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Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 879-889
Signaling Through the Interaction of Membrane-Restricted Stem
Cell Factor and c-kit Receptor Tyrosine Kinase: Genetic
Evidence for a Differential Role in Erythropoiesis
By
Reuben Kapur,
Manus Majumdar,
Xiangli Xiao,
Monica McAndrews-Hill,
Karen Schindler, and
David A. Williams
From the Howard Hughes Medical Institute, Indiana University School
of Medicine; and the Section of Pediatric Hematology/Oncology, Herman B
Wells Center for Pediatric Research, James Whitcomb Riley Hospital for
Children, Indiana University School of Medicine, Indianapolis, IN.
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ABSTRACT |
Mutations of the receptor tyrosine kinase c-kit or its
ligand stem cell factor (SCF), which is encoded as a soluble and
membrane-associated protein by the Steel gene in mice, lead to
deficiencies of germ cells, melanocytes, and hematopoiesis, including
the erythroid lineage. In the present study, we have used genetic
methods to study the role of membrane or soluble presentation of SCF in
hematopoiesis. Bone marrow-derived stromal cells expressing only a
membrane-restricted (MR) isoform of SCF induced an elevated and
sustained tyrosine phosphorylation of both c-kit and
erythropoietin receptor (EPO-R) and significantly greater proliferation
of an erythrocytic progenitor cell line compared with stromal cells
expressing soluble SCF. Transgene expression of MR-SCF in
Steel-dickie (Sld) mutants
resulted in a significant improvement in the production of red blood
cells, bone marrow hypoplasia, and runting. In contrast, overexpression
of the full-length soluble form of SCF transgene had no effect on
either red blood cell production or runting but corrected the myeloid
progenitor cell deficiency seen in these mutants. These data provide
the first evidence of differential functions of SCF isoforms in vivo
and suggest an abnormal signaling mechanism as the cause of the severe
anemia seen in mutants of the Sl gene.
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INTRODUCTION |
THE PHOSPHORYLATION of proteins on
specific tyrosine residues is critical for regulating cell
proliferation and differentiation in eukaryotes.1,2 The
activation of protein tyrosine kinases (PTKs), resulting in the
tyrosine phosphorylation of downstream signaling proteins, is an
important event in many signal transduction cascades critical for
growth and development. The level of phosphorylation at specific
tyrosine residues and the amplification of downstream signals initiated
by PTKs to a large extent determine the above cellular outcomes.
The c-kit receptor tyrosine kinase (RTK) encoded by the murine
Dominant white spotting (W) locus and its ligand
stem cell factor (SCF), encoded by the Steel (Sl)
locus, define a signaling pathway essential for murine
hematopoiesis.3,4 The c-kit RTK is a member of the
platelet-derived growth factor (PDGF) receptor family.5,6
The interaction of RTKs that belong to the PDGF receptor family and
their cognate ligands induce receptor dimerization or oligomerization
followed by activation of intrinsic tyrosine kinase and receptor
transphosphorylation.7 Many lines of evidence suggest that
bivalent binding of ligands is key for inducing receptor dimerization
and subsequently receptor activation.8 Although SCF is
dimeric in solution and has been shown to induce the dimerization of
c-kit,9,10 low levels (3 ng/mL) exist in
serum,11 and based on the dimerization Ka, it has been
predicted that greater than 90% of the circulating SCF would exist in
monomeric form.11 Recent studies using radiolabeled SCF
added to serum have shown that 49% to 72% of the circulating SCF
exists in monomeric form.11 Further, a detailed examination
of dimerization defective variants of SCF showed substantially reduced
mitogenic activity, whereas the activity of disulfide-linked SCF dimer
was 10-fold higher than that of wild-type (wt) SCF. These results
suggest a correlation between dimerization affinity and biological
activity. Based on these findings, one would predict that SCF and other
cytokines or growth factors that are expressed as membrane-associated
(MA) proteins may be more efficient at forming dimers because of an enhanced probability of monomers to encounter each other in the plasma
membrane lipid bilayer as compared serum. This may subsequently result
in a significantly greater mitogenic activity of MA protein in
comparison with its soluble counterpart.
Many cytokines and/or growth factors exist as both MA and
soluble isoforms. These proteins may participate in a novel mode of
intercellular communication restricted to adjacent cells, termed juxtacrine stimulation.12 Examples of proteins that exist
as both soluble and MA forms include transforming growth factor- (TGF-),12 tumor necrosis factor (TNF),13 and
colony stimulating factor-1 (CSF-1).14,15 Two major
isoforms of SCF also exist in both mice and humans as a result of mRNA
splicing. A glycoprotein of 248 (SCF248) amino acids (aa)
is rapidly cleaved to release a biologically active soluble (S) protein
of 164 aa. In contrast, a glycoprotein of 220 aa
(SCF220), which lacks the proteolytic cleavage site
encoded by differentially spliced exon 6 sequences, remains
predominantly MA.16-18 This isoform can also be slowly
released from the cell surface through the use of an alternative
proteolytic cleavage site in exon 7. Our laboratory has shown that
site-directed mutagenesis of the SCF cDNA to ablate both proteolytic
cleavage sites results in the generation of a membrane-restricted (MR)
and biologically active form of SCF
(SCFX9/D3).19 In spite of significant
molecular and biochemical characterization of SCF, little is known
regarding the physiological role(s) of these isoforms.
Steel-dickie (Sld), a viable Sl
mutant, is the result of an intragenic 4-kb genomic deletion that
removes the exons encoding the transmembrane and cytoplasmic domains of
Sld-protein and impairs the ability of the protein
to anchor in the plasma membrane.20 Thus,
Sld mice appear to be capable of producing
biologically active soluble SCF, although this truncated protein lacks
both exon 7- and exon 8-encoded membrane-proximal aa and therefore
differs from the wt isoform of soluble SCF.21 The severe
hematologic deficiencies in compound heterozygous
Sl/Sld and homozygous
Sld/Sld mice have suggested that the MA
isoform of SCF is critical for normal mouse development. Indeed, the
severe nature of the erythroid deficiency in Sl/Sld
mice in spite of the presence of truncated soluble protein has continued to be an enigmatic feature of the Sl/Sld
phenotype years after the cloning of the Sl gene.
The most overt hematopoietic phenotypes of W and Sl
mutant mice are severe macrocytic anemia and mast cell deficiency. In addition, W and Sl mutants have defects at the level of
hematopoietic stem and progenitor cells.3,4,22 In vitro
culture assays have shown that soluble SCF synergizes with a number of
lineage-restricted hematopoietic growth factors, including
erythropoietin (EPO), granulocyte macrophage-colony stimulating factor
(GM-CSF), and interleukin-7 (IL-7).23,24 In addition, our
laboratory has previously shown differences in the
proliferation/survival of human hematopoietic progenitor cells when
exposed in vitro to the MA verses soluble isoforms of human
SCF.25 The wide range of cellular responses observed
following c-kit receptor activation has made it difficult to
dissect the molecular events that provide biological specificity to the
c-kit/SCF signaling pathway. Recently, the phenotype of mice
deficient in EPO suggest that the survival and proliferation of late
erythroid progenitors depends on EPO.26 Sl/Sld mutants also show a profound deficiency in
the erythroid lineage, despite of the presence of high levels of
circulating EPO.4 These data suggest that the normal
proliferation and/or differentiation of erythroid progenitors
to produce mature red cells requires both a functional SCF and EPO
signaling pathway. However, to date the isoform of SCF responsible
physiologically for the proliferation and/or differentiation of
erythroid progenitors to mature red blood cells remains unknown.
In the studies presented here, we have elected to use the MR form of
SCF to more clearly elucidate the role of membrane presentation in
vitro and in vivo because the wt form of MA SCF (SCF220),
at least in vitro, is slowly secreted from the cell
surface.18,19 We show that stromal cells expressing only
the MR form of SCF induce a more sustained and elevated tyrosine
phosphorylation of the EPO receptor (EPO-R) in erythroid cells and
stimulate a significantly greater proliferation of an erythrocytic cell
line as compared with soluble SCF. In vivo, using
Sl/Sld mutant and transgenic mice that overexpress
either the soluble or the MR form of SCF, we show a significant
increase in the production of red cells, correction of runting, and
bone marrow hypocellularity in Sl/Sld mice that
express MR SCF as compared with mice that overexpress soluble SCF only.
In contrast, overexpression of soluble SCF in Sl/Sld mice resulted in complete correction of
myeloid progenitor cell deficiency. These in vitro and in vivo
observations suggest distinct biological role(s) for SCF isoforms.
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MATERIALS AND METHODS |
Cell lines.
The murine growth factor-dependent cell line, HCD57, was a gift from
Dr H. Lodish (MIT, Boston, MA). This is an EPO-dependent cell line that
responds to murine soluble SCF. The biological characteristics of this
cell line and culture conditions have been previously
described.27,28 HCD57 cells were maintained in Iscove's
modified Dulbecco's medium (IMDM) supplemented with 20% fetal calf
serum (FCS) and 1 µ/mL of recombinant human (rh) EPO (Amgen, Thousand
Oaks, CA). For growth and survival assays, HCD57 cells were
factor-starved by washing three times with IMDM and culturing for 18 hours without EPO. Subsequently, starved cells were plated at 1 × 104 cells per mL in medium without growth factor (control),
supplemented with rhEPO (1 µ/mL), or cocultured on mitomycin
C-treated Sl/Sl4 stromal cells expressing
either isoform of SCF in the presence or absence of 1 µ/mL of rhEPO.
The culture plates were gently centrifuged for 1 minute at 100g
to ensure direct cell-cell contact and further cocultured for 48 hours
at 37°C. Viable cells were counted after 48 hours.
Sl/Sl4-SCF transfectants were prepared by treating with
mitomycin C (5 µg/mL; Sigma, St Louis, MO) for 2 hours
at 37°C, washed three times with phosphate-buffered saline (PBS),
treated with trypsin, and plated to confluency
(1 × 106/well) on 6-well gelatin-coated tissue culture
plates (Falcon, Lincoln Park, NJ), and cultured overnight.
Flow cytometric analysis.
To determine the cell surface expression of SCF on Sl/Sl4
stromal cells transfected with cDNAs encoding either the soluble or the
MR form of SCF, flow cytometric analysis with a rat anti-murine SCF
monoclonal antibody (MoAb) was performed. Briefly, 1 × 106 stromal cells were stained separately with either 1 µg of primary rat anti-mouse SCF MoAb (Genzyme, Cambridge, MA) or a
rat IgG2a isotype (PharMingen, San Diego, CA) control
antibody for 30 minutes at 4°C. Afterward, the cells were washed
twice with PBS/0.1% bovine serum albumin (BSA) and subsequently
stained with 1 µg of secondary fluorescein-isothiocyanate
(FITC)-conjugated goat (F(ab )2 anti-rat IgG (GIBCO-BRL,
Gaithersburg, MD) under identical conditions and analyzed by
fluorescence-activated cell sorter (FACS).
Immunoprecipitation and Western blot analysis.
Immunoprecipitation (IP) and Western blot (WB) analyses were performed
as previously described.29 Briefly, Sl/Sl4
cells expressing either isoform of SCF were treated as described above. These cells were washed and plated on 6-well gelatin-coated plates (1 × 106/well) and cultured for 36 to 48 hours.
Factor-starved HCD57 cells (10 × 106/well) were loaded
as described above and further cocultured for various periods at
37°C. Thereafter, the supernatants were removed, and cells were lysed
in lysis buffer (10 mmol/L K2HPO4, 1 mmol/L EDTA, 5 mmol/L EGTA, 10 mmol/L MgCl2, 1 mmol/L
Na2VO4, 50 mmol/L beta-glycerol-phosphate, 10 µg/mL leupeptin, 1 µg/mL pepstatin, and 10 µg/mL aprotinin) at
4°C for 30 minutes. Cell lysates were clarified by centrifuging for
30 minutes at 10,000g at 4°C. IPs were performed by
incubating equivalent amounts of cell lysates with either an
anti-EPO-R antibody (5 µL per 10 × 106 cells; kindly
provided by Dr G. Krystal) or with an anti-c-kit antibody (2 µLs per 10 × 106 cells; PharMingen) for 3 hours at
4°C. Protein A- or protein G-Sepharose beads (Pierce, Rockford, IL)
were used to collect the antigen-antibody complexes. The IPs were
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), and proteins were electrophoretically transferred onto
Immobilon-P membranes (Millipore, Bedford, MA). After blocking residual
binding sites on the transfer membrane by incubating the membrane with 2% BSA/TBST (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 0.05% Tween-20), for 12 hours at 37°C, Western blot analysis using an anti-phosphotyrosine antibody (1:2,000 dilution;
Transduction Laboratories, Lexington, KY) and the enhanced
chemiluminescence detection system (Amersham, Arlington Heights, IL)
were performed according to manufacturer's instructions.
Generation of transgenic mice.
Experiments involving mice described here were reviewed and approved by
Animal Use Committee of Indiana University School of Medicine. C3H/HeJ
mice were obtained from Jackson Laboratories (Bar Harbor, ME).
Transgenic mice were generated by microinjecting into the pronuclei of
fertilized C3H/HeJ eggs a 1.3-kb Ndel/Kpnl fragment comprising either
the hPGK-mSCF248 or the hPGK-mSCFX9/D3 minigene
(Fig 1). Microinjected eggs
were transferred to the oviducts of pseudo-pregnant outbred
Swiss-Webster females. Offspring were tested for the presence of
transgene by analyzing tail DNA. Briefly, tail DNA from mice was
digested overnight in digestion buffer (100 mmol/L NaCl, 10 mmol/L Tris
base pH 8, 25 mmol/L EDTA pH 8, 1% SDS, and 150 µg/mL proteinase K)
at 50°C and extracted the next day with phenol and chloroform. The
high molecular weight DNA was subsequently digested with EcoRl,
electrophoresed, transferred to filters, and probed using a full-length
32P-labeled cDNA murine SCF probe. To examine the in vivo
role of the two isoforms of SCF on various lineages in naturally
occurring Sl mutants (ie, Sl/Sld), we
first crossed transgenic mice overexpressing either the soluble or the
MR form of SCF to WC/ReJ-Sl/+ mice to obtain
Sl/+ mice that overexpress either isoform of SCF.
These mice were identified based on their phenotype (ie, forehead blaze
and diluted belly) and Southern blot analysis. Transgene positive
Sl/+ male mice were further crossed to
C57Bl6-Sld/+ (Jackson Laboratory) mice
to obtain Sl/Sld transgene positive or negative
mice.
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| Fig 1.
SCF transgene constructs. Transgene expression of the
mSCF248 and mSCFX9/D3 cDNAs in founder mice was
achieved using a minigene cassette consisting of mSCF248 or
mSCFX9/D3 cDNA expressed from the human PGK promoter. The
PGK promoter, consisting of 514 bp 5-flanking sequence from the
X-linked human phosphoglycerate kinase-1 gene,54 up to but
excluding the translational start codon, was subcloned into pGEM-7 as
an Aatll/Sph l fragment. The mSCF248 or
mSCFX9/D3 cDNA in addition to a splice donor/acceptor and
poly A site was subcloned from the expression plasmid
V19.8mSCF248 or V19.8mSCFX9/D3 36 as a
Xho l/Kpn l fragment 3 to the PGK promoter in the
pGEM-7 plasmid vector to generate the final transgene plasmid. hPGKpr: human phosphoglycerate kinase promoter: SD/SA: splice donor/splice acceptor sequences; mSCF248 cDNA: murine SCF cDNA encoding
full-length soluble SCF protein: mSCFX9/D3 cDNA: murine SCF
cDNA encoding full-length MR19 SCF protein; poly A:
polyadenylation sequence. (X) denotes the location of an inserted
Xho l at the proteolytic cleavage site in exon 6, and ( )
denotes a 12-bp deletion of the secondary proteolytic cleavage site in
exon 7.
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Expression of the transgene.
Total cellular RNA was purified from tissues using Tri Reagent
(Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions and used as template. Equal amount of RNA
(using actin as an internal control) was used to synthesize single-stranded cDNA by reverse transcriptase (RT) and random hexamers
(Perkin Elmer, Branchburg, NJ). cDNA was amplified in a
100 µL reaction mixture by polymerase chain reaction (PCR) using AmpliTaq DNA polymerase in 35 cycles of 1 minute denaturation at 94°C, 2 minutes of annealing at 55°C, and 3 minutes of synthesis at 72°C using a 5-GGAGATCTGCGGGAATCC-3 sense primer and
5-GGCTGCAGTCCACAATTACACCTCTTG-3 antisense primer based on published
sequence.21 These primers fail to amplify a PCR product
from Sl/Sld-encoded mRNA.21 The
predicted amplification product from both mSCF248 and
mSCFX9/D3 transgene expression is 733 bp. PCR (RT-PCR)
products were examined on 1% agarose gel (Fig
2). As a control for loading and integrity of total RNA, primers specific for actin
(5-TGGTGGGAATGGGTCAGAAGGACTC-3 sense primer and
5-TTGGCATAGAGGTCTTTACGGATGT-3 antisense primer) were used to
amplify cDNA using the described conditions.30 The
predicted amplification product of these primers is 732 bp. To further
confirm the identity of SCF and actin mRNAs, RT-PCR gels were
transfered to nylon membranes (MSI, Westboro, MA), and reverse
transcribed mRNAs were detected by hybridization using a
32P random-primed full-length mSCF and  -actin cDNA.
Hybridizations were performed using ExpressHyb hybridization solution
(Clontech Laboratories, Palo Alto, CA) according to manufacturer's
instructions.
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| Fig 2.
Analysis of transgene expression in bone marrow-derived
stromal cells from Sl/Sld,
Sl/Sld-S, and
Sl/Sld-MR mice. Total cellular RNA was
extracted as described in Materials and Methods. SCF-specific primers
(upper panel) were used that recognize only the full-length transcript
of SCF. As a loading control actin specific primers (lower panel) were
used on the same RNA sample as described in Materials and Methods.
RT-PCR products were examined on 1% ethidium bromide containing
agarose gel and subsequently probed with SCF and actin specific probes. Upper arrow, SCF-specific primers (733 bp); lower panel, actin-specific primers (732 bp). Molecular weight (MW) marker is shown on the left.
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Peripheral blood analysis.
Total peripheral RBC and WBC counts were analyzed on tail vein bleeds
with a hemocytometer and Coulter Model ZM electronic particle counter
(Coulter Electronics, Hialeah, FL). For WBC counts, RBCs were lysed
using Zapoglobin (Coulter Electronics) according to manufacturer's
recommendations. Peripheral blood hematocrits were performed by
spinning capillary tubes for 5 minutes in a model MB microcapillary
centrifuge (IEC, Boston, MA).
Hematopoietic progenitor cell assays.
BM was obtained as described previously,31 and cellularity
was determined using a Coulter Model ZM as described. Bone marrow cells
to be evaluated for committed progenitor colony formation were plated
in IMDM (GIBCO) with 0.9% methylcellulose (Fluka, Hauppage, NY), 30%
FCS (GIBCO), 2 U/mL human EPO (Amgen), 100 ng/mL recombinant rat SCF
(Amgen), 10 U/mL murine IL-3 (Genzyme), 0.1 mmol/L hemin (Eastman
Kodak, Rochester, NY), 2 × 10 3 mol/L L-glutamine
(GIBCO), and 1 × 105 mol/L beta-mercaptoethanol
(Sigma). Cultures were incubated at 37°C in a humidified environment
with 5% O2 and 5% CO2 and were scored after 7 to 10 days of incubation as CFU-MIX, CFU-GM, or BFU-E. Primitive colony
forming cells with high proliferative potential (HPP-CFC) were also
assayed because these progenitors have been shown to be responsive to
added recombinant SCF in multiple studies.32-34
Double-layer HPP-CFC agar cultures were prepared as
described.35 The bottom agar (1%) layer contained 100 ng/mL rat SCF (Amgen), 200 U/mL murine IL-3 (Genzyme), 500 U/mL murine IL-1 (Genzyme), and 1,600 U/mL murine macrophage colony-stimulating factor (Genetics Institute, Cambridge, MA) BM cells
(50,000/mL) were plated in the top agar (0.6%) and incubated at 37°C
in a humidified environment at 5% O2, 10%
CO2, and 85% N2. HPP-CFC colonies were scored
as dense, macroscopic colonies measuring greater than 0.5 mm after 14 days of incubation. LPP-CFC were scored as colonies measuring less than
0.5 mm in diameter and containing greater than 50 cells.
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RESULTS |
MR isoform of SCF enhances the growth and survival of a
factor-dependent erythrocytic progenitor cell line.
Previously it has been shown that c-kit and EPO-R physically
associate in an EPO-dependent and SCF-responsive erythrocytic progenitor cell line, HCD57.27 In the present study we have used this cell line as an in vitro model system to delineate the role
of MR and soluble isoforms of SCF in erythropoiesis. We have examined
the growth and survival of HCD57 cells and signaling events downstream
from c-kit in response to soluble or MR SCF stimulation. A
stromal cell line (Sl/Sl4) derived from the fetal liver
hematopoietic microenvironment (HM) of animals deficient in SCF as a
result of genomic deletion of Sl coding sequences (Sl/Sl homozygotes) and stable transfectants of this line
expressing either the MR (Sl/Sl4-MR) or the soluble
(Sl/Sl4-S) isoform of SCF were used. These stromal cell
lines were treated with mitomycin C to inhibit proliferation,
thoroughly washed, then cultured for 24 hours as described in Materials
and Methods. Thereafter, factor-starved HCD57 cells were cocultured
with subconfluent stromal cell layers for another 48 hours. We have
previously shown by immunoprecipitation of soluble and cell-associated
SCF that the expression of each isoform is similar in the
stromal cell lines used in experiments described here.19
Moreover, because of the absence of proteolytic cleavage sites in the
cDNA encoding the MR form of SCF, no soluble SCF is detectable in the
supernatant of stromal cell cultures.19 However, this
isoform was readily detectable on the stromal cell surface by flow
cytometry (Fig 3C). In contrast, no detectable expression of SCF was
observed on stromal cells that express either the soluble form of SCF
(Fig 3B) or that are
completely devoid of SCF expression (Fig 3A).
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| Fig 3.
Flow cytometric analysis of the cell surface expression
of SCF isoforms on stromal cells. Stromal cells stably transfected with
cDNAs encoding either the soluble (Sl/Sl4-S) or the MR
(Sl/Sl4-MR) form of SCF were stained for flow cytometric
analysis as described in Materials and Methods. (A) Parental
Sl/Sl4 cells were stained with either isotype control
(dotted line) or rat anti-mouse SCF (solid line) MoAb followed by
FITC-conjugated goat F(ab)2 anti-rat IgG secondary; (B)
Sl/Sl4-S cells were stained with either isotype control
(dotted line) or rat anti-mouse SCF (solid line) MoAb followed by
FITC-conjugated goat F(ab)2 anti-rat IgG secondary; (C)
Sl/Sl4-MR cells were stained with either isotype control
(dotted line) or rat anti-mouse SCF (solid line) MoAb followed by
FITC-conjugated goat F(ab)2 anti-rat IgG secondary.
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As shown in Fig 4, both
Sl/Sl4-S and Sl/Sl4-MR stromal cells support
the survival of HCD57 cells in the absence of exogenous EPO. However,
Sl/Sl4-MR stromal cells stimulate the proliferation of
these cells at significantly higher levels compared with
Sl/Sl4-S (Fig 4). Addition of EPO to stromal cell cultures
expressing the MR form of SCF showed a significantly greater effect on
the growth of HCD57 cells compared with the soluble form of SCF (Fig 4).
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| Fig 4.
Survival and proliferation of factor-dependent HCD57
cells in response to in vitro stimulation by SCF isoforms or by a
combination of SCF isoforms and rhEPO. HCD57 cells were factor-starved,
and stromal cells expressing various forms of SCF were mitomycin
C-treated as described in Materials and Methods. HCD57 cells were then
plated at 1 × 104 cells per mL on day 1 (Input cells) in
medium without growth factor (No Epo), supplemented with rhEPO (1 µ/mL Epo), or cocultured with parental Sl/Sl4 cells
(Sl/Sl4) or with stromal cells expressing either the
soluble (Sl/Sl4-S) or the MR (Sl/Sl4-MR) form
of SCF alone or in combination with rhEPO (Sl/Sl4-S + 1
µ/mL rhEPO or Sl/Sl4-MR + 1 µ/mL rhEPO). Viable
cells were counted after 48 hours of culture. Data represent the mean ± SEM (bars) for each group on one of the two representative
experiment done in triplicate. (*)P < .05 Sl/Sl4-MR versus Sl/Sl4-S and (*)P < .05 Sl/Sl4-MR + 1 µ/mL rhEPO versus
Sl/Sl4-S + 1 µ/mL rhEPO.
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MR SCF induced c-kit activation results in sustained
phosphorylation of the EPO-R in HCD57 cells.
To determine if coculturing HCD57 cells with stromal cells expressing
only the MR form of SCF was associated with differences in activation
of c-kit and/or downstream signaling from
c-kit, mitomycin C-treated confluent stromal cells were
further incubated for 24 to 48 hours. Factor-starved HCD57 cells were
loaded on these stromal layers and cocultured for various periods of
time, then lysed in lysis buffer. Soluble cellular protein derived from the coculture were analyzed by IP using an anti-c-kit antibody followed by Western blot analysis with an anti-phosphotyrosine antibody
(Fig 5A). In the presence of
SCF ligand expressed in stromal cells, IP with an anti-c-kit
antibody resulted in coimmunoprecipitation of c-kit and EPO-R.
Consistent with previous observations of this cell line, only the
slower-migrating glycosylated form of c-kit was tyrosine
phosphorylated upon SCF stimulation (Fig 5A). Despite high level
expression of c-kit on HCD57 cells (data not shown), no
detectable c-kit tyrosine phosphorylation was observed upon stimulation of HCD57 cells with EPO only (Fig 5B). Tyrosine
phosphorylation of both c-kit and EPO-R was induced within 10 minutes of coculture with stroma expressing either form of SCF (Fig 5A,
lanes 2 and 3). However, tyrosine phosphorylation of both c-kit
and EPO-R was greater in cells cocultured on stroma expressing only the MR form of SCF (Fig 5A, lane 3). This increase was noted as early as 10 minutes after coculture and became more apparent after 60 and 120 minutes. As shown in Fig 5A, lanes 5 and 7, coculture of HCD57 cells
with stromal cells expressing only the MR form of SCF resulted in
elevated and sustained phosphorylation of both c-kit and EPO-R.
In contrast, as noted in Fig 5A, lanes 4 and 6, phosphorylation of
EPO-R by stromal cells expressing only the soluble form of SCF was
consistently lower than that induced by MR SCF throughout the course of
coculture in two separate experiments. An apparent decrease in the
phosphorylation of c-kit and EPO-R observed after 60 minutes of
coculture with stromal cells expressing the soluble form of SCF (Figure
5A, lane 4) was caused by under loading of the protein as seen in Fig
5A, bottom panel. As a loading control, Western blot probed with an
anti-phosphotyrosine antibody was stripped and reprobed with an
anti-c-kit antibody (Fig 5A, bottom panel). In summary, these
data suggest that MR form of SCF induces a greater level of
phosphorylation of both c-kit and EPO-R in HCD57 cells, which
is significantly prolonged compared with soluble SCF.
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| Fig 5.
Coimmunoprecipitation and phosphorylation of
c-kit and EPO-R in HCD57 cells. (A) Stimulation with MR and
soluble SCF. Factor-starved HCD57 cells were cocultured with mitomycin
C-treated stromal cells expressing either the soluble
(Sl/Sl4-S) or the MR (Sl/Sl4-MR) form of SCF
for various periods of time at 37°C as described in Materials and
Methods. Subsequently at various times, cell lysates were collected and
subjected to IP with an anti-c-kit antibody and WB analysis
with an anti-phosphotyrosine antibody and the enhanced
chemiluminescence detection system. Coimmunoprecipitated and
tyrosine-phosphorylated Golgi-processed c-kit (slow
migrating)27 and EPO-R are indicated. Lane 1 corresponds to
parental Sl/Sl4 cells cocultured for 10 minutes; lanes 2, 4, and 6 correspond to Sl/Sl4-S cells cocultured for 10, 60, and 120 minutes, respectively; lanes 3, 5, and 7 correspond to
Sl/Sl4-MR cells cocultured for 10, 60, and 120 minutes,
respectively. (B) Stimulation with EPO. Factor-starved HCD57 cells were
exposed to no growth factor (lane 1) or stimulated with 10 µ/mL rhEPO for 5 minutes (lane 2) and for 10 minutes (lane 3). Cell
lysates were subjected to IP using an anti-EPO-R antibody and WB using an anti-phosphotyrosine antibody as described above.
Tyrosine-phosphorylated EPO-R is indicated.
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These in vitro observations, along with the fact that
Sl/Sld mutants show severe deficiencies in the
erythroid lineage, suggest that membrane presentation of SCF may be
important in the normal development of these cells in vivo. Therefore,
we hypothesized that transgene expression of MR-SCF would have more
profound effects than soluble SCF on the development of erythroid
lineage in Sl mutants in vivo. We tested this hypothesis using
transgenic mice expressing either the soluble or the MR form of SCF on
a Sl/Sld genetic background.
Generation of Sl/Sld mice that overexpress
soluble SCF or express the MR isoform of SCF.
Transgene positive male founders were obtained following pronuclear
injection of the human phosphoglycerate kinase (hPGK)-murine (m)
SCF248 and hPGK-mSCFX9/D3 expression plasmids,
respectively, (Fig 1) into C3H/HeJ fertilized eggs. The hPGK promoter
(pr) was used to express the SCF transgene, because we and others have
successfully shown long-term and stable expression of transgenes,
including the human SCF cDNA, using this promoter in transduced
hematopoietic cells and in transgenic mice.36,37 Selected
on the basis of comparable levels of expression of transgenes, two
founders, PGK 248-F4 (hereafter referred to as mSCF248)
and PGK X9/D3-2 (hereafter referred to as
mSCFX9/D3) were bred to C3H/HeJ to establish
transgenic lines. As expected the transgene was inherited by 50% of
the offspring (data not shown). Founders, as well as their progeny
carrying the transgene, appeared normal and fertile with no gross
phenotypic abnormalities (data not shown). Very similar levels of
expression of both transgenes were observed in stromal cells derived
from bone marrow of transgenic mice (Fig 2) and several other primary
tissues (data not shown). The steady state level of each transgene mRNA
is similar in multiple tissues examined using comparison to actin mRNA
derived from the same cells (data not shown). In contrast to transgenic
mice and as expected based on the primers used, no SCF transcript was
amplified from mRNA derived from bone marrow cells (Fig 2) or primary
tissue (data not shown) from Sl/Sld mutant mice.
Compound heterozygous Sl/Sld mutant mice, in which
only soluble truncated SCF protein is produced, are viable but show
severe hematological abnormalities, including anemia. In an effort to determine the in vivo role of membrane presentation of SCF in hematopoiesis, we performed genetic crosses of transgenic animals expressing the soluble and MR isoforms of SCF into
Sld mutants to generate
Sl/Sld-mSCFX9/D3
(Sl/Sld-MR) and
Sl/Sld-mSCF248
(Sl/Sld-S) mice. Briefly, mice carrying either of
the transgenes were first bred to Sl/+ mice.
Crosses involving these mice produced progeny in the expected ratios,
with approximately 25% having the genotype Sl/+
and carrying the transgenes (ie,
Sl/+-mSCF248 or
Sl/+-mSCFX9/D3). These mice were
identified on the basis of their phenotype (white forehead blaze and a
diluted belly) and Southern blot analysis (data not shown).
Sl/+-mSCF248 or
Sl/+-mSCFX9/D3 male mice were chosen
for further breedings and were mated to Sld/+ female mice. All black-eyed white
mice were further characterized along with their wt littermates for the
presence or absence of the transgene by Southern blot analysis. As
expected, approximately 8% of the progeny were of the genotype
Sl/Sld-mSCF248
(Sl/Sld-S) or
Sl/Sld-mSCFX9/D3
(Sl/Sld-MR) (data not shown), and these animals and
their wt littermates were studied in detail. All mice were killed and
analyzed completely at 12 weeks of age.
Expression of MR SCF as a transgene in Sl/Sld
mice is critical for erythropoiesis.
To ascertain if the expression of either transgene in these mice
rescued deficiencies in the production of mature red cells, we analyzed
Sl/Sld, Sl/Sld-MR, and
Sl/Sld -S mice for total red cell production. Our
results show a 40% increase in total red cell production in
Sl/Sld-MR mice as compared with
Sl/Sld mice (P = .01) (Fig
6). Peripheral red cell counts (Table 1) and hematocrits (data not shown) in Sl/Sld-MR mice
were also significantly higher than Sl/Sld or
Sl/Sld-S mice. In contrast, red cell production was
not increased in Sl/Sld-S mice when compared with
Sl/Sld. As seen in Fig 6 and Table
1, in comparison with wt mice the expression of MR SCF transgene in Sld mutants does
not completely correct the red cell deficiency, a result which may
relate to the level or timing of transgene expression during
development.
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| Fig 6.
Expression of MR SCF in Sl/Sld mice
improves red cell production. Total red cells were compared between
Sl/Sld (n = 16), Sl/Sld-S
(n = 10), Sl/Sld-MR (n = 13), and wt
(n = 20) mice. All black-eyed white mice and wt mice at 12 weeks of
age were tail bled, and their RBC counts/mL were determined as
described in Materials and Methods. Total red cells in each animal were
calculated by multiplying the mean blood volume of an adult animal;
(3.15 mL/100 g)55 by the total body weight
(in grams) and the RBC counts per mL. The results show mean (×10
9) ± SEM (bars). (*) P = .01 , Sl/Sld-MR versus Sl/Sld
and Sl/Sld-S.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1.
Comparison of Peripheral Blood Parameters, Bone Marrow
Cellularity (BMC), and Multilineage Progenitors Between Transgenic and
Nontransgenic Sl/Sld Mice
|
|
Expression of MR SCF as a transgene improves runting associated with
Sl/Sld mice.
The most obvious phenotypic difference between
Sl/Sld and Sl/Sld transgenic
mice, the presence of severe runting, was observed shortly after birth.
As seen in Fig 7, left panel, 12-week-old Sl/Sld-MR mice appeared larger and healthier than
either Sl/Sld or Sl/Sld-S mice.
Quantitatively, the expression of MR SCF as a transgene significantly
improved the total body weight of these animals in comparison to
nontransgenic mice (P = .01; Fig 7, right panel). In
contrast, no increase in body weight was observed in
Sl/Sld mice that overexpressed the soluble form of
SCF as a transgene (Sl/Sld-S).
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| Fig 7.
Expression of MR SCF improves runting associated with
Sl/Sld mice. Shown in the left panel are
pictures of 12-week-old mutant mice that either express the MR form of
SCF or overexpress the soluble form. The right panel shows the weights
of these mice and their comparison with wt littermates. Weight
comparison between Sl/Sld (n = 16),
Sl/Sld-S (n = 10),
Sl/Sld-MR (n = 14), and wt (n = 20)
mice. All black-eyed white mice and wt mice at 12 weeks of age were
weighed. The results show mean ± SEM (bars). (*) P = .01, Sl/Sld-MR versus Sl/Sld
and Sl/Sld-S.
|
|
Overexpression of soluble SCF in Sl/Sld mice
increases the frequency of myeloid progenitors in the bone marrow.
In addition to anemia, Sl/Sld mice show bone marrow
hypocellularity and myeloid cell deficiency. To determine if expression of either isoform of SCF as a transgene affected these abnormalities, we compared the bone marrow cellularity of Sl/Sld
mice with that of Sl/Sld-S and
Sl/Sld-MR mice. A significant increase in the BM
cellularity was evident in Sl/Sld-MR mice compared
with Sl/Sld animals (Table 1). A modest, but
nonsignificant increase in the total BM cellularity was observed in
Sl/Sld mice overexpressing the soluble isoform of
SCF. Because marrow cellularity does not necessarily reflect more
immature clonogenic compartments, we compared progenitor cell numbers,
including high proliferative potential (HPP) and low proliferative
potential (LPP) colony-forming cells (CFC), and lineage-restricted
progenitors in Sl/Sld,
Sl/Sld-S, and Sl/Sld-MR mice.
In contrast to the lack of effect on erythropoiesis, transgene
overexpression of soluble SCF (mSCF248) in
Sl/Sld mice completely corrected the deficiency of
myeloid progenitors in the bone marrow (Fig
8). Total bone marrow myeloid progenitor content (ie, HPP-CFC + LPP-CFC) in Sl/Sld-S was
significantly greater than that observed in either
Sl/Sld or Sl/Sld-MR mice
(P = .005). The increase in myeloid progenitors in
Sl/Sld-S mice represented both primitive HPP-CFC
(67,601 ± 11,172 v 40,519 ± 4,336,
Sl/Sld-S v Sl/Sld,
respectively) and more mature LPP-CFC (286,034 ± 33,212 v
208,598 ± 23,421, Sl/Sld-S v
Sl/Sld, respectively). This correction in myeloid
progenitor content occurred in spite of the BM hypocellularity seen in
these animals (Table 1). The expression of either transgene completely
corrected the deficiency of multipotential clonogenic cells, colony
forming unit-mix (CFU-MIX), which contain cells of both erythroid and myeloid lineages (Table 1). No significant differences were noted in
the number of primitive erythroid progenitor blast forming unit-erythroid (BFU-E) between the transgenic mice expressing either
isoform of SCF (data not shown). In addition, the peripheral white
blood cell counts were improved by expression of either isoform (Table
1). This increase in peripheral leukocyte count was mainly caused by an
increase in neutrophils (data not shown). Thus, both
transgene-expressed isoforms of SCF are biologically active in vivo and
can differentially rescue specific hematologic defects in
Sl/Sld mice.
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| Fig 8.
Overexpression of soluble SCF increases the frequency of
myeloid progenitors in the bone marrow. HPP-CFC and LPP-CFC assays were
performed on bone marrow cells derived from various transgenic and
nontransgenic white mice. Shown are total myeloid progenitors (ie, sum
of all HPP and LPPs) from Sl/Sld (n = 22),
Sl/Sld-S (n = 13),
Sl/Sld-MR (n= 14), and wt (n = 20). HPP
and LPP content were determined by multiplying the concentration of
HPP-CFC or LPP-CFC by the total bone marrow cellularity. (*) P
= .005, Sl/Sld-S versus
Sl/Sld and Sl/Sld-MR.
The results show mean (×103) ± SEM (bars).
|
|
| |
DISCUSSION |
The phosphorylation of RTKs on tyrosine by their cognate ligands is
central to the regulation of the pathways of signal transduction that
control cellular proliferation and differentiation.1,2 The
net levels of protein phosphorylation depend on the strength of the
receptor-ligand interaction. In the present study we have examined in
vitro and in vivo the interaction between the soluble and MR isoforms
of SCF and the RTK, c-kit. We show that MR SCF plays an
important role in the maturation of erythroid progenitors to mature red
cells. In contrast, soluble SCF may be more important for the normal
development of myeloid lineage. These results show that expression of
different isoforms of SCF may result in different cellular outcomes
both in vitro and in vivo depending on the hematopoietic cell lineages
in which ligand/receptor interaction occurs.
A large number of studies have shown that recombinant soluble SCF
(rSCF) in combination with other cytokines significantly enhances the
growth of clonogenic cells in vitro.38-43 Soluble SCF plus
EPO primarily increase the number and size of BFU-Es, whereas SCF plus
granulocyte colony-stimulating factor result in enhanced neutrophil
colonies.23,24 Soluble SCF has also been shown to synergize
with other cytokines, such as IL-7, in the development of lymphoid
lineage. Mutations in the RTK c-kit and the common cytokine
gamma chain reduce cellularity, but are permissive to thymocyte
development.44,45 However, mice that lack both
c-kit and the gamma chain show complete abrogation of thymocyte
development.46 EPO and EPO-R knock out mice have been reported and show that neither EPO nor the EPO-R is required for erythroid lineage commitment.26 However, EPO and EPO-R are
crucial in vivo for the proliferation and survival of later erythroid progenitors.26 Despite increased circulating levels of EPO
in Sl/Sld mice, these animals still show
deficiencies in the erythroid lineage, suggesting that EPO by itself is
not sufficient to induce normal proliferation and/or
differentiation of committed progenitors to mature red cells in
Sl/Sld mutants.4,22 Recent observations
showing the activation of one receptor by a second receptor of a
different cytokine family in response to ligation provides a potential
explanation for the diversity of effects seen with different growth
factors, including soluble rSCF. In this regard, it has also been shown
that soluble SCF can activate the IL-3 receptor in a myeloid cell line
in an IL-3- independent fashion.47 More recently, it has
been shown that very high concentrations of soluble rSCF (1,000 ng/mL)
can lead to transient phosphorylation of the EPO-R and stimulate
proliferation of an erythrocytic progenitor cell line in an
EPO-independent fashion.27 Although useful, these in vitro
and in vivo studies, using high concentrations of soluble rSCF, do not
completely define the role of the cytokine in normal hematopoietic
microenvironment. In addition, because naturally occurring murine
Sl mutants are deficient in SCF/c-kit interactions
throughout development, the function of the differentially spliced
isoforms of SCF in vivo in definitive hematopoiesis is not well
understood.
Many proteins exist as both MA and soluble isoforms, and considerable
interest has been generated by the recent finding that certain growth
factors, classically thought of as autocrine factors, not only exist as
MA proteins but also are active as such.12 These factors
may participate in a novel mode of intercellular communication
restricted to adjacent cells, termed juxtacrine stimulation.12 For instance, cleavage of pro-TGF- does
not result in the generation of an active form of the factor from a
precursor, but rather results in the conversion of one active form (MA)
to another (soluble).12 For TNF it has been shown that TNF
receptor (TNF-R) can be strongly stimulated by MA form of TNF compared
with soluble TNF. Moreover, upon activation of the TNF-R by MA TNF, a
phenotypic switch of the cellular response pattern to TNF can be
observed. In this case, cells fully resistant to the cytotoxic action
of soluble TNF become susceptible and are killed upon contact with
membrane TNF.13
Previously, we have shown that primary human progenitor cells and
hematopoietic cell lines proliferate and differentiate in distinct
patterns when exposed to stromal cells expressing either MA or soluble
SCF and that these differences are correlated with distinct patterns of
c-kit activation.25,48 We have shown that the
activation of c-kit tyrosine kinase persists longer when
stimulated with MA SCF compared with soluble SCF, and that the length
of activation of c-kit could be shortened upon adding soluble
SCF to cultures producing MA SCF.48 These data suggest that
differences in the kinetics of c-kit activation are dependent
on the isoform of SCF presented. However, in vitro stromal cultures
favor myeloid over erythroid differentiation, making the interpretation
of these experiments with respect to the function of SCF in erythroid
cell development difficult. This is a major shortcoming, because anemia is the most prominent hematopoietic phenotypic abnormality seen in
Sl and W mutants in vivo. To further study the function
of each SCF isoform in vitro and in vivo, we have combined in vitro cellular and biochemical studies with an in vivo genetic approach. Our
data in vitro using an erythrocytic progenitor cell line shows that MR
isoform of SCF induces an elevated and sustained tyrosine phosphorylation not only of c-kit but also of EPO-R and
significantly greater proliferation of this cell line compared to
soluble SCF. Membrane presentation of SCF in vivo also resulted in the
production of significantly more red blood cells. In contrast,
expression of either SCF isoform appeared to be sufficient for the
generation of multilineage progenitors, CFU-MIX, and for early
erythroid BFU-E. Finally, soluble SCF appeared to be a potent growth
factor in the development of myeloid progenitors, such as HPP-CFC and LPP-CFC. This finding is not surprising, because at least one group of
investigators used HPP-CFC colony stimulation in the cloning of soluble
SCF.38 Of relevance to these observations, the differential
findings of erythroid and myeloid lineage cells, the similar effects on
CFU-MIX, and the similarities in expression of the transgenes would
argue against the level of expression of the two transgenes per se
being responsible for the observed phenotypic differences.
Previously it has been shown that stimulation of cells expressing
c-kit with soluble SCF induces rapid downmodulation of cell surface c-kit expression and degradation of metabolically
radiolabeled c-kit protein.49,50 Degradation after
internalization of the ligand-receptor complex has been reported in
many other receptor systems including the PDGF and the CSF-1 receptors,
which are structurally related to c-kit and may be a mechanism
of modulation of signaling pathways and cellular
responses.50,51 Therefore, we hypothesize that the
regulation of EPO-R activation, at least in part, may be caused by the
length of time activated c-kit remains membrane-associated in
erythroid progenitors in vivo. Alternatively, MR SCF may be more
efficient than soluble SCF in forming dimers and, subsequently,
activating EPO-R. Recent reports have indicated that EPO activates
EPO-R by inducing dimerization. However, c-kit activates the
EPO-R probably through phosphorylation of tyrosine residues in the
cytosolic domain rather than EPO-R dimerization.52 Therefore, during erythroid differentiation, activation of the EPO-R
may take place by two different mechanisms, which may lead to different
intracellular signals essential for survival, proliferation, and
terminal differentiation of committed erythroid progenitors to produce
mature red cells. At least one signal emanating from c-kit must
use the EPO-R as a downstream signal-transduction protein, because
EPO-R/ fetal liver cells after transduction and
expression of exogenous EPO-R require SCF in vitro to further
differentiate.52 Thus, our in vitro and in vivo data would
suggest that the terminal differentiation and/or proliferation
of late erythroid progenitors to mature red cells requires MA
SCF/c-kit-mediated activation, presumably because of
phosphorylation of the EPO-R both by EPO-mediated and
c-kit-mediated ligation. In this regard, we have observed a
significant increase in the production of late erythroid progenitors (CFU-Es) in Sl/Sld mice expressing the MR isoform
of SCF (R.K. and D.A.W., unpublished observation, August
1997).
Despite the significant improvement in various hematopoietic lineages
in Sl/Sld mice expressing the two isoforms of SCF,
neither transgene resulted in the rescue of germ cells, mast cells, and
melanocytes in these mutants. Characterization of the SCF promoter and
subsequent use of this promoter to generate transgenic mice in the
future should help to ascertain the in vivo role of SCF isoforms in
other lineages. SCF has been shown to be expressed at high levels
during embryogenesis in cells associated with both the migratory
pathways of melanoblasts and germ cells.53 In this regard,
either the dysregulation or level of expression of SCF via the PGK
promoter may be the reason for the lack of affects on other lineages as
observed in this study. Indirect evidence to support this explanation
comes from our previous report of mild melanocyte deficiencies caused
by interference of endogenous SCF/c-kit interactions in vivo by
PGK-expressed human SCF during development.36,37 On the
other hand, once present in the dermis, melanocytes can respond to SCF
transgene expression because pigmentation is enhanced when expression
of mSCFX9/D3 is directed by an epidermal-specific promoter
in wt transgenic mice.56 In this regard, we have
occasionally seen mild and scattered pigmentation in
Sl/Sld mice overexpressing soluble SCF (R.K. and
D.A.W., unpublished observation, January 1997).
In summary, we have shown that both soluble and MR SCF can enhance the
production of multilineage progenitors. Soluble SCF plays a significant
role in the production of myeloid progenitors, however, MA SCF may be
more important for the normal development of the erythroid lineage. SCF
is a unique example of modulation of target cell responses based on the
provision of alternatively spliced forms acting through a common
receptor, c-kit.
| |
FOOTNOTES |
Submitted September 8, 1997;
accepted October 1, 1997.
Supported by R01 DK48605.
Address reprint requests to David A. Williams, Howard Hughes Medical
Institute, Herman B Wells Center for Pediatric Research, James Whitcomb
Riley Hospital for Children, Cancer Research Building, 1044 W Walnut
St, Room 406C, Indianapolis, IN 46202.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank the members of our laboratory and Drs M. Dinauer, W. Clapp,
and M. Yoder of the Herman B Wells Center for Pediatric Research for
critical reading of the manuscript. We thank Drs Frank Martin and Keith
Langley for originally furnishing mSCF cDNAs and Dr Ian McNeice for
supplying rrSCF. We also thank Dr H. Lodish for providing HCD57 cell
line and Dr G. Krystal for supplying anti-EPO-R antibody.
| |
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Abstract
Introduction
Abstract