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Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1104-1118
RAPID COMMUNICATION
Erythropoietin Receptor and STAT5-Specific Pathways Promote SKT6
Cell Hemoglobinization
By
Richard C. Gregory,
Ning Jiang,
Kazuo Todokoro,
Jill Crouse,
Robert E. Pacifici, and
Don M. Wojchowski
From the Department of Biochemistry and Molecular Biology, the Center
for Gene Regulation, the Graduate Program in Genetics, and the
Department of Veterinary Science, The Pennsylvania State Univeristy,
University Park, PA; the Tsukuba Life Science Center, Tsukuba, Ibaraki,
Japan; and Amgen, Inc, Thousand Oaks, CA.
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ABSTRACT |
Erythrocyte production in mammals is known to depend on the exposure
of committed progenitor cells to the glycoprotein hormone erythropoietin (Epo). In chimeric mice, gene disruption experiments have demonstrated a critical role for Epo signaling in development beyond the erythroid colony-forming unit (CFU-e) stage. However, whether this might include the possible Epo-specific induction of red
blood cell differentiation events is largely unresolved. To address
this issue, mechanisms of induced globin expression in Epo-responsive
SKT6 cells have been investigated. Chimeric receptors containing an
epidermal growth factor (EGF) receptor extracellular
domain and varied Epo receptor cytoplasmic domains first were expressed
stably at physiological levels in SKT6 cells, and their activities in
mediating induced hemoglobinization were assayed. While activity was
exerted by a full-length chimera (EE483), truncation to remove 7 of 8 carboxyl-terminal tyrosine sites (EE372) markedly enhanced
differentiation signaling. Moreover, mutation of a STAT5 binding site
in this construct (EE372-Y343F) inhibited induced globin expression and
SKT6 cell hemoglobinization, as did the ectopic expression of
dominant-negative forms of STAT5 in parental SKT6 cells. As in normal
CFU-e, SKT6 cells also were shown to express functional receptors for
stem cell factor (SCF). To further define possible
specific requirements for differentiation signaling, effects of SCF on
SKT6 cell hemoglobinization were tested. Interestingly, SCF not only
failed to promote globin expression but inhibited this Epo-induced
event in a dose-dependent, STAT5-independent fashion. Thus, effects of
Epo on globin expression may depend specifically on STAT5-dependent
events, and SCF normally may function to attenuate terminal
differentiation while promoting CFU-e expansion.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
DURING RED BLOOD CELL development,
stringent control is exerted over the proliferation, survival, and
maturation of erythroid progenitor cells. An early step in this process
is the commitment of pluripotent hematopoietic progenitor cells to the
erythroid lineage, and this is thought to be dictated by the
coexpression of select lineage-restricted trans-factors, including
GATA1,1 Friend of GATA1
(FOG),2 and erythroid
Krüppel-like factor (EKLF).3 Subsequently, the development of these committed
erythroid progenitor cells as erythrocytes has been shown to depend on
the lineage- and stage-restricted expression of the single
transmembrane receptor for erythropoietin (Epo).4,5 Epo has
long been recognized to be required for the efficient formation of red
blood cells from erythroid colony-forming units (CFU-e) in
vitro6 and, recently, this role for Epo has been confirmed
in chimeric mice via disruption of the Epo7 and Epo
receptor genes.7-9 Specifically, in the presence of stem
cell factor and pokeweed mitogen-stimulated spleen cell conditioned
medium, the development of CFU-e from Epo
receptor / fetal hepatocytes proceeds
efficiently.7,8 However, development beyond this stage is
either undetectable9 or markedly inhibited in that, in
studies by Lin et al,8 limited frequencies of
benzidine-positive fetal liver cells were detected. These findings and
observations that Epo blocks DNA fragmentation in normal erythroid
progenitor cells10 provide clear evidence that Epo
critically supports progenitor cell survival and expansion beyond the
CFU-e stage.
By comparison, whether Epo might also selectively promote
differentiation is an unresolved issue. Using Epo
receptor/ fetal hepatocytes as a model, two
types of experiments recently have been performed in which heterologous
receptors have been demonstrated to promote CFU-e differentiation in
the absence of Epo. First, based on the observed expression of
thrombopoietin (Tpo) receptor transcripts in these cells, the ability
of Tpo to support erythroid differentiation has been
tested.9 In the presence of either stem cell factor (SCF)
or interleukin-3 (IL-3) plus IL-11, limited levels of Tpo-dependent red
blood cell formation could be demonstrated. Second, Epo
receptor/ fetal hepatocyte differentiation
also has been shown to be supported after the ectopic overexpression
and activation of a full-length rabbit prolactin receptor
form.11 These studies provoke two alternate
interpretations. As a strict interpretation, Epo might be considered to
be nonessential for differentiation signaling and to be important only
as a general survival factor for preprogrammed erythroid progenitor
cells. However, high conservation exists in signaling events that are
activated by Epo,12 Tpo,13 and prolactin14 via their structurally related
receptors,15 and Tpo and prolactin receptors therefore may
specifically compensate for the Epo receptor upon their expression and
activation in late erythroid progenitor cells.
Consistent with the notion that Epo can specifically promote at least
certain late erythroid differentiation events, in several murine,16-18 human,19 and avian20
cell lines, Epo has been shown to induce globin gene expression. Murine
erythroleukemic SKT6 cells comprise one such example,17 and
in recent studies we have isolated stable SKT6 cell sublines that
readily hemoglobinize in response to Epo at physiological
concentrations.21 In addition, we have demonstrated that
chimeric receptors composed of the human epidermal growth factor
(EGF) receptor extracellular domain and defined Epo
receptor cytoplasmic domains support ligand-induced hemoglobinization,
and in preliminary studies we21 as well as Wakao et
al22 and Iwatsuki et al18 have provided
evidence that STAT5, a signal transducer and
activator of transcription,23 may
regulate this Epo response pathway. In the present study, we now show
that chimeric receptors containing highly truncated Epo or prolactin
Nb2 receptor cytoplasmic domains in fact mediate ligand-induced SKT6
cell hemoglobinization significantly more efficiently than do
endogenous Epo receptors. In addition, the point mutation of tyrosine
sites for STAT5 binding in these minimal chimeras as well as the
ectopic expression of dominant-negative forms of STAT5 are demonstrated
to effectively inhibit induced SKT6 cell differentiation. Thus,
extended evidence is provided that STAT5 may function as a regulator of
Epo-induced globin expression and hemoglobinization. STAT5 is 1 of 8 related transcription factors that have been shown to be activated by
most hematopoietic cytokines.23 As defined first in the
interferon- receptor system, cytokine activation of STATs proceeds
from the SH2 domain-mediated binding of STATs to phosphotyrosine sites
within activated receptor complexes.24 Receptor-associated
Jak kinases then phosphorylate STATs at a conserved C-terminal tyrosine
residue, and phosphorylated STATs then self-associate. Subsequently
dimeric or multimeric STATs undergo nuclear translocation, bind to
conserved cis-elements via central DNA binding domains, and modulate
transcription at targeted promoters and enhancers via C-terminal
transactivation domains. Epo has been shown to selectively activate
Jak2 and to recruit primarily STAT5 A and B (2 highly related isoforms
in mice)25 to a receptor Y343
site.26 However, Epo activation of STATs 1 and/or 3 also has been observed in normal rat fetal liver CFU-e,27
Friend virus-infected murine erythroid splenocytes,28 HCD57
cells,29 and SKT6 cells.21
In addition, Epo effects on red blood cell production are augmented by
several alternate hematopoietic growth factors. Specifically, IL-3,30 granulocyte-macrophage colony-stimulating factor
(GM-CSF),31 IL-9,32 and SCF33
promote the expansion of early erythroid progenitor cells
(burst-forming units-erythroid), whereas Tpo,34
IL-6,35 and again SCF36 have been shown to
exert effects at later developmental stages. SCF has perhaps been
best-defined as a coregulator. In mice with mutations in the genes for
SCF37 or c-Kit,38 anemias are prevalent, and
SCF and Epo synergistically promote red blood cell production ex
vivo.39 Furthermore, in HCD57 cells, signaling via c-Kit
has been suggested to depend on the trans-phosphorylation of the Epo
receptor40; in at least certain cell lines c-Kit may occur
in constitutive association with Jak2,41,42 and in
marrow-derived mast cells SCF may activate STAT5.43 Thus,
in the context of red blood cell development, important questions are
raised regarding possible effects of SCF on erythropoietic
proliferation versus differentiation events. Like normal CFU-e, SKT6
cells presently are shown to express functional receptors for SCF, and
the effects of SCF on induced globin expression and SKT6 cell
hemoglobinization also have been investigated. Interestingly, SCF
proved to inhibit Epo-induced SKT6 cell differentiation in a
concentration-dependent, STAT5-independent fashion. Overall,
investigations provide novel evidence that specific signaling events
may underlie the ability of Epo to advance the development of CFU-e and
indicate that SCF may attenuate this pathway to terminal
differentiation.
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MATERIALS AND METHODS |
Chimeric receptor and STAT cDNA constructs.
Receptor constructs studied include the wt Epo receptor and the
chimeric constructs EE483, EE372, and EENb2. In each chimera, the
extracellular domain is derived from the EGF receptor
(Leu1-Cys620) and the transmembrane domain from
the Epo receptor (Pro225-Leu247).44
EE483 contains the full-length cytoplasmic domain of the Epo receptor,
EE372 contains Epo receptor residues
Ser248-Met372,45 and EENb2 contains
8 membrane cytoplasmic residues of the Epo receptor
(Ser248-Lys256) fused to cytoplasmic residues
Ile243-His393 of the rat prolactin Nb2
receptor.46 cDNAs encoding these receptor forms were
constructed and cloned into pCIneo (Promega, Madison, WI) as follows.
For pCIneo-EE483, an EGF receptor/Epo receptor chimeric
cDNA47 was cloned into pGEM5Zf+ (to acquire a
5 Spe I site) and then into a pCIneo vector
(pCIneo BII)21 at Nhe I and Sal I sites.
pCIneo-EE372 was prepared by restricting pCIneo-EE483 with Bgl
II and Sal I (to excise codons encoding Ile257-Ser483 of the Epo receptor) and by
replacing this fragment with an Epo receptor cDNA encoding residues
Ile257-Met372.47 pCIneo-EENb2 was
constructed by first using polymerase chain reaction (PCR) to prepare a
cDNA fragment from a wild-type Nb2 construct.21,46 This
modified 3 Nb2 cDNA then was cloned into pCIneoEE483 at
Bgl II and Sal I sites. pCINeo-EE372Y343F was
constructed by cloning the Bgl II-Xho I fragment of
pSL1180-ER396 into pSP72 and by mutating Y343 to
phenylalanine by overlap extension.48 In overlap extension, the primers 5-CCAGGACACCTTCTTGGTATTGGAT-3 and
5-ATCCAATACCAAGAAGGTGTCCTGG-3 were used together with M13
reverse and forward sequencing primers, respectively. The derived PCR
product was then cloned into pSP72, excised as a Bgl
II-Xho I fragment, and cloned stepwise into pCINeo-EE483 at
Bgl II and Sal I sites. pMK1059-EE375Y343F
was constructed using pXM190 as a template together with the following
PCR primers: 5-GATCGGGCCCTTACTGGAGCCGGTGGGCAGTGAGCATGCCCAGGACACCTTCTTGGTATTGGATAAGTGG-3 and 5-GCTCTAGACTAAGCTTCATCCATAGTCACAGGGTCCAC-3. This PCR
product was cloned into pSP72 at Apa I and Xba I sites,
was excised as a 360-bp Bgl II-Xba I fragment, and was
used to construct the expression vector pMK1059-EE375Y343F.
In the constructs S5D and S3D, the predicted DNA binding domains
of murine STAT5A (Thr409-Arg523) and STAT3
(Asn400-Leu508) were deleted using a Pfu
polymerase, PCR-based method (Exsite System; Stratagene, La Jolla, CA).
The PCR primers used were 5-GGGCTGGTGGTACTCCATGACGCAA-3, 5-GGGTTGACCAAGGAGAACCTCGTGTTC-3 (S5D), and
5-AGCCTCCTCCATGTTCATCACTTTTGTGTTCG-3, 5-AGCTGGCAGTTCTCGTCCACCACC-3 (S3D). For S5C, a stop
codon was inserted after Ala713 and a 3 Xba
I site was introduced using
5-CAGCAACCACCTCGAGGACTACAACA-3 and
5-CGTCAATGCATCCACAGATGCCTGATCTAGAGC-3 as PCR primers. All PCR products were sequenced to confirm deletions, recovery of reading
frames, and insertions. cDNAs encoding S5D, S5C, and S3D then
were cloned to pCINeo. In addition, S5D and S5C were cloned into the dicistronic expression vector
pMK1059.49
SKT6 cell culture, transfections, and derived cell lines.
The ability of the above-described chimeric receptor forms to mediate
mitogenic and differentiation signaling was studied via their stable
expression in SKT6 cells. SKT6 cells were maintained and
electrotransfected as described previously.21 Stably
transfected lines were selected in G418 (1.2 mg/mL), and low complexity
subclones were isolated by dilution. Expression of chimeric receptor
forms in derived cell lines was assayed by Northern blotting.
Expression of STAT5 mutants from pCINeo and pMK1059 vectors
was assayed by Western blotting of cell lysates with antibodies to
STAT5A (-S5#1; Santa Cruz Biotechnology, Santa Cruz, CA) or STAT5A/B
(-S5#2; Transduction Laboratories, Lexington, KY).
Assays of ligand-induced SKT6 cell proliferation.
In assays of ligand-induced proliferation, exponentially growing SKT6
cells were cultured for 10 hours in OptiMem I supplemented with 1%
fetal bovine serum (FBS), penicillin (1 U/mL),
streptomycin (1 µg/mL), amphotericin B (2.5 ng/mL), and
105 mol/L  -mercaptoethanol. Cells then were
adjusted to 3 × 105 cells/mL in 96-well plates (50 µL/well). Epo or EGF was added (50 µL) at 48 hours of culture.
[methyl-3H] thymidine (1 µCi) was added. At 2 hours of
incubation, rates of incorporation were assayed by scintillation
counting (Beckman 1205 Betaplate reader; Beckman
Instruments, Palo Alto CA).
Assays of ligand-induced SKT6 cell hemoglobinization and globin
expression.
In assays of ligand-induced SKT6 cell hemoglobinization, cultures were
initiated at 1.5 × 105 cells/mL (1.5 mL/well, 6-well
plates) and were exposed to Epo (±10 U/mL) or EGF (±15 ng/mL).
At 36 hours, an equal volume of media was added, and at 48 and 72 hours, hemoglobin-positive cells were assayed by staining with 2, 7 diaminofluorene (DAF).21 In assays of globin expression,
ligand-exposed cells were collected (1,500g for 8 minutes),
washed in 1.5 mL phosphate-buffered saline (PBS; 137 mmol/L NaCl, 2.7 mmol/L KCl, 4.3 mmol/L NaHPO4-7H2O, 1.4 mmol/L
KH2PO4, pH 7.3), and lysed by gentle vortexing
and incubation (10 minutes at 4°C) in RIPA buffer (1% nonidet-P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 0.5 mmol/L phenylmethylsulfonyl fluoride, 0.5 µg/mL leupeptin, 0.7 µg/mL pepstatin A, and 2.2 µg/mL aprotinin). Cleared lysates were assayed for protein (BCA assay; Pierce, Rockford, IL) and were denatured for SDS-polyacrylamide gel electrophoresis (1.7% SDS, 0.1 mol/L dithiothreitol, 0.001 mmol/L bromophenyl blue, 5% glycerol, and
0.06 mol/L Tris-HCl, pH 6.8). Electrophoresis (15% gels) and Western
blotting was performed as described previously21 using 0.1-µm nitrocellulose membranes (Protran; Schleicher & Schuell, Keene, NH).
Assays of STAT5 binding activity and tyrosine phosphorylation.
STAT5 activation was based on binding to a biotinylated prolactin
response element (PRE; 5-TTAGATTTCTAGGAATTCAAATC-3, and 5-biotin-GATTTGAATTCCTAGAAATCT-3).21
SKT6-EE372 and SKT6-EENb2 cells were exposed to Epo (20 U/mL) or EGF
(33 ng/mL), chilled immediately to 0°C, washed in PBS, and lysed in
10 mmol/L CHAPS, 2 mmol/L Na2EDTA, 0.1 mmol/L
Na2VO4, 5 mmol/L NaF, 1 mmol/L dithiothreitol, 0.5 mmol/L phenylmethylsulfonyl fluoride, 0.5 µg/mL leupeptin, 0.7 µg/mL pepstatin A, 2.2 µg/mL aprotinin, 50 mmol/L Tris, pH 8.0, at
0°C with sonication (30 seconds, 50% duty; Branson Sonifier 250; Branson Ultrasonics, Danbury, CT). Cleared lysates
(175 µL of 600 µL total per 1.5 × 107 cells) were
then combined with 10 µg of poly dIdC (Pharmacia, Piscataway, NJ),
biotinylated PRE cassette (250 µmol/L final concentration), and
binding buffer to yield 300 µL of sample in 2% glycerol, 60 mmol/L
KCl, 0.5 mmol/L Na2EDTA, 1 mmol/L dithiothreitol, 4 mmol/L Tris, 12 mmol/L HEPES, pH 7.9. Samples were incubated stepwise for 20 minutes at 4°C and at 25°C and then were adsorbed to
streptavidin Agarose CL4B (40 µL of gel per sample). Gels were washed
four times in binding buffer, and bound STAT5 was eluted in sample buffer for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and assayed by ECL Western blotting (STAT5A antibodies; Santa Cruz Biotech). For assay of STAT5 tyrosine phosphorylation, cleared lysates
from above were incubated stepwise with antibodies to STAT5A (-S5#1)
at 4°C for 3 hours and with 25 µL of Protein-A Agarose CL4B
(Boehringer Mannheim Biochemicals, Indianapolis, IN) at 4°C for 1 hour. STAT5/gel complexes were then washed four times in 5 mmol/L
Na2EDTA, 5 mmol/L NaF, 0.05% SDS, 0.05% Na-deoxycholate, 0.1 mmol/L Na2VO4, and 40 mmol/L Tris, pH 7.4, and samples were eluted and electrophoresed (7.5% polyacrylamide, SDS
gels). Western blotting was performed using antibodies to
phosphotyrosine (UBI, Lake Placid, NY) and ECL (Amersham, Arlington
Heights, IL).
Northern blot analyses.
Total RNA was isolated from SKT6 cells using the method of Chomczynski
and Sacchi50 and TRIzol reagent (1 × 107 cells/mL; Life Technologies, Gaithersburg, MD). Total
RNA was electrophoresed in 1.5% agarose gels containing 5.8%
formadehyde, was transferred to Nytran membranes (Schleicher & Schuell), and was UV and heat-fixed. Hybridizations were performed in
Quick-Hyb solution, as described previously.51
32P-labeled probes were prepared by random priming using
the following cDNA fragments: EGF receptor, EcoRI-Bgl
II fragment of pCINeo-EE483; Epo receptor, Bgl II-Xba I
of pUC19-EpoR429; Cis, EcoRI-Not I of pCRV-Cis; Myb,
Bgl II-Not I of pBluescript-h-cMyb; c-Myc, Xho I fragment of pCINeo-c-myc; c-Kit, Xba I fragment of
pCDM8-m-cKit; GAPDH, Kpn I-Xho I fragment of pSP-GAPDH.
The 7S rRNA probe was generated by PCR of pSP-7S using
5-TGTAGTTCCAGCTACTCGGGAGGCT-3 and
5-TCCCGCCTGGTCGTTCACCCCT-3 primers.
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RESULTS |
Chimeric receptor signaling of SKT6 cell differentiation: Elevated
activities of carboxyl-terminal truncated receptor forms.
In studies aimed at defining Epo receptor subdomains and associated
effectors that regulate induced globin expression in SKT6 cells,
chimeric receptor cDNAs were constructed that encode the extracellular
domain of the human EGF receptor and cytoplasmic domains of either the
Epo receptor (EE483), an Epo receptor form truncated to remove 7 of 8 tyrosine sites for effector binding (EE372), or a naturally occurring
truncated form of the rat prolactin Nb2 receptor (EENb2)
(Fig 1A). These chimeric constructs were cloned into pCINeo vectors and were expressed stably in SKT6 cells. In
derived cell lines, expression of EE483, EE372, and EENb2 receptor transcripts first was assayed by Northern blotting using a human EGF
receptor 32P-cDNA probe, and sublines expressing
transcripts at comparable levels were isolated (Fig 1B, upper panel).
In addition, to compare chimera expression levels with levels of
endogenous Epo receptor transcripts, blots were stripped and probed
with a 32P-Epo receptor 3 cDNA fragment (Fig 1B,
lower panel). As assayed by phosphor-imaging, levels of chimeric
receptor transcript expression were shown to be approximately fivefold
lower than endogenous Epo receptor transcript expression levels. In
addition, levels of chimeric receptor expression at the cell surface
were assayed by fluorescence-activated cell sorting (FACS)
using nonactivating phycoerythrin (PE)-labeled antibody
to the human EGF receptor extracellular domain. Via this approach,
receptor densities were shown to be in the range of 200 to 600 receptors per cell (N. Jiang, data not shown). These initial
experiments served to confirm the expression of receptor chimeras at
uniform and physiological levels in selected SKT6-EE483, -EE372, and
-EENb2 sublines. Previously, overexpression of mutated Epo receptor
forms or chimeras has been indicated to activate effectors and response
pathways that otherwise would be uncoupled,52,53 and
expression levels therefore are a nontrivial consideration.
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| Fig 1.
Chimeric receptor constructs and expression in
erythroleukemic SKT6 cells. (A) Diagrammed are wild-type Epo, prolactin
Nb2, and EGF receptors together with derived chimeric constructs
composed of the human EGF receptor extracellular domain, and
cytoplasmic domains of the Epo receptor (full-length EE483,
C-truncation EE372) or the Nb2 receptor (EENb2). (B) SKT6 cells were
transfected with pCIneo vectors encoding EE483, EE372, or EENb2, and
expression of chimeric receptor transcripts in derived cell lines was
assayed by Northern blotting. Hybridization was to either a human EGF receptor probe (32P-EGFR) or to a murine Epo receptor probe
(32P-EpoR). Equivalence in RNA loading was assessed by
hybridization to a GAPDH cDNA.
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In initial experiments, the abilities of the above chimeric receptor
forms to modulate SKT6 cell growth were assessed. Parental SKT6 cells
or derived SKT6-EE483, -EE372, and -EENb2 cell lines were cultured for
10 hours in the presence of 1% serum to limit proliferation and were
exposed to either Epo or EGF at increasing concentrations. At 48 hours
of cytokine exposure, stimulated rates of [3H] thymidine
incorporation were assayed. Exposure of each derived SKT6 cell line to
Epo led to an apparent inhibition of growth (Fig 2A). In SKT6-EE372 and SKT6-EENb2
cells, exposure to EGF likewise resulted in a significant inhibition of
[3H] thymidine incorporation (Fig 2B). In contrast,
EGF-activation of the full-length chimeric receptor form EE483 in
SKT6-EE483 cells affected only a modest inhibition of proliferation.
Thus, Epo-induced differentiation of parental SKT6 cells is associated with a growth-inhibitory effect, and this response was mediated efficiently in SKT6-EE372 and -EENb2 cells expressing
carboxyl-truncated chimeric receptor forms.
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| Fig 2.
Inhibition of SKT6 cell proliferation via C-terminal
truncated chimeric receptor forms. To test possible effects of EGF (or Epo as an internal control) on SKT6-EE483, -EE372, and -EENb2 cell
proliferation, cells were cultured for 48 hours in the presence of Epo
(upper panel, solid symbols) or EGF (lower panel, open symbols) at
increasing concentrations. Rates of [methyl-3H] thymidine
([3H]dT) incorporation then were assayed. Graphed are the
normalized mean rates of [3H]dT incorporation (n = 3).
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In SKT6-EE483, -EE372, and -EENb2 cells, the ability of EGF to induce
differentiation next was assayed. Initially, this was based on
EGF-dependent induction of hemoglobinization
(Fig 3A). Here, three independent sublines
of SKT6-EE483, -EE372, and -EENb2 cells were analyzed, and cells also
were exposed in parallel to Epo as an internal positive control. In
SKT6-EE483 cells, the chimeric receptor EE483 was shown to mediate
EGF-induced hemoglobinization at frequencies (11% on average)
approaching those supported by Epo (17% on average; Fig 3A, upper
panel). By comparison, the truncated chimeric receptor forms EE372 and
EENb2 each proved to be significantly more active in mediating
EGF-induced hemoglobinization, with mean frequencies of 46% and 45%
hemoglobin-positive cells observed, respectively, upon exposure to EGF
(Fig 3A, lower panels). Among SKT6-EENb2 sublines, somewhat lower
frequencies of induced hemoglobinization were observed for one subline.
However, in this subline, Northern blot analyses showed an apparent
inefficiency in chimeric receptor transcript processing (R.C.G., data
not shown). Also, and as shown in previous
studies,21 exposure of parental SKT6 cells to h-EGF did not
affect differentiation (or any other assayed signaling events).
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| Fig 3.
Activities of EE483, EE372, and EENb2 chimeras in
mediating induced SKT6 cell hemoglobinization and globin expression.
(A) In SKT6 cells stably expressing the above chimeric receptor forms (see Fig 1), hemoglobinization as induced by EGF first was analyzed. SKT6-EE483, -EE372, and -EENb2 cell lines (3 sublines for each designated by open, closed, and shaded histograms, respectively) were
exposed to either EGF (15 ng/mL) or Epo (10 U/mL) and, at 72 hours of
cytokine exposure, hemoglobin-positive cells were stained with 2, 7 diaminofluorene (DAF) and scored (>200 cells per sample). Graphed are
mean frequencies of DAF-positive cells ± standard deviations (n = 3). (B) EGF-induced globin expression in SKT6-EE483, -EE372, and -EENb2
cell lines. Cells were exposed to EGF (15 ng/mL) or Epo (10 U/mL) for
the indicated intervals (0, 48, and 72 hours), and levels of globin
expression were assayed by direct Western blotting of cell lysates
using purified antibodies to murine hemoglobin.
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Activities of the above-mentioned chimeric receptor forms in supporting
ligand-induced SKT6-EE483, -EE372, and -EENb2 cell differentiation also
were assayed by Western blotting of globins. Results are shown in Fig
3B and are presented quantitatively in Table 1 (together with the overall results
of the above assays of induced hemoglobinization and proliferation).
For each cell line and assay, data are normalized versus mean
Epo-response values. These data show that, despite their expression at
levels below those of endogenous wt Epo receptors in SKT6 cells, the
truncated chimeric receptor forms EE372 and EENb2 nonetheless promote
these differentiation events at enhanced efficiencies as compared with endogenous Epo receptors. Therefore, the membrane proximal cytoplasmic region of the Epo receptor (Ser248-Met372)
mediates induced hemoglobinization, and effectors whose activation normally depends on C-terminal receptor domains may attenuate this
differentiation response.
Roles for STAT5 in Epo-induced globin expression and SKT6 cell
hemoglobinization.
Based on the elevated activity of the truncated chimeric Epo receptor
form EE372 in mediating ligand-induced SKT6 cell differentiation and on
the retention of a Y343 site for STAT5 recruitment in this
construct,26,54 possible roles for STAT5 in regulating this
response were next investigated. First, in analyses of EGF- versus
Epo-induced STAT5 DNA-binding activity, SKT6-EE372 cells were exposed
to cytokines and, at defined intervals, cells were rapidly chilled to
0°C and lysed. Lysates then were incubated with a biotinylated PRE
element to quantitatively retrieve activated STAT5 complexes and bound
STAT5 was assayed by adsorption to streptavidin agarose, elution from
washed gels, and Western blotting. As shown in
Fig 4, the duration of STAT5 activation in
response to EGF (via EE372) was sustained and, unlike Epo, persisted
beyond 6 minutes to nominally 18 minutes. In independent experiments
(and in independent SKT6-EE372 and SKT6-EENb2 sublines), this was
observed reproducibly and is at least consistent with the notion that
sustained STAT5 activation might contribute to the enhanced activity of
C-terminal truncated receptor forms in promoting SKT6 cell
differentiation.
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| Fig 4.
Sustained activation of STAT5 via the truncated chimeric
receptor form EE372. In SKT6-EE372 cells, the time course of STAT5 activation as induced by EGF versus Epo was assayed as follows. Cells
were exposed to either EGF or Epo at concentrations shown to promote
mitogenesis of myeloid FDCW2-wtER and FDCW2-EE483 cells at 50% maximal
rates (35 ng/mL or 90 nmol/L and 20 U/mL or 120 nmol/L, respectively;
R.C.G., unpublished data). At the indicated intervals of
cytokine exposure, SKT6 cell lysates were prepared by sonication in the
presence of CHAPS. Activated STAT5 then was bound to a biotinylated PRE
cassette, adsorbed to streptavidin agarose, eluted from washed gels,
and assayed by Western blotting. Data shown are representative of two
independent experiments.
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Next, to test the extent to which STAT5 might regulate Epo-induced SKT6
cell hemoglobinization, two approaches were used. First, effects on
Epo-induced SKT6 cell differentiation of expressing dominant-negative
forms of STAT5 were assessed. For these experiments, two distinct
dominant-negative forms of STAT5A were prepared: S5D, in which the
predicted DNA-binding domain was deleted, and S5C, in which the
carboxyl-terminal transactivation domain was removed
(Fig 5A). Towards assessing
specificity, a DNA-binding domain deletion mutant of STAT3 (S3D)
also was prepared. These STAT constructs each were expressed in SKT6
cells, and in stably transfected and derived sublines (SKT6-pCI-S5D,
-pMK-S5D, and -pMK-S5C cells) expression was assessed (Fig 5B).
For STAT5D, expression first was accomplished using pCINeo as a
vector. Based on Western blotting with C-terminal directed antibodies
(-S5#1), expression levels were appreciable, yet were somewhat below
levels of endogenous wt STAT5A. Therefore, STAT5D also was expressed stably in SKT6 cells using pMK as a vector. This increased expression to levels approximating those of endogenous STAT5A (Fig 5B, upper panel). Using pMK as a vector, STAT5C independently was expressed in
SKT6 cells at comparable levels (Fig 5B, lower panel). For STAT5C,
Western blotting was accomplished using antibodies directed against a
more central epitope (-S5#2). Cells stably expressing these deletion
constructs next were tested for their ability to support Epo-induced
hemoglobinization. SKT6-pCI-S5D, -pMK-S5D, -pMK-S5C, and
-pCI-S3D cells were exposed to Epo for 48 or 72 hours, and
frequencies of induced hemoglobinization were assayed. As shown in Fig
5C, ectopic expression of either STAT5D or STAT5C inhibited
Epo-induced hemoglobinization by approximately 10-fold, and in cells
expressing these constructs only low frequencies of hemoglobin-positive
cells were detected (ie, <2 %). In addition, lysates from the
above-mentioned cell lines were prepared and levels of globin
expression at 72 hours of Epo exposure (±Epo) were assessed by
Western blotting (Fig 5D). Here, STAT5D- and STAT5C-dependent
inhibition of Epo-induced differentiation likewise was observed.
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| Fig 5.
Dominant-negative forms of STAT5A inhibit
Epo-induced SKT6 cell hemoglobinization and globin expression. SKT6
cells were transfected stably with expression vectors encoding forms of
STAT5A in which either the predicted DNA binding domains
(pCIneo-S5 D, pMK-S5D) or the C-terminal transactivation domains
were deleted (pMK-S5C) (Wang-Ihle). As a control, a STAT3
DNA binding domain deletion mutant (S3D) also was constructed and
was expressed stably (SKT6-pCIneo-S3D cells). Effects of the
expression of these STAT mutants on Epo-induced SKT6 cell
differentiation were then assessed. (A) Shown diagrammatically are wt
STAT5A, wt STAT3, and the derived deletion constructs S5D, S5C,
and S3D. (B) Levels of expression of STAT5A deletion constructs in
SKT6-pCIneo-S5D, -pMK-S5D, and -pMK-S5C cells as assayed by
Western blotting of total cell lysates. The designated antibody S5#1
recognizes a C-terminal epitope of murine STAT5A
(Asp774-Ser793), whereas S5#2 recognizes an
epitope within a central domain (Phe451-Tyr649)
of STAT5-A and -B. As controls, lysates from parental SKT6 cells as
well as STAT5A immunoprecipitated from SKT6 cells were coanalyzed. (C)
In assays of induced hemoglobinization, SKT6 cells expressing either
STAT5D, STAT5C, or STAT3D (as a control) were exposed to Epo
(2 U/mL) and, at the indicated intervals (48 and 72 hours), hemoglobinized cells were stained with DAF and scored (>200 cells per
sample). Values are the mean frequencies of DAF-positive cells ± standard deviations (n = 3). Frequencies of DAF staining-positive cells scored in the absence of Epo were subtracted as background from
these numbers. (D) In assays of induced globin expression, SKT6-pCIneo-S5D and SKT6-pCIneo-S3D cells (top panel) or
SKT6-pMK-S5C and SKT6-pMK-S5D cells (lower panel) were exposed to
Epo (2 U/mL) and, at 72 hours, cell lysates were prepared. Globin
levels then were assayed by Western blotting. As a positive control,
lysates from parental SKT6 cells (±Epo exposure) were coanalyzed.
|
|
To test more directly roles for STAT5 in Epo receptor-induced SKT6 cell
hemoglobinization, the STAT5 binding site Y343 within the
chimeric receptor form EE372 was mutated to phenylalanine, and effects
of this mutation on receptor activity in mediating SKT6 cell
differentiation were assayed. In addition, based on its previous use in
studies of mitogenic signaling in DA-3 cells,54 a related
receptor form EE375-Y343F was prepared (as an EGF receptor chimera) and
also was expressed in SKT6 cells. Northern blot analyses of derived
SKT6-EE372-Y343F and -EE375-Y343F cells first were performed, and
sublines were selected that expressed chimera transcripts at levels
approximating those of the endogenous Epo receptor
(Fig 6A). Next, the ability of these
receptor forms to mediate the ligand-induced transcription of an
established STAT5 target gene, cis,55 was assessed. As
shown in Fig 6B, cis gene transcription was induced by Epo in each of
these SKT6 cell lines. However, among the above chimeric constructs,
only EE372 (but neither EE372-Y343F nor EE375-Y343F) mediated this
STAT5-dependent response. EGF-induction of SKT6 cell
hemoglobinization likewise was supported only in SKT6-EE372 cells, and
not in SKT6-EE372-Y343F or -EE375-Y343F cells (R.C.G., data not shown).
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| Fig 6.
Mutation of the STAT5 binding site Y343 in
chimeric EE372 and EE375 receptor forms inhibits EGF-induced SKT6 cell
hemoglobinization. (A) To further test roles for STAT5 during
ligand-induced SKT6 cell differentiation, the Y343 STAT5
binding site within two distinct truncated chimeric receptor forms,
EE372-Y343F and EE375-Y343F, was mutated to phenylalanine. These
point-mutated chimeras are illustrated, and levels of transcript expression for these chimeras and endogenous Epo receptors in SKT6-EE372, EE372-Y343F, and -EE375-Y343F cell lines are presented. For
EE375-Y343F, increased transcript size (*) is due to expression from
the dicistronic vector. (B) To functionally confirm loss of STAT5
signaling via the receptor forms EE372-Y343F and EE375-Y343F, derived
SKT6 cell lines were exposed to either EGF (±35 ng/mL) or Epo (±20
U/mL) for 180 minutes, and induced transcription of the STAT5-regulated
gene, cis, was assayed by Northern blotting. Equivalence in loading was
confirmed by hybridization to a 32P-GAPDH cDNA.
|
|
SCF-inhibition of Epo-induced SKT6 cell differentiation.
SCF is known to act synergistically with Epo in promoting red blood
cell production,36 and c-Kit function recently has been suggested to depend on Epo receptor expression and possibly on receptor
trans-activation events.40 Also, because SCF and Epo activate at least an overlapping set of signaling events, possible effects of SCF on SKT6 cell differentiation were investigated. Initial
experiments served to establish that, like CFU-e, SKT6 cells express
functional SCF receptors. In these experiments, SKT6-EE372 cells were
cultured in 0.2% FBS for 10 hours and were then exposed to either Epo,
EGF, or SCF. Levels of c-myb, c-myc, and cis transcripts then were
assayed by Northern blotting (Fig 7A). Each
cytokine detectably induced the expression of c-myb transcripts with
the highest levels of induction by c-Kit. c-myc transcription also was
induced by SCF, Epo, and EGF. In contrast, cis gene transcription was
activated strongly by EGF and Epo, but not by SCF. In addition, levels
of c-Kit transcript expression in Epo-exposed SKT6 cells also were
assayed by Northern blotting to assess any possible effects on c-Kit
expression (Fig 7B). Predicted increases in levels of globin
transcripts were observed, but levels of c-Kit (or Epo receptor)
transcripts were not modulated significantly during this period of
induction.
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| Fig 7.
SCF activation of c-kit response pathways in SKT6-EE372
cells. (A) SCF/c-kit induced transcription of c-myc and c-myb genes in
SKT6-EE372 cells. SKT6-EE372 cells were cultured for 10 hours in 0.2%
FBS and were exposed to Epo (20 U/mL), EGF (25 ng/mL), or SCF (100 ng/mL) for 0, 90, or 180 minutes. At the indicated intervals, cells
were lysed, total RNA was isolated, and levels of c-myc, c-myb, and cis
transcript expression were analyzed by Northern blotting. Equivalence
in loading was assessed by hybridization to a 7S rRNA cDNA probe. (B)
c-kit transcript expression levels are not downmodulated during
Epo-induced SKT6 cell hemoglobinization. SKT6 cells were exposed to Epo
at 10 U/mL and, at the indicated intervals (0, 48, and 72 hours),
levels of c-kit, Epo receptor, and maj-globin
transcripts were assayed by Northern blotting. Equivalence in loading
was assessed by hybridization to a 32P-GAPDH cDNA.
|
|
Given the above-indicated occurrence of functional c-Kit receptors in
SKT6-EE372 and SKT6 cells, possible effects of SCF on induced
hemoglobinization next were tested. SKT6-EE372 cells first were
cultured for 72 hours in the presence of either SCF, EGF, or Epo and
hemoglobinization was assayed by staining with DAF and by Western
blotting of globins (Fig 8A and B). Whereas
Epo and EGF each efficiently induced these differentiation responses, no such effects were induced by SCF. Based on these results, whether SCF might act to modulate Epo-induced SKT6 cell differentiation next
was tested. SKT6 cells were exposed to SCF at 10, 30, or 100 ng/mL for
8 hours and subsequently to Epo for 72 hours. Interestingly, DAF
(Fig 8C) and Western blot analyses (R.C.G., data not
shown) showed that SCF exerted a dose-dependent
inhibition of Epo-induced globin expression and SKT6 cell
hemoglobinization.
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| Fig 8.
SCF-dependent inhibition of Epo-induced SKT6 cell
hemoglobinization. (A and B) SKT6-EE372 cell hemoglobinization is
induced by Epo and EGF, but not by SCF. SKT6-EE372 cells were exposed to Epo (20 U/mL), EGF (25 ng/mL), or SCF (100 ng/mL) and, at 72 hours
of culture, hemoglobin-positive cells were stained with DAF and scored
(>200 cells per sample). Graphed (A) are mean frequencies of
hemoglobin-positive cells ± standard deviation (n = 3). Also analyzed by Western blotting were levels of globin expression in Epo-,
EGF-, and SCF-exposed SKT6 EE372 cells (B). (C) Concentration-dependent SCF-inhibition of Epo-induced SKT6 cell hemoglobinization. SKT6 cells
were exposed for 8 hours to SCF at the concentrations indicated and
subsequently were stimulated with Epo (2 U/mL) for 72 hours. As
controls, SKT6 cells also were exposed independently to either SCF (100 ng/mL) or Epo (2 U/mL). Hemoglobin-positive cells were stained with
DAF, and positive cells were scored (> 200 cells per sample). Mean
frequencies of DAF-positive cells ± standard deviations (n = 3) are
illustrated.
|
|
Finally, experiments were performed to initially investigate possible
mechanisms of SCF-inhibition of SKT6 cell differentiation. Specifically, based on the apparent roles for STAT5 during Epo-induced globin expression and hemoglobinization, whether SCF might modulate STAT5 tyrosine phosphorylation or DNA-binding activity was tested. SKT6
cells were exposed first to SCF for 0, 7.5, and 15 minutes and
subsequently to Epo (±) for 7.5 minutes. Cells then were rapidly chilled to 0°C and lysed. Phosphotyrosine Western blotting of immunoprecipitated STAT5 from these lysates first showed that SCF did
not detectably induce STAT5 tyrosine phosphorylation and did not
detectably modulate this response as induced by Epo
(Fig 9A). Furthermore, in STAT5 DNA-binding
assays, no effects of SCF on Epo-induced PRE cassette binding were
observed (Fig 9B). Therefore, SCF does not stimulate but rather
inhibits SKT6 cell differentiation, and mechanisms underlying this
inhibition apparently do not involve direct effects of SCF and c-Kit on
Jak2-STAT5 activation.
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| Fig 9.
Epo-induced activation of STAT5 in SKT6 cells is not
modulated by SCF. To test whether SCF-inhibition of Epo-induced SKT6 cell differentiation might involve effects on STAT5 activation, SKT6
cells were pre-exposed to SCF at 100 ng/mL for increasing intervals and
subsequently were exposed to Epo at 20 U/mL for 7.5 minutes. Lysates
were then prepared by sonication in the presence of CHAPS. (A) Levels
of cytokine-induced tyrosine phosphorylation of STAT5 were analyzed by
immunoprecipitation and ECL-Western blotting using antibodies to
phosphotyrosine. (B) Activated STAT5 was assayed by binding to a
biotinylated PRE-element, adsorption to streptavidin-agarose, elution
from washed gels, and Western blotting using antibodies to STAT5A.
|
|
| |
DISCUSSION |
One central issue in hematopoietic growth factor (HGF) signaling
concerns the extent to which HGFs may affect lineage-specific blood
cell differentiation, and an important yet unresolved example is
provided by Epo. As introduced above, the ability of at least certain
heterologous receptors of the type 1 superfamily to substitute for the
Epo receptor in supporting terminal differentiation
events9,11,21,22 suggests that Epo receptor-derived signals
may be generic ones that serve only to support survival during a
preprogrammed course of terminal differentiation. However, in several
in vitro systems, Epo readily activates at least select late erythroid
differentiation events,16-19 and in transgenic mice the
enforced expression of Bcl-2 in erythroid progenitor cells recently has
been shown to support BFU-e and CFU-e survival but not red blood cell
formation.56 Thus, these latter observations suggest that
at least some degree of specificity for differentiation signaling is
provided by Epo. To investigate mechanisms associated with such
Epo-induced differentiation responses, responsive SKT6 cells presently
have been used together with a chimeric receptor approach to define Epo
receptor domains and effectors that regulate induced globin expression
and hemoglobinization. In these investigations in this model system,
three findings merit discussion: the enhanced activity of highly
truncated receptor forms, apparent roles for STAT5 in this
differentiation response pathway, and an observed repression of
Epo-induced SKT6 cell differentiation that occurs upon SCF activation
of c-Kit.
With regards first to the observed abilities of truncated chimeric
receptor forms to mediate SKT6 cell differentiation at enhanced levels,
negative roles for Epo receptor C-terminal domains previously have been
defined first in the context of proliferation. A murine Epo receptor
construct lacking 40 C-terminal amino acids (including tyrosine
residues 443, 460, 464, and 479) originally was shown in BaF/3 cells to
support mitogenesis at increased efficiencies.44 Mitogenesis since has been shown to also be attenuated by more proximal
Y429 and Y431 receptor sites for the
recruitment of hematopoietic cell phosphatase (HCP).57 In
the truncated chimeric receptor form EE372, each of the above-noted
negative regulatory domains is lacking and one possible explanation for
the enhanced activity of this chimera in SKT6 cells is that effectors
that negatively regulate mitogenesis may act similarly to dampen
differentiation signaling (Fig
10). For HCP, this appears to be the case, because its
forced expression in SKT6 cells recently has been shown to inhibit
Epo-induced hemoglobinization, possibly by attenuating Jak2-STAT5
signaling.58 However, evidence exists to suggest that
certain other effectors may act to differentially regulate growth
versus differentiation events. For example, the expression of an Epo
receptor form retaining only Y464 has been shown to
efficiently support BaF/3 cell proliferation, but not CFU-e
differentiation in Epo receptor/ fetal
hepatocytes.59 Therefore, the enhanced activity of EE372 and EENb2 chimeras in mediating hemoglobinization in SKT6 cells may
depend additionally on an uncoupling of effectors that normally favor a
mitogenic response. Consistent with these findings in SKT6 cells, in
ELM-I-1 cells (a murine erythroleukemic cell line that likewise
hemoglobinizes in response to Epo),18 a chimeric receptor
form truncated at K348 recently has been reported to
mediate ligand-induced hemoglobinization at frequencies somewhat above
those supported by a full-length control chimera. However, in studies
of the activity of retrovirally expressed Epo receptor mutants in fetal
hepatocytes, conflicting results have been observed. Specifically, Wu
et al59 observed that an Epo receptor form mutated at all
cytoplasmic tyrosine sites except Y343 was attenuated in
its ability to support CFU-e maturation, whereas Socolovsky et
al11 observed enhanced activity for an Epo receptor form
truncated at E374. Within this system, these discrepancies
may derive from varied retroviral overexpression of heterologous
receptor forms or possibly from partial misfolding of the former Epo
receptor mutant.
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| Fig 10.
Candidate effectors of Epo- and SCF-regulated SKT6 cell
hemoglobinization. In this model, STAT5 may act to promote globin gene
expression via one (or more) primary target genes. By comparison, inhibitory effects of SCF may dependent on the downmodulation of Jak2
by HCP.
|
|
With regards to possible roles for STAT5 during Epo-induced erythroid
differentiation, mutation of a defined Y343 site for STAT5
binding (Y343F) in the C-terminal truncated chimera EE372 (and the
related chimera EE375) resulted in a loss in signaling of SKT6 cell
hemoglobinization. In addition, induced differentiation via endogenous
Epo receptors likewise was inhibited efficiently upon the expression of
either of two dominant negative forms of STAT5A, S5D, or S5C.
While these investigations were ongoing, Wakao et al22
reported an observed attenuated differentiation signaling in SKT6 cells
for a receptor form point-mutated at Y343 and an inhibition
of Epo-induced globin expression upon the expression of a point mutated
form of ovine STAT5A (Y694F). Also, the expression of a C-terminally
truncated form of STAT5A recently has been shown to diminish
frequencies of hemoglobinization in ELM-I-1 cells.18 Thus,
the present investigation and these related latter studies each
demonstrate at least a partial failure in Epo-induced globin expression
and hemoglobinization upon the disruption of STAT5 activation. Also, in
the case of exogenously expressed STAT5A mutants, this is presumed in
each study to include dominant-negative inhibition of any contributions
by STAT5B based on the ability of A and B isoforms to
heterodimerize.25 Therefore, these observations consistently suggest that STAT5 normally may mediate Epo effects on
globin gene expression and possibly to additional events involved in
the terminal differentiation of red blood cells. Beyond this, it is
tempting to at least speculate that these responses might involve the
STAT5-dependent activation of select targeted genes within late
erythroid progenitor cells. However, these possibilities are tempered
by recent investigations of STAT5 function in three alternate systems.
First, in human erythroleukemic TF-1 cells, a mutated form of the Epo
receptor has been shown to be expressed and to limit levels of STAT5
activation.19 Expression of the wt murine Epo receptor in
these cells increased levels of STAT5 activation, provided for
prolonged proliferation in the presence of Epo, and detectably reduced
frequencies of benzidine-positive cells. Based on these observations,
STAT5 was suggested to promote mitogenesis and to attenuate
differentiation signaling by Epo in this model. Second, in studies in
Epo receptor/ fetal hepatocytes Epo receptor
forms retaining any 1 of 8 cytoplasmic tyrosine residues each has been
demonstrated upon retroviral overexpression to support CFU-e
maturation.59 Although differences in the apparent activities of these Epo receptor mutants were limited, relatively high
activities were exerted by receptor forms retaining either Y479 (a site for PI3-kinase binding) or Y343
(STAT5 binding site). Therefore, findings in this system are at least
consistent with the notion that STAT5 may contribute in an important
fashion to Epo-induced differentiation. Finally, effects of disrupting
the expression of either STAT5A or STAT5B genes in chimeric mice
recently have been reported. In STAT5A/ mice,
mammary gland development is inhibited, and this is consistent with a
previously defined role for STAT5 in mediating prolactin-induced whey
acidic protein gene expression as a direct differentiation response.60 Also, in STAT5A/
mice, macrophage proliferative responsiveness to GM-CSF is attenuated detectably.61 By comparison,
STAT5B/ mice display growth hormone-related
defects in liver gene expression.62 However, no overt
defects in hematopoiesis or erythropoiesis have been reported to date
in STAT5-deficient mice. In the context of erythropoiesis, this can be
interpreted to mean that either no important roles are exerted by STAT5
or that these roles are compensated for either by alternate Epo
receptor-activated effectors and possibly other STATs. Interestingly,
in STAT5B/ mice, an increased
expression of STAT1 and possibly STAT3 is apparent in at least certain
tissues.62 Although this has not yet been analyzed in
erythroid progenitor cells, it is noteworthy that Epo-activation of
STAT1 and 3 has been reported in normal erythroid rat fetal
hepatocytes27 and in SKT6 cells.21
Finally, with regards to c-Kit signaling, the present studies of
effects of SCF on SKT6 cell differentiation were promoted by the
consideration that c-Kit function recently has been suggested to
involve trans-signaling through Epo receptor complexes40 and by the predication that if signals for differentiation in fact are
generic ones, then coactivation of c-Kit and Epo receptor complexes
might augment differentiation. However, contrary to this prediction,
SCF was shown to effectively inhibit Epo-induced SKT6 cell
hemoglobinization. This finding first raises the question as to nature
of SCF effects on erythroid progenitor cell development. Although SCF
has been demonstrated to act as a mitogen and survival factor for
erythroid progenitor cells,33,36 SCF interestingly also has
been shown to retard Epo-dependent hemoglobinization in normal
erythroid colony-forming cells.63 These observations indicate that, although SCF supports the expansion of late erythroid progenitor cells, c-Kit signaling also may attenuate terminal differentiation events. Second, given the demonstrated ability of SCF
to act as both a mitogen and viability factor,33,36,51 this
observed SCF-dependent inhibition of late erythroid differentiation also indirectly supports the notion that Epo may provide signals beyond
those required simply for survival. Indicated specific effects of Epo
on late differentiation might depend on the activation of discrete
effectors (such as STAT5) or possibly on factors that differentially
regulate cell growth and/or cycle status. Further investigations using SKT6 cells expressing minimal chimeric receptor forms should serve to advance mechanistic resolutions of this problem
in red blood cell development.
| |
FOOTNOTES |
Submitted December 11, 1997;
accepted May 27, 1998.
First authorship is merited for N.J. and R.C.G.
Supported by National Institutes of Health Grants No.
HL44491 and RCDA HL 03042 to D.M.W. and Sigma Xi Grants-in-Aid of
Research to R.C.G. and N.J.
Address reprint requests to Don M. Wojchowski, PhD, 115 William L. Henning Bldg, The Pennsylvania State University, University Park, PA
16802; e-mail: dmw1{at}email.psu.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Amgen, Inc (Thousand Oaks, CA) for the generous
provision of recombinant human Epo and Drs Akihiko Yoshimura, Kenneth
Lord, and Peter Besmer for the generous provision of cis, c-myb, and
c-Kit cDNAs, respectively.
| |
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Abstract
Introduction
Abstract