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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1163-1172
The Anemic Friend Virus gp55 Envelope Protein Induces Erythroid
Differentiation in Fetal Liver Colony-Forming
Units-Erythroid
By
Stefan N. Constantinescu,
Hong Wu,
Xuedong Liu,
Wendy Beyer,
Amy Fallon, and
Harvey F. Lodish
From the Whitehead Institute for Biomedical Research, Cambridge, MA;
and the Department of Biology, Massachusetts Institute of Technology,
Cambridge, MA.
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ABSTRACT |
The gp55 envelope proteins of the spleen focus-forming virus
initiate erythroleukemia in adult mice. Because the gp55 from the
polycythemic strain (gp55-P), but not from the anemic strain (gp55-A),
activates the erythropoietin receptor (EpoR) for proliferation of
hematopoietic cell lines, the mechanism by which gp55-A initiates erythroleukemia has remained a mystery. We show here that gp55-A activates the EpoR in fetal liver cells. In contrast to previous studies using bone marrow cells from phenylhydrazine-treated, anemic
mice, we find that both gp55-A and gp55-P induce erythroid differentiation from colony-forming unit-erythroid (CFU-E)
progenitors in fetal liver cells. The effects on CFU-Es of both gp55-A
and -P are mediated by the EpoR, because no colonies are seen upon expression of either gp55 in EpoR / fetal liver cells.
However, only gp55-P induces erythroid bursts from burst-forming
unit-erythroid progenitors and only gp55-P induces Epo
independence in Epo-dependent cell lines. Using chimeric gp55 P/A
proteins, we extend earlier work showing that the transmembrane sequence determines the capacity of gp55 proteins to differentially activate EpoR signaling. We discuss the possibilities for different signaling capacities of gp55-A and -P in fetal liver and bone marrow-derived erythroid progenitor cells.
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INTRODUCTION |
THE PROLIFERATION, survival, and
differentiation of erythroid progenitors into mature red blood cells
(RBCs) absolutely requires erythropoietin (Epo) and the corresponding
Epo receptor (EpoR).1,2 Epo/ and
EpoR/ knock out embryos die around embryonic
day 13 due to failure of fetal liver hematopoiesis. They contain normal
numbers of both burst-forming unit-erythroid (BFU-E) and colony-forming
unit-erythroid (CFU-E) progenitors, but the transition from CFU-E to
mature RBCs is abolished. Thus, the EpoR is not required for generation
of committed erythroid BFU-E and CFU-E progenitors, but is essential for terminal proliferation and differentiation of CFU-Es.1
The EpoR is a member of the type I cytokine receptor
superfamily3,4 and is activated by ligand induced
homo-dimerization.2,5-9 The EpoR activates a number of
intracellular signal transduction pathways that are also activated by
other cytokines and growth factor receptors, including JAK2, STAT5,
PI-3 kinase, and the protein tyrosine phosphatases SHP-1 and
SHP-2.2,10 Exactly which signals or combination of signals
are required for proliferation, survival, and differentiation of the
various erythroid progenitors is not clear; however, these signals are
likely to be shared by several cytokine receptors.11
The spleen focus-forming virus (SFFV) induces erythroleukemia in adult
mice12 and the envelope glycoprotein of SFFV, gp55, is the
only component required for oncogenicity.13,14 Two
different strains of SFFV have been identified: the polycythemic (P)
and the anemic (A).15 Although both induce erythroleukemia,
SFFV-P induces polycythemia, a condition reminiscent of human
Polycythemia vera, and characterized by a very high number of
mature RBCs. Early stages of disease are characterized by a dramatic
increase in the number of erythroid BFU-E and CFU-E progenitors, which retain the capacity to terminally differentiate to mature RBCs and are
not transplantable.16-18 Malignant clones emerge at 4 to 6 weeks after infection as a result of further genetic events. Specific
to SFFV-induced leukemia is the activation of expression of the Spi-1
gene due to proviral insertional mutagenesis.19
In contrast, SFFV-A induces erythroleukemia with anemia. Because anemia
is due to hemodilution and not to absolute loss of RBCs, the anemia
strain may be called nonpolycythemic.20 gp55 sequences
entirely account for the oncogenicity of both SFFV-A and -P, because
they are oncogenic in the absence of other viral proteins.21 The phenotype of leukemia (P or A) is dependent on sequences at the 3 end of the gp55 gene, which encodes the gp55 membrane spanning domain.22 The mechanism by which
SFFV-A initiates erythroleukemia has remained a mystery,18
because there is no evidence for a functional interaction between
gp55-A and the EpoR, whereas several studies indicate that gp55-P
likely activates the EpoR. For example, expression of gp55-P but not of
gp55-A in interleukin-3 (IL-3)-dependent Ba/F3 cells results in
growth-factor independence provided that the cells are expressing the
EpoR.23 Interestingly, both gp55-P and gp55-A
coimmunoprecipitate with the EpoR in these cells.24
Similarly, gp55-P but not gp55-A renders Epo-dependent HCD 57 cells
growth factor-independent.25 Bone marrow and spleen
erythroid precursors isolated from mice infected with SFFV-A require
Epo for in vitro CFU-E differentiation, whereas those infected by
SFFV-P are Epo independent.26 Also, bone marrow-derived
erythroid cells infected in vitro with SFFV-P can form erythroid bursts
in the absence of Epo, whereas bone marrow-derived erythroid precursors
infected with SFFV-A need Epo for maximal proliferation and for
differentiation.27 We use wild-type (EpoR+/+)
as well as EpoR/ mouse embryos here to show
that, in primary fetal liver cells, gp55-A indeed activates the EpoR
and induces erythroid differentiation. Both gp55-A and gp55-P induce
erythroid differentiation from CFU-E progenitors, and no colonies are
formed upon expression of either gp55 in
EpoR/ fetal liver cells. However, only gp55-P
induces erythroid bursts from BFU-E progenitors and only gp55-P induces
Epo independence in Epo-dependent cell lines. Using chimeric gp55 P/A
proteins, we extend earlier work showing that the transmembrane
sequence determines the capacity of gp55 proteins to differentially
activate EpoR signaling. Thus, within the fetal liver-derived CFU-E,
signals delivered by the gp55-A through the EpoR are sufficient to
support the terminal stages of erythroid proliferation and
differentiation.
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MATERIALS AND METHODS |
Plasmids.
cDNAs encoding SFFV gp55 proteins, all cloned into SFFV cDNA, were
kindly provided by Dr Sandra K. Ruscetti (National Cancer Institute,
Frederick, MD). Restriction sites for EcoRI and Fok I
divide the gp55 sequence into three fragments,22 labeled
either A or P (Fig 1A). Thus, we denoted
SFFVAP-L, which exerts effects identical to
SFFV-P,14 as gp55-APP or, abbreviated, gp55-P. The
construct denoted gp55-AAP contains only one residue (Ser 376) in the
extracellular domain unique to gp55-P and the entire membrane-spanning
domain of gp55-P (Fig 1A). Using primers encoding Bgl II and
Sal I restriction sites, gp55 sequences were polymerase chain
reaction (PCR)-amplified and cloned into SFFV or into the BamHI
and Sal I sites of pBABE and pMX retroviral
vectors.28,29
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| Fig 1.
(A) Diagram of the gp55 constructs. The EcoRI and
Fok I restriction sites were used to divide the coding sequence
into three segments as described by Chung et al.22 P,
sequences derived from gp55-P; A, sequences derived from gp55-A.
gp55-APP was abbreviated as gp55-P because it is coded by
SFFVAP-L, which induces polycythemic effects
indistinguishable from those of SFFV-P.14 Construct gp55-AAP contains the entirety of gp55-A except for the
membrane-spanning domain and one unique residue (Ser 376) in the
exoplasmic domain derived from gp55-P. (B) Induction of erythroid
colony formation from day-12.5 fetal liver CFU-E progenitors. Fetal
liver cells were infected with retroviruses encoding gp55-P, gp55-A,
gp55-AAP, or gp55-APA or with virus encoding  -galactosidase
(control). Formation of CFU-E colonies was scored in the absence of Epo
after 72 hours by staining with benzidine. Data represent the mean of the indicated number of assays (N) ± 1 standard deviation. Treatment with 1 U/mL Epo induced 957 ± 99 CFU-E colonies/50,000 fetal liver cells (N = 6).
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Generation of retroviral supernatants.
To generate high-titer replication-free retroviral supernatants, we
used transient transfection of the BOSC23 packaging cell line.30 BOSC cells were transfected using the calcium
phosphate method with 5 µg of plasmid DNA encoding various gp55
constructs. As controls for transfection and infection, empty
retroviral vectors or retroviruses encoding -galactosidase, green
fluorescence protein (GFP), or the cell-surface CD2 protein were
transfected in parallel. Viral supernatants were collected 48 to 72 hours posttransfection and stored at  70°C. Viral titers were
measured by infecting NIH 3T3 cells or Ba/F3 cells with serial
dilutions of viral supernatant in the presence of 4 µg/mL polybrene
and assaying by fluorescence-activated cell sorting (FACS) for cells
exhibiting GFP fluorescence. Titers of 1 × 106 to 4 × 106 CFU/mL were routinely obtained. After
collection of viral supernatants, expression of various gp55 proteins
in the BOSC cells was assayed by Western blotting using the anti-gp55
7C10 monoclonal antibody (a gift of Dr Sandra Ruscetti) or with goat
polyclonal anti-Rauscher gp70 envelope protein.
Erythroid colony formation in fetal liver cells.
Day-12.5 to -13.5 mouse fetal livers, which contain a majority of
erythroid progenitors, were harvested and single-cell suspensions were
prepared as described.1,11 Cells were infected with various retroviruses in the presence of 4 µg/mL polybrene for 4 hours.1,11 For CFU-E assays, 105 cells were
resuspended in semisolid 1% methylcellulose medium containing 20%
plasma depleted serum (Animal Technologies Inc, Tyler, TX),
antibiotics, and -mercaptoethanol or in MethoCult 3230 medium
containing 0.9% methylcellulose and 20% fetal bovine serum (Stem Cell
Technologies, Vancouver, British Columbia, Canada) in the presence or
absence of 1 to 3 U/mL Epo (a generous gift from Dr Joan Egrie, Amgen
Corp, Thousand Oaks, CA). Erythroid colonies generated by CFU-E
progenitors were scored 72 hours after seeding by staining with
diaminobenzidine (Sigma, St Louis, MO). As previously
described,11 an aliquot of fetal liver cells infected in
parallel with retroviruses encoding GFP or CD2 was incubated in liquid
culture in Iscove's modified Dulbecco's medium (IMDM) containing 20% fetal calf serum and 1 U/mL Epo and analyzed 36 hours
after infection using FACS scanning (Becton Dickinson, Mountain View,
CA) for CD2 expression (using phycoerythrin-conjugated
antimurine CD2 antibodies) or GFP expression to have a control for the
efficiency of infection by a particular set of retroviruses. Colonies
induced by expression of gp55 or Epo were photographed and scanned, and the surface occupied by CFU-E colonies was quantitated using the NIH
Image program.
For BFU-E assays, 105 fetal liver progenitors were
centrifuged after infection and resuspended in semisolid 1%
methylcellulose medium containing 20% plasma derived serum, 1% spleen
conditioned medium, and 50 ng/mL Steel factor (SF; a generous gift from
Dr Joan Egrie) in the presence or absence of 3 U/mL Epo. Hemoglobinized BFU-E colonies were scored 7 to 9 days after
infection.31,32 The capacity of gp55 proteins to replace SF
or spleen-conditioned medium (SCM) was tested by omitting either one in
the semisolid medium and testing the effects of Epo or gp55 on BFU-E
formation in the presence or absence of Epo.
Assay for growth factor independence.
IL-3-dependent Ba/F3 cells stably transfected with the murine
EpoR5 were grown in RPMI supplemented with 10% fetal calf serum and either 5% WEHI supernatant (as a source of IL-3) or 1 U/mL
Epo. HCD 57 cells were grown in IMDM supplemented with 20% fetal calf
serum and Epo.25 Cells were infected in the presence of 4 µg/mL polybrene with retroviral supernatants generated in BOSC cells
and cultured in medium containing 1 U/mL Epo or 5% WEHI supernatant
(as a source of IL-3) for 48 hours after infection. They were then
extensively washed in RPMI and plated in growth medium without Epo or
IL-3 in 24 wells of a microtiter plate. Viable growing cells were
usually identified 4 to 6 days after growth factor withdrawal and cell
pools were amplified and tested for expression of gp55 proteins by
Western blotting. A minimum of three pools was analyzed for each
construct. For viruses encoding GFP or CD2, FACS analysis was performed
24 and 48 hours after infection to have a control for the efficiency of
a particular set of retrovirus infections.
Retroviral bicistronic expression of gp55 and GFP proteins.
To achieve high levels of expression of transduced gp55 proteins that
can be followed by FACS, we used GFP as a reporter gene. We generated a
novel bicistronic retroviral vector, pMX-IRES-GFP, which will be
described in detail elsewhere. Briefly, the encephalomyocarditis virus
internal ribosome entry sequence (IRES) was inserted in front of the
GFP gene in the pMX vector.29 Sequences for gp55-P (APP),
gp55-A, or gp55-APA were cloned in front of the IRES sequence (see Fig
5). The pMX-IRES-GFP constructs encoding gp55 proteins were transfected
into BOSC23 cells and their expression was documented by FACS for GFP
fluorescence and by Western blotting for gp55 expression. Retroviral
supernatants generated from the transfected BOSC cells were used to
infect Ba/F3 EpoR cells. The cells were cultured in WEHI (as a source
of IL-3), and usually about 17% to 20% of the infected cells became
fluorescent 24 hours after infection. The 1% most GFP fluorescent
cells were sorted by FACS and then cultured in WEHI. As described in
the text, these sorted cells were then assayed for their content of
Epo- and IL-3-independent cells by culture in the absence of any
growth factors. Epo-independent clones were analyzed by FACS and shown
to express high levels of GFP. This technique allowed the isolation of
cells stably expressing high levels of various gp55 proteins.
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| Fig 5.
Bicistronic viruses encoding gp55 proteins and GFP.The
pMX retroviral vector was modified to include the encephalomyocarditis virus IRES and, downstream, the GFP coding sequence. The cDNAs coding
for gp55-P, gp55-APA, and gp55-A were inserted upstream of the IRES
generating pMX-gp55-IRES-GFP vectors.
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RESULTS |
Activation of erythroid colony formation from CFU-Es by gp55 proteins.
Results obtained using Epo/ and
EpoR/ mice showed that Epo is absolutely
required for terminal proliferation and differentiation of fetal liver
CFU-Es to mature erythrocytes.1 This led us to investigate
whether expression of gp55 proteins can replace Epo and activate the
EpoR to promote terminal proliferation and differentiation of CFU-E
progenitors. We prepared retroviruses encoding gp55-P (or APP) and
gp55-A (or AAA) as well as several gp55 chimeras, ie, gp55-APA and
gp55-AAP (Fig 1A); gp55-AAP contains the entirety of gp55-A except for
the membrane-spanning domain and one unique residue in the exoplasmic
domain derived from gp55-P. The gp55 cDNAs were either cloned in
SFFV22 or in the retroviral vector pBABE. We generated
high-titer retroviral suspensions using the BOSC23 packaging cell
system30 and used the virus to infect day-12.5 fetal liver
cells. The fetal liver cells were assayed 2 to 3 days postinfection for
erythroid colony formation from CFU-Es in the absence of Epo. This
assay measures a combination of survival, proliferation, and
differentiation in that a scored CFU-E colony has to contain at least 8 hemoglobinized cells.32
Figure 1B shows that expression of either gp55-P, gp55-A, gp55-AAP, or
gp55-APA results in CFU-E differentiation in the absence of Epo. Two
types of controls indicated that essentially all of the CFU-E
progenitors infected by retroviruses expressing any of these gp55
proteins went on to form an erythroid colony. First, parallel cultures
were infected with retroviruses encoding -galactosidase or CD2 that
were generated in parallel to those encoding gp55. An aliquot of cells
from each infection was cultured for 36 hours in liquid medium in the
presence of Epo and then analyzed by FACS scan for the fraction of
infected cells. In repeated experiments, the ratio of the number of
erythroid CFU-E colonies promoted by gp55 to that promoted (in
uninfected cultures) by Epo was the same or higher than the estimated
rate of retrovirus infection (15% to 25%); the latter was measured by
the fraction of cells expressing transfected CD2 or -galactosidase.
Specifically, Epo supported the formation of 957 ± 99 CFU-E
colonies per 50,000 nucleated fetal liver cells (N = 6; Fig 1); thus,
the number of colonies (200 to 300 per 50,000 nucleated fetal liver
cells; Fig 1B) induced by expression of gp55 proteins correlated very
well with the frequency of infection.
As another means of determining the efficiency of retroviral infection,
fetal liver cells infected with retroviruses encoding -galactosidase
were incubated in methylcellulose culture in the presence of Epo, and
the resultant colonies were stained for -galactosidase activity. The
number of Epo-induced CFU-E colonies that expressed -galactosidase
(or CD2 in similar experiments) was the same (200 to 250 per 50,000 nucleated fetal liver cells) as the number of colonies induced by gp55
expression in the absence of Epo; again, the estimated frequency of
infection was 15% to 25%. We conclude that most of the CFU-E
progenitors infected by a retrovirus expressing either gp55-P or gp55-A
undergo terminal erythroid differentiation in the absence of Epo.
Erythroid colonies induced by either gp55-P or gp55-A were
significantly smaller than those induced by Epo. Most Epo-induced CFU-E
colonies contained between 32 and 48 cells, whereas gp55-induced colonies contained between 12 and 16 cells. To better quantify these
differences, we randomly photographed colonies induced by Epo or by
gp55 and measured their surface using the NIH Image program. Colonies
induced by gp55 had one half of the area of Epo-induced colonies.
Specifically, the surface of Epo-induced colonies (N = 11) was 5,105 ± 662 arbitrary square pixels, whereas the surface of gp55-P
promoted colonies was 2,649 ± 500 square pixels. In
methylcellulose, the colonies are spherical; thus, it is possible that
the twofold difference in surface area may represent an even more
significant difference in volume.
Importantly, no difference in morphology, staining, or number of cells
per colony was detected between those promoted by gp55-P and by gp55-A.
Thus, gp55-P and gp55-A deliver an equivalent similar signal to fetal
liver CFU-E progenitors that is sufficient to promote their terminal
proliferation and differentiation into erythroid colonies.
Activation of erythroid burst formation from BFU-Es by gp55 proteins.
The in vitro assay for BFU-E erythroid differentiation measures the
progression to CFU-Es and then to RBCs, a process that takes 7 to 9 days.32 In culture, differentiation of BFU-Es to CFU-Es
requires Epo as well as IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF; both contained in SCM), or SF (KIT ligand). Thus, we
infected day-12.5 to -13.5 fetal liver progenitors with retroviral suspensions encoding various gp55 proteins and then assayed for BFU-E
erythroid differentiation by plating the cells in 1% methylcellulose in the presence of plasma-derived serum, SCM, and SF. Typical BFU-E
colonies were scored 7 to 9 days after infection and stained with
benzidine; alternatively, they were analyzed by Wright-Giemsa stain
after cytospinning.
Figure 2A shows that expression of gp55-P
was able stimulate BFU-E colony formation in the absence of Epo. As
judged by the number of Epo-induced BFU-E colonies that expressed
-galactosidase, the estimated frequency of infection was 15% to
25%. The number of colonies induced by gp55-P infection and in the
absence of Epo (~80 per 100,000 nucleated fetal liver cells) was
about one-third of that induced in control cultures treated with 3 U/mL
Epo (230 colonies per 100,000 fetal liver cells). Thus, virtually every BFU-E infected by a retrovirus expressing gp55-P was able to form an
erythroid burst. Figure 2B shows that the BFU-E colonies induced by
gp55-P were significantly smaller than those induced by Epo; however,
cytospin analysis and benzidine staining did not point to any
differences other than in the numbers of cells, a result analogous to
the situation with gp55-induced CFU-E colonies.
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| Fig 2.
Induction of erythroid burst formation from day-12.5
fetal liver BFU-E progenitors. (A) Fetal liver cells were infected with retroviruses encoding gp55-P, gp55-A, gp55-AAP, or gp55-APA or with
virus encoding -galactosidase (control). Formation of BFU-E colonies
was scored 7 to 9 days after infection in complete methylcellulose medium containing plasma-derived serum, SCM, and SF (see the Materials and Methods). Data represent the mean of the indicated number of assays ± 1 standard deviation. Treatment with 3 U/mL Epo resulted in the
formation of 230.5 ± 16.1 BFU-E colonies per 100,000 fetal liver
cells (N = 4). (B) BFU-E colonies induced either by 3 U/mL Epo or
after infection with SFFV-gp55-P and culture in the absence of Epo.
Scale bar = 50 µm.
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Infection with viruses encoding gp55-A did not result in erythroid
burst formation (Fig 2A and Table 1). Thus,
the BFU-E assay clearly differentiates between the polycythemic (P) and anemic (A) phenotype. Because gp55-A and gp55-APA did not induce BFU-E
differentiation and because gp55-P and AAP did induce normal BFU-E
differentiation (Fig 2A and Table 1), sequences located within the
membrane-spanning domain are responsible for the differences between
gp55-P and gp55-A. This correlates very well with the capacity of
several chimeric gp55 proteins to induce polycythemia in adult
mice.25 Furthermore, because the gp55-AAP chimera contains only one unique residue from the exoplasmic domain of gp55-P (at position 376, Fig 1A) and the entire membrane-spanning domain of
gp55-P, the notion that the transmembrane domain is crucial for the
observed effects between gp55-P and gp55-A is strengthened.
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Table 1.
Fetal Liver Erythroid Colony Formation and Induction of
Epo Independence in Cell Lines by Native and Chimeric gp55 Proteins
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For Epo to stimulate maximum BFU-E colony formation from fetal liver
progenitors, the medium must contain SF and SCM, which provides a
mixture of IL-3 and GM-CSF. Whereas Epo alone cannot support BFU-E
colony formation (not shown), a combination of Epo and SF (lacking SCM)
induced formation of 76% of the number of BFU-E colonies induced by
complete medium (Epo + SF + SCM; Fig 3),
and a combination of Epo and SCM induced 40% of the number of colonies
induced by complete medium.Thus, Epo can induce a significant number of
BFU-E colonies in the presence of only SF or only SCM. In contrast,
gp55-P requires the presence of both SF and SCM in the plating medium
to promote BFU-E differentiation. (Fig 3). The smaller size of BFU-E
colonies induced by gp55-P and the absolute requirement for both SF and
SCM for gp55-P to stimulate BFU-E differentiation suggest that gp55-P
can only partially activate the EpoR in BFU-Es.
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| Fig 3.
Induction of erythroid burst formation from day-12.5
fetal liver BFU-Es by gp55-P require the presence of both SF and SCM. Fetal liver cells were infected with retroviruses encoding gp55-P or,
as control, -galactosidase and then plated in methylcellulose containing the indicated growth factors. Data represent the average of
two typical experiments, each performed in duplicate; the variation was
less than 20%. Cells plated solely in Epo without SF and SCM did not
produce any BFU-E colonies.
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Expression of the EpoR and the effects of gp55 proteins on erythroid
differentiation.
Because expression of either gp55-P or gp55-A can replace Epo for the
activation of CFU-E differentiation in fetal liver cells, we
investigated whether expression of the EpoR was required for this
function. To this end, we infected day-12.5 fetal liver cells from
homozygous EpoR/ embryos with viruses
encoding gp55 proteins and then assayed for erythroid colony formation
from CFU-E progenitors as previously described.1,33 In this
system, retroviral-mediated expression of the EpoR results in Epo- and
SF-dependent formation of erythroid colonies from CFU-E and BFU-E
progenitors at 72 hours and 7 to 9 days postinfection,
respectively.1,33 Figure 4A
shows that none of the gp55-expressing viruses (cloned in SFFV or
pBABE) was able to induce erythroid colony formation from
EpoR/ CFU-E progenitors. In these
experiments, SF was added to all samples as a precaution, because an
interaction between the KIT and Epo receptors is essential for terminal
differentiation of EpoR/ CFU-E progenitors
expressing recombinant EpoR.33 In one control experiment,
infection of the EpoR/ progenitors with virus
encoding the EpoR resulted in Epo-dependent formation of a large number
of erythroid colonies in the presence of SF (Fig 4A, right column). As
another control, the same gp55-encoding retroviral suspensions were
able to induce CFU-E differentiation from wild-type
(EpoR+/+) fetal liver cells (Fig 4B). Similar results (not
shown) were obtained when differentiation from BFU-E progenitors was
assayed, showing that expression of gp55-P results in erythroid colony formation only in the presence of the EpoR.
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| Fig 4.
Induction of erythroid colony formation from fetal liver
CFU-E progenitors requires the expression of the EpoR. Day-12.5 fetal liver cells dissected from EpoR/ embryos (A) or from
wild-type embryos (B) were infected with viruses encoding
-galactosidase (control), gp55-P, gp55-A, and murine EpoR. The
retroviral vector used is indicated (SFFV or pBABE). Cells were assayed
in CFU-E assays in the absence or presence of EPO or (in the right-most
panel of [A]) in the presence of Epo and SF. Data represent the
average of two experiments, each performed in duplicate; the variation
was less than 20%.
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gp55-P and not gp55-A induce Epo-independent growth of Epo-dependent
cells: bicistronic expression of gp55 and GFP.
Expression of gp55-P but not gp55-A induces growth-factor independence
of certain Epo-dependent hematopoietic cell lines.23,25,34 We tested viruses encoding gp55-P, gp55-A, and gp55-APA for their ability to induce Epo-independence in Ba/F3-EpoR or HCD 57 cells. Whereas gp55-P, and gp55-AAP induced Epo-independence, gp55-PPA, gp55-A, and gp55-APA did not (Table 1). Thus, sequences located primarily within the membrane-spanning domain of gp55 are crucial for
induction of Epo-independence, confirming previous work,25 as well as for fetal liver BFU-E differentiation.
Because only gp55-P and chimeras of gp55 that contain the
membrane-spanning domain of gp55-P can render cell lines Epo
independent, we were not able to analyze signaling events induced by
gp55-A or gp55-APA. Furthermore, in factor-independent cells expressing gp55-P, one is studying events that may be a consequence of the selection for Epo independence and not those due only to expression of
gp55-P. Indeed, whereas virtually all BFU-E and CFU-E progenitors infected with a retrovirus encoding gp55-P were able to form an erythroid colony or burst in the absence of Epo (Figs 1 and 2), our
preliminary experiments indicated that only a minority of EpoR-expressing Ba/F3 cells infected with a retrovirus encoding gp55-P
was able to proliferate in the absence of Epo.
To understand better the relationship between the efficiency of
infection and the induction of Epo independence in cell lines, we used
a bicistronic retroviral vector encoding a gp55 protein and GFP,
separated by an IRES (Fig 5 and the
Materials and Methods). In this system, the translation of the two
proteins is tightly linked, in that expression of GFP (initiated by the
IRES) is proportional over a 100-fold range to the level of expression
of the protein coded by the cDNA directly downstream of the left
LTR.35 We generated retroviruses encoding gp55-P, gp55-A,
and gp55-APA by packaging pMX-gp55-IRES-GFP constructs in BOSC 23 cells
and used these to infect Ba/F3 EpoR cells
(Fig 6). Cells were scanned by FACS for GFP
fluorescence and sorted for high levels of GFP fluorescence. Pools of
sorted cells expressing the same amounts of gp55-A-GFP, gp55-APA-GFP,
and gp55-P-GFP (Fig 6B, C, and D, respectively) were assayed for the
capacity to grow in the absence of Epo. Only gp55-P-GFP cells were able
to grow after Epo withdrawal. As an important control, the levels of
gp55 protein were similar in cells sorted for GFP expression and
expressing gp55-P, gp55-A, or gp55-PPA; these levels were comparable to
that in gp55-P-infected cells and selected for growth in the absence
of Epo (Fig 6, lower panel).
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| Fig 6.
Bicistronic expression of gp55 proteins and GFP. Ba/F3
EpoR cells were infected with pMX-gp55-IRES-GFP retroviruses encoding gp55-A (B), gp55-APA (C), and gp55-P (D) cloned in the pMX-IRES-GFP retroviral vector or with a similar pMX retrovirus without an insert
(A). The FACS analysis shows the percentage of cells that express
different levels of GFP at 48 hours after infection. Cells with high
GFP fluorescence (bars in B, C, and D) were sorted and then cultured in
the presence of Epo. Greater than 78% of these cells retained high GFP
expression, as indicated by the panels in the middle column depicting
the sorted cells. These sorted cells were also assayed for Epo
independence. Only cells infected with pMX-gp55-P-IRES-GFP virus could
grow in the absence of Epo; FACS analysis of these is shown in (D),
Selected. The level of expression of gp55 proteins in the respective
cell lines was shown in the lower panel by a Western blot of whole cell
lysates with the anti-gp55 antibody.
|
|
This system allowed us to determine that the fraction of
gp55-P-expressing cells that eventually becomes Epo independent is only 1 in 1,000 to 1 in 5,000. This explains the lag phase of 4 to 6 days required for generation of Epo-independent clones of Ba/F3 cells
after infection with gp55-P coding viruses.23,36 In
contrast, virtually all fetal liver progenitors infected with a
gp55-P-expressing virus formed erythroid colonies and bursts (Figs 1
and 2).
| |
DISCUSSION |
Our most important result is that both types of SFFV envelope proteins,
polycythemic (gp55-P) and anemic (gp55-A), promote erythroid colony
formation from fetal liver CFU-Es in the absence of Epo. Colony
formation requires expression of the EpoR, a result that implies that
gp55-A indeed can activate the EpoR and that any quantitative or
qualitative differences in signaling between gp55-P and gp55-A are
irrelevant at this stage of erythroid differentiation. We also showed
that gp55-P but not gp55-A can promote erythroid colony formation from
BFU-E progenitors and that only gp55-P can induce growth factor
independence in Epo-dependent cell lines. To insure that the inability
to obtain factor-independent cells expressing gp55-A was not due to
absence of gp55-A expression, we used a bicistronic retrovirus vector
expressing GFP under control of an IRES to generate populations of
Ba/F3 EpoR cells stably expressing gp55-P and gp55-A at high levels;
again, only gp55-P-expressing cells became Epo independent. Consistent
with these observations, recent studies showed that, in transfected
Ba/F3 cells expressing the EpoR, the levels of cell-surface gp55-P and
gp55-A proteins are similar but that the interaction of the two gp55
proteins with the Epo receptor is different; only gp55-P could be
cross-linked to radioiodinated Epo.37 Finally, by using
chimeric gp55 A and P proteins, we showed that only the
membrane-spanning segment of gp55-P is essential for promoting
erythroid colony formation from BFU-E progenitors as well as for
induction of growth factor independence in Epo-dependent cell lines.
To study the functional interaction between gp55 proteins and the EpoR,
we used fetal liver erythropoiesis. In this system, we can follow
erythroid differentiation from BFU-E to CFU-E and finally to RBCs and
we can assess the role of the EpoR by parallel experiments in
EpoR/ embryos. Earlier studies were performed
with bone marrow progenitors taken from adult mice that had been
rendered anemic by phenylhydrazine27,38 or with bone marrow
and spleen erythroid precursors derived from mice infected with SFFV-A
or SFFV-P.26 These studies showed that gp55-A stimulated
the proliferation but not the terminal differentiation of BFU-Es,
whereas gp55-P was able to replace Epo for both proliferation and
differentiation.27,38 No effect on CFU-E differentiation
was reported when gp55-expressing retroviruses were used to infect, in
vitro, bone marrow cells.27,38 However, bone marrow CFU-Es
isolated from SFFV-P-infected animals were shown to form erythroid
colonies in the absence of Epo, whereas precursors isolated from
SFFV-A-infected animals still required Epo for erythroid
differentiation.26 We can suggest several explanations for
the discrepancies between these studies and our present results.
First, although Epo induces CFU-E differentiation in bone marrow
progenitors, the effects of in vitro infection with SFFV-P seem to be
uniquely on the BFU-Es, which form bursts earlier (by day 5) than do
the Epo-induced bursts (day 8).38 Second, the experiments
on bone marrow progenitors were performed using replication competent
Friend virus; we used replication-incompetent viruses. Third, there may
be important overlooked differences between EpoR signaling in fetal
liver versus adult bone marrow progenitor cells. Indeed, the
transcription factor PU.1 is required for erythropoiesis in adult bone
marrow progenitors but not in fetal liver cells; ES cells homozygous
for a PU.1 deletion (PU.1/ knock out cells),
when injected into PU.1+/+ embryos, contribute to fetal
liver-derived erythrocytes but not to bone marrow-derived erythrocytes,
and PU.1/ fetal liver cells can reconstitute
the erythroid compartment of lethally irradiated adult
mice.39 Furthermore, fetal liver hematopoietic stem cells
(HSC) are functionally and phenotypically different from bone marrow
HSC.40 Fetal liver HSC include a much higher frequency of
cycling cells and have higher reconstitution ability in lethally
irradiated mice when compared with bone marrow HSC.40
Together with the data from previous studies in bone marrow,26,27,38 our data show that differences between
fetal liver and bone marrow progenitors can also be noted at the later, committed CFU-E stage.
Our studies, using high-titer replication-defective recombinant
retroviruses and fetal liver progenitor cells derived from normal mouse
embryos, indicate that gp55-P and gp55-A are equally able to activate
the EpoR for proliferation and terminal differentiation of CFU-E
progenitors. Thus, the suggestion27 that gp55-A cannot activate a differentiation-specific pathway is unlikely, at least in
the context of fetal liver CFU-Es. These results point to interesting differences between fetal liver and bone marrow progenitors that deserve further study.
The CFU-E and the BFU-E colonies induced by gp55 proteins were smaller
than those induced by Epo, due to the presence of lower numbers of
cells per colony. Epo-stimulated proerythroblasts both proliferate (ie,
generate more proerythroblasts) and divide to progress toward terminal
differentiation. The cell divisions required for progression towards
reticulocytes are also called differentiation divisions, because
proerythroblasts normally divide 4 to 5 times to generate progressively
smaller erythroblast intermediates and eventually to generate
reticulocytes.41 Our cytospin analysis showed the presence
of the expected erythroblast intermediates in the gp55-A- and
gp55-P-induced colonies. In contrast, Epo-induced colonies apparently
contain erythroblast intermediates that also undergo proliferation
before dividing to yield the next intermediate in the differentiation
program. We hypothesize that such proliferation of CFU-Es is stimulated
by Epo but not by gp55-P or gp55-A proteins. Our study does show that
the effect of gp55 in primary cells is due to the EpoR because, in
EpoR/ knock out embryos, neither gp55-P nor
gp55-A can induce CFU-E colonies, irrespective of the presence of SF in
the plating medium.
Interestingly, expression in EpoR/ fetal
liver cells of a mutant EpoR containing only 1 of the 8 cytosolic
tyrosines (F7Y401) resulted in the formation of a normal number of
small CFU-E colonies33 similar to the ones induced by
gp55-A and gp55-P. Similar to gp55-A, EpoR mutant F7Y401 cannot support
Epo-dependent proliferation of Ba/F3 cells.33 We suggest
that, in CFU-E cells, gp55 delivers an incomplete signal through the
EpoR that activates some signal transduction pathways (hypothetically,
one[s] activated through phosphorylated tyrosine 401) but not others,
yet still triggers the differentiation divisions. Whether gp55
activates tyrosine phosphorylation of EpoR tyrosine 401 in CFU-E cells
is under investigation in our laboratory. Erythroid differentiation
requires a fine balance between the levels of GATA transcription
factors, and this level may be differentially affected by Epo or gp55
activation of the EpoR. A low level of expression of the GATA2
transcription factor (or a low GATA2/GATA1 ratio) was associated with
erythroid differentiation and small-sized bursts.42
A high level of expression of gp55-P is not sufficient to induce
autonomous cell growth of Ba/F3 EpoR cells. Using a bicistronic retrovirus to coexpress gp55 proteins with GFP, we showed that only a
tiny minority of cells expressing gp55-P became Epo independent. This
finding is consistent with the several-day lag period required for
detection of factor-independent growth after infecting EpoR-expressing cell lines with viruses expressing gp55-P. This bicistronic retrovirus system may be useful to study the initial stages of receptor activation by gp55 proteins, in the absence of selection for Epo-independent growth. For example, we recently showed that cells sorted for high
levels of gp55-P expression indeed exhibit factor-independent activation of STAT5, whereas cells expressing equivalent high levels of
gp55-A or gp55-PPA do not, suggesting that activation of STAT5 is a
direct consequence of gp55-P expression (unpublished results). This system will enable further study of the
initial events required for gp55-expressing cells to escape dependence on growth factors.
Finally, although both gp55-P and gp55-A activate the EpoR, the nature
of these interactions is different. Because gp55-A activated the
combination of cell proliferation and differentiation required for
colony formation from CFU-E progenitors, we suggest that the
differences between gp55-A and gp55-P are quantitative rather than
qualitative. At the CFU-E stage, the proliferation and differentiation
programs are coupled and can be initiated by much weaker signals from
the EpoR than those required at the earlier late BFU-E stage. This is
in agreement with the significantly lower concentrations of Epo
required for erythroid differentiation in vitro of CFU-Es than from
BFU-Es.43 EpoR-expressing cell lines appear to be quite
insensitive to gp55-P, because only a minority of cells expressing
gp55-P eventually became autonomous for cell growth. Thus, our new data
provide novel insights into the mechanisms of EpoR signaling and also
point to important differences in EpoR signaling between fetal liver
and bone marrow-derived erythroid progenitors.
| |
FOOTNOTES |
Submitted October 20, 1997;
accepted November 28, 1997.
Supported by Grant No. HL 32262 from National Institute of Health and
by a grant from Amgen Corporation to H.F.L. S.N.C. held a fellowship
from the Anna Fuller Fund and is now a fellow of the Medical
Foundation/Charles A. King Trust. X.L. is supported by a postdoctoral
fellowship from the National Institutes of Health. H.W. was supported
by a postdoctoral fellowship from the Damon Runyan-Walter Winchell
Cancer Research Fund.
Address reprint requests to Harvey F. Lodish, PhD, Whitehead Institute
for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142.
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 thank Dr Sandra K. Ruscetti (Laboratory of Molecular
Oncology Frederick Cancer Research and Development Center, National
Cancer Institute, Frederick, MD) for gp55 cDNA constructs and for
monoclonal antibody 7C10. We thank Dr Merav Socolovsky for advice on
retroviral infection of fetal liver cells and for many suggestions.
| |
REFERENCES |
1.
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]
2.
Watowich SS,
Wu H,
Socolovsky M,
Klingmuller U,
Constantinescu SN,
Lodish HF:
Cytokine receptor signal transduction and the control of hematopoietic cell development.
Annu Rev Cell Dev Biol
12:91,
1996 [Medline]
[Order article via Infotrieve]
3.
D'Andrea AD,
Lodish HF,
Wong GG:
Expression cloning of the murine erythropoietin receptor.
Cell
57:277,
1989[Medline]
[Order article via Infotrieve]
4.
D'Andrea AD,
Fasman GD,
Lodish HF:
Erythropoietin receptor and interleukin-2 receptor beta chain: A new receptor family.
Cell
58:1023,
1989[Medline]
[Order article via Infotrieve]
5.
Yoshimura A,
Longmore G,
Lodish HF:
Point mutation in the exoplasmic domain of the erythropoietin receptor resulting in hormone-independent activation and tumorigenicity.
Nature
348:647,
1990[Medline]
[Order article via Infotrieve]
6.
Watowich SS,
Yoshimura A,
Longmore GD,
Hilton DJ,
Yoshimura Y,
Lodish HF:
Homodimerization and constitutive activation of the erythropoietin receptor.
Proc Natl Acad Sci USA
89:2140,
1992[Abstract/Free Full Text]
7.
Watowich SS,
Hilton DJ,
Lodish HF:
Activation and inhibition of erythropoietin receptor function: role of receptor dimerization.
Mol Cell Biol
14:3535,
1994[Abstract/Free Full Text]
8.
Wrighton NC,
Farrell FX,
Chang R,
Kashyap AK,
Barbone FP,
Mulcahy LS,
Johnson DL,
Barrett RW,
Jolliffe LK,
Dower WJ:
Small peptides as potent mimetics of the protein hormone erythropoietin.
Science
273:458,
1996[Abstract]
9.
Livnah O,
Stura EA,
Johnson DL,
Middleton SA,
Mulcahy LS,
Wrighton NC,
Dower WJ,
Jolliffe LK,
Wilson IA:
Functional mimicry of a protein hormone by a peptide agonist: The EPO receptor complex at 2.8 A.
Science
273:464,
1996[Abstract]
10.
Ihle JN,
Witthuhn BA,
Quelle FW,
Yamamoto K,
Silvennoinen O:
Signaling through the hematopoietic cytokine receptors.
Annu Rev Immunol
13:369,
1995[Medline]
[Order article via Infotrieve]
11.
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]
12.
Friend C:
Cell-free transmission in adult Swiss mice of a disease having the character of a leukemia.
J Exp Med
105:307,
1957[Abstract]
13.
Linemeyer DL,
Menke JG,
Ruscetti SK,
Evans LH,
Scolnick EM:
Envelope gene sequences which encode the gp52 protein of spleen focus-forming virus are required for the induction of erythroid cell proliferation.
J Virol
43:223,
1982[Abstract/Free Full Text]
14.
Wolff L,
Ruscetti S:
Malignant transformation of erythroid cells in vivo by introduction of a nonreplicating retrovirus vector.
Science
228:1549,
1985[Abstract/Free Full Text]
15.
Mirand EA,
Steeves RA,
Lange RD,
Grace JT Jr:
Virus-induced polycythemia in mice: erythropoiesis without erythropoietin.
Proc Soc Exp Biol Med
128:844,
1968[Medline]
[Order article via Infotrieve]
16.
Ruscetti SK:
Erythroleukaemia induction by the Friend spleen focus-forming virus.
Clin Haematol
8:225,
1995
17.
Kabat D:
Molecular biology of Friend viral erythroleukemia.
Curr Top Microbiol Immunol
148:1,
1989[Medline]
[Order article via Infotrieve]
18.
Ben-David Y,
Bernstein A:
Friend virus-induced erythroleukemia and the multistage nature of cancer.
Cell
66:831,
1991[Medline]
[Order article via Infotrieve]
19.
Moreau-Gachelin F,
Ray D,
de Both NJ,
van der Feltz MJ,
Tambourin P,
Tavitian A:
Spi-1 oncogene activation in Rauscher and Friend murine virus-induced acute erythroleukemias.
Leukemia
4:20,
1990[Medline]
[Order article via Infotrieve]
20.
Tambourin PE,
Wendling F,
Jasmin C,
Smadja-Joffe F:
The physiopathology of Friend leukemia.
Leuk Res
3:117,
1979[Medline]
[Order article via Infotrieve]
21.
Wolff L,
Kaminchik J,
Hankins WD,
Ruscetti SK:
Sequence comparisons of the anemia- and polycythemia-inducing strains of Friend spleen focus-forming virus.
J Virol
53:570,
1985[Abstract/Free Full Text]
22.
Chung SW,
Wolff L,
Ruscetti SK:
Transmembrane domain of the envelope gene of a polycythemia-inducing retrovirus determines erythropoietin-independent growth.
Proc Natl Acad Sci USA
86:7957,
1989[Abstract/Free Full Text]
23.
Li J-P,
D'Andrea AD,
Lodish HF,
Baltimore D:
Activation of cell growth by binding of Friend spleen focus-forming virus gp55 glycoprotein to the erythropoietin receptor.
Nature
343:762,
1990[Medline]
[Order article via Infotrieve]
24.
Showers MO,
De Martino JC,
Saito Y,
D'Andrea AD:
Fusion of the erythropoietin receptor and the Friend spleen focus-forming virus gp55 glycoprotein transforms a factor-dependent hematopoietic cell line.
Mol Cell Biol
13:739,
1993[Abstract/Free Full Text]
25.
Ruscetti SK,
Janesch NJ,
Chakraborti A,
Sawyer ST,
Hankins WD:
Friend spleen focus-forming virus induces factor independence in an erythropoietin-dependent erythroleukemia cell line.
J Virol
64:1057,
1990[Abstract/Free Full Text]
26.
MacDonald ME,
Reynolds FH Jr,
Van de Ven WJ,
Stephenson JR,
Mak TW,
Bernstein A:
Anemia- and polycythemia-inducing isolates of Friend spleen focus-forming virus. Biological and molecular evidence for two distinct viral genomes.
J Exp Med
151:1477,
1980[Abstract/Free Full Text]
|
Abstract
Introduction
Abstract