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Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 1915-1925
The Presence of Novel Amino Acids in the Cytoplasmic Domain of Stem
Cell Factor Results in Hematopoietic Defects in
Steel17H Mice
By
Reuben Kapur,
Ryan Cooper,
Xingli Xiao,
Mitchell J. Weiss,
Peter Donovan, and
David A. Williams
From The Section of Pediatric Hematology/Oncology, Department of
Pediatrics, Herman B Wells Center for Pediatric Research, James
Whitcomb Riley Hospital for Children, Indiana University School of
Medicine, The Howard Hughes Medical Institute, Indiana University
School of Medicine, Indianapolis, IN; Ontogeny, Inc, Cambridge, MA; and
Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA.
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ABSTRACT |
Stem cell factor (SCF) is expressed as an integral membrane growth
factor that may be differentially processed to produce predominantly
soluble (S) (SCF248) or membrane-associated (MA)
(SCF220) protein. A critical role for membrane presentation
of SCF in the hematopoietic microenvironment (HM) has been suggested
from the phenotype of the Steel-dickie
(Sld) mice, which lack MA SCF, and by studies
performed in our laboratory (and by others) using long-term bone marrow
cultures and transgenic mice expressing different SCF isoforms.
Steel17H (Sl17H) is an SCF
mutant that demonstrates melanocyte defects and sterility in males but
not in females. The Sl17H allele contains a
intronic mutation resulting in the substitution of 36 amino acids
(aa's) in the SCF cytoplasmic domain with 28 novel aa's. This
mutation, which affects virtually the entire cytoplasmic domain of SCF,
could be expected to alter membrane SCF presentation. To investigate
this possibility, we examined the biochemical and biologic properties
of the Sl17H-encoded protein and its impact in vivo
and in vitro on hematopoiesis and on c-Kit signaling. We demonstrate
that compound heterozygous Sl/Sl17H mice manifest
multiple hematopoietic abnormalities in vivo, including red blood cell
deficiency, bone marrow hypoplasia, and defective thymopoiesis. In
vitro, both S and MA Sl17H isoforms of SCF exhibit
reduced cell surface expression on stromal cells and diminished
biological activity in comparison to wild-type (wt) SCF isoforms. These
alterations in presentation and biological activity are associated with
a significant reduction in the proliferation of an SCF-responsive
erythroid progenitor cell line and in the activation of
phosphatidylinositol 3-Kinase/Akt and mitogen-activated protein-Kinase signaling pathways. In vivo, transgene
expression of the membrane-restricted (MR) (SCFX9/D3) SCF
in Sl/Sl17H mutants results in a significant
improvement in peripheral red blood cell counts in comparison to
Sl/Sl17H mice.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
STEM CELL FACTOR (SCF; also known as
Kit-ligand [KL], mast cell growth factor [MGF], and Steel
[Sl] factor) is the ligand for the receptor tyrosine kinase
encoded by the c-kit proto-oncogene.1,2 The c-Kit receptor
is a membrane spanning protein that is a member of the platelet-derived
growth factor (PDGF) receptor family and possesses intrinsic kinase
activity.3 Mutations of c-Kit are associated with the
dominant White Spotting (W) phenotype in mice, whereas
mutations of the c-Kit ligand are associated with the Sl mutant
phenotype.2,4-8 Mutations at either locus produce similar
abnormalities in mice, with many mutations resulting in embryonic
lethality due to severe anemia.9 Viable mice homozygous for
mutations at either locus typically are deficient in erythrocytes, mast
cells, progenitors for multiple hematopoietic lineages, and melanocytes
(resulting in black-eyed white mice) and these mice are
sterile.9
Several cytokines and/or growth factors, such as colony-stimulating
factor-1 (CSF-1), interleukin-1 (IL-1), and tumor necrosis factor
(TNF), have been described that function as both membrane and soluble proteins.10,11 The majority of these
transmembrane growth factors are proteolytically processed to release
soluble protein.12,13 The membrane-anchored counterparts of
these proteins also possess biological activity and can promote several
distinct biological functions.13-15 SCF can induce
proliferation, survival, adhesion/migration, and differentiation in
c-Kit-expressing cells of hematopoietic and nonhematopoietic lineages.
Both soluble (S) (SCF248) and
membrane-associated (MA) (SCF220) SCF can mediate direct
cell-cell contact with c-Kit-positive cells.16-19 Our
laboratory has demonstrated that the generation of soluble SCF is
dependent on 3 distinct proteolytic cleavage sites. The primary site in
exon 6 is preferentially used, in vitro, in stromal cells expressing
SCF. A secondary site located in exon 7 is used only in the absence of
the primary site.15 Chymase digestion of a third site
(encoded in exon 6) results in a protein product of 158 amino acids
(aa's).20 This site is sensitive to chymase digestion by
mast cells but not by stromal cells.20 Proteolytic cleavage
at all 3 sites results in biologically active SCF
protein.14,15,20 Additionally, we have shown that
mutagenesis of the primary cleavage site in exon 6 and the secondary
site in exon 7 in the SCF cDNA leads to the generation of
membrane-restricted (MR) and biologically active form of SCF
(SCFX9/D3), when expressed in murine stromal cells in vitro
and in vivo.15,21
Some viable Sl alleles (eg, Steel-panda
[Slpan] and Steel-contrasted
[Slcon]) exhibit alterations in the enhancer
regions of the Sl gene that affect the levels of SCF mRNA in
various tissues.22 A small number of Sl mutations
disrupt the coding sequence of SCF, which changes the structure of the
expressed SCF protein. Steel-dickie (Sld) is the
result of an intragenic 4-kb deletion that removes
sequences encoding transmembrane and cytoplasmic
domains.23 Sld mice
produce only a soluble mutant form of the SCF protein.16,23 The fact that Sld mice are viable, but display
multiple hematopoietic cell deficiencies, are sterile, and lack coat
color suggests that the Sld SCF protein retains
some biological activity and that the MA isoform must mediate
proliferation, survival, or migration of several hematopoietic and
nonhematopoietic lineages in vivo. In this regard, we have recently
shown that transgene expression of the MR SCF but not soluble SCF in
Sld mutants significantly improves the hematologic
abnormalities associated with these mice.20 Also, exclusive
expression in vivo of the MA SCF (SCF220) results in mast
cell deficiency, but no other hematopoietic or nonhematopietic
abnormalities.24 These observations suggest that sequences
located in the cytoplasmic domain of SCF or that membrane presentation
or both is pivotal for c-Kit activation.
Another viable Sl allele, Steel17H
(Sl17H), contains a mutation in the polypyrimidine
tract of intron 7, leading to abnormal splicing and the absence of exon
8 encoded sequences that comprise the cytoplasmic domain of SCF. This
results in the substitution of 28 novel aa's with only the first
cytoplasmic aa (lysine) read in the correct frame.25 The
first 3 aa's of the cytoplasmic domain of native SCF are lysines.
Positively charged aa's within the cytoplasmic domain located
immediately adjacent to the transmembrane region have been shown to be
important for anchoring transmembrane proteins in the
membrane.26,27 Mutations that reduce the net positive
charge in this region can lead to instability of protein in the cell
membrane.26 In addition, mutations in the cytoplasmic tail
of membrane growth factors, particularly the removal of the COOH-terminal valine (which is also present in SCF), lead to reduced cell surface expression of these growth factors.28
Therefore, we hypothesized that the mutation in the
Sl17H would affect stromal cell membrane
presentation of SCF. In this study, we cloned both the soluble and MA
isoforms of Sl17H and studied the presentation,
biological activity, and in vivo consequences of expression of this
mutant in blood cell development.
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MATERIALS AND METHODS |
Generation of Sl/Sl17H transgenic mice.
Experiments involving mice described here were reviewed and approved by
Animal Use Committee of Indiana University School of Medicine
(Indianapolis, IN). C3H-Sl17H/+ mice were a kind
gift of the ABL-Basic Research Program, National Cancer Institute
(Frederick, MD). These mice were maintained by breeding
into C3H background. 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
Nde I/Kpn I fragment comprising either the human
phosphoglycerate kinase promoter (hPGK) and cDNA encoding the soluble
(hPGK-SCF248) or the MR (hPGK-SCFX9/D3) form of
SCF, as described previously.21 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% sodium dodecyl sulfate [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 EcoRI, electrophoresed, transferred to filters, and probed using a full-length 32P-labeled cDNA murine SCF
probe. To examine the in vivo role of the 2 isoforms of SCF on
hematopoietic lineages in Sl17H mutants, 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. We have previously shown that these mice
express equivalent amounts of SCF transgene.21 Transgene positive Sl/+ male mice were further crossed to
C3H-Sl17H/+ mice to obtain
Sl/Sl17H transgene-positive or -negative mice.
Peripheral blood analysis.
Total peripheral red blood cell (RBC) and white blood cell (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 the manufacturer's recommendations. Peripheral blood hematocrits were performed by spinning capillary tubes
for 5 minutes in a model MB micro-capillary centrifuge (IEC, Boston, MA).
Cell preparations, antibodies, and flow cytometric analysis.
Mice were killed by cervical dislocation, and thymus glands and bone
marrow (BM) were surgically excised from adult wild-type (wt) and
Sl17H transgenic and nontransgenic mice. Thymus
suspensions were prepared and filtered through nylon-mesh to remove
debris. BM was harvested and cellularity was determined using a Coulter
Model ZM. Phycoerythrin (PE)-conjugated monoclonal antibodies (MoAbs)
were directed against CD4, CD8, and CD69. All the PE- and fluorescein
isothiocyanate (FITC)-conjugated MoAbs, including the
isotype control antibodies, were purchased from Pharmingen (San Diego,
CA). Cells (1 × 106) were incubated at
4°C for 30 minutes with 1 µg of the first MoAb. Cells were washed
3 times with phosphate-buffered saline (PBS) containing 0.1% bovine
serum albumin (BSA). Cells were then incubated with 1 µg of the
second MoAb for 30 minutes at 4°C, subsequently washed 3 times with
PBS containing 0.1% BSA, and analyzed by fluorescence-activated cell
sorter (FACS; Becton Dickinson, San Jose, CA). To determine the cell
surface expression of SCF on Sl/Sl4 stromal
cells transfected with cDNAs encoding either the soluble or the MA
isoforms of wt and Sl17H protein, flow cytometric
analysis with an anti-SCF antibody was performed. Briefly, 1 × 106 stromal cells were stained separately with either 1 µg/mL of primary anti-SCF antibody (Genzyme, Cambridge, MA) or a
control antibody for 30 minutes at 4°C. Afterwards, the cells were
washed twice with PBS/0.1% BSA and subsequently stained with 1 µg of secondary PE-conjugated anti-IgG (Santa Cruz Biotechnology, Santa Cruz,
CA) under identical conditions and analyzed by FACS.
Cloning of the Sl17H soluble
(Sl17H-SCF248) and MA
(Sl17H-SCF220) cDNAs.
Murine wt-SCF248 and
Sl17H-SCF248 cDNAs were blunt-end
subcloned into the HincII site of Bluescript (Stratagene, La
Jolla, CA). Subsequently, BamHI/Xho I fragment
containing the entire coding sequence of SCF was cloned into the PSG5
expression vector (Stratagene). wt-SCF220 and
Sl17H-SCF220 cDNAs were cloned into the
V19.8 expression vector.29 Briefly, polymerase chain
reaction (PCR) was performed on reverse-transcribed (RT) total RNA
derived from testes of a Sl/Sl17H mouse.
Amplification and cDNA synthesis was performed according to the
manufacturer's instructions (Perkin-Elmer, Branchburg, NJ). PCR
fragments were purified by agarose gel electrophoresis before cloning.
The upstream SCF primer was flanked at its 5' end by an
artificial BamHI site, and the downstream primer was flanked by
a Pst I site. This allows cDNAs amplified by PCR to be cloned
between the BamHI and Pst I sites of bluescript and subsequently into the expression vector V19.8. Nucleotide sequencing was performed to determine the absence of exon 8 encoding sequences on
double-stranded DNA encoding both
Sl17H-SCF248 and
Sl17H-SCF220.
Generation of stable transfectants of Sl/Sl4 stromal
cells expressing wt and Sl17H-SCF cDNAs.
Transfection of plasmid DNA into Sl/Sl4 stromal
cells devoid of endogenous SCF mRNA and protein was performed using
DOTAP (Boehringer Mannheim, Indianapolis, IN) according to the
manufacturer's recommendations. Sl/Sl4 cells were
grown to subconfluence in Dulbecco's modified Eagle's medium
(DMEM) with 10% calf serum (CS). Plasmid
mixtures containing cDNAs of wt or Sl17H SCF and a
hygromycin-resistance gene in p48 (10:1 ratio) were incubated with
DOTAP at room temperature for 10 minutes and then diluted with fresh
medium.30 The medium was then removed from the cells and
replaced with the transfection mixture, and the cells were allowed to
grow overnight. The following day, fresh medium containing hygromycin
(300 U/mL; Boehringer Mannheim) was added to the cells. Well-isolated
hygromycin-resistant colonies arising in 10 to 14 days were transferred
to 24-well plates and expanded. Total cellular RNA was prepared from
each clone using Tri-reagent (Molecular Research Center, Cincinnati,
OH) and clones were screened for SCF expression by RT-PCR.
Expression of wt-SCF248, wt-SCF220,
Sl17H-SCF248, and
Sl17H-SCF220 cDNAs in
Sl/Sl4 stromal cells.
Total cellular RNA from at least 3 stromal cell transfectants
expressing either wt or Sl17H SCF was purified
using Tri Reagent (Molecular Research Center) according to the
manufacturer's instructions and was used as template. Semiquantitative
RT-PCR was performed on at least 2 clones of each stromal cell
transfectants expressing wt-SCF248, wt-SCF220,
Sl17H-SCF248, and
Sl17H-SCF220 cDNAs using actin as an
internal control. Briefly, RNA was used to synthesize single-stranded
cDNA by RT and random hexamers (Perkin-Elmer). cDNA was amplified in a
100 µL reaction mixture by PCR using ampliTaq DNA polymerase
in 35 cycles of 1 minute of 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'-GTCCACAATTACACCTCTTG-3' antisense primer based on
published sequences.16 RT-PCR products were examined on a
2.5% agarose gel. As a control for integrity of total RNA, primer
specific for actin (5'-TGGTGGGAATGGGTCAGAAGGACTC-3' sense
primer and 5'-TTGGCATAGAGGTCTTTACGGATGT-3' antisense
primer) were used to amplify cDNA using the described
conditions.31 The predicted amplification product of these
primers is 732 bp.
Metabolic labeling and immunoprecipitation.
For protein analysis, subconfluent cells were starved for 30 minutes in
methionine-free DMEM, 10% dialyzed CS. Afterwards, the cells were
labeled with 0.5 mCi/mL of [35S]Met for 30 minutes and
chased with cold media for various time points. Conditioned medium was
collected from each cell line after 1, 2, and 3 hours, filtered through
a 0.45-µm filter, and stored at  80°C until used. Labeled
cells were washed with PBS and lysed in lysis buffer (0.5%
sodium-deoxycholate, 0.5% Nondiet P-40, 50 mmol/L NaCl, 25 mmol/L
Tris-Cl [pH 8.0], and 1 mmol/L phenylmethylsulfonyl fluoride).
Immunoprecipitation was performed using a rabbit polyclonal antibody
raised against rat-SCF (kindly supplied by Dr Larry Bennett, Amgen,
Thousand Oaks, CA). Two milliliters of polyclonal antisera was
conjugated to 1 mL protein A-agarose using the Affinica purification kit (Schleicher and Schuell, Keene, NH) according to the
manufacturer's instructions. The antibody-conjugated protein A-agarose
was resuspended in 3 mL of PBS with 0.05% sodium azide and stored at
4°C. For immunoprecipitation, labeled cells were thawed overnight
at 4°C concentrated 2× for 30 minutes using a Centriprep-10
(Amicon, Beverly, MA) device according to the
manufacturer's instructions and transferred to 1.5-mL microfuge tubes.
Twenty-five microliters of conjugated antibody was added to each sample
and allowed to incubate overnight at 4°C. The samples were
centrifuged for 3 minutes at 4°C, the supernatant was discarded,
and the immunoprecipitates were washed 3 times in wash buffer (0.5 mol/L NaCl, 20 mmol/L Tris-HCl [pH 7.5], and 1% Triton) and once
with 20 mmol/L Tris-Cl (pH 7.5). The protein was released from the
beads by boiling for 5 minutes. An equal volume of 2× sample
loading buffer (100 mmol/L Tris-HCl [pH 6.8], 200 mmol/L
dithiothreitol, 4% SDS, 0.02% bromophenol blue, and 20% glycerol)
was added to each sample and boiled for 5 minutes, and the proteins
were analyzed on 12% or 15% SDS-polyacrylamide gel electrophoresis
(SDS-PAGE).
Biological activity of transfected cell lines.
The effect of Sl17H-SCF248 and
Sl17H-SCF220 protein on proliferation
of an SCF-dependent erythrocytic progenitor cell line,
G1E-ER2,32 was assayed using thymidine incorporation. The
presence of soluble and MA SCF activity in stable stromal cell
transfectants was analyzed using a coculture assay. On the day before
assay, stromal cells were treated with 5 µg/mL mitomycin C, then
washed 3 times in PBS, counted, and seeded at 3 × 104
cells/well in 0.1% gelatin-coated 96-well plates. These cultures were
incubated in DMEM, 10% CS at 37°C. After 24 hours, 5 × 104 G1E-ER2 cells were added to the stromal cells and
cultured for 24 to 48 hours. Subsequently, 1.0 µCi of
[3H] thymidine was added to each well for 6 to 8 hours at
37°C. Cells were then harvested using an automated cell harvester
(96-well harvester; Brandel, Gaithersburg, MD), and thymidine
incorporation was determined in a scintillation counter.
MAP-Kinase and Akt activation by stromal cells
expressing either the wt-SCF220 or the
Sl17H-SCF220 isoform of SCF.
Activation of mitogen-activated protein (MAP)-Kinase (Erk-1 and Erk-2)
was determined by utilizing a phospho-specific MAP-Kinase antibody
(Thr202/Tyr204; New England Biolabs, Beverly, MA). The antibody detects
Erk-1 and Erk-2 MAP-Kinase only when they are catalytically activated
by phosphorylation at Thr202 and Tyr204. Activation of the
phosphatidylinositol 3 (PI-3) Kinase/Akt pathway was
determined by using a phospho-specific Akt (Ser 473) antibody (New
England Biolabs). Akt is a downstream signaling molecule from
PI-3Kinase.33-35 Briefly, Sl/Sl4 cells
expressing either the wt-SCF220 or the
Sl17H-SCF220 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. A c-Kit+, SCF-responsive erythroid progenitor cell
line, G1E-ER2, was factor-starved for 6 to 8 hours in medium containing
1 mg/mL BSA and Iscove's modified Dulbecco's medium
(IMDM). Subsequently, 6 to 8 × 106
cells were loaded onto stromal cells expressing either the
wt-SCF220 or the
Sl17H-SCF220 SCF and were further
cocultured for various time points at 37°C. Thereafter, cells were
harvested and 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
 -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.
Western blot analyses were performed according to the manufacturer's
instructions (New England Biolabs).
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RESULTS |
Impaired hematopoiesis in Sl17H mutant mice.
Previous work (Brannan et al25) has shown that the
Sl17Hallele results in impaired melanocyte and germ
cell development. At the molecular level, this result is due to a
splicing defect caused by a mutation in the 3' splice acceptor
site of intron 7, specifically, a T A transversion within the
polypyrimidine tract in the 3' splice acceptor site of intron 7 (Fig 1A).25 This point mutation
impedes splicing; as a consequence, exon 8 is skipped and exon 7 is
spliced directly to exon 9 (Fig 1B). A comparison of the wt and
Sl17H sequences showed that exon 8 is completely
missing from Sl17H mRNA (Fig 1).25 Exon
8 begins 1 aa carboxy-terminal to the transmembrane domain and encodes
23 of 36 aa's of the SCF cytoplasmic domain. The result of this
deletion is a frameshift; only the first aa (lysine) of the
Sl17H cytoplasmic domain is read in frame; the next
27 aa's are read in an alternative reading frame before a stop codon
is encountered (Fig 1B).25
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| Fig 1.
Schematic representation of the Sl17H
genomic mutation. (A) Sl17H contains a TA
transversion in the 3' splice acceptor site of intron 7. The
mutation is marked with an asterisk (*). Lowercase letters indicate
intron 7 sequences that are flanked on the 5' end with exon 7 ( ) and at the 3' end with exon 8 ( ). The short dotted line
indicates abnormal splicing, which skips exon 8 sequences in
Sl17H. (B) Schematic representation of the wt and
Sl17H SCF protein. N, amino terminus;
EC, extracellular domain; TM, transmembrane domain
( ); CD, cytoplasmic domain (); C, carboxy
terminus. Boundaries for exons 7, 8, and 9 are indicated by arrows
( ). The gap ( ) in the Sl17H protein due to
the absence of exon 8 sequences is indicated below the wt SCF protein.
The last 36 and 28 aa's encoded by the wt and the
Sl17H cDNAs, respectively, are shown below each of
the schematic diagrams.
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To examine the in vivo consequence(s) of the absence of normal
cytoplasmic domain SCF aa's and the presence of 28 novel aa's in the
cytoplasmic domain of the Sl17H-encoded SCF protein
on hematopoiesis, we compared peripheral blood and BM in Sl/+
versus Sl/Sl17H compound heterozygotes. As shown in
Fig 2, the Sl17H allele
has a deleterious effect on hematopoiesis. Sl/Sl17H
mice demonstrate significantly lower hematocrit levels (Fig 2A), peripheral RBCs (Fig 2B), and BM cellularity (Fig 2D) compared with
Sl/+ mice. In contrast, peripheral WBC counts were comparable (Fig 2C).
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| Fig 2.
A comparison of hematologic values between 12-to
14-week-old Sl/Sl17H and control Sl/+
mice. (A) Hematocrit levels, (B) peripheral RBC counts, (C) peripheral
WBC counts, and (D) BM cellularity. A minimum of 3 mice were examined
in each group. Data shown are the mean ± SEM. *P < .05.
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Recent studies have shown that c-Kit is expressed on thymocytes and
that SCF/c-Kit interactions play an important role in T-cell
development but not in mature T cells.9,36-38 In addition, our laboratory has shown that membrane presentation of SCF is critical
for normal T-cell development.38 To determine if the Sl17H-encoded protein affects thymopoiesis, we
compared total thymic cellularity and thymocyte subset distribution in
Sl/+ and Sl/Sl17H mice. As seen in
Fig 3A, Sl/Sl17H mice
demonstrate a 70% decrease in total thymic cellularity in comparison
to Sl/+ mice. Thymocytes from mutant mice were examined for
maturation using the expression of CD4, CD8, and CD3. In comparison to
control mice, mutant mice exhibit a significant reduction in the
percentage of immature CD4+CD8+ double-positive
(DP) thymocytes (Fig 3B). In addition, a small but significant increase
in the percentage of CD4 CD8
double-negative (DN) thymocytes is also seen in these mice (data not
shown). The percentage of CD4+ single-positive (SP) and
CD8+ SP thymocytes is also significantly increased in these
mice (data not shown). A reduction in the
CD4+CD8+ T-cell subset and an increase in the
SP thymocytes is consistent with a more mature phenotype (ie,
CD4+CD3hi or CD8+CD3hi)
of thymocytes in the mutant mice. To further examine T-cell maturation,
we performed 2-color flow cytometric analysis on thymocytes using MoAbs
directed against CD4 and CD3 or CD8 and CD3. As shown in Fig 3C and D,
in comparison to controls, thymocytes from mutant mice
demonstrate a significantly elevated percentage of cells of a mature
CD4+CD3hi or CD8+CD3hi
phenotype. These data suggest that the expression of
Sl17H-SCF results in abnormal T-cell development.
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| Fig 3.
A comparison of thymic cellularity and T-cell subset
distribution between Sl/Sl17H and control
Sl/+ mice. (A) Thymocytes were harvested and counted as
described in Materials and Methods. At least 5 mice from each group
were examined. Data shown are the mean ± SEM. *P < .05. Thymocytes from mutant and control mice were harvested and analyzed by
flow cytometry for the expression of (B) CD4 and CD8, (C) CD4 and CD3,
and (D) CD8 and CD3 antigens. The percentage of various T-cell subsets
are indicated.
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Expression of Sl17H-SCF248 and
Sl17H-SCF220 cDNAs in stromal cells:
Comparison with wt SCF cDNAs.
To further examine the biochemical, biological, and cellular
abnormalities associated with the Sl17H-encoded
protein, we isolated and cloned cDNAs representing the Sl17H-SCF248 (soluble) and the
Sl17H-SCF220 (MA) SCF as described in
Materials and Methods. wt and Sl17H cDNAs
were cotransfected into Sl/Sl4 cell line with the
hygromycin expression plasmid, p48. Hygromycin-resistant colonies were
selected and analyzed for the expression of the introduced cDNAs using RT-PCR. The presence of RT-PCR transcripts of the predicted size was
documented in at least 2 clones of each genotype. As shown in
Fig 4 (and as expected), no band is present
in Sl/Sl4stromal cells derived from homozygous mice
deleted of SCF coding sequence (Sl/Sl). Bands of 733 bp and 623 bp representing wt-SCF248 and wt-SCF220 can be
seen in Fig 4 (lanes 3 and 4). Representative clones expressing shorter
PCR products encoding Sl17H-SCF248 (665 bp; lane 5) and Sl17H-SCF220 (555 bp;
lane 6) can also be seen in Fig 4. The bottom panel demonstrates RT-PCR
products amplified using actin primers for RNA loading and integrity.
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| Fig 4.
Analysis of wt and Sl17H mutant cDNA
expression in stably transfected in stromal cells.
Sl/Sl4 stromal cells were transfected with cDNAs
encoding the wt-SCF248 (soluble) and wt-SCF220
(MA) or the Sl17H-SCF248 (soluble) and
Sl17H-SCF220 (MA) mutant isoforms of
SCF. SCF-specific primers (upper panel) were used that amplify both the
wt and the Sl17H mutant forms of SCF. For
semiquantitative analysis and RNA integrity, actin-specific primers
(lower panel) were used. RT-PCR products were examined on a 2.5%
ethidium bromide-containing agarose gel. Upper panel, SCF-specific
primers. Lane 1, water control; lane 2, parental
Sl/Sl4 cell line; lane 3, wt-SCF248
(733 bp); lane 4, wt-SCF220 (623 bp; exon 6); lane 5, Sl17H-SCF248 (665 bp; exon 8); and lane
6, Sl17H-SCF220 (555 bp; exons 6 and
8). Lower panel, actin-specific primers (732 bp). The molecular weight
(MW) marker is shown on the left.
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To examine if the Sl17H-encoded protein is secreted
from stromal cells, we compared the biosynthesis of the
wt-SCF248 and Sl17H-SCF248
proteins. Sl/Sl4 stable stromal cell transfectants
expressing similar levels of RNA (shown in Fig 4) were pulsed with
[35S]-Met and chased for various time points.
Subsequently, conditioned medium consisting of secreted SCF and cell
lysates were harvested for immunoprecipitation. The immunoprecipitated
proteins were analyzed on SDS-PAGE gels.
Figure 5 compares the time course of protein synthesis of secreted (left panel) and cell-associated (right
panel) SCF from stromal cells expressing either the wt or the
Sl17H protein. As seen in the left panel, a 30-kD
glycosylated SCF product is secreted from cells expressing both the wt
and the Sl17H SCF. As previously reported and as
shown in Fig 5 (right panel), the cell-associated primary SCF
translation products of both wt and Sl17HcDNAs are
progressively processed primarily by carbohydrate modification as they
are transported through the endoplasmic reticulum and the Golgi
compartments.14 In addition to small molecular mass proteins representing the unglycosylated forms of SCF, with time, a
43-kD mature cell-associated SCF product is observed in both the wt and
the Sl17H stromal cell transfectants, as seen in
the right hand panel (Fig 5). These data suggest that the
Sl17H-SCF protein is proteolytically processed and
secreted by stromal cells in a fashion similar to wt protein, whereas
the cell-associated form of Sl17-encoded protein is
clearly made and contained in stromal cells.
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| Fig 5.
Comparison of the biosynthesis of wt-SCF248
(soluble) and Sl17H-SCF248 (soluble)
isoforms of SCF. Stable stromal cell transfectants were starved of
methionine-free medium, labeled with 0.5 mCi/mL of
[35S]-methionine, and chased for 0, 1, 2, and 3 hours
with cold medium. Conditioned medium was collected from each cell line.
Immunoprecipitation was performed using a rabbit polyclonal antibody.
Synthesis of soluble (left panel) and cell-associated (right panel)
fractions of SCF from stromal cells transfected with cDNAs listed on
the left are shown. A 30-kD glycosylated SCF product is secreted from
cells expressing the two cDNAs (left panel). In addition to small
molecular mass proteins representing the unglycosylated forms of SCF,
with time, a 43-kD mature cell-associated SCF product is observed in
both the wt and the Sl17H stromal cell
transfectants, as seen in the right-hand panel.
|
|
We and others have previously demonstrated that both soluble and MA
isoforms of SCF can be detected on the cell surface by flow
cytometry.14,21,39 However, the levels of cell surface expression of the 2 isoforms differ significantly, due to rapid proteolytic processing of the soluble SCF in comparison to the MA
isoform.14,15,39 To examine if the presence of novel aa in
the cytoplasmic domain of the Sl17H protein affects
the cell surface expression of SCF, we compared wt and
Sl17H stromal cell transfectants by flow cytometry.
At least 2 clones of stromal cell lines expressing either the wt or the
Sl17H SCF isoforms were stained with an anti-SCF
antibody and analyzed. The left panels in
Fig 6 demonstrate that the cell surface
expression of soluble Sl17H-SCF248
(lower panel) is undetectable in comparison to soluble
wt-SCF248 (upper panel) in representative clones of each
genotype. Because we have previously demonstrated a critical role for
MA SCF in normal erythropoiesis, we next compared the cell surface
expression of the MA wt-SCF220 and
Sl17H-SCF220 isoforms. Compared with
wt-SCF220 expression (upper panel), a significant reduction
in the surface expression of
Sl17H-SCF220 (lower panel) is seen in
Sl/Sl4 stromal cell transfectants. Shown are
representative clones of each genotype. These data suggest that the
surface presentation of Sl17H is defective, despite
apparently normal biosynthesis and half-life.
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| Fig 6.
Flow cytometric analysis of the stromal cell surface
expression of wt-SCF248 and wt-SCF220 or
Sl17H-SCF248 and
Sl17H-SCF220 isoforms of SCF. (Top left
panel) wt-SCF248 (soluble), (bottom left panel)
Sl17H-SCF248 (soluble), (top right
panel) wt-SCF220 (MA), and (bottom right panel)
Sl17H-SCF220 (MA) stromal cell lines
were stained with either isotype control (solid lines) or antimouse SCF
(dotted lines) antibody. Shown is 1 of 2 clones for each genotype
showing similar results.
|
|
Impaired biological activity of the Sl17H protein
expressed in hematopoietic microenvironment derived stromal cells.
These data suggest that impaired erythropoiesis in vivo in
Sl17H mice may be due to reduced membrane SCF
presentation. To examine the biological consequences of expression of
the Sl17H protein in more detail, we compared the
proliferation of an SCF-dependent erythrocytic progenitor cell line,
G1E-ER2, in response to stromal cell presentation of wt and
Sl17H SCF isoforms. Analysis of the SCF-induced
proliferation was performed using a thymidine incorporation assay.
G1E-ER2 cells were cocultivated with mitomycin C-treated stromal cell
transfectants expressing either the wt or the Sl17H
soluble or MA protein. Figure 7A and B
demonstrate increased proliferation of G1E-ER2 cells in response to
stromal cell transfectants expressing either wt or
Sl17H isoforms compared with
Sl/Sl4 stromal cells. However, expression of either
the soluble or the MA Sl17H protein in stromal
cells results in significantly less proliferation of G1E-ER2 cells in
comparison to wt SCF isoforms (Fig 7A and B). These data suggest that
presentation of the Sl17H-encoded protein by
stromal cells is associated with diminished proliferation due to either
defective SCF/c-Kit interactions, reduced stromal cell presentation, or
both.
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| Fig 7.
A comparison of the biological activity of
wt-SCF248 and wt-SCF220 with
Sl17H-SCF248 and
Sl17H-SCF220 isoforms of SCF. G1E-ER2
cells were cocultured for 24 to 48 hours with parental
Sl/Sl4 stromal cells or (A) stable stromal cell
transfectants expressing wt-SCF248 or
Sl17H-SCF248 SCF or (B) stable stromal
cell transfectants expressing wt-SCF220 or
Sl17H-SCF220 SCF. Proliferation was
measured by thymidine incorporation assay. Results show the mean ± SEM of a representative experiment performed at least twice with more
than 1 clone in replicates of 6. *P < .05 Sl/Sl4 v wt (SCF248 and
SCF220); **P < .05 Sl/Sl4 v Sl17H (SCF248 and
SCF220); and P < .05 wt (SCF248 and
SCF220) v Sl17H
(SCF248 and SCF220). Note difference in scale
between (A) and (B).
|
|
Impaired MAP-Kinase and PI-3Kinase activation of
c-Kit+ erythroid progenitors by MA isoform of
Sl17H.
We have previously demonstrated prolonged activation of c-Kit in
response to stromal cell presentation of MA SCF.21,40 To
examine the consequence(s) of impaired membrane presentation of the
Sl17H protein by stromal cells on c-Kit signaling,
we examined the activation of the downstream signaling pathways,
MAP-Kinase and PI-3Kinase/Akt, in G1E-ER2 cells. Activation of the
MAP-kinase pathway was determined by examining the phosphorylation of
Erk-1 and Erk-2. Activation of PI-3Kinase was determined by examining the phosphorylation of Akt, a downstream effector of
PI-3Kinase.33-35 The G1E-ER2 cells were cocultured on
stromal cells expressing either the wt-MA or the
Sl17H-MA SCF for various time points, and cellular
lysates were analyzed. As shown in Fig 8,
coculture of erythroid progenitors on wt-MA results in enhanced and
sustained activation of Akt (upper panel) and of Erk-1 and Erk-2
(middle panel) over a period of 120 minutes. Coculturing G1E-ER2 cells
on stromal cells expressing the Sl17H-MA results in
significant reduction of Akt activation (upper panel) and Erk-1 and
Erk-2 (middle panel) activation in comparison to wt SCF. The bottom
panel represents total protein in each lane. These experiments were
performed at least 3 times using 2 different clones of each genotype
with similar results.
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| Fig 8.
Activation of MAP-Kinase and PI-3Kinase/Akt in erythroid
progenitors by stromal cells expressing either the
wt-SCF220 (MA) or
Sl17H-SCF220 (MA) isoform of SCF.
Factor-starved erythroid progenitors were cocultured with mitomycin
C-treated stromal cells expressing either the wt or the
Sl17H MA SCF for various time points.
Subsequently, at various times, cell lysates were collected and
subjected to Western blot analysis with a rabbit anti-phospho Akt
antibody and a rabbit anti-phospho MAP-Kinase antibody and the enhanced
chemiluminescence detection system. The phosphorylated form of Akt
(upper panel) and MAP-Kinase (Erk-1 and Erk-2) (middle panel) are
indicated. In both the upper and middle panels, lane 1 corresponds to
unstimulated starved erythroid progenitor cells and lanes 2, 3, and 4 correspond to stromal cells expressing the wt-SCF220 (MA)
form of SCF cocultured with erythroid progenitor cells for 10, 60, and
120 minutes, respectively. Lanes 5, 6, and 7 correspond to stromal
cells expressing the Sl17H-SCF220 (MA)
form of SCF cocultured with erythroid progenitor cells for 10, 60, and
120 minutes, respectively. The bottom panel demonstrates the loading
control for total protein in each lane. These experiments were
performed at least 3 times with 2 different clones of each genotype.
|
|
Expression of membrane-restricted SCF improves erythroid deficiency
in Sl/Sl17H mice.
Because the above-mentioned studies demonstrate impaired cell surface
presentation of the Sl17H-SCF, we hypothesized that
the expression of the MR form of SCF (SCFX9/D3) should
improve the phenotypic abnormalities in these mice. To test this
hypothesis, we performed genetic crosses of transgenic mice expressing
SCFX9/D3 cDNA into Sl/Sl17H mutants to
generate Sl/Sl17H-MR mice. We have previously used
this approach to examine the effects of SCFX9/D3 transgene
is Sl/Sld mutants.21 As shown in
Fig 9, expression of the MR-SCF transgene in Sl/Sl17H mice significantly improves their
hematocrits (Fig 9A) and peripheral RBC counts (Fig 9B) in comparison
to Sl/Sl17H mice. However, in comparison to
Sl/+ mice, the expression of SCFX9/D3 transgene did
not completely correct these deficiencies, a result that may relate to
the level or timing of transgene expression during development. No
effects were seen on the coat color, a result similar to our findings
when wt SCF isoforms were expressed in Sld
mice.21
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| Fig 9.
Rescue of erythroid lineage deficiencies by transgene
expression of SCFX9/D3 (MR) in Sl/Sl17H
mice. A comparison of (A) hematocrit levels and (B) peripheral RBC
counts between Sl/+, Sl/Sl17H, and
Sl/Sl17H-MR mice. A minimum of 5 Sl/+, 3 Sl/Sl17H, and 6 Sl/Sl17H-MR
mice were examined in each group. Results show the mean ± SEM.
*P < .05.
|
|
| |
DISCUSSION |
Despite significant molecular and biochemical characterization of SCF,
little is known regarding the physiological role(s) of soluble and MA
SCF isoforms. Sld, a viable Sl mutant,
arose as result of an an intragenic deletion removing the exons
encoding the transmembrane and cytoplasmic domains of
SCF.23 The Sld allele appears to be
capable of producing biologically active secreted protein, although the
truncated protein lacks both exon 7- and exon 8-encoded
membrane-proximal aa's and is therefore not proteolytically processed
in the same fashion as the wt isoform of soluble SCF.16 The
severe hematologic deficiencies in compound heterozygous
Sl/Sld mice have suggested that the MA isoform of
SCF is critical in c-Kit function, and the cytoplasmic domain of SCF is
potentially important in mediating juxtracrine signaling.
We have used the naturally occurring mutation Sl17H
to analyze the biological importance of the SCF cytoplasmic domain as
it relates to cell surface presentation, biosynthetic processing, bioactivity, and juxtracrine signaling by using in vitro and in vivo
genetic approaches. The major conclusions of our study are (1) the
cytoplasmic domain sequences of SCF are important for cell surface
presentation and is abnormal in Sl17H mice; (2)
membrane presentation is important for c-Kit/SCF-mediated juxtacrine
signaling, which is impaired in Sl17H mice; (3)
membrane presentation of SCF is critical for the proliferation/survival of c-Kit-expressing hematopoietic cells and is also defective in
Sl17H mice; and (4) Sl/Sl17H
mice are deficient in RBC production, suggesting a critical role for
membrane presentation in normal erythroid blood cell development.
Our findings that the wt sequences in the cytoplasmic domain of SCF are
required for expression of SCF on the cell surface are in agreement
with reports showing several soluble and membrane-anchored proteins in
which cytoplasmic domain mutations interfere with cell surface
expression. A single point mutation in the cytoplasmic domain of both
the membrane-anchored P-glycoprotein and the c-Kit receptor causes
these proteins to be inefficiently processed.41,42 In
addition, mutations of the COOH-terminal cytoplasmic residues of the
 -proteinase inhibitor or the thyroxine-binding globin also causes
these proteins to be inefficiently processed.43 Proteins
with cytoplasmic domain mutations may not interact properly with
molecules involved in transport from the endoplasmic reticulum (ER) to
the cell surface. Also, incorrect folding as a consequence of mutations
in the cytoplasmic domain may be responsible for the observed defects
on maturation.44,45
We find soluble SCF in the supernatant of HM-derived stable stromal
cell transfectants expressing the Sl17H mutant SCF
by IP at levels comparable to that of wt SCF. However, our in vitro
biological data suggest that stromal cells making soluble SCF are less
capable of stimulating the proliferation/survival of c-Kit+
cells. These data suggest that the altered cytoplasmic domain sequences
of the mutant protein do not eliminate the secretion of soluble SCF,
although they do impair the biological activity of the mutant protein.
In this regard, it is possible that the presence of novel aa's in the
cytoplasmic tail of the mutant protein prevents SCF monomers from
efficiently forming dimers. Some evidence suggests that bivalent
binding of ligands is key for inducing receptor dimerization and
subsequently receptor activation.46 Recent studies using
radiolabeled SCF added to serum have shown that 49% to 72% of the
circulating SCF exists in monomeric form.47 Furthermore, a
detailed examination of dimerization-defective variants of SCF show
substantially reduced mitogenic activity, whereas the activity of
disulfide-linked SCF dimer was 10-fold higher than that of wt
SCF.47 These results suggest a correlation between
dimerization capacity and biological activity.
Based on these findings, one would predict that SCF and other cytokines
that are synthesized as membrane-anchored proteins are expressed on the
cell surface as dimers to efficiently activate their cognate receptors.
Indeed, recent biochemical studies have shown that most of the membrane
SCF exists as a dimeric protein, and the presence of novel aa's in the
Sl17Hmutant may result in inefficient dimerization
and reduced adhesion of mast cells to COS cells, in comparison to wt
SCF COS cell transfectants.48 However, the interpretation
of these data may not be straightforward. These experiments were
performed on membrane fractions derived from COS cell transfectants
that already expressed 50% less cell surface SCF, making it difficult
to conclude if the diminished Sl17H dimerization in
COS cell transfectants is due to a reduction in total cell surface
protein or due to an actual impairment in dimerization as a result of
the presence of 28 novel aa's in the cytoplasmic domain.
An important determinant for anchoring proteins in the membrane is the
presence of positively charged aa's located immediately adjacent to
the transmembrane region within the cytoplasmic
domain.26,27 Mutations that reduce the net positive charge
in this region can lead to membrane protein instability.27
In the case of SCF, the first 3 aa's of the cytoplasmic domain are
Lys-Lys-Lys (+3). In the Sl17H protein, these aa's
are Lys-Tyr-Ala (+1), a net reduction in positive charge. This
reduction in positive charge could significantly reduce the stability
of SCF within the cell membrane and, consequently, its ability to
interact with c-Kit. Our flow cytometric data demonstrating reduced
cell surface expression and biological data showing reduced activity by
stromal cell transfectants expressing the soluble and MA
Sl17H SCF would support this notion. In this
regard, our results are consistent with previously published studies in
which up to 50% reduction in the amount of cell surface
Sl17H mutant SCF in COS cells and in primary
BM-derived stromal cells was reported.48,49 Our data, along
with the observations made by others, would suggest that a reduction in
the function of cell surface expressed Sl17H mutant
SCF is due to reduced SCF membrane presentation. Importantly, the
apparent decrease in PI-3Kinase/Akt activation and MAP-Kinase activation in erythroid progenitor cell line by the mutant MA SCF
further supports the notion that appropriate presentation of SCF is
necessary for full activation of 2 important signaling pathways
downstream from Kit involved in cell proliferation.
The Sl17H mutation has previously been shown to
affect melanogenesis and gametogenesis.25 In addition,
Sl17H homozygous mutants
(Sl17H/Sl17H) also demonstrate reduced
mast cells and impaired homing of stem/progenitor cells to the
spleen.48 In this report, we demonstrate that BM cellularity, peripheral RBC counts, hematocrit levels, and T-cell development were appreciably affected in Sl/Sl17H
compound heterozygous mice. The reduced cell surface presentation we observed likely contributes to these hematopoietic deficiencies. We
and others have previously shown that Sld mice also
suffer from impaired erythropoiesis. It has been hypothesized that this
deficiency is due to the lack of MA SCF in Sld
mice. In this regard, we have recently shown that
Sld mice expressing MR SCF as a transgene produce
significantly more RBCs in comparison to Sld mice
or Sld mice expressing the soluble form of
SCF.21 Recent work by Tajima et al24 have
confirmed that soluble SCF is not required for RBC production. We
observed reduced cell surface presentation of the
Sl17H-encoded protein, and a significant decrease
in the proliferation and activation of downstream signaling molecules,
in an erythrocytic progenitor cell line by Sl17H MA
SCF. These data, along with the correction in RBC deficiency seen in
Sl/Sl17H mice expressing the MR form of SCF,
suggest a critical role for the cell-surface presentation of SCF in
normal erythropoiesis.
| |
FOOTNOTES |
Submitted February 18, 1999; accepted May 4, 1999.
R.K. was initially supported by a postdoctoral fellowship from the
Leukemia Society of America and is currently being supported by a
National Institutes of Health (NIH) postdoctoral fellowship (NRSA F32
DK09752). Supported by grants from the NIH to D.A.W. (RO1DK48605).
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.
Address reprint requests to Reuben Kapur, PhD, Herman B Wells Center
for Pediatric Research, Cancer Research Building, 1044 W Walnut, Room
402C, Indianapolis, IN 46202-5225; e-mail: rkapur{at}iupui.edu.
| |
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