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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 475-482
Developmental Expression of Plasminogen Activator Inhibitor-1
Associated With Thrombopoietin-Dependent Megakaryocytic Differentiation
By
Seiji Madoiwa,
Norio Komatsu,
Jun Mimuro,
Kouzoh Kimura,
Michio Matsuda, and
Yoichi Sakata
From Division of Hemostasis and Thrombosis, Institute of Hematology
and Division of Hematology, Department of Medicine, Jichi Medical
School, Tochigi-ken, Japan; and Kimura Maternity Hospital,
Tochigi-ken, Japan.
 |
ABSTRACT |
Plasminogen activator inhibitor-1 (PAI-1) is present in the platelet
 -granule and is released on activation. However, there is some
debate as to whether the megakaryocyte and platelet synthesize PAI-1,
take it up from plasma, or both. We examined the expression of PAI-1 in
differentiating megakaryocytic progenitor cells (UT-7) and in
CD34+/CD41 cells from cord blood. UT-7
cells differentiated with thrombopoietin (TPO) resembled megakaryocytes
(UT-7/TPO) with respect to morphology, ploidy, and the expression of
glycoprotein IIb-IIIa. PAI-1 messenger RNA (mRNA) expression was
upregulated and PAI-1 protein synthesized in the UT-7/TPO cells
accumulated in the cytoplasm without being released spontaneously. In
contrast, erythropoietin (EPO)-stimulated UT-7 cells (UT-7/EPO) did not
express PAI-1 mRNA after stimulation with TPO because they do not have
endogenous c-Mpl. After cotransfection with human wild-type
c-mpl, the cells (UT-7/EPO-MPL) responded to phorbol
12-myristate 13-acetate (PMA), tumor necrosis factor- (TNF-), and
interleukin-1 (IL-1) with enhanced PAI-1 mRNA expression within
24 to 48 hours. However, induction of PAI-1 mRNA in UT-7/EPO-MPL cells
by TPO required at least 14-days stimulation. UT-7/EPO cells expressing
c-Mpl changed their morphology and the other characteristics similar to
the UT-7/TPO cells. TPO also differentiated human cord blood
CD34+/CD41 cells to
CD34/CD41+ cells, generated
morphologically mature megakaryocytes, and induced the expression
of PAI-1 mRNA. These results suggest that both PAI-1 mRNA and de novo
PAI-1 protein synthesis is induced after differentiation of immature
progenitor cells into megakaryocytes by TPO.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PLATELETS MAY AFFECT clot lysis by a
variety of mechanisms. Platelets contain factor XIII, which, on
activation, cross-links fibrin monomer and  2-plasmin inhibitor
(2-PI), thereby rendering it more resistant to digestion by
plasmin.1,2 Activated platelets also secrete plasminogen
activator inhibitor-1 (PAI-1) and 2-PI, which inhibit plasmin
formation and activity, respectively.3,4 PAI-1 is a member
of serine protease inhibitor super family, and is the major
physiological inhibitor of fibrinolysis. Platelets are the main
reservoir of PAI-1, with approximately 85% of circulating PAI-1
contained within platelet -granules.5 It has been
reported that platelet PAI-1 exists predominantly in a latent or
inactive form,6,7 suggesting that its effect on
fibrinolysis may be limited. However, Fay et al reported
that PAI-1-deficient platelets inhibited tissue-type plasminogen
activator (t-PA)-mediated clot lysis to a substantially lesser extent
than normal platelets.8 Positive PAI-1 immunostaining of
human platelets and megakaryocytes has also been shown.9
However, PAI-1 messenger RNA (mRNA) was neither detected by Northern
blot analysis of human platelet RNA, nor amplified from
reverse-transcribed human platelet RNA.10 Therefore we
questioned whether a relationship may exist between thrombopoietin
(TPO)-induced megakaryocytic differentiation from progenitor cells and
PAI-1 synthesis.
TPO, the recently isolated and cloned ligand for the cytokine receptor
Mpl,11-15 is a hematopoietic growth factor that regulates platelet production. TPO stimulates the proliferation of megakaryocyte progenitor cells, promotes megakaryocyte terminal differentiation, and
is essential for the production and maintenance of normal levels of
thrombopoiesis.16-19 In the past, the study of
megakaryocytic differentiation was limited because of the rarity of
megakaryocytes in normal bone marrow, the poorly defined cell
population, and inadequate assay methods. The UT-7 cell line was
established from bone marrow cells of a patient with acute
megakaryoblastic leukemia.20 In particular, recently
isolated UT-7/TPO cells have an absolute dependence on TPO and show
mature megakaryocytic features.21 In contrast, the
UT-7/erythropoietin (EPO) cell line shows erythroid development without
TPO dependency due to a lack of endogenous c-Mpl
expression.22 Therefore, comparison of these cell lines will be useful for evaluating the PAI-1 response during megakaryocyte development induced by TPO.
In the present study, we used these UT-7 cell lines and their
transfectants with or without transfection with c-mpl
complementary DNA (cDNA) as a model system and show expression of PAI-1
mRNA and de novo synthesis of PAI-1 protein with TPO-dependent
differentiation. We also confirm these results using CD34+
progenitor cells isolated from human cord blood.
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MATERIALS AND METHODS |
Hematopoietic growth factors and reagents.
Recombinant human TPO was provided by the Kirin Brewery Co, Ltd (Gumma,
Japan). Recombinant human EPO was a gift from the Life Science Research
Institute of Snow Brand Milk Company (Tochigi, Japan). Recombinant
human granulocyte-macrophage colony stimulating factor (GM-CSF) was
provided by Sumitomo Pharmaceutical Company (Osaka, Japan). Human cDNA
clones for full-length c-mpl P (wild type) and
c-mpl K (truncated) were kindly provided by Dr M. Okada (Eisai, Tsukuba, Japan) and Dr S. Gisselbrecht (INSERM, Paris, France), respectively.
Cell culture.
The original UT-7 cell line (UT-7/OR) was established from bone marrow
cells obtained from a patient with acute megakaryocytic leukemia20 and maintained in liquid culture with Iscove's
modified Dulbecco's medium (IMDM; GIBCO Laboratories, Grand Island,
NY) containing 10% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT) and 1 ng/mL of GM-CSF. UT-7/GM was isolated after long-term culture
of UT-7 cells and maintained as described for the UT-7 cells.23 The UT-7/EPO cell line, which is a subclone of
UT-7, was maintained continuously in IMDM containing 10% FCS and 1 U/mL of EPO.22 The UT-7/TPO cell line was maintained in
IMDM containing 10% FCS and 10 ng/mL of TPO.21 Primary
human umbilical cord-derived endothelial cells (HUVEC) were harvested
from human umbilical cord veins treated with 0.1% collagenase as
described elsewhere,24 and grown on fibronectin-precoated
culture plate in Medium-199 (GIBCO Laboratories), containing 15% FCS,
2 mmol/L of glutamine, 15 mmol/L of HEPES, 100 µg/mL of heparin, and
60 µg/mL of endothelial cell growth supplement (Equitech-Bio Inc,
Ingram, TX).
Reverse-transcriptase polymerase chain reactions (RT-PCR) and
Southern blotting analysis.
Total RNA was isolated from cells according to the methods of
Chomczynski and Sacchi.25 RT-PCR was performed using
oligonucleotide primers as follows. The PAI-1 forward
5'-GAACAAGGATGAGATCAGCACC-3' (nucleotides 402-423) and
reverse 5'-ACTATGACAGCTGTGGATGAGG-3' (nucleotides
1151-1172) primers; GP-Ib forward
5-AAGCTGGAGAAGCTCAGTCTGG-3' (nucleotides 535-556) and reverse
5'-CTCCTTAGTGGATTCTTGTGTTGG-3' (nucleotides 1072-1095);
PF-4 forward 5'-GCTGAAGCTGAAGAAGATGGG-3' (nucleotides
98-108) and reverse 5'-TAGCAAATGCACACACGTAGG-3'
(nucleotides 324-344); urokinase-type plasminogen activator (u-PA)
forward 5'-GATCTGATGCTCTTCAGCTGG-3' (nucleotides 389-409)
and reverse 5'-CTGCTCCGGATAGAGATAGTCG-3' (nucleotides
1038-1059); Protease-activated receptor 1 (PAR-1) forward
5'-TGTCTGTGTCAGCAGCATAAGC-3' (nucleotides 1292-1313) and
reverse 5'-CTTGGAATAACACCGTCATCTCG-3' (nucleotides 1688-1710); PAR-3 forward 5'-GGTAACATGTGGACTGGTGTGG-3'
(nucleotides 777-798) and reverse
5'-AATGGAGCTCCTTGCACTATGC-3' (nucleotides 1334-1355);
c-mpl P forward 5'-CAAGGCTTCTTCTACCACAGC-3'
(nucleotides 934-954) and reverse
5'-TCAGTCTCCTGTAGTGTGCAGG-3' (nucleotides 1552-1573);
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward 5'-CCCATGGCAAATTCCATGGCA-3' (nucleotides 215-235) and
reverse 5'-GGTGGACCTGACCTGCCGTCTAGA-3' (nucleotides
759-782). Each first-strand cDNA was synthesized by RT using a
commercial kit (Roche Molecular System, Branchburg, NJ). Total cellular
RNA (1 µg) was RT using oligo-dT16 primers, and amplified
by PCR (94°C for 60 seconds, primer annealing at 56°C for 60 seconds, extension at 72°C for 60 seconds) for 30 cycles in a
Program Temp Control System PC-700 (Astec, Tokyo, Japan), and a final
incubation at 72°C for 10 minutes. Amplification products were
separated on 1.5% agarose gels stained with ethidium bromide and
photographed. The RT-PCR products were transferred to nylon membranes
and incubated with PAI-1 and c-mpl P cDNA labeled with
32P-CTP by the random-priming method. After an overnight
incubation at 65°C in 5× SSPE (150 mmol/mL NaCl,
10 mmol/mL sodium phosphate, pH 7.4, containing 1 mmol/mL EDTA) with
5× Denhardt's solution, 20 µg/mL of nonhomologous salmon sperm
DNA, and 0.5% (wt/vol) sodium dodecyl sulfate (SDS), the blots were
washed three times with 2× SSC (150 mmol/mL NaCl, 15 mmol/mL
sodium citrate, pH 7.0), 0.5× SSC, 0.1× SSC,
plus 0.1% SDS for 15 minutes each. The membranes were autoradiographed
using Kodak XAR-5 film (Eastman Kodak Co, Rochester, NY)
with an intensifying screen at  80°C.
Preparation of c-mpl transfectant.
A c-Mpl expression vector was generated by ligation of full-length
c-mpl cDNA into the pRc cytomegalovirus (CMV) mammalian expression vector. Electroporation was used for stable transfection of
the plasmid into UT-7/EPO cells, as described previously.26 In brief, 20 µg of pRcCMV containing c-mpl cDNA was
introduced into 1 × 107 UT-7/EPO cells resuspended in
0.25 mL of RPMI 1640 medium containing 10% fetal bovine solution (FBS)
by electropulse at 250V, 960 µFD. Transfected cells were seeded at 3 to 5 × 107 cells/mL in IMDM medium
containing 10% FBS and 1 U/mL of EPO and neomycin- (Life Technologies
Inc, Grand Island, NY) resistant clones were selected.
Metabolic labeling and immunoprecipitation.
UT-7/OR, UT-7/TPO cells, and HUVEC (1 × 107 cells/mL)
were metabolically labeled for 15 minutes with 250 µCi/mL
35S-methionine (EXPRE35S35S; Du
Pont Co, Wilmington, DE) in 2.5 mL of methionine-free medium, as
previously described.27 The cells were further incubated in
serum-free medium supplemented with unlabeled methionine, and were
harvested at various time intervals. Culture media was separated from
cell pellets by centrifugation, and stored at 80°C
immediately. The cells were washed with phosphate-buffered saline (PBS)
and lysed on ice in lysis buffer composed of 20 mmol/L Tris-HCl (pH 7.4), 135 mmol/L NaCl, 20% glycerol, 1% NP-40, 1 mmol/L
phenylmethylsulfonylfluoride (PMSF), 15 µg/mL aprotinin, and 2 mmol/L
sodium orthovanadate. The culture media and cell lysates were
precleared with protein A-Sepharose (Pharmacia Biotech), and incubated
at 4°C for 1 hour with shaking with mouse antihuman PAI-1
monoclonal antibodies (MoAb) (JTI-3 and JTI-4) and nonimmune mouse
immunoglobulin G (IgG) (Cappel ICN Pharmaceuticals Inc, Aurora, OH)
bound to protein A-Sepharose beads.28 The
immunoprecipitates were washed to remove unbound proteins and the bound
proteins were eluted from the Sepharose by heating at 100°C for 5 minutes and then subjected to electrophoresis on 10%
SDS-polyacrylamide gels.29 The gels were dried and exposed to radiograph film for autoradiography at 80°C.
Separation of human CD34-positive cells and megakaryocytic colony
formation.
Human cord blood was obtained with informed consent from women who
underwent normal vaginal delivery, and approximately 50 mL of cord
blood was collected. To deplete adherent cells, cord blood cells were
incubated with silica beads (KAC-2, Japan Antibody Institute, Gumma,
Japan) at 37°C for 30 minutes. The cells were separated as the
interface mononuclear FH cells by centrifugation (400g, 30 minutes at 25°C) over Ficoll Hypaque (FH; 1.077 g/cm3;
Pharmacia Biotech, Uppsala, Sweden). The nonadherent mononuclear cells
were adjusted to 5 × 107 cells/mL in PBS without
Ca2+ and Mg2+, and CD34+ progenitor
cells were isolated using a magnetic cell sorting system (Dynal CD34
progenitor cell selection system; Dynal, Oslo, Norway) according to the
manufacturer's instructions. CD34+-enriched cells were
plated at 1 × 106 cells/mL in megakaryocyte culture
medium containing 20% heparinized plasma (from the human cord blood)
in 24-well tissue culture plates (Costar Corp, Cambridge, MA) as
described previously.30-32 They were cultured at 37°C
with 5% CO2 for 12 days.
Flow cytometry.
Cell-surface antigens were detected by immunofluorescence using
fluorescein isothiocyanate (FITC; Becton Dickinson, Mountain View, CA)
conjugated mouse antihuman CD34 and phycoerythrin (PE)-conjugated antihuman CD41 MoAb. In brief, UT-7 cells or isolated cord blood CD34+
progenitor cells were incubated for 30 minutes at 4°C with the
appropriately diluted antibodies. After washing, cell-bound fluorescence on 5,000 to 10,000 cells/sample was determined with a flow
cytometer (FACScan; Becton Dickinson).
| |
RESULTS |
Expression of GPIb-, PF-4, u-PA, and PAI-1 in UT-7 megakaryocytic
cell lines.
We examined the expression of GPIb-, PF-4, and u-PA in the UT-7 cell
lines by means of RT-PCR. The GPIb- transcript was detected in the
original UT-7 cell lines and in UT-7/TPO cells, but was undetectable in
UT-7/EPO or UT-7/GM cells. PF-4, a protein specific to megakaryocytes
and platelets, was detected solely in UT-7/TPO cells, and was entirely
absent in UT-7/OR, UT-7/EPO, or UT-7/GM cells. In contrast, the u-PA
mRNA was scarcely detectable UT-7/TPO cells as well as in platelets
(data not shown). These results and previous studies21
suggest that the UT-7/TPO cells have mature megakaryocyte
characteristics with respect to morphology, ploidy, and the expression
of megakaryocyte-specific proteins. Thus, we examined the effect of
differentiation into megakaryocytes on PAI-1 expression using these
cell lines. As shown in Fig 1, the PAI-1
expression level was detected in UT-7/OR and UT-7/GM cells, with
abundant expression in the UT-7/TPO cells, but the transcript was
scarcely detected in UT-7/EPO cells.
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| Fig 1.
Expression of PAI-1 mRNAs in UT-7 cell lines. Total
cellular RNAs were extracted from UT-7 cell lines (UT-7/OR, UT-7/EPO,
UT-7/GM, and UT-7/TPO) and expressions of PAI-1 mRNA were evaluated by
RT-PCR (see Materials and Methods). The RT-PCR products were resolved
by agarose gel electrophoresis, and the bands were transferred to a
membrane and hybridized to 32P-labeled PAI-1 or -actin
cDNA probes.
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Pulse-chase analysis of PAI-1 protein in UT-7/TPO cells.
We evaluated the synthesis of PAI-1 and its secretion from UT-7/OR,
UT-7/TPO cells, and HUVECs by pulse-chase analysis. The cells were
pulse-labeled with [35S]-methionine for 15 minutes and
were chased with cell lysate and culture medium for various periods. As
shown in Fig 2, PAI-1 protein was not
synthesized in UT-7/OR cells. In contrast, the protein produced for 15 minutes was apparent in the UT-7/TPO cell lysate and did not appear
in the culture medium. Although UT-7/TPO cells express
protease-activated receptor-1 (PAR-1) and PAR-3, a recently identified
thrombin receptor of platelets and megakaryocytes,33 addition of thrombin to the pulse-labeled cells had no effect on the
release of PAI-1 protein (data not shown).
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| Fig 2.
Pulse-chase analysis of PAI-1 in UT-7/OR, UT-7/TPO, and
HUVEC. UT-7/OR, UT-7/TPO, and HUVEC were incubated for 60 minutes in
methionine-free medium and then pulse labeled with
35S-methionine for 15 minutes, followed by a chase with
excess of unlabeled methionine for the indicated period. Cells and
media were harvested at appropriate intervals, and PAI-1 was
immunoprecipitated from each sample with mouse antihuman PAI-1 MoAb
(JTI-3 and JTI-4) or normal mouse IgG. Subsequently, they were
subjected to SDS-polyacrylamide gel electrophoresis (10% separating
gels) and autoradiography.
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The effect of TPO and other ligands on PAI-1 mRNA expression.
Because UT-7/EPO cells are committed to the erythroid
lineage,22 and do not express endogenous c-Mpl, we
introduced full-length c-mpl cDNA into the UT-7/EPO cells (Fig
3A). We selected neomycin-resistant clones
expressing high levels of c-Mpl on the surface of the cells as
determined by flow cytometry with a polyclonal antibody against the
extracellular domain of c-Mpl. These cells were designated as
UT-7/EPO-MPL cells. Using UT-7/OR, UT-7/TPO, UT-7/EPO, and UT-7/EPO-MPL
cells, we examined the effect of tumor necrosis factor (TNF)-,
interleukin (IL)-1, phorbol 12-myristate 13-acetate (PMA), and TPO on the expression of PAI-1 mRNA. TNF-
and IL-1 induced the expression of PAI-1 mRNA within 48 hours in all
cell lines (Fig 4). Incubation with 10 nmol/mL PMA resulted in a more-rapid increase in the PAI-1 mRNA
expression. Although the expressions of GP-Ib and PF-4 mRNA were
also upregulated by PMA for 24 hours, UT-7/EPO-MPL cells did not
obviously change their morphology, except for mild adhesion to the
plastic dishes and extension of pseudopadia (data not shown). In
contrast, TPO did not increase PAI-1 mRNA expression until day 7 (Fig
3B). The effect of TPO on the growth of UT-7/EPO-MPL cells was similar
to that of EPO when incubated for several days. However, over a
long-term culture of 14 to 28 days, the TPO-stimulated UT-7/EPO-MPL
cells gradually enlarged and became nearly identical with UT-7/TPO
cells. In parallel with these morphological changes, PAI-1 transcript
was increased and visible by RT-PCR on day 14.
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| Fig 3.
Effect of TPO on the expression of PAI-1 mRNA in UT-7/EPO
transfected with c-Mpl (UT-7/EPO-MPL). (A) Total cellular RNAs
extracted from UT-7/EPO, UT-7/EPO transfected with c-Mpl
(UT-7/EPO-MPL), and UT-7/TPO were RT and amplified by PCR. The RT-PCR
products were resolved by agarose gel electrophoresis, and the bands
were transferred to a membrane and hybridized to
32P-labeled c-mpl P cDNA probes. (B) UT-7/EPO-MPL
cells were cultured in the presence of 1 U/mL EPO or 10 ng/mL TPO. The
cells were harvested at the indicated time points (0 to 28 days) and
subjected to RT-PCR analysis for PAI-1 and GAPDH mRNA expression. PAI-1
RT-PCR products were analyzed on 2% agarose gels and transferred to
membranes and hybridized to 32P-labeled PAI-1 cDNA
probes.
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| Fig 4.
Effect of TNF-, IL-1, and PMA on the expression of
PAI-1 mRNA in UT-7 cell lines. At the initiation of (A) UT-7/OR, (B)
UT-7/TPO, (C) UT-7/EPO, and (D) UT-7/EPO-MPL culture, any one of 10 ng/mL TNF-, 10 ng/mL IL-1, and 10 nmole/mL PMA was
added to the culture. The cells were harvested at the indicated times
(0, 24, and 48 hours) and examined by RT-PCR followed by Southern blot
analysis. Each value (mean ± SD, n = 3) shows the ratio of PAI-1
expression after 24 and 48 hours of culture versus the respective
control (0 hour) expression.
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Expression of PAI-1 during short-term liquid culture of
CD34+ cord blood cells.
To verify the parallel increase of PAI-1 mRNA expression with apparent
morphological change to a more megakaryocytic-like cell, we examined
the expression of c-mpl P and PAI-1 mRNA transcripts in
the presence of TPO in a short-term liquid culture of cord blood.
CD34+ cells were isolated from normal human cord blood
using a magnetic cell sorting system and cultured for 6 to 12 days in a
megakaryocyte culture medium as previously
described.31,32,34 As shown in the upper panel of Fig 5,
cells with morphologic features of immature megakaryocytes, including
basophilic cytoplasm and budding, began to appear after 3 days of
culture. Mature megakaryocytes-like features with polyploid nuclei were
observed after 6 days. Total cellular RNA was isolated on days 1, 3, and 6, and the expression of c-mpl P and PAI-1 mRNA was
examined by semiquantitative RT-PCR analysis. On day 1, CD34+ cells expressed neither c-mpl P nor
PAI-1 mRNA transcript just after isolation, whereas GAPDH mRNA, which
was used as an internal control, was readily detectable (Fig 5, lower
panel, day 1). The expression of c-mpl P was observed
after 3 days of culture, which is apparently preceded by
polyploidization. In contrast, PAI-1 mRNA transcript was not present on
day 3, but was apparent only after 6 days of culture in parallel with
the appearance of mature megakaryocytes. This chronological order of
PAI-1 expression is consistent with the findings obtained with the UT-7
cell lines.
| |
DISCUSSION |
Platelets can modify fibrinolysis through several mechanisms. Platelet
arterial thrombi are more resistant to lysis by plasminogen activators
than platelet-poor thrombi.35 Several studies suggest that
PAI-1, which inhibits fibrinolysis by binding irreversibly to the
active site of t-PA and u-PA, is a major determinant of the resistance
of platelet-rich clots to lysis by t-PA.36,37 Activation of
platelets results in release of PAI-1 from the -granules. In this
study, we have investigated the mechanism of production of PAI-1 during
the process of megakaryocyte differentiation. Among the UT-7 cell
lines, the UT-7/OR cells have some properties of megakaryocytes,
including polyploidy and positive staining of platelet
peroxidase.20 In contrast, UT-7/EPO cells have progressed further in erythroid development than the parent UT-7
cells,22 and UT-7/GM cells are of the
erythroid-megakaryocytic bipotential lineage, because they have the
capacity to differentiate into erythroid or megakaryocytic lineages by
treatment with EPO and TPO, respectively.23
Morphologically, UT-7/TPO cells have mature megakaryocytic
characteristics, such as a developed demarcation membrane in the
cytoplasm and a multinucleated appearance with a high level of DNA
content.21 In addition, UT-7/TPO cells contain high levels
of GP-Ib, PF4 and PAI-1 mRNA (Fig 1). Therefore, we initially
hypothesized that PAI-1 expression might be closely related to
megakaryocyte differentiation induced by TPO. In contrast, u-PA mRNA
transcript was decreased in the UT-7/TPO cells and not detected in
platelets. Because immature cells and many cell lines originated from
cancer cells are apt to have both PAI-1 and u-PA mRNAs,38,39 this result may also support the
differentiation of UT-7, developed from megakaryoblastic leukemia, into
a more megakaryocytic cell line, UT-7/TPO.
PAI-1 protein was synthesized de novo in UT-7/TPO cells as shown by the
[35S]-methionine-labeled pulse-chase analysis (Fig 2).
Interestingly, unlike HUVECs and other cell lines,40
UT-7/TPO cells did not secrete PAI-1 immediately after synthesis (Fig
2). This may represent a storage pool of PAI-1, although whether or not
it is eventually secreted awaits further experimentation. Furthermore,
PAI-1 was not secreted after thrombin stimulation, although UT-7/TPO
cells express both PAR-1 and PAR-3 protease receptors capable of
binding thrombin (data not shown). This indicates that the signal
transduction system for these receptors probably differ between
megakaryocytes and platelets.
To clarify the mechanism of TPO-dependent induction, we compared the
induction of PAI-1 by other agents such as PMA, IL-1, and TNF-.
Analyses of the mechanism involved in megakaryocytic differentiation
and the expression of megakaryocytic genes were performed with
PMA-induced human megakaryocytic cell line models.41 Hill40 and Konkle42 showed that PMA-induced
PAI-1 mRNA expression and the accumulation of PAI-1 protein in Dami
cells and CHRF-288 cell lines, respectively. However, because PMA is a
chemical agent and not a physiological regulator, its action on
megakaryoblastic cell lines may not always mimic normal
megakaryocytopoiesis. Because c-Mpl specifically regulates
megakaryocytopoiesis and thrombopoiesis through activation by its
ligand TPO,18 we forced c-Mpl expression in UT-7/EPO cells,
which do not express endogenous c-Mpl, and studied the effect of TPO
stimulation on the transfected cells (UT-7/EPO-MPL). UT-7/EPO-MPL cells
not only depended on EPO for growth and survival, but they also
acquired the ability to proliferate and differentiate in the presence
of TPO. PAI-1 mRNA expression was induced by PMA in UT-7/EPO-MPL cells
within 12 to 48 hours (Fig 4). In addition, the proinflammatory
cytokines, IL-1 and TNF-, which are known to enhance PAI-1
production through increased transcription rate, also induced PAI-1
mRNA in UT-7/EPO-MPL for 48 hours without inducing apparent
morphological changes. Furthermore, the comparisons of PAI-1 mRNA
expression between UT-7/OR and UT-7/TPO or UT-7/EPO and UT-7/EPO-MPL
show that upregulations of PAI-1 production by these cytokines are
independent of c-Mpl presentation. In contrast, PAI-1 mRNA transcripts
were detectable in UT-7/EPO-MPL cells only after 14 days stimulation
with TPO, a time when these cells changed morphology to mature
megakaryocytes-like feature (Fig 3). Our data suggest that
megakaryocytes response to TPO through c-Mpl is essential for their
maturation, differentiation, and constitutive expression of PAI-1 mRNA.
However, because PAI-1 message is not detected in platelets but the
protein is,9,10 it is also possible that mRNA turnover
might contribute in part to the regulation of PAI-1 message
level.43 Recently, PU.1/Spi-1, an Ets-related transcription
factor, was found to be selectively induced by TPO and it increased the
transcription activity of megakaryocyte-related gene promoters, whereas
PMA did not.44 Because the PAI-1 gene promoter contains
several Ets-binding core sequences GGAA (GGAA 621, 460,
396, and 61) and a predicted GATA-binding site (GATA
426),45 these elements could be responsible for the
TPO action.
In normal cord blood CD34/CD41+ colonies
induced by TPO, c-mpl mRNA expression was induced before the
detection of PAI-1 mRNA transcript and PAI-1 mRNA expression peaked at
6 days (Fig 5), and reached a plateau on
day 12 (data not shown). Although PAI-1 mRNA appeared faster in these
colonies than in the UT-7/EPO-MPL cells, a difference in the maturation
stage between these two groups may be a contributing factor. Thus, the
expression of PAI-1 was first shown at the transcriptional level both
in leukemic and normal megakaryocytes.
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| Fig 5.
Expression of PAI-1 and c-Mpl mRNA during short-term
culture of normal human cord blood CD34+ progenitor cells
with TPO. The CD34+ cells were isolated from human cord
blood using antihuman CD34 MoAb bound to magnetic microspheres and
cultured in the presence of 10 ng/mL TPO. The cells were harvested at
the indicated time points and subjected to morphologic examination on
Wright-Giemsa staining cytospin slides (upper panel) and RT-PCR
analysis for PAI-1, c-mpl P and GAPDH mRNA expression
(lower panel). Amplified products were analyzed on 2% agarose gels
followed by ethidium bromide staining. M, molecular size marker.
|
|
In summary, we report here that PAI-1 mRNA is expressed in
megakaryocytes and is accompanied by de novo PAI-1 protein synthesis. This endogenous PAI-1 synthesis may be closely related to TPO-dependent megakaryocyte maturation.
| |
ACKNOWLEDGMENT |
We thank Dr Hiroshi Tomizuka for helpful discussion regarding the flow
cytometry analyses of cultured cells.
| |
FOOTNOTES |
Submitted September 1, 1998; accepted March 10, 1999.
Supported in part by a Grant-in-Aid for Scientific Research, No.
09470234 from the Ministry of Education, Science, Sports, and Culture
of Japan, by a grant from Nippon Foundation, and by a grant from Jichi
Medical School Young Investigator Award.
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 correspondence to Yoichi Sakata, MD, PhD, Department of
Hemostasis and Thrombosis, Institute of Hematology, Jichi Medical
School, Minamikawachi-machi, Tochigi-ken, 329-0498, Japan.
| |
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