1 Departments of Medicine and Cell Biology, 2 Pathology,
3 Center for Irnmunology, Washington University School of Medicine,
St. Louis, MO 63110 USA
4 The Walter and Eliza Hall Institute of Medical Research, 3050
5 Ralph H. Johnson Department of Veterans Affairs Medical Center,
Charleston, SC 29401 USA
*corresponding author: Telephone 314-362-8800; FAX 314-362-8826;
The receptor for erythropoietin is restricted to cells of mature
erythroid and possibly megakaryocyte lineages. Studies in cell lines
have suggested that cytokine receptors share a conserved signaling
pathway for proliferation. We retrovirally transduced the erythropoietin
receptor or a constitutively activated form of the EPOR into normal
hematopoietic progenitors, including blast cell colonies. The EPO-R
was able to support the proliferation and differentiation of early
erythroid, early megakaryocytic, and macrophage progenitors, but
not granulocyte progenitors. Blast cell colonies transduced with
the EPO-R proliferate in response to EPO but the development of
erythroid cells was not favored over other lineages. These results
with normal cells suggest that some but not all cytokine receptors
exhibit shared signaling pathways, and that EPO signaling alone
is not sufficient to drive erythroid development. The Janus family
of cytosolic tyrosine kinases mediate cytokine initiated mitogenic
signals. We have determined that the EPO-R box 1 cytoplasmic motif
is required for the binding and activation of JAK2. However, sequences
outside the box 1 domain most likely regulate the specificity of
JAK kinase association.
Erythropoietin (EPO) is a serum glycoprotein hormone required
for the survival, proliferation and differentiation of committed
erythroid progenitor cells, and is the principal hormone regulating
the level of circulating red blood cells. The administration of
recombinant human EPO to anemic patients suffering from chronic
renal failure, AIDS, or bone marrow suppression due to chemotherapy
has dramatically alleviated their need for blood transfusions. In
contrast to many other hematopoietic growth factors, EPO acts primarily
on relatively mature erythroid progenitors, and to a significantly
lesser extent megakaryocyte progenitors, within the fetal liver
and adult bone marrow (reviewed in (Krantz, 1991). The receptor
for EPO is normally restricted in its expression to relatively mature
cells of the erythroid and megakaryocyte lineage(Youssoufian, Longmore
et al., 1993), and has also been reported to be expressed in the
placenta(Sawyer, Krantz et al., 1989), by embryonic stem cells(Keller,
Kennedy et al., 1993), on endothelial cells(Anagnostou, Lee et al.,
1990), and on some neuronal-like celllines(Masuda, Nagao et al.,
1993). The functional relevance of this developmentally diverse
EPO-R gene expression is not clear, and quite apart from its function
in hematopoiesis, EPO-Rs may play other roles in non-hematopoietic
cells. In the case of the erythroid lineage, expression of the EPO-R
is low or absent on immature progenitors such as BFU-E and is increased
by the CFU-E stage of differentiation, before decreasing as the
erythroid cells undergo terminal differentiation(Sawada, Krantz
et al., 1990). Stimulation of adult BFU-E with EPO as a single stimulus
fails to support the formation of erythroid colonies(Oai, Krantz
et al., 1991). Whether EPO directly affects differentiation as well
as proliferation is not clear. A murine EPO-R cONA, which stimulates
EPO-dependent proliferation of several hematopoietic cell lines,
was isolated by expression cloning and encodes a type I membrane-spanning
protein of 66kO which is a member of the cytokine receptor superfamily(O'Andrea,
Lodish et al., 1989). Studies have detected a single binding affinity
for EPO (kO = 300-800pM) in heterologous hematopoietic cells, fibroblasts,
or COS cells transfected with the EPO-R cONA. Binding studies on
human and murine bone marrow and fetal erythroid progenitor cells
have detected either one or two affinities for radioiodinated EPO
(Youssoufian, Longmore et al., 1993). Two affinities for EPO may
suggest the presence of other components which modulate the binding
activity of the cloned EPO-R. However, it is unresolved whether
there are indeed EPO-Rs of multiple affinities. A constitutively
active (cytokine-independent) form of the EPO-R has been isolated
from a retroviral transduction system and found to contain a single
point mutation, resulting in an Arg to Cys change at residue 129
of the exoplasmic domain (Yoshimura, Longmore et al., 1990). The
R129C receptors form disulfide-linked homodimers independent of
hormone, yet retain the capacity to bind EPO with a single affinity
(kD= 700pM)(Watowich, Yoshimura et al., 1992). Since several members
of the cytokine receptor family are active as ligand-induced homo-
or heteromultimers, the disulfide-linked dimers most likely mimics
the structure of the hormone-bound form of the EPO-R and thus transmit
a constitutive proliferative signal. Chimeric c-kit/EPO-R receptors
transmit mitogenic signals in response to added c-kit ligand (Ohashi,
Maruyama et al., 1994). When an EPO-R lacking the entire cytoplasmic
region is coexpressed, in excess, with wild type EPO-R EPO-induced
mitogenic signals are blocked ("dominant negative" receptor form)
(Watowich, Hilton et al., 1994). Taken together these observations
strongly suggest that the functional cell surface form of the EPO-R
is a multimeric protein complex consisting of, at least, a homodimer
of the cloned cDNA. Although the cytosolic domain of the EPO-R does
not contain an obvious protein kinase domain, ligand induced multimerization
of the EPO-R stimulates rapid and transient tyrosine phosphorylation
of a number of cellular proteins, which are essential for EPO-induced
proliferation (Miura, D'Andrea et al., 1991). Sequence analysis,
and functional mutagenesis studies of members of the cytokine receptor
superfamily of receptors have identified two small conserved motifs,
box 1 and box 2, in the membrane proximal cytoplasmic domains, which
are required for proliferative signals (Murakami, Narakaki et al.,
1991). This membrane proximal domain has been shown to be necessary
for binding and phosphorylation of Janus kinase family members (cytosolic,
nonreceptor tyrosine kinases). There is some specificity for the
JAK kinase member utilized by different receptors. For example,
the EPO-R activates only JAK2, whereas the 11-6 receptor signal
transducing component, gp130, activates either or both JAK1 and
JAK2, depending upon the cell type expressing gp130 (Witthuhn, Quelle
et al., 1993)(Stahl and Yancopoulos, 1993).
Results and Discussion
To identify features of the EPO-R that mediate its interaction
with JAK2, we generated chimeric receptor proteins that contained
the cytoplasmic domain of the EPO-R or gp130 fused to the extracellular
and transmembrane domain of the VSV G protein. This modification
allowed us to immunoprecipitate and immunoblot both EPO-R and gp130
receptors with the same monoclonal antibody and eliminated the variability
of using different receptor antibodies for our comparison. Because
the kinase activity of JAK2 is not detectable in the absence of
cytokine stimulation we generated constitutively active forms of
JAK1 and JAK2 by replacing the tyrosine kinase domain of the JAKs
with an epitope tagged (myc) tyrosine kinase domain from p59fyn
.Thus anti-myc monoclonal antibodies will immunoprecipitate the
JAKs. The chimeric receptors and JAK kinases were transiently coexpressed
in HeLa cells using the vaccinia-17 expression system to determine
if cytokine receptors and JAK kinases could form stable complexes.
The membrane proximal domain of the EPO-R was required for association
with JAK2, and JAK1 did not associate with the EPO-R (Table 1).
Specifically box 1, not box 2, was required for the association
between the EPO-R and JAK2. Similarly the membrane proximal box
1 domain of gp130 was required for association with JAK2, but also
associated with JAK1. This suggests that box 1 sequences are required
for both JAK1 and JAK2 association with cytokine receptors but that
sequences outside the box 1 domain regulate the specificity of JAK
kinase association. IL-6 and CNTF, cytokines whose receptors utilize
gp130, can stimulate the phosphorylation of JAK1, JAK2, and tyk2
depending on the cell line examined(Stahl and Yancopoulos, 1993).
In contrast only JAK2 has been shown to associate with the EPO-R,
in vivo(Miura, Nakamura et al., 1994). Our results demonstrated
that the EPO-R and gp130 behave differently with regards JAK activation,
in a heterologous cell line. Thus, receptor interactions with JAK
kinases could be regulated in two ways; receptors for cytokines
such as EPO encode the specificity of association within receptor
protein sequence whereas JAK kinase interaction with receptors like
gp130 must be influenced by cell-specific factors. It will be important
to determine what structural features of the receptors regulate
the specific interaction of JAK2 with the EPO receptor protein.
Expression of exogenous EPO-R in some IL-3- and GM-CSF-dependent
cell lines (BaF3, DA-1, 32D, and FDCP-1) confers upon these cells
the capacity to proliferate in response to EPO(Youssoufian, Longmore
et al., 1993). This is not the case for most IL-2-dependent cell
lines (CTLL-2, HT-2)(Yamamura, Kageyama et al., 1992). Similarly,
studies of the biochemical events following ligand binding to members
of the cytokine receptor family have suggested conservation of signal
transduction mechanisms between some members of the cytokine receptor
family(Youssoufian, Longmore et al., 1993)(Ihle, Witthuhn et al.,
1994). One possible interpretation for these observations is that
specificity of response to a growth factor is obtained at the level
of receptor expression rather than at the level of signal transduction.
Given the normally limited expression of the EPO-R, we were interested
to determine if the EPO-R was capable of generating a proliferative
and differentiative signal in cells normally responsive to the ligands
of other members of the cytokine receptor family. Retroviral vectors
expressing EPO-R(wt) and EPOR(R129C) were constructed, primary hematopoietic
progenitor cells (d12.5 fetal liver and 5-fluorouracil treated adult
bone marrow) were infected, and cultured in methylcellulose in the
absence or presence of added EPO(McArthur, Longmore et al., 1995)(Pharr,
Hankins et al., 1993).
Table 1. The association of IAK2 or JAKl
with the EPO-R or gp130
(A) Requirement of the membrane proximal box 1 of the EPO-R for
JAK2 association. (8) Boxl but not box2 is critical for JAK association
with the cytoplasmic domain of gp130. Open and closed boxes represent
the extracellular and transmembrane domain of the VSV G protein,
respectively. Hatched boxes represent the intracellular domain of
the indicated cytokine receptor. HeLa cells were transfected with
cDNAs corresponding to the indicated cytokine receptor chimeras
and either JAKl or JAK2. Cells were lysed in 1% 8rij 96 and the
detergent soluble extract was immunoprecipitated with anti-VSV G
or anti-myc antibodies. Association of the cytokine receptor chimeras
with the kinases was determined by in vitro kinase reactions, in
the presence of 32p-gATP, upon the immunoprecipitated pellets. The
products of the in vitro kinase reactions were separated on a 7.5%
acrlamide-SDS gel and analyzed by autoradiography. Expression of
cytokine receptor chimeras was confirmed by anti-VSV G protein immunoblot,
and JAK kinase by anti-myc IP kinase reactions.
Table 2 shows the effect of EPO upon erythroid progenitors transduced
with wild type EPO-R or an activated form of the EPO-R, EPO-R(R129C).
Cultures of cells infected with the EPO-R retrovirus demonstrated
erythroid colony formation in response to EPO as a single stimulus
when analyzed at day 2 or day 8. Normally stimulation of BFU-E by
EPO does not support the development of day 8 colonies (Table 1
NeoR experiment). The finding of frequent day 8 erythroid colonies
in EPO stimulated cultures of fetal liver cells transduced with
EPO-R(wt) suggest that the signal transduction mechanism allowing
fior EPO-induced cell proliferation and differentiation are present
in some murine BFU-E. Unstimulated cultures of fetal liver cells
transduced with EPO-R(R129C) contained erythroid colonies when analyzed
at day 2 but not day 8. However, the same cell population stimulated
by EPO did contain day 8 erythroid colonies. This suggests that
there may be some functional differences in signals generated by
EPOR(R129C) in the unstimulated versus the EPO-stimulated state.
Analysis of cell surface EPO-Rs in BaF3 expressing EPO-R(R129C)
detects a minority of receptor dimers as opposed to monomers(Watowich,
Yoshimura et al., 1992). Thus EPOstimulation may be able to generate
additional EPO-R dimers leading to an enhanced signal. When fetal
liver or post 5-FU bone marrow cells transduced with EPO-R or .
EPO-R(R129C) were stimulated by EPO there was an increased number
of megakaryocyte colonies (Table 3). This appeared to be due to
the recruitment of additional progenitors cells rather than altered
committment of existing progenitors as there was no change in non-megakaryocyte
colony numbers. The recruitment of additional megakaryocyte clones
was of both the BFU-Meg and CFU-Meg type. These observations suggest
the presence of signal transduction mechanisms alowing EPOinduced
proliferation and differentiation in cells of both erythroid and
megakaryocyte lineages at stages of differentiation prior to the
expression of significant numbers of EPO-Rs. The current experiments
also demonstrated a direct stimulation of individual clones of the
macrophage lineage by EPO, when these cells are transduced with
EPO-R or EPO-R(R129C) (Table 3). In contrast, the data did not demonstrate
EPO-induced proliferation of isolated clones of the granulocyte
lineage. Cells of the granulocyte lineage transduced with a c-fms
(CSF-1R) also fail to proliferate in response to CSF-1(McArthur,
Rohrschneider et al., 1994). These results, taken together, suggest
that granulocyte progenitors may express a restricted range of signal
transduction molecules influencing their response to specific cytokines.
Table 2. Erythroid colony formation
in cultures of fetal liver cells induced to express EPO-R or EPO-R(R129C)
Day 12.5 fetal liver cells were infected with retroviruses expressing
NeoR, EPOR/NeoR, or EPO-R(R129C)/NeoR by cocultivation with retroviral
producing cells. Cultures contained 5 x 104 fetal liver cells. All
cultures contained 1.5 mg/ml of G418. Results are from quadruplicate
cultures and expressed as number of colonies relative to NeoR cultures.
Cultures were stimulated by 2 U /ml of hEPO or 10% spleen conditioned
media (SCM). Maximal responses are depicted in cultures of NeoR
transduced cells containing EPO and SCM.
Hematopoietic cells at different stages in development are thought
to have different complements of cytokine receptors. Whether the
appearance of specific receptors initiates a particular developmental
sequence is not known: Does the acquisition of a lineage-specific
receptor induce differentiation? To address this question we utilized
retroviral-mediated gene transfer to express the EPO-R in multilineage
blast cell progenitors (Pharr, Ogawa et al., 1994) (Table 4). By
DNA PCR we demonstrated that pluripotent blast cell clones could
be infected with EPO-R(R129C)-expressing retroviruses. Blast cells
and their progeny, CFU-GM and CFU-mix, express retrovirally derived
EPO-R(R129C) as determined by PCR of cDNA prepared from these colonies
(not shown). We observed no evidence that blast cells transduced
with EPO-R(R129C) could induce erythroid differentiation, even following
the addition of EPO. These results are consistent with in vivo experiemtns
in which EPO has been shown to regulate the rate at which committed
erythrocyte progenitors become erythroblasts.
Table 3. Colony formation in cultures
of post 5-FU bone marrow cells induced to express EPO-R or EPO-R(R129C).
Adult mice had their bone marrow harvested 4 days after treatment
with 5fluorouracil, 150 mg/kg. Cells were infected by cocultivation
with retroviral producing cells. Cultures contained 5 x 104 post
5FU bone marrow cells. All cultures contained 2 mg/ml G418. Cultures
were stimulated by 2 U/ml of hEPO, and 1000 U/ml of mIL-3. Results
are from triplicate plates, scored after 7 days of culture, and
reported as colony numbers relative to NeoR infected plates. G,
granulocytes; GM, granulocyte-macrophage; M, macrophage; MEG, megakaryocyte.
The initiation of differentiation could be induced by an exogenous
stimulus or could be a spontaneous random event with survival of
committed cells dependent on the availability of a supportive milieu.
Metcalf analyzed the progenitor content of developing blast cell
colonies and found that GM-CSF or IL-3 with SCF increased the relative
frequency of granulocytic progenitors. Others have demonstrated
that primitive progenitors derived from human cord blood divide
asymmetrically and that this was not affected by different combinations
of cytokines. The results presented here are more consistent with
a supportive role for EPO in the differentiation of red cells. Thus
expression of lineage-specific receptors for EPO may be a consequence
rather than a cause of differentiation.
Table 4. Effect of EPO-R(R129C) on the
composition of mixed colonies derived from infected blast cells
Spleen cells were harvested from mice treated with 5-FU (150 mg/ml)
4 days earlier. These were cultured in methylcallulose. Blast cell
colonies, identified on day 6 or 7, were picked, washed and resuspended
in retroviral supernatant or control. Following infection samples
of blast cells were plated at 50-100 per plate. Mixed colonies were
identified, picked and cytospin preparations counted after Wrights
staining. acultures contained Stem Cell Factor (SCF) (3 U/rnl),
IL-3 (100 U/ml), and EPO (1 U/ml) and were scored on day 9. G, granulocytes;
M, macrophage; E, erythroid; MEG, megakaryocyte.
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