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Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 870-878
Redundant and Selective Roles for Erythropoietin Receptor
Tyrosines in Erythropoiesis In Vivo
By
Gregory D. Longmore,
Yun You,
Jaime Molden,
Kathleen D. Liu,
Aki Mikami,
Stephen Y. Lai,
Pamela Pharr, and
Mark A. Goldsmith
From the Departments of Medicine and Cell Biology, Washington
University School of Medicine, St Louis, MO; the Gladstone Institute of
Virology and Immunology, San Francisco, CA; the Department of Medicine,
School of Medicine, University of California, San Francisco; and Ralph
H. Johnson Department of Veterans Affairs Medical Center and Department
of Medicine, Medical University of South Carolina, Charleston.
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ABSTRACT |
Cytokine receptors have been shown in cell culture systems to use
phosphotyrosine residues as docking sites for certain signal transduction intermediates. Studies using various cellular backgrounds have yielded conflicting information about the importance of such residues. The present studies were undertaken to determine whether or
not tyrosine residues within the erythropoietin receptor (EPOR) are
essential for biologic activity during hematopoiesis in vivo. A variant
of the EPOR was constructed that contains both a substitution (R129C)
causing constitutive receptor activation as well as replacement of all
eight cytoplasmic tyrosines by phenylalanines (cEPORYF). A comparison
between animals exposed to recombinant retroviruses expressing cEPOR
and cEPORYF showed that efficient red blood cell (RBC) development in
vivo is dependent on the presence of tyrosine residues in the
cytoplasmic domain of the EPOR. In addition, an inefficient EPOR
tyrosine independent pathway supporting RBC development was detected.
Tyrosine add-back mutants showed that multiple individual tyrosines
have the capacity to restore full erythropoietic potential to the EPOR
as determined in whole animals. The analysis of primary erythroid
progenitors transduced with the various cEPOR tyrosine mutants and
tyrosine add-backs showed that only tyrosine 343 (Y1) and tyrosine 479 (Y8) were capable of supporting immature burst-forming unit-erythroid
progenitor development. Thus, this receptor is characterized by
striking functional redundancy of tyrosines in a biologically relevant
context. However, selective tyrosine residues may be uniquely important
for early signals supporting erythroid development.
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INTRODUCTION |
MAMMALIAN BLOOD CELL development depends
on exquisite timing, critical cell fate decisions, and remarkable
renewal capacity.1 These features are coordinated in vivo
to produce an orderly sequence of steps by which stem cell populations
differentiate into mature erythroid, myeloid, and lymphoid cell types.
Hematopoietic cytokines are critical for the development and expansion
of blood cells. However, whether or not cell fate decisions during
blood cell development are directly affected by hematopoietic cytokines
is not entirely clear.2 If so, the specific signaling
pathways controlling the distinct biological outcomes resulting from
cytokine actions are not known.
A prototypical hematopoietic cytokine is erythropoietin (EPO), a
34-kD glycoprotein that is a major determinant of
erythroid cell development.3 Extensive cell culture and
genetic analyses have shown that EPO exerts survival, proliferative,
and differentiative effects on red blood cell (RBC)
progenitors.3-8 These actions are mediated by the
erythropoietin receptor (EPOR),9 which is believed to
operate as a noncovalent complex containing two identical 64-kD
transmembrane proteins. Engagement by EPO apparently promotes
dimerization of these receptor subunits, thereby initiating transmembrane signaling that leads to cellular responses.10 Indeed, a mutant EPOR (EPOR(R129C) or cEPOR) that homodimerizes constitutively in the absence of ligand also causes ligand-independent growth of cell lines containing this form of the EPOR.11
Like other cytokine receptors, the native EPOR has been the subject of
extensive molecular characterization in immortalized cell lines to
define its functional domains and the signal transduction circuitry linked to these domains.
An important scientific goal is to define the molecular mechanisms by
which specificity is maintained in signaling responses mediated by
hematopoietic cytokine receptors such as the EPOR, and to relate these
mechanisms to blood cell development within the whole animal context.
Studies of structure-function relationships within the EPOR in
immortalized cell culture models collectively implicate phosphorylation
of specific tyrosine residues within the cytoplasmic tail of the EPOR
as a key determinant of growth signaling and/or selective
activation of various signaling intermediates.12-15 Although such studies have been quite instructive, the information derived is confounded somewhat by limitations inherent to studies using
immortalized cell culture systems, such as variability caused by
differences in cell context, and the inability of cell lines to
recapitulate all cellular responses ascribed to EPO. For example, one
major signaling pathway engaged by the EPOR is the JAK-STAT system. The
tyrosine kinase JAK2 becomes activated rapidly upon receptor
engagement16 followed by induction of the transcription factor STAT-5.12,13,17-21 Studies with selective EPOR
cytoplasmic tyrosine mutants in cell culture systems have suggested a
surprising degree of redundancy in specific tyrosine use in the
activation of STAT-5.12,13,17-21 More importantly, these
results offer little information about how these and other early
signaling events are linked to downstream cellular consequences (eg,
expansion of erythroid progenitor pools, maturation of RBCs, and
survival effects) that are important for normal RBC physiology in vivo.
The present studies employed a novel strategy to investigate
structure-function relationships of the tyrosine residues in the
cytoplasmic tail of the EPOR as they relate to complete erythropoiesis in vivo. This approach exploits the previous report that the
constitutively active mutant cEPOR11 causes dramatic
expansion of the erythroid cell compartment in mice upon introduction
via recombinant spleen focus-forming viruses.22 We have
used this "dominant" genetic system to test the signal
transduction and biological activities of site-directed mutants of the
EPOR in vivo. The results showed both the tyrosine-dependence of these
processes for efficient RBC development, and a remarkable degree of
functional redundancy in tyrosine use.
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MATERIALS AND METHODS |
Constructs and viruses.
A synthetic DNA fragment encoding a mutant cytoplasmic tail of the
murine EPOR in which all eight tyrosine residues were replaced by
phenylalanines was constructed by oligonucleotide annealing, and this
DNA fragment was subcloned into the corresponding region of the
cEPOR-cDNA to generate cEPORYF. Tyrosine add-back mutants (Fig 1A) were
prepared by polymerase chain reaction-based amplification of short
segments using primers containing the desired tyrosine replacement
substitutions, and subsequent insertion of these segments into the
corresponding region of the cEPORYF-cDNA backbone. All constructs were
verified by automated DNA sequencing. For studies in cell lines, these
cDNAs were inserted into the pCMV4Neo expression vector. For studies in
vivo, these cDNAs were subcloned into the previously described proviral
plasmid pSFF. Retroviruses were generated from these plasmids and
characterized as previously described22,23 and as described
below, and adult NIH/Swiss mice subsequently were inoculated as
described.
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| Fig 1.
Tyrosine mutants of the EPOR and effects in cell culture.
(A) Schematic of the parental cEPOR containing an intact cytoplasmic tail and the R129C substitution in the extracellular domain (WT), the
cEPORYF mutant in which all eight cytoplasmic tyrosines have been
replaced by phenylalanines (YF), and various mutants retaining selected
tyrosine residues as shown (Y1F, YF:1Y, YF:1-4Y, YF:5-8Y, and YF:8Y).
For convenience, specific tyrosine positions in the cytoplasmic segment
have been numbered 1-8 as indicated. (B) Immunoblot analysis showing
comparable levels of the cEPOR variants in transfected BaF3 cell lines.
Arrow, EPOR. (C) Titers of SFFV-cEPOR viruses containing the tyrosine
variants described above, as determined in infection analyses with
fibroblasts. The schematic represents a genetic map of these
recombinant retroviruses. The immunoblot displays detection of the
cEPOR proteins from the indicated viruses.
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Cell lines and growth factors.
The interleukin-3 (IL-3)-dependent pro-B cell line BaF3 has been
described previously.24 Growth factor independence was assessed by transfecting cell cultures with the indicated plasmids by
electroporation as previously described, culturing the cells in IL-3
and G418 for 1 week, and then changing the medium to exclude IL-3.
After 10 days it was readily apparent by visual inspection and trypan
blue staining which cultures were viable and expanding (designated
herein as factor-independent) compared with those that had expired. The
cEPORYF cell line required the continuous presence of IL-3.
Hematopoietic cell lines were established from spleens of infected mice
(see below) as described previously.22,23 Briefly, splenic
cells from infected mice exhibiting splenomegaly were dispersed and
placed in culture medium (Iscove's modified Dulbecco's medium/20%
fetal calf serum, containing  -mercaptoethanol) lacking any added
growth factors. Such cell lines from animals infected with each of the
viruses were maintained indefinitely in culture, except those from mice
infected with SFFV-cEPORYF (see text). Culture supernatants from
WEHI-3B cells (American Type Culture Collection) were used as a source
of IL-3. Recombinant human EPO was provided as a gift by Ortho Biotech
(Raritan, NJ).
Expression analysis and immunoblotting.
Whole cell lysates prepared from transfectants or from the murine cell
lines described above were analyzed by immunoblotting with an antisera
directed against the C-terminal segments of the EPOR as described
previously.25,26 For analysis of viral titers, approximately 5 × 105 NIH 3T3 fibroblast cells in a P100
plate were infected with 4 mL of culture supernatent from retroviral
producer cells. After infection, cells grown to confluence were
harvested and lysed. From the detergent soluble extract 150 µg of
protein was resolved by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE; 8% gels), transferred to nitrocellulose,
and immunoblotted with antisera raised against the C-terminal tail of
the EPOR (see above). For splenic extracts, single cell suspensions of
virally infected or uninfected spleen cells were prepared. The cells
were washed once with phosphate-buffered saline (PBS) and lysed in 1%
Triton X-100; 150 mmol/L NaCl; 20 mmol/L Tris.Cl, pH 7.4; 1 mmol/L
EDTA; 0.5% NP-40, containing aprotinin; and phenylmethylsulfonyl
fluoride (PMSF) at 4°C for 15 minutes. Samples were clarified by
centrifugation at 10,000g for 15 minutes. Soluble protein
concentration was determined using the Pierce Chemical BCA protein
determination kit (Pierce Chemical Corp, Rockford, IL).
From each extract 150 µg of protein was applied to an 8% SDS-PAGE
and products were resolved under reducing conditions, transfered to
nitrocellulose, and immunoblotted with antisera against the EPOR.
Signal transduction assays.
JAK2 phosphorylation was measured by preparing whole cell lysates from
resting or stimulated cells and conducting immunoprecipitation with an
anti-JAK2 antiserum followed by immunoblot analysis as described
previously.27 Electrophoretic mobility shift assays (EMSAs)
were performed as described previously.28 Mitogen-activated kinase (MAPK) assays were performed using a nonradioactive MAP kinase
assay kit (New England Biolabs, Beverly, MA) according to
the manufacturer's instructions. In brief, 30 to 50 × 106 cells per sample were washed twice in
calcium-magnesium-free PBS and stripped of growth factors by
incubation in 150 mmol/L NaCl and 10 mmol/L Na citrate, pH 4, for 1 minute at room temperature. Cells were starved in RPMI 1430 supplemented with 1% bovine serum albumin (BSA; Sigma Chemical Co, St
Louis, MO) for 6 hours at 37°C. Cells were stimulated
with the indicated cytokines for 10 minutes and lysed in 220 to 300 ml
of lysis buffer. Immunoprecipitations were performed with the
anti-phosphoMAPK antibody. In vitro kinase assays were performed using
GST-Elk1 as an exogenous substrate; these kinase assays were subjected
to SDS-PAGE, followed by immunoblotting with an anti-phosphoElk1
antibody.
Infection and culture of hematopoietic progenitor cells.
Single cell suspensions of fetal liver were prepared from day 13 pregnant DBA-2 mice (Charles River Laboratories, Bar Harbor, ME). Cells
were washed three times in  medium. Cells (106) were
resuspended in medium containing fresh virus, and 4 µg/mL polybrene was added; cells were then incubated at
37°C for 3 hours. After infection, samples were washed in medium
and replated in medium containing 30% fetal bovine serum (Sterile
Systems, Logan, UT), 1% crystallized BSA, 1.2% 1,500 centipoise
methylcellulose (1 poise = 0.1 Pa.sec; Fischer Scientific Corp,
Pittsburgh, PA), recombinant murine stem cell factor (20 ng/mL; Genzyme Corp, Boston, MA) and 50 µmol/L
2-mercaptoethanol (Sigma) at a cell concentration of 2 × 104/mL. Partially purified human urinary EPO (specific
activity = 250 U/mg), a generous gift from M. Kawakita (Kumamoto
University, Japan), was used where indicated.
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RESULTS |
Tyrosine-dependent signaling by the EPOR.
Studies of mutants of the EPOR expressed in various cellular
backgrounds have yielded somewhat conflicting information about the
importance of tyrosine residues for receptor
function.12,13,15,29 In addition, the actual sites of
EPO-dependent tyrosine phosphorylation of the EPOR in either erythroid
progenitors cells in vivo or established erythroid cell lines have not
been identified. Therefore, we tested the capacity of cytoplasmic
tyrosine mutants of the EPOR to support RBC development in vivo by
using a receptor backbone containing the dominant EPO-independent
mutation (R129C) of the EPOR (referred to here as cEPOR).11
To establish the feasibility of such an approach, we constructed an
EPOR variant that contains the R129C mutation as well as replacement of
all eight cytoplasmic tyrosines by phenylalanines (cEPORYF) (Fig
1A). Stable transfectants of the
IL-3-dependent cell line BaF3 were prepared that express either cEPOR
or cEPORYF, and expression of each receptor was verified by
immunoblotting (Fig 1B). Cells expressing cEPOR grew continuously in
culture even on complete withdrawal of cytokines (IL-3 and EPO),
whereas cells expressing cEPORYF ceased to grow in the absence of
cytokines; these cells did exhibit modest growth with the addition of
exogenous EPO levels (10 U/mL). These observations suggested that
cEPORYF was not entirely devoid of growth-signaling potential but
rather was much less efficient compared with wild-type cEPOR. These
findings are consistent with earlier reports of a shifted dose response to EPO by tyrosine-negative mutants of the EPOR.12
To evaluate this function further in a more biologically relevant
cellular background, genes encoding cEPOR or cEPORYF were transferred into primary hematopoietic progenitor cells via recombinant erythroleukemic spleen focus-forming virus (SFFV; Fig 1C). Both viruses
were of comparable titer, as they infected and expressed their cEPOR
transgene products in fibroblasts with equal efficiency (Fig 1C, lanes
2 and 3). These viruses were then used to infect day 13 murine fetal
liver cells, and the development of erythroid colonies was measured. To
negate any contribution of endogenous wild-type EPOR to the outcome,
cultures were performed in the absence of added EPO (Table
1). As previously reported,30
when transduced into erythroid progenitors, the parental cEPOR potently stimulated EPO-independent mature colony-forming unit-erythroid (CFU-E) progenitor development and immature burst-forming
unit-erythroid (BFU-E) progenitor development (Table 1).
Interestingly, expression of the cEPORYF gene in erythroid progenitors
supported EPO-independent CFU-E development at a level above background
but significantly less than that of cEPOR (Table 1). Furthermore, in
contrast to cEPOR, cEPORYF failed to support any detectable
EPO-independent immature BFU-E progenitor development (Table 1). This
distinction between BFU-E and CFU-E responses implies somewhat
different requirements for tyrosines at different stages of erythroid
maturation (see below).
To assist in delineating signaling processes linked to the receptor
that may contribute to these cellular responses, the BaF3 transfectants
were evaluated for several recognized signal transduction pathways.
Immunoprecipitation of whole cell lysates with anti-JAK2 antibody
followed by immunoblotting with anti-phosphotyrosine antibody showed
comparable levels of constitutive tyrosine phosphorylation of JAK2 in
both transfectants with none detectable in the untransfected parental
line (Fig 2A); these constitutive levels of
phosphorylation in the transfectants were modestly increased with the
addition of low-dose (1 U/mL) or high-dose (10 U/mL) EPO to the
cultures. Thus, despite quantitative differences in the overall
expression of cEPOR and cEPORYF protein in the transfectants, such
differences were not reflected in important distinctions at the level
of this early marker of signal transduction.
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| Fig 2.
Signal transduction characteristics of cEPOR mutants in
BaF3 cells. (A) Whole cell lysates of the BaF3 transfectants described in Fig 1 were subjected to immunoprecipitation with anti-JAK2 antiserum
followed by separation by polyacrylamide gel electrophoresis and
immunoblotting analysis with anti-phosphotyrosine antibody. Cells were
either resting (0) or stimulated with EPO at low-dose (1, 1 U/mL) or
high-dose (10, 10 U/mL) EPO for 15 minutes before lysis. The blots were
reprobed with anti-JAK2 antibody to verify the identity of the
phosphoprotein band and equivalent gel loading (not shown). (B) Nuclear
extracts prepared from resting or EPO-stimulated BaF3 cell lines were
prepared and subjected to EMSA with an oligonucleotide probe containing
a STAT-binding sequence (Fc RI). Supershift analysis with anti-STAT5
antisera was used to confirm the identity of the STAT within the
retarded nucleoprotein complex (not shown). (C) Whole cell extracts
prepared from resting (0) or EPO-stimulated (1 U/mL or 25 U/mL, as
indicated) BaF3 cell lines were prepared and subjected to in vitro
kinase assays as described in Materials and Methods.
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Despite comparable levels of JAK2 phosphorylation, EMSAs showed a
marked difference in the induction of STAT5 by these receptors. Constitutive STAT5 DNA-binding activity was very faintly detectable in
cEPOR cells in the absence of exogenous EPO, and strongly induced by
low or high levels of exogenous EPO (Fig 2B). STAT DNA-binding activity
was nominal in cEPORYF cells even at high levels of EPO (Fig 2B), as
has been reported previously by others using EPORYF constructs without
the R129C backbone.12,15,29 The fact that some markers of
signaling via the cEPOR can be detected in transfected cells only after
the addition of EPO has been reported previously,31 and may
simply reflect limitations in the sensitivity among various assays.
Because the activation of MAPKs has been linked to cytokine-dependent
regulation of cell cycle progression, the cEPOR and cEPORYF
transfectants were compared for their abilities to couple to MAPK
activation as measured by in vitro kinase assays. As observed in the
STAT analyses, activation of MAPK by the cEPOR was detectable with the
addition of low- or high-dose EPO, but was negligible with cEPORYF (Fig
2C). Therefore, by multiple signal transduction parameters, cEPORYF
was found to be largely uncoupled from downstream signaling responses.
Tyrosine-dependent erythropoiesis mediated by the EPOR in
vivo.
To evaluate the relative capacity of these receptors to support
hematopoiesis in vivo, adult mice were infected with a mixture of the
defective SFFV-cEPOR virus and helper Rauscher (R)-MuLV.25 R-MuLV does not affect hematopoiesis of infected mice within the time
course of these experiments; rather, it simply supplies the capacity
for defective virus replication within the host.23 Weekly
hematocrits were determined (Fig 3A), and
when severe elevations in hematocrit were persistent, or at 6 weeks
following infection, mice were killed and spleen weight determined (Fig
3B). As expected, by 5 weeks after infection with SFFV-cEPOR, all
animals exhibited a marked expansion of mature RBCs (elevation in
hematocrit), indicative of stimulated erythroid
differentiation.22 In addition, massive splenomegaly
developed during this period, indicative of marked expansion of
erythroid progenitor populations (erythroid
proliferation).23,25 In contrast, SFFV-cEPORYF virus was
inefficient at stimulating either phase of erythropoiesis, as indicated
by only slight elevation in hematocrit or spleen size (Fig 3). Infected
diseased spleens were found to express cEPOR or cEPORYF proteins at
comparable levels when the differences in cellularity of diseased and
normal spleens were considered. Cell lysates prepared from spleen cell suspensions from these animals were tested by immunoblotting with the
anti-EPOR antiserum, which showed strong receptor expression in mice
infected with either construct (Fig 4B). As
determined in spleens from uninfected control animals, endogenous EPOR
protein was expressed at levels below the detection limits of the
assay. It is important to note that in animals infected with SFFV-cEPOR virus the enlarged spleens have been previously shown to be markedly and specifically enriched for erythroid cells compared with normal spleens from animals not manifesting disease. Therefore, the modest apparent differences in expression of cEPOR compared with cEPORYF in
infected spleens (with equivalent loading of extracts on a total
protein basis) most likely reflects differences in the degree of
replacement of splenic tissue with erythroid progenitors rather than
differences in receptor expression on a per-erythroid cell basis.
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| Fig 3.
Hematopoietic consequences of SFFV-cEPOR viruses in
infected mice. (A) Hematocrits of mice infected with SFFV-cEPOR ( ,
left) or SFFV-cEPORYF ( , right) measured serially following
inoculation. Each symbol represents a value from an individual animal,
and the dashed line indicates the normal hematocrit of an adult mouse. (B) Spleen weights of mice infected with SFFV-cEPOR or SFFV-cEPORYF determined after euthanizing the mice at the end of the experiment. Each symbol represents a value from an individual animal, and the
dashed line indicates the normal spleen weight of an adult mouse.
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| Fig 4.
Stimulated erythropoiesis in mice infected with viruses
encoding tyrosine add-back mutants of the EPOR. (A) Immediately before termination, hematocrits were determined for each animal infected with
viruses encoding the indicated cEPOR variants. Each symbol represents a
value from an individual animal, and the dashed line indicates the
normal hematocrit of an adult mouse. (B) Immunoblot analysis for
expression of the transduced EPOR protein in primary spleen cells
isolated from infected mice.
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One of the five mice injected with SFFV-cEPORYF developed a late, acute
rise in hematocrit and splenomegaly. However, in contrast to mice
infected with SFFV-cEPOR, no immunologically detectable EPOR was
present in the enlarged spleen or in primary splenic cells isolated
from this animal and grown in culture for 7 days (data not shown).
Furthermore, it was not possible to establish permanent growth
factor-independent cell lines from this diseased spleen, whereas such
lines were readily established from animals infected with cEPOR and
other constructs (see below). All other mice infected with SFFV-cEPORYF
developed a slight increase in hematocrit, and a small increase in
splenic weight after 6 weeks. Detectable cEPORYF protein was present in
splenic extracts from all these mice (data not shown). Therefore, the
mechanism of disease in this single animal remains unclear and appears
to be distinct from that produced by the cEPOR and other related
variants (see Discussion). Nonetheless, a comparison between animals
exposed to viruses expressing cEPOR and cEPORYF shows that efficient
RBC development in vivo is dependent on the presence of tyrosine
residues in the cytoplasmic domain of the EPOR. In addition, the
results suggest that an inefficient EPOR tyrosine-independent pathway supporting RBC development is active.
Reconstitution of EPOR-mediated signaling by multiple tyrosines.
This inefficiency of cEPORYF in stimulating erythropoiesis allowed us
to begin to determine the contribution of specific tyrosine residues in
the EPOR cytoplasmic tail to RBC development in vivo. Selected tyrosine
add-back mutants were constructed in the cEPOR backbone (Fig 1A) and
expressed in BaF3 cells to assess cellular responses. Unlike the
cEPORYF construct, each of the add-back constructs was capable of
supporting continuous growth factor-independent growth of transfected
BaF3 cells, indicating that they had more pronounced potential for
stimulating proliferation compared with cEPORYF.
The selective functioning of cEPORYF in CFU-E development but not in
BFU-E development suggested a differential role for tyrosines at
specific stages of erythroid maturation. For further analyses in
primary erythroid progenitor cell assays and in vivo, recombinant SFFV
viruses containing these EPOR tyrosine add-back mutants were generated
and found to exhibit equivalent titers as compared with SFFV-cEPOR and
SFFV-cEPORYF viruses (Fig 1C). When expressed in primary CFU-E
progenitors, each of these tyrosine add-back mutants strongly supported
mature CFU-E development on introduction into day 13 fetal liver cells
(Table 1), indicating that multiple individual tyrosine residues or
combinations of tyrosines had the capacity to enhance the biological
signaling potential of the cEPORYF. Interestingly, of the tyrosine
add-backs tested, only Y1 and Y8 restored the capacity of cEPORYF to
support immature BFU-E development when expressed in primary progenitor
cells (Table 1).
Various aspects of signaling function were evaluated in the
cytokine-independent transfectants expressing cEPORYF tyrosine add-back
mutants. With regard to the JAK-STAT pathway, cEPORY1F, cEPORYF:1Y, and
cEPORYF:1-4Y all exhibited constitutive or readily inducible STAT
DNA-binding activity, whereas cEPORYF:5-8Y and cEPORYF:8Y failed to
show detectable activity even with the addition of high-dose EPO (Fig
2B); these findings are consistent with those reported by others using
EPOR variants lacking R129C in BaF3.12,14,15 In contrast,
only cEPORY1F was a robust inducer of MAPK, whereas cEPORYF:1Y,
cEPORYF:1-4Y, cEPORYF:5-8Y, and cEPORYF:8Y showed MAPK induction only
with the addition of high-dose EPO (Fig 2C). Therefore, some degree of
selectivity was evident in the pattern of coupling to STAT5 and MAPK by
these tyrosines, but in each case functional redundancy was evident
among two or more tyrosine positions.
Reconstitution of EPOR-mediated erythropoiesis by multiple tyrosines.
To examine these properties in vivo, adult mice were subsequently
infected with selected viruses and the hematopoietic compartment was
evaluated. As in the progenitor cell assay, multiple independent tyrosines were observed to be capable of restoring RBC development signals to cEPORYF in whole animals. Preterminal hematocrits, indicative of effective RBC differentiation, were elevated in at least
75% of mice infected with each of these add-back constructs (Fig 4A).
Spleen weight, indicative of expansion of RBC progenitor populations,
was also increased in all diseased mice (not shown). Moreover, from the
enlarged spleens we were readily able to establish permanent growth
factor-independent cell lines ex vivo representing each of the
add-back mutants. Immunoblot studies of cellular lysates from infected
spleens showed that all expressed proteins derived from the transduced
cEPOR variant, although at somewhat varying levels (Fig 4B). Therefore,
multiple tyrosine residues of the EPOR cytoplasmic tail individually
and in combinations appear competent to mediate the biological signals
regulating both expansion of erythroid progenitors and formation of
mature RBCs in the whole animal.
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DISCUSSION |
Extensive information has been developed regarding the
structure-function relationships that govern signal transduction by cytokine receptors such as the EPOR in cell culture models. Likewise, gene deletion methods have facilitated delineation of the major functions of cytokine/cytokine receptor systems such as the EPO/EPOR complex in animals.32,33 The present studies were
undertaken as a first effort to integrate these scientific areas by
relating molecular features of the EPOR to biologic functions to which it is coupled in vivo.
The experimental strategy was to employ a "dominant" system in
which a constitutively active mutant of the EPOR (cEPOR)11 was used as a receptor backbone on which site-directed mutations selectively affecting signaling pathways were constructed. Based on
prior experience with the parental cEPOR construct,22 it was anticipated that delivery of such constructs to hematopoietic progenitor cells via recombinant spleen focus-forming viruses would
permit these receptors to engage their signal transduction programs
(and associated biological outcomes) without the necessity of silencing
the endogenous EPO/EPOR system by gene disruption. Because animals
infected with the parental cEPOR virus reliably develop marked
expansion of the hematopoietic compartment with predictable kinetics,
this biologic response served as a useful readout of receptor activity.
In this setting, proliferation of erythropoietic precursors was
manifested as marked splenomegaly, whereas RBC maturation was
represented by striking increases in peripheral hematocrits. While
these conditions per se are certainly pathological, they
represent predictable exaggerations of normal EPOR-dependent processes.
Therefore, they serve as useful experimental markers of
receptor-dependent function in this animal model.
Based on our prior observations of redundancy within other cytokine
receptors,34 we initiated the present studies with a mutant
EPOR in which all eight cytoplasmic tyrosines had been silenced by
substitution with phenylalanine (cEPORYF). Consistent with the findings
of others,12,15,29 this mutant showed marked impairment of
its capacity to cause growth factor-independent proliferation of cell
lines compared with the parental cEPOR. Likewise, it appeared to be
substantially uncoupled from signal transduction pathways typically
linked to the EPOR. Importantly, when introduced into mice via the SFFV
system, this receptor variant likewise exhibited drastically reduced
capacity to expand erythroid progenitor cells or to promote
differentiation leading to mature RBCs in the periphery. These findings
provide strong evidence that these biological processes in vivo depend
substantially upon tyrosine residues, and indicate that tyrosines
within such a receptor type represent relevant functional sites in the
whole organism context. It is nonetheless notable that, as has been
observed in prior studies in various cell line
systems12,15,29 and in erythroid progenitors from
EPOR( /) mice,35 the present studies confirm that such
tyrosines are not absolutely essential for receptor activity; both the
cellular studies (with transfectants and transduced primary
progenitors) and the animal infections (see below) revealed measurable
but clearly diminished erythopoietic responses to cEPORYF. These
results suggest that less efficient tyrosine-independent pathways of
signaling are also represented in the biological program driven by the
EPOR.
The observation that one animal in five underwent hematopoietic
expansion on introduction of the cEPORYF variant is intriguing and
indicates the complexity of undertaking such physiological studies.
Three lines of evidence suggest that the process manifested by this
animal may not be identical to that of the reference cEPOR animals.
First, the disease was delayed significantly in this animal compared
with the wild-type cEPOR and cEPORYF add-back animals, suggesting that
the mechanism underlying it may differ. Second, we were unable to
establish permanent cell lines from the spleen of this animal, despite
the ease of sustaining lines from the other infected animals. Third,
immunoblotting of primary splenocyte cultures or whole splenic tissue
from this animal showed no detectable cEPORYF expression, whereas
strong expression was readily detected in spleen and splenic cell lines
from animals infected with viruses carrying the parental cEPOR gene or
other spleens from other animals infected with SFFV-cEPORYF that did not exhibit disease. Further investigation is warranted to determine whether this delayed disease represents the inefficient
tyrosine-independent signaling function.
Further insights were derived by analyzing add-back mutants in which
various tyrosine residues were reconstituted individually or in
combinations in the cEPORYF backbone. Interestingly, growth signaling
as measured by induction of growth factor-independent proliferation of
transfected cell lines was restored by multiple and diverse tyrosines
within the cytoplasmic segment of the EPOR. Additionally, several
tyrosines apparently restored receptor coupling to STAT5 or MAPK in
specific patterns characterized by a degree of redundancy. Various
tyrosines also restored the erythropoietic response to cEPORYF when
these add-back mutants were expressed in primary mature CFU-E
progenitors. These results indicate that this process is driven by
functionally redundant tyrosine-containing elements within the
cytoplasmic tail of the EPOR, a general phenomenon which has been
reported previously in the interleukin-2 receptor system36
and which has been observed recently in the EPOR system in studies
employing transfected cell lines15 or in hematopoietic progenitor cells derived from EPOR(/) mice.35
Although numerous individual tyrosines were capable of supporting
mature CFU-E development in the progenitor cell assay, only
tyrosine-343 (Y1) and tyrosine-479 (Y8) supported complete
erythropoiesis as measured by CFU-E and more immature BFU-E progenitor
development. Because gene disruption experiments have indicated that
the EPOR is not essential for definitive BFU-E formation, the
physiological importance of forced expression of cEPOR or various
mutants within developing BFU-E is questionable. Nonetheless, using
this approach we have identified specific EPOR cytoplasmic tyrosine
residues that exhibit differential functional capacities; this
observation would not have been possible in studies with
nondifferentiating, nonerythroid cell lines in culture. However, it is
not possible to conclude definitively which signaling pathways are
responsible for the cellular response because there was no clear
association between the erythropoietic or growth response in cell
culture and the apparent efficiency of linkage to STAT5 or MAPK per
se. That the functional difference observed between CFU-E and BFU-E
regarding specific tyrosine-dependence was not manifest in the whole
animal context is not surprising because expansion of the CFU-E pool
alone would be expected to result in erythrocytosis and splenomegaly,
which, as has been observed in Friend virus disease, is a retrovirus
that affects predominantly CFU-E development.37
The possibility that endogenous EPO may have contributed to the effects
observed in the present system in vivo must also be considered. Indeed,
it has been established that progenitor cells transduced with cEPOR
exhibit an enhanced response with the addition of exogenous EPO despite
the fact that they develop in an EPO-independent manner in
methylcellulose culture.38 In any event, the effect of
cEPORYF is markedly lower than that of cEPOR in vivo.
Furthermore, the ex vivo progenitor assays were performed in the
absence of added EPO. Finally, each of the tyrosine add-back receptors
promoted massive eythroid expansion an outcome not observed when mice
were infected with SFFV viruses expressing wild-type EPOR
lacking the critical R129C mutation.23 Therefore, this
system appears to be useful as a first step toward dissecting these
receptor-dependent processes in animals.
The present studies employed both primary hematopoietic progenitor
cells and whole animals as a first step toward defining the
structure-function relationships of the EPOR as they relate to
erythropoiesis. In view of the variability often seen among various
established cell line systems, these strategies were employed to
evaluate these features in the biological context in which they
normally are manifested. The central findings generally correspond well
with those observed in prior cell culture systems; however, some
specificity of EPOR cytoplasmic tyrosine residue requirement for
supporting immature erythroid progenitor development was detected. Further studies using the present system and other strategies should
reveal more about the molecular mechanisms of specificity by these
receptors, the role of cellular context in determining signal
transduction processes and outcomes, and the connections of biological
outcomes to various intracellular signaling pathways.
| |
FOOTNOTES |
Submitted May 5, 1997;
accepted September 30, 1997.
Supported by the James S. McDonnell Foundation and The American Cancer
Society (I.R.G.-36-37); the office of Research and Development, Medical
Research Service Department of the Veterans Administration; and the J. David Gladstone Institutes and the National Institutes of Health
(GM54531 and CA75315).
Address reprint requests to Mark A. Goldsmith, MD, PhD, Gladstone
Institute of Virology and Immunology, PO Box 419100, San Francisco, CA
94141-9100.
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.
| |
ACKNOWLEDGMENT |
The authors acknowledge the excellent assistance of Jessica Diamond,
John Carroll, and Amy Corder in the preparation of this manuscript.
Recombinant human EPO was the generous gift of Ortho Biotech.
| |
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Abstract
Introduction
Abstract