/mpl cells were washed twice in culture medium lacking GM-CSF, resuspended in this medium at 1 × 105 cell/mL, and
dispensed in aliquots of 0.1 mL into round bottom 96-well tissue
culture plates (Falcon, London, UK). After preincubation for 4 hours at 37°C under 5% CO2/95% air, growth
factors were added at the appropriate dilution, and then the plates
were incubated for a further 48 hours. At the end of this period, and
unless otherwise indicated, cells were pulsed with 0.05 µCi
[3H]thymidine (1 µCi/nmol) for 6 hours, and then
harvested onto glass fiber filtermats (Skatron Instruments, Suffolk,
UK) as previously described.44 All assays were performed in
triplicate.
Analysis of megakaryocytic differentiation.
To study megakaryocytic differentiation, UT-7/mpl cells were washed
free of GM-CSF and grown for 4 days with 10 nmol/L TPO, in culture
medium lacking GM-CSF. Alternatively, cells were incubated in culture
medium supplemented with GM-CSF in the presence of 10 nmol/L PMA.
Antigen expression was determined by flow cytometry using the
fluorescein isothiocyanate (FITC)-conjugated MoAb Tab, which recognizes
the platelet glycoprotein (Gp)IIb (CD41a), a generous gift from Dr S. Burstein (Oklahoma City, OK). Cells (1 to 2 × 106) were
harvested by centrifugation at 150g for 5 minutes, washed with
ice-cold PBS containing 1% FCS, and incubated in 0.1 mL PBS/1% FCS
with a 1:50 dilution of FITC-conjugated Tab or an equivalent amount of
control FITC-conjugated mouse IgG1 (Becton Dickinson, Le Pont-de-Claix,
France) for 30 minutes at 4°C. Then, cells were washed once and
resuspended in 0.5 mL PBS/1% FCS. Antigen expression was determined
using a FACSort instrument (Becton Dickinson, Mountain View, CA). Data
were acquired and analyzed using the Cell Quest software (Becton
Dickinson). 7-Amino actinomycin D staining was used to eliminate dead
cells from the analysis. Relative antigen expression was estimated on
live cells using the median fluorescent intensity. Nonspecific
fluorescence of cells stained with the control antibody was subtracted
from all determinations. Ploidy was determined by flow cytometric
analysis of DNA content using propidium iodide. After harvesting,
aliquots of 1 to 2 × 106 cells were fixed in 80% ethanol
for at least 4 hours at 20°C, then washed twice with ice-cold PBS,
and permeabilized for 5 minutes with 0.25% Tween 20 in PBS at 4°C.
Finally, samples were incubated in PBS containing 50 µg/mL propidium
iodide (Sigma) and 0.1 mg/mL RNAse A (Merck, Damstadt, Germany) for at
least 2 hours in the dark. DNA fluorescence distributions were analyzed
as previously described,45 using pulse processing to
exclude cell clumps.
Statistical analysis.
Experiments were performed at least three times and unless otherwise
indicated, results from one representative experiment are shown. Where
indicated, levels of statistical significance were determined using the
Student's t-test.
| |
RESULTS |
Stimulation of PKC translocation by TPO.
To assess the effect of TPO on the stimulation of PKC in intact cells
we measured the "translocation" of the enzymatic activity from
the cytosolic to the membrane-rich subcellular fraction. This
redistribution is regarded as one of the hallmarks of PKC activation.33 The levels of total PKC activity found in the cytosolic and membrane fractions of quiescent UT-7/mpl 5.1 cells were
19.8 ± 1.8 U/105 cells and 2.7 ± 0.2 U/105
cells, respectively (average of four independent
determinations ± SD). Treatment of UT-7/mpl 5.1 cells for 15 minutes with PMA resulted in a larger than fourfold increase in the
total content of PKC activity in the membrane fraction, and a
corresponding decrease in the cytosolic fraction (Fig
1A). A similar, albeit less
pronounced, effect was observed when these cells were treated with 40 ng/mL TPO (Fig 1A). The TPO-induced rise of PKC activity in the
membrane fraction was detectable as early as 1 minute after addition of TPO, reached a maximum (2.3-fold) after 15 minutes, and persisted for
at least 60 minutes (Fig 1B). This effect of TPO was dose-dependent, with a half maximal stimulation observed at 7 ng/mL, and maximal stimulation seen above 40 ng/mL (Fig 1C).
View larger version (13K):
[in this window]
[in a new window]
| Fig 1.
Stimulation of PKC translocation by TPO in
UT-7/mpl 5.1 cells. (A) Effect of TPO or PMA on the subcellular
distribution of total PKC activity. Quiescent cells were treated for 15 minutes with 40 ng/mL TPO or 50 nmol/L PMA and then PKC activity was
measured in the membrane and cytosolic fractions. Values represent the mean ± SD of four independent experiments. ( ), control; ( ), TPO; ( ), PMA. *P < .05, **P < .01. (B) Time
course of TPO-induced increase in membrane-associated PKC activity.
Cells were treated with 40 ng/mL TPO for the indicated times and then
PKC activity was measured in the membrane fraction. (C) Dose response
for the increase in membrane-associated PKC activity induced by TPO.
Cells were treated with the indicated concentrations of TPO for 15 minutes and then PKC activity was measured in the membrane fraction.
PKC activity is expressed relative to the level present in unstimulated cells incubated in parallel. All other experimental details were as
described in Materials and Methods.
|
|
To address the possibility that the effect of TPO on PKC might have
been an artificial characteristic of a particular clone, we examined
two additional independent UT-7/mpl clones that express different
levels of c-Mpl on their surface. As shown in Table 1, TPO was unable to significantly affect
the subcellular distribution of PKC in parental UT-7 cells, which do
not express sufficient amounts of c-Mpl.18 In contrast,
this factor stimulated a marked translocation of PKC in all the
c-Mpl-expressing clones. Furthermore, there was a positive correlation
between the degree of TPO-induced PKC translocation and the relative
level of c-Mpl expressed on the surface of each clone (Table 1). To
extend this finding to cells that express an endogenous c-mpl,
we also examined the effect of TPO on the translocation of PKC in CMK
cells. This cell line was previously reported to display a functional
response to TPO.4 Table 1 shows that in CMK cells, TPO
stimulated the translocation of PKC to an extent that was similar to
that found in UT-7/mpl cells. Thus, the TPO-induced translocation of
PKC was a common feature of every c-Mpl-expressing cell examined,
including a cell line that expressed the endogenous gene.
Effect of TPO on the translocation of individual PKC isoforms.
PKC consists of a family of at least 11 different isoforms, classified
in three groups according to their cofactor
requirements.32,33 Immunoblot analysis using PKC
isotype-specific MoAbs showed that UT-7/mpl 5.1 cells expressed
moderate to high levels of the conventional isoforms and , the
novel PKC isoforms  and  , and the atypical isoform  (data not
shown). These cells also expressed the PKC isoform µ (also known as
PKD), which respond to phorbol esters but cannot be ascribed to any of
the above three groups (data not shown). Except for PKC  , which was
not tested, other isoforms (,  ,  , and  ) were either absent
or barely detectable. In the present study we characterized the effect
of TPO on the translocation of the classical isoforms and .
Figure 2 shows that in untreated UT-7/mpl 5.1 cells these isoforms were
found preferentially in the cytosolic compartment, their presence in
the membrane fraction being hardly detectable. This figure also shows
that treatment with PMA resulted in the translocation of PKC- and
from the cytosol to the membrane compartment. Similarly, a marked
increase (threefold to sixfold) of these isoforms was observed in the
membrane fraction after the cells were treated with TPO (Fig 2).
A corresponding decrease of
immunoreactivity in the cytosolic fraction was noticeable in the case
of PKC-, but not in the case of PKC-. In contrast to the effects
of PMA or TPO, when the cells were treated with GM-CSF, the
subcellular distribution of PKC- and was not affected (Fig 2).
View larger version (12K):
[in this window]
[in a new window]
| Fig 2.
Effect of TPO on the subcellular localization of PKC-
and - isoforms as detected by immunoblotting. Quiescent UT-7/mpl
5.1 cells were treated for 15 minutes with 40 ng/mL TPO, 2 ng/mL GM-CSF or 50 nmol/L PMA as indicated. Cytosolic and membrane fractions were
prepared from each sample and aliquots equivalent to 5 × 105 cells in case of the membrane fraction, or half that
amount in case of the cytosolic fraction, were analyzed by
immunoblotting using MoAbs against the indicated PKC isoforms, as
described in Materials and Methods.
|
|
Effect of GF109203X on the membrane-associated PKC activity
translocated by TPO.
To obtain further evidence that the activity elevated in the membrane
fraction following treatment with TPO belonged to PKC, we tested the
effect of the bisindolylmaleimide GF109203X, a specific protein kinase
inhibitor that acts selectively against the PKC family, both in
cell-free systems and in intact cells.46 As shown in Fig
3A, addition of 3 µmol/L GF109203X to
intact UT-7/mpl 5.1 cells resulted in a complete inhibition of the
enzymatic activity measured in the membrane fraction, following
treatment of the cells with either TPO or PMA. Furthermore, addition of
GF109203X directly to the PKC assay inhibited this activity at
nanomolar concentrations (IC50 = 23 nmol/L; average from
two experiments; range, 19 to 26 nmol/L), with a maximal effect
observed at 100 nmol/L (Fig 3B). These results were entirely consistent
with previous reports showing a selective and specific effect of this
inhibitor on PKC.46
View larger version (15K):
[in this window]
[in a new window]
| Fig 3.
Effect of GF109203X on the membrane-associated PKC
activity translocated by TPO. (A) Inhibition of PMA-dependent or
TPO-dependent membrane-associated PKC activity by GF109203X in intact
cells. Quiescent UT-7/mpl 5.1 cells were preincubated for 15 minutes in
the absence () or presence () of 3 µmol/L GF109203X, and then
treated for another 15 minutes with 40 ng/mL TPO or 50 nmol/L PMA. At
the end of this period PKC activity was measured in the membrane
fraction as described in Materials and Methods. Results represent the
mean of four independent experiments ± SD. PKC activity is expressed
relative to the level present in unstimulated cells incubated in
parallel. (B) Inhibition of membrane-associated PKC activity as a
function of GF109203X concentration in the assay. Cells were treated
for 15 minutes with 40 ng/mL TPO and then PKC activity was measured in
the membrane fraction in the presence of the indicated concentrations
of GF109203X. Results are expressed as the percentage of PKC activity
in the sample with no GF109203X added.
|
|
Correlation between the stimulation of PKC activity by TPO and the
induction of mitogenesis.
GF109203X was also used to investigate the role of PKC in the mitogenic
action of TPO. Mitogenesis was examined by [3H]thymidine
incorporation into DNA. As shown in Fig 4A,GF109203X inhibited the TPO-induced mitogenic response. This inhibitory effect was dose-dependent (IC50 = 0.5 µmol/L), with
complete inhibition observed at 3 to 4 µmol/L. Figure 4B shows the
dose response for the stimulation of mitogenesis induced by TPO in the
absence or presence of GF109203X. TPO stimulated mitogenesis in
UT-7/mpl 5.1 cells in a dose-dependent manner with an EC50
of 2 ng/mL (Fig 4B,  ). An optimal dose of GF109203X (3 µmol/L)
shifted this dose-response curve to the right (Fig 4B,  ). Thus, at
concentrations of TPO below 10 ng/mL, GF109203X caused a
complete suppression of the mitogenic response, whereas above 10 ng/mL
TPO, the inhibition reached 70%.
View larger version (13K):
[in this window]
[in a new window]
| Fig 4.
Inhibition of the TPO-induced mitogenic response by
GF109203X. (A) Inhibition of the TPO-induced mitogenic response as
function of GF109203X concentration. UT-7/mpl 5.1 cells were treated
for 48 hours with 5 ng/mL TPO and the indicated concentrations of GF109203X. [3H]Thymidine incorporation into DNA was
measured as described in Materials and Methods. Results are expressed
as a percentage of the response observed in the cells treated with TPO
alone. (B) Effect of GF109203X on the dose response for the induction
of mitogenesis stimulated by TPO. Cells were treated for 48 hours with
various concentrations of TPO, in the absence () or presence ()
of 3 µmol/L GF109203X. [3H]Thymidine incorporation into
DNA was measured as described in Materials and Methods. Results are
expressed as a percentage of the maximal response. Mean values from one
representative experiment performed in triplicate are shown in each
case. Error bars (SDs), which were smaller or equal to the size of the
symbol, are not visible.
|
|
Another powerful approach to study the role of PKC in cellular
responses is to downregulate its level by prolonged pretreatment with
high concentrations of phorbol esters.28,33 Hence, PKC downregulation was also used to obtain further evidence for the role of
this enzyme in the mitogenic action of TPO. Figure
5A shows that in UT-7/mpl 5.1 cells a
6-hour pretreatment with 500 nmol/L phorbol 12,13-dibutyrate (PDBu)
was sufficient to cause a substantial downregulation of
PKC isoforms and (>75%). A longer pretreatment reduced PKC
levels even further. However, because prolonged incubations of
hematopoietic cells with phorbol esters induce terminal
differentiation,39,47 and hence, irreversibly arrest
proliferation, the shorter 6-hour preincubation time was chosen. Figure
5B shows that this short pretreatment with PDBu inhibited the
TPO-induced mitogenic response by greater than 70% (P = .01). In contrast, the same pretreatment had a marginal, statistically nonsignificant effect on the mitogenic response induced
by GM-CSF (P > .05). Therefore, this result showed that the
inhibition of the mitogenic response to TPO was not the result of a
toxic effect or of a more general inhibition of mitogenesis.
View larger version (16K):
[in this window]
[in a new window]
| Fig 5.
Inhibition of the TPO-induced mitogenic response by PKC
downregulation. (A) Time course for the downregulation of PKC isoforms and . UT-7/mpl 5.1 cells were incubated in culture medium
lacking GM-CSF, in the absence or presence of 500 nmol/L PDBu, for the indicated lengths of time. At the end of the incubations whole cell
lysates were prepared and aliquots equivalent to 0.3 × 106 cells were analyzed by immunoblotting using MoAbs
against the indicated PKC isoforms, as described in Materials and
Methods. (B) Effect of PKC downregulation on the TPO-induced or
GM-CSF-induced mitogenic response. After a pretreatment of 6 hours with
500 nmol/L PDBu, cells were washed three times with culture medium
lacking GM-CSF to remove the PDBu, and then incubated for a further 48 hours with 10 ng/mL TPO or 2 ng/mL GM-CSF in culture medium containing 0.1 µCi/mL [3H]thymidine. At the end of this period
cells were harvested onto glass fiber filtermats as described in
Materials and Methods. Radioactivity values represent the mean ± SD
of three replicates. (), Untreated cells; (), cells treated with
PDBu.
|
|
Effect of GF109203X on the induction of megakaryocytic
differentiation.
Previously we have shown that GF109203X can be used as a tool to assess
the involvement of PKC in phorbol-ester-induced megakaryocytic differentiation.35 Therefore, in the present study this
inhibitor was also used to investigate the significance of PKC
activation for TPO-induced differentiation of UT-7/mpl cells. As shown
in Fig 6 (top), treatment of UT-7/mpl 1101C
cells for 4 days with either PMA or TPO resulted in a marked increase
(30- and 25-fold, respectively) in the expression of GpIIb, a surface
marker of megakaryocytic differentiation. Addition of 3 µmol/L
GF109203X almost completely suppressed the increase in GpIIb expression induced by PMA, but had no significant effect when expression of this
antigen was induced by TPO. Similar results were obtained in UT-7/mpl
5.1 cells (data not shown). Analysis of DNA content (Fig 6, bottom)
revealed that PMA treatment caused a marked increase in the proportion
of polyploid cells (cells with DNA content greater than 4n). This
response was also inhibited by GF109203X. In contrast to PMA, TPO
caused only a marginal increase in ploidy. For this reason, it was
difficult to ascertain what was the effect of GF109203X on TPO-induced
polyploidization. Culture in the presence of TPO for longer than a week
caused a more substantial increase in the number of polyploid cells.
However, under these conditions the effect of GF109203X could not be
assessed, as it induced apoptosis of a large proportion of cells in the
culture (data not shown).
View larger version (26K):
[in this window]
[in a new window]
| Fig 6.
Lack of inhibition of the TPO-induced differentiation
response by GF109203X. UT-7/mpl 1101C cells were incubated for 4 days with either 2 ng/mL GM-CSF (left), 2 ng/mL GM-CSF + 10 nmol/L PMA TPO
(center), or with 10 nmol/L TPO (right), in the absence or presence of
3 µmol/L GF109203X. GpIIb expression and DNA content were analyzed by
flow cytometry as described in Materials and Methods. In the top panels
the open traces correspond to the fluorescence distributions of samples
stained with a control IgG1, and the filled traces correspond to
parallel samples stained with the MoAb Tab.
|
|
| |
DISCUSSION |
The PKC family has been implicated in intracellular mechanisms that
transduce signals for survival, proliferation, and differentiation of
hematopoietic cells.28,33-36,48 In this study we have
investigated the involvement of PKC in both the mitogenic and the
differentiation responses induced by TPO. For this purpose we have used
the human pluripotent hematopoietic cell line UT-7, after it has been
modified by a retroviral strategy to express the TPO receptor. UT-7/mpl proliferates and differentiates in response to TPO in a similar fashion
to hematopoietic progenitors, and thus it provides a useful model
system to study the signaling pathways through which TPO might
function.
The results reported here show that engagement of the c-Mpl receptor by
TPO leads the translocation of PKC from the cytosol to membrane sites.
This translocation, which reflects the activation of various isoforms
of this enzyme,33 was found not only in each of the three
c-mpl-transfected UT-7 clones examined, but also in CMK cells,
which express a functional TPO receptor from the endogenous
gene.4 Thus, activation of PKC by TPO cannot be attributed
to an artifact of the transfection, or to a property of one particular
cell line. Furthermore, the TPO-induced translocation of PKC was
comparable in magnitude to that seen in other systems in which PKC is
known to be activated by physiological stimuli, acting through
receptor-mediated mechanisms.49-51 Recently, using a myelin
basic protein-derived synthetic peptide substrate of PKC, Kunitama et
al52 reported a TPO-stimulated increase of protein kinase
activity in low speed supernatants of detergent-made lysates from
UT-7/mpl cells. This increase was attributed to stimulation of PKC via
diacylglycerol generation. However, since these investigators measured
the total cellular content of this kinase activity rather than its
translocation, and the assay included all the necessary cofactors to
produce full activation of PKC, the meaning of this increase remains
unclear.
Hematopoietic cells express several PKC isoforms that may be activated
by various independent mechanisms.31,32 In this study we
have focused particularly on the
Ca2+/diacylglycerol-sensitive isoforms and , and we
have shown that both are translocated to the membrane following TPO
treatment. However, at this stage, we cannot rule out the possibility
that other PKC isoforms are also activated. We should also emphasize that the antibody used here to recognize PKC- cannot distinguish between I and II, the two isoforms produced by alternative
splicing. The pathway leading from c-Mpl receptor occupancy to PKC
activation also remains to be elucidated. Signal transduction
mechanisms involving tyrosine phosphorylation couple this receptor to
intracellular effectors.19,20,22,23,26,27 Studies with
other hematopoietic growth factor receptors have suggested that
tyrosine phosphorylation may result in the activation of a
phosphatidylcholine-specific phospholipase C that generates the
necessary diacylglycerol for PKC activation.36 In the case
of c-Mpl, there is some evidence indicating that TPO stimulates the
phosphorylation of phospholipase C,19,21 raising the
possibility that this enzyme might participate in the pathway leading
to PKC activation.
A major finding of this study is that GF109203X blocked the TPO-induced
mitogenic response of UT-7/mpl cells (Fig 4). In this context, it is
important to note that the IC50 and the optimal dose of
GF109203X for the inhibition of TPO-induced mitogenesis were similar to
the values reported in other cellular systems for the inhibition of
receptor-mediated biological responses in which PKC is known to be
involved.46 However, it is also noteworthy that at
concentrations of TPO above 10 ng/mL, GF109203X did not block the
mitogenic response completely. This suggests that high concentrations
of TPO could induce proliferation through an additional signaling
pathway which is independent of PKC. This possibility is consistent
with the notion that mitogenic signals may be transduced via redundant
intracellular pathways.53 Additional support for the
involvement of PKC in the mitogenic effects of TPO came from results of
PKC downregulation experiments (Fig 5). These experiments clearly
showed that a reduction of 75% to 80% in the levels of PKC- and
- led to a 75% inhibition of the TPO-induced mitogenic response. In
this case the lack of complete inhibition could be attributed either to
the presence of residual PKC, or to the existence of an additional
TPO-stimulated mitogenic pathway that operates independently of PKC, as
discussed above. Whatever the reason for the lack of complete
inhibition, both approaches to interfere with PKC, namely inhibition of
the enzymatic activity or the downregulation of its expression,
strongly suggest that the mitogenic response induced by TPO in UT-7/mpl
cells is mediated predominantly by a pathway involving the activation
of PKC.
As shown in the present study, engagement of the GM-CSF receptor in
UT-7/mpl cells did not result in the translocation of PKC- or -.
Furthermore, downregulation of these isoforms had no significant effect
on the mitogenic response induced by GM-CSF. Thus, in UT-7/mpl cells,
PKC- and do not appear to be required by GM-CSF for the
induction of a mitogenic response. In agreement with our findings,
Shearman et al54 reported that also in multipotential FDCP-Mix A4 cells, GM-CSF was unable to stimulate the translocation of
these isoforms to the membrane. In contrast to these results, several
studies had previously indicated that GM-CSF stimulates the activation
of PKC in various hematopoietic cells.36 However, in the
majority of those cases the individual PKC isoforms were not
identified. Thus, our findings are not necessarily incompatible with
previous work suggesting that GM-CSF can indeed stimulate PKC
activation. It is possible, for example, that GM-CSF activates PKC
isoforms other than or , and that this depends also on the cell
type in question. Support for this interpretation is provided by
studies in TF-1 cells, a multipotent human hematopoietic cell line with
properties similar to UT-7. In these cells, PKC-55 and
PKC-,56 but not PKC-,56 were reported to
be involved in the signal transduction pathways activated by GM-CSF.
Thus, taken together, our findings and those of other laboratories
suggest that in UT-7/mpl cells, GM-CSF and TPO would require different PKC isoforms to mediate a proliferative response.
The present study clearly shows that in contrast to its inhibitory
effect on TPO-induced mitogenesis, GF109203X did not affect the
induction of GpIIb expression by TPO (Fig 6). Therefore, our data
indicate that in UT-7/mpl cells, PKC is not required by TPO for
transmission of signals that regulate the induction of megakaryocytic differentiation antigens. This conclusion is compatible with previous findings suggesting that c-Mpl-generated signals for differentiation are transmitted via Shc and Grb2, through the Ras-MAP kinase
cascade,26 of which PKC is not a component.57
On the other hand, our data also show that GF109203X completely
prevented PMA-induced differentiation (Fig 6), suggesting that PKC does
play a role in the process. These apparently conflicting results can be
explained if one considers that MAP kinases can also be activated by
PKC via Raf-1, independently of Ras activation.58 In
addition, evidence has now been provided that MAP kinases are
downstream mediators of PMA-induced megakaryocytic differentiation.59 Thus, taken together, our findings and
those of other laboratories fit a model in which megakaryocytic
differentiation may be activated through PKC-dependent and
PKC-independent pathways converging on the MAP kinase cascade.
Recently, it has been shown that nuclear polyploidization and
cytoplasmic maturation may be regulated independently.60
Therefore, the evidence provided here showing that PKC is not involved
in the induction of GpIIb expression by TPO does not exclude the
possibility that a PKC-dependent mechanism might be involved in TPO
induction of polyploidization. The fact that TPO caused a very small
increase in the number of polyploid cells made it impracticable to
assess this possibility fully. Nonetheless, consistent with the
inhibitory effect of GF109203X on TPO-induced DNA synthesis (Fig 4), it
would appear that this compound also somewhat reduced the small
increase in ploidy induced by TPO (Fig 6 and data not shown). Whether
PKC plays a role in TPO-induced events associated with
polyploidization, other than DNA replication, remains to be explored.
Although the present work clearly shows the dominant role of a
PKC-independent pathway in the induction of megakaryocytic
differentiation antigens by TPO in UT-7/mpl cells, it does not exclude
the possibility that PKC might play a role in TPO-induced
differentiation of primary hematopoietic progenitors.
| |
FOOTNOTES |
Submitted April 23, 1997;
accepted September 23, 1997.
Supported in part by Research Grant No. PG/95001 from the British Heart
Foundation.
Address reprint requests to Jorge D. Erusalimsky, PhD, Cruciform
Project, Rayne Institute, University College London, 5 University Street, London WC1E 6JJ, UK.
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.
| |
REFERENCES |
1.
Kaushansky K:
Thrombopoietin: The primary regulator of platelet production.
Blood
86:419,
1995[Free Full Text]
2.
Eaton DL,
de Sauvage FJ:
Thrombopoietin: The primary regulator of megakaryocytopoiesis and thrombopoiesis.
Exp Hematol
25:1,
1997[Medline]
[Order article via Infotrieve]
3.
de Sauvage FJ,
Hass PE,
Spencer SD,
Malloy BE,
Gurney AL,
Spencer SA,
Darbonne WC,
Henzel WJ,
Wong SC,
Kuang WJ,
Oles KJ,
Hultgren B,
Solberg LA Jr,
Goeddel DV,
Eaton DL:
Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand.
Nature
369:533,
1994[Medline]
[Order article via Infotrieve]
4.
Kaushansky K,
Lok S,
Holly RD,
Broudy VC,
Lin N,
Bailey MC,
Forstrom JW,
Buddle MM,
Oort PJ,
Hagen FS,
Roth GJ,
Papayannopoulou T,
Foster DC:
Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin.
Nature
369:568,
1994[Medline]
[Order article via Infotrieve]
5.
Debili N,
Wendling F,
Katz A,
Guichard J,
Breton Gorius J,
Hunt P,
Vainchenker W:
The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors.
Blood
86:2516,
1995[Abstract/Free Full Text]
6.
Kaushansky K,
Broudy VC,
Lin N,
Jorgensen MJ,
McCarty J,
Fox N,
Zucker Franklin D,
Lofton Day C:
Thrombopoietin, the Mp1 ligand, is essential for full megakaryocyte development.
Proc Natl Acad Sci USA
92:3234,
1995[Abstract/Free Full Text]
7.
Gurney AL,
Carver Moore K,
de Sauvage FJ,
Moore MW:
Thrombocytopenia in c-mpl-deficient mice.
Science
265:1445,
1994[Abstract/Free Full Text]
8.
de Sauvage FJ,
Carver Moore K,
Luoh SM,
Ryan A,
Dowd M,
Eaton DL,
Moore MW:
Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin.
J Exp Med
183:651,
1996[Abstract/Free Full Text]
9.
Kobayashi M,
Laver JH,
Kato T,
Miyazaki H,
Ogawa M:
Recombinant human thrombopoietin (Mpl ligand) enhances proliferation of erythroid progenitors.
Blood
86:2494,
1995[Abstract/Free Full Text]
10.
Kaushansky K,
Broudy VC,
Grossmann A,
Humes J,
Lin N,
Ren HP,
Bailey MC,
Papayannopoulou T,
Forstrom JW,
Sprugel KH:
Thrombopoietin expands erythroid progenitors, increases red cell production, and enhances erythroid recovery after myelosuppressive therapy.
J Clin Invest
96:1683,
1995
11.
Alexander WS,
Roberts AW,
Nicola NA,
Li R,
Metcalf D:
Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietic receptor c-Mpl.
Blood
87:2162,
1996[Abstract/Free Full Text]
12.
Ku H,
Yonemura Y,
Kaushansky K,
Ogawa M:
Thrombopoietin, the ligand for the Mpl receptor, synergizes with steel factor and other early acting cytokines in supporting proliferation of primitive hematopoietic progenitors of mice.
Blood
87:4544,
1996[Abstract/Free Full Text]
13.
Carver Moore K,
Broxmeyer HE,
Luoh SM,
Cooper S,
Peng J,
Burstein SA,
Moore MW,
de Sauvage FJ:
Low levels of erythroid and myeloid progenitors in thrombopoietin- and c-mpl-deficient mice.
Blood
88:803,
1996[Abstract/Free Full Text]
14.
Borge OJ,
Ramsfjell V,
Veiby OP,
Murphy MJ Jr,
Lok S,
Jacobsen SE:
Thrombopoietin, but not erythropoietin promotes viability and inhibits apoptosis of multipotent murine hematopoietic progenitor cells in vitro.
Blood
88:2859,
1996[Abstract/Free Full Text]
15.
Ritchie A,
Vadhan Raj S,
Broxmeyer HE:
Thrombopoietin suppresses apoptosis and behaves as a survival factor for the human growth factor-dependent cell line, M07e.
Stem Cells
14:330,
1996[Abstract]
16.
Souyri M,
Vigon I,
Penciolelli JF,
Heard JM,
Tambourin P,
Wendling F:
A putative truncated cytokine receptor gene transduced by the myeloproliferative leukemia virus immortalizes hematopoietic progenitors.
Cell
63:1137,
1990[Medline]
[Order article via Infotrieve]
17.
Skoda RC,
Seldin DC,
Chiang MK,
Peichel CL,
Vogt TF,
Leder P:
Murine c-mpl: A member of the hematopoietic growth factor receptor superfamily that transduces a proliferative signal.
EMBO J
12:2645,
1993[Medline]
[Order article via Infotrieve]
18.
Debili N,
Wendling F,
Cosman D,
Titeux M,
Florindo C,
Dusanter Fourt I,
Schooley K,
Methia N,
Charon M,
Nador R,
Bettaieb A,
Vainchenker W:
The Mpl receptor is expressed in the megakaryocytic lineage from late progenitors to platelets.
Blood
85:391,
1995[Abstract/Free Full Text]
19.
Drachman JG,
Griffin JD,
Kaushansky K:
The c-Mpl ligand (thrombopoietin) stimulates tyrosine phosphorylation of Jak2, Shc, and c-Mpl.
J Biol Chem
270:4979,
1995[Abstract/Free Full Text]
20.
Tortolani PJ,
Johnston JA,
Bacon CM,
McVicar DW,
Shimosaka A,
Linnekin D,
Longo DL,
O'Shea JJ:
Thrombopoietin induces tyrosine phosphorylation and activation of the Janus kinase, JAK2.
Blood
85:3444,
1995[Abstract/Free Full Text]
21.
Yamada M,
Komatsu N,
Okada K,
Kato T,
Miyazaki H,
Miura Y:
Thrombopoietin induces tyrosine phosphorylation and activation of mitogen-activated protein kinases in a human thrombopoietin-dependent cell line.
Biochem Biophys Res Commun
217:230,
1995[Medline]
[Order article via Infotrieve]
22.
Sasaki K,
Odai H,
Hanazono Y,
Ueno H,
Ogawa S,
Langdon WY,
Tanaka T,
Miyagawa K,
Mitani K,
Yazaki Y,
Hirai H:
TPO/c-mpl ligand induces tyrosine phosphorylation of multiple cellular proteins including proto-oncogene products, Vav and c-Cbl, and Ras signaling molecules.
Biochem Biophys Res Commun
216:338,
1995[Medline]
[Order article via Infotrieve]
23.
Pallard C,
Gouilleux F,
Benit L,
Cocault L,
Souyri M,
Levy D,
Groner B,
Gisselbrecht S,
Dusanter Fourt I:
Thrombopoietin activates a STAT5-like factor in hematopoietic cells.
EMBO J
14:2847,
1995[Medline]
[Order article via Infotrieve]
24.
Morita H,
Tahara T,
Matsumoto A,
Kato T,
Miyazaki H,
Ohashi H:
Functional analysis of the cytoplasmic domain of the human Mpl receptor for tyrosine-phosphorylation of the signaling molecules, proliferation and differentiation.
FEBS Lett
395:228,
1996[Medline]
[Order article via Infotrieve]
25.
Miyakawa Y,
Oda A,
Druker BJ,
Miyazaki H,
Handa M,
Ohashi H,
Ikeda Y:
Thrombopoietin induces tyrosine phosphorylation of Stat3 and Stat5 in human blood platelets.
Blood
87:439,
1996[Abstract/Free Full Text]
26.
Alexander WS,
Maurer AB,
Novak U,
Harrison Smith M:
Tyrosine-599 of the c-Mpl receptor is required for Shc phosphorylation and the induction of cellular differentiation.
EMBO J
15:6531,
1996[Medline]
[Order article via Infotrieve]
27.
Gurney AL,
Wong SC,
Henzel WJ,
de Sauvage FJ:
Distinct regions of c-Mpl cytoplasmic domain are coupled to the JAK-STAT signal transduction pathway and Shc phosphorylation.
Proc Natl Acad Sci USA
92:5292,
1995[Abstract/Free Full Text]
28.
Whetton AD,
Monk PN,
Consalvey SD,
Huang SJ,
Dexter TM,
Downes CP:
Interleukin 3 stimulates proliferation via protein kinase C activation without increasing inositol lipid turnover.
Proc Natl Acad Sci USA
85:3284,
1988[Abstract/Free Full Text]
29.
Mason Garcia M,
Weill CL,
Beckman BS:
Rapid activation by erythropoietin of protein kinase C in nuclei of erythroid progenitor cells.
Biochem Biophys Res Commun
168:490,
1990[Medline]
[Order article via Infotrieve]
30.
DeVito WJ,
Avakian C,
Stone S,
Okulicz WC:
Prolactin-stimulated mitogenesis of cultured astrocytes is mediated by a protein kinase C-dependent mechanism.
J Neurochem
60:832,
1993[Medline]
[Order article via Infotrieve]
31.
Nishizuka Y:
Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C.
Science
258:607,
1992[Abstract/Free Full Text]
32. Nishizuka Y: Protein kinase C and lipid signaling for sustained
cellular responses. FASEB J 9:484, 1995
33.
Hug H,
Sarre TF:
Protein kinase C isoenzymes: Divergence in signal transduction?
Biochem J
291:329,
1993
34.
Whetton AD,
Heyworth CM,
Nicholls SE,
Evans CA,
Lord JM,
Dexter TM,
Owen Lynch PJ:
Cytokine-mediated protein kinase C activation is a signal for lineage determination in bipotential granulocyte macrophage colony-forming cells.
J Cell Biol
125:651,
1994[Abstract/Free Full Text]
35.
Hong Y,
Martin JF,
Vainchenker W,
Erusalimsky JD:
Inhibition of protein kinase C suppresses megakaryocytic differentiation and stimulates erythroid differentiation in HEL cells.
Blood
87:123,
1996[Abstract/Free Full Text]
36. Mufson RA: The role of serine/threonine phosphorylation in
hematopoietic cytokine receptor signal transduction. FASEB J 11:37,
1997
37.
Nishizuka Y:
Studies and perspectives of protein kinase C.
Science
233:305,
1986[Abstract/Free Full Text]
38.
Long MW,
Smolen JE,
Szczepanski P,
Boxer LA:
Role of phorbol diesters in in vitro murine megakaryocyte colony formation.
J Clin Invest
74:1686,
1984
39. Erusalimsky JD, Martin JF: Cellular model systems to study
megakaryocyte differentiation, in Watson SP, Authi KS (eds): Platelets,
a Practical Approach. Oxford, UK, IRL, 1996, p 27
40.
Komatsu N,
Nakauchi H,
Miwa A,
Ishihara T,
Eguchi M,
Moroi M,
Okada M,
Sato Y,
Wada H,
Yawata Y,
Suda T,
Miura Y:
Establishment and characterization of a human leukemic cell line with megakaryocytic features: Dependency on granulocyte-macrophage colony-stimulating factor, interleukin 3, or erythropoietin for growth and survival.
Cancer Res
51:341,
1991[Abstract/Free Full Text]
41.
Hermine O,
Mayeux P,
Titeux M,
Mitjavila MT,
Casadevall N,
Guichard J,
Komatsu N,
Suda T,
Miura Y,
Vainchenker W,
Breton-Gorius J:
Granulocyte-macrophage colony-stimulating factor and erythropoietin act competitively to induce two different programs of differentiation in the human pluripotent cell line UT-7.
Blood
80:3060,
1992[Abstract/Free Full Text]
42.
Goncalves F,
Lacout C,
Villeval JL,
Wendling F,
Vainchenker W,
Dumenil D:
Thrombopoietin does not induce lineage-restricted commitment of Mpl-R expressing pluripotent progenitors but permits their complete erythroid and megakaryocytic differentiation.
Blood
89:3544,
1997[Abstract/Free Full Text]
43.
Markowitz D,
Goff S,
Bank A:
Construction and use of a safe and efficient amphotropic packaging cell line.
Virology
167:400,
1988[Medline]
[Order article via Infotrieve]
44.
Erusalimsky JD,
John J,
Hong Y,
Moore M:
A glass fiber/diethylaminoethyl double filter binding assay that measures apoptotic internucleosomal DNA fragmentation.
Anal Biochem
242:187,
1997
45.
van der Loo B,
Hong Y,
Hancock V,
Martin JF,
Erusalimsky JD:
Antimicrotubule agents induce polyploidization of human leukaemic cell lines with megakaryocytic features.
Eur J Clin Invest
23:621,
1993[Medline]
[Order article via Infotrieve]
46.
Toullec D,
Pianetti P,
Coste H,
Bellevergue P,
Grand Perret T,
Ajakane M,
Baudet V,
Boissin P,
Boursier E,
Loriolle F,
Duhamel L,
Charon D,
Kirilovsky J:
The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C.
J Biol Chem
266:15771,
1991[Abstract/Free Full Text]
47.
Collins SJ:
The HL-60 promyelocytic leukemia cell line: proliferation, differentiation, and cellular oncogene expression.
Blood
70:1233,
1987[Abstract/Free Full Text]
48.
Rossi F,
McNagny M,
Smith G,
Frampton J,
Graf T:
Lineage commitment of transformed haematopoietic progenitors is determined by the level of PKC activity.
EMBO J
15:1894,
1996[Medline]
[Order article via Infotrieve]
49.
Farrar WL,
Thomas TP,
Anderson WB:
Altered cytosol/membrane enzyme redistribution on interleukin-3 activation of protein kinase C.
Nature
315:235,
1985[Medline]
[Order article via Infotrieve]
50.
Farese RV,
Standaert ML,
Francois AJ,
Ways K,
Arnold TP,
Hernandez H,
Cooper DR:
Effects of insulin and phorbol esters on subcellular distribution of protein kinase C isoforms in rat adipocytes.
Biochem J
288:319,
1992
51.
Xia P,
Aiello LP,
Ishii H,
Jiang ZY,
Park DJ,
Robinson GS,
Takagi H,
Newsome WP,
Jirousek MR,
King GL:
Characterization of vascular endothelial growth factor's effect on the activation of protein kinase C, its isoforms, and endothelial cell growth.
J Clin Invest
98:2018,
1996[Medline]
[Order article via Infotrieve]
52.
Kunitama M,
Shimizu R,
Yamada M,
Kato T,
Miyazaki H,
Okada K,
Miura Y,
Komatsu N:
Protein kinase C and c-myc gene activation pathways in thrombopoietin signal transduction.
Biochem Biophys Res Commun
231:290,
1997[Medline]
[Order article via Infotrieve]
53.
Rozengurt E:
Early signals in the mitogenic response.
Science
234:161,
1986[Abstract/Free Full Text]
54.
Shearman MS,
Heyworth CM,
Dexter TM,
Haefner B,
Owen PJ,
Whetton AD:
Haemopoietic stem cell development to neutrophils is associated with subcellular redistribution and differential expression of protein kinase C subspecies.
J Cell Sci
104:173,
1993[Abstract]
55.
Ettinger SL,
Lauener RW,
Duronio V:
Protein kinase C delta specifically associates with phosphatidylinositol 3-kinase following cytokine stimulation.
J Biol Chem
271:14514,
1996[Abstract/Free Full Text]
56.
Baxter GT,
Miller DL,
Kuo RC,
Wada HG,
Owicki JC:
PKC epsilon is involved in granulocyte-macrophage colony-stimulating factor signal transduction: Evidence from microphysiometry and antisense oligonucleotide experiments.
Biochemistry
31:10950,
1992[Medline]
[Order article via Infotrieve]
57.
Avruch J,
Zhang XF,
Kyriakis JM:
Raf meets Ras: Completing the framework of a signal transduction pathway.
Trends Biochem Sci
19:279,
1994[Medline]
[Order article via Infotrieve]
58.
Marquardt B,
Frith D,
Stabel S:
Signalling from TPA to MAP kinase requires protein kinase C, raf and MEK: Reconstitution of the signalling pathway in vitro.
Oncogene
9:3213,
1994[Medline]
[Order article via Infotrieve]
59.
Whalen AM,
Galasinski SC,
Shapiro PS,
Nahreini TS,
Ahn NG:
Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.
Mol Cell Biol
17:1947,
1997[Abstract]
60.
Kikuchi J,
Furukawa Y,
Iwase S,
Terui Y,
Nakamura M,
Kitagawa S,
Kitagawa M,
Komatsu N,
Miura Y:
Polyploidization and functional maturation are two distinct processes during megakaryocytic differentiation: Involvement of cyclin-dependent kinase inhibitor p21 in polyploidization.
Blood
89:3980,
1997[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
W. Nogami, H. Yoshida, K. Koizumi, H. Yamada, K. Abe, A. Arimura, N. Yamane, K. Takahashi, A. Yamane, A. Oda, et al.
The effect of a novel, small non-peptidyl molecule butyzamide on human thrombopoietin receptor and megakaryopoiesis
Haematologica,
October 1, 2008;
93(10):
1495 - 1504.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. S. Hitchcock, M. M. Chen, J. R. King, and K. Kaushansky
YRRL motifs in the cytoplasmic domain of the thrombopoietin receptor regulate receptor internalization and degradation
Blood,
September 15, 2008;
112(6):
2222 - 2231.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Giraudier, H. Chagraoui, E. Komura, S. Barnache, B. Blanchet, J. P. LeCouedic, D. F. Smith, F. Larbret, A.-L. Taksin, F. Moreau-Gachelin, et al.
Overexpression of FKBP51 in idiopathic myelofibrosis regulates the growth factor independence of megakaryocyte progenitors
Blood,
September 26, 2002;
100(8):
2932 - 2940.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M.-D. Filippi, F. Porteu, F. L. Pesteur, V. Schiavon, G. A. Millot, W. Vainchenker, F. J. de Sauvage, A. Dubart Kupperschmitt, and F. Sainteny
Requirement for mitogen-activated protein kinase activation in the response of embryonic stem cell-derived hematopoietic cells to thrombopoietin in vitro
Blood,
February 15, 2002;
99(4):
1174 - 1182.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Eisbacher, L. M. Khachigian, T. H. Khin, M. L. Holmes, and B. H. Chong
Inducible Expression of the Megakarocyte-specific Gene Glycoprotein IX Is Mediated through an Ets Binding Site and Involves Upstream Activation of Extracellular Signal-regulated Kinase
Cell Growth Differ.,
August 1, 2001;
12(8):
435 - 445.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Shiroshita, M. Musashi, K. Sakurada, K. Kimura, Y. Tsuda, S. Ota, H. Iwasaki, T. Miyazaki, T. Kato, H. Miyazaki, et al.
Involvement of Protein Kinase C-epsilon in Signal Transduction of Thrombopoietin in Enhancement of Interleukin-3-Dependent Proliferation of Primitive Hematopoietic Progenitors
J. Pharmacol. Exp. Ther.,
June 1, 2001;
297(3):
868 - 875.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
P. Rojnuckarin and K. Kaushansky
Actin reorganization and proplatelet formation in murine megakaryocytes: the role of protein kinase C{alpha}
Blood,
January 1, 2001;
97(1):
154 - 161.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Jandrot-Perrus, S. Busfield, A.-H. Lagrue, X. Xiong, N. Debili, T. Chickering, J.-P. L. Couedic, A. Goodearl, B. Dussault, C. Fraser, et al.
Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily
Blood,
September 1, 2000;
96(5):
1798 - 1807.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Dorsch, N. N. Danial, P. B. Rothman, and S. P. Goff
A Thrombopoietin Receptor Mutant Deficient in Jak-STAT Activation Mediates Proliferation But Not Differentiation in UT-7 Cells
Blood,
October 15, 1999;
94(8):
2676 - 2685.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Haslauer, K. Baltensperger, and H. Porzig
Erythropoietin- and Stem Cell Factor-Induced DNA Synthesis in Normal Human Erythroid Progenitor Cells Requires Activation of Protein Kinase Calpha and Is Strongly Inhibited by Thrombin
Blood,
July 1, 1999;
94(1):
114 - 126.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-C. Mamputu and G. Renier
Differentiation of Human Monocytes to Monocyte-Derived Macrophages Is Associated With Increased Lipoprotein Lipase–Induced Tumor Necrosis Factor-{alpha} Expression and Production : A Process Involving Cell Surface Proteoglycans and Protein Kinase C
Arterioscler. Thromb. Vasc. Biol.,
June 1, 1999;
19(6):
1405 - 1411.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Miyakawa, P. Rojnuckarin, T. Habib, and K. Kaushansky
Thrombopoietin Induces Phosphoinositol 3-Kinase Activation through SHP2, Gab, and Insulin Receptor Substrate Proteins in BAF3 Cells and Primary Murine Megakaryocytes
J. Biol. Chem.,
January 19, 2001;
276(4):
2494 - 2502.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract