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Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3381-3387
A Minimal Cytoplasmic Subdomain of the Erythropoietin Receptor Mediates
Erythroid and Megakaryocytic Cell Development
By
Chris P. Miller,
Zi Y. Liu,
Constance T. Noguchi, and
Don M. Wojchowski
From the Programs in Genetics and Cell and Developmental Biology, The
Pennsylvania State University, University Park, PA; and the Laboratory
of Chemical Biology, NIDDK, National Institutes of Health, Bethesda,
MD.
 |
ABSTRACT |
Signals provided by the erythropoietin (Epo) receptor are essential
for the development of red blood cells, and at least 15 distinct
signaling factors are now known to assemble within activated Epo
receptor complexes. Despite this intriguing complexity, recent investigations in cell lines and retrovirally transduced murine fetal
liver cells suggest that most of these factors and signals may be
functionally nonessential. To test this hypothesis in erythroid progenitor cells derived from adult tissues, a truncated Epo receptor chimera (EE372) was expressed in transgenic mice using a GATA-1 gene-derived vector, and its capacity to support colony-forming unit-erythroid proliferation and development was analyzed. Expression at physiological levels was confirmed in erythroid progenitor cells
expanded ex vivo, and this EE372 chimera was observed to support
mitogenesis and red blood cell development at wild-type efficiencies
both independently and in synergy with c-Kit. In addition,
the activity of this minimal chimera in supporting megakaryocyte development was tested and, remarkably, was observed to approximate that of the endogenous receptor for thrombopoietin. Thus, the box 1 and
2 cytoplasmic subdomains of the Epo receptor, together with a tyrosine
343 site (each retained within EE372), appear to provide all of the
signals necessary for the development of committed progenitor cells
within both the erythroid and megakaryocytic lineages.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIESIS and hemostasis involve the
continuous production of 6 major types of blood cells (erythrocytes,
megakaryocytes, monocytes, granulocytes, B lymphocytes, and T
lymphocytes) at tightly controlled rates. This is regulated in part
through the differential expression and activation of lineage- and
stage-specific hematopoietic growth factor (HGF) receptors of the type
1 superfamily.1 For example, lymphocyte production is known
to depend on interleukin-7 (IL-7) receptor expression and
signaling,2,3 whereas erythropoietin (Epo) receptor
expression and activation is required for the development of erythroid
progenitor cells beyond the colony-forming unit-erythroid (CFUe)
stage.4 One key effect exerted by HGFs and their receptors is an inhibition of programmed cell death. This effect is
well-illustrated in the IL-7 receptor system, in which transgenic
expression of the survival factor Bcl-2 in IL-7 receptor  -deficient
mice has been shown to rescue T-cell development.5,6 By
comparison, certain HGFs appear to affect more than the survival of
targeted lineage-restricted progenitor cells. Epo, the prime hormonal
regulator of red blood cell production,7 provides one such
example and, interestingly, neither Bcl-2 nor Bcl-xL
expression in erythroid progenitor cells is able to rescue red blood
cell development in the absence of Epo signaling.8-10 Based
on these considerations and on clinical significance,11
significant efforts have focused on establishing the nature of key Epo
receptor-induced signals that promote progenitor cell survival, growth,
and/or differentiation.
One approach to dissecting Epo signaling has been to identify effectors
that associate directly with activated wild-type Epo receptor
complexes. For example, our laboratory has demonstrated that binding of
Jak2 kinase to the Epo receptor box 1 domain and its rapid activation
are generally important for signaling,12,13 whereas others
have worked to establish the identity of a complex set of SH2
domain-encoding factors and associated cofactors that associate at
phosphorylated tyrosine sites within the carboxyl-terminal region of
the murine Epo receptor. This includes the commonly activated cytokine
effectors Grb2/mSos/Raf/Ras,14 PI3 kinase,15 and phospholipase C- (PLC-)16; the
tyrosine phosphatases Syp17 and HCP18; the
putative nucleotide exchange factors Vav19 and C3G (via the
SH2 adaptor Cbl)20; the inositol phosphatase SHIP (via the
adaptor Shc)21; Cis as an inhibitor of Jak2
kinase22; the latent signal transducers and activators of
transcription STATs 5 a and b23,24; the Src family tyrosine
kinase Lyn25; and at least 1 additional molecular adaptor,
Gab1.26 However, recent studies of tyrosine point-mutated
and truncated receptor forms in cell lines and fetal liver have
provided provocative evidence that a membrane proximal cytoplasmic
subdomain linked to a single phosphotyrosine (PY) site may be necessary
and sufficient to support essentially all biological responses normally
relayed via endogenous wild-type Epo receptors.27 Studies
ex vivo using fetal liver cells are of particular interest. However,
fetal liver is a short-term site of hematopoiesis28 and
only transiently supports heightened nonhemostatic
erythropoiesis.29 Thus, the extent to which fetal
progenitor cells might correspond to permanent erythroid progenitor
cells in adult marrow and spleen is unclear.28-33 For these
reasons, and to quantitatively determine the extent to which signals
derived from the above-delimited Epo receptor cytoplasmic subdomain
support red blood cell development, we presently have used a GATA-1
gene-derived expression vector to express a minimal human epidermal
growth factor (hEGF) receptor/murine Epo receptor chimera (EE372) in
transgenic mice and have investigated the ability of this receptor
construct to support proliferative signaling and red blood cell
formation in adult erythroid tissues ex vivo. In addition, based on a
direct structural relatedness between the receptors for Epo and
thrombopoietin (Tpo)34 and upon the prediction that this
EE372 transgene also should be expressed in megakaryocytic progenitor
cells,35 the ability of this minimal chimera to support
megakaryocyte development also has been investigated.
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MATERIALS AND METHODS |
Transgenic mice.
For expression in mice, a cDNA encoding a minimal hEGF receptor/murine
Epo receptor chimera (EE372)8 was subcloned into a GATA-1
gene-derived vector that contains an upstream activating region, exons
1a and 1b, and the nontranslated region of exon 2.36 A 17-kb Kpn I-Cla I restriction
fragment containing this linked cDNA construct was injected into
pronuclei of fertilized eggs, and eggs were implanted into
pseudo-pregnant BDF1 females. Transgene-positive founders were
identified initially by polymerase chain reaction (PCR) using primers
specific to the hEGF receptor (5'-TCC ATA CAG TGC CAC CCA
GAG-3') and murine Epo receptor (5'-AGC AGC CAC AGC TGG AAG
TTA-3'). Southern blotting was performed on Bgl II
digests of genomic DNA using a 770-bp Bgl II-Xba I
fragment of a murine Epo receptor cDNA.37
Erythroid progenitor cell preparations and proliferation assays.
Enriched populations of erythroid progenitor cells were prepared from
the spleens of mice treated subcutaneously with either thiamphenicol
(TAP)38 or phenylhydrazine (PHZ)39 and from bone marrow. Briefly, TAP was administered as an implant on day 1 (14 g/kg), mice were phlebotomized on days 2 through 4, TAP was withdrawn
on day 6, and splenocytes were prepared on day 9.5. PHZ was injected
subcutaneously on days 1, 2, and 4 (50 mg/kg), and splenocytes were
prepared on day 5. Mice were anesthetized with metofane
(Schering-Plough, Union, NJ) during all procedures and prior to
sacrifice. Disrupted spleen or bone marrow preparations were passed
through a 70-µm cell strainer (Fisher, Pittsburgh, PA),
collected, exposed for 4 minutes to a freshly combined solution of 50 mmol/L NH4Cl/phosphate-buffered saline (9:1) to lyse mature red blood cells (phosphate-buffered saline, 140 mmol/L NaCl, 0.27 mmol/L KCl, 0.43 mmol/L Na2HPO4, 0.14 mmol/L
KH2PO4, pH 7.4), centrifuged through 50% fetal
bovine serum in phosphate-buffered saline, washed, and adjusted to 1 × 107 cells/mL in 0.5% fetal bovine serum, OptiMEM 1 medium (GIBCO/BRL, Grand Island, NY) supplemented with
penicillin (100 U/mL), streptomycin (100 ng/mL), and amphotericin B
(250 ng/mL) (PSF). For assays of hEGF or Epo-stimulated proliferation,
splenocytes (5 × 106 cells/mL in 10% fetal bovine
serum, OptiMEM 1 medium supplemented with PSF) were exposed to
cytokines and were cultured at 37°C, 7.5% CO2 for 48 hours. Rates of methyl [3H] thymidine incorporation then
were determined as described.37
Flow cytometry.
Marrow cells from transgenic and normal mice were cultured ex vivo
under conditions recently shown to selectively support the erythroid
expansion of CD34+ cells.40 At day 3 of
culture, cells were washed in phosphate-buffered saline and 0.5%
bovine serum albumin and were incubated sequentially for 1 hour at
2°C with the Fc fragment of murine IgG (5 µg/mL; Pierce,
Rockford, IL) and for 1.5 hours (in the presence of 0.02% NaN3) with a monoclonal antibody (EGFR.1; PharMingen, San
Diego, CA) to the hEGF receptor extracellular domain (3.3 µg/mL). Cells (2 × 106 cells/sample) were then
washed, incubated for 30 minutes at 2°C with a
phycoerythrin-conjugated antibody specific to murine IgG F(ab')2 (Jackson Research Labs, West Grove,
PA), washed, and analyzed by flow cytometry (Coulter
XL-MCL system; Coulter, Miami, FL). EE372 receptor density
was estimated in a regression analysis by comparing fluorescence values
from EE372-positive cells with values obtained for
phycoerythrin-molecular equivalent beads (Spherotech, Libertyville, IL). Estimates accounted for a stoichiometry of 1 molecule of phycoerythrin per conjugated secondary antibody and the
binding of 2 phycoerythrin antibodies per primary receptor-bound antibody.
Assays of erythroid and megakaryocyte colony formation.
In CFUe assays, splenocytes and bone marrow cells were prepared as
described above and were cultured at 37°C, 7.5% CO2
for 48 hours in Methocult HCC3242 media (Stem Cell Technologies,
Vancouver, British Columbia, Canada) at 3 × 105 cells/mL. Cultures contained either Epo (5 U/mL) or
hEGF (5 to 10 ng/mL) and, when specified, murine stem cell factor (50 ng/mL; Peprotech, Rocky Hill, NJ). Hemoglobin-positive
colonies were stained with a freshly combined solution (1:1) of
benzidine (3% in 90% glacial acetic acid) plus 5%
H2O2. Megakaryocyte development from marrow
progenitor cells was assayed using a serum-free MegaCult collagen
system (Stem Cell Technologies). Cells were plated at both 1 × 105 and 3 × 105 cells/mL in
the presence of murine IL-3 (50 ng/mL) and either hEGF (10 ng/mL) or
Tpo (50 ng/mL). At day 7 of culture, cultures were dried, fixed, and
stained for acetylcholinesterase-positive colonies.
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RESULTS |
Transgenic expression and proliferative activity of the minimal
hEGF/Epo receptor chimera EE372 in erythroid progenitor cells.
To test the ability of a minimal membrane-proximal cytoplasmic domain
of the Epo receptor to support erythropoiesis in adult tissues, this
domain was linked to the extracellular domain of the hEGF receptor and
the resulting hEGF-activatable chimera was expressed in transgenic
mice. In this construct (EE372; Fig 1A), cytoplasmic features of the murine Epo receptor include conserved box 1 and 2 motifs and a single (P)Y343 site that has been shown to bind
STAT523 and to contribute significantly to
growth41,42 and differentiation signaling8,27
in cell line models. To provide for expression of this EE372 receptor
form in erythroid progenitor cells, a GATA-1 gene-derived vector was
used that recently has been shown to direct the expression of a
 -galactosidase reporter gene in erythroid cells in
vivo.43 Two EE372-positive founder mice were identified by
PCR, and Southern blot analyses served to confirm the reliable
transmission of this transgene at low copy number in mice derived from
these 2 founders (Fig 1B, representative results shown for f3 and f4
generations).
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| Fig 1.
The hEGF receptor (hEGF R)-murine Epo receptor (mEpo R)
chimera, GATA-1 gene-derived transgenic expression vector, and
integration of pG1-EE372 in transgenic mice. (A) Diagrammed are the
wild-type (wt) murine Epo receptor and the minimal chimeric construct
EE372. In EE372, the extracellular domain is that of the human EGF
receptor, and the murine Epo receptor cytoplasmic domain is truncated
to delete 7 of 8 sites of tyrosine (Y) phosphorylation. Also diagrammed
is the GATA-1 gene-derived vector used to express EE372 in transgenic
mice (pG1-EE372). uas, upstream activating sequence. pA,
polyadenylation signal. (B) Southern blot analyses shown are for
representative litters from f3 and f4 generations. Indexed are
Bgl II products from the endogenous Epo receptor gene and the
pG1-EE372 transgene.
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The capacity of this minimal EE372 chimera to signal hEGF-induced
proliferation in erythroid progenitor cells from adult mice was assayed
in primary tests of function. To provide for an enriched population of
erythroid cells, mice were treated with either TAP or PHZ under
regimens shown in pilot experiments to maximize CFUe production in
spleen. TAP in particular was used to generate a developmentally
synchronized population of splenic CFUe while limiting the
representation of megakaryocytic progenitor cells (see below). In
[3H]dT incorporation assays, hEGF proved to support the
proliferation of erythroid splenocytes from EE372 mice at rates that
paralleled those induced by Epo via the endogenous wild-type Epo
receptor (Fig 2). Data shown are for 3 transgenic mice (and nontransgenic control animals) and are
representative of results obtained for 6 animals from 3 distinct
generations at 5 to 20 weeks of age. These results confirmed the
expression of functional EE372 receptors and indicated an essentially
wild-type activation of proliferative response pathways. To directly
confirm that EE372 receptors were expressed at high frequencies with
low variegation (and to discount the possibility that observed
proliferative response profiles might be accounted for by EE372
receptor overexpression within subpopulations), the density and
distribution of EE372 transgene expression were assayed in erythroid
progenitor cells expanded ex vivo. Marrow cells from transgenic and
control mice were cultured under conditions recently shown to
selectively support erythroid progenitor cell expansion from human
CD34+ cells.40 At day 3 of culture,
colony-forming assays showed that approximately 50% of expanded cells
corresponded to CFUe (data not shown). For cells at this stage, an
antibody to the human EGF receptor extracellular domain was used to
assay EE372-positive cells (Fig 3). In
cells expanded from transgenic mice, 47% of size-gated cells were
positive for EE372 expression and mean densities of EE372 expression
among gated cells approximated 5,000 receptors per cell (calculated as
described in Materials and Methods). Thus, this EE372 transgene is
expressed at high frequencies among erythroid progenitor cells at
levels within the same order of magnitude as reported for endogenous
Epo receptors.44
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| Fig 2.
EE372 efficiently supports the ex vivo proliferation of
adult splenic erythroid progenitor cells. Shown are the rates of hEGF-
and Epo-stimulated [3H]dT incorporation for erythroid
splenocytes from EE372 transgenic versus nontransgenic (wt, wild-type)
control TAP-treated mice. In each panel, responses for 1 representative
EE372 mouse and 1 nontransgenic mouse are presented. Values are means
of duplicate samples expressed as a percentage of maximal
[3H]dT-Incorporation induced by Epo. Standard error
values are indicated by error bars for all data points. Values for
maximal [3H]dT-incorporation induced by Epo for mice no.
37, 47, 50, 178, and 199 are 37.9 ± 3.5, 36.9 ± 4.6, 29.7 ± 2.0, 9.9 ± 1.44, and 41.2 ± 0.7 (×103 cpm),
respectively. *Unavailable data points.
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| Fig 3.
Expression of EE372 receptors on adult erythroid
progenitor cells expanded ex vivo. (A) Using conditions developed by
Panzenbock et al,40 erythroid progenitor cells were
expanded from the marrow of transgenic (EE372) and control mice (wt,
wild-type). EE372 expression was assayed using an antibody specific to
the hEGF receptor extracellular domain. PE, phycoerythrin fluorescence
intensity. FALS, forward angle light scatter. (B) Example estimate of
EE372 receptor densities. Phycoerythrin molecular equivalent microbeads
were used to generate a calibration profile (3,800, 12,000, 34,000, 124,000, and 300,000, reading left to right). This profile (and
regression analyses) then were used to estimate EE372 receptor
densities on marrow cells expanded ex vivo (see inset).
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The minimal chimera EE372 promotes red blood cell development
independently and in synergy with c-Kit.
Given the uniform expression of mitogenically competent chimeric
receptors in EE372 mice described above, investigations next assessed
the ability of the above EE372 receptor form to support red blood cell
production. Specifically, the ability of hEGF to support the
differentiation of CFUe within marrow and in splenocytes from TAP or
PHZ treated EE372 mice was tested. For erythroid progenitor cells from
each source and among all EE372 transgenic mice assayed, hEGF promoted
red cell colony formation at efficiencies essentially equivalent to
levels promoted by Epo at equimolar concentrations (ie, via endogenous
Epo receptors; Table 1). Colony morphology (typically 16-cell hemoglobinized colonies) was indistinguishable from
CFUe differentiated from control or transgenic mice in the presence of
Epo (data not shown), and hEGF failed to support any detectable
differentiation or outgrowth of CFUe from nontransgenic animals.
The development of CFUe at high efficiencies is known to depend on
coexposure to stem cell factor (SCF),27 and
some evidence has been generated to suggest that SCF signaling via its
receptor tyrosine kinase c-Kit may even depend on lateral signaling
through Epo receptor complexes.45 Based on these
considerations, whether the truncated chimera EE372 might act in
synergy with SCF/c-Kit to support CFUe development also was tested. In
control experiments, red cell formation due to Epo was enhanced
markedly in the presence of SCF (Fig 4).
Interestingly, this effect also was exerted at full efficiency for
erythroid splenocytes from EE372 transgenic mice after their exposure
to hEGF plus SCF (Fig 4). Thus, the membrane- proximal cytoplasmic
subdomain of the Epo receptor efficiently mediates the synergistic
effects of SCF/c-Kit on red blood cell development from adult erythroid
progenitor cells ex vivo.
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| Fig 4.
EE372 mediates hEGF-dependent CFUe development and
synergizes with c-Kit. The ability of erythroid splenocytes from
TAP-treated EE372 mice to form hemoglobinized colonies was assayed in
the absence of cytokines or the presence of either Epo (5 U/mL), hEGF
(20 ng/mL), SCF (50 ng/mL), Epo plus SCF, or hEGF plus SCF.
Benzidine-positive colonies (CFUe) were scored at 48 hours of culture.
Values are the means (± standard error) of 3 assays and are
representative of 2 independent experiments.
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Megakaryocyte development is supported efficiently via the minimal
Epo receptor chimera EE372.
Structurally, the single transmembrane receptor for thrombopoietin
(Mpl) is related closely to the Epo receptor, and each is known to
signal via Jak2 and to activate STAT5.34 In addition, substantial overlap exists between lineage-restricted transcription factors known to dictate commitment to megakaryocytic and erythroid lineages.35,46 This includes GATA-1, and the GATA-1
gene-derived vector used in these studies has been shown to direct
expression in megakaryocytes.35 Based on these
considerations and on the prediction that EE372 mice should also
express this receptor chimera in megakaryocytic cells, the ability of
the EE372 receptor to support the development of megakaryocytic
colonies (colony-forming unit-megakaryocyte [CFU-meg])
was assayed. Remarkably, colony-forming assays in serum-free medium
showed that CFU-meg development was supported efficiently by this
minimal receptor form in response to hEGF at levels approximating that
supported by Tpo (Fig 5A). Also, the
morphology of acetylcholinesterase-positive megakaryocytic colonies was
essentially indistinguishable from those of CFU-meg colonies cultured
in the presence of Tpo (Fig 5B). Thus, the box 1 and 2 domains of the
Epo receptor together with tyrosine 343 appear to provide all signals
necessary to support both erythroid and megakaryocytic development.
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| Fig 5.
EE372 mediates hEGF-dependent megakaryocyte colony
formation. Bone marrow cells from adult nontransgenic control (wt,
wild-type) and EE372 transgenic mice were used to test the ability of
hEGF and EE372 to support CFU-meg colony formation. (A) Cultures were
exposed to either hEGF (EGF, solid histograms, 20 ng/mL) or Tpo (open
histograms, 50 ng/mL) in the presence of IL-3 (10 mg/mL). At day 7, acetylcholinesterase-positive CFU-meg colonies were scored (mean values ± standard error for n = 4 replicate assays). In this experiment,
numbers of Tpo-induced colonies appear to be greater for the EE372
mouse. However, this effect was not observed in repeated independent
analyses. (B) Shown are the similar morphologies of
acetylcholinesterase-positive (brown) CFU-meg colonies propagated from
EE372 mice in the presence of Tpo (top panel) or hEGF (lower panel).
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DISCUSSION |
Novel features of the present investigation that merit discussion
include the following: (1) the nature of signals provided by the
receptor form EE372 that support erythroid and megakaryocytic cell
development from adult hematopoietic tissues and (2) advantages of this
unique transgenic model for investigations of receptor-derived signals
that promote CFUe and CFU-meg survival, proliferation, and terminal
differentiation. With regards first to erythropoiesis, previous studies
of the activities of carboxyl terminal-truncated and (P)Y-mutated Epo
receptor forms in cell lines and fetal liver have provided compelling
evidence that the minimal cytoplasmic subdomain contained in EE372
efficiently supports proliferation and differentiation signaling in
these models.8,27,41,42 However, whether this receptor
subdomain and derived signals might also support red blood cell
development from CFUe in adult tissues has not been studied to date.
This is an important issue, because fetal and adult erythropoiesis are
known to differ in several notable ways. Direct evidence that Epo
receptor activation mechanisms in fetal liver differ from those in
adult tissues recently has been provided based on the ability of the
gp55 protein of the anemia-inducing strain of Friend virus to promote
Epo receptor-dependent erythropoiesis from fetal liver but not from
marrow.30 In addition, progenitor cells in fetal liver are
known to reconstitute hematopoiesis in irradiated mice at efficiencies
higher than progenitor cells from marrow,31 and Epo
produced by fetal liver per se may target stromal cells and affect
their activity in supporting progenitor cell development.32
In at least certain ways, programming of an erythroid fate also appears
to differ in that ES cells nullizygous for the transcription factor
PU.1 contribute to fetal liver but not adult marrow erythropoiesis in
chimeric mice.33 Despite these differences, the present
studies clearly show that, when expressed at levels comparable with the
endogenous Epo receptor, the minimal Epo receptor cytoplasmic subdomain
containing only the box 1 and box 2 domains and 1 Y343 site exhibits
wild-type activity in promoting proliferation and hemoglobinization of
erythroid progenitor cells derived from adult spleen and marrow.
The observed ability of the minimal Epo receptor chimera EE372 to also
support megakaryopoiesis raises interesting questions concerning the
nature of key signals that this and the endogenous receptors for Tpo
and Epo transduce. In each, membrane proximal box 1 and 2 cytoplasmic
domains are conserved37 and signaling depends critically on
Jak2.47 However, in the Tpo receptor, tyrosine
phosphorylation is limited to only 2 carboxyl terminal sites (ie, Y599,
Y604 and Y626,Y631 in the murine and human receptors, respectively).47 In murine Mpl, Y599 and Y604 each appear
to contribute to the activation of STAT5a/b and possibly
STAT3.47 The Epo receptor and EE372 also selectively
activates STAT5,23 and this signal transducer and activator
of transcription therefore comprises 1 shared effector. Moreover, in
the Tpo receptor, Epo receptor, and EE372, the selective uncoupling of
STAT5 activation has been demonstrated to blunt proliferative and/or
differentiation signaling in at least certain cell line
models.8,27,41,42,48 However, in mice nullizygous for STAT5
a and b, erythropoiesis and megakaryopoiesis are essentially
unperturbed.49 Thus, this latter result either is the
consequence of compensatory mechanisms or no essential roles exist for
STAT5a or b in these lineages. In the Tpo receptor system, Shc recently
has emerged as an apparently important transducer and is recruited to
PY599 of murine Mpl.50 Mutation of this site inactivates
differentiation signaling in megakaryocytic F36P cells48 as
well as in myeloid WEHI3B-D, M1, and 32D cells.51,52 Beyond
this, Shc is known to be linked to Grb2/mSos/Raf/Ras and SHIP
signaling,53 and Tpo-induced polyploidy and gpIIb/IIIa
expression in F36P cells is inhibited by a dominant-negative form of
c-H-Ras (S17N) and is stimulated by a dominant-active form
(G12V).48 Thus, the intensity of Mpl and Shc-mediated
activation of Ras has been suggested to regulate megakaryocyte
development. In EE372, candidate PY sites for Shc recruitment are
deleted. However, He et al54 previously have described an
Epo receptor and Jak2-dependent pathway to Shc activation that
efficiently proceeds in the absence of receptor tyrosine
phosphorylation. Thus, Shc is proposed to comprise an attractive
candidate effector of EE372, Epo, and Tpo receptor-mediated
erythropoiesis and megakaryopoiesis. In addition, it is considered
likely that a number of as yet undiscovered targets likely lie
downstream of Jak2 per se, and it is proposed that certain of these
also play important roles in Epo and Tpo response pathways. The
observed ability of the minimal Epo receptor domain to support
megakaryocyte formation in the absence of Tpo is inconsistent with an
instructive model of Tpo signaling and is in keeping with studies by
Stoffel et al55 in which the intact cytoplasmic domain of
the granulocyte colony-stimulating factor receptor was shown to support
megakaryopoiesis and platelet formation in Tpo receptor-deficient mice.
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ACKNOWLEDGMENT |
The authors thank Dr S.H. Orkin (Children's Hospital, Boston, MA) for
generously providing pA2GATA as a transgene expression vector, Dr T.L.
Blankenship-Paris for expert veterinary advice, and Dr E. Kunze for
directing flow cytometric analyses.
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FOOTNOTES |
Submitted May 10, 1999; accepted July 15, 1999.
Supported by National Institutes of Health Grant No. DK40242 (D.M.W.).
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 Don M. Wojchowski, PhD, 115 Henning Bldg,
The Pennsylvania State University, University Park, PA 16802; e-mail:
dmw1{at}psu.edu.
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REFERENCES |
1.
Ihle JN, Thierfelder W, Teglund S, Stravapodis D, Wang D, Feng J, Parganas E:
Signaling by the cytokine receptor superfamily.
Ann NY Acad Sci
865:1, 1998[Medline]
[Order article via Infotrieve]
2.
Peschon JJ, Morrissey PJ, Grabstein KH, Ramsdell FJ, Maraskovsky E, Gliniak BC, Park LS, Ziegler SF, Williams DE, Ware CB:
Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice.
J Exp Med
180:1955, 1994[Abstract/Free Full Text]
3.
von Freeden-Jeffry U, Vieira P, Lucian LA, McNeil T, Burdach SE, Murray R:
Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine.
J Exp Med
181:1519, 1995[Abstract/Free Full Text]
4.
Wu H, Liu X, Jaenisch R, Lodish HF:
Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor.
Cell
83:59, 1995[Medline]
[Order article via Infotrieve]
5.
Akashi K, Kondo M, von Freeden-Jeffry U, Murray R, Weissman IL:
Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor-deficient mice.
Cell
89:1033, 1997[Medline]
[Order article via Infotrieve]
6.
Maraskovsky E, O'Reilly LA, Teepe M, Corcoran LM, Peschon JJ, Strasser A:
Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant rag-1 / mice.
Cell
89:1011, 1997[Medline]
[Order article via Infotrieve]
7.
Krantz SB:
Erythropoietin.
Blood
77:419, 1991[Free Full Text]
8.
Gregory RC, Jiang N, Todokoro K, Crouse J, Pacifici RE, Wojchowki DM:
Erythropoietin receptor and STAT5-specific pathways promote SKT6 cell hemoglobinization.
Blood
92:1104, 1998[Abstract/Free Full Text]
9.
Lacronique V, Varlet P, Mayeux P, Porteu A, Gisselbrecht S, Kahn A, Lacombe C:
Bcl-2 targeted overexpression into the erythroid lineage of transgenic mice delays but does not prevent the apoptosis of erythropoietin-deprived erythroid progenitors.
Blood
90:3050, 1997[Abstract/Free Full Text]
10.
Chida D, Miura O, Yoshimura A, Miyajima A:
Role of cytokine signaling molecules in erythroid differentiation of mouse fetal liver hematopoietic cells: Functional analysis of signaling molecules by retrovirus-mediated expression.
Blood
93:1567, 1999[Abstract/Free Full Text]
11.
Fisher JW:
Erythropoietin: Physiologic and pharmacologic aspects.
Proc Soc Exp Biol Med
216:358, 1997[Medline]
[Order article via Infotrieve]
12.
Zhuang H, Patel SV, He TC, Sonsteby SK, Niu Z, Wojchowski DM:
Inhibition of erythropoietin-induced mitogenesis by a kinase-deficient form of Jak2.
J Biol Chem
269:21411, 1994[Abstract/Free Full Text]
13.
Jiang N, He TC, Miyajima A, Wojchowski DM:
The box1 domain of the erythropoietin receptor specifies Janus kinase 2 activation and functions mitogenically within an interleukin 2 beta-receptor chimera.
J Biol Chem
271:16472, 1996[Abstract/Free Full Text]
14.
Boguski MS, McCormick F:
Proteins regulating Ras and its relatives.
Nature
366:643, 1993[Medline]
[Order article via Infotrieve]
15.
Damen JE, Cutler RL, Jiao H, Yi T, Krystal G:
Phosphorylation of tyrosine 503 in the erythropoietin receptor (EpR) is essential for binding the P85 subunit of phosphatidylinositol (PI) 3-kinase and for EpR-associated PI 3-kinase activity.
J Biol Chem
270:23402, 1995[Abstract/Free Full Text]
16.
Ren HY, Komatsu N, Shimizu R, Okada K, Miura Y:
Erythropoietin induces tyrosine phosphorylation and activation of phospholipase C-gamma 1 in a human erythropoietin-dependent cell line.
J Biol Chem
269:19633, 1994[Abstract/Free Full Text]
17.
Tauchi T, Damen JE, Toyama K, Feng GS, Broxmeyer HE, Krystal G:
Tyrosine 425 within the activated erythropoietin receptor binds Syp, reduces the erythropoietin required for Syp tyrosine phosphorylation, and promotes mitogenesis.
Blood
87:4495, 1996[Abstract/Free Full Text]
18.
Klingmuller U, Lorenz U, Cantley LC, Neel BG, Lodish HF:
Specific recruitment of SH-PTP1 to the erythropoietin receptor causes inactivation of JAK2 and termination of proliferative signals.
Cell
80:729, 1995[Medline]
[Order article via Infotrieve]
19.
Miura O, Miura Y, Nakamura N, Quelle FW, Witthuhn BA, Ihle JN, Aoki N:
Induction of tyrosine phosphorylation of Vav and expression of Pim-1 correlates with Jak2-mediated growth signaling from the erythropoietin receptor.
Blood
84:4135, 1994[Abstract/Free Full Text]
20.
Barber DL, Mason JM, Fukazawa T, Reedquist KA, Druker BJ, Band H, D'Andrea AD:
Erythropoietin and interleukin-3 activate tyrosine phosphorylation of CBL and association with CRK adaptor proteins.
Blood
89:3166, 1997[Abstract/Free Full Text]
21.
Damen JE, Liu L, Cutler RL, Krystal G:
Erythropoietin stimulates the tyrosine phosphorylation of Shc and its association with Grb2 and a 145-Kd tyrosine phosphorylated protein.
Blood
82:2296, 1993[Abstract/Free Full Text]
22.
Yoshimura A, Ohkubo T, Kiguchi T, Jenkins NA, Gilbert DJ, Copeland NG, Hara T, Miyajima A:
A novel cytokine-inducible gene CIS encodes an SH2-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors.
EMBO J
14:2816, 1995[Medline]
[Order article via Infotrieve]
23.
Damen JE, Wakao H, Miyajima A, Krosl J, Humphries RK, Cutler RL, Krystal G:
Tyrosine 343 in the erythropoietin receptor positively regulates erythropoietin-induced cell proliferation and Stat5 activation.
EMBO J
14:5557, 1995[Medline]
[Order article via Infotrieve]
24.
Klingmuller U, Bergelson S, Hsiao JG, Lodish HF:
Multiple tyrosine residues in the cytosolic domain of the erythropoietin receptor promote activation of STAT5.
Proc Natl Acad Sci USA
93:8324, 1996[Abstract/Free Full Text]
25.
Chin H, Arai A, Wakao H, Kamiyama R, Miyasaka N, Miura O:
Lyn physically associates with the erythropoietin receptor and may play a role in activation of the Stat5 pathway.
Blood
91:3734, 1998[Abstract/Free Full Text]
26.
Lecoq-Lafon C, Verdier F, Fichelson S, Chretien S, Gisselbrecht S, Lacombe C, Mayeux P:
Erythropoietin induces the tyrosine phosphorylation of GAB1 and its association with SHC, SHP2, SHIP, and phosphatidylinositol 3-kinase.
Blood
93:2578, 1999[Abstract/Free Full Text]
27.
Wu H, Klingmuller U, Acurio A, Hsiao JG, Lodish HF:
Functional interaction of erythropoietin and stem cell factor receptors is essential for erythroid colony formation.
Proc Natl Acad Sci USA
94:1806, 1997[Abstract/Free Full Text]
28.
Yu H, Bauer B, Lipke GK, Phillips RL, Van Zant G:
Apoptosis and hematopoiesis in murine fetal liver.
Blood
81:373, 1993[Abstract/Free Full Text]
29.
Emerson SG, Thomas S, Ferrara JL, Greenstein JL:
Developmental regulation of erythropoiesis by hematopoietic growth factors: Analysis on populations of BFU-E from bone marrow, peripheral blood, and fetal liver.
Blood
74:49, 1989[Abstract/Free Full Text]
30.
Constantinescu SN, Wu H, Liu X, Beyer W, Fallon A, Lodish HF:
The anemic Friend virus gp55 envelope protein induces erythroid differentiation in fetal liver colony-forming units-erythroid.
Blood
91:1163, 1998[Abstract/Free Full Text]
31.
Morrison SJ, Hemmati HD, Wandycz AM, Weissman IL:
The purification and characterization of fetal liver hematopoietic stem cells.
Proc Natl Acad Sci USA
92:10302, 1995[Abstract/Free Full Text]
32.
Ohneda O, Yanai N, Obinata M:
Erythropoietin as a mitogen for fetal liver stromal cells which support erythropoiesis.
Exp Cell Res
208:327, 1993[Medline]
[Order article via Infotrieve]
33.
Scott EW, Fisher RC, Olson MC, Kehrli EW, Simon MC, Singh H:
PU.1 functions in a cell-autonomous manner to control the differentiation of multipotential lymphoid-myeloid progenitors.
Immunity
6:437, 1997[Medline]
[Order article via Infotrieve]
34.
Moutoussamy S, Kelly PA, Finidori J:
Growth-hormone-receptor and cytokine-receptor-family signaling.
Eur J Biochem
255:1, 1998[Medline]
[Order article via Infotrieve]
35.
Shivdasani RA, Fujiwara Y, McDevitt MA, Orkin SH:
A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocyte growth and platelet development.
EMBO J
16:3965, 1997[Medline]
[Order article via Infotrieve]
36.
Skoda RC, Tsai SF, Orkin SH, Leder P:
Expression of c-MYC under the control of GATA-1 regulatory sequences causes erythroleukemia in transgenic mice.
J Exp Med
181:1603, 1995[Abstract/Free Full Text]
37.
Quelle DE, Wojchowski DM:
Localized cytosolic domains of the erythropoietin receptor regulate growth signaling and down-modulate responsiveness to granulocyte-macrophage colony-stimulating factor.
Proc Natl Acad Sci USA
88:4801, 1991[Abstract/Free Full Text]
38.
Nijhof W, Wierenga PK:
Isolation and characterization of the erythroid progenitor cell: CFU-E.
J Cell Biol
96:386, 1983[Abstract/Free Full Text]
39.
Kozlov VA, Zhuravkin IN, Coleman RM, Rencricca NJ:
Splenic plaque-forming cells (PFC) and stem cells (CFU-s) during acute phenylhydrazine-induced enhanced erythropoiesis.
J Exp Zool
213:199, 1980[Medline]
[Order article via Infotrieve]
40.
Panzenbock B, Bartunek P, Mapara MY, Zenke M:
Growth and differentiation of human stem cell factor/erythropoietin-dependent erythroid progenitor cells in vitro.
Blood
92:3658, 1998[Abstract/Free Full Text]
41.
Gobert S, Porteu F, Pallu S, Muller O, Sabbah M, Dusanter-Fourt I, Courtois G, Lacombe C, Gisselbrecht S, Mayeux P:
Tyrosine phosphorylation of the erythropoietin receptor: Role for differentiation and mitogenic signal transduction.
Blood
86:598, 1995[Abstract/Free Full Text]
42.
Damen JE, Wakao H, Miyajima A, Krosl J, Humphries RK, Cutler RL, Krystal G:
Tyrosine 343 in the erythropoietin receptor positively regulates erythropoietin-induced cell proliferation and Stat5 activation.
EMBO J
14:5557, 1995
43.
McDevitt MA, Fujiwara Y, Shivdasani RA, Orkin SH:
An upstream, DNase I hypersensitive region of the hematopoietic-expressed transcription factor GATA-1 gene confers developmental specificity in transgenic mice.
Proc Natl Acad Sci USA
94:7976, 1997[Abstract/Free Full Text]
44.
Shinjo K, Takeshita A, Higuchi M, Ohnishi K, Ohno R:
Erythropoietin receptor expression on human bone marrow erythroid precursor cells by a newly devised quantitative flow-cytometric assay.
Br J Haematol
96:551, 1997[Medline]
[Order article via Infotrieve]
45.
Wu H, Klingmuller U, Besmer P, Lodish HF:
Interaction of the erythropoietin and stem-cell-factor receptors.
Nature
377:241, 1995
46.
Tsang AP, Fujiwara Y, Hom DB, Orkin SH:
Failure of megakaryopoiesis and arrested erythropoiesis in mice lacking the GATA-1 transcriptional cofactor FOG.
Genes Dev
12:1176, 1998[Abstract/Free Full Text]
47.
Drachman JG, Kaushansky K:
Dissecting the thrombopoietin receptor: Functional elements of the Mpl cytoplasmic domain.
Proc Natl Acad Sci USA
94:2350, 1997[Abstract/Free Full Text]
48.
Matsumura I, Nakajima K, Wakao H, Hattori S, Hashimoto K, Sugahara H, Kato T, Miyazaki H, Hirano T, Kanakura Y:
Involvement of prolonged ras activation in thrombopoietin-induced megakaryocytic differentiation of a human factor-dependent hematopoietic cell line.
Mol Cell Biol
18:4282, 1998[Abstract/Free Full Text]
49.
Teglund S, McKay C, Schuetz E, van Deursen JM, Stravopodis D, Wang D, Brown M, Bodner S, Grosveld G, Ihle JN:
Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses.
Cell
93:841, 1998[Medline]
[Order article via Infotrieve]
50.
Gurney AL, Wong SC, Henzel WJ, de Sauvage FJ:
Distinct regions of c-Mpl cytoplasmic domain are coupled to the JAK-STAT signal transduction pathway and Shc phosphorylation.
Proc Natl Acad Sci USA
92:5292, 1995[Abstract/Free Full Text]
51.
Alexander WS, Maurer AB, Novak U, Harrison-Smith M:
Tyrosine-599 of the c-Mpl receptor is required for Shc phosphorylation and the induction of cellular differentiation.
EMBO J
15:6531, 1996[Medline]
[Order article via Infotrieve]
52.
Hill RJ, Zozulya S, Lu YL, Hollenbach PW, Joyce-Shaikh B, Bogenberger J, Gishizky ML:
Differentiation induced by the c-Mpl cytokine receptor is blocked by mutant Shc adaptor protein.
Cell Growth Differ
7:1125, 1996[Abstract]
53.
Pronk GJ, de Vries-Smits AM, Buday L, Downward J, Maassen JA, Medema RH, Bos JL:
Involvement of Shc in insulin- and epidermal growth factor-induced activation of p21ras.
Mol Cell Biol
14:1575, 1994[Abstract/Free Full Text]
54.
He TC, Jiang N, Zhuang H, Wojchowski DM:
Erythropoietin-induced recruitment of Shc via a receptor phosphotyrosine-independent, Jak2-associated pathway.
J Biol Chem
270:11055, 1995[Abstract/Free Full Text]
55.
Stoffel R, Ziegler S, Ghilardi N, Ledermann B, de Sauvage FJ, Skoda RC:
Permissive role of thrombopoietin and granulocyte colony-stimulating factor receptors in hematopoietic cell fate decisions in vivo.
Proc Natl Acad Sci USA
96:698, 1999[Abstract/Free Full Text]
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