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Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 1961-1970
Transforming Growth Factor- 1 (TGF-1) Induces Thrombopoietin From
Bone Marrow Stromal Cells, Which Stimulates the Expression of TGF-
Receptor on Megakaryocytes and, in Turn, Renders Them Susceptible to
Suppression by TGF- Itself With High Specificity
By
Sumio Sakamaki,
Yasuo Hirayama,
Takuya Matsunaga,
Hiroyuki Kuroda,
Toshiro Kusakabe,
Takehide Akiyama,
Yuichi Konuma,
Katsunori Sasaki,
Naoki Tsuji,
Tetsuro Okamoto,
Masayoshi Kobune,
Katsuhisa Kogawa,
Junji Kato,
Rishu Takimoto,
Ryuzo Koyama, and
Yoshiro Niitsu
From the 4th Department of Internal Medicine, Sapporo Medical
University School of Medicine, Sapporo, Japan; and the Hokkaido
Prefectural Sapporo Kitano Hospital, Sapporo, Japan.
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ABSTRACT |
The present study was designed to test the concept that platelets
release a humoral factor that plays a regulatory role in megakaryopoiesis. The results showed that, among various
hematoregulatory cytokines examined, transforming growth factor- 1
(TGF-1) was by far the most potent enhancer of mRNA expression of
bone marrow stromal thrombopoietin (TPO), a commitment of lineage
specificity. The TPO, in turn, induced TGF- receptors I and II on
megakaryoblasts at the midmegakaryopoietic stage; at this stage,
TGF-1 was able to arrest the maturation of megakaryocyte
colony-forming units (CFU-Meg). This effect was relatively specific
when compared with its effect on burst-forming unit-erythroid
(BFU-E) or colony-forming unit-granulocyte-macrophage
(CFU-GM). In patients with idiopathic thrombocytopenic
purpura (ITP), the levels of both TGF-1 and stromal TPO mRNA were
correlatively increased and an arrest of megakaryocyte maturation was
observed. These in vivo findings are in accord with the aforementioned
in vitro results. Thus, the results of the present investigation
suggest that TGF-1 is one of the pathophysiological feedback
regulators of megakaryopoiesis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THROMBOPOIETIN (TPO), one of the most
potent stimulators of platelet production, had been shown to be
produced by various organs such as the liver, kidney, spleen, and
lung.1-5 Because the mode of TPO production by these organs
is constitutive, serum levels of TPO had been proposed to be regulated
by adsorption of TPO on the surface of platelets (sponge
theory).6 However, we have recently found that bone marrow
(BM) stromal cells nonconstitutively produced it, relative to the
platelet counts, ie, higher levels of TPO mRNA were found in patients
with idiopathic thrombocytopenic purpura (ITP) than in normal subjects,
and these high levels were normalized upon treatment.7
Therefore, it was speculated that there must be some sensor(s) on
stromal cells to detect the platelet counts in the circulation,
although the mechanism was unknown.
Alternatively, regulation of platelet formation by growth suppressors
acting directly on megakaryopoiesis has also been proposed. Among
these, transforming growth factor-1 (TGF-1) has attracted particular interest as a candidate for a mediator of feedback signal
from the end product of megakaryopoiesis (platelets), because it is
highly concentrated in platelets.8 Several investigators have indeed demonstrated a dose-dependent suppression of megakaryocyte colony-forming units (CFU-Meg) by TGF-1.9,10 However,
the fact that a similar growth inhibition by TGF-1 has reportedly been observed with progenitors of erythroid, myeloid, and primitive hematopoiesis, although the inhibition was relatively intense on
CFU-Meg, raised a question as to the specificity of TGF-1 action on
megakaryopoiesis. Contrarily, some in vivo experiments using murine
models are suggestive of selectivity of TGF-1 on megakaryopoiesis.
Systemic administration of TGF-1 was shown to evoke thrombocytopenia
but not suppress the neutrophil and lymphocyte counts.11 In
TGF-1 gene knockout mice, an excess of megakaryopoiesis with
increased platelet count in circulation was reported.12,13
Taking these facts into account, we postulated that, in vivo, TGF-1,
in addition to its direct inhibitory action on CFU-Meg, specifically
regulates the production of TPO by stromal cells and thereby indirectly
defines its specificity on megakaryocyte lineage.
In the present investigation, therefore, we examined the effect of
TGF-1 on expression of TPO in stromal cells obtained from normal
volunteers as well as its effect on CFU-Meg induced by TPO. We also
elucidated the concentrations of BM TGF-1, levels of stromal TPO
mRNA, and profiles of BM megakaryopoiesis in normal subjects and
patients with ITP.
The results obtained clearly indicated that TGF-1 is indeed a
feedback regulator of megakaryopoiesis in humans.
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MATERIALS AND METHODS |
Patients and normal subjects.
Six patients with ITP admitted to our hospital from 1996 to 1997 were
examined after informed consent was obtained. Five of the patients
underwent steroid therapy. Normal subjects were volunteer doctors in
our department and were also fully informed of the experimental protocol.
Cytokines.
Recombinant human granulocyte colony-stimulating factor (G-CSF), TPO,
erythropoietin (EPO), interleukin-3 (IL-3), and IL-6 were kindly
provided by Kirin Brewery Co, Ltd (Maebashi, Japan). Recombinant human
TGF-1 (rhTGF-1) in active form and recombinant human tumor
necrosis factor- (TNF-) were purchased from Genzyme Corp (Boston, MA).
Treatment of BM stromal cells with TGF-1, TNF, IL-3, IL-6, TPO,
G-CSF, or EPO.
BM stromal cells were prepared according to the previously published
Dexter's method.14-16 One milliliter of BM blood was taken from the iliac bone crest of patients with ITP and normal subjects using a heparinized syringe as described elsewhere. To examine the
modulation of TPO mRNA expression by various hematoregulatory cytokines, BM stromal cells (1 × 106 cells) obtained
from a normal subject (Y.H.) were incubated with various cytokines in
RPMI 1640 medium containing 10% fetal calf serum (FCS) and 10% horse
serum (HS) at 37°C in humidified 5% CO2 for 24 hours
and then collected in RPMI 1640. The concentration range for each
cytokine (TGF-1, 0.1 to 2.0 ng/mL; TNF-, 50 to 500 U/mL; IL-3, 5 to 50 ng/mL; IL-6, 5 to 50 ng/mL; TPO, 10 to 100 ng/mL; EPO, 0.2 to 2 U/mL; and G-CSF, 10 to 100 ng/mL) was determined to cover their upper
limit of pathophysiological concentrations in biological
fluid.17,18 In certain pathological conditions, circulating
TGF-1 often reach concentrations as high as 1.0 ng/mL. TNF- at
500 U/mL has been previously used to stimulate growth factors mRNA
expressions such as granulocyte-macrophage colony-stimulating factor
(GM-CSF), IL-6, and IL-1 in normal human BM stromal
cells.19 In some pathological conditions, the concentration
of IL-3 or IL-6 in biological fluid may reach as high as
50 ng/mL. TPO at 100 ng/mL, 100 ng/mL of G-CSF, and 2 U/mL of EPO have
been routinely used for stimulation of CFU-Meg,20,21
colony-forming unit-granulocyte-macrophage (CFU-GM),22 and burst-forming unit-erythroid
(BFU-E).23
Assay of hematopoietic progenitors (CFU-Meg, CFU-GM, and BFU-E).
CD34+ cells from normal subjects were collected from a
suspension of BM mononuclear cells using immunomagnetic beads according to the method of Debili et al.24 The recovery rate (65.6% ± 3.8%) and purity (93.1% ± 2.9%) of CD34+ cells
were sufficiently high for fluorescence-activated cell sorting (FACS).
CD34+ cells (1 × 103) were cultured in a
35-mm diameter plate (Corning, Corning, NY) in the
presence of rhTGF-1 (0.1 or 1.0 ng/mL) and TPO (1.0, 10.0, or 100 ng/mL), G-CSF (100 ng/mL), or EPO (2 U/mL) in 20% FCS containing 0.8%
methylcellulose following the previous reported method.24 The plates were then incubated at 37°C in an atmosphere of 5% CO2 in air in a humidified incubator. After 14 days, the
cultures were examined microscopically and the number of colonies
formed in the presence of each TPO, G-CSF, or EPO were scored as
CFU-Meg (>4 cells), CFU-GM (>50 cells), or BFU-E (>50 cells),
respectively. To determine at which stage of megakaryopoiesis TGF-1
acts, CD34+ cells from normal subject (Y.H.) were incubated
with 1.0 ng/mL rhTGF-1 in the presence of TPO (100 ng/mL) for 14 days and the number of CFU-Meg colonies were scored at days 0, 2, 4, 6, 8, 10, 12, and 14, respectively. CD34+ cells that were
preincubated with 1.0 ng/mL of rhTGF-1 for 24 hours were also
cultured with 100 ng/mL of TPO and CFU-Meg colony numbers were scored
at day 14.
Preparation of standard mRNAs of TPO, G-CSF, and GAPDH for TaqMan
polymerase chain reaction (PCR).
To quantitate an absolute amount of TPO and G-CSF mRNA in stromal cells
by TaqMan PCR, standard curves for both cytokine mRNA were
prepared.25 Briefly, total mRNA was prepared from stromal cells of Y.H. using RNA Sol B (Tel-Test, Inc, Friendswood, TX) according to the acid guanidium isothiocyanate extraction
method.3,26 The first cDNA strand was synthesized using
total mRNA (1 µg) at 42°C for 30 minutes in buffer containing 100 ng of oligo (dT), 500 mmol/L dNTPs, 10 mmol/L dithiothreitol
(DTT), 200 U of SuperScript II RNase H-reverse
transcriptase (Life Technologies, Inc, Gaithersburg, MD), and 1×
reverse transcriptase buffer with a 20 µL reaction volume. The
mixture was then denatured at 94°C for 3 minutes and cooled on ice.
For amplification of the open reading frame, the following primers were
used for TPO, G-CSF, and GAPDH mRNA, respectively: 5' (102-126 nt), 3' (1138-1163 nt); 5' (32-53 nt), 3' (636-655 nt); and 5' (112-132 nt), 3' (708-744 nt),
respectively.1,27 These oligomers (1 µg) were
phosphorylated at 5'-end by incubation with 10 U of T4
polynucleotide kinase (Takara Shuzo, Kyoto, Japan) at 37°C for 1 hour. PCR amplification was performed with 35 cycles of 94°C for 30 seconds and 68°C for 3 minutes by using Advantage-GC cDNA PCR kit
(Clontech Laboratories, Inc, Palo Alto, CA). The PCR products were
electrophoresed on a 1% agarose gel and a fragment of 1,062 bp (TPO)
or 624 bp (G-CSF) in size was extracted by Geneclean kit (BIO 101, Inc,
Vista, CA). TPO or G-CSF cDNA was subcloned into the EcoRV site
of pPCR-Script Amp SK(+) cloning vector (Stratagene, La Jolla, CA). To
prepare the linear template DNA, the cloning vector containing TPO or
G-CSF cDNA was digested with HindIII. Sense RNA of
TPO or G-CSF was synthesized using T3 RNA polymerase. The linearized
vector (0.5 µg) was incubated at 37°C for 1 hour with 10 U of T3
RNA polymerase (Takara Shuzo), 10 U of placental ribonuclease inhibitor
(Takara Shuzo), 500 mmol/L NTPs, and 10 mmol/L DTT. After incubation,
10 U of RNase-free DNase I was added and incubated at 37°C for 30 minutes to digest out the template DNA. The transcribed RNA was
purified by extraction with phenol/chloroform and concentrated by
ethanol precipitation. The pellet was rinsed with 75% ethanol, dried,
and dissolved in diethyl pyrocarbonate (DEPC)-water. The
concentration of RNA was calculated by absorbance at 260 nm. Serially
diluted RNAs with DEPC-water containing 100 µg/mL of yeast tRNA as a
carrier were subjected to TaqMan reverse transcriptase-PCR (RT-PCR)
followed by the method as described below. The TPO and G-CSF mRNA were
amplified linearly in a range of 4 log dilutions of input molecules.
TaqMan real time quantitative RT-PCR assay for mRNAs of TPO, G-CSF,
and GAPDH.
According to our previous report,7 TaqMan RT-PCR assay was
conducted. In brief, oligonucleotides for TaqMan RT-PCR assay were
labeled with FAM (6-carboxyfluorescein), JOE
(6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein), or TAMRA
(6-carboxytetramethylrodamine). RNA from stromal cells incubated with
or without cytokines as described above was applied for reverse
transcription and amplification using TaqMan EZ RT-PCR kit (Perkin
Elmer, Foster City, CA) according to the manufacturer's protocol. A
master mixture that contained all reagents required for RT-PCR was
prepared to give a final concentration of 1× TaqMan EZ buffer,
0.3 mmol/L dNTPs, 3 mmol/L manganese acetate, 0.01 U/mL AmpErase UNG,
and 0.1 U/mL rTth DNA polymerase. Total RNA extracts (containing
unknown amounts of target TPO, G-CSF, and GAPDH mRNA from BM stromal
cells) were added to the master mixture. This mixture was used to
generate two sets of tubes, set I and set II. To detect the amount of
the TPO, G-CSF, and GAPDH mRNA RT-PCR amplicon, a target hybridization
probe and primers were added to set I and an internal control
hybridization probe and primers were added to set II to give final
probe concentrations of 100 and 200 nmol/L primers. Each mixture was
transferred to a set of thermocycler tubes. The increase in
fluorescence ( Rn) was proportional to the concentration of template
in the PCR. Threshold Rn is calculated by multiplying the standard
deviation of 3 Rn-values (no template controls) by 6.965 according to
the manufacturer's protocol for TaqMan RT-PCR Kit. The PCR cycle
number at the threshold line represents cycle of threshold (CT).
RT-PCR for TGF- receptor I and II mRNA.
BM CD34+ cells of normal subjects were cultured in
-minimum essential medium (-MEM) containing TPO
(100 ng/mL) and 20% plasma. The cells were harvested on days 0, 2, 4, 7, and 14 and mRNA was extracted on each day. An aliquot of 1.0 µg
RNA from each sample was reverse transcribed according to the
above-mentioned method. Using an oligonucleotide primer of TGF-
receptor I (5', 1342-1360 nt; 3', 1672-1690 nt)28 and II (5', 1722-1740 nt; 3', 2009-2027 nt),29 40 cycles of PCR were conducted. The amplified
products were electrophoresed on 5% polyacrylamide gels and stained
with ethidium bromide.
Preparation of platelet lysate.
Platelet pellets were obtained by centrifugation at 800g for 30 minutes from 100 mL of platelet-rich plasma from a healthy volunteer,
followed by 5 cycles of freezing and thawing of the pellet in
phosphate-buffered saline. Acidification of lysate (0.5 mL) was
performed by incubation with 1 mL of 0.23 mol/L HCl.30 After 12 hours, the concentrations of latent and active TGF-1 were
measured and stored at  20°C until use.
Assay for TGF-.
The concentrations of TGF-1 (both active and latent forms) in BM and
peripheral blood (PB) plasma of patients with ITP and normal subjects
and those of platelet lysates prepared as described below were measured
using an enzyme-linked immunosorbent assay (ELISA) kit (Promega Co,
Madison, WI).31 Briefly, 1 mL of BM and PB
samples were diluted with normal saline (3-fold) containing 100 U/mL of
heparin; BM and PB samples (containing 100 U/mL heparin) were then
placed on ice and centrifuged within 1 hour after collection at 2°C
to 8°C for 30 minutes at 1,000g. The supernatants were collected as platelet-poor plasma for the following assay. The total
(active + latent) TGF-1 was assayed after acid activation of the
plasma or platelet lysate by adding 1 µL of 1 N HCl to 50 µL
diluted plasma (1:5 in Dulbecco's phosphate-buffered saline [DPBS]). The reaction solution was mixed and incubated
at room temperature for 15 minutes before it was neutralized by 1 µL
of 1 N of NaOH. It was further diluted to 1:150 in DPBS buffer
(containing K , Na+,
Cl, HPO4,
Ca2+, and Ma2+, pH 7.35) before ELISAs. To
measure the amount of active TGF-1, the acidification procedure was
omitted. Concentration of latent TGF-1 was calculated as a
concentration of total TGF-1 minus active TGF-1. The color change
of the final reaction was measured at a wavelength of 450 nm for the
optimal density, and the standard curve (TGF-1 concentration
v absorbances) was also a linear in a linear-linear scale.
Assay for TPO.
Concentrations of TPO in conditioned media from BM stromal cells
stimulated with TGF-1 or platelet lysates were measured using an
ELISA kit (R&D Systems, Rochester, MN) following the instructions of the provided manufacturer's manual as previously described.7
Measurement of megakaryocyte numbers.
BM aspirates of patients with ITP or normal subjects were
obtained from iliac bone crest during diagnostic procedures. After BM
aspirates were 20-fold diluted with Turk medium, and the megakaryocyte number was counted with Fuchs-Rosenthal calculation glass (Kayagaki, Tokyo, Japan). Although counting megakaryocytes from aspirate may not
be as accurate as counting from biopsy samples, the latter technique
was not used in this particular investigation because it was difficult
to obtain informed consent for biopsy from normal volunteers. The
morphology of megakaryocytes was studied on
May-Grunwald-Giemsa-stained smears by light microscopy. Immature
megakaryocytes with a low cytoplasm-to-nucleus ratio, nongranular
cytoplasm, and a nucleus that appears uniformly stippled were
identified following the method reported by Odell and
Jacson.32
Statistical analysis.
Means and standard errors were assessed using the Mann-Whitney U-tests.
A P value of less than .05 was accepted as statistically significant.
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RESULTS |
Suppression of CFU-Meg by TGF-1 at midmegakaryopoiesis.
The effects of TGF-1 on CFU-Meg from 3 normal subjects were examined
in the presence of TPO. As shown in Fig 1,
CFU-Meg was suppressed by TGF-1 in a dose-dependent manner. TGF-1
also suppressed CFU-GM and BFU-E, but to a lesser extent. For example,
at 0.1 ng/mL of TGF-1, CFU-Meg were suppressed 5 times more than
CFU-GM and twice more than BFU-E. Therefore, suppression by TGF-1
was considered to be relatively specific for CFU-Meg as compared with CFU-GM or BFU-E.
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| Fig 1.
Dose-dependent suppression of CFU-Meg, CFU-GM, and BFU-E
colony formation by TGF-1. CD34+ cells for colony
assays were obtained from 3 different normal volunteers (cases no. 1, 2, and 3). CFU-Meg colony assay from 1 × 103 of
CD34+ cells was performed with TPO at a concentration of
100 ng/mL ( ), 10 ng/mL ( ), or 1.0 ng/mL ( ) in the presence of
0.1 and 1.0 ng/mL of TGF-1. CFU-GM and BFU-E colony assay was
performed with 100 ng/mL of G-CSF and 2 U/mL of EPO, respectively, in
the presence of TGF-1 (0.1 or 1.0 ng/mL). Data are shown as the mean
of triplicate colony number with error bars as SD.
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We next determined if the suppression of CFU-Meg by TGF-1 was
limited to a specific stage of megakaryopoiesis. TGF-1 was added at
various times to CFU-Meg formed in the presence of TPO (Fig 2). CD34+ cells that were
not exposed to TPO stimulation showed no susceptibility to TGF-1.
The effect of TGF-1 was weak at early stages (days 0 to 2), became
apparent between days 4 and 6, and then at later stages returned to low
levels (days 8 to 14). The results indicate that the suppressive effect
of TGF-1 is seen primarily at mid-megakaryopoiesis.
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| Fig 2.
Stage-specific suppression of CFU-Meg by TGF-1.
CFU-Meg colony assay was performed with CD34+ cells
incubated with TGF-1 (1.0 ng/mL) in a delayed addition fashion at
days 0, 2, 4, 6, 8, 10, 12, and 14 ([ ] presence of TPO in culture
medium; [ ] presence of TGF-1 in the culture medium). CFU-Meg
colony assays, without TGF-1 and with TGF-1 preincubation for 24 hours, were also performed.
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Induction of TGF- receptor I and II by TPO at midmegakaryopoietic
stage.
The above-noted observations prompted us to examine the expression of
receptors for TGF- during megakaryopoiesis. The results are shown in
Fig 3. The expression(s) of TGF-
receptors I and II was demonstrated to be evident only on days 2 to 4 and not on earlier or later days. This finding was compatible with the above-noted results showing that the maximal suppression of CFU-Meg by
TGF-1 is observed at the midmegakaryopoietic period.
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| Fig 3.
Serial expression of mRNAs for TGF- receptors I and II
in culture. CD34+ cells were cultured in  -MEM
containing TPO (100 ng/mL). mRNAs for TGF- receptors I and II were
examined by RT-PCR.
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Modulation of TPO expression in BM stromal cells by cytokines.
If TGF-1 is indeed a feedback regulator for megakaryopoiesis, there
should be a certain mechanism(s) that defines strict specificity of
TGF- action on megakaryopoiesis besides its relatively selective
effect on CFU-Meg. We hypothesized that TGF-1, as compared with
other hematoregulatory cytokines, such as IL-3, IL-6, G-CSF, EPO, and
TNF, might preferentially induce the stromal production of TPO, which,
in turn, promotes commitment of stem cells into specific
differentiation of megakaryolineage. Because, at present, there is no
method to obtain large quantities of stromal cells directly from BM, we
used the Dexter culture method that provides stromal cells comparable
to their in vivo antecedents.33-35 Quantification of mRNA
in stromal cells was performed using TaqMan RT-PCR. This method has
been recently proven to be far more sensitive than RNAse protection
assay and more accurate than competitive RT-PCR. The absolute amounts
of mRNA in stromal cells were assessed by using standard curves of
synthesized mRNA (Fig 4A). When normal BM
stromal cells were treated with TGF-1, expression of TPO mRNA showed
an apparent and dose-dependent increment up to 7 times that of
untreated cells (Fig 4B). TNF also showed an increment but to a much
lesser degree, almost one fourth at maximal level than that of
TGF-1-treated stromal cells. TPO itself and IL-6 rather suppressed
stromal TPO mRNA. G-CSF, EPO, and IL-3 showed essentially no effect on
TPO mRNA expression.
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| Fig 4.
(A) Standard curves of mRNAs for TPO (A), G-CSF (B), and
GAPDH (C) in TaqMan RT-PCR analysis. Each standard mRNA preparation,
synthesized using the method as shown in the text, was applied to the
TaqMan RT-PCR system. The actual weight of each mRNA inputted to the
system were plotted versus each cycle of threshold (CT). Absolute
amounts of mRNA in stromal samples was determined by extrapolation to
the x-axis. (B) Analysis of TPO mRNA expression in BM stromal cells
treated with various cytokines. BM stromal cells obtained using Dexter
culture method were cultured in -MEM with various cytokines for 24 hours. One microgram of total cellular RNA was isolated and TaqMan
RT-PCR was performed with oligonucleotide primer for TPO. GAPDH was
used as the internal control. Threshold Rn value and CT were obtained
as described in Materials and Methods. TPO mRNA level and GAPDH mRNA
level can be determined by standard curve of applied RNA weight and CT
value. (C) Analysis of G-CSF mRNA expression in BM stromal cells
treated with TGF-1. BM stromal cells, obtained using the Dexter
culture method, were cultured in -MEM with 0.3, 1.0, and 2.0 ng/mL
of TGF-1 for 24 hours. One microgram of total cellular RNA was
isolated and TaqMan RT-PCR was performed with oligonucleotide primer
for G-CSF. GAPDH was used as the internal control. G-CSF mRNA level and
GAPDH mRNA level can be determined by standard curve of applied RNA
weight and CT values.
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The increment of TPO expression in stromal cells by TGF-1 was
further verified by the measurement of TPO protein. As shown in
Table 1, TPO concentrations in conditioned
medium of stromal cells cultured for 24 hours and 48 hours with
TGF-1 were 37.5 ± 4.6 pg/mL and 162.7 ± 25.4 pg/mL,
respectively. These were significantly higher than those (18.3 ± 2.5 pg/mL and 30.4 ± 3.7 pg/mL) of culture supernatants without
TGF-1.
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Table 1.
Concentrations of TPO Protein in the Supernatant of BM
Stromal Cells Stimulated With TGF-1 or Platelet Lysate
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To rule out the effect of agents other than TGF-1 originating from
platelets, we investigated the effect of anti-TGF- antibody on TPO
production of BM stromal cells by platelet lysates (Table 1). TPO
concentrations in culture supernatant were 36.5 ± 3.1 pg/mL and 147.2 ± 18.4 pg/mL for 24 and 48 hours after incubation, respectively. This increment was blocked when the lysate was incubated with 100 µg/mL of anti-TGF-1 polyclonal antibody (Selotec,
Oxford, UK) but not with an irrelevant mouse Ig G1. These results
clearly showed that TGF-1, and no other factors originating from
platelets, stimulates BM stromal TPO production.
We further studied the effect of TGF-1 on stromal mRNA of 2 other
essential hematopoietic factors: G-CSF and EPO. As shown in Fig 4C, the
levels of G-CSF mRNA in TGF-1-treated cells did show an increment,
but only twice those of nontreated cells when treated at the same
concentrations used for stimulation of TPO mRNA. EPO mRNA was not
detectable in the stromal cell preparation; therefore, no effect due to
TGF-1 treatment was observed. Taken collectively, TGF-1 was found
to be a potent and considerably specific upregulator of TPO mRNA in
stromal cells.
Increased expression levels of TPO mRNA in stromal cells and TGF-
concentrations in BM from the patients with ITP.
The pathophysiological significance of the previously stated in vitro
findings were validated by examining the levels of BM TGF-1 and
stromal TPO mRNA expression in normal subjects and patients with ITP.
Quantification of the stromal TPO mRNA was similarly performed using
TaqMan RT-PCR. In Table 2 are shown the
mean concentrations of active TGF-1 together with those of latent
form in BM and PB of normal subjects and patients with ITP. Both active
and latent values in ITP were higher (P < .05) than those in
normal subjects and returned to near-normal values after steroid
therapy. As shown in Fig 5, in each case
without exception, the level of active and latent TGF-1 in BM was
higher than that in PB. There was a strong positive correlation between values of TGF-1 in BM and those in PB of these patients (active TGF-1, r = .82; latent TGF-1, r = .85). The
inclination is steeper in latent TGF-1 (y = 2.05x 0.34) than in active TGF-1 (y = 1.23x + 0.032). When the data of these results were plotted
(Fig 6), a positive correlation between
these concentrations of active TGF-1 and levels of TPO mRNA was
observed.
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Table 2.
Laboratory Findings on Megakaryopoiesis of Normal
Subjects and the Patients With ITP Before and After PSL Treatment
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| Fig 5.
Relationship between BM and PB TGF- concentrations.
The concentrations of TGF-1 (both active and latent forms) in BM and
PB plasma were measure by ELISA. () Normal subject; ( ) patient
with ITP; ( ) patient with ITP after PSL treatment. A positive
correlation was observed in both active and latent TGF-1 (r
= .82 and .85, respectively). In each subject, the TGF-1
concentrations in BM were consistently higher than those in the PB. (A)
Active TGF-1; (B) latent TGF-1.
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| Fig 6.
Relationship between the concentration of BM TGF-1 and
TPO mRNA expression in BM stromal cells. () Normal subject; ()
patient with ITP; () patient with ITP after PSL treatment. Positive
correlation was observed (r = .72).
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Maturation arrest of megakaryocytes in BM of ITP patients.
BM trephines of ITP patients showed a higher number of megakaryocytes
than normal subjects with hypolobulation (maturation arrest) that
tended to normalize as the TGF-1 concentration returned to normal in
those who underwent steroid therapy (Table 2), consistent with the in
vitro finding that TGF--treated CFU-Meg is impaired at
middifferentiation stages but not at earlier stages
(Fig 7).
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| Fig 7.
Regulation of megakaryopoiesis. TPO mRNA expression
in BM stromal cells is enhanced by TGF-1, which suppresses
the TPO-promoted CFU-Meg with relative specificity at
midmegakaryopoiesis by interacting with receptors I and II, which were
induced by TPO itself in normal subjects (A). Increased
TGF-1 from destroyed platelets or megakaryocytes markedly
upregulates TPO mRNA expression in stromal cells. The subsequent
production of TPO stimulates stem cells to commit to the megakaryocyte
lineage. The expression of the TGF- receptor on megakaryoblasts
renders them susceptible to suppression by TGF-1 in patients with
ITP (B).
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DISCUSSION |
Inhibition of CFU-Meg by TGF- has been previously shown by several
investigators. However, in their investigations, relatively nonspecific
stimulators for CFU-Meg, such as combined preparations of plasma of
aplastic anemia, phytohemagglutinin (PHA)-stimulated lymphocyte conditioned medium, IL-3, or IL-6, were used because of the
unavailability of TPO; therefore, the inhibitory effects were not
discussed in reference to genuine TPO activity.9,10
In the present study, we first demonstrated that TGF- did strongly
suppress TPO-induced CFU-Meg in a dose-dependent manner. The magnitude
of suppression on CFU-Meg was greater than on G-CSF-induced CFU-GM or
on EPO-induced BFU-E. Interestingly, the suppression was rather
stage-specific (middifferentiation), with no effect on primitive stem
cells, resulting in a maturational arrest of megakaryopoiesis. This
observation is consistent with previous reports by Roberts and
Sporn,36 who found a lack of responsiveness to TGF- in
matured megakaryocyte, and Kuter et al,37 who found the
inhibition by TGF- on endomitosis of megakaryocytes. In support of a
stage-specific suppression of megakaryopoiesis, we also found that TPO
induced the expression of TGF- receptors on megakaryoblasts at
midmegakaryopoiesis (Fig 3).
However, to establish the concept of feedback regulation, it may be
required to prove further specificity of TGF- action on
megakaryopoiesis. In this context, because we have recently disclosed
the fact that BM stromal TPO is a key regulator of megakaryopoiesis, we
hypothesized that TGF- may enhance expression of stromal TPO, which
specifically induced proliferation and differentiation of megakaryocytes. To analyze the effect of TGF- on TPO mRNA expression of BM stromal cells, we used TaqMan real time RT-PCR, because the
expression level of TPO mRNA in BM stromal cells was far lower than the
detection limit of RNAse protection assay. TGF- was found to
apparently stimulate stromal TPO mRNA expression, whereas other
hemato-regulatory cytokines were shown to either suppress it (TPO and
IL-6), slightly enhance it (TNF), or have no effect (G-CSF, EPO, and
IL-3). Furthermore, when the effect of TGF-1 on the other major
hematopoietic cytokine, G-CSF, which is produced by stromal cells and
defines the myeloid lineage specificity, was compared with that on TPO,
the latter was stronger by far. Because another lineage-defining
cytokine, EPO, was not produced by stromal cells, the effect of
TGF-1 on this particular cytokine was not examined in the present
study. Thus, the specificity of TGF-1 action on
megakaryopoiesis is considered to be doubly assured at 2 steps: (1) an
induction of stromal TPO and (2) at suppression of CFU-Meg. TGF-1
selectively enhances the stromal TPO, which commits the stem cell
specifically to the megakaryocyte lineage and, in turn, suppresses
megakaryopoiesis at the middifferentiation period. We then verified
these in vitro experimental results by in vivo comparison of the
concentrations of BM TGF-1, expression of stromal TPO mRNA, and BM
profiles of megakaryopoiesis between patients with ITP and normal
subjects. We focused our interest on ITP because, in this pathological
state, TGF-1 is speculated to be released from megakaryocytes or
platelets that have been degradated by autoantibodies, and potency of
megakaryopoiesis itself is not deteriorated. In patients with ITP, both
active and latent TGF-1 were higher (P < .05) than those
in normal subjects and returned to near-normal values after steroid
therapy. The levels of active and latent TGF-1 in BM were higher
than those in PB in each case without exception. This indicates that
TGF-1 in BM were mainly derived from cells in BM, most likely
megakaryocytes and platelets.38-41 A strong
positive correlation between values of TGF-1 in BM and PB was
observed; however, the inclination was steeper in latent TGF-1 than
in active TGF-1. This may be explained by assuming the faster
clearance of active TGF-1, which interacts with TGF-1 receptors
expressed on megakaryocytes, than of latent TGF-1, which normally
exist as a complex with 2-macroglobulin (receptor-inaccessible form)
in plasma. These results clearly suggest that active
TGF-1 is one of the key factors defining the TPO production by
stromal cells. Nevertheless, the above-mentioned in vitro findings were
compatible with the subsequently made in vivo observations that
megakaryocytes in ITP patients were increased but hypo-lobulated
(immature), had a profile of maturation arrest without
any particular change in other hematopoietic lineages, and tended to
become normal after steroid therapy. This unique BM feature is in
agreement with that previously documented for ITP.42,43 In
addition to ITP, there are some pathological conditions in which the
relationship between levels of circulation TPO and platelet counts is
unexplainable by the sponge theory. For example, in busulfan-treated
mice, plasma TPO after platelet transfusion are elevated to such a high
level as to be unaccountable for by the sponge theory.6
However, this may be explained by assuming that transfused platelets
supplied sufficient TGF- to the stromal cells to produce TPO.
Another example is found with NF-E2/ mice in
which normal serum thrombopoietin levels are seen in the face of
profound thrombocytopenia.44 It may be that stromal cells
had not been exposed to TGF- due to innate platelet insufficiency. Measurement of TGF- levels in these mice will confirm the
above-noted assumption. Another pathological condition that indicates a
relationship between TGF- and megakaryopoiesis may be essential
thrombocythemia. We have recently found that, in this particular
disease, there is a mutation as kinase domain of type II receptor for
TGF- (data not shown). Elucidation as to whether this mutation has
pathognomonic meaning is warranted.
As for the physiological test of the animal model, Carlino et
al11 have already reported that, when TGF- (0.1%
wt/vol) was administered daily subcutaneously from day 0 to day 13 to C3H/He mice, a highly significant decrease was seen in
platelet count in circulation, and the megakaryocyte count increased
inversely. With regard to the gene knock-out effect, Shull et
al12 have already observed that, in TGF-
(/) mice, there was an increase of platelets in the
circulation as well as of neutrophils and monocytes. However, this
phenomenon may not be solely ascribed to the direct effect of TGF-1
deletion on hematopoiesis but possibly to the hyperimmunoreactive state
caused by TGF-1 depletion. In this context, Letterio et
al,13 using TGF- (/) major
histocompatibility complex (MHC) class II
(/) double knockout mice, demonstrated an excess of
megakaryopoiesis accompanied by a slight increase of granulocyte count.
These previous observations in TGF--administered or TGF-
knockout mice strongly support our model of megakaryopoiesis regulated by TGF- and TPO.
In conclusion, we propose the concept of feedback regulation on
megakaryopoiesis by TGF-1 released from platelets or megakaryocytes; TGF-1 from destroyed platelets or megakaryocytes stimulates TPO synthesis in stromal cells and, in turn, stimulates stem cells (CD34+ cells) to commit to the megakaryocyte lineage and to
express TGF-1 receptors that render them susceptible to suppression
by TGF-1 (Fig 7).
| |
ACKNOWLEDGMENT |
The authors thank Dr Susan Feldman for her editorial assistance; Dr
Irving Listowsky for helpful discussion and critical reading of our
manuscript; Dr Kohei Miyazono for helpful discussion; and Kevin Litton
for critical reading of our manuscript.
| |
FOOTNOTES |
Submitted December 8, 1998; accepted May 17, 1999.
Supported in part by a Grant-in-Aid for Scientific Research from the
Ministry of Education, Science and Culture of Japan.
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 Yoshiro Niitsu, MD, PhD, Chief and
Professor, 4th Department of Internal Medicine, Sapporo Medical
University School of Medicine, South-1, West-16, Chuo-ku, Sapporo,
060-8543, Japan; e-mail: niitsu{at}sapmed.ac.jp.
| |
REFERENCES |
1.
Lok S, Kaushansky K, Holly RD, Kuijper JL, Foster DC:
Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo.
Nature
369:565, 1994[Medline]
[Order article via Infotrieve]
2.
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]
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.
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.
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, Elliott G, Izumi R, Covey T, Crouse J, Garcia A, Xu W, Castillo J Del, Biron J, Cole S, Hu MCT, 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]
6.
Kuter D, 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]
7.
Hirayama Y, Sakamaki S, Matsunaga 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]
8.
Asssoian RK, Komoriya A, Meyers CA, Miller DM, Sporn MB:
Transforming growth factor in human platelets: Identification of a major storage site, purification, and characterization.
J Biol Chem
258:7155, 1983[Abstract/Free Full Text]
9.
Mitjavilla MT, Villeval JL, Kieffer N, Henri N, Testa U, Breton-Gorious J, Veinchenker W:
Human platelet alpha granules contain a nonspecific inhibitor of megakaryocyte colony formulation: Its relationship to type transforming growth factor (TGF-).
J Cell Physiol
134:93, 1988[Medline]
[Order article via Infotrieve]
10.
Ishibashi T, Miller SL, Burstein SA:
Type transforming growth factor is a potent inhibitor of murine megakaryocytopoiesis in vitro.
Blood
69:1737, 1987[Abstract/Free Full Text]
11.
Carlino JA, Higley HR, Creson JR, Avis PD, Ogawa Y, Ellinsworth LR:
Transforming growth factor 1 systemically modulates granuloid, erythroid, lymphoid, and thrombocytic cells in mice.
Exp Hematol
20:943, 1992[Medline]
[Order article via Infotrieve]
12.
Schull MM, Ormsby I, Kier AB, Pawlowski S, Diebold RJ, Yin M, Allen R, Sidman C, Proetzel B, Calvin D:
Targetted disruption of the mouse transforming growth factor-1 gene results in multifocal inflammatory disease.
Nature
359:693, 1992[Medline]
[Order article via Infotrieve]
13.
Letterio JJ, Geisei AG, Kulkarni AB, Dang H, Kong L, Nakabayashi T, Mackall CL, Gress RE, Roberts AB:
Autoimmunity associated with TGF-1-deficiency in mice is dependent on MHC class II antigen expression.
J Clin Invest
98:2109, 1996[Medline]
[Order article via Infotrieve]
14.
Dexter TM, Allen TD, Lajtha LG:
Conditions controlling the proliferation of hematopoietic stem cells in vitro.
J Cell Physiol
91:335, 1979
15.
Hirayama Y, Kohgo Y, Sakamaki S, Matsunaga T, Ohi S, Niitsu Y:
Cytokine mRNA expression of bone marrow stromal cells from patients with aplastic anemia and myelodysplastic syndrome.
Br J Haematol
85:676, 1993[Medline]
[Order article via Infotrieve]
16.
Kohgo Y, Hirayama Y, Sakamaki S, Matsunaga T, Ohi S, Niitsu Y:
Chronic idiopathic neutropenia associated with abnormal expression of granulocyte-colony stimulating factor mRNA of bone marrow stromal cells.
Int J Hematol
59:177, 1994[Medline]
[Order article via Infotrieve]
17.
Tahara T, Usuki K, Sato H:
A sensitive sandwich ELISA for measuring thrombopoietin in human serum: Serum thrombopoietin levels in healthy volunteers and in patients with haemopoietic disorders.
Br J Haematol
93:783, 1996[Medline]
[Order article via Infotrieve]
18.
Kosugi S, Kurata Y, Tomiyama Y, Tahara T, Kato T, Tadokoro S, Shiraga M, Honda S, Kanakura Y, Matsuzawa Y:
Circulating thrombopoietin level in chronic immune thrombocytopenic purpura.
Br J Haematol
93:704, 1996[Medline]
[Order article via Infotrieve]
19.
Rennick D, Yang G, Gemmell L, Lee F:
Control of hemopoiesis by a bone marrow stromal cell clone: Lipopolysaccharide-and Interleukin-1-inducible production of colony-stimulating factors.
Blood
69:682, 1987[Abstract/Free Full Text]
20.
Farese AM, Hunt P, Boone T, MacVittie TJ:
Recombinant human megakaryocyte growth and development factor stimulates thrombocytopoiesis in normal nonhuman primates.
Blood
86:54, 1995[Abstract/Free Full Text]
21.
Bruno E, Hoffman R:
Effect of interleukin 6 on in vitro human megakaryopoiesis: Its interaction with other cytokines.
Exp Hematol
17:1038, 1989[Medline]
[Order article via Infotrieve]
22.
Quesenberry PJ, Ihle JN, McGrath E:
The effect of interleukin-3 and GM-CSA-2 on megakaryocyte and myeloid clonal colony formation.
Blood
65:214, 1985[Abstract/Free Full Text]
23.
Ogawa M, Parnley TR, Bank LH, Spicer SS:
Human marrow erythropoiesis in culture I. Characterization of methylcellulose colony assay.
Blood
48:407, 1976[Abstract/Free Full Text]
24.
Debili N, Wendling AK, Guichard J, Gorius JB, Hunt P, Vainchenkerm 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, 1996[Abstract/Free Full Text]
25.
Gibson UEM, Heid CA, Williams PM:
A novel method for real time quantitative RT-PCR.
Genome Res
6:995, 1996[Abstract/Free Full Text]
26.
Gough NM:
Rapid and quantitative preparation of cytoplasmic RNA from small numbers of cells.
Anal Biochem
173:93, 1988[Medline]
[Order article via Infotrieve]
27.
Nagata S, Tsuchiya M, Asano S, Yamamoto O, Hirata Y, Kubota N, Oheda M, Nomura H, Yamazaki T:
The chromosomal gene structure and two mRNAs for human granulocyte colony-stimulating factor.
EMBO J
5:575, 1986[Medline]
[Order article via Infotrieve]
28.
Franzen P, Djike PT, Ichijo H, Yamasita H, Miyazono K:
Cloning of a TGF- type I receptor that forms a heteromeric complex with the TGF- type II receptor.
Cell
75:681, 1993[Medline]
[Order article via Infotrieve]
29.
Lin HY, Wang XF, Ng-Eaton E, Weinberg RA, Lodish HF:
Expression cloning of the TGF- type II receptor, a functional transmembrane serine/threonine kinase.
Cell
68:775, 1992[Medline]
[Order article via Infotrieve]
30.
Terui T, Niitsu Y, Mahara K, Fujisaki Y, Urusizaki Y, Mogi Y, Kohgo Y, Watanabe N, Oguma M, Saito H:
The production of transforming growth factor- in acute megakaryoblastic leukemia and its possible implications in myelofibrosis.
Blood
75:1540, 1990[Abstract/Free Full Text]
31.
Wang XL, Liu SX, Wilken DEL:
Circulating transforming growth factor- and coronary artery disease.
Cardiovasc Res
34:404, 1997[Abstract/Free Full Text]
32.
Odell TT, Jacson CW:
Polyploidy and maturation of rat megakaryocytes.
Blood
32:102, 1968[Abstract/Free Full Text]
33.
Marsh JCW, Chung J, Testa NG, Hows JM, Dexter TM:
In vitro assessment of bone marrow `stem cell' and stromal cell function in aplastic anemia.
Br J Haematol
78:258, 1991[Medline]
[Order article via Infotrieve]
34.
Kojima S, Matsunaga T, Kodera Y:
Hematopoietic growth factor released by marrow stromal cells from patients with aplastic anemia.
Blood
79:256, 1992
35.
Tisdale JF, Stewart AK, Dickstein B, Little RF, Dube I, Cappe D, Dunbar CE, Brown KE:
Molecular and serological examination of the relationship of human herpesvirus 8 to multiple myeloma.
Blood
92:2681, 1998[Abstract/Free Full Text]
36.
Roberts AB, Sporn MB:
The transforming growth factor-, in
Sporn MB,
Roberts AB
(eds):
The Handbook of Experimental Pharmacology, vol 95. Heidelberg, Germany, Springer-Verlag, 1990, p 419.
37.
Kuter DJ, Gminski DM, Rosenberg RD:
Transforming growth factor inhibits megakaryocyte growth and endomitosis.
Blood
79:619, 1992[Abstract/Free Full Text]
38.
Childs CB, Proper JA, Tucker RF, Moses HL:
Serum contains a platelet-derived transforming growth factor.
Proc Natl Acad Sci USA
79:5312, 1982[Abstract/Free Full Text]
39.
Associan RK, Komoriya A, Meyers CA, Miller DM, Sporn MB:
Transforming growth factor-beta in human platelets. Identification of a major storage site, purification, and characterization.
J Biol Chem
258:7155, 1983
40.
Fava RA, Casey TT, Wilcox J, Pelton RW, Moses HL, Nanney LB:
synthesis of transforming growth factor-beta 1 by megakaryocytes and its localization to megakaryocyte and platelet alpha-granules.
Blood
76:1946, 1990[Abstract/Free Full Text]
41.
Roberts AB, Anzano MA, Meyers CA, Wideman J, Blacher R, Pan YE, Stein S, Lehrman SR, Smith JM, Lamb LL, Sporn MB:
Purification and properties of a type transforming growth factor from bovine kidney.
Biochemistry
22:5692, 1983[Medline]
[Order article via Infotrieve]
42.
Pistiotta AV, Stefanini M, Dameshek W:
Studies on platelet X. Morphologic characteristics of megakaryocytes by phase contrast microscopy in normals and in patients with idiopathic thrombocytopenic purpura.
Blood
8:703, 1953[Abstract/Free Full Text]
43.
Queissr U, Queiser W, Spiertz B:
Polyploidization of megakaryocytes in normal humans, in patients with idiopathic thrombocytopenic purpura.
Br J Haematol
20:489, 1971[Medline]
[Order article via Infotrieve]
44.
Shivdasani RA, Rosenblatt MF, Zucker D, Kacson CW, Hint P, Saris M, 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]
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ABSTRACT
INTRODUCTION