|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1219-1224
The Distal Cytoplasmic Domain of the Erythropoietin Receptor Induces
Granulocytic Differentiation in 32D Cells
By
Kevin W. Harris,
Xian-Jue Hu,
Suzanne Schultz,
Murat O. Arcasoy,
Bernard G. Forget, and
Nanette Clare
From the South Texas Veterans Health Care System, San Antonio; the
Department of Medicine, Division of Hematology, and Department of
Pathology, University of Texas Health Science Center, San Antonio, TX;
and the Department of Medicine, Hematology Section, Yale University
School of Medicine, New Haven, CT.
 |
ABSTRACT |
The role of hematopoietic growth factors in lineage commitment and
differentiation is unclear. We present evidence that heterologous expression of an erythroid specific receptor allows granulocytic differentiation of a myeloid cell line. We have previously
characterized a truncation mutant of the erythropoietin receptor
(EpoR), which is associated with familial erythrocytosis (Blood
89:4628, 1997). This truncated EpoR lacks the distal 70 amino acids of
the cytoplasmic domain. To study the functional role of this distal
receptor domain, 32D cells, a murine interleukin-3 (IL-3)-dependent
myeloid line, were transfected with the wild-type EpoR (32D/EpoR
WT) or the truncated EpoR (32D/EpoR FE). 32D
cells expressing either the full-length or truncated EpoR display
equivalent proliferative rates in saturating concentrations of Epo.
There is a dramatic difference in maturational phenotype between the
two cell lines, however. The 32D/EpoR FE cells and mock transfected 32D
cells have an immature, monoblastic morphology and do not express the primary granule protein myeloperoxidase. The 32D/EpoR WT cells, on the
other hand, demonstrate granulocytic differentiation with profuse
granulation, mature, clumped chromatin, and myeloperoxidase expression.
There is no evidence of erythroid differentiation in 32D cells
transfected with either the full-length or truncated EpoR. Treatment of
the cells with the specific Jak2 inhibitor tyrphostin AG 490 inhibits
myeloid differentiation driven by the distal EpoR. We conclude that:
(1) the distal cytoplasmic domain of the EpoR is able to induce a
specific myeloid differentiation signal distinct from mitogenic
signaling, and (2) these data extend to myelopoiesis the growing body
of evidence that the cellular milieu, not the specific cytokine
receptor, determines the specificity of differentiation after cytokine
receptor activation.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
HEMATOPOIETIC GROWTH factors such as
interleukin-3 (IL-3), granulocyte colony-stimulating factor
(G-CSF), and erythropoietin (Epo) are potent mitogens for
their target cells. The mitogenic effects of these growth factors are
mediated through homo or hetero-dimerization of their cognate
receptors1 and subsequent activation of several signal
transduction pathways including the JAK/STAT pathway,2 and
the mitogen-activated protein (MAP) kinase
pathway.3 In addition, these factors are able to suppress
apoptosis. For instance, Epo is able to prevent apoptosis of erythroid
precursors.4
Several lines of evidence suggest that hematopoietic growth factors are
also capable of inducing differentiation of appropriate immature
precursor cells. For instance, hematopoietic cells that normally do not
express  globin can be induced to express this erythroid specific
protein in an Epo-dependent manner after overexpression of the
erythropoietin receptor.5,6
Other data are consistent with a more passive role for hematopoietic
growth factors, ie, the growth factor merely allows a progenitor cell
that is already "predetermined" to proceed with its
differentiation program. Suppression of apoptosis by overexpression of
Bcl-2 allowed spontaneous, cytokine independent differentiation of a
multipotent progenitor cell line into several hematopoietic lineages.7 In another example, the permissive effect of Epo in erythropoiesis can be mimicked by prolactin in transfected erythroid
cells that overexpress the prolactin receptor.8
We have recently described a family with a novel truncation mutation of
the human EpoR associated with the disease familial erythrocytosis
(FE).9 FE is a rare disease of red blood cell overproduction caused by EpoR mutations resulting in truncation of the
cytoplasmic domain of the EpoR. This truncation deletes a negative
regulatory domain of the EpoR and results in hyperactivity of the
receptor.10 This mutation (EpoR FE) was characterized in
the erythroid precursors of affected individuals and in murine myeloid
32D cells transfected with the mutant EpoR. EpoR FE was shown to result
in hypersensitivity to Epo9 and to increased activation of
the JAK/STAT pathway (Arcasoy M, Harris K, Forget B, manuscript
submitted). These effects are thought to be due to
deletion of the binding site on the EpoR for the negative regulatory tyrosine phosphatase SHP-1.10 It has been shown that the
proximal cytoplasmic region of the EpoR containing the Jak binding
domains are sufficient to support mitogenesis of transfected
hematopoietic cells.6,8
During these studies, we noticed that 32D cells transfected with the
native, full-length EpoR had several obvious morphologic differences
from mock transfected cells and cells transfected with EpoR FE, despite
identical growth rates and mitogenic signaling. We demonstrate here
that the full-length human EpoR is capable of inducing granulocytic
differentiation in this myeloid cell line. This differentiative signal
requires the distal 70 amino acids of the receptor, as well as intact
Jak2 kinase activity.
| |
MATERIALS AND METHODS |
Cell lines.
Murine myeloid IL-3-dependent 32D cells (clone 3, a gift of Dr Arati
Khanna-Gupta, Yale University) have been previously
described.11,12 Cells were grown in Iscove's modified
Dulbecco's medium (IMDM; GIBCO Life Technologies, Gaithersburg, MD)
with 10% fetal calf serum (FCS) and 2 ng/mL murine IL-3 (StemCell
Technologies, Vancouver, Canada) at 37°C in 5% CO2.
Cloning and expression of the mutant human EpoR cDNA and generation of
32D cells stably transfected with pRc/CMV expression
vector (Invitrogen, San Diego, CA) containing the wild-type (32D/EpoR
WT cells) or mutant (32D/EpoR FE cells) EpoR cDNAs have been
described.9 As a control, 32D cells were similarly
transfected with empty pRc/CMV vector (32D/neo cells). The stably
transfected pools of 32D/EpoR WT and 32D/EpoR FE cells 32D were
maintained in 2 U/mL Epo (a gift of Ortho Biotech, Raritan, NJ) and 0.2 mg/mL G418 (Sigma, St Louis, MO) or 2 ng/mL IL-3 and 0.4 mg/mL G418 (32D/neo cells). For some experiments, the transfected cells
were grown in 100% defined serum-free media (Aim-V; GIBCO BRL, Grand
Island, NY) containing the above growth factors. The experiments
reported here were all performed with pools of transfected clones.
Identical results were obtained with several single cell clones of each
transfectant obtained by limiting dilution (see Fig 4 and data not
shown).
Cell growth assays.
32D EpoR transfectants (5,000 cells per well) were cultured in 96-well
plates in IMDM/10% FCS containing 2 U/mL Epo. After 3 days, the viable
cells were assayed in triplicate with MTT
(dimethylthiazol-2-yl-2,5-diphenyltetrazolium) as previously
described.13 Aliquots were removed from similarly treated
wells and assayed for viable cells by the trypan blue exclusion
technique. For thymidine incorporation assays, 20,000 cells were
cultured in 1.5 mL of IMDM/10% FCS containing 2 U/mL Epo. 0.2 µCi of
3H-thymidine was added and, after an additional 4 hours of
culture, trichloroacetic acid precipitable radioactivity was measured.
AG 490 inhibition of cells.
32D transfectants (10,000 cells per mL) were cultured in 24-well plates
in 2 mL of IMDM/10% FCS containing 2 ng/mL IL-3 (32D/neo) or 2 U/mL
Epo (32D/EpoR WT and 32D/EpoR FE). The Jak2 inhibitor AG 490 (Calbiochem, San Diego, CA) was added at varying concentrations (0 to
10 µmol/L) at 0 hours and again at 24 and 48 hours. At 72 hours,
myeloperoxidase (Mpo) cytochemical stains were performed on cytospin
preparations.
Immunoblots.
The 32D parent line and the 32D EpoR transfectants were grown for 3 days in IMDM/10% FCS containing 10% WEHI conditioned media (as a
source of IL-3) with varying concentrations of Epo. 1 × 105 cells were then electrophoresed on a 10% sodium
dodecyl sulfate (SDS) gel, transferred to nitrocellulose, and
immunoblotted with polyclonal rabbit antibody against mouse hemoglobin
(Cappel, Durham, NC). Blots were developed with chemiluminescent
reagent (Sigma). A total of 1 × 104 mouse
reticulocytes were used as a positive control. Reticulocytes were
prepared from phenylhydrasine-treated mice and represented about 30%
of the total number of red blood cells in the specimen used.
Cytochemical staining of cells.
Mpo cytochemical assay was performed on cytospin preparations with
p-phenylenediamine/catechol reagent (Sigma) as described by the
manufacturer. For assay of percent Mpo positive cells, 10,000 cells
were stained for Mpo as described above. At least 200 cells per slide
were counted and only unequivocally stained cells were considered
positive. Each point was performed in triplicate using cytospins from
three independent wells. Wright-Giemsa staining was performed by
standard methods.14
| |
RESULTS |
32D cells transfected with the full-length (32D/EpoR WT) and truncated
(32D/EpoR FE) human EpoR constructs (Fig 1)
were analyzed for growth in the presence of Epo. 32D cells were chosen
for these experiments because they do not express endogenous murine
EpoR, as judged by ligand binding studies using
125I-Epo.9 At 2 U/mL of Epo, both cell lines
grow at an equivalent rate as indicated by 3H-thymidine
incorporation (Fig 2). Similar
results were obtained using MTT cell proliferation assay or viable cell
counts using trypan blue (data not shown). This differs from the
Epo-stimulated growth at lower Epo concentrations (0.01 to 0.1 U/mL) at
which the 32D/EpoR FE cells proliferate much faster than the 32D/EpoR WT cells.9 When analyzed with Hoescht staining, fewer than 1% apoptotic cells were noted during Epo-induced log phase growth in
both 32D/EpoR WT and 32D/EpoR FE cells (data not shown). In the absence
of Epo, both 32D/EpoR WT and 32D/EpoR FE cells undergo apoptosis,
although 32D/EpoR FE cells initiate apoptosis at a slower rate than
EpoR WT cells after growth factor withdrawal (M. Arcasoy, K. Harris,
and B. Forget, manuscript submitted). Thus, at 2 U/mL
Epo, both 32D/EpoR WT and 32D/EpoR FE cells have similar mitogenic and
antiapoptotic signaling. Neither parental 32D nor 32D/neo cells will
survive in Epo alone, presumably as a result of the absence of
endogenous EpoR.9
View larger version (30K):
[in this window]
[in a new window]
| Fig 1.
Structures of normal EpoR and EpoR truncation mutant. The
deletion found in the familial erythrocytosis mutation studied here is
shown (EpoR FE). There is a frameshift at the coding sequence of amino
acid 433 resulting in 17 novel amino acids (black box) followed by a
premature stop codon. The 70 terminal amino acids of the normal EpoR
(EpoR WT) are deleted in this mutation. The deletion includes 6 of the
9 cytoplasmic tyrosine residues (short vertical lines). The region
required for mitogenesis (which includes the box 1 and box 2 domains)
is shown in the open box.
|
|
View larger version (13K):
[in this window]
[in a new window]
| Fig 2.
32D cells transfected with either the full-length or
truncated EpoR demonstrate equivalent growth at high Epo
concentrations. Cells were grown in 2 U/mL Epo for 3 days and then
assayed in triplicate for 3H-thymidine incorporation.
32D/neo cells do not proliferate in Epo because they lack Epo
receptors. Standard error bars are indicated.
|
|
Despite similar proliferation under these conditions, the two
EpoR-transfected cell lines have a markedly different morphology. Wright-Giemsa-stained preparations (Fig
3A) demonstrate that the mock transfected 32D/neo cell line, as well as
the 32D/EpoR FE cells, have a monocytoid appearance with large vacuoles
and little evidence of granules. The nuclei have frequent nucleoli and
an open chromatin pattern. This morphology for 32D cells is very similar to that described in previous reports.12,15,16
View larger version (66K):
[in this window]
[in a new window]
| Fig 3.
Morphology of 32D transfectants. Cells were grown for 3 days in either 2 ng/mL IL-3 (32D/neo), or 2 U/mL Epo (32D/EpoR WT and
FE cells). Cytospin preparations were prepared for Wright-Giemsa stain
or Mpo stain. Original magnification is 500X.
|
|
The 32D/EpoR WT cells, on the other hand, have a dimorphic morphology
on passage in Epo. About 50% of the cells are similar to the 32D
parent line and the 32D/EpoR FE cells. The other 50% have a
granulocytic morphology with a decreased number of vacuoles and many
granules. The nucleus has a more mature morphology with condensed
chromatin and infrequent nucleoli. The morphology of these cells
indicates differentiation to the stage of metamyelocytes and bands.
This morphology for 32D/EpoR WT is consistent with previously described
G-CSF-induced granulocytic maturation of 32D cells.12 In
our hands, G-CSF induces similar granulocytic differentiation in 32D,
32D/neo, 32D/EpoR WT, and 32D/EpoR FE cells (data not shown). The
increased granulation of the 32D/EpoR WT cells in the presence of Epo
was also demonstrated by an increase in side scattering on flow
cytometric analysis (data not shown).
To verify that the 32D/EpoR WT cells were undergoing granulocytic
differentiation, the cell lines were stained for the granulocytic enzyme myeloperoxidase. As shown in Fig 3B, there are numerous Mpo
positive cells among the 32D/EpoR WT cells. 32D/EpoR FE and 32D/neo
routinely stains negative (Fig 3B), as do parental 32D cells (data not
shown). Analogous results were obtained using northern blots, ie,
strong expression of Mpo mRNA in 32D/EpoR WT cells and no detectable
expression in 32D and 32D/EpoR FE cells (data not shown).
To ensure that the results described above were not due to a spurious
integration event in a subpopulation of cells in the pools tested,
single cell clones of the 32D transfectants were tested for Mpo
expression. Figure 4 demonstrates that in
multiple single cell clones approximately 40% 32D/EpoR WT cells
express Mpo. Both 32D/neo and 32D/EpoR FE express Mpo in 1% to 3% of
cells. Similar results were obtained when cells were grown in either 10% fetal calf serum (Fig 4, open bars) or in 100% defined serum-free media (Fig 4, dotted bars).
View larger version (20K):
[in this window]
[in a new window]
| Fig 4.
Expression of Mpo in single cell clones of 32D
transfectants. Single cell clones of the 32D transfectants (obtained by
limiting dilution) were grown in either 10% FCS (open bars) or 100%
serum-free media (dotted bars) in the presence of 2 ng/mL IL-3
(32D/neo) or 2 U/mL Epo (32D/EpoR WT and 32D/EpoR FE) for 3 days and
then cytochemical stains for Mpo were performed. A total of 200 cells were counted for each individual clone tested. The number of clones tested was 2 for neo, 5 for EpoR WT, and 5 for EpoR FE. The bars indicate standard error.
|
|
Neither the full-length nor truncated EpoR was capable of inducing
erythroid differentiation of 32D cells.
Figure 5 demonstrates a Western blot for
murine hemoglobin of lysates from 32D, 32D/EpoR WT, and 32D/EpoR FE
cells grown at various concentrations of Epo. There is no expression of
globin proteins in any of the three cell lines.
View larger version (33K):
[in this window]
[in a new window]
| Fig 5.
32D transfectants do not express globin. Cells were grown
for 3 days in 10% WEHI conditioned media (as a source of IL-3)
containing 0 (lane 1), 0.1 (lane 2), 0.5 (lane 3), 1 (lane 4), or 5 U/mL Epo (lane 5). The lysate from 1 × 105 cells was
electrophoresed in a 10% SDS gel and then immunoblotted with antimouse
hemoglobin. Lane labeled "Ret" indicates mouse reticulocytes (1 × 104 cells) as a positive control.
|
|
Regulation of the Jak/Stat pathway after Epo stimulation differs in
32D/EpoR FE compared with 32D/EpoR WT cells (M. Arcasoy, K. Harris, and
B. Forget, manuscript submitted), probably as a result of
deletion of the C terminal negative regulatory domain of the
EpoR.10,17 In 32D/EpoR FE cells, Jak2 and Stat5 remain phosphorylated and active for a longer time after Epo withdrawal than
in 32D/EpoR WT cells. Because these two cell lines also differ in their
ability to differentiate after Epo exposure, we wondered whether the
granulocytic differentiation driven by the distal EpoR cytoplasmic
domain is mediated by the level of activation of the Jak/Stat pathway.
The specific Jak2 inhibitor tyrphostin AG 49018 was used to
inhibit Jak2 signaling in the 32D/neo cell line and the cells
transfected with the full-length and truncated EpoR. As expected, there
was a dose-dependent effect of the inhibitor on cellular proliferation of all three cell lines (Fig 6A), although
there was some cell proliferation at all concentrations of inhibitor
used except the 10 µmol/L concentration (note the dotted line
indicating the initial cell number). At 10 µmol/L AG 490, an
increased number of cells demonstrated nuclear fragmentation (data not
shown) suggesting that at this concentration of inhibitor, cellular
proliferation was approximately balanced by cell death.
View larger version (57K):
[in this window]
[in a new window]
| Fig 6.
Effect of the Jak2 inhibitor AG 490 on the growth and
differentiation of 32D transfectants. Cells were cultured in IMDM/10% FCS containing 2 ng/mL IL-3 (32D/neo cells) or 2 U/mL Epo (32D/EpoR WT
and 32D/EpoR FE cells). The indicated concentration of the Jak2
inhibitor AG 490 was added at 0 hours and again at 24 and 48 hours. The
cells were studied at 72 hours. AG 490 concentrations were 0, 0.1, 1, 2, 5, and 10 µmol/L. 32D/neo ( ), 32D/EpoR WT ( ), 32D/EpoR FE
( ). (A) The viable cells were counted and expressed as a function of
increasing AG 490. The initial cell number (10,000 cells) is indicated
by the dotted line. Each point was done in triplicate. Standard error
at each point was 10% or less of indicated value and has been omitted
for clarity. (B) Cytospins were stained for Mpo. Mpo positive cells
were counted and expressed as a function of increasing AG 490. Each
point was done in triplicate with standard error bars shown. (C)
Representative fields of Mpo-stained cytospins of 32D/EpoR WT cells at
the indicated AG 490 concentrations.
|
|
In addition to these effects on cell growth, there was a dose-dependent
inhibition of granulocytic differentiation, as determined by Mpo
cytochemical staining (Fig 6B). In this experiment, there was a
dose-dependent decrease in Epo-induced Mpo positivity in the 32D/EpoR
WT cells from 49% ± 4% to 16% ± 2% over the range of 0 to
10 µmol/L AG 490. The inhibitor had little effect on Mpo positivity
of the 32/EpoR FE or 32D/neo cells (0% to 3% positivity at all
concentrations of inhibitor). Figure 6C shows representative Mpo stains
of 32D/EpoR WT cells at two concentrations of inhibitor. Similar
results were obtained in two other experiments. Wright-Giemsa-stained preparations of this experiment confirmed the inhibition of
granulocytic differentiation in 32D/EpoR WT, ie, there was a
dose-dependent decrease in the number of morphologically recognizable
granulocytic cells with increasing AG 490 (data not shown).
| |
DISCUSSION |
The results we present here demonstrate that myeloid differentiation in
32D cells does not require stimulation by G-CSF. Signal transduction
via an hematopoietic growth factor receptor from another cell lineage
is able to effectively substitute for G-CSF. When stimulated with Epo,
transfected EpoR was able to induce myeloid differentiation of this
cell line, as judged by Mpo expression, granule formation, and
characteristic nuclear condensation and clumping (Figs 3 and 4). The
Epo/EpoR interaction was sufficient to induce granulocytic
differentiation of 32D cells, as equivalent levels of differentiation
were obtained in either FCS or serum-free media (Fig 4). This indicates
that other maturation factors are not required for this effect. The
Epo/EpoR interaction was not able to induce erythroid differentiation
in this cell line, as judged by morphology (Fig 3) or globin expression
(Fig 5).
These results are similar to previous reports with other hematopoietic
lineages. The prolactin receptor is able to substitute for the EpoR in
mediating erythroid differentiation,8 and the G-CSFR
cytoplasmic domain is able to substitute for the thrombopoietin receptor cytoplasmic domain in megakaryocyte
differentiation.19 We have now extended these observations
to the myeloid lineage. It seems clear from these data that growth
factors are not "instructive" in hematopoietic differentiation.
Nor do our results support a simple permissive model of the role of
growth factors in myeloid differentiation. Both the 32D/EpoR WT and
32D/EpoR FE cell lines grow at equivalent rates in the presence of 2 U/mL Epo (Fig 2). Very few apoptotic cells were noted in either cell
line in the presence of Epo. Upon Epo withdrawal, 32D/EpoR FE cells
actually initiate apoptosis at a slower rate than 32D/EpoR WT cells.
Clearly, the full-length EpoR is providing signals that do more than
support the cell long enough to allow expression of a predetermined
endogenous program. This conclusion is consistent with similar work in
32D cells demonstrating that suppression of apoptosis by overexpression
of Bcl-2 is not sufficient to support myeloid
differentiation.15 Likewise, Pless et al20 have
shown that erythroid differentiation of BaF3 cells requires dimerization of two full-length hematopoietic receptor subunits. In
their study, combinations of receptor subunits that did not include two
distal receptor domains were not able to induce differentiation, despite intact mitogenic signaling.
32D cells were chosen for our experiments because they represent the
most commonly studied cell line for in vitro myeloid differentiation11,12,15,16 (and the references contained therein). 32D cells have been extensively used because they are growth
factor-dependent, nontumorigenic, and have a normal
karyotype.11,12 They may thus more accurately reflect the
molecular events leading to granulocytic differentiation than other
nongrowth factor-dependent tumor cell lines such as HL-60 or M1.
Our results indicate that the distal 70 amino acids of the EpoR are
required for myeloid differentiation in this system. This is similar to
results described for the G-CSFR. For instance Fukunaga et
al21 demonstrated that the distal half of the G-CSFR is
required for myeloid differentiation in the FDCP cell
line. This differentiation domain, like the domain we have described
here, is dispensable for mitogenesis. Interestingly, similar results
have not been forthcoming with the EpoR. In TSA8 cells6 and
primary erythroid precursor cells,8 the distal EpoR is not
required for erythroid differentiation. It is possible that myeloid
differentiation is intrinsically different than erythroid
differentiation in the requirement for distal receptor signaling. A
suggestion that this is the case comes from the human mutations that
result in the truncation of hematopoietic growth factor receptors. In
these conditions, the EpoR truncation results in
erythrocytosis,22 while the G-CSFR truncation results in
neutropenia.16 The role of the Jak/Stat pathway in
hematopoietic differentiation is controversial. Some investigators have
seen a direct correlation between increased Stat activation by the EpoR
and erythroid differentiation,23 while others have seen an
inverse relationship.20 Our results suggest that intact Jak
signaling is required, but not sufficient for Epo-induced myeloid
differentiation in 32D/EpoR WT cells. It is important to keep in mind
that Jak2 may have other physiological substrates in addition to the
Stat proteins.
The EpoR binds and activates Jak2, while the G-CSFR binds and activates
numerous members of this family including Jak1, Jak2, and
Tyk2.24 Jak1 activation, however, may be critical in G-CSFR signaling.24 If Jak signaling is indeed required for
myeloid differentiation, our results would suggest that Jak2 can
substitute for Jak1 in this process.
The findings presented here, in conjunction with those of others
mentioned above, suggest that it is likely that under most situations
an hematopoietic growth factor-induced signal is required for
differentiation of hematopoietic precursor cells. This signal is
distinct from mitogenic and antiapoptosis signaling and, at least in
the case of myeloid differentiation, requires the distal domain of an
hematopoietic receptor. The specificity of the signal delivered,
however, is determined by the cell, presumably through some unknown
molecular mechanism associated with commitment. The availability of
numerous well characterized EpoR mutants, as well as the recent rapid
development of other tools for the study of signal transduction
pathways, should allow identification of the mechanism by which the
distal 70 amino acids of the EpoR are able to induce myeloid
differentiation in this system.
| |
FOOTNOTES |
Submitted January 21, 1998;
accepted April 15, 1998.
Supported by a Veterans Administration Merit Review grant, San Antonio
Cancer Institute grant, and a University of Texas Health Science Center
at San Antonio Howard Hughes Medical Institute Institutional Resources
grant (to K.W.H.), and Grant No. DK 44058 from the National Institutes
of Health, Bethesda, MD (to B.G.F.).
Address reprint requests to Kevin W. Harris, MD, PhD, Department of
Medicine, Division of Hematology, University of Texas Health Science
Center, 7703 Floyd Curl Dr, San Antonio, TX 78284-7880.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Qing Shen for technical help, David Haile for the reticulocyte
lysate, Yair Gazitt for help with flow cytometry, Katri Selander for
performing the Hoescht stain analysis of apoptosis, and Ortho Biotech
for a gift of human Epo.
| |
REFERENCES |
1.
Wells JA,
de Vos AM:
Hematopoietic receptor complexes.
Annu Rev Biochem
65:609,
1996[Medline]
[Order article via Infotrieve]
2.
Ihle JN:
Cytokine receptor signalling.
Nature
377:591,
1995[Medline]
[Order article via Infotrieve]
3.
Miura Y,
Miura O,
Ihle JN,
Aoki N:
Activation of the mitogen-activated protein kinase pathway by the erythropoietin receptor.
J Biol Chem
269:29962,
1994[Abstract/Free Full Text]
4.
Koury M,
Bondurant M:
Erythropoietin retards DNA breakdown and prevents programmed cell death in erythroid progenitor cells.
Science
248:378,
1990[Abstract/Free Full Text]
5.
Krosl J,
Damen JE,
Krystal G,
Humphries RK:
Erythropoietin and interleukin-3 induce distinct events in erythropoietin receptor-expressing BA/F3 cells.
Blood
85:50,
1995[Abstract/Free Full Text]
6.
Maruyama K,
Miyata K,
Yoshimura A:
Proliferation and erythroid differentiation through the cytoplasmic domain of the erythropoietin receptor.
J Biol Chem
269:5976,
1994[Abstract/Free Full Text]
7.
Fairbairn L,
Cowling G,
Reipert B,
Dexter T:
Suppression of apoptosis allows differentiation and development of a multipotent hemopoietic cell line in the absence of added growth factors.
Cell
74:823,
1993[Medline]
[Order article via Infotrieve]
8.
Socolovsky M,
Dusanter-Fourt I,
Lodish HF:
The prolactin receptor and severely truncated erythropoietin receptors support differentiation of erythroid progenitors.
J Biol Chem
272:14009,
1997[Abstract/Free Full Text]
9.
Arcasoy MO,
Degar BA,
Harris KW,
Forget BG:
Familial erythrocytosis associated with a short deletion in the erythropoietin receptor gene.
Blood
89:4628,
1997[Abstract/Free Full Text]
10.
Klingmüller 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]
11.
Greenberger JS,
Sakakeeny MA,
Keith HR,
Eaves CJ,
Eckner RJ:
Demonstration of permanent factor-dependent multipotential (erythroid/neutrophil/basophil) hematopoietic progenitor cell lines.
Proc Natl Acad Sci USA
80:2931,
1983[Abstract/Free Full Text]
12.
Migliaccio G,
Migliaccio AR,
Krieder BL,
Rovera G,
Adamson JW:
Selection of lineage-restricted cell lines immortalized at different stages of hematopoietic cell differentiation from the murine cell line 32D.
J Cell Biol
109:833,
1989[Abstract/Free Full Text]
13.
Mosmann T:
Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.
J Immunol Methods
65:55,
1983[Medline]
[Order article via Infotrieve]
14.
Williams WJ:
Polychrome staining
, in Williams WJ,
Beutler E,
Erslev AJ,
Lichtman MA
(eds):
Hematology
New York, NY, McGraw-Hill
, 1990
, p 1699
15.
Rodel JE,
Link DC:
Suppression of apoptosis during cytokine deprivation of 32D cells is not sufficient to induce complete granulocytic differentiation.
Blood
87:858,
1996[Abstract/Free Full Text]
16.
Dong F,
Brynes RK,
Tidlow N,
Welte K,
Löwenberg B,
Touw IP:
Mutations in the gene for the granulocytic colony-stimulating factor receptor in patients with acute myeloid leukemia preceded by severe congenital neutropenia.
N Engl J Med
333:487,
1995[Abstract/Free Full Text]
17.
D'Andrea AD,
Yoshimura A,
Youssoufian H,
Zon LI,
Koo J-W,
Lodish HF:
The cytoplasmic region of the erythropoietin receptor contains nonoverlapping positive and negative growth-regulatory domains.
Mol Cell Biol
11:1980,
1991[Abstract/Free Full Text]
18.
Meydan N,
Grunberger T,
Dadi H,
Shahar M,
Arpala E,
Lapidot Z,
Leeder JS,
Freedman M,
Cohen A,
Gazit A,
Levitzki A,
Roifman CM:
Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor.
Nature
379:645,
1996[Medline]
[Order article via Infotrieve]
19. (abstr)
Stoffel R,
Ledermann B,
Sauvage FJd,
Skoda RC:
Evidence for a selective-permissive role of cytokine receptors in hematopoietic cell fate decisions.
Blood
90:535a,
1997
20.
Pless M,
Norga K,
Carroll M,
Heim MH,
D'Andrea A,
Mathey-Prevot B:
Receptors that induce erythroid differentiation of Ba/F3 cells: Structural requirements and effect on STAT5 binding.
Blood
89:3175,
1997[Abstract/Free Full Text]
21.
Fukunaga R,
Ishizaka-Ikeda E,
Nagata S:
Growth and differentiation signals mediated by different regions in the cytoplasmic domain of the granulocyte colony-stimulating factor receptor.
Cell
74:1079,
1993[Medline]
[Order article via Infotrieve]
22.
de La Chappelle A,
Traskelin A,
Juvonen E:
Truncated erythropoietin receptor causes dominantly inherited benign human erythrocytosis.
Proc Natl Acad Sci USA
90:4495,
1993[Abstract/Free Full Text]
23.
Iwatsuki K,
Endo T,
Misawa H,
Yokouchi M,
Matsumoto A,
Ohtsubo M,
Mori KJ,
Yoshimura A:
STAT5 activation correlates with erythropoietin receptor-mediated erythroid differentiation of an erythroleukemia cell line.
J Biol Chem
272:8149,
1997[Abstract/Free Full Text]
24.
Shimoda K,
Feng J,
Murakami H,
Nagata S,
Watling D,
Rogers NC,
Stark GR,
Kerr IM,
Ihle JN:
Jak1 plays an essential role for receptor phosphorylation and Stat activation in response to granulocyte colony-stimulating factor.
Blood
90:597,
1997[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
R. Thorpe and S. J Swanson
Current Methods for Detecting Antibodies against Erythropoietin and Other Recombinant Proteins
Clin. Vaccine Immunol.,
January 1, 2005;
12(1):
28 - 39.
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. K. Kaulsay, H. C. Mertani, K.-O. Lee, and P. E. Lobie
Autocrine Human Growth Hormone Enhancement of Human Mammary Carcinoma Cell Spreading Is Jak2 Dependent
Endocrinology,
April 1, 2000;
141(4):
1571 - 1584.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Zhu and P. E. Lobie
Janus Kinase 2-dependent Activation of p38 Mitogen-activated Protein Kinase by Growth Hormone. RESULTANT TRANSCRIPTIONAL ACTIVATION OF ATF-2 AND CHOP, CYTOSKELETAL RE-ORGANIZATION AND MITOGENESIS
J. Biol. Chem.,
January 21, 2000;
275(3):
2103 - 2114.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract