Institute for Molecular Pathology, A-1030 Vienna, Austria
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cytokine Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) regulates proliferation, differentiation, and apoptosis during myelopoiesis
and erythropoiesis. Structure-function relationships of
GM-CSF interactions with its receptor (GM-R), the
biochemistry of GM-R signal transduction, and GM-CSF action in vivo are relatively well understood. Much
less is known, however, about GM-R function in primary hematopoietic cells. In this paper we show that
expression of the human GM-R in a heterologous cell
system (primary avian erythroid and myeloid cells)
confirms respective results in murine or human cell
lines, but also provides new insights how the GM-R regulates progenitor proliferation and differentiation.
As expected, the hGM-CSF stimulated myeloid progenitor proliferation and differentiation and enhanced
erythroid progenitor proliferation during terminal differentiation. In the latter cells, however, the hGM-R
only partially substituted for the activities of the erythropoietin receptor (EpoR). It failed to replace the
EpoR in its cooperation with c-Kit to induce long-term
proliferation of erythroid progenitors. Furthermore,
the hGM-R chain specifically interfered with EpoR signaling, an activity neither seen for the
c subunit of
the receptor complex alone, nor for the
chain of the
closely related Interleukin-3 receptor. These results
point to a novel role of the GM-R
chain in defining
cell type-specific functions of the GM-R.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE tight regulation of proliferation, differentiation, and apoptosis required for homeostasis during hematopoiesis is to a large extent effected by numerous humoral factors (cytokines). These polypeptide factors induce and maintain the production of the correct amounts of immature and mature hematopoietic cells. Furthermore, cytokines are responsible for the fast responses of the hematopoietic system to specific needs arising from immune responses, blood loss, or during disease.
One of the best-studied cytokines is the Granulocyte-
Macrophage Colony-Stimulating Factor (GM-CSF).1 It
represents a major regulator at different stages of hematopoiesis (Metcalf, 1980). GM-CSF induces a large variety of
biological effects, such as proliferation induction in early
progenitors, stimulation of differentiation along various
myeloid lineages depending on factor concentration (Metcalf, 1989
), cooperation with or even replacement of erythroid cytokines (Nishijima et al., 1995
), as well as regulation
of mature cell function (Metcalf, 1980
). Finally, GM-CSF
also stimulates the proliferation of multipotent CD34-positive progenitors (Sonoda et al., 1994
).
GM-CSF binds to a heterodimeric receptor complex
(GM-R) consisting of an chain (GM-R
) and a common
chain (
c). Whereas the
chain confers specific binding
to GM-CSF,
c by itself does not bind the ligand, but alters
the affinity of the receptor complex for GM-CSF from low
to high affinity. Furthermore,
c associates with the intracellular tyrosine kinase Jak2 (Quelle et al., 1994
). Since
neither the GM-R
, nor
c have an intrinsic kinase activity, this association is thought to be a necessary step in triggering signal transduction. Interfering with the association
between
c and Jak2 renders respective GM-R mutant
proteins unable to confer a mitogenic signal (Watanabe et
al., 1996
). After ligand activation of the GM-R Jak2 phosphorylates several tyrosine residues on
c. These subsequently interact with the src homology region 2 (SH-2) domains of signaling intermediates like Stat5 (Ihle et al., 1994
;
Mui et al., 1995
; Wakao et al., 1995
), as well as phosphatidylinositol-3-kinase (PI-3-kinase), Grb2, Shc, and PTP-1D
(Lanfrancone et al., 1995
; Rao and Mufson, 1995
; Pratt et
al., 1996
).
In contrast to c, the GM-R
chain has a very small cytoplasmic domain that lacks tyrosine residues phosphorylated by Jak-2 or other kinases. Nevertheless, the
chain
contributes to the activity of the GM-R, since a receptor
complex with an
chain lacking its cytoplasmic tail is unable to promote proliferation and differentiation (Sakamaki et al., 1992
; Weiss et al., 1993
; Matsuguchi et al.,
1997
). How the
chain contributes to receptor function is
still obscure, but obviously important to understand receptor-specific signal transduction.
The c receptor subunit is not only used by the GM-R,
but also by the interleukin (IL)-3 and IL-5 receptors. Like
the GM-R, both of these receptors have a receptor-specific
chain, but they also require interaction with
c for
signal transduction and biological activity. Despite this
shared use of
c, all three receptor complexes have distinct
biological activities when activated by their respective
ligands. IL-5 induces differentiation of eosinophils (Dent
et al., 1990
), a specificity explained mainly by the restricted expression pattern of the IL-5 receptor
chain. In contrast, IL-3 functions on a similar subset of hematopoietic
progenitors as GM-CSF. However, its activity is much less
biased to myeloid progenitors as the activity of GM-CSF
(Nimer and Uchida, 1995
). Thus, receptor specificity may
be dependent on signaling events mediated by the
chains
of the receptors.
In contrast to this rather detailed knowledge on receptor structure and in vivo function, much less is known
about how ligand activation of the GM-R affects cell proliferation, differentiation, and apoptosis in primary, differentiating hematopoietic progenitors. So far, mainly immortalized cell lines of human or murine origin were used
in such studies (Spooncer et al., 1986; Jubinsky et al., 1993
;
Mui et al., 1995
). However, many of these lines are altered
in their response to cytokines and sometimes exhibit incomplete and/or aberrant differentiation (for review see
Beug et al., 1995).
Clones of chicken erythroid and myelomonocytic progenitors capable of sustained proliferation offer an alternative. Long-term proliferation can be induced by transformation with temperature-sensitive (ts) oncoproteins
(ts-v-sea, ts-gag-myb-ets; Beug et al., 1984; Knight et al.,
1988) orin the case of erythroid progenitors
by combinations of "self renewal factors," i.e., receptor tyrosine kinase ligands plus steroid hormones (Hayman et al., 1993
;
Beug et al., 1995). In both cases, the proliferating progenitors can be induced to terminal differentiation into erythrocytes or macrophages either by ts oncoprotein inactivation at 42°C or by replacement of "self-renewal factors" by
cytokines required for differentiation.
In this paper, we sought to clarify the action of the GM-R
and the cooperation between its and
chains by expressing these proteins in the above-described heterologous cell systems. Since avian hematopoietic progenitors are
unresponsive to mammalian GM-CSF or other cytokines,
e.g., those present in serum (Steinlein et al., 1994
; Wessely
et al., 1997b
; Beug, H., and E.M. Deiner, unpublished observations), this allowed to test for the specific contributions of the GM-R. The usefulness of such an approach to
analyze mammalian hematopoietic regulators and oncoproteins has recently been demonstrated (Tran Quang
et al., 1997). Here, we show that GM-CSF promotes differentiation of hGM-R-expressing myelomonocytic progenitors and cooperates with the EpoR in regulating erythroid
differentiation. Interestingly, however, non-liganded exogenous hGM-R renders erythroid progenitors unresponsive
to signaling via the endogenous aEpoR. This quenching of
Epo-responsiveness is specifically caused by the hGM-R
chain, while neither
c nor the hIL-3 receptor
chain induce this effect. Epo responsiveness of the GM-R-expressing
cells can be restored by an exogenously expressed mEpoR.
However, the hGM-R cannot substitute for all functions of
the mEpoR, in particular it failed to cooperate with c-Kit
to induce long-term proliferation in erythroblasts.
These results constitute the first demonstration that the
chain of the GM-R may contribute to lineage commitment by differential signal transduction in erythroid and
myeloid hematopoietic cell types.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Viruses and Cells
The cDNAs encoding the hGM-R was a gift of Genentech Inc. (South
San Francisco, CA), the hIL3-R
and
c were generously provided by
DNAX (Palo Alto, CA). The construction of the pCRNCM/hGM-R retroviral vector will be described in detail elsewhere. In general, the strategy
used was similar to that described in Tran Quang et al. (1997), namely separating the two cDNAs for GM-R
and
c by an internal ribosome entry
site (IRES; Elroy-Stein et al., 1989). Additionally, to express the single
chains of the receptor complex,
c was cloned into the avian retroviral expression vector pOli (Wessely et al., 1997b
) and the hGM-R
, as well as
the hIL3-R
, were inserted behind the cytomegalovirus (CMV) promoter
of pCRNCM (Steinlein et al., 1994
). RCAS/EpoR was a kind gift of J. Ghysdael (Institute Curie, Orsay, France).
Chicken embryo fibroblasts expressing the retroviral constructs
pCRNCM/hGM-R, pCRNCM/hGM-R, pCRNCM/hIL3-R
and pOli/
c
were generated as described earlier (Zenke et al., 1990
; Wessely et al.,
1997b
). All fibroblasts were grown in standard growth medium (Graf,
1973
). Ts21-E26 transformed myeloblasts, SCF progenitors, and SCF/
TGF
progenitors were grown from the bone marrow of 3-10-d-old Spafas chicks as described previously (Hayman et al., 1993
; Schroeder et al.,
1993
; Woldmann et al., 1997
). The HD3 erythroblast cell line HD3-EpoR-E22 has been described previously (Mellitzer et al., 1996
). Steroids (estradiol, dexamethasone) were used at a final concentration of 10
6 M, cMGF
at 10 ng/ml, aSCF at 100 ng/ml (Bartunek et al., 1996
), TGF
(Promega,
Heidelberg, Germany) at 5 ng/ml, IGF-1 (Sigma Chemical Co., St. Louis,
MO) at 40 ng/ml, hGM-CSF (Promega) at 10 ng/ml, hEpo at 10 U/ml, insulin at 1.4 nM, and PD 153035 at 2.5 µM (Fry et al., 1994
).
Infection of Primary Erythroblasts and Myeloblasts with Retroviruses
To infect erythroblasts, freshly prepared chicken bone marrow cells were
cocultivated with mitomycin C-treated CEF expressing the respective retroviral constructs for 2 d (Fuerstenberg et al., 1992). The cells were then
either further propagated as a mass culture or seeded in CFU-E-methocel
containing aSCF, TGF
, E2, and Dex. Outgrowing colonies were isolated
and expanded in CFU-E medium plus aSCF, TGF
, E2, and Dex (Hayman et al., 1993
) Myeloid progenitors were obtained by coculturing bone
marrow cells with both mitomycin C-treated, ts21-E26-transformed myeloblasts and CEF expressing the respective retroviral constructs. Subsequently, the myeloid progenitors were maintained in the presence of
cMGF (Beug et al., 1984
).
Proliferation Kinetics
To determine the growth kinetics of erythroblast mass cultures, cells were
subjected to daily partial medium changes plus re-addition of fresh factors. They were kept at densities of 2-4 × 106 cells/ml, and aliquots
counted in an electronic cell counter (CASY-1, Schärfe-System, Reutlingen,
FRG). Cumulative cell numbers were determined as described in Fuerstenberg et al. (1992).
Differentiation of Myeloid Progenitors
Cells were washed twice in PBS and seeded at 1-2 × 106 cells/ml into 35-mm
dishes containing 2 ml of differentiation medium (Woldmann et al., 1997)
and were incubated at 42°C. When indicated, cMGF or hGM-CSF was
added. The cells were maintained at densities of 2-4 × 106 cells/ml,
counted daily, and aliquots were removed at days 3-5 for morphological
analysis by cytocentrifugation onto slides and staining with histological
dyes (Beug et al., 1984
).
Differentiation of Erythroid Progenitors
Cells were washed twice in PBS and seeded at 1-2 × 106 cells/ml into
35-mm dishes containing 2 ml of differentiation medium (Dolznig et al.,
1995). When indicated, 3-5% high titer anemic serum (as a source for
aEpo), insulin, hGM-CSF, and/or hEpo were added. Cells were counted
daily and maintained at densities of 2-4 × 106 cells/ml. Aliquots were removed for hemoglobin determination and morphological analysis at the
days indicated.
Analysis of Differentiation by Cell Morphology and Staining
Cells were cytocentrifuged onto slides and subsequently stained with histological dyes and neutral benzidine for hemoglobin as described in (Beug
et al., 1982). Images were taken using a CCD camera (Photometric, Tucson, AZ) and a blue filter (480 nm), so that mature cells (stained yellow to
brownish) appear darkly stained. Images were processed with Photoshop
(Adobe Systems Inc., Mountain View, CA).
Photometric Hemoglobin Assay
Three 50-µl aliquots of the cultures were removed and processed for photometric determination of hemoglobin as described (Kowenz et al., 1987).
Values obtained were normalized to cell number.
Northern Blot Analysis
Cells were lysed in GITC buffer (Chomczynski and Sacchi, 1987) and
NaAc, pH 4.0, was added to 25 mM. The solution was extracted with H2O-saturated phenol, chloroform, and isoamylalcohol. After precipitation
with isopropanol, the pellet was dissolved in 10 mM Tris/HCl, pH 7.0, 1 mM
EDTA, 0.2% SDS, and proteinaseK was added to 200 µg/ml. After a 30-min incubation at 37°C, the solution was extracted with phenol/chloroform/isoamylalcohol (25:24:1), pH 7.0, and then precipitated with ethanol.
10-20 µg of RNA was run on a formaldehyde-containing agarose gel and
transferred to nylon filters (Gene Screen, Dupont NEN, Boston, MA)
using conventional procedures. Single-stranded DNA probes were radioactively labeled with 32P by using an Oligolabeling Kit (Pharmacia Biotechnology Inc., Piscataway, NJ) and hybridized at 65°C in 7% SDS, 0.5 M
Na phosphate, pH 7.0, 1 mM EDTA. Membranes were washed in 1%
SDS, 40 mM Na phosphate at 65°C, and then autoradiographed.
Growth Factor Treatment and Cell Lysis
For growth factor stimulation primary myeloid and erythroid progenitors
were washed once with PBS and then withdrawn from factors by cultivation for 12 h in differentiation medium (Dolznig et al., 1995). Afterwards
the cells were washed twice with PBS, suspended in differentiation medium, and then treated as indicated with either 50 ng/ml TGF
, 50 U/ml
hEpo, 100 ng/ml hGM-CSF, 100 ng/ml cMGF, or without factors for 15 min at 37°C. Stimulation of HD3-EpoR-E22 cells was performed similarly
except that the cells were withdrawn from growth factors using serum-free
differentiation medium using 2.5 µM PD153035 to inhibit v-ErbB activity
(Mellitzer et al., 1996
) and were shifted to 42° instead of 37°C.
After stimulation, 106 cells were lysed in 10 µl EMSA lysis buffer (20 mM Hepes, pH 7.9, 140 mM NaCl, 1.5 mM MgCl2, 1.0% NP-40, 1 mM sodium orthovanadate, 10 µg/ml aprotinin, and 2 µg/ml leupeptin), or in 20 µl immunoprecipitation buffer (1% Triton-X-100, 50 mM Tris/HCl, pH 8.0, 100 mM NaCl, 1 mM sodium orthovanadate, 10 µg/ml aprotinin, and 2 µg/ml leupeptin), respectively. Cell lysates were cleared by centrifugation for 15 min at 15,000 rpm before use.
Mobility Shift (EMSA) Assay
This assay was performed as described earlier (Mellitzer et al., 1996) using
the IFP53-GUS oligonucleotide to detect complex formation with Stat5b.
Phosphotyrosine Blot
Lysates from growth factor-stimulated cells were subjected to Western
blot analysis as described earlier (Hayman et al., 1993; Mellitzer et al.,
1996
). Samples were run on an 8% SDS-PAGE gel, transferred to nitrocellulose membranes (Dupont-NEN, Boston, MA), and then probed with
an anti-phosphotyrosine antibody (4G10; Upstate Biotechnology Inc.,
Lake Placid, NY).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
hGM-R Functionally Expressed in ts21-E26-transformed Myeloid Progenitors
To demonstrate the functionality of the hGM-R in chicken
cells, we introduced it into myelomonocytic progenitors
transformed by the ts21 mutant of the E26 virus, expressing a thermolabile p135gag-myb-ets oncoprotein. These ts21-E26 myeloblasts proliferate at 37°C, if supplied with the
avian cytokine cMGF (Leutz et al., 1988, 1989
). After inactivation of the ts21-E26 oncoprotein at 42°C, the cells
differentiate into macrophages, again requiring cMGF (Beug et al., 1984
; Woldmann et al., 1997
).
Stable expression of both chains of the hGM-R was
achieved by constructing a retrovirus that coexpresses the
and the
c chain (for a schematic drawing see Fig. 1 A).
Both chains were inserted behind an internal CMV promoter, separated from each other by an IRES (Elroy-Stein
et al., 1989
). Chicken embryo fibroblasts producing this virus were cocultivated with a mixture of ts21-E26-transformed myeloblasts and freshly prepared chicken bone
marrow cells. Methocel colonies growing in the presence
of cMGF and G418 were expanded and analyzed for the
expression of the hGM-R by Northern analysis. Fig. 1 B
shows that these clones express mRNA for both the
and the
c chain. Also, treatment with hGM-CSF caused only
in hGM-R-expressing clones the phosphorylation of a
specific 130-kD protein in phosphotyrosine blots, whereas
cMGF treatment caused phosphorylation of a slightly smaller protein in both hGM-R expressing and control
ts21-E26 cells (Fig. 1 C). Finally, recombinant hGM-CSF
and cMGF evoked a similar, short-term proliferation response in hGM-R expressing ts21 cells (Fig. 2 B; see below). Control cells only responded to recombinant cMGF
(Fig. 2 A). This clearly shows that the exogenous hGM-R
is bioactive.
|
|
Next, we analyzed whether hGM-CSF could replace
cMGF in factor-dependent differentiation of ts21-E26
cells. Differentiation was analyzed by three parameters.
First, daily cell counting shows that ts21-E26 myeloblasts
differentiating at 42°C undergo a few cell divisions before
becoming stationary after 2-3 d. Second, they become adherent and acquire a typical macrophage morphology in
cytospin preparations stained with histological dyes. And
thirdly, they express typical macrophage antigens (MC22/3,
MC47/83) and lose myeloblast antigens (MC4M12/26;
Beug et al., 1984). When ts21-E26 cells expressing the
hGM-R were induced to differentiate at 42°C, hGM-CSF
fully replaced cMGF. After initial transient proliferation
(Fig. 2 B; somewhat prolonged in case of hGM-CSF treatment), hGM-R-expressing ts21-E26 cells differentiated
into macrophage-like cells in both hGM-CSF and cMGF
(Fig. 2 C, right panels). In contrast, control cells lacking
the hGM-R showed the expected differentiation behavior
in cMGF, but underwent apoptosis in hGM-CSF (Fig. 2 C,
left panels). Similar to the cMGF-treated control cells, the
hGM-R-expressing cells differentiating in the presence
of hGM-CSF gained expression of macrophage surface
markers detected by the monoclonal antibodies MC22-3
and MC47/83, while they lost the immature cell marker
MC4M12/26 (data not shown; Beug et al., 1984
).
Taken together, these results indicate that the hGM-R can be functionally expressed in avian myelomonocytic progenitors, inducing typical differentiation accompanied by transient proliferation.
Cooperation of the hGM-R with the EpoR during Erythroid Differentiation Requires hGM-CSF
In human or murine bone marrow, GM-CSF acts as a
burst-promoting activity (BPA), supporting Epo-dependent erythroid differentiation (Metcalf, 1989). So far, we
have been unable to find a similar activity for cMGF
(Beug, H., and E.M. Deiner, unpublished observations). Sequence comparisons suggested cMGF to be homologous to IL-6 or G-CSF rather than to GM-CSF (Boulay
and Paul, 1993
). It was therefore of interest whether erythroid
progenitors expressing the hGM-R would respond to
hGM-CSF in a fashion analogous to early mammalian erythroid progenitors (BFU-E). To isolate normal erythroblast clones expressing the hGM-R, avian bone marrow
cells growing in aSCF, TGF
, estradiol, and dexamethasone (Wessely et al., 1997a
) were infected with the hGM-R
retrovirus and seeded into methocel containing G418. Resistant colonies were picked and expanded. After confirming expression of both hGM-R chains by Northern and phosphotyrosine blot analysis (data not shown; see Fig. 1),
hGM-R-expressing clones as well as empty vector control
clones were induced to differentiate in insulin alone (control), hGM-CSF plus insulin, aEpo-containing anemic
chicken serum (AS) plus insulin, and in both AS and
hGM-CSF plus insulin. After 4 d, cells were subjected to
cytocentrifugation followed by histochemical staining for
hemoglobin. In addition, we quantitated hemoglobin (normalized to cell number) in these cell populations (see Materials and Methods).
As expected, control cells lacking the hGM-R differentiate in AS/insulin regardless of the presence or absence of hGM-CSF (Fig. 3, A and B, left panels). In the absence of AS (i.e., in insulin alone or hGM-CSF plus insulin) these cells undergo abortive differentiation (production of aberrant, partially mature cells with reduced hemoglobin levels, followed by apoptosis). This is evident both from hemoglobin accumulation (Fig. 3 A, left) and cytospin analysis (data not shown). The slight decrease in hemoglobin content caused by hGM-CSF in the presence of AS is most likely caused by endotoxin present in the hGM-CSF preparation used, since it could be avoided by using purer batches of hGM-CSF (data not shown).
|
Erythroblast clones expressing the hGM-R differentiated in the presence of insulin plus both hGM-CSF and AS, but showed abortive differentiation in hGM-CSF plus insulin alone (Fig. 3, A and B, right panel). This confirmed, that hGM-CSF cannot induce erythroid differentiation on its own, rather exhibiting a BPA-like activity in hGM-R-expressing chicken erythroblasts. Surprisingly however, the hGM-R-expressing cells no longer differentiated in the presence of AS plus insulin, showing only abortive differentiation as with insulin alone. They expressed only slightly elevated levels of hemoglobin (Fig. 3 A, right panel) and appeared as either dead cells or partially mature cells expressing little or no hemoglobin in cytospins (Fig. 3 B, right panels).
These results show that the ligand-activated hGM-R supports AS-dependent erythroid differentiation and displays a BPA-like activity on erythroid progenitors. However, expression of the non-liganded receptor seems to interfere with erythroid differentiation, preventing a normal response to differentiation factors like aEpo.
The Chain of the hGM-R Is Sufficient to Interfere
with Erythroid Differentiation
The chain of the GM-R is mainly responsible for ligand
binding, whereas the
chain is necessary for interaction
with JAK2 and other signal transduction intermediates
(Quelle et al., 1994
; Watanabe et al., 1996
). Thus we sought
to determine, whether the interference with AS-induced
differentiation caused by the complete hGM-R receptor
heterodimer could also be induced by one of the two
chains alone. For this, retroviral vectors were constructed which either expressed the
chain or the
c chain of the
hGM-R (see Fig. 4 A for schematic drawings).
|
Erythroblast clones infected with these constructs were
generated and expression of the hGM-R proteins verified
(data not shown). Well-expressing clones were then induced to differentiate in the presence of AS plus insulin or
insulin alone and analyzed for erythroid differentiation parameters as above. Erythroblasts expressing the c chain
behaved exactly as clones containing empty vector. In the
presence of AS/insulin they expressed the same, high levels of hemoglobin (Fig. 4 B) and differentiated erythrocytes were visible in the stained cytospins (data not shown).
In insulin alone, both cell types showed the expected abortive differentiation (see above; Fig. 3 B), characterized by
much lower hemoglobin levels.
In contrast, the chain of the hGM-R heavily interfered
with erythroid differentiation. In both insulin alone and
AS plus insulin, these erythroblasts accumulated only
heavily reduced hemoglobin levels (Fig. 4 B) and failed to
differentiate into mature erythrocytes, giving rise to malformed, partially mature cells instead (abortive differentiation; data not shown).
To test whether this suppression of AS-induced differentiation was specific for the hGM-R chain, we also generated clones expressing the
chain of a related cytokine
receptor, the hIL-3R (see Fig. 4 A). Surprisingly, the hIL-3R
chain did not affect the responsiveness of erythroblasts
to AS/insulin. Rather, the hIL-3R
chain expressing clones
differentiated normally into mature erythrocytes expressing high hemoglobin levels (Fig. 4 B; data not shown).
These data clearly demonstrate that the chain of the
hGM-R specifically interferes with erythroid differentiation. Neither
c nor the related hIL-3R
chain could substitute for the hGM-R
chain in causing this effect. Thus,
at least part of the lineage-specific effects of the GM-R are
dependent on the
chain.
Exogenous Expression of the Murine EpoR Prevents Interference with AS-induced Differentiation by the Non-liganded hGM-R
The EpoR and the GM-R share several structural and
functional characteristics, suggesting that overexpression
of one receptor may interfere with signaling of the other.
Unfortunately this question could not be addressed directly, since neither Epo nor the EpoR have been cloned
in the chicken. However, aEpo shares most biochemical
properties with mammalian Epo (Kowenz et al., 1987) and
is neutralized by a polyclonal antibody to human Epo (Wessely, O., E.M. Deiner, and H. Beug, unpublished observations), even though aEpo fails to bind and activate
the mammalian EpoR and vice versa (Kowenz et al., 1987
;
Beug, H., unpublished results). We therefore decided to
ectopically express the murine EpoR in hGM-R-expressing erythroblasts. If the exogenously expressed hGM-R would interfere with the activity of the endogenous avian
EpoR (presumably expressed at much lower levels) by
quenching signals emanating from it, exogenous expression of both the mEpoR and the hGM-R at similar levels
should neutralize this effect.
To allow simultaneous expression of three proteins (i.e.,
hGM-R,
c, and mEpoR) in the same cell, we used an approach described previously (Tran Quang et al., 1997). Fibroblasts were co-transfected with the hGM-R retrovirus
plus a replication competent retroviral vector expressing
the mEpoR (RCAS-mEpoR; Fig. 5 A) and used to infect
avian bone marrow cells. G418-resistant erythroblast clones were isolated from methocel, expanded, and then tested
for expression of the three proteins (data not shown). Well
expressing clones were analyzed for erythroid differentiation as above. 4 d after differentiation induction, aliquots
of the cultures were processed for hemoglobin determination (Fig. 5 B) and histochemical staining for hemoglobin
(Fig. 5 C). Similar to erythroblasts expressing the hGM-R
only, mEpoR/hGM-R expressing cells terminally differentiated in the presence of hGM-CSF plus AS. In AS/insulin
alone, the cells expressed only low levels of hemoglobin
(Fig. 5 B) and developed into abortively differentiated, apoptotic cells (Fig. 5 C). Interestingly, and in clear contrast to
AS, activation of the exogenous mEpoR by hEpo in the
mEpoR/hGM-R-expressing cells induced their terminal differentiation into erythrocytes expressing high hemoglobin levels. Addition of hGM-CSF plus hEpo/insulin only
slightly increased hemoglobin levels beyond those induced
by the single ligands.
|
These results show that the interference of the exogenous hGM-R with AS-induced differentiation could be overcome, if a mEpoR was expressed in an exogenous fashion as well, and therefore suggest that the GM-R interacts with the EpoR signaling pathway.
The Ectopic hGM-R Interferes with Signaling of the Endogenous aEpoR
The EpoR and the GM-R share several signal transduction pathways, for instance activation of the Ras/MapK
pathway and the Stat5 pathway (Ihle et al., 1994; Mui et al.,
1995
; Wakao et al., 1995
). Recently, activation of Stat5b by
AS has also been reported for avian erythroid cells (Mellitzer et al., 1996
). Therefore we used this signaling pathway to study the effects of exogenous hGM-R on EpoR
signaling. However, primary erythroblasts do not tolerate
complete withdrawal of growth factors before re-activation with ligand (Mellitzer et al., 1996
, Wessely et al.,
1997b
). We therefore expressed the hGM-R and
as a
control
empty vector in the HD3 erythroblast cell line
HD3-EpoR-E22. These cells express low levels of a bioactive mEpoR (<1,000 receptors per cell; Mellitzer, G., unpublished observations) and undergo terminal differentiation in response to both avian and mammalian Epo, if
exogenous (v-ErbB) and endogenous receptor tyrosine
kinases (c-ErbB, c-Kit) causing sustained proliferation of
these cells are inactivated by kinase inhibitors (PD 153.035)
and ligand withdrawal (Mellitzer et al., 1996
). Furthermore, these cells show the same interference of the hGM-R with aEpoR signaling as primary cells (data not shown). They are thus much more suitable for biochemical analysis
of signal transduction pathways (Mellitzer et al., 1996
).
HD3-EpoR-E22 cells were withdrawn from growth factors overnight and re-stimulated with hGM-CSF or hEpo.
Extracts normalized to protein content were then analyzed
by phosphotyrosine blot and EMSA (see Materials and
Methods; Mellitzer et al., 1996). As a positive control,
Stat5b activation was induced in the same cells by the
c-ErbB ligand TGF
. Control HD3 cells lacking exogenous hGM-R showed specific tyrosine phosphorylation of
the 80-kD EpoR after stimulation with hEpo (and of the
170-kD c-ErbB protein after TGF
stimulation), whereas
no protein was specifically phosphorylated after stimulation with hGM-CSF (Fig. 6, top panel). Consequently, specific Stat5b DNA binding in EMSA assays was induced by
hEpo and TGF
but not by hGM-CSF (Fig. 6, bottom
panel). In contrast, the hGM-R-expressing HD3-EpoR
cells showed the expected, hGM-CSF-induced phosphorylation of the 130-kD
c protein, but surprisingly hEpo now
failed both to induce EpoR autophosphorylation and to
give rise to phosphorylated substrates. This impaired
signal transduction could be confirmed with respect to
activation of Stat5b; hGM-CSF (and TGF
) could induce
Stat5b/DNA complex formation, but hEpo could not (Fig. 6).
|
Taken together, these results show that exogenous overexpression of the hGM-R actively interferes with the signaling from the EpoR.
The hGM-R Cannot Replace the EpoR in Its Cooperation with c-Kit and Steroid Hormone Receptors to Induce Sustained Erythroblast Self-Renewal
The ability of the hGM-R chain to interfere with differentiation induction by the endogenous aEpoR prompted
us to study whether the hGM-R would exhibit similar
properties with respect to EpoR-dependent proliferation
control. In mouse bone marrow cultures, erythroid colonies induced by Epo plus SCF are more numerous and bigger than those induced by Epo/Insulin, suggesting that a
cooperation of c-Kit and the EpoR causes enhanced erythroid progenitor proliferation (Broxmeyer et al., 1991
;
McNiece et al., 1991
; Muta et al., 1995
). Furthermore, the
EpoR is phosphorylated by ligand-activated c-Kit in certain cell lines (Wu et al., 1995a
). These findings were recently extended by our finding that the EpoR cooperates
with c-Kit and steroid hormone receptors in primary erythroid progenitors derived from chickens, humans and
mice, causing their sustained proliferation (Wessely, O.,
E.M. Deiner, and H. Beug, unpublished observations;
Beug, H., unpublished observations; Wessely, O., and H. Beug, unpublished observations). In the chicken system,
this was shown by the fact that expression of the mEpoR (cooperating with c-Kit and the glucocorticoid receptor)
allowed sustained proliferation of erythroid progenitors
(see Fig. 7; Wessely, O., E.M. Deiner, and H. Beug, unpublished observations).
|
This system allowed us to test, if the activated hGM-R could replace the mEpoR in its contribution to self-renewal induction by c-Kit and steroid hormone receptors. Erythroid progenitors were infected as described above with retroviruses expressing the mEpoR or the hGM-R complex. Empty vector served as a control. Cells were cultivated in hEpo and hGM-CSF plus the other factors required to allow sustained proliferation (see Materials and Methods) and cell proliferation assayed by daily counting (Fig. 7). As expected, the cells expressing the mEpoR were capable of sustained proliferation, while control cells lacking the mEpoR ceased to proliferate around day 6-10. Interestingly, the cells expressing the hGM-R were completely unable to self renew. They disintegrated even faster than the control cells.
These results clearly show, that the cooperation between EpoR and c-Kit leading to erythroblast self-renewal is dependent on specific features of the EpoR and cannot be substituted by a related cytokine receptor mainly active in another hematopoietic lineage.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this paper we have analyzed the function of the hGM-R
in a heterologous cell system. These are primary erythroid
and myeloid progenitors of the chicken that can be induced at will to either proliferate or differentiate. The
need for such experiments was obvious, since GM-CSF-
dependent immortalized cell lines like TF-1 or FDCP-mix
are extremely useful to analyze the biochemistry of GM-R
action and contribution to proliferation control, but are
less suitable to clarify how the GM-R controls the balance between proliferation and differentiation or how it causes
lineage-specific effects. These effects of GM-CSF, however, are important in vivo. For instance, exogenously expressed GM-CSF in transgenic mice led to elevated numbers of macrophages, but did not cause leukemia (Elliott
et al., 1991). This suggested that one of the major tasks of
the GM-R is to control the balance between myeloid progenitor proliferation and differentiation (Metcalf, 1989
).
The Ligand-activated hGM-R Promotes Myeloid Differentiation, but Does Not Contribute to Self-Renewal Induction
Expression of the hGM-R in ts21-E26-transformed myeloid progenitors was used to show that the GM-R can
have similar functions in cultured primary cells as in vivo.
These cells express a thermolabile p135gag-myb-ets oncoprotein and proliferate at 37°C, but are induced to differentiation into macrophages when shifted to 42°C (Beug et al.,
1984). For both proliferation and differentiation the presence of the avian cytokine cMGF is absolutely required
(Woldmann et al., 1997
). Interestingly, in hGM-R-expressing ts21-E26 myeloblasts hGM-CSF induced the typical
differentiation response of these progenitors i.e. terminally differentiating into macrophages accompanied by a
limited number of cell divisions. However, hGM-CSF differed from cMGF in that it failed to induce long-term proliferation at 37°C, i.e., in the presence of a fully active
p135gag-myb-ets oncoprotein (not shown). This is in clear
contrast to GM-CSF-dependent myeloid cell lines, in
which GM-CSF is required for long-term proliferation, but
has no clear effects on differentiation. Thus, the action of
GM-CSF in suspension cultures of hGM-R-expressing primary avian myeloid progenitors closely corresponds to
the established cytokine responses of mammalian bone
marrow in colony assays, i.e., induction of differentiation
in immature cells, stimulation of transient proliferation
during differentiation and regulation of cell function in
mature cells.
The fact that the ligand-activated hGM-R could not induce long-term proliferation at 37°C clearly sets GM-CSF
apart from cMGF. This is important, since the relationship
of cMGF to mammalian cytokines is still unclear. On the
sequence and structural level, cMGF is most homologous
to mammalian IL-6 with slightly less homology to G-CSF
(Sterneck et al., 1992; Boulay and Paul, 1993
). On the other hand, the in vivo activity of cMGF (i.e., a marked increase of functionally activated monocytes in the peripheral blood of chickens constitutively expressing high levels
of cMGF) (York et al., 1996
) is more easily comparable to
the biological activities of M-CSF or GM-CSF. Our result
that the ectopically expressed, ligand-activated GM-R was
unable to substitute for all activities of cMGF now rules out
the possibility that cMGF is the avian version of GM-CSF.
Function of the hGM-R in Erythroid Cells
The biological activity of GM-CSF is not restricted to the
myeloid compartment. GM-CSF also supports erythroid
differentiation (Metcalf, 1989; Nishijima et al., 1995
) but in
contrast to Epo is unable to induce or maintain it on its
own. Such a BPA is also typical for other cytokines, e.g.,
IL-3. In the case of GM-CSF, it has been explained by the
loss of ligand responsiveness resulting from downregulation of the GM-R during differentiation. However, extending these findings, we demonstrate here that the activated hGM-R is unable to promote terminal erythroid
differentiation even when it is exogenously expressed in
avian erythroblasts and thus cannot be downregulated.
Rather, GM-CSF must cooperate with an activity present
in anemic chicken serum. This activity is most likely Epo
since it behaves like mammalian Epo during biochemical purification (Kowenz et al., 1987
) and is neutralized by a
polyclonal antibody to mammalian Epo (Wessely, O., unpublished observations). However, formal proof of this
notion has to await molecular cloning of avian Epo and/or
its receptor.
The cooperative action of the EpoR and the GM-R
seems to be easily explainable in mechanistic terms, since
both receptors mediate signaling via the intracellular tyrosine kinase JAK2 (Witthuhn et al., 1993; Quelle et al.,
1994
). They also activate a comparable subset of signaling
molecules like STAT5, Ras, or PI-3-kinase (Mui et al.,
1995
; Pallard et al., 1995
) and both receptors activate marker genes characteristic for mature erythrocytes like
carbonic anhydrase II (CAII; Wessely, O., unpublished
observations). But why does GM-CSF only act as a BPA
even when its receptor is ectopically expressed? A possible answer to this question could be that the EpoR has
multiple activities. In addition to inducing terminal erythroid differentiation, the EpoR physically associates with the tyrosine kinase receptor c-Kit and even to induce tyrosine phosphorylation of the latter receptor tyrosine kinase (Wu et al., 1995). As shown elsewhere (Beug, H., unpublished observation; Wessely, O., E.M. Deiner, and H. Beug, unpublished observations; Wessely, O., and H. Beug,
unpublished observations) the combination of c-Kit and
the EpoR also seem to be key players to induce long-term proliferation in human, murine, and avian erythroblasts.
However when the hGM-R was tested in conjunction with
ligand-activated c-Kit, it failed to induce long-term outgrowth. Thus the GM-R cannot substitute for the EpoR in
its cooperation with c-Kit.
Our finding of a partial functional overlap between the
EpoR and the GM-R agree with data obtained from cytokine/cytokine receptor knockout mice (Dent et al., 1990;
Dranoff and Mulligan, 1994
; Robb et al., 1995
; Lin et al.,
1996
), showing that the various cytokine receptors exhibit
a partial functional redundancy. Ablation of these genes
usually does not lead to complete ablation of a given lineage, but shows much more subtle phenotypes. Only in case of specialized, cell type-specific receptors such as the EpoR or the IL-5 receptor (Wu et al., 1995b
; Lin et al.,
1996
; Yoshida et al., 1996
), the more generally expressed
cytokine receptors such as the GM-R are unable to functionally substitute and thus hematopoiesis is more severely
affected.
The Chain of the hGM-R May Confer
Myeloid Cell Specificity by Interfering with EpoR
Signal Transduction
Many functions of the GM-R are similar to those of the
IL-3 receptor. Both receptors can stimulate proliferation
of immature myeloid and erythroid progenitors, support
differentiation along these lineages, and act via similar signal transduction pathways (McNiece et al., 1991; Quelle et
al., 1992
; Sonoda et al., 1994
; Mui et al., 1995
). This is not
surprising, since both receptors have to interact with
c
and Jak2 to initiate signal transduction. In this paper, however, we identified a biological activity specific for the
GM-R and not shared by related cytokine receptors. In the absence of GM-CSF, the hGM-R was incompatible
with erythroid differentiation. hGM-R-expressing erythroblasts disintegrated rather than differentiated in AS/Ins,
whereas hIL-3R-expressing erythroblasts differentiate normally under the same conditions (Steinlein, P., E.M. Deiner,
and H. Beug, unpublished observations). Furthermore,
isolated expression of the
or
chains of the GM-R receptor complex showed that the
chain was sufficient to
interfere with erythroid differentiation. In contrast, overexpression of neither
c nor the IL-3R
chain exerted this
effect. These biological observations were matched by biochemical data. When the hGM-R was expressed in the
erythroblast cell line HD3, hGM-CSF caused both tyrosine phosphorylation and DNA binding of Stat5b. However, the non-liganded hGM-R inhibited both EpoR autophosphorylation and induction of Stat5b DNA binding in
response to hEpo. In HD3-EpoR control cells lacking the
exogenous hGM-R, hEpo induced both effects as expected. These results support the idea of a crucial role of
the hGM-R
chain in lineage-specific signal transduction. Mutation analysis of the cytoplasmic tail of the GM-R
chain recently revealed that this molecule is required together with the
c chain for the activation of JAK2 (Matsuguchi et al., 1997
). Therefore, interference by GM-R
with signal transduction intermediates such as Jak-2 or
Stat5b in a fashion distinct from the
c chain is one possible explanation for this cross talk. Finally, experiments by
others show that overexpression of the EpoR in the murine cell line FDCP interfered specifically with signaling
from the GM-R (Quelle and Wojchowski, 1991
). This corresponded to the finding that a protein of 100 kD could no
longer be tyrosine phosphorylated in response to hGM-CSF, when the EpoR was exogenously expressed as well
(Quelle et al., 1992
).
These results raise the interesting possibility that non-liganded cytokine receptors may contribute to lineage
commitment by specific effects mediated via their lineage-specific chains. Thus, cytokine receptors may use different mechanisms to regulate the balance between proliferation and apoptosis on the one hand and differentiation
versus proliferation/apoptosis on the other. Whereas the
former mechanism (the one easily studied in immortalized cell lines) is always ligand dependent, the latter mechanisms may be less ligand dependent, being mainly driven
by tissue-specific expression of specific types of receptor
chains.
![]() |
Footnotes |
---|
Address all correspondence to Hartmut Beug, Institute for Molecular Pathology, Dr. Bohr Gasse 7, A-1030 Vienna, Austria. Tel.: +43-1-79730-621. Fax: +43-1-798-7153. E-mail: beug{at}nt.imp.univie.ac.at
Received for publication 2 January 1998 and in revised form 23 March 1998.
O. Wessely and E.-M. Deiner contributed equally to this work.The authors thank Genentech Inc. and DNAX for their gifts of hGM-R
and hIL3-R
and
c, respectively. J. Ghysdael, Institute Curie, Orsay,
France provided us with the RCAS/EpoR retroviral construct. The authors also thank Drs. M. von Lindern, C. Tran-Quang, and J. Ghysdael for
stimulating discussions; Dr. T. Decker for critical reading of the manuscript and I.G. Stengl for expert technical assistance.
![]() |
Abbreviations used in this paper |
---|
AS, anemic serum;
c, common
chain;
BPA, burst-promoting activity;
cMGF, chicken myelomonocytic
growth factor;
Epo, erythropoietin;
EpoR, erythropoietin receptor;
GM-CSF, Granulocyte-Macrophage Colony-Stimulating Factor;
GM-R, GM-CSF receptor;
GM-R
, GM-R
chain;
IL, interleukin;
IRES, internal ribosome entry site;
ts, temperature sensitive.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Bartunek, P., L. Pichlikova, G. Stengl, G. Boehmelt, F.H. Martin, H. Beug, M. Dvorak, and M. Zenke. 1996. Avian stem cell factor (SCF): production and characterization of the recombinant, His-tagged SCF of chicken and its neutralizing antibody. Cytokine 8: 14-20 |
2. | Beug, H., S. Palmieri, C. Freudenstein, H. Zentgraf, and T. Graf. 1982. Hormone-dependent terminal differentiation in vitro of chicken erythroleukemia cells transformed by ts mutants of avian erythroblastosis virus. Cell 28: 907-919 |
3. | Beug, H., A. Leutz, P. Kahn, and T. Graf. 1984. Ts mutants of E26 leukemia virus allow transformed myeloblasts, but not erythroblasts or fibroblasts, to differentiate at the nonpermissive temperature. Cell 39: 579-588 |
4. | Beug, H., T. Metz, E.W. Müllner, and M. Hayman. 1996. Self renewal and differentiation in primary avian hematopoietic cells: An alternative to mammalian in vitro models? Curr. Top. Microbiol. Immunol. 211: 29-39 |
5. | Boulay, J.L., and W.E. Paul. 1993. Hematopoietin subfamily classification based on size, gene organization and sequence homology. Curr. Biol. 3: 573-581 . |
6. | Broxmeyer, H.E., G. Hangoc, J.R. Zucali, A. Mason, R. Schwall, C. Carow, and S. Cooper. 1991. Effects in vivo of purified recombinant human activin and erythropoietin in mice. Int. J. Hematol. 54: 447-454 |
7. | Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid ganidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159 |
8. | Dent, L.A., M. Strath, A.L. Mellor, and C.J. Sanderson. 1990. Eosinophilia in transgenic mice expressing interleukin 5. J. Exp. Med. 172: 1425-1431 [Abstract]. |
9. | Dolznig, H., P. Bartunek, K. Nasmyth, E. Müllner, and H. Beug. 1995. Terminal differentiation of normal chicken erythroid progenitors: Shortening of G1 correlates with loss of D cyclin/CDK-4 expression and altered cell size control. Cell. Growth Differ. 6: 1341-1352 [Abstract]. |
10. | Dranoff, G., and R.C. Mulligan. 1994. Activities of granulocyte-macrophage colony-stimulating factor revealed by gene transfer and gene knockout studies. Stem Cells (Dayt.). 1: 173-182 . |
11. |
Elliott, M.J.,
A. Strasser, and
D. Metcalf.
1991.
Selective up-regulation of macrophage function in granulocyte-macrophage colony-stimulating factor
transgenic mice.
J. Immunol.
147:
2957-2963
|
12. | Elroy-Stein, O., T.R. Fuerst, and B. Moss. 1989. Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc. Natl. Acad. Sci. USA. 86: 6126-6130 [Abstract]. |
13. | Fry, D.W., A.J. Kraker, A. McMichael, L.A. Ambroso, J.M. Nelson, W.R. Leopold, R.W. Connors, and A.J. Bridges. 1994. A specific inhibitor of the epidermal growth factor receptor tyrosine kinase. Science 265: 1093-1095 |
14. | Fuerstenberg, S., I. Leitner, C. Schroeder, H. Schwarz, B. Vennström, and H. Beug. 1992. Transcriptional repression of band 3 and CAII in v-erbA transformed erythroblasts accounts for an important part of the leukemic phenotype. EMBO (Eur. Mol. Biol. Organ.) J. 11: 3355-3366 [Abstract]. |
15. | Graf, T.. 1973. Two types of target cells for transformation with avian myelocytomatosis virus. Virology 54: 398-413 |
16. |
Hayman, M.J.,
S. Meyer,
F. Martin,
P. Steinlein, and
H. Beug.
1993.
Self renewal and differentiation of normal avian erythroid progenitor cells: regulatory roles of the c-erbB/TGF![]() |
17. | Ihle, J.N., B.A. Witthuhn, F.W. Quelle, K. Yamamoto, W.E. Thierfelder, B. Kreider, and O. Silvennoinen. 1994. Signaling by the cytokine receptor superfamily: JAKs and STATs. Trends Biochem. Sci. 19: 222-227 |
18. | Jubinsky, P.T., D.G. Nathan, D.J. Wilson, and C.A. Sieff. 1993. A low-affinity human granulocyte-macrophage colony-stimulating factor/murine erythropoietin hybrid receptor functions in murine cell lines. Blood 81: 587-591 [Abstract]. |
19. | Knight, J., M. Zenke, C. Disela, E. Kowenz, P. Vogt, J.D. Engel, M.J. Hayman, and H. Beug. 1988. Temperature-sensitive v-sea transformed erythroblasts: a model system to study gene expression during erythroid differentiation. Genes Dev. 2: 247-258 [Abstract]. |
20. | Kowenz, E., A. Leutz, G. Döderlein, T. Graf, and H. Beug. 1987. ts-oncogene- transformed erythroleukemic cells: a novel test system for purifying and characterizing avian erythroid growth factors. Hamatol. Bluttransfus. 31: 199-209 |
21. | Lanfrancone, L., G. Pelicci, M.F. Brizzi, M.G. Aronica, C. Casciari, S. Giuli, L. Pegoraro, T. Pawson, P.G. Pelicci, and M.G. Arouica. 1995. Overexpression of Shc proteins potentiates the proliferative response to the granulocyte-macrophage colony-stimulating factor and recruitment of Grb2/SoS and Grb2/p140 complexes to the beta receptor subunit. Oncogene 10: 907-917 |
22. |
Leutz, A.,
H. Beug,
C. Walter, and
T. Graf.
1988.
Hematopoietic growth factor
glycosylation. Multiple forms of chicken myelomonocytic growth factor.
J.
Biol. Chem.
263:
3905-3911
|
23. | Leutz, A., K. Damm, E. Sterneck, E. Kowenz, S. Ness, R. Frank, H. Gausepohl, Y.C. Pan, J. Smart, and M. Hayman. 1989. Molecular cloning of the chicken myelomonocytic growth factor (cMGF) reveals relationship to interleukin 6 and granulocyte colony stimulating factor. EMBO (Eur. Mol. Biol. Organ.) J. 8: 175-181 [Abstract]. |
24. | Lin, C.S., S.K. Lim, V. D'Agati, and F. Costantini. 1996. Differential effects of an erythropoietin receptor gene disruption on primitive and definitive erythropoiesis. Genes Dev. 10: 154-164 [Abstract]. |
25. |
Matsuguchi, T.,
Y. Zhao,
M.B. Lilly, and
A.S. Kraft.
1997.
The cytoplasmic domain of Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF) receptor ![]() |
26. | McNiece, I.K., K.E. Langley, and K.M. Zsebo. 1991. Recombinant human stem cell factor synergises with GM-CSF, G-CSF, IL-3 and epo to stimulate human progenitor cells of the myeloid and erythroid lineages. Exp. Hematol. 19: 226-231 |
27. |
Mellitzer, G.,
O. Wessely,
T. Decker,
A. Eilers,
M.J. Hayman, and
H. Beug.
1996.
Activation of Stat 5b in erythroid progenitors correlates with the ability of ErbB to induce sustained cell proliferation.
Proc. Natl. Acad. Sci. USA.
93:
9600-9605
|
28. | Metcalf, D.. 1980. Clonal analysis of proliferation and differentiation of paired daughter cells: Action of granulocyte-macrophage colony stimulating factor on granulocyte-macrophage precursors. Proc. Natl. Acad. Sci. USA. 77: 5327-5330 [Abstract]. |
29. | Metcalf, D.. 1989. The molecular control of cell division, differentiation, commitment and maturation in haematopoietic cells. Nature 339: 27-30 |
30. | Mui, A.L., H. Wakao, A.M. O'Farrell, N. Harada, and A. Miyajima. 1995. Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5 transduce signals through two STAT5 homologs. EMBO (Eur. Mol. Biol. Organ.) J. 14: 1166-1175 [Abstract]. |
31. |
Muta, K.,
S.B. Krantz,
M.C. Bondurant, and
C.H. Dai.
1995.
Stem cell factor retards differentiation of normal human erythroid progenitor cells while stimulating proliferation.
Blood
86:
572-580
|
32. | Nimer, S.D., and H. Uchida. 1995. Regulation of granulocyte-macrophage colony-stimulating factor and interleukin 3 expression. Stem Cells (Dayt.). 13: 324-335 [Abstract]. |
33. | Nishijima, I., T. Nakahata, Y. Hirabayashi, T. Inoue, H. Kurata, A. Miyajima, N. Hayashi, Y. Iwakura, K. Arai, and T. Yokota. 1995. A human GM-CSF receptor expressed in transgenic mice stimulates proliferation and differentiation of hemopoietic progenitors to all lineages in response to human GM-CSF. Mol. Biol. Cell 6: 497-508 [Abstract]. |
34. |
Pallard, C.,
F. Gouilleux,
M. Charon,
B. Groner,
S. Gisselbrecht,
I. Dusanter, and
Fourt.
1995.
Interleukin-3, erythropoietin, and prolactin activate a
STAT5-like factor in lymphoid cells.
J. Biol. Chem.
270:
15942-15945
|
35. |
Pratt, J.C.,
M. Weiss,
C.A. Sieff,
S.E. Shoelson,
S.J. Burakoff, and
K.S. Ravichandran.
1996.
Evidence for a physical association between the Shc-PTB
domain and the beta c chain of the granulocyte-macrophage colony-stimulating factor receptor.
J. Biol. Chem.
271:
12137-12140
|
36. | Quelle, D.E., and D.M. Wojchowski. 1991. Localized cytosolic domains of the erythropoietin receptor regulate growth signaling and down-modulate responsiveness to granulocyte-macrophage colony-stimulating factor. Proc. Natl. Acad. Sci. USA. 88: 4801-4805 [Abstract]. |
37. |
Quelle, F.W.,
D.E. Quelle, and
D.M. Wojchowski.
1992.
Interleukin 3, granulocyte-macrophage colony-stimulating factor, and transfected erythropoietin
receptors mediate tyrosine phosphorylation of a common cytosolic protein
(pp100) in FDC-ER cells.
J. Biol. Chem.
267:
17055-17060
|
38. | Quelle, F.W., N. Sato, B.A. Witthuhn, R.C. Inhorn, M. Eder, A. Miyajima, J.D. Griffin, and J.N. Ihle. 1994. JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region. Mol. Cell. Biol. 14: 4335-4441 [Abstract]. |
39. |
Rao, P., and
R.A. Mufson.
1995.
A membrane proximal domain of the human
interleukin-3 receptor beta c subunit that signals DNA synthesis in NIH 3T3
cells specifically binds a complex of Src and Janus family tyrosine kinases
and phosphatidylinositol 3-kinase.
J. Biol. Chem.
270:
6886-6893
|
40. | Robb, L., C.C. Drinkwater, D. Metcalf, R. Li, F. Kontgen, N.A. Nicola, and C.G. Begley. 1995. Hematopoietic and lung abnormalities in mice with a null mutation of the common beta subunit of the receptors for granulocyte-macrophage colony-stimulating factor and interleukins 3 and 5. Proc. Natl. Acad. Sci. USA. 92: 9565-9569 [Abstract]. |
41. | Sakamaki, K., I. Miyajima, T. Kitamura, and A. Miyajima. 1992. Critical cytoplasmic domains of the common beta subunit of the human GM-CSF, IL-3 and IL-5 receptors for growth signal transduction and tyrosine phosphorylation. EMBO (Eur. Mol. Biol. Organ.) J. 11: 3541-3549 [Abstract]. |
42. |
Schroeder, C.,
L. Gibson,
C. Nordström, and
H. Beug.
1993.
The estrogen receptor cooperates with the TGF![]() |
43. |
Sonoda, Y.,
H. Sakabe,
Y. Ohmisono,
S. Tanimukai,
S. Yokota,
S. Nakagawa,
S.C. Clark, and
T. Abe.
1994.
Synergistic actions of stem cell factor and
other burst-promoting activities on proliferation of CD34+ highly purified
blood progenitors expressing HLA-DR or different levels of c-kit protein.
Blood
84:
4099-4106
|
44. | Spooncer, E., C.M. Heyworth, A. Dunn, and T.M. Dexter. 1986. Self-renewal and differentiation of interleukin-3-dependent multipotent stem cells are modulated by stromal cells and serum factors. Differentiation 31: 111-118 |
45. | Steinlein, P., E.M. Deiner, A. Leutz, and H. Beug. 1994. Recombinant murine erythropoietin receptor expressed in avian progenitors mediates terminal erythroid differentiation in vitro. Growth Factors 10: 1-16 |
46. | Sterneck, E., C. Blattner, T. Graf, and A. Leutz. 1992. Structure of the chicken myelomonocytic growth factor gene and specific activation of its promoter in avian myelomonocytic cells by protein kinases. Mol. Cell. Biol. 12: 1728-1735 [Abstract]. |
47. |
Tran Quang, C., O. Wessely, M. Pironin, H. Beug, and J. Ghysdael.
1997.
Cooperation of SPI-1/PU.1 with an activated erythropoietin receptor inhibits apoptosis and EPO-dependent differentiation in primary erythroblasts and induces their KIT-ligand dependent proliferation.
EMBO (Eur. Mol. Biol.
Organ.) J.
16:
5639-5653
|
48. | Wakao, H., N. Harada, T. Kitamura, A.L. Mui, and A. Miyajima. 1995. Interleukin 2 and erythropoietin activate STAT5/MGF via distinct pathways. EMBO (Eur. Mol. Biol. Organ.) J. 14: 2527-2535 [Abstract]. |
49. |
Watanabe, S.,
T. Itoh, and
K. Arai.
1996.
JAK2 is essential for activation of
c-fos and c-myc promoters and cell proliferation through the human granulocyte-macrophage colony-stimulating factor receptor in BA/F3 cells.
J.
Biol. Chem.
271:
12681-12686
|
50. | Weiss, M., C. Yokoyama, Y. Shikama, C. Naugle, B. Druker, and C.A. Sieff. 1993. Human granulocyte-macrophage colony-stimulating factor receptor signal transduction requires the proximal cytoplasmic domains of the alpha and beta subunits. Blood 82: 3298-3306 [Abstract]. |
51. |
Wessely, O.,
E.M. Deiner,
H. Beug, and
M. von Lindern.
1997a.
The glucocorticoid receptor is a key regulator of the decision between self renewal and differentiation in erythroid progenitors.
EMBO (Eur. Mol. Biol. Organ.) J.
16:
267-280
|
52. | Wessely, O., G. Mellitzer, M. von Lindern, A. Levitzki, A. Gazit, I. Ischenko, M.J. Hayman, and H. Beug. 1997b. Distinct regulatory roles of the receptor tyrosine kinases c-ErbB and c-Kit in regulating the balance between erythroid cell proliferation and differentiation. Cell. Growth Differ. 8: 481-493 [Abstract]. |
53. | Witthuhn, B.A., F.W. Quelle, O. Silvennoinen, T. Yi, B. Tang, O. Miura, and J.N. Ihle. 1993. JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell 74: 227-236 |
54. | Woldmann, I., G. Mellitzer, M. Kieslinger, D. Buchhart, A. Meinke, H. Beug, and T. Decker. 1997. STAT5 involvement in the differentiation response of primary chicken myeloid progenitor cells to chicken myelomonocytic growth factor. J. Immunol. 159: 877-886 [Abstract]. |
55. | Wu, H., U. Klingmuller, P. Besmer, and H.F. Lodish. 1995a. Interaction of the erythropoietin and stem-cell-factor receptors. Nature 377: 242-246 |
56. | Wu, H., X. Liu, R. Jaenisch, and H.F. Lodish. 1995b. Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell 83: 59-67 |
57. | York, J.J., A.D. Strom, T.E. Connick, P.G. McWaters, D.B. Boyle, and J.W. Lowenthal. 1996. In vivo effects of chicken myelomonocytic growth factor: delivery via a viral vector. J. Immunol. 156: 2991-2997 [Abstract]. |
58. | Yoshida, T., K. Ikuta, H. Sugaya, K. Maki, M. Takagi, H. Kanazawa, S. Sunaga, T. Kinashi, K. Yoshimura, J. Miyazaki, et al . 1996. Defective B-1 cell development and impaired immunity against Angiostrongylus cantonensis in IL-5R alpha-deficient mice. Immunity 4: 483-494 |
59. | Zenke, M., A. Munoz, J. Sap, B. Vennstrom, and H. Beug. 1990. v-erbA oncogene activation entails the loss of hormone-dependent regulator activity of c-erbA. Cell 61: 1035-1049 |