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Thrombopoietin Stimulates Colony-Forming Unit-Megakaryocyte
Proliferation and Megakaryocyte Maturation Independently of Cytokines
That Signal Through the gp130 Receptor Subunit
By Virginia C. Broudy, Nancy L. Lin, Norma Fox, Tetsuya Taga, Mikiyoshi Saito, and Kenneth Kaushansky
Thrombopoietin (Tpo), the ligand for thec-Mpl receptor, is
a major regulator of megakaryopoiesis. Treatment of mice
with Tpo raises the platelet count fourfold
within a few days.
Conversely, c-mpl knock-outmice have platelet counts that
are 15% that of normal. The subunit structure of the c-Mpl
receptor is not fully
understood. Some cytokines that stimulate megakaryopoiesis (IL-6, IL-11, leukemia inhibitory factor, and oncostatin M) bind t o receptors that use gp130 as
a signal transduction subunit. For these reasons, we determined whethergp130 function wasrequired for Tpo-induced
signal transduction. Murine marrow cells were cultured in
semi-solid media in the presence of Tpo or IL-3, with or
without a neutralizing anti-gpl30 monoclonalantibody
(RX187) or a soluble form of c-Mpl receptor (soluble Mpl)
that blocks Tpo bioactivity, and thenumbers of colony-forming unit-megakaryocyte (CFU-Meg) colonies were counted
on day 5. Murine marrowcells were also cultured in suspen-
sion under serum-free conditions for5 days, and megakaryocyte DNA content was measured by flow cytometry, as an
index of nuclear maturation. The addition of RX187 did not
block Tpo-induced CFU-Meg colony growth nor CFU-Meg
nuclear maturation in suspension culture. However, IL-3induced CFU-Meg colony growth and megakaryocyte nuclear maturation decreased in the presence of RX187. Soluble Mpl completely ablated Tpo-induced CFU-Meg growth,
andpartiallyblockedIL-3-stimulated
CFU-Meg growth.
Thus the effects of Tpo on megakaryopoiesis in vitro do not
depend on cytokines that signal through gp130. Furthermore, it is unlikely that gp130 serves as a beta chain for
the c-Mpl receptor, as Tpo signalling is unimpaired in the
presence of RX187. In contrast, the effects of IL-3 on CFUMeg growthare mediated in part throughTpo and through
gp130-signalling cytokines.
0 1996 b y The American Society of Hematology.
P
then initiation of signal transduction. Thus the gp130receptor subunit, which is ubiquitously expressed in vivo,'5 plays
a critical role in the transmission of signals of the IL-6family
of cytokines across the cell membrane.
Thrombopoietin, also known as Mpl ligand, megapoietin.
or megakaryocyte growth and development factor, is a key
regulator of megakaryopoiesis.'" In vitro, Tpo synergizes
with erythropoietin, SCF, and IL-I 1 to promote growth of
CFU-Meg colonies, and is a potent stimulant of megakaryocyte nuclear endoredupli~ation.~"'~~'~
Treatment of mice with
Tpo increases the platelet count fourfold," while mice engineered to eliminate c-Mpl receptor expression exhibit platelet counts that are 15% that of normal levels.I9 Tpo is likely
to be used clinically to speed platelet recovery after myelosuppressive chemotherapy, so an understanding of its mechanism of action is important.
Thec-Mpl receptorisamember
of the hematopoietic
growth factor
receptor
as
are
receptors
the
for the IL-6 family of cytokines. Members of this receptor
superfamily share structuralfeaturesincludingconserved
cysteine residues and a characteristic WSXWS motif in the
extracellular domain, and an absence of recognizable kinase
domains in the intracellular portion of the receptor. Certain
of the receptors in the superfamily homodimerize after ligand
binding: others require association of a signal transducing P
chain, which may be shared by several members of the family. The subunit structure of the functional c-Mpl receptor
complex has not been fully defined.
We investigated whether signal transduction via thegp 130
receptor subunit wasrequired forCFU-Meg proliferation
and megakaryocyte nuclear maturation in response
to Tpo
or to IL-3. Our results demonstrate that the effects of Tpo
on megakaryopoiesis in vitro do not requiregp130. Conversely,theeffects
of IL-3 on CFU-Meg proliferation irz
r'itro are dependent in part on Tpo andoncytokines
that
signal through the gp 130 receptor subunit.
LATELET PRODUCTION is anorderly
process in
which committed megakaryocyte progenitor cells (colony-forming unit-megakaryocyte [CFU-Meg]) proliferate
and differentiate into immature megakaryocytes,which then
undergo cytoplasmic maturation and nuclear endoreduplication to generate mature,polyploid megakaryocytes. The mature megakaryocytes release platelets.',' These processes are
governed by a flotillaof cytokines. Someof these hematopoieticgrowthfactors
(including IL-3 and stemcell factor
[SCF]) predominantly affect CFU-Meg proliferation, while
others (including IL-6, IL- 1 l , leukemia inhibitoryfactor
[LIF], and oncostatin M) mainlyinfluence megakaryocyte
maturation.'.' Thrombopoietin
enhances
both CFU-Meg
growth and megakaryocyte
Interleukin-6, IL- 1 1, LIF, oncostatin M, and ciliary neurotropic factor have also been called neuropoietic cytokines,' I
because of their diverse biological effects on hematopoiesis,
B lymphocyte differentiation, induction of acute phase proteins, and survival and differentiation of neurons. The neuropoietic cytokines. together with cardiotrophin-I , l 2 use gpl30
asa component of the cell surface receptor complex.".'4
Ligand binding triggers association of gp 130, and formation
of gp 130 homodimers or gp 130-LIFreceptor heterodimers,
From the University of Washington. Seattle. WA; and Osaka University, Osaka, Japan.
Submitted December 21. 1995; accepted May 17, 1996.
Supported by National Institutes rgHealtlz Grunts No.DK 44194,
CA 31615, and DK 49855 and an American Cancer Society Faculo
Research Award to Virginia C. Broudy.
Address reprint requests to Virginiu C. Broudy, MD, University
of Washington, Division of Hematology. Bo.x 357710, Seattle, WA
98195-7710.
The publication costs of this article were defrayed in part by page
churge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 17.34 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8806-0031$3.00/0
2026
MATERIALSANDMETHODS
Megakaryocyte colony assays. BhD2FI mice (JacksonLaboratories, Bar Harbor. ME), 8 to 10 weeks old, were killed by rapid
Blood, Vol 88, No 6 (September 15). 1996:pp 2026-2032
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TPOACTSINDEPENDENTLYOF
2027
~ ~ 1 3 0
Table 1. Effect of Blocking gp130 Activity on CFU-Meg
cervical dislocation, andmarrow cells were obtained by flushing
Colony Growth
the femurs with Iscove’s Modified Dulbecco’s Medium (IMDM,
GIBCO, Grand Island, NY) supplemented with 2% fetal calf serum
CFU-Meg Colonies/
Experiment
(FCS; Hyclone, Logan, UT). The marrow cells were cultured in
2 x 10’ Cells
Addition
No.
IMDM supplemented with 15% horse serum (Hyclone) and 5 X
0
1
0
IO-’ molar p mercaptoethanol (Sigma Chemical CO,St Louis, MO)
35.0 2 4.0
TPO
in 0.285% agar (DIFCO, Detroit, MI) in triplicate plates as pre37.7 2 2.4
Tpo + RX187
viously described.8 Megakaryocyte colonies were counted after a 5Tpo + Sol Mpl
0
day incubation at 37°C in a humidified atmosphere containing 5%
IL-3
22.7 2 0.3
COz. The growth factors used included recombinant murine Tpo
11-3 + RX187
12.7 2 0.3
and recombinant murine IL-3, both expressed in BHK cell^.'^^^^ Fifty
10.0 2 1.5
IL-3 + Sol Mpl
units of Tpo or IL-3 activity were defined as that quantity that gave
0
2
0
one-half maximal stimulation in the BaF3Mpl proliferation assay,
41.0 2 2.2
TPO
or in the murine marrow CFU-GM colony assay, respectively.” In
41.1 2 4.3
Tpo + RX187
some experiments, recombinant human IL-l1 (Genzyme, Boston,
Tpo + Sol Mpl
0
MA) was also used. A soluble form of c-Mpl receptor (soluble Mpl)
IL-3
33.0 2 1.4
was generated by truncation at the interface of the extracellular and
IL-3 + RX187
18.0 t 1.2
and
transmembrane domains, was purified as previously de~cribed?~
15.3 2 2.6
IL-3 + Sol Mpl
was provided by ZymoGenetics, Inc (Seattle, WA). Soluble Mpl is
IL-3 + RX187 + Sol Mpl
18.3 2 3.9
a specific competitive antagonist of Tpo a~tivity.’~
Murine marrow cells were cultured in 0.285% agar in IMDM suppleThe RX187 rat antimouse monoclonal antibody (IgG2a) recogmol/L p mercaptoethanol
mented with 15% horse serum and 5 x
nizes the extracellular domain of gp130, and neutralizes the bioactivand Tpo (2,000 U/mL) or IL-3 (100 U/mL). The RX187 antibody was
ity of IL-6 and IL-I I in vitro. To generate the M 1 8 7 antibody, a
added at a concentration of 1.5 pg/mL, and soluble Mpl was added
soluble form of the extracellular domain of murine gp130 was preat a concentration of 5 pg/mL. Thenumber of CFU-Meg-derived colopared by introducing a stop codon just above the transmembrane
nies was counted on day 5. The data are presented as the mean
domain. Soluble gp130 was used to immunize a rat, and the resultant
2 SEM of triplicate plates. Five additional experiments gave similar
hybridomas were screened by flow cytometry for ability to recognize
results.
the native transmembrane form of murine gp130. The m 1 8 7 antibody was able to immunoprecipitate a 130 kD protein from
methionine-labeled cells into which cDNA encoding murine gp130
had been introduced, but not from untransfected parental cells; congoat antirat IgG (Jackson Immunoresearch Labs, Inc, West Grove,
trol rat IgG2a did not immunoprecipitate the 130 kD protein. The
PA) for 1 hour at 4°C. The cells were then incubated with propidium
M 1 8 7 antibody blocked the ability of the T1165 mouse plasmacyiodide (50 pg/mL; Molecular Probes, Inc. Eugene, OR) in a hypotoma cell line to proliferate in the presence of IL-6 or IL-11.25 Further
tonic citrate solution, and analyzed in a Coulter Epics Elite flow
evidence of the neutralizing activity of the m 1 8 7 antibody is procytometer.’
vided in the Results section. The control rat IgG2a antibody was
obtained from PharMingen, San Diego, CA.
RESULTS
To determine if the horse serum used for the megakaryocyte colMegakaryocytic colony growth. We examined the ability
ony assays contained Tpo, a Tpo-responsive murine cell line, DAof the neutralizing anti-gpl30 monoclonal antibody W 1 8 7
1-TpoZ6was cultured in the presence of horse serum with or without
to block growth of CFU-Meg colonies in the presence of
soluble Mpl, and cell proliferation was quantitated. Parallel cultures
Tpo or IL-3 (Table 1). In seven independent experiments,
of the DA-l-Tpo cells contained known quantities of Tpo. These
experiments showed that the quantity of horse serum used for the
the addition of RX187 antibody did not affect the number
CFU-Meg colony assays would contribute approximately 2 U/mL
of CFU-Meg colonies found in the presence of 2,000 U/mL.
of Tpo activity to the culture. Megakaryocyte colony assays conof Tpo. In contrast to the results with Tpo, the addition of
taining 15% horse serum but no added growth factor had no CFUM 1 8 7 antibody blocked IL-3-stimulated CFU-Meg colony
Meg colonies, demonstrating that this quantity of Tpo alone was
growth by approximately 40% to 45%. The addition of coninsufficient to support CFU-Meg colony growth.
trol IgG2a had no effect on CFU-Meg colony growth in the
Analysis of megakaryocyte ploidy. Murine marrow cells were
~ ~ ~ ~ of Tpo or of IL-3 (data not shown). Soluble Mpl,
cultured in serum-free suspension as previously d e s ~ r i b e d . ~ ~ ’presence
which competes with cell surface c-Mpl for binding Tpo,
Briefly, the marrow cells were cultured at a concentration of 1 X
completely inhibited Tpo-stimulated megakaryocyte colony
lo6cells/mL in IMDM supplemented with 1% Nutridoma-SP (Boehm o m p mercapringer Mannheim COT, Indianapolis, IN), 5 X
growth, and inhibited XL-3-stimulated megakaryocyte coltoethanol, and growth factors with or without M 1 8 7 antibody as
ony growth by approximately 50% (Table 1). The horse
described above. The cells were incubated at 37°C for 5 days. The
serum used in the CFU-Meg colony assay contributed apDNA content of the megakaryocytes was analyzed by flowcytometry
proximately 2 U/mL of Tpo activity to the culture; this may
after labeling with the 4A5 monoclonal antibody and withpropidium
account in part for the ability of soluble Mpl to inhibit CFUiodide as previously des~ribed.~,~’
The 4A5 monoclonal antibody
Meg colony growth in cultures containing IL-3.
recognizes a 74 kD glycoprotein present on murine megakaryocytes
To determine whether R X I 87 antibody would inhibit
and
and
was
generously provided by Dr Sam Burstein
CFU-Meg colony growth in the presence of lower concentra(University of Oklahoma, Oklahoma City, OK). Briefly,the cells
tions of Tpo, we cultured murine marrow cells with Tpo (0,
were labeled with the 4A5 monoclonal antibody (15 &mL) or with
15, 60,350, 1,OOO, or 2,000 U/mL) with or without RX187
control rat IgG2a antibody in 0.15 moVL NaCl (pH 7.4) containing
antibody (Fig 1). The addition of RX187 antibody did not
0.5% bovine serum albumin (Sigma) and0.05% sodium azide
(Sigma) for 30 minutes at 4”C, then incubated with FITC-conjugated
block Tpo-induced CFU-Meg colony growth in the presence
’%-
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BROUDYET AL
2028
45
T
40
Y)
3 35
r'
g 30
0
-1J1
0
2._ 25
c
20
I I
0
350
0
15
l000
U.
04
0
3
10
D
Concentrationof IL-l1 (@mi)
1
-
rm
2000
60
Concentrationof Tpo (Ulml)
Fig 1. Effect of neutralizing gp130 activity on growthof CFU-Meg
colonies in the presence of Tpo. Murine marrow cells (2 x 105/mL)
were culturedin semi-solid media in the presence of various concentrations of Tpo (0 t o 2,000 UlmL) without (0)or with (M) RX187
antibody (1.5 pglmL) for 5 days. The data represent the number of
CFU-Megcolonies (mean f SEM of triplicate plates)from one of two
similar experiments.
of lower concentrations of Tpo (Fig l ) . The combination of
Tpo plus IL-3 is a very potent stimulant of megakaryocytic
colony growth (Fig 2). The addition of RX 187 antibody did
not diminish CFU-Meg colony growth in thepresence of
Tpo plus TL-3 (Fig 2). suggesting that signal transduction
via gp130 is not required for megakaryocytic colony growth
under these conditions.
Although IL-l1 exerts its predominant effects on megakaryocyte maturation rather than on CFU-Meg proliferation,
IL-l I can synergize with Tpo to augment the growth of
megakaryocytic colonies.x The addition of IL- I 1 ( I O to 100
ng/mL) to a submaximal concentration of Tpo ( I O 0 U/mL)
T
Fig 3. Effect of neutralizing gp130 activity on growthof CFU-Meg
colonies. Murine marrow cells were cultured as in Fig 1 in the presence of Tpo (100 UlmL) plusvarious concentrations of IL-l1 ( 0 t o 100
nglmL) withor without RX187 antibody (1.5 pg/mLI for 5 days. The
data represent the number of CFU-Meg colonies (mean f SEM of
triplicate plates) from one of two similar experiments.
enhanced CFU-Meg colony growth (Fig 3 ) . The RX I87 antibody was able to ablate the incremental effect of IL-l 1 on
growth of CFU-Meg colonies (Fig 3). directly demonstrating
the neutralizing activity of this antibody. Consistent with the
data in Table 1 andFig I , the RX187 antibody did not
diminish CFU-Meg colony growth below that seen withTpo
alone.
To further define the neutralizing activity of RXI 87 antibody, murine marrow cells were cultured in the presence of
a range of concentrations of IL-l I , with or without RX187
antibody or control IgG2a antibody (Fig 4).The addition of
RX I X7 antibody blocked the effects of IL-l1 on CFU-Meg
colony growth. At high concentrations of IL-l I , a modest
number of CFU-Meg colonies was found even in the presence of RX I87 antibody (Fig 4).This suggests that IL-1 1 and
RXI X7 antibody compete for binding tothe gp130 subunit of
T
11 1
15
20(
3
100
Concentrationof IL-3 (Ulml)
20$8.')
0
1
5
Concentration of IL-l1 (ng/ml)
Fig 2. Effect of neutralizing gp130 activity on growth ofCFU-Meg
colonies in the presence of Tpo plus 11-3. Murine marrow cells were
cultured as in Fig 1 in thepresence of various concentrations of IL-3
(0).
11-3 plus Tpo l100 UlmL, O), or 11-3 plus Tpo plus
RX187 (1.5 p g l
mL, M) for 5 days. The data represent the number of CFU-Meg colonies (mean f SEM of triplicate plates) from one of two similar experiments.
Fig 4. Effect of neutralizinggp130 activity on growth of
CFU-Meg
colonies in the presence of IL-11. Murine marrowcells were cultured
as in Fig 1 in the presence of various concentrations of IL-l1 ( 0 t o 80
nglmL) in the presence of control ratlgG2a 11.5 pglmL, 0 )or RX187
antibody (1.5 pg/mL, M) for 5 days. The data represent the mean f
SEM of triplicate plates from
one experiment.
)
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2029
TPO ACTS INDEPENDENTLY OF ~ ~ 1 3 0
the receptor, and also demonstrates that the effects of m 1 8 7
antibody are not due to nonspecific toxicity. To ensure that
appropriate concentrations of M 1 8 7 antibody were used for
these experiments, an additional experiment examined the
effects of varying amounts of the M 1 8 7 antibody on CFUMeg colony growth in the presence of Tpo plus IL-11 (Table
2). The RX187 antibody at a concentration of 0.5 to 1.5 pg/
mL completely blocked the incremental effect of IL-l1 on
CFU-Meg colony growth. The control IgG2a antibody had
no effect (Table 2).
Megakulyocytenuclearendoreduplication.
We examined the DNA content of the megakaryocytes generated in
serum-free suspension cultures in the presence of Tpo or IL3, with or without RX187 antibody, to assess the effects of
these cytokines on nuclear endoreduplication. Some megakaryocytes were produced even in the absence of exogenous
growth factor, and the modal ploidy of these megakaryocytes
was 16N (Fig 5). When m 1 8 7 antibody was added to the
cultures containing no exogenous growth factor, megakaryocyte ploidy decreased (data not shown), suggesting that endogenous production of cytokines that require gp130 as a
signal transduction subunit is contributing to megakaryocyte
endoreduplication in these cultures. IL-6 and IL-11, which
are produced by stromal ~ e l l s , ~are
~ . ~likely
'
candidate cytokines; IL-6 can also be produced by megakaryocyte^.^',^^
The addition of Tpo profoundly increased megakaryocyte
nuclear endoreduplication over that seen withno added
growth factor (Fig 5). In the presence of Tpo, the majority
of megakaryocytes were of 32-64N ploidy (Fig 5). The addition of RX187 did not diminish the proportion of 32N, 64N,
and 128N megakaryocytes produced in the presence of Tpo
(Fig 5). A relatively small proportion of the megakaryocytes
generated in the presence of IL-3 exhibited 32N, 64N, or
128N DNA content (Fig 6), in contrast to megakaryocytes
produced in the presence of Tpo. Neutralizing gp130 activity
decreased megakaryocyte nuclear endoreduplication in the
presence of IL-3, and virtually eliminated the production of
64N megakaryocytes (Fig 6). We interpret these results to
mean that the modest effect of IL-3 to stimulate megakaryocyte nuclear endoreduplication is dependent on the presence
of endogenous IL-6 or IL-11 in these c u l t ~ r e s . ~ ~ - ~ *
Table 2. Effect of RX187 Antibody on CFU-Meg Colony Growth
Addition
CFU-Meg Colonies1
2 X 10' Cells
0
(100
Tpo
ng/mL)
Tpo(25 IL-l1
IL-l1
Tpo
Tpo + I L - l 1
L11
Tpo
IL-l1
Tpo
IL-l1
Tpo
IL-l1
Tpo
IL-l1
Tpo
+
+
+
+
+
+
+
+ RX187 (0.05 pg/mL)
+ RX187 (0.15 pg/mL)
+ RX187 (0.5 Fg/mL)
+ RX187 (1.5 pg/mL)
+ IgG2a (0.15 pg/mL)
+ IgG2a (0.5pg/rnL)
+ lgG2a (1.5 pg/mL)
0
41.0
59.0
63.7
55.7
41.0
43.0
58.0
62.0
56.7
2 2.2
2 5.1
2 7.8
i 0.3
1.4
t 0.6
2 1.2
t 2.2
t 2.3
C
Murine marrow cells were cultured as described in Table 1 in the
presence or absence of RX187 antibody (0.05 t o 1.5 pg/mL). The data
represent the mean 2 SEM of triplicate plates from one experiment.
2N
TII
DNA CONTENT
Fig 5. DNA histogram ofmegakaryocytes generated in wspension culture. Murine marrow cells l1 x 106/mL) were cultured in
IMDM supplementedwith 1% Nutridoma-SP with no added growth
factor (A), Tpo (100 U l m l B), or Tpo plus RX187 IC). After 5 days of
culture, the cells were labeled with the 4A5 antimouse megakaryocyte monoclonal antibody, stained with propidium iodide, and analyzed by flow cytometry. The results of one of four similar experiments are shown.
DISCUSSION
Hematopoiesis in vivo is intricately controlled to maintain
thenumbers of circulating erythrocytes, leukocytes, and
platelets with a narrow range under normal circumstances.
Much is understood about the physiology of the key growth
factors that regulate the terminal stages of erythropoiesis and
myelopoiesis, and several of these cytokines have been in
clinical use for approximately 10 years. In contrast, the cytokine that predominantly controls megakaryopoiesis had remained elusive. The cloning of the c-mpl proto-~ncogene,~~
and the subsequent use of c-Mpl receptor affinity chromatography to isolate its ligand 34,35 in parallel to a functional
cloning strategy,36 and other purification appro ache^,^'.^'
yielded Tpo. Contrary to predictions from experimental
models of thrombocytopenia, and previous studies with partially purified molecules, recombinant Tpo was found to promote both the proliferation of CFU-Meg and the maturation
of their progeny.
The network of hematopoietic cytokines is characterized
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BROUDY ET AL
2030
f
I
l
32N
2N
I 4N
1
C
2N
..L
I..
DNA CONTENT
Fig 6. Effect of W187 antibody on nuclear endoreduplication of
megakaryocytes generated in the presence of IL-3. Murine marrow
cells were cultured as describedin Fig 2 in thepresence of no added
growth factor (A), IL-3 (100 UlmL, B), or IL-3 plus RX187 (C). After 5
days of culture, the DNA content of the WNS was analyzed as described in Fig 2. The data from one of four similar experiments are
shown.
by functional redundancy. Previous work had shown that IL3 is a potent stimulant of murine CFU-Meg proliferation,"*
and that the IL-6 family of cytokines including IL-6, IL-11,
LIF, and oncostatin M stimulate megakaryocyte maturat i ~ n . ~ ,Some
~ , ' of the members of the hematopoietic growth
factor receptor superfamily share a common p chain, which
plays a critical role in signal transduction.** In this study,
we used a neutralizing anti-gpl30 monoclonal antibody to
determine if gp130 was a component of the c-Mpl receptor
complex, and to explore the extent to which the IL-6 family
of cytokines contribute to Tpo-stimulated and to IL-3-stimulated megakaryopoiesis in vitro.
Ablation of signal transduction through gp130 did notalter
the ability of Tpo to stimulate the cytoplasmic maturation of
rnegakary~cytes~~
or the proliferation of CFU-Meg or the
nuclear endoreduplication of megakaryocytes. These results
demonstrate that gp130 is not a component of the functional
receptor complex for Tpo. Expression of a chimeric receptor
consisting of the extracellular and transmembrane domains
of the IL-4 receptor with the cytoplasmic domain of c-mpl
in a lymphoid cell line had shown that the cytoplasmic region
of c-mpl is sufficient for signal transd~ction.~'Subsequent
studies have dissected the regions of the c-mpl cytoplasmic
domain that are required for activation of specific JAK and
STAT protein~.~',~'
However, because of the ubiquitous expression of gp13O,ls these studies do not rule out its participation in Tpo signal transduction. Furthermore, the present
results with the anti-gp130 monoclonal antibody U 1 8 7
show that the activity of IL-11 is not required for the full
spectrum of Tpo biological effects to be realized in vitro.
These data confirm and extend a report that a polyclonal
antibody to IL-11 did not impair the ability of Tpo to stimulate CFU-Meg growth."
In contrast to the results with Tpo, the effects of IL-3 on
megakaryopoiesis do require the cooperation of cytokines
that signal through the gp130 receptor subunit. A monoclonal
antibody that neutralizes the effects of IL-6 blocked the ability of IL-3 to enhance murine megakaryop~iesis.~~
IL-3 can
also induce megakaryocytes to produce IL-6,"whichcan
then stimulate megakaryocyte cytoplasmic maturationand
nuclear endoreduplication via an autocrine loop mechanism.
Studies with soluble Mpl have demonstrated that full megakaryocyte cytoplasmic maturation inthe presence of IL-3
also requires TPO.'~
Taken together, the presentresults establish that the effects
of Tpo on CFU-Meg colony growth and on megakaryocyte
nuclear endoreduplication arenot dependent on cytokines
that signal through gp130, and it is unlikelythat gpl30 serves
as a p chain for the c-Mpl receptor complex. However, the
effects of IL-3 on CFU-Meg proliferation and on megakaryocyte nuclear maturation are mediated in part by Tpo and by
the IL-6 family of cytokines.
ACKNOWLEDGMENT
The authorsthankDr
C. Lofton-Day (ZymoGenetics, Inc) for
providing soluble Mpl, Dr S . Burstein (University of Oklahoma)
for providing the 4A5 monoclonal antibody, and Z . Sisk and A.
Dimaunahan for manuscript preparation.
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1996 88: 2026-2032
Thrombopoietin stimulates colony-forming unit-megakaryocyte
proliferation and megakaryocyte maturation independently of
cytokines that signal through the gp130 receptor subunit
VC Broudy, NL Lin, N Fox, T Taga, M Saito and K Kaushansky
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