From the Department of Veterinary Science and
Graduate Programs in § Genetics and Biochemistry & Molecular
Biology Pennsylvania State University,
University Park, Pennsylvania 16802
Received for publication, August 16, 2000, and in revised form, December 1, 2000
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ABSTRACT |
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Erythroid homeostasis depends critically upon
erythropoietin (Epo) and stem cell factor cosignaling in late
progenitor cells. Epo bioresponses are relayed efficiently by minimal
receptor forms that retain a single Tyr-343 site for STAT5 binding,
while forms that lack all cytoplasmic Tyr(P) sites activate JAK2 and
the transcription of c-Myc plus presumed additional target genes. In
FDCER cell lines, which express endogenous c-Kit, the signaling
capacities of such minimal Epo receptor forms (ER-HY343 and ER-HY343F)
have been dissected to reveal: 1) that Epo-dependent
mitogenesis, survival, and bcl-x gene expression via
ER-HY343 depend upon the intactness of the Tyr-343 STAT5 binding site;
2) that ER-HY343-dependent bcl-xL
gene transcription is enhanced markedly via c-Kit; 3) that socs-3, plfap, dpp-1, and
cacy-bp gene transcription is induced via ER-HY343, whereas
dpp-1 and cacy-bp gene expression is also supported by ER-HY343F; 4) that ectopically expressed SOCS-3 suppresses proliferative signaling by not only ER-HY343 but also c-Kit; and 5)
that in FDCER and primary erythroid cells, c-Kit appears to provide the
primary route to MAPK activation. Thus, integration circuits exist in
only select downstream pathways within Epo and stem call factor
receptor signaling.
Epo,1 the prime hormonal
regulator of red cell development, initiates its effects by binding to
receptor dimers on the surface of erythroid burst- and colony-forming
units and activating the tethered Janus family kinase (JAK) 2 (1, 2).
JAK2 then mediates the phosphorylation of eight cytoplasmic tyrosine
sites within the Epo receptor, and via these sites, a complex set of
Src homology 2 domain-encoding effectors (and associated cofactors) are
engaged. These include STAT 5A and B; Grb2/mSOS/Raf/Ras;
phosphatidylinositol 3-kinase, phospholipase- Despite the complexity of this signaling network, studies of
tyrosine-mutated and -truncated Epo receptors in cell lines (9, 10),
murine fetal liver (11, 12), and adult murine marrow and spleen (13)
have established that signals necessary for Epo
receptor-dependent erythroid development are supported by receptor forms retaining only a membrane-proximal box domain for JAK2
binding plus a single phosphotyrosine Tyr(P)-343 STAT5 binding site.
Additionally, in JAK2 Epo Receptor, SOCS-3, Pim-1, and Bcl-xL Expression
Constructs--
pMKwtER was prepared by the stepwise cloning of a wild
type Epo receptor cDNA (20) to pSL1180 (Amersham Pharmacia Biotech) at SpeI and SalI sites, and to the dicistronic
pMK1059 vector (21) as a 1.8-kb SpeI to
XbaI fragment. pMKER-HY343 is an Epo receptor form truncated
at Ala-375 and was prepared by PCR using the primers 5'-TGG TCC TCA TCT
CGC TGT TGC TGA-3' and 5'-AAG CTT CAT CCA TAG TCA CAG GGT CCA C-3'.
This 436-base pair PCR product was cloned stepwise to pCR-ScriptSK(+)
(Stratagene), to pSL1180wtER as a BglII to XbaI
fragment, and to pMK1059 as a 5' extended 1.2-kb SpeI to XbaI fragment. In ERH-Y343F, a Tyr-343
site for STAT5 binding was mutated (to phenylalanine) by overlap
extension using the primers 5'-GAT CGG GCC CTT ACT GGG AGC CGG TGG GCA
GTG AGC ATG CCC AGG ACA CCT TCT TGG TAT TGG ATA AGT GG -3' and 5'-CTA AGC TTC ATC CAT AGT CAC AGG TCC AC-3'. The resulting 158-base pair
product was cloned stepwise to pSP72 (Promega, Madison, WI) at
ApaI and XbaI, to pSL1180wtER at BglII
to XbaI, and to pMK1059 as a 5' extended 1.2-kb
SpeI to XbaI fragment. All products were confirmed by sequencing. SOCS-3 was expressed using a pEF vector (22).
For Pim-1 and Bcl-xL expression, cDNAs (23, 24) were cloned as 1.2-kb EcoRI to NotI and 0.8-kb
EcoRI fragments, respectively, to pEFNeo.
Cell Lines and Primary Erythroid Progenitor
Cells--
FDCW2-(pMK)wtER, -(pMK)ER-HY343, and -(pMK)ER-HY343F
cells were maintained at 37 °C, 7.5% CO2 in Opti-MEM I
(Life Technologies, Inc.), 7% fetal bovine serum (FBS), and 4%
conditioned medium from WEHI-3B cells as a source of IL-3 (25), and
prepared by electrotransfection (26) and selection in G418 (0.9 mg/ml). The parental line, FDCW2, in a well characterized
IL-3-dependent murine myeloid cell line (26). For the
expression of Pim-1, Bcl-xL, and SOCS-3, FDC cell lines
were cotransfected with pEFNeo-Pim-1, pEFNeo-Bcl-xL,and
pEF-SOCS-3, respectively (and with pAPuro) (27). Cells were selected in
puromycin (0.4 µg/ml), cloned by dilution, and screened by Northern
and/or Western blotting. Antisera used were to Pim-1 (see
"Acknowledgments") or Bcl-xL (Transduction Laboratories, Lexington, KY). Erythroid progenitor cells were prepared
from the spleens of mice treated with thiamphenicol as described (13)
or from marrow cells cultured for 72 h under conditions shown to
support the selective expansion of CFU-e (28).
Assays of Mitogenesis and Apoptosis--
In assays of
mitogenesis, exponentially growing cells were washed three times in
Dulbecco's modified Eagle's medium, and were cultured with cytokines
in Opti-MEM I, 8% FBS at 1.5 × 105 cells/ml. At
48 h of culture, methyl [3H]thymidine incorporation
was assayed (20). In assays of apoptosis, cells were washed twice in
Dulbecco's modified Eagle's medium and cultured at 5 × 105 cells/ml in Opti-MEM I, 1% FBS in Epo and/or SCF. At
the intervals indicated, cells were stained with propidium iodide (5 µg/ml for 2 min) or with annexin-V detection reagent (Roche Molecular
Biochemicals) prior to flow cytometry.
Northern Blotting, RNase Protection, RT-PCR, and cDNA Array
Assays--
RNA was isolated using TRIzol reagent (Life Technologies,
Inc.) and Northern blotting was performed as described (26) using the
following 32P-labeled murine cDNA probes:
cis (1-kb EcoRI to NotI fragment of
pCRV) (5), c-Myc (1.5-kb XhoI fragment of pSVLc-Myc) (29), bcl-xL (0.8-kb EcoRI fragment of
pBlueScriptSK(+)bcl-xL) (24), Epo receptor (1.5-kb
XhoI fragment of pXMwtER) (30), pim-1 (1.3-kb EcoRI to XbaI fragment of pCMP2 Assays of ERK Activation--
In assays of ERKs from FDCER cell
lines, washed cells were cultured at 5 × 105 cells/ml
in OptiMEM I, 1% FBS for 12 h prior to cytokine stimulation. For
erythroid splenocytes, cells were pre-cultured at 4 × 106 cells/ml for 8 h, while progenitor cells expanded
from marrow were pre-cultured for 10 h at 2 × 106 cells/ml prior to a 5-min exposure to Epo (25 units/ml)
and/or SCF (50 ng/ml). Cells then were adjusted to 0 °C, washed, and lysed in 0.2% Triton X-100, 10 mM NaCl, 6 mM
MgCl2, 10 mM NaF, 0.2 mM
NaVO4, 10 mM Tris, pH 7.4, containing
phenylmethylsulfonyl fluoride (50 µg/ml), aprotinin (3 µg/ml),
pepstatin (0.7 µg/ml), and leupeptin (0.5 µg/ml). Protein
concentrations of cleared lysates were adjusted to equivalence, and
samples were analyzed by Western blotting with an antibody to
phosphorylated forms of ERK-1 and -2 (catalog no. V803A, Promega,
Madison, WI) or a pan-specific antibody to MAPKs (catalog no. 06182, Upstate Biotechnology Inc., Lake Placid, NY). In vitro
assays of ERK activity were performed using myelin basic protein and
[ Electrophoretic Mobility Shift Assays (EMSA)--
Cells were
washed, cultured for 8 h in Opti-MEM I, 1% FBS at 8 × 105 cells/ml, and exposed for 5 min to Epo (25 units/ml)
and/or SCF (50 ng/ml). Cells then were adjusted to 0 °C, washed, and
lysed in three volumes of 10 mM NaCl, 6 mM
MgCl2, 1 mM dithiothreitol, 0.1 mM
Na3VO4, 0.1% Triton X-100, 10 mM
Tris, pH 7.4, plus protease inhibitors. Following a 20-min incubation,
lysates were centrifuged (15 s at 8,000 × g) and
resuspended in three volumes of 20% glycerol, 420 mM NaCl,
1.5 mM MgCl2, 0.2 mM
EDTA, 1 mM dithiothreitol, 0.1 mM
Na3VO4, 20 mM HEPES, pH 7.9, plus
protease inhibitors. Samples then were incubated on ice for 30 min, and
centrifuged (10 min at 8,000 × g). Supernatants were
assayed for protein (BCA assay, Pierce) and flash-frozen. EMSAs were
performed using a GAS-like element from the FDCW2-TVA Cells and Retroviruses--
For use in transduction
experiments, FDCW2 cells were electrotransfected with
pCDNA3.1-TVA950, selected in G418 (1 mg/ml), and cloned by limited
dilution. Clones that efficiently supported transduction were
identified by infection with an RCAS-GFP virus (32) and flow cytometry.
Virus encoding STAT5A-1*6 (33) was prepared by constructing an
RCAS-STAT5A-1*6 plasmid and transfecting chicken DF1 packaging cells
(32) with this construct.
Signals Relayed via JAK2/STAT5 Versus JAK2-activating Epo Receptor
Forms--
Epo receptor (ER) forms used in the present investigation
include the wtER, ER-HY343 in which a single cytoplasmic Tyr(P)-343 site is retained (and seven additional PY sites are deleted) within a
minimal Epo receptor form truncated at Ala-375, and ER-HY343F in which
this Tyr-343 site for STAT5 binding (within this truncated receptor
form) is mutated (Fig. 1A).
For expression in factor-dependent FDCW2 cells, a
dicistronic vector (pMK1059) was used to obviate the use of Epo as a
selection agent. As assayed by Northern blotting, each of the above Epo
receptor forms was expressed at highly comparable levels in stably
transfected, G418-selected FDCW2 cells (Fig. 1B).
125I-Epo binding assays of FDCER-HY343, FDCER-HY343F and
FDCER-wt cells also demonstrated Epo receptor densities to be uniformly on the order of 800-1000
receptors/cell.2 In addition
(and as shown below), each of these Epo receptor forms proved to
support c-Myc expression at comparable rates. In FDCER cells, the
requirement for Tyr-343 in STAT5 activation has not been demonstrated
previously and this therefore also was assayed in initial experiments.
Mitogenic activities of the above Epo receptor forms in FDCER cells
next were assessed. wtER and ER-HY343 receptor forms supported
Epo-induced [3H]dT incorporation at comparable rates
while ER-HY343F was essentially inactive (Fig. 1C). These
results were confirmed in repeated experiments using polyclonal lines
from independent transfections (as well as in direct viable cell
counting assays) and strongly suggest that in this strictly
hematopoietic growth factor-dependent model, Tyr-343 (and
STAT5) target proliferative effectors. Finally, EMSAs using a
The abilities of ER-HY343 and ER-HY343F receptor forms to transduce
Epo-induction of several known immediate response genes was next
studied. As shown in Fig. 2, c-Myc
transcripts were induced via HY343 and HY343F receptor forms at
comparable efficiencies, whereas in FDCER-HY343F cells, Epo induction
of cis and pim-1 transcripts was blocked (as was
predicted by the loss of STAT5 activation). More notably, Epo signaling
of bcl-xL gene transcription in FDCER-HY343F
cells was discovered to also be inhibited sharply (i.e.
These results led us to investigate whether the above minimal Epo
receptor forms might support the induction of additional response
genes. Specifically, FDCER-HY343 and FDCER-HY343F cells were exposed to
Epo (±20 units/ml), [32P]cDNAs were prepared from
poly(A)+-enriched RNA, and were hybridized to duplicate
microarrays of known murine cDNAs. In independent hybridizations
using [32P]cDNAs prepared from independent RNA
samples, we identified four additional genes not previously known to be
regulated via ER-HY343 or ER-HY343F, i.e. SOCS-3
(22), proliferation-associated protein I (plfap; p38-2G4)
(36), dipeptidyl-peptidase I precursor (dpp-1) (37), and
calcyclin-binding protein gene (cacy-bp) (38) (Fig. 4). Although each was induced via
ER-HY343, dpp-1 and cacy-bp transcript expression
also was activated efficiently via Tyr-343-independent routes in
FDCER-HY343F cells. With the exception of c-Myc, these latter two genes
are the first in the Epo receptor system to be shown to lie downstream
of JAK2 per se. How PLFAP, DPP-1, and Cacy-bp might function
in proliferative signaling is under investigation, while effects of
SOCS-3 on not only Epo- but also SCF-dependent growth are
described below. For SOCS-3, the possible role of STAT5 in regulating
its transcription also was tested functionally in the following way. To
allow for analyses of short term effects, FDCW2 cells first were
transfected stably with the avian leukosis virus receptor, TVA. Next,
avian retroviruses encoding the constitutively active form of STAT5A
(STAT5A1*6) (or GFP as a control) were prepared and transduced into
FDCW2-TVA cells. In these STAT5A1*6-transduced cells, levels of SOCS-3
transcripts were elevated following the withdrawal of IL-3 as
determined by Northern blot and phosphorimaging (Fig.
5). Without correction for the estimated
efficiency of transduction (~20% of cells; Fig. 5, lower
panel), this effect was severalfold. Although this result
does not demonstrate direct transcriptional activation of SOCS-3 by
STAT5, it does provide function evidence in situ for
STAT5's involvement in this response pathway.
Integration of Epo Receptor and c-Kit Growth and Survival
Signals--
As mentioned in the Introduction, coactivation of the Epo
receptor and c-Kit is essential for the expansion of progenitor cells
at normal rates. Previously, we have shown that FDCW2 cells and derived
sublines express endogenous, functional c-Kit (19). Combined effects of
Epo and SCF on FDCER cell responses therefore were investigated next
(Fig. 6). With regard to proliferation, (and as predicted) (19), c-Kit synergized with Epo receptor forms in
FDCER-wt and FDCER-HY343 cells (Fig. 6). More interestingly, c-Kit also
synergized with ER-HY43F, and this result defines at least a basal Epo
receptor (phospho)tyrosine-independent mechanism of c-Kit cosignaling
as compared with FDCER-HY343 cells. Maximal effects of Epo plus SCF on
proliferation in FDCER-HY343F cells, however, were blunted somewhat as
compared with FDCER-HY343 cells. Whether Epo and SCF might possibly
cotarget bcl-xL or alternate bcl-2-related genes next was assessed. Initially, RNase
protection assays were used to inventory levels of Epo- and/or
SCF-dependent transcription of
bcl-xL, bcl-w, bak,
bax, bad, and bcl-2 (Fig. 7). These assays indicated that only
bcl-xL and bcl-2 transcript levels
were modulated significantly, and this was observed only in FDCER-HY343
cells and only in the presence of Epo. In addition, in the presence of
Epo plus SCF, a detectable increase in bcl-xL and bcl-2 transcription was observed. Based on these
apparent effects on bcl-xL gene transcription,
Northern blot analyses also were performed (Fig.
8). For bcl-xL
transcripts in FDCER-HY343 cells, marked synergistic effects
(i.e. severalfold above additive values as estimated by
scanning and imaging) were observed. Additionally, cis was
observed to be a clear target of Epo and SCF synergy, but such synergy
was selective and SCF was not observed to significantly affect
Epo-induced pim-1 expression, for example. SCF alone was a
poor inducer of bcl-xL transcript accumulation,
and did not detectably stimulate pim-1 or cis
gene transcription. Thus, SCF specifically augments the transcription
of select Epo response genes, and as one such target
bcl-xL may contribute to the combined proliferative effects of Epo and SCF.
Like bcl-xL, cis, and
pim-1, the expression of socs-3 also was shown
(above) to be induced by ER-HY343 (but not ER-HY343F). Possible
coordinate regulation of SOCS-3 expression by Epo plus SCF therefore
was assessed by Northern blotting. As shown in Fig. 9A, however, SCF proved to
have little effect on Epo-dependent SOCS-3 expression in
FDCER-HY343 cells. In an extended functional context, effects on Epo-
and SCF-dependent growth of exogenously expressed SOCS-3
were also assessed in stably transfected FDCER-HY343 cells (Fig.
9B). In clones ectopically expressing SOCS-3 at increased levels (and as predicted), Epo receptor-dependent growth
was attenuated sharply. Importantly, 32P RT-PCR served to
confirm that this was not due to possible differences in levels of
ectopically expressed ER-HY343 receptors among clones (Fig.
9B). More remarkably, exogenous SOCS-3 also proved to
significantly inhibit proliferative signaling via c-Kit (as well as the
proliferation supported by Epo-plus-SCF) (Fig. 9C) while
effects on IL-3-dependent growth were relatively minor
(
Finally, in an expanded population of human peripheral erythroid
progenitor cells, Epo and SCF recently have been shown to cotarget ERKs
(16), and in FDCER cell lines ERK activation therefore also was
investigated. In FDCER-HY343 cells (Fig.
10A, top
panel) ERKs (especially ERK2) were observed (based on
Western blotting with an antibody specific to phosphorylated ERKs 1 and
2) to be activated efficiently by SCF, but much less so by Epo. FDCW2
cells expressing the wt Epo receptor also were assayed, and similar results were obtained. To test whether this bias in signaling perhaps
reflected an unusual property of FDCER cells, Epo and SCF activation of
ERKs also was assayed in primary erythroid cells expanded from marrow,
and in splenocytes from mice treated with thiamphenicol. For each
preparation, using transgenic mice expressing an EGF receptor-Epo
receptor chimera from a GATA-1 gene-derived expression vector, we have
shown previously that erythroid progenitor cells comprise Aspects of the present work that merit discussion include
signaling defects associated with the mutation of a STAT5 binding site
in the Epo receptor form ER-HY343, and mechanisms underlying the
ability of this minimal Epo receptor form to synergize with c-Kit.
These newly defined signaling routes are summarized in the model
presented in Fig. 11. The efficient
induction of bcl-x gene expression by ER-HY343 (but not
HY343F) first is of physiological interest since, among Epo-regulated
survival factors, Bcl-xL has been shown in several model
systems to be affected most in its expression (24, 40, 41). However,
relatively little is understood concerning underlying mechanisms, and
conflicting results have been reported. In particular, a
phosphotyrosine-deficient Epo receptor form EpoR-S previously has been
reported to support bcl-xL (and
bcl-2) transcript expression as expressed in 32D cells, and in this system possible roles for STAT5 in this response pathway were
largely discounted (42). In FDCW2 cells, similar results were obtained
for at least certain sublines transfected with monocistronic ER-HY343F
expression vectors and selected in Epo (19).2 However,
responsive clones were rare, and proliferative responses were
attenuated. In part, this provided the impetus for presently preparing
FDCW2 cell lines stably expressing HY343 and HY343F receptor forms from
dicistronic vectors. As shown in Figs. 1, 2, and 6, FDCER-HY343F cells
failed to grow or survive in the presence of Epo yet efficiently
supported c-Myc transcript induction. Notably, this result is
consistent with the reported inactivity of cytoplasmic Tyr(P)-deficient
chimeric receptor forms as assayed for CFU-e forming activity in
transduced fetal cells (12), with the limited activity of a
Tyr(P)-deficient Epo receptor form as assayed in fetal liver cells from
Epo receptor
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1; and SHIP; Lyn, Syk,
and Tec; SHPTP-1 and 2; the nucleotide exchange factors Vav and C3G
(via Cbl); Cis and SOCS-3; and the adaptors Shc, Gab1, Gab2, CrkL, APS,
and IRS-2 (reviewed by Wojchowski et al. (Ref. 3)). Although
many of these are proto-oncogenic growth regulators, others are
negative effectors whose action in terminating Epo-stimulated events is likewise crucial to regulated erythropoiesis. These include Cis, which
appears to compete with STAT5 for binding at Tyr-343 (4, 5); SHPTP-1,
which acts to dephosphorylate JAK2 (6), and the suppressor
of cytokine signaling, SOCS-3,
which also binds and inhibits JAK2 (7). In
SOCS-3
/
mice, in fact, a fatal
erythrocytosis is precipitated (8).
/
mice (14) (as in Epo
receptor
/
mice (Ref. 2)), definitive
erythropoiesis fails and lethal embryonic anemias are engendered. In at
least certain systems, however, Epo receptor forms that lack all
cytoplasmic Tyr(P) sites (yet activate JAK2) also have been reported to
retain significant bioactivity (4), and among STAT5
a
/
and b
/
mice
those which survive embryonic stress later sustain no apparent defects
in adult erythropoiesis (14). Together, these observations raise basic
questions concerning how JAK2, and possibly STAT5 plus SOCS-3,
integrate key Epo receptor-derived signals. As evidenced by severe
anemias in mice sustaining mutations in the receptor tyrosine kinase
c-Kit or its ligand (15), SCF also is known to exert important effects
on red cell production. Through colony-forming assays (11, 13, 16) and
in vivo analyses (17), SCF has been shown to act in marked
synergy with Epo on cotargeted progenitor cells. In fact, this has been
proposed to involve the trans-phosphorylation of the Epo receptor
by c-Kit (18). In the present investigation, FDCW2-derived cell lines
(which express endogenous functional c-Kit) (19) have been used to
dissect signaling events that are relayed via Tyr(P)-343-retaining
versus Tyr(P)-deficient Epo receptor forms and c-Kit. Data
reveal roles for Tyr(P)-343 in mediating bcl-xL
and socs-3 transcription, and in augmenting novel effects of
c-Kit on Epo-stimulated bcl-xL and
cis transcription. In addition, SOCS-3 is shown to inhibit
not only Epo receptor but also c-Kit-dependent
proliferation; several new ER-HY343 (and ER-HY343F) target genes are
identified; and a primary role for SCF in MAPK activation is described.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3) (23), SOCS-3
(0.5-kb SmaI to XbaI fragment of pEF-SOCS-3)
(22), and GAPDH (0.8-kb KpnI to XhoI fragment of
pSP-GAPDH). RNase protection assays were performed using a RiboQuant
multi-probe system according to the manufacturer (PharMingen, San
Diego, CA). cDNAs for PCR analysis were synthesized using an
oligo(dT) primer (Life Technologies, Inc.) and Superscript II RNase
H(
) reverse transcriptase. ER-HY343 and HPRT were amplified using the
following primer pairs: 5'-GAG CCG CCC CCT GGA CAC C-3' and 5'-GAG GGG
GTC CCT GGA GGC G-3' for ER-HY343; 5'-CAC AGG ACT AGA ACA CCT GC-3' and
5'-GCT GGT GAA AAG GAC CTC T-3' for HPRT. In each reaction, 1 µCi of
[
-32P]ATP was included. Products were electrophoresed
in 5% acrylamide gels and were analyzed by phosphorimaging (Storm
system, Molecular Dynamics, Sunnyvale, CA). Atlas cDNA expression
arrays were performed according to the manufacturer
(CLONTECH, Palo Alto, CA).
-32P]ATP according to the manufacturer (Upstate
Biotechnology Inc.).
-casein promoter
(TGCTTCTTGGAATT) as a probe (31). Extracts (25 µg/assay) were
incubated for 10 min at 23 °C with the above 32P-labeled
GAS element in 4% Ficoll, 0.1 mM EDTA, 1 mM
dithiothreitol, 12 mM Hepes, 4 mM Tris-HCl, pH
7.9 (plus 2 µg of poly(dI-dC)/20-µl assay) prior to electrophoresis
in 0.25× TBE.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-casein promoter-derived GAS element and extracts from FDCER-HY343
versus ER-HY343F cells were performed and clearly confirmed
an essential role for Tyr-343 in this response (Fig. 1D).
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Fig. 1.
Epo receptor forms ER-HY343 and ER-HY343F
expression and proliferative signaling in FDCW2 cells. Panel
A, the Epo receptor form "H" (i.e. ER-HY343) is a
deletion construct truncated to remove C-terminal residues including
seven of eight Tyr(P) sites. Retained are box-1 and -2 domains, and a
Tyr-343 STAT5 binding site. In ER-HY343F, Tyr-343 is mutated to
phenylalanine. Panel B, cDNAs encoding the above Epo
receptor forms (and the wild type Epo receptor) were cloned to
pMK1059, and were expressed from this dicistronic vector in
stably transfected FDCW2 cells. As illustrated by Northern blotting,
this allowed for expression of receptor forms in polyclonal lines at
highly comparable levels in the absence of Epo selection. Panel
C, FDCW2 cell lines stably expressing HY343, HY343F or wtER forms
were assayed for proliferative signaling activity based on stimulated
rates of Epo-induced [3H]dT incorporation (mean
values ± S.D.). Wild type and ER-HY343 forms displayed comparable
activities while ER-HY343F was essentially inactive. Panel
D, in FDCER-HY343 but not FDCER-HY343F cells, Epo (but not
SCF) was observed to rapidly activate STAT5 as assayed by EMSA using a
-casein-derived [32P]GAS cassette.
4-fold based on phosphorimaging). This result was confirmed in three
independent experiments and is the first analysis to directly link
Tyr-343 of the Epo receptor to endogenous bcl-xL gene transcription. Parallel effects were observed for
bcl-2, but in FDC cell lines levels of bcl-2
transcript expression (and induction by either Epo or IL-3) were
10%
of bcl-x transcripts. Based on these results, whether
exogenous Bcl-xL might rescue the Epo-dependent
growth of FDCER-HY343F cells next was tested (Fig.
3). Cells were transfected with
pEFNeo-Bcl-xL, and clones stably expressing exogenous
Bcl-xL were identified (Fig. 3, upper right panel). In FDCER-HY343F-Bcl-xL
cells, however, ectopic expression of Bcl-xL at elevated
levels failed to affect growth (Fig. 3, upper
left panel). As a second proto-oncogene whose
Epo-induced expression likewise depends upon Tyr-343 (see above),
pim-1's ability to rescue FDCER-HY343F-Pim-1 cell growth
also was tested (Fig. 3, lower panels). Effects, however, were limited
to relatively high level expressing clones in which a modest increase
in background rates of [3H]dT incorporation was observed.
Each ectopically expressed factor, however, did protect FDCER-HY343F
cells from apoptosis due to cytokine withdrawal by as much as 3-fold,
respectively (data not shown). These results are consistent with those
of related in vivo studies (34, 35) and suggest the
existence of important Tyr(P)-343-dependent effectors
besides bcl-xL or pim-1.
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Fig. 2.
Tyr-343 of ER-HY343 is required for efficient
Epo-induced bcl-x gene transcription. FDCER-HY343
and ER-HY343F cells were cultured for 10 h in the absence of
hematopoietic growth factors and then exposed to Epo (25 units/ml). At
the intervals indicated, RNA was isolated and analyzed by Northern
blotting. Transcription of c-Myc was induced via both ER-HY343 and
ER-HY343F, while induced transcription of bcl-x was
attenuated markedly via ER-HY343F (and the induction of cis
and pim-1 transcription was essentially blocked).
Equivalence in RNA loading was confirmed by hybridization to a
[32P]GAPDH cDNA.
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Fig. 3.
Neither Bcl-xL nor Pim-1 rescue
mitogenesis via ER-HY343F. Right panels, exogenous
Bcl-xL or Pim-1 was expressed in FDCER-HY343F cells via
stable transfection with pEFNeo vectors. In clones isolated by limiting
dilution, Bcl-xL or Pim-1 expression was assayed by
Northern blotting (and was confirmed by Western blotting, data not
shown). Clones expressing bcl-xL were screened
following 8 h of culture in the absence of hematopoietic growth
factors. Left panels, illustrated are Epo-induced mitogenic
response profiles (based on induced rates of [3H]dT
incorporation) (mean values ± S.D.) for FDCER-HY343 and
FDCER-HY343F cells, and for representative FDCER-HY343F clones stably
expressing exogenous Bcl-xL (clones 19, and 23) or
exogenous Pim-1 (clones 7 and 11).
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Fig. 4.
Epo-induced gene transcription via
Tyr-343-dependent and Tyr(P)-independent routes.
FDCER-HY343 and ER-HY343F cells were cultured for 10 h in 0.9%
FBS, OptiMEM medium in the absence of cytokines. Cells then were
exposed to Epo (± 25 units/ml for 90 min), and RNA was isolated.
Differential cDNA expression was analyzed using a cDNA
expression arrays. Transcription of socs-3 and
plfap were induced only in ER-HY343, while dpp-1
and cacy-bp were induced via both ER-HY343 and ER-HY343F.
Results were analyzed by phosphorimaging and are represented as
Epo-induced -fold induction (signals were against
gapdh).
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Fig. 5.
SOCS-3 transcript levels are increased due to
ectopic expression of a constitutively active STAT5A-1*6.
Upper panel, to treat possible effects of STAT5 on
SOCS-3 gene expression, FDCW2-TVA cells were transducted
with retroviruses encoding either STAT5A-1*6 or GFP as a negative
control. In derived FDCW2-TVA-STAT5-1*6 cells, levels of SOCS-3
transcript were elevated severalfold upon the withdrawal of IL-3
(i.e. cytokine withdrawal). Assays were by Northern blotting
and phosphorimaging at 0, 4, and 8 h of IL-3 withdrawal, and are
normalized to levels of GAPDH. Data shown are representative of two
independent experiments. No such effect on SOCS-3 transcript levels was
observed in FDCW2-TVA-GFP cells (data not shown). Lower
panel, to confirm transduction efficiency, FDCW2-TVA cells were
infected in parallel with a GFP-encoding virus at approximately
equivalent high titer and at 24 h after infection were analyzed
for the frequency of infected cells. In this (and in three independent
experiments), ~20% of cells reproducibly were transduced.
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Fig. 6.
SCF and Epo mitogenic synergy is relayed via
Epo receptor Tyr-343-dependent, as well as
Tyr(P)-independent mechanisms. In FDCER-HY343 and FDCER-HY343F
cells, the ability of receptor forms HY343 and HY343F to act in synergy
with c-Kit was assayed based on rates of [3H]dT
incorporation induced by Epo in the presence (or absence) of SCF (100 ng/ml). Data (mean values ± S.D.) are normalized
versus maximal responses (to Epo plus SCF) and are
representative of three independent experiments.
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Fig. 7.
Epo and/or SCF-induced expression of
bcl-x-related transcripts in FDCER-HY343 and HY343F
cells. FDCER-HY343 and FDCER-HY343F cells were cultured for
10 h in the absence of hematopoietic growth factors, and were
stimulated for 90 min with Epo (25 units/ml), SCF (25 ng/ml), or both
factors. Levels of bcl-xL, bcl-2,
bcl-w, bak, bax, bad, and
gapdh transcripts then were estimated by RNase protection
assays. No cytokine corresponds to FDCER-HY343
cells at 10 h of culture.
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Fig. 8.
SCF selectively enhances
Epo-dependent bcl-x and cis
gene transcription. FDCER-HY343 and FDCER-HY343F cells were
cultured for 10 h in the absence of hematopoietic growth factors,
and then were exposed to Epo (25 units/ml) and/or SCF (50 µg/ml). At
the intervals indicated, RNA was isolated and levels of c-Myc,
pim-1, bcl-xL, cis, and
gapdh transcripts were assayed by Northern blotting.
20% attenuation).2 c-Kit therefore is revealed to
comprise a lateral target for SOCS-3.
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Fig. 9.
Ectopic expression of SOCS-3 in FDCER-HY343
cells markedly inhibits mitogenic responses to both Epo and SCF.
Panel A, to confirm the ability of Epo to induce endogenous
SOCS-3 mRNA expression, FDCER-HY343 cells were cultured for 10 h in 1% FBS, OptiMEM in the absence of cytokines. Cells were then
exposed to Epo (2.5 units/ml, experiment 1; 25 units/ml, experiment 2),
SCF (50 ng/ml), or the combination of both factors and at the intervals
indicated levels of endogenous SOCS-3 transcripts were assayed by
Northern blotting. Panel B, FDCER-HY343-SOCS-3 cells were
prepared by the stable transfection of FDCER-HY343 cells with a pEF
vector encoding SOCS-3 and clonal sublines were isolated by limiting
dilution. In the subclones indexed, levels of exogenous SOCS-3
transcripts were assayed by Northern blotting. To confirm equal
loading, blots were hybridized to a [32P]GAPDH cDNA
(upper panel). To confirm similar levels of
ER-HY343 expression among clones studied, transcript levels were
assayed by RT-PCR (lower panel). Panel
C, illustrated are cytokine-induced mitogenic response profiles
(as induced rates of [3H]dT incorporation) for
FDCER-HY343-SOCS-3 clones 1 and 9, and for parental FDCER-HY343 cells.
Values are percentage of maximal FDCER-HY343 response and are
representative of two independent experiments.
50% of
expanded populations (13, 39). As shown in Fig. 10A
(center panels), ERKs were activated efficiently
by SCF but once again much less so by Epo. For progenitor cells from spleen, this result also was confirmed in vitro kinase
assays using myelin basic protein as a substrate (Fig. 10A,
lower panel). Finally, to confirm Epo sensitivity
in these erythroid progenitor cells, levels of cis
transcript induction and STAT5 activation were assayed. As shown by
Northern blotting and EMSA, respectively, Epo efficiently activated
each response (Fig. 10B). Thus, the activation of ERKs in
each of the above model systems is proposed to occur primarily via
SCF/c-Kit rather than Epo-dependent routes.
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Fig. 10.
ERKs are activated primarily via
c-Kit-dependent routes in Epo- and SCF-exposed FDCER cell
lines and in primary erythroid progenitor cells. Panel
A, SCF and Epo activation of ERKs was assayed in FDCER cell lines
(upper panels), marrow-derived erythroid
progenitor cells (upper center), and erythroid
splenocytes from mice treated with thiamphenicol (lower
center). For each preparation, cells were cultured for
10 h in the absence of hematopoietic growth factors and then were
exposed for 5 min to Epo (E, 25 units/ml), SCF
(S, 50 µg/ml), or both factors. Lysates then were prepared
and analyzed directly for levels of activated ERKs by Western blotting.
Blots were stripped and re-probed with a pan-specific MAPK antibody.
ERK activity in erythroid splenocytes also was determined by
immunoprecipitating MAPKs, and assaying levels of myelin basic protein
phosphorylation (lower subpanel). Panel
B, Epo responsiveness of primary erythroid progenitor cell
populations was confirmed based on the efficient activation by Epo (but
not SCF) of STAT5 DNA binding activity, and cis gene
transcription. Activated STAT5 was assayed by EMSA using a -casein
promoter-derived [32P]GAS element, and cis
transcripts were assayed by Northern blotting. Shown are results for
erythroid splenocytes used in the above ERK assays. Essentially
equivalent results were obtained for marrow-derived erythroid
progenitor cells (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice (11), and with the loss
in CFU-e forming activity observed upon the mutation of Tyr-343 within
a truncated prolactin receptor - Epo receptor chimera (12). Based on
the suggestion that Tyr-343 and STAT5 therefore may comprise important
transducing components, Socolovsky et al. (43) also recently
evaluated embryonic erythropoiesis in mice deficient in STAT5A and
STAT5B. Unlike surviving adult animals in which erythropoiesis appears
normal (44), embryos displayed marked anemia and apoptosis appeared to
be a frequent fate of erythroid progenitor cells. Furthermore, based on
experiments in Ba/F3 and HCD-57 cells using a bcl-x promoter
reporter construct plus dominant-negative and activated forms of STAT5,
bcl-xL was proposed to comprise a STAT5 target
gene (33, 43). Present findings are not only consistent with this
model, but also provide a specific link between the Epo receptor STAT5
binding site Tyr(P)-343 and endogenous bcl-xL
gene transcription.
View larger version (24K):
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Fig. 11.
Schematic summary of newly observed Epo and
SCF signaling pathways. Depicted are the four major
pathways/routes characterized by the present investigation.
A illustrates a primary route from c-Kit (rather than the
Epo receptor) to ERK-1 and ERK-2 activation; B illustrates
specific synergistic effects exerted by c-Kit on
Epo-dependent bcl-xL and
cis transcription; C illustrates Epo-directed,
STAT5-dependent pathways to pim-1,
socs-3, and plfap gene transcription;
D illustrates JAK2-dependent, ERHY343F- (and
possibly STAT5-) independent pathways to c-myc,
dpp-1, and cacy-bp.
The forced expression of Bcl-xL in FDCER-HY343F cells, however, did not rescue proliferative activity (even upon Epo activation of ER-HY343F). This result is consistent with studies of exogenously expressed Bcl-xL in fetal liver cells (34), and prompted the screening via arrays of known genes of additional ER-HY343 and/or ER-HY343F targeted genes. Four ER-HY343 response genes were identified, i.e. socs-3, plfap, dpp-1, and cacy-bp. Among these, socs-3 previously has been reported to be induced via the full-length Epo receptor (22). Beside this, Epo induction of socs-3 and plfap were observed to depend upon intactness of Tyr-343 and therefore are suggested to perhaps constitute STAT5 target genes. Interestingly, a human homologue of PLFAP, EBP-1 (ErbB-3-binding protein) recently has been shown to interact with ErbB-3 in a two-yeast hybrid screen (45), and ErbB-3 has been shown to interact with Shc/Grb-2 complexes in response to neuregulin (46). dpp-1 and cacy-bp, in contrast, were induced via both ERY343 and ERY343F and to our knowledge are the first target genes other than c-myc to be mapped downstream of JAK2-dependent, Epo receptor Tyr(P)-independent routes in the Epo receptor system. Interestingly, DPP-1 gene expression also has been discovered to be induced by IL-2 in primary human lymphocytes, and may act as a central coordinator of serine proteases in hematopoietic cells (47). cacy-bp previously was cloned from a mouse brain library using a cDNA probe deduced from the partial amino acid sequences of calcyclin (38). The sequence of this clone was novel, and based on its Ca2+-dependent interaction with calcyclin, Cacy-bp also has been proposed to function in signal transduction (47).
With regards to the capacities of the above Epo receptor forms to
synergize with c-Kit, four novel effects are described. First, an Epo
receptor Tyr(P)-independent mechanism of mitogenic synergy with c-Kit
is dissected. Based both on the inactivity of directly related Epo
receptor forms in other model systems (11, 12) and on a proposed
mechanism for synergy wherein c-Kit acts to phosphorylate Epo receptor
Tyr(P) sites (11), the observed ability of the Tyr(P)-deficient
receptor form ER-HY343 to synergize with c-Kit in stimulating
mitogenesis was somewhat unexpected. Mechanistically, studies by Sui
et al. (16) argue that this is not a direct consequence of
Epo receptor-associated JAK2 phosphorylation of c-Kit. Rather, it is
speculated that other downstream JAK2-dependent events
account for this effect, and these are the subjects of ongoing studies.
Second, and in contrast, effects of SCF on bcl-x gene
expression appear to depend upon Tyr-343 since this response is not
induced by SCF in Epo-exposed FDCER-HY343F cells. Epo-activated STAT5
therefore is likely to be one possible target of SCF effectors. Although ectopically expressed c-Kit has been reported in certain systems to activate STAT5 (48), this possible direct mechanism was
discounted in assays of STAT5 activation in FDCER cell lines and
primary erythroid cells. Additionally, c-Kit previously has been shown
in FDCER lines to fail to activate JAK2 (19). In other type 1 cytokine
receptor systems, STAT activity has been shown to be enhanced via
ERK-dependent S/T phosphorylation (49, 50) and such events
therefore may underlie c-Kit effects on certain STAT5 target genes
(including cis (Ref. 5)). Alternatively, it is possible that
c-Kit targeted transcription factors might act in concert with STAT5,
and in melanocytes the transcription factor Mi, for example, previously
has been discovered as a specific target for c-Kit activated MAPKs
(51). Third, it previously has been observed in human erythroid
colony-forming cells (as expanded from peripheral BFU-e) that Epo and
SCF act synergistically to stimulate ERKs (16). In FDCER cell lines, at
least additive effects of SCF plus Epo on ERK activation were observed
in certain experiments,2 yet ERK activation via c-Kit was a
more potent effect in all model systems examined. In FDCW2 cell lines,
this was not due to levels of receptor expression, as 125I
ligand binding assays have shown c-Kit and Epo receptor forms to be
expressed at similar low densities.2 Also, results were not
attributable to distinct kinetics of Epo versus SCF
signaling, since ERK activation by each was shown in extended
time-course experiments to peak sharply at 5 min of cytokine exposure.2 Therefore, the more efficient coupling between
c-Kit and ERKs also may underlie SCF and Epo's combined effects on
progenitor cell expansion. Fourth and finally, evidence is provided
that SOCS-3, as induced via an ER-HY343 (but not ER-HY343F nor
c-Kit)-dependent pathway, acts to inhibit not only Epo
receptor but also c-Kit induced proliferation. For at least certain
SOCS factors, their discovery was based on an ability to bind JAKs
(52-54) and interactions between both the NH2-terminal
kinase inhibitory and Src homology 2 domains in SOCS-3 and the kinase
domain in JAK2 have been described (55). Additional mechanisms of
suppression also appear to exist, and in the growth hormone receptor
system, JAK2 suppression by SOCS-3 is dependent on tyrosine
phosphorylation of the growth hormone receptor (7, 56). By comparison,
in the c-Kit system SOCS-1 has been shown to affect proliferation by
binding competitively to Grb-2 (57). In repeated experiments in FDC
cell lines and in primary erythroid cells, no significant induction of
SOCS-1 expression by SCF or Epo was detected.2 This does
not discount important suppressive effects, however, and this is
illustrated by persistent expression of SOCS-3 in Epo
receptor/
mice (8) as well as by the
presently discovered suppression of c-Kit signaling by SOCS-3.
Suppression of SCF/c-Kit-dependent proliferation in
FDCER-HY343 cells, in fact, was almost as efficient as suppression of
Epo-induced mitogenesis. IL-3-dependent proliferation, however, was relatively unaffected but this might be due to the maintenance of FDCER-HY343-SOCS-3 cells in IL-3. Since SCF dramatically enhances BFU-e and CFU-e production (18), inhibition of c-Kit activity
by Epo-induced SOCS-3 may comprise an important growth inhibitory
mechanism during normal erythropoiesis. In addition, the absence of
this lateral inhibition also may contribute to severe erythrocytosis
and lethality in embryonic SOCS-3
/
mice
(8).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank R. C. Gregory for efforts in Epo receptor cDNA manipulations and expression. We also thank the following investigators for providing the cDNA and/or plasmids indicated: Dr. Alice Mui (pXM-STAT5-1*6), Dr. Robyn Starr (pEF-FLAG-I-SOCS-3), and Dr. Harold E. Varmus (pRCAS-GFP and pCDNA6-TVA950).
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant RO1 DK44491.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: 115 Henning Bldg.,
Pennsylvania State University, University Park, PA 16802. Tel.: 814-865-0657; Fax: 814-863-6140; E-mail: dmw1@psu.edu.
Published, JBC Papers in Press, December 20, 2000, DOI 10.1074/jbc.M007473200
2 T. J. Pircher, J. N. Geiger, C. P. Miller, D. Zhang, P. Gaines, and D. M. Wojchowski, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
Epo, erythropoietin;
ER, erythropoietin receptor;
wt, wild type;
EMSA, electrophoretic
mobility shift assay;
JAK, Janus kinase;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
kb, kilobase pair(s);
FBS, fetal bovine serum;
PCR, polymerase chain reaction;
RT, reverse
transcription;
MAPK, mitogen-activated protein kinase;
SCF, stem cell
factor;
GFP, green fluorescent protein;
IL, interleukin;
TVA, tumor virus-A;
GAS, interferon- activated sequence;
STAT, signal
transducers and activators of transcription.
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