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Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2859-2866
High-Level Expression of Mpl in Platelets and Megakaryocytes Is
Independent of Thrombopoietin
By
Karine Cohen-Solal,
Natacha Vitrat,
Monique Titeux,
William Vainchenker, and
Françoise Wendling
From the INSERM U 362, Institut Gustave Roussy, Villejuif, France.
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ABSTRACT |
Thrombopoietin (TPO) is a hematopoietic growth factor that regulates
megakaryocytopoiesis and platelet production through binding to its
receptor, Mpl, encoded by the c-mpl proto-oncogene. Circulating
levels of TPO are regulated by receptor-mediated uptake and
degradation. To better understand this mode of TPO regulation, we
examined whether expression of Mpl was regulated by its ligand. Using
RNase protection analysis, we found no differences in the levels of
c-mpl transcripts in megakaryocytes (MKs) produced in vitro
either in the presence or absence of TPO and in platelets (PLTs)
obtained from mice hyperstimulated in vivo by ectopic secretion of TPO.
Similarly, Western blot analysis of MKs produced in the presence or
absence of TPO showed no difference in Mpl levels. Levels of Mpl,
GpIIb, or P-selectin were virtually identical in platelet lysates
obtained from normal, TPO knockout and mildly TPO-stimulated mice. In
contrast, the expression of Mpl was significantly reduced in PLTs from
severely thrombocythemic mice. These results show that TPO does not
have a major effect on the transcription or translation of Mpl.
However, they do suggest that an excess of circulating TPO can lead to
the disappearance of Mpl from PLTs via catabolism.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THROMBOPOIETIN (TPO), the ligand for the
Mpl receptor, is the major physiologic regulator of
megakaryocytopoiesis and platelet production.1-4 The major
sites of TPO production are the parenchymal and sinusoidal endothelial
cells of the liver and the proximal convoluted tubules of the
kidney.5-8 TPO is released into blood and circulates to
reach target cells present in the hematopoietic organs. It has been
clearly shown that circulating TPO levels are inversely correlated to
platelet mass.9 By analogy with the transcriptional
regulation of erythropoietin (EPO) mRNA levels by
anemia,10 it has been suggested that TPO gene transcription might be upregulated in response to a decrease in platelet mass. Alternatively, it has been proposed that TPO production is constant, with TPO activity regulated by binding to platelets (PLTs) and catabolism. In recent studies of mice made profoundly thrombocytopenic or thrombocythemic, no changes were observed in liver or kidney TPO
mRNA levels. In addition, no variations were detected in the major
alternative splice forms encoding nonsecreted or inactive TPO
proteins.11-15 It is still a matter of debate, however,
whether TPO synthesis in stromal cells is regulated by platelet
mass.16,17 Definitive evidence that there is no modulation
of TPO mRNA levels in response to platelet demand has been provided by
mice genetically altered to be defective in TPO.14,18 In
contrast, PLTs have been shown to play a key role in the plasma
clearance of TPO, as they actively bind TPO via Mpl, and internalize
and degrade the protein.19,20 Megakaryocytes (MKs) may also
have a role in the regulation of TPO. NF-E2 knockout mice and patients
with idiopathic thrombocytopenic purpura (ITP) are both markedly
thrombocytopenic, exhibit increased numbers of MKs in their marrow, and
have normal or only slightly elevated plasma levels of
TPO.21-25 Together, these results support a model in which
plasma TPO levels are regulated both by circulating PLTs, the most
mature cells of the megakaryocytic lineage, and by maturing MKs present
in the hematopoietic organs. Thus, the number of Mpl receptors present
on the surface of PLTs and MKs is an important parameter in the
regulation of circulating TPO levels.
Upregulation of receptor by ligand has been shown in cells from
different hematopoietic lineages. One well-documented example is the
interleukin-2 receptor (IL-2R). It has been reported that IL-2 augments
expression of the receptor  chain in human T and B
cells26 and  and  chain mRNA levels in human
monocytes.27,28 Upregulation of IL-2R transcripts is
associated with increased IL-2 binding activity on the surface of
monocytes.28 Similarly, granulocyte colony-stimulating
factor (G-CSF) specifically upregulates transcripts of its own receptor
in normal murine bone marrow cells and in a granulocytic cell
line,29 and granulocyte-macrophage (GM)-CSF positively
regulates GM-CSF receptors in murine macrophages.30 Such a
mechanism has also been suggested for the M-CSF receptor.31
The aim of this study was to analyze whether TPO could positively
regulate the transcription and/or translation of its receptor, Mpl, on
MKs and PLTs, a mechanism that would amplify Mpl-mediated uptake of
TPO. c-mpl transcript levels were first compared in cultured
murine MKs grown either in the presence or absence of murine
recombinant TPO, and in PLTs isolated from normal and TPO-stimulated mice. Second, the level of Mpl protein was analyzed in MKs obtained in
the presence and absence of TPO and in PLTs isolated from normal, TPO-stimulated, and TPO knockout mice. To allow comparison between the
different samples, the expression of two glycoproteins highly expressed
in PLTs, P-selectin and GpIIb, was also analyzed. The data strongly
argue against regulation of Mpl by TPO in PLTs and MKs, either at the
transcriptional or translational level. However, we observed that
expression of the Mpl protein was significantly reduced in PLTs
obtained from mice with severe thrombocythemia.
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MATERIALS AND METHODS |
Experimental murine models of thrombocytosis.
Two different approaches were used to produce thrombocythemic mice. In
the first model, (C57Bl/6 × DBA/2) F1 mice (Janvier, Lyon,
France) were subcutaneously transplanted with 1 × 106 tumorigenic factor-dependent cell-P1 (FDC-P1) cells
engineered to secrete murine TPO.13 PLTs were increased
twofold to fourfold 3 to 4 weeks after the graft. In the second model,
C57Bl/6 mice were lethally irradiated and hematologically reconstituted
by a syngeneic transplantation of hematopoietic progenitor cells infected with a MPZen retrovirus carrying the murine TPO cDNA, as
previously described.32 The number of PLTs in blood from these reconstituted mice sharply increased and reached eightfold to
10-fold baseline levels between 4 and 10 weeks posttransplant.
Platelet preparation.
Mice were bled by cardiac puncture under ether anesthesia. Blood was
collected on sodium citrate (3.8%, 1 vol citrate for 9 vol blood) and
diluted sixfold in buffered saline-glucose-citrate (BSGC, pH 7.3).
Diluted blood was first centrifuged at room temperature for 15 minutes
at 200g to eliminate nucleated and red blood cells. Supernatant
was then centrifuged for 15 minutes at 1,700g to spin down the PLTs.
Culture and purification of megakaryocytes.
Both femurs and tibias from C57Bl/6 adult mice (10 to 12 weeks old)
were flushed in Mem medium (-MEM) (GIBCO-BRL,
Eragny, France) containing 2% fetal bovine serum (FBS). Cells (6 × 108 in 6 mL) were incubated for 30 minutes at
4°C with a cocktail of monoclonal antibodies containing 10 µg/mL
of CD45R-B220, 5 µg/mL of TER 119 and 10 µg/mL of GR-1, Mac-1
(1:3,000), Ly+1 (1:1,000), and GK 1.5 (1:10). CD45R-B220, TER 119, and
GR-1 antibodies were obtained from Pharmingen and Becton Dickinson (Le
Pont de Claix, France). Mac-1 and Ly+1 were obtained from ascites and
GK 1.5 from culture supernatant. After one wash, cells were incubated for 30 minutes at 4°C with immunomagnetic beads coated with sheep anti-rat IgG (Dynal, Compiègne, France) in a ratio of 3 beads/mononuclear target cell. The Lin cell fraction
was isolated using a Dynal separator (Oslo, Norway). To ensure higher
purity of the preparation, a second round of bead separation was used.
The overall yield was 10%. All washing, incubation, and purification
procedures were performed in phosphate-buffered saline (PBS) containing
1% bovine serum albumin (BSA) (Sigma, St Quentin Fallavier, France).
The Lin cellular fraction was cultured in suspension
at 1 × 106 cells/mL in -MEM medium supplemented
with 10% FBS and stimulated either with 10 ng/mL recombinant murine
TPO (r-mu TPO; R & D Systems, Minneapolis, MN) or in
the presence of a cocktail of recombinant cytokines composed of r-mu
IL-3 (R & D Systems; 50 U/mL), r-hu IL-6 (10 ng/mL; a generous gift
from Dr S. Burstein, William K. Warren Medical Research Institute,
University of Oklahoma, Oklahoma City, OK), and r-mu
stem cell factor (SCF) (R & D Systems; 10 ng/mL). On day 4 of culture,
MKs were enriched (50% to 70% purity) on a BSA gradient as previously
described.33
Ribonuclease protection assay.
A 32P uridine triphosphate (UTP)-labeled
antisense probe was transcribed from the T3 promoter of an XbaI
linearized plasmid (KS MPL 500) containing a murine c-mpl cDNA.
To construct KS MPL 500, an SphI-StuI fragment
containing nucleotides 230 to 734 of the Mpl coding sequence
was inserted into the polylinker site of the pBluescript II KS vector
(Stratagene, La Jolla, CA). A 32P-labeled antisense murine
GpIIb probe transcribed from the T7 promoter of the HindIII
linearized pCR GpIIb 185 plasmid was used as an internal control for
each reaction sample. The plasmid pCR GpIIb 185 (kindly provided by J. Starck [CNRS UMR 5534, Villeurbanne, France] and G. Uzan [INSERM
U506, Villejuif, France]) was constructed by inserting a polymerase
chain reaction (PCR) product, containing exons 24 and 25 of GpIIb
cDNA,34 into the polylinker site of the pCR TM II
vector (Invitrogen, Groningen, The Netherlands). Total
RNAs (2 µg) from PLTs and MKs were hybridized with radioactive probes
at 50°C overnight. Nonhybridizing RNAs were digested with RNase A
(10 µg/mL) and RNase T1 (1,000 U/mL) for 1 hour at 37°C. To
inhibit RNase activity, sodium dodecyl sulfate (SDS) (0.6%) and
proteinase K (145 µg/mL) were added for 15 minutes at 37°C. Protected fragments were extracted in the presence of 15 µg of carrier transfer RNA with phenol/chloroform/isoamyl alcohol and precipitated with absolute ethanol at  20°C. Fragments were
resolved on a 4% polyacrylamide, 7 mol/L urea gel and autoradiographed on hyperfilm MP (Amersham, Buckinghamshire, UK).
Fragment sizes were determined by labeled MspI-digested pBR322
(New England Biolabs, Beverly, MA). Quantification of
protected fragments was performed with a phosphorimager system (MACBAS
program; Fuji Photo Film Co, Japan).
Western blots.
Cells were lysed at 4°C in lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% NP40, 5% glycerol, and 1 mmol/L dithiothreitol) containing protease inhibitors (Complete; Boehringer Mannheim, Meylan, France). A colorimetric assay was
used to determine the concentration of proteins in the different
lysates (Bio-Rad DC Protein Assay; Bio-Rad, Hercules,
CA). Proteins from total lysates (30 µg) were
analyzed on 8% polyacrylamide gels with prestained molecular weight
markers (Bio-Rad). Proteins were transferred to nitrocellulose
membranes (Amersham). The membranes were probed successively with a
rabbit anti-mouse Mpl polyclonal antibody (kindly provided by Dr S. Lok, ZymoGenetics, Seattle, WA), a rabbit anti-human P-selectin
polyclonal antibody cross-reacting with murine P-selectin (Pharmingen),
a rabbit anti-human GpIIb polyclonal antibody cross-reacting with
murine GpIIb35 (obtained from Dr D. Pidard, Institut
Pasteur, Paris, France) and, finally, a goat anti-mouse actin
polyclonal antibody (TEBU, le Perray en Yvelines, France).
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RESULTS |
Quantitative measurement of c-mpl transcripts in MKs and PLTs.
RNase protection assays were used to quantify the levels of
c-mpl transcripts in MKs and PLTs. To determine the threshold of the technique, a series of experiments was performed. A murine c-mpl antisense riboprobe was constructed that contained exon 3 (without the first 18 nucleotides), all nucleotides from exon 4, and 44 nucleotides from exon 5.36 To normalize c-mpl
transcripts, an antisense riboprobe specific for the mouse
megakaryocytic marker GpIIb spanning exons 24 and 2534 was
introduced into the reaction samples. Various concentrations of total
RNA prepared from MKs and PLTs were analyzed to measure the relative
intensities of the protected fragments. Two protected fragments were
obtained with the c-mpl riboprobe
(Fig 1). The 440-bp protected fragment corresponds to an alternatively spliced form of c-mpl
characterized by a 24-nucleotide deletion in exon 4. The 506-bp
protected fragment corresponds to the wild-type c-mpl
mRNA.37 Two protected fragments were also detected with the
GpIIb riboprobe that probably corresponds to splice variants.
As shown in Fig 1, the intensity of protected fragments varies in
parallel with the concentration of total RNA from PLTs or MKs. We also
confirmed that the ratio between c-mpl mRNA and GpIIb mRNA was
independent of the total RNA concentration used in the protection
assay. These results showed that this method could be used to compare
levels of c-mpl mRNA obtained from different sources.
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| Fig 1.
Ribonuclease protection analysis of RNA from MKs and
PLTs. Decreasing amounts of RNA from 10 to 2 µg were used for MKs and
PLTs. Transfer RNA (tRNA) was used as a negative control to verify
specificity of protected fragments. The size of the markers is
indicated on the left of the panel. Arrows on the right indicate the
position of Mpl (top) and GpIIb (bottom) protected fragments.
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c-mpl mRNA levels in MKs grown in the presence or absence of TPO.
To study the effects of TPO on the stabilization of c-mpl
transcripts and transcription of the c-mpl gene, two different
protocols were used. For the first set of experiments, immature
Lin cells were grown in liquid medium in the
presence of r-mu TPO. After 3 days of stimulation, cells were washed
and MKs were enriched on a BSA gradient. The MK population was then
divided into two samples. One was grown for an additional 24 hours in
the presence of r-mu TPO, while the other one was stimulated with a
cocktail of three cytokines that did not contain TPO. Three independent MK preparations were analyzed for each condition. Total RNA was extracted and RNase protection assay was performed. Results
(Fig 2A and
Table 1) show that the ratios between
c-mpl- and GpIIb-protected fragments were similar in the two
culture conditions, suggesting that TPO has no major effect on
c-mpl or GpIIb mRNA levels.
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| Fig 2.
Comparison of c-mpl transcript levels in MKs in
the presence or absence of TPO, by ribonuclease protection analysis (2 µg of RNA). (A) MKs obtained in the presence of TPO (3 days) were
either maintained in TPO or shifted for 24 additional hours to a medium
deprived of TPO, but containing a combination of SCF, IL-6, and IL-3.
For each condition, three independent MK preparations were made. (B)
MKs were obtained either in the presence of TPO or in the presence of a
combination of SCF, IL-6, and IL-3 (4 days). Two independent MK
preparations were used for each condition. Arrows on the right indicate
the position of the protected fragments.
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Table 1.
Ratios of c-mpl mRNA to GpIIb mRNA Levels in
PLTs From Normal and TPO-Stimulated Mice and in MKs in
the Presence and Absence of TPO
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To exclude the possibility that stimulation by TPO for 3 days had
irreversibly induced c-mpl expression in MKs, we analyzed the
levels of c-mpl transcripts in MKs grown for 4 days either in
the presence of TPO alone or in the presence of the three-cytokine cocktail. Two independent MK samples were analyzed in each case. The
levels of c-mpl and GpIIb mRNA remained similar whether
MKs were grown in the presence or absence of TPO (Fig 2B and Table 1).
In these studies, TPO and the three-cytokine cocktail had exactly the
same effect on the level of expression of c-mpl transcripts. Together, these results strongly argue against positive regulation by
TPO of c-mpl gene transcription or transcript stabilization in MKs.
c-mpl transcript levels in PLTs from control and TPO-stimulated mice.
To study whether in vivo long-term stimulation of megakaryocytopoiesis
by suprapharmacologic doses of TPO would affect c-mpl transcript levels, we used different animal models. In the first model,
normal mice were subcutaneously injected with FDC-P1 tumorigenic cells
engineered to secrete mu TPO. Three to 4 weeks after grafting, PLT
counts were increased twofold to fourfold above baseline values. In the
second model, the hematopoietic system of lethally irradiated mice was
reconstituted with a bone marrow transplant infected with a retrovirus
encoding mu TPO. Two months posttransplant, PLT counts were increased
8- to 10-fold, and the plasma TPO level was augmented by a factor of
1,000 to 5,000.32 In each model, independent PLT total RNA
samples were analyzed by RNase protection assays. No variation in the
ratios between c-mpl and GpIIb transcripts was detected when
PLTs from normal mice were compared with PLTs from mildly or
hyperstimulated animals (Fig 3A and B and
Table 1). These data show that long-term in vivo stimulation of PLTs by
high concentrations of TPO did not have a positive effect on the
stabilization of c-mpl transcripts.
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| Fig 3.
Comparison of c-mpl transcript levels in PLTs
from control and TPO-stimulated mice by means of ribonuclease
protection assays (2 µg). PLTs were isolated from mice grafted
subcutaneously with FDC-P1 cells producing TPO ( PLT; A) or from
mice in which the hematopoietic cells produce TPO ( PLT; B).
Three (A) or two (B) independent platelet RNA preparations have been
used.
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Levels of Mpl, GpIIb, P-selectin, and actin proteins in MKs grown in
the presence or absence of TPO.
To determine whether TPO had any regulatory effect on the level of Mpl
protein, we performed immunoblot experiments. The specificity of the
anti-mouse Mpl polyclonal antibody was demonstrated on lysates prepared
from Ba/F3 cells stably transfected with a murine c-mpl
expression vector (Fig 4). MKs were
prepared according to our first protocol (3 days with TPO followed by
24 hours with either TPO or IL-3, IL-6, and SCF) and the levels of Mpl
in both samples were compared. Protein concentrations in MK lysates
were determined by a colorimetric assay and equal amounts of protein (50 µg) were loaded on the gel. After protein transfer, membranes were successively incubated with antibodies directed against Mpl, GpIIb
(CD41), P-selectin (CD62P) and, to ensure equivalent protein loading,
actin. As shown in Fig 5, no significant
difference in the levels of Mpl protein was detectable
between MKs maintained in TPO and those shifted to the combination of
SCF, IL-6, and IL-3. These data suggest that TPO does not have a major
effect on the translation of Mpl.
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| Fig 4.
Western blot analysis of the specificity of the rabbit
polyclonal antibody directed against murine Mpl. The same amount of
protein lysate from Ba/F3 and Ba/F3-mu-Mpl cells was analyzed, and two
concentrations of the anti-Mpl antibody were used. Nonrelevant rabbit
purified immunoglobulins were used as a control. The size of protein
markers is indicated on the right of the figure.
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| Fig 5.
Comparison of Mpl levels in MKs in the presence or
absence of TPO. MKs obtained in the presence of TPO (3 days) were
either maintained in TPO (right lane) or shifted to a medium without
TPO and containing SCF, IL-6, and IL-3 (left lane). Immunoblot analysis
of MK lysates using antibodies directed against Mpl, GpIIb, P-selectin,
and actin, as described in Materials and Methods.
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Levels of Mpl, GpIIb, P-selectin, and actin proteins in PLTs from
normal, TPO-stimulated, and TPO knockout mice.
We further examined levels of Mpl protein in PLTs from normal, TPO
knockout, and TPO-stimulated mice. Mpl expression was analyzed using
PLT lysates prepared from heterozygous (TPO+/) and
homozygous (TPO/) TPO knockout mice and
compared with normal mice. Whatever the phenotype of the mice, no
significant difference was found in Mpl expression after normalization
with actin and the two megakaryocytic markers (CD41 and CD62P). These
results further indicate that TPO has no direct effect on the
translation or stabilization of Mpl (Fig
6A). The level of Mpl in TPO/ mice argues
strongly against TPO being necessary for high-level expression of its
receptor. Similarly, the level of Mpl protein in PLTs from mice made
moderately thrombocythemic (twofold to fourfold increase) by ectopic
TPO expression was not different from that in PLTs from normal mice
(Fig 6B). In contrast, when PLTs from severely thrombocythemic mice (8- to 10-fold increase) were analyzed, a reproducible and significant
reduction in Mpl levels was seen, while the expression of GpIIb or
P-selectin remained unchanged (Fig 6B).
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| Fig 6.
Comparison of Mpl levels in PLTs isolated from control,
TPO+/, TPO/, and TPO-stimulated
thrombocythemic mice. Immunoblot analysis of platelet lysates from (A)
TPO+/ and TPO/ mice; (B) the two
types of TPO-stimulated mice, using antibodies directed against Mpl,
GpIIb, P-selectin, and actin. For PLT and PLT, see legend to
Fig 3. Two independent lysates were used for each TPO stimulation
model.
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DISCUSSION |
Circulating levels of cytokines can be regulated by uptake and
degradation when mature blood cells express the corresponding receptor.38-40 One well-documented example is the
G-CSF/G-CSF-R model in which neutrophils play a key role in the
clearance of G-CSF from plasma.40 G-CSF-R downregulation
at the neutrophil membrane has been shown in vitro, as has
internalization of G-CSF after interaction with its
receptor.41 The mechanism of receptor downregulation and
ligand-receptor complex internalization is a general feature of
cytokine receptors,42 but in the case of G-CSF, it seems
that this mechanism is largely involved in the in vivo clearance of
G-CSF. It has also been reported that G-CSF upregulates G-CSF-R mRNA
levels in a granulocytic precursor cell line.29 This
process may be relevant to the in vivo increase of G-CSF clearance by
neutrophils.40 Similar to the role played by neutrophils in
the G-CSF/G-CSF-R system, PLTs and MKs are needed for clearance of
circulating TPO.22,23,25,43 Binding and degradation of TPO
by PLTs and MKs have also been documented,19,20,24 but
downregulation of the Mpl receptor has not yet been shown.
In the Mpl/TPO system, precise regulation of platelet production is
necessary to avoid significant fluctuations in platelet counts. In a
manner similar to the G-CSF/G-CSF-R model, receptor-mediated TPO
uptake by PLTs and MKs may be tightly controlled. Upregulation of Mpl
by TPO could provide a mechanism for increased clearance of circulating
TPO. To test this hypothesis, we determined if TPO was able to
positively regulate c-mpl transcript and/or Mpl protein levels
in MKs and PLTs, the two cell types involved in its clearance.
In this study, the megakaryocyte preparations had a purity of 50% to
70% with varying numbers of nonmegakaryocytic cells present. Therefore, we used two different megakaryocyte markers, GpIIb and
P-selectin, to normalize the levels of c-mpl transcripts and Mpl protein in MK preparations.
We first investigated by RNase protection assay whether c-mpl
transcripts could be modulated by TPO. Using MKs grown in vitro, we
found that the levels of c-mpl transcripts were not modified by
the presence of TPO in the culture medium. The same results were
obtained when the bone marrow Lin fraction cultured
for 3 days in TPO was shifted to a TPO-containing or deprived medium
for 1 additional day, or the Lin fraction was
cultured for 4 days in the presence or absence of TPO.
We next analyzed c-mpl transcript levels in PLTs stimulated by
high doses of TPO. In two in vivo models of TPO overexpression, c-mpl transcript levels were similar to those found in normal PLTs. These results strongly suggest that TPO does not positively regulate c-mpl transcripts in PLTs and MKs, either by
stabilization or by increased transcription. Surprisingly, the levels
of GpIIb transcripts were the same whether MKs were obtained in the
presence or absence of TPO, and whether PLTs were from normal or
TPO-stimulated mice. These findings differ from previous studies
looking at several human and murine GM-CSF or IL-3-dependent cell
lines in which a shift to TPO induces expression of GpIIb. This
increase in GpIIb expression is generally associated with an induction
of MK maturation, including morphologic changes and
polyploidization.44-46 Commitment to MK maturation appears
to be related to prolonged activation of the ras
pathway44,45 and to modification in the expression of
transcription factors such as Spi1/PU1.47 In normal
CD34+ cells, a three-cytokine combination (IL-3, IL-6, and
SCF) mimics the effects of TPO on MK differentiation, inducing
polyploidization, high-level expression of GpIIb, and cytoplasmic
maturation.48 As recently reported, use of a constitutively
activated H-Ras has similar effects on differentiation.45
Our findings and those from these other studies imply that TPO does not
directly affect the expression of individual MK-specific genes, such as
c-mpl or GpIIb. Rather, TPO may allow the development of a
complete megakaryocytic differentiation program, as does a combination of cytokines or an activated H-Ras.
To investigate if TPO has a regulatory effect on translation of Mpl,
protein levels were compared in PLTs from normal,
TPO/, TPO+/, and mildly
thrombocythemic mice. The data indicate that in these different
samples, similar amounts of Mpl protein were present. In contrast, PLTs
from thrombocythemic mice with very high and persistent levels of TPO
expression had markedly decreased Mpl levels relative to PLTs from
normal mice (Fig 6B). However, no significant differences were seen at
the level of c-mpl transcripts (Fig 3B). In this model, the
concentration of TPO in the plasma reached values 1,000-fold to
5,000-fold above normal.32 These results suggest that
hyperstimulation of PLTs by very high TPO concentrations may result in
increased internalization of Mpl followed by active degradation. TPO
has been shown to be internalized and degraded after binding to Mpl
expressed on platelet membranes.14,19 Both our results and
these data suggest that the Mpl/TPO complex may be internalized and
degraded. However, the hypothesis of Mpl recycling or shedding under
physiologic conditions, as described for the IL-6
receptor,49 cannot be excluded.
In conclusion, our study strongly argues against TPO-mediated
regulation of Mpl transcription or translation in MKs and PLTs.
| |
ACKNOWLEDGMENT |
We thank Drs S. Lok (ZymoGenetics, Seattle, WA), D. Pidard (Institut
Pasteur, Paris, France), and S. Burstein (Oklahoma City, OK) for their
gifts of antimouse Mpl polyclonal antibody, antihuman GpIIb polyclonal
antibody, and recombinant human IL-6, respectively. We thank Dr F. de
Sauvage for the TPO-deficient mice, and Drs J. Starck and G. Uzan for
the plasmid pCR GpIIb 185.
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FOOTNOTES |
Submitted July 29, 1998; accepted December 14, 1998.
Supported by the INSERM, the Institut Gustave Roussy, and by grants
from the Association de Recherche contre le Cancer, La Ligue Nationale
contre le Cancer and La ligue de Paris contre le Cancer. K.C.-S. was
supported by a fellowship from La Ligue Nationale contre le Cancer.
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 Karine Cohen-Solal, PhD,
INSERM U 362, Institut Gustave Roussy, PR1, 39 Rue Camille Desmoulins,
94805 Villejuif, France; e-mail: kcohen{at}igr.fr.
| |
REFERENCES |
1.
Lok S, Kaushansky K, Holly RD, Kuijper JL, Lofton-Day CE, Oort PJ, Grant FJ, Heipel MD, Burkhead SK, Kramer JM, Bell LA, Sprecher CA, Blumberg H, Johnson R, Prunkard D, Ching AFT, Mathewes SL, Bailey MC, Fostrom JW, Buddle MM, Osborn SG, Evans SJ, Sheppard PO, Presnell SR, O'Hara PJ, Hagen FS, Roth GJ, Foster DC:
Cloning and expression of murine thrombopoietin and stimulation of platelet production in vivo.
Nature
369:565, 1994[Medline]
[Order article via Infotrieve]
2.
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 LAJ, Goeddl DV, Eaton DL:
Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-MPL ligand.
Nature
369:533, 1994[Medline]
[Order article via Infotrieve]
3.
Bartley TD, Bogenberger J, Hunt P, Li YS, Lu HS, Martin F, Chang MS, Samal B, Nichol JL, Swift S, Johnson MJ, Hsu RY, Parker VP, Suggs S, Skrine JD, Merewether LA, Clogston C, Hsu E, Hokom MM, Hornkohl A, Choi E, Pangelinan M, Sun Y, Mar V, McNinch J, Simonet L, Jacobsen F, Xie C, Shutter J, Chute H, Basu R, Selander L, Trollinger D, Sieu L, Padilla D, Trail G, Elliot G, Izumi R, Covey T, Crouse J, Garcia A, Xu W, Del Castillo J, Biron J, Cole S, Hu M-T, Pacifici R, Ponting I, Saris C, Wen D, Yung YP, Lin H, Bosselman RA:
Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl.
Cell
77:1117, 1994[Medline]
[Order article via Infotrieve]
4.
Wendling F, Maraskovsky E, Debili N, Florindo C, Teepe M, Titeux M, Methia N, Breton-Gorius J, Cosman D, Vainchenker W:
c-Mpl ligand is a humoral regulator of megakaryocytopoiesis.
Nature
369:571, 1994[Medline]
[Order article via Infotrieve]
5.
Shimada Y, Kato T, Ogami K, Horie K, Kokubo A, Kudo Y, Maeda E, Shoma Y, Akahori H, Kawamura K, Miyazaki H:
Production of thrombopoietin (TPO) by rat hepatocytes and hepatoma cell lines.
Exp Hematol
23:1388, 1995[Medline]
[Order article via Infotrieve]
6.
Nomura S, Ogami K, Kawamura K, Tsukamoto I, Kudo Y, Kanakura Y, Kitamura Y, Miyazaki H, Kato T:
Cellular localization of thrombopoietin mRNA in the liver by in situ hybridization.
Exp Hematol
25:565, 1997[Medline]
[Order article via Infotrieve]
7.
Sungaran R, Markovic B, Chong BH:
Localization and regulation of thrombopoietin mRNA expression in human kidney, liver, bone marrow and spleen using in situ hybridization.
Blood
89:101, 1997[Abstract/Free Full Text]
8.
Cardier JE, Dempsey J:
Thrombopoietin and its receptor, c-mpl, are constitutively expressed by mouse liver endothelial cells: Evidence of thrombopoietin as a growth factor for liver endothelial cells.
Blood
91:923, 1998[Abstract/Free Full Text]
9.
Kuter DJ, Rosenberg RD:
The reciprocal relationship of thrombopoietin (c-Mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit.
Blood
85:2720, 1995[Abstract/Free Full Text]
10.
Bondurant MC, Koury M:
Anemia induces accumulation of erythropoietin mRNA in the kidney and liver.
Mol Cell Biol
6:2731, 1986[Abstract/Free Full Text]
11.
Gurney AL, Kuang WJ, Xie MH, Malloy BE, Eaton DL, de Sauvage FJ:
Genomic structure, chromosomal localization, and conserved alternative splice forms of thrombopoietin.
Blood
85:981, 1995[Abstract/Free Full Text]
12.
Chang MS, McNinch J, Basu R, Shutter J, Hsu RY, Perkins C, Mar V, Suggs S, Welcher A, Li L, Lu H, Bartley T, Hunt P, Martin F, Samal B, Bogenberger J:
Cloning and characterization of the human megakaryocyte growth and development factor (MGDF) gene.
J Biol Chem
270:511, 1995[Abstract/Free Full Text]
13.
Cohen-Solal K, Villeval J-L, Titeux M, Lok S, Vainchenker W, Wendling F:
Constitutive expression of Mpl ligand transcript during thrombocytopenia and thrombocytosis.
Blood
88:2578, 1996[Abstract/Free Full Text]
14.
Fielder PJ, Gurney AL, Stefanich E, Marian M, Moore MW, Carver-Moore K, de Sauvage FJ:
Regulation of thrombopoietin levels by c-mpl-mediated binding to platelets.
Blood
87:2154, 1996[Abstract/Free Full Text]
15.
Stoffel R, Wiestner A, Skoda RC:
Thrombopoietin in thrombocytopenic mice: Evidence against regulation of the mRNA level and for a direct regulatory role of platelets.
Blood
87:567, 1996[Abstract/Free Full Text]
16.
McCarty JM, Sprugel KH, Fox NE, Sabath DE, Kaushansky K:
Murine thrombopoietin mRNA levels are modulated by platelet count.
Blood
86:3668, 1995[Abstract/Free Full Text]
17.
Hirayama Y, Sakamaki S, Matsunaga T, Kuga T, Kuroda H, Kusakabe T, Sasaki K, Fujikawa K, Kato J, Kogawa K, Koyama R, Niitsu Y:
Concentrations of thrombopoietin in bone marrow in normal subjects and in patients with idiopathic thrombocytopenic purpura, aplastic anemia, and essential thrombocythemia correlate with its mRNA expression of bone marrow stromal cells.
Blood
92:46, 1998[Abstract/Free Full Text]
18.
de Sauvage FJ, Carver-Moore K, Shiuh-Ming L, 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]
19.
Fielder P, Hass P, Nagel M, Stefanich E, Widmer R, Bennett G, Keller G-A, de Sauvage F, Eaton D:
Human platelets as a model for the binding and degradation of thrombopoietin.
Blood
89:2782, 1997[Abstract/Free Full Text]
20.
Broudy V, Lin N, Sabath D, Papayannopoulou T, Kaushansky K:
Human platelets display high-affinity receptors for thrombopoietin.
Blood
89:1896, 1997[Abstract/Free Full Text]
21.
Shivdasani RA, Rosenblatt MF, Zucker-Franklin D, Jackson CW, Hunt P, Saris CJM, Orkin SH:
Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development.
Cell
81:695, 1995[Medline]
[Order article via Infotrieve]
22.
Mukai HY, Kojima H, Todokoro K, Tahara T, Kato T, Hasegawa Y, Kobayashi T, Ninomiya H, Nagasawa T, Abe T:
Serum thrombopoietin (TPO) levels in patients with amegakaryocytic thrombocytopenia are much higher than those with immune thrombocytopenic purpura.
Thromb Haemost
76:675, 1996[Medline]
[Order article via Infotrieve]
23.
Ichikawa N, Ishida F, Shimodaira S, Tahara T, Kato T, Kitano K:
Regulation of serum thrombopoietin levels by platelets and megakaryocytes in patients with aplastic anaemia and idiopathic thrombocytopenia purpura.
Thromb Haemost
76:156, 1996[Medline]
[Order article via Infotrieve]
24.
Shivdasani RA, Fielder P, Keller GA, Orkin SH, de Sauvage FJ:
Regulation of the serum concentration of thrombopoietin in thrombocytopenic NF-E2 knockout mice.
Blood
90:1821, 1997[Abstract/Free Full Text]
25.
Nagata Y, Shozaki Y, Nagahisa H, Nagasawa T, Abe T, Todokaro K:
Serum thrombopoietin level is not regulated by transcription but by the total counts of both megakaryocytes and platelets during thrombocytopenia and thrombocytosis.
Thromb Haemost
77:808, 1997[Medline]
[Order article via Infotrieve]
26.
Kumaki S, Armitage R, Ahdieh M, Park L, Cosman D:
Interleukin-15 up-regulates interleukin-2 receptor chain but down-regulates its own high-affinity binding sites on human T and B cells.
Eur J Immunol
26:1235, 1996[Medline]
[Order article via Infotrieve]
27.
Espinoza-Delgado I, Longo DL, Luca-Gusella G, Varesio L:
Regulation of IL-2 receptor subunit genes in human monocytes: Differential effects of IL-2 and IFN-gamma.
J Immunol
149:2961, 1992[Abstract]
28.
Bosco MC, Espinoza-Delgado I, Schwabe M, Luca Gusella G, Longo DL, Sugamura K, Varesio L:
Regulation by interleukin-2 (IL-2) and interferon of IL-2 receptor chain gene expression in human monocytes.
Blood
83:2995, 1994[Abstract/Free Full Text]
29.
Steinman RA, Tweardy DJ:
Granulocyte colony-stimulating factor receptor mRNA upregulation is an immediate early marker of myeloid differentiation and exhibits dysfunctional regulation in leukemic cells.
Blood
83:119, 1994[Abstract/Free Full Text]
30.
Fan K, Ruan G, Sensenbrenner L, Chen BD-M:
Up-regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in murine peritoneal exudate macrophages by both GM-CSF and IL-3.
J Immunol
149:96, 1992[Abstract]
31.
Gliniak BC, Rohrschneider LR:
Expression of the M-CSF receptor is controlled posttranscriptionally by the dominant actions of GM-CSF and multi-CSF.
Cell
63:1073, 1990[Medline]
[Order article via Infotrieve]
32.
Villeval J-L, Cohen-Solal K, Tulliez M, Giraudier S, Guichard J, Burstein S, Cramer E, Vainchenker W, Wendling F:
High thrombopoietin production by hematopoietic cells induces a fatal myeloproliferative syndrome in mice.
Blood
90:4369, 1997[Abstract/Free Full Text]
33.
Drachman JG, Sabath DF, Fox NE, Kaushansky K:
Thrombopoietin signal transduction in purified murine megakaryocytes.
Blood
89:483, 1997[Abstract/Free Full Text]
34.
Chen YQ, Gao X, Timar J, Tang D, Grossi IM, Chelladurai M, Kunicki TJ, Fligiel SE, Taylor JD, Honn KV:
Identification of the alpha IIb beta 3 integrin in murine tumor cells.
J Biol Chem
267:17314, 1992[Abstract/Free Full Text]
35.
Cramer EM, Savidge GF, Vainchenker W, Berndt MC, Pidard D, Caen JP, Masse JM, Breton-Gorius J:
Alpha-granule pool of glycoprotein IIb-IIIa in normal and pathologic platelets and megakaryocytes.
Blood
75:1220, 1990[Abstract/Free Full Text]
36.
Vigon I, Florindo C, Fichelson S, Guenet JL, Mattei MG, Souyri M, Cosman D, Gisselbrecht S:
Characterization of the murine Mpl protooncogene, a member of the hematopoietic cytokine receptor family: Molecular cloning, chromosomal location and evidence for a function in cell growth.
Oncogene
8:2607, 1993[Medline]
[Order article via Infotrieve]
37.
Alexander WS, Dunn AR:
Structure and transcription of the genomic locus encoding murine c-Mpl, a receptor for thrombopoietin.
Oncogene
10:795, 1995[Medline]
[Order article via Infotrieve]
38.
Bartocci A, Mastrogiannis DS, Migliorati G, Stockert RJ, Wolkoff AW, Stanley ER:
Macrophages specifically regulate the concentration of their own growth factor in the circulation.
Proc Natl Acad Sci USA
84:6179, 1987[Abstract/Free Full Text]
39.
Layton JE, Hockman H, Sheridan WP, Morstyn G:
Evidence for a novel in vivo control mechanism of granulopoiesis: Mature cell-related control of a regulatory growth factor.
Blood
74:1303, 1989[Abstract/Free Full Text]
40.
Ericson SG, Gao H, Gericke GH, Lewis LD:
The role of polymorphonuclear neutrophils (PMNs) in clearance of granulocyte colony-stimulating factor (G-CSF) in vivo and in vitro.
Exp Hematol
25:1313, 1997[Medline]
[Order article via Infotrieve]
41.
Khwaja A, Carver J, Jones HM, Paterson D, Linch DC:
Expression and dynamic modulation of the human granulocyte colony-stimulating factor receptor in immature and differentiated myeloid cells.
Br J Haematol
85:254, 1993[Medline]
[Order article via Infotrieve]
42.
Ihle JN:
The Janus protein tyrosine kinases in hematopoietic cytokine signaling.
Semin Immunol
7:247, 1995[Medline]
[Order article via Infotrieve]
43.
Kuter DJ, Rosenberg RD:
Appearance of a megakaryocyte growth-promoting activity, megapoietin, during acute thrombocytopenia in the rabbit.
Blood
84:1464, 1994[Abstract/Free Full Text]
44.
Rouyez MC, Boucheron C, Gisselbrecht S, Dusanter-Fourt I, Porteu F:
Control of thrombopoietin-induced megakaryocytic differentiation by the mitogen-activated protein kinase pathway.
Mol Cell Biol
17:4991, 1997[Abstract]
45.
Matsumura I, Nakajima K, Wakao H, Hattori S, Hashimoto K, Sugahara H, Kato T, Miyazaki H, Hirano T, Kanakura Y:
Involvement of prolonged ras activation in thrombopoietin-induced megakaryocytic differentiation of a human factor-dependent hematopoietic cell line.
Mol Cell Biol
18:4282, 1998[Abstract/Free Full Text]
46.
Goncalves F, Lacout C, Féger F, Cohen-Solal K, Guichard J, Cramer E, Vainchenker W, Duménil D:
Inhibition of erythroid differentiation and induction of megakaryocytic (MK) differentiation by thrombopoietin (TPO) are regulated by two different mechanisms in TPO dependent UT-7/c-mpl and TF1/c-mpl cell lines.
Leukemia
12:1355, 1998[Medline]
[Order article via Infotrieve]
47.
Doubeikovski A, Uzan G, Doubeikovski Z, Prandini MH, Porteu F, Gisselbrecht S, Dusanter-Fourt I:
Thrombopoietin-induced expression of the glycoprotein IIb gene involves the transcription factor PU.1/Spi-1 in UT7-Mpl cells.
J Biol Chem
272:24300, 1997[Abstract/Free Full Text]
48.
Norol F, Vitrat N, Cramer E, Guichard J, Burstein SA, Vainchenker W, Debili N:
Effects of cytokines on platelet production from blood and marrow CD34+ cells.
Blood
91:830, 1998[Abstract/Free Full Text]
49.
Müllberg J, Schooltink H, Stoyan T, Günther M, Graeve L, Buse G, Mackiewicz A, Heinrich PC, Rose-John S:
The soluble interleukin-6 receptor is generated by shedding.
Eur J Immunol
23:473, 1993[Medline]
[Order article via Infotrieve]
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