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Xenogeneic Expression of Human Stem Cell Factor in Transgenic Mice
Mimics Codominant c-kit Mutations
By Manas K. Majumdar, Eric T. Everett, Xiangli Xiao, Ryan Cooper, Keith Langley, Reuben Kapur, Terry Vik,
and David A. Williams
Mutations of c-kit, which encodes a transmembrane receptor tyrosine kinase, have been identified in mice by abnormal
coat color, anemia, and germ cell defects. Mice heterozygous
for mutations of c-kit have a white forehead blaze and a
white ventral spot, leading these mutants t o be termed dominant White spotting ( W). We have previously demonstrated
that the membrane-associated isoform of human stem cell
factor (hSCpO,the ligand for c-kit) is inefficiently processed
in murine stromal cell transfectants. Thus, in murine cell
lines analyzed in vitro, h S C p otransfectants present SCF as
a membrane restricted protein in contrast t o the murine
SCFmocDNA protein product, which is slowly cleaved and
secreted. We show here that transgenic mice expressing the
human SCFZo isoform in vivo display a phenotype indistinguishable from some alleles of W. Specifically, hSCFmO-expressing transgenic mice display a prominent forehead blaze
and a white ventral spot. Generations of doubly heterozygous animals that carry both a mutated c-kit allele and the
h S C f2o transgene display a more severe coat color abnormality. This phenotype appears t o be due t o occupancy of
murine c-kit by human SCF and diminished cell surface expression of endogenous murine SCF. Normal signaling
events that lead t o cell survival or proliferation appear t o be
disrupted in vivo in these transgenic mice.
0 1996 by The American Society of Hematology.
R
although distinct tissue distribution of each isoform exists."
Furthermore, we have recently noted that the protein product
of hSCFZZfl
cDNA is inefficiently cleaved in the context of
murine stromal cells, and thus the presentation of the MA
protein of hSCF is altered in murine cells in vitro."
Viable mutant alleles of the SZ gene have contributed to
our understanding of SCF function. Two alleles, Steel-Dickie
(SZd) and SteeZ-17H (SZ'7H) suggest that the MA isoform of
SCF is critical for all three affected cell lineages. SZd results
from a small intragenic deletion of sequences encoding the
transmembrane and carboxy terminus of the SCF protein
leading to the generation of a biologically active, obligately
secreted mutant protein. The SZ'7H mutation has been shown
to arise as a result of a splicing defect that results in skipping
of exon 8 and a mutant cytoplasmic tail derived from a
frame-shift in the reading frame." Compound heterozygous
SZ/SZd mice are viable, but sterile, with white coat color and
severe anemia. Homozygous SZ'7H/SZ'7H mice are mostly
white with mild anemia. Males with this genotype are sterile,
whereas females have reduced fertility. The persistence of
phenotypic abnormalities associated with S1 mutants that
lack proper expression or function of MA SCF suggest the
importance of this isoform of the protein in normal cellular
functions in the affected lineages.
Our laboratory has also demonstrated important differences in proliferation and/or survival of human hematopoi-
ECEPTOR TYROSINE kinases and their growth factor
ligands play important roles during mammalian development, including regulation of cell proliferation, differentiation, survival, and possibly migration. The proto-oncogene
c-kit encodes a transmembrane receptor tyrosine kinase that
is a member of the platelet-derived growth factor (PDGF)
receptor subfamily. The ligand for c-kit has been identified
and is termed stem cell factor (SCF), kit ligand (KL), steel
factor, or mast cell growth factor (MGF).3-6In mice, SCF is
encoded by the Steel (SI) gene; and White spotting ( W )
and Steel (SZ) mutations are allelic with c-kit and S1 genes,
respectively. Mutations at either locus affect the development of several cell lineages, including primordial germ
cells, neural crest-derived melanoblasts, and hematopoietic
cells, particularly stem, erythroid, and mast cells.' W and SZ
homozygous mice have similar phenotypes, which include
sterility, severe macrocytic anemia, hypopigmentation, and
mast cell deficiency; these deficiencies often culminate in
prenatal and perinatal lethality.' However, some mutant alleles (eg, W 4 ' ) demonstrate milder defects that appear to
differentially affect melanocytes leading to black-eyed white
homozygous mice with near normal hematopoiesis and variable fertility. Depending on the specific mutant allele, heterozygous mice also demonstrate varying phenotypes, including
white spots with normal or only slightly deficient hematopoiesis and fertility; these mutations are considered codominant.
The product of the SI gene is expressed in supporting cells,
such as stromal cells, which interact with target c-kit expressing cells.
Multiple isoforms of SCF arise in both mice and humans
via alternatively spliced transcripts, which result in membrane associated glycoproteins of 248 (SCFZ4') and 220
(SCFZz0)amino acids (aa).'.'" SCFZ4' is rapidly cleaved to
release a soluble (S) protein of 164/165 amino acids. SCFZz0,
which lacks the proteolytic cleavage site encoded by the
differentially spliced exon 6 sequences, remains predominantly membrane-associated (MA), but can be slowly released from the cell surface via the use of an alternative
proteolytic cleavage site in exon 7.'"." Little is known regarding the physiologic roles of these two isoforms during
embryonic development or postnatal biology of the mouse,
Blood, Vol 87, No 8 (April 15). 1996:pp 3203-3211
From the Section of Pediatric Hematology/Oncology, Herman B
Wells Center for Pediatric Research, James Whitcomb Riley Hospital for Children, and the Howard Hughes Medical Institute, Indiana
University School of Medicine, Indianapolis: and AMGEN Inc, AMGEN Center, Thousand Oaks, CA.
Submitted September 8, 1995; accepted November 30. 1995.
Address reprint requests to David A. Williams, MD, Herman B
Wells Centerfor Pediatric Research, Howard Hughes Medical Institute, 702 Barnhill Dr, Indianapolis, IN 46202-5225.
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.
0 1996 by The American Society of Hematology.
0006-4971/96/8708-O$3.00/0
3203
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3204
MAJUMDAR ET AL
etic stem cells when exposed in vitro to either the MA or
secreted (S) isoforms of human SCF.I3 Other investigators
have noted similar differences in the response of murine
germ cells to either MA or S isoforms of mouse SCF.I4.l5
To further understand the biology of MA and S isoforms of
SCF we have generated transgenic mice expressing either
MA or S isoforms of both murine and human cDNAs.
Founder mice expressing either murine (m) SCF''" or
mSCF"* demonstrated no gross phenotypic abnormalities.16
Since the secreted form of human SCF has very low affinity
for rodent c-kit4*I7and, compared with the secreted form of
rodent SCFs, stimulates rodent c-kit positive cells only at
high concentrations, we predicted no phenotypic abnormalities in mice expressing human (h)SCF. Remarkably, founder
mice expressing the human (SCF220)isoform (and to a lesser
degree hSCFZ4*)demonstrated coat color deficiencies that
mimic heterozygous W mice.
MATERIALS AND METHODS
Generation of transgenic mice. All mice were obtained from
Jackson Laboratories (Bar Harbor, ME). Transgenic mice were generated by microinjecting into the pronuclei of fertilized C3IUHe.l
eggs a 1.3 kb Nde VKpn I fragment comprising either the hPGKhSCF2*' or the hFGK-hSCFN* minigene (Fig l).'* Microinjected
eggs were transferred to the oviducts of pseudopregnant outbred
Swiss-Webster females. Offspring were tested for the presence of
the transgene by analyzing tail DNA. Tail DNA was digested with
Nde I and used as a template for PCR. Primer pairs specific for
hPGK (5'-CACGTCGGCAGTCGGCTCCCTCG'ITGACCG) and
hSCF (5'-ATGACTTGGCAAAACATCCATCCCGGGGAC) were
used to amplify a 0.8 kb product. The PCR products were transferred
by Southern blot and probed using the hPGK-hSCFZZ0
cDNA construct. To confirm the presence of the transgene, DNA from PCRpositive mice was digested with EcoRI, electrophoresed, transferred
to filters, and probed using the hPGK-hSCFZZ'cDNA construct.
Protein analysis. Immortalized stromal cell lines were generated
from long-term marrow cultures derived from transgenic mice as
previously described" and analyzed for the expression of hSCF.
Metabolic labeling and immunoprecipitation were performed as previously described." Briefly, cells were labeled overnight with ['%IMet-Cys (ICN Radiochemicals, Irvine, CA). Conditioned medium
and cell lysates were collected and immunoprecipitation was performed using a rabbit polyclonal antibody raised against human SCF
(kindly provided by Dr Lany Bennett, Amgen, Thousand Oaks,
CA). Proteins were analyzed by a 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). To determine the
effect of expression of the transgene on endogenous cell surface
SCF expression, these same cell lines were examined by flow analysis with a rat anti-murine SCF monoclonal antibody that recognizes
murine SCF. Briefly, 0.5 X lo6 stromal cell were stained separately
with 0.5 pg of primary rat antimouse SCF monoclonal antibody
(Genzyme, Cambridge, MA), rabbit antihuman SCF polyclonal antibody (kindly provided by Dr Larry Bennett, Amgen) or pre-immune
rabbit serum for 30 minutes at 4°C. Afterward, the cells were washed
twice with phosphate-buffered saline (PBS)/O.1% bovine serum albumin (BSA) and subsequently stained with 2 pg of secondary
fluorescein-isothiocyanate (FlTC) conjugated goat F(ab' )2 antirat
IgG (GIBCOISRL, Gaithersburg, MD) or goat F(ab')z antirabbit
IgG (Sigma, St Louis, MO) under identical conditions. In addition,
total cellular RNA was isolated from these cells followed by Northem blot analysis using the mSCF cDNA (a gift of F. Martin, Amgen)
as a probe.
Cell proliferation, receptor, and MAP kinase studies. Low density bone marrow cells from C57B116 mice were obtained by flushing
femur cavities as previously described?' followed by separation on
Histo-Paque (Sigma) (1,650 rpm for 25 minutes at 15°C). Resulting
low density cells were washed and incubated at 4°C with 20% (vol/
vol) goat serum (GIBCO). These cells were washed twice, resuspended in cold PBS with 0.1% BSA and depleted of plastic-adherent
cells for 30 minutes at 37°C. The resulting low density, nonadherent
cells were then stained for surface expression of c-kit using the
ACK-2 rat monoclonal antibody (MoAb; GIBCOISRL) (2 pg/106
cells) (or IgG2b isotype control) for 30 minutes at 4°C in PBS/O.l%
BSA, followed by a secondary FITC conjugated goat antirat IgG
(GIBCOISRL) under identical conditions. Stained cells were sorted
using fluorescence activated cell sorting. Post-sort c-kit" cells were
then maintained in alpha-minimum essential medium (MEM) with
5% fetal calf serum and recombinant mIL-3 (100 U/mL; Genzyme,
Boston, MA) overnight at 37°C in 5% COz before further use.
The effect of hSCF on HPP-CFC survival was performed by
incubating c-kit' murine bone marrow cells for 3 days with recombinant (r) human (h) or rat (r) SCF (Amgen) at the concentrations
noted below. Following this incubation, all cells were removed and
plated for HPP-CFC as previously described.2"
The effect of rhSCF or rrSCF on c-kit receptor down-modulation
in c-kit+ murine hematopoietic cells was performed on these same
cell populations. Since we have previously demonstrated that the
interaction of human MA SCF with c-kit substantially delays receptor down-modulation,2' we used soluble SCF for these studies. ckit' cells were removed from IL-3 and washed twice with PBS/
0.1% BSA at 4°C. Cells were incubated with ACK-2 MoAb, or
IgG2b isotype control, no SCF, I O 0 ng/mL rrSCF or 100 ng/mL
rhSCF for 15 minutes at 37°C or 4°C. Following this incubation
both groups of cells were washed twice in ice cold PBS/O.I% BSA
and stained with ACK-2 antibody as noted above.
The effects of stromal cell lines generated from transgenic mice
on the survival and proliferation of murine c-kit' bone marrow cells
were studied by using c-kit' and stromal cells as described above.
Stromal cells were pretreated with 10 pg/mL mitomycin C as previously de~cribed'~
to inhibit continued proliferation and plated at a
density of 3 x lo5 cellslwell on gelatin-coated (Sigma) 6-well tissue
culture plates (Falcon, Lincoln Park, NJ). c-kit' murine bone marrow
cells were cocultured on each stromal cell line for 3 days. Subsequently, all cells were harvested and plated in HPP-CFC assays as
described elsewhere.2"
Mitogen activated protein (MAP) kinase studies were performed
on c-kit+ bone marrow cells isolated as described above and incubated overnight in mIL-3. Cells were stimulated with rrSCF or rhSCF
at 100 ng/mL for 15 minutes. Unstimulated cells were also analyzed.
Cell lysates were prepared as previously described." MAP kinase
activity was measured by in vitro kinase assay with 10 pL lysate in
30 pL reaction with 20 mmoVL Tris-HC1 (pH 7.2), 10 mmol/L
MgC12, 100 mmol/L ATP with 5 pCi [gan~ma-'~P]ATP, and 5
pg myelin basic protein (MBP) (all from Sigma). Reactions were
incubated at 30°C for 1.5 minutes, then stopped by adding SDSPAGE sample buffer. Assays were performed in triplicate and resolved by SDS-PAGE. MBP bands were visualized by Comassie
Blue staining, excised from the gel, and ["PI incorporation was
measured by scintillation spectroscopy.
RESULTS
Human SCFzZotransgenic mice express MA protein and
exhibit a W phenotype. Sixteen mice were obtained following pronuclear injection of the hPGK-hSCF"' expression
plasmid (Fig 1) and screened using polymerase chain
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TRANSGENIC HUMAN SCF MIMICS C-KIT MUTATIONS
3205
hSCF220
Aat II
AatII,
Nde I
Ndel
-1
Sph If
Sphlf
r
Hind Ill
Sac II
HindIII
0.8kb1 -
Fig 1. P G K h S C P and PGKhSCfa transgene constructs. Transgene expression of the h S C P and h S C f a cDNAs in founder mice was
achieved using a minigene cassette consisting of the human SCFn0 or hSCFza cDNA expressed from the human PGK promoter. The PGK
promoter, consisting of a 514 bp 5'-flanking sequence from the X-linked human phosphoglycerate kinase-1 gene" up t o but excluding the
translational start codon was subcloned into pGEM-7 as an Ast ll/Sph Ifragment. The hSCFUo or hSCF2" cDNA in addition t o a splice donor/
acceptor and poly A site was subcloned from the expression plasmid v19.8hSCFm or vl9.8hSCf" as a Xho IIKpn Ifragment 3' t o the PGK
promoter in the pGEM-7 plasmid vector to generate the final transgene plasmid. hPGKpr: human phosphoglycerate kinase promoter; SD/SA
splice donorlsplice acceptor sequences; hSCFzzocDNA human SCF cDNA (exon 6 3 encoding membrane-associated protein; hSCFza cDNA
human SCF cDNA (exon 6') encoding secreted protein; poly A polyadenylation sequence. Arrows show location of PCR primers used for
analysis of transgenic founders.
reaction (PCR) and Southern blot analysis. Six mice were
found to carry the hSCF22n transgene. One female
founder TgN(PGKhSCFZzn)452Dawand one male founder
TgN(PGKhSCF22n)441
Daw [hereafter referred to as TgN
(PGKhSCF22n)]each had a large white spot on the ventral
surface, diamond-shaped white spot on the forehead, white
feet, and a banded tail (Fig 2, animal number 4). The remaining mice had no phenotype and were not studied further.
One transgenic mouse obtained from injection of the PGKhSCF248plasmid (Fig 1) had a faint forehead blaze and very
mildly diluted belly spot. Subsequent breeding of this animal
demonstrated that this phenotype was not stable and this line
was not studied further. The phenotype seen in TgN
(PGKhSCF22n)mice is very similar to the coat-color deficiency seen in W/+mice. Transgenic lines have been established from TgN(PGKhSCF2") following mating of this
founder to C3WHeJ mice. The transgene and unique coat
color were passed on to 50% of the offspring. Coat color
and transgene were found not to segregate, nor was there
variation in the penetrance of the coat color deficiency
through six generations. Founders as well as their progeny
canying the transgene appeared both hematologically normal and fertile. The present studies were conducted using
offspring from the TgN(PGKhSCF2") line. Indirect evidence
that the phenotype is related to the level of transgene expression was obtained by generating homozygous transgene animals from TgN(PGKhSCFZzn)mice. Mice homozygous for
the transgene demonstrated a more prominent forehead
blaze, with secondary white spots over the cervical region
and occasionally elsewhere as well as significantly larger
belly spot (E.T.E. and D.A.W., unpublished results). These
animals also demonstrated abnormal thymic development
(R.K. and D.A.W., manuscript in preparation). Homozygosity for the human transgene was confirmed by backcrossing
to normal mice.
The expression and membrane-restricted localization of
hSCF protein in transgenic mice was determined by immunoprecipitation (IP) (Fig 3) of conditioned medium (lanes 1
through 6) and cell lysates (lanes 7 through 12) of immortalized bone marrow stromal cell lines generated from the F,
offspring of TgN(PGKhSCF2*")founder." Immunoprecipitations were performed with a polyclonal antibody generated
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MAJUMDAR ET AL
3206
1
2
3
4
A
B
C
M
Fig 2. F, Progeny from crosses between TgN(PGKhSCFzz01
and W!
mice. TgN(PGKhSCFZz0)
transgenic mice were bred to dominant
White Spotting mutant mice I W / + ) and the resulting progeny were
analyzedusing coat color as a marker. (1I Wild-type agouti coat color;
(2) typical W / + markings; (3)typical markings of TgNIPGKhSCFZZoI
x W / + compound; (4) TgN(PGKhSCFUo)founder. ( A X ) Dorsal, lateral,
and ventral view, respectively.
+
against hSCF, which effectively distinguished human and
mouse proteins." Stromal cell lines expressing hSCF (lanes
8, 1 I , and 12) give rise to multiple bands presumably due
to posttranslational glycosylation of the protein. The major
bands (approximately 35 kD) immunoprecipitated with this
antisera are almost entirely cell associated since little secreted human protein is detected in conditioned medium
(lanes 2, 5 , or 6) collected from these cells. Lanes 5, 6, 1 I ,
and 12 are immunoprecipitations derived from the
TgN(PGKhSCF"?")line. Other lanes contain immunoprecipitations of murine SCF expressing stromal cells and no
cross-hybridizing hands are seen in these lanes. Thus the
expression of hSCF"" in vivo in transgenic mice is primarily
membrane-bound.
Intemction of PGKhSCF-"" with W. These data suggested that expression of normal hSCF"" in murine cells and
its localization to the membrane in vivo mimic codominant
c-kit mutations. Since founder animals were derived from
mice expressing normal levels of endogenous c-kit, we hypothesized that coexpression of the hSCF"" transgene in the
setting of a mutated or deficient c-kit protein would result
in a more severe phenotype. To test this hypothesis, we
crossed hSCF"" transgenic mice with W/+ mice.
Crosses involving W/+ (Fig 2, animal no. 2) and
TgN(PGKhSCF"") (Fig 2, animal no. 4) produced progeny
of which 29% ( I 6/56) demonstrated wild-type (+/+) agouti
coloration (Fig 2, animal no. I). Forty-six percent (26/56)
of the mice demonstrated coat coloration similar to either
parent (Fig 2, animal no. 2) [on an agouti background
TgN(PGKhSCF"") and W/+ appear very similar and are
difficult to distinguish]. The remaining 25% (14/56) of the
progeny demonstrated a more severe deficiency in coat color
pigmentation (Fig 2, animal no. 3) than either parent (characterized by large areas of hypopigmentation). Doubly heterozygous TgN(PGKhSCF""/+, W/+)
mice were fertile and not
anemic. However these mice demonstrated greatly reduced
numbers of connective tissue mast cells (data not shown).
Similar results were seen when TgN(PGKhSCF"") mice
were crossed with SI/+ mice (data not shown). Progeny from
genetic crosses with SI/+ were produced in the following
proportion: 27% (6/22) wild-type, +/+; 27% (6/22), SI/+;
27% (6/22), TgN(PGKhSCF""); and the remaining 18% (41
22) progeny exhibited a more severe pigment loss. In these
crosses SI/+ and transgenic animals have distinguishable
phenotypes, since SI results in diluted ventral pigment, while
the transgene results in a pure white belly spot. Genetic
crosses between TgN(PGKhSCF"") transgenic mice and SI/
+ mice offer further confirmation that the phenotypic abnormalities seen in transgenic mice were due to disruption of
the SCF/c-kit signaling pathway.
Previous work has demonstrated that binding of SCF to ckit leads to receptor dimerization, autophosphorylation, and
signaling followed by receptor internalization and degradation.?l.'"2" However, hSCF has previously been shown (by
Scatchard analysis) to bind murine c-kit (mc-kit) with low
affinity (kd > 700 nM)."" We hypothesized that expression
of hSCF'"' in transgenic mice may occupy mc-kit due to
high local concentration of the membrane-associated human
protein, that hSCF"" may form cell-associated heterodimers
with mSCF or that expression of the transgene may lead to
modulation of expression of the endogenous SCF protein,
any of which might interfere with normal mSCF/mc-kit interaction. To determine if the deficiency in coat color observed in TgN(PGKhSCF'")) mice was a result of altered
interaction of murine c-kit and hSCF'"', functional studies
using c-kit primary murine bone marrow cells were performed. Since receptor down modulation studies are difficult
to perform with MA protein (see above)," we elected to
perform these studies with soluble recombinant human SCF
protein. As previously noted," treatment of c-kit' murine
hematopoietic cells with increasing concentrations of hSCF
demonstrated an inability of hSCF to induce cell prolifera-
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3207
TRANSGENIC HUMAN SCF MIMICS C-KIT MUTATIONS
1 2 3 4 5 6 7 8 9 1011 12
MW
97 -
5035 29,
21 -
Fig. 3. Immunoprecipitation of stromal cell lines derived from TgN(PGKhSCF? transgenic mice. Immunoprecipitation of conditioned
medium (lanes 1 through 61 and cell lysates (lanes 7 through 12). Molecular weight markers are shown on the left. Immortalized bone marrow
stromal cells from TgN(PGKhSCFzz0Iwere generated from long-term bone marrow cultures as previously described” and analyzed for the
expression of hSCF. A homozygous SIISI lie, SCF-deficient) stromal line‘ and stable transfectants expressing hSCF and mSCF cDNAs”.’~ were
used as positive and negative controls, respectively. Metabolic labeling and immunoprecipitation were performed as previously described,”
see the Materials and Methods. Lanes 1 and 7, SIISI‘, a stromal cell line expressing no SCF (negative control]; lanes 2 and 8, Sl‘Ih220, a
stromal cell line derived from SI/SI‘ expressing only human membrane-associated SCF’’; lanes 3 and 9, SI‘Im220, a stromal cell line derived
from SIISI‘ expressing only murine membrane-associated SCF”; lanes 4 and 10, U2, a bone marrow stromal cell line generated from normal
mouse2’; lanes 5, 11, 6, and 12, both stromal cell lines (clones 2 and 5, respectively1 generated from adult transgenic TgN(PGKhSCFml mice
expressing the hSCFno transgene (and endogenous mSCF). Arrow at left (around 28 t o 30 kD] shows the position of secreted protein, which
is not present in lanes 5 or 6; bracket on right shows position of human cell-associated SCF, not present in lanes 7, 9, or 10. X-ray film was
exposed at -80°C for 1 week.
tion, except at very high concentrations (data not shown).
In addition, the survival or proliferation of primitive high
proliferative potential-colony forming cells (HPP-CFC) was
unaffected by hSCF at low to moderate concentrations (Fig
4). whereas rrSCF induced an increase in HPP-CFC (Fig 4)
when compared with no growth factor or human SCF. Similar results were obtained using low proliferative potential
(LPP)-CFC (data not shown). In contrast, these same concentrations of hSCF (100 ng/mL) resulted in a substantial downmodulation of murine c-kit expression even though they are
insufficient to induce cell proliferation (Fig 5A to C). These
data imply that hSCF physically interacts with murine c-kit
at concentrations insufficient to trigger downstream signaling events.
These data were further confirmed by examining MAP
kinase activity. Bone marrow cells isolated from mice and
selected for expression of cell surface c-kit have a low endogenous MAP kinase activity following overnight culture
in mIL-3. MAP kinase activity has previously been shown
to be increased in cells stimulated with SCF when compared
with controls.27The assay for MAP kinase is very sensitive,
and measures the down stream effect of c-kit activation. This
provided a convenient model to look as signals generated
by rodent vs. human SCF. We measured MAP kinase activity
in whole cell extracts (Fig 6), as well as by immunoprecipitation kinase assays and consistently found significant eleva-
tion of MAP kinase activity in cells treated with rat, but not
human, SCF (Fig 6).
Further, direct evidence of hSCF”” interference with the
function of murine SCF was seen when c-kit’ murine hematopoietic cells were cocultured on stromal cell lines derived
from transgenic mice expressing both membrane-associated
hSCF and endogenous mSCF. Survival or proliferation of
primitive HPP-CFC was reduced on stromal cell lines expressing hSCF compared with wild-type stromal cells or
stromal cells expressing high levels of only membrane-associated mSCF (Fig 7). In additional experiments using recombinant SCF, co-incubation of high concentrations of hSCF
(up to 500 ng/mL) with rrSCF led to decreased growth of
HPP-CFC from purified c-kit’ murine bone marrow cells in
response to rodent SCF (data not shown). These data provide
indirect evidence that the expression of h-SCF and the maintenance of this protein on the cell surface may interfere with
SCF/c-kit signaling in vivo.
Because the phenotypic abnormalities seen in these
transgenic mice could also be explained by the replacement
of endogenous murine SCF with less active human SCF, we
examined the expression of endogenous mSCF in cell lines
derived from the transgenic mice. Northern blot analysis of
total RNA from stromal cell lines used in the proliferation
assays described above demonstrated comparable levels of
the major -5 kb-mSCF mRNA transcript (data not shown).
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MAJUMDAR ET AL
3208
U
0
10
100
SCF (ngfml)
Fig 4. Effect of human SCF on survival of e-kit' murine bone marr o w cells. Low density bone marrow cells from C57B116 mice were
obtained by flushing femur cavities as described (see the Materials
and Methods). Low density, nonadherent cells were then stained
for cell surface expression of c-kit using the ACK-2 rat monoclonal
antibody, followed by staining with an FlTC conjugated goat antirat
lgG under identical conditions (see the Materials and Methods). Cells
were sorted on a FACStar Plus (Becton Dickinson) flow sorter after
gating for forward and side scatter. c-kit' cells were collected based
on fluorescence intensity 95% higher than isotype control. The percentage of c-kit+ bone marrow cells ranged from 5% t o 10% of the
gated population and post-sort purity was >go%. Four hundred
eighty c-kit+ cells were incubated in rhSCF or rrSCF at the concentrations noted for 3 days. Subsequently all cells were removed and
plated for HPP-CFC as previously described?" Results show one of
three independent experiments giving similar results as mean f SD,
n = 4. Differences between hSCF and rSCF, P < ,0001.
with abnormal coat color, but normal fertility and hematopoiesis. In addition, Ray et al have demonstrated coat color
abnormalities when the W42mutant form of c-kit was expressed in transgenic mice, although inheritance of this phenotype did not appear stable.2yWe surmise that the tremendous cell amplification possible in hematopoietic and germ
cell lineages may compensate for mild abnormalities in these
lineages in TgN(PGKhSCF'20) mice. Alternatively, the coat
color deficiency may be due to increased sensitivity of melanoblasts to alterations in c-kit signaling.
In this study, we demonstrate that expression of normal
human membrane-associated SCF in transgenic mice mimics
a codominant mutation of the receptor for SCF, c-kit. The
phenotypic abnormalities seen in these mice suggest that
expression of hSCF in vivo can lead to c-kit receptor occupancy but less than optimal activation of the receptor. The
mechanism of suboptimal mc-kit activation could be displacement of endogenous mSCF. This mechanism requires
0
I
- lsotype
--
Flow cytometric analysis demonstrated significant levels of
human SCF on the surface of a representative stromal cell
line, hm220-2, derived from a transgenic mouse (Fig 8A).
The antihuman SCF antibody does not crossreact with endogenous mSCF, as demonstrated by the absence of cell
surface staining on murine U2 bone marrow stromal cells
(Fig 8A). However, significant levels of membrane associated endogenous mSCF is detected on U2 stromal cells upon
staining these cells with an antimurine SCF MoAb (Fig 8B).
In contrast, expression of the MA isoform of endogenous
mSCF is significantly reduced on stromal cell lines (hm2202 and hm220-5 cells) derived from the TgN(PGKhSCF22")
mice, as shown for hm220-2 in Fig 8C. Thus, expression
of hSCF on the surface of transgenic-derived stromal cells
appears to be associated with diminished presentation of
endogenous MA protein.
DISCUSSION
Multiple mutants of SI and W have been characterized at
the molecular level. Several mutants of c-kit are associated
with residual receptor kinase activity (reviewed in Blovin et
aI2'). These mutants often demonstrate milder phenotypes
compared with mutants lacking any kinase activity. Phenotypes of these mutants, particularly v4
and v7,
are remarkably similar to that of TgN(PGKhSCFZZ0)
mice reported here,
.'.
rSCF
.-.:.
0' -." 101 ;02''1bS
;4
,
Fluorescence
lnfensity
Fig 5. Effect of SCF on cell surface expression of murine c-kit by
antibody staining. For receptor studies, c-kit' cells were removed
from IL-3, washed at 4°C. and resuspended in no SCF, 100 ng/mL
rrSCF, or 100 ng1mL rhSCF. Cells without ligand were maintained at
4°C or 37%. while cells incubated with rrSCF or rhSCF were incubated
at 37°C for 15 minutes, cooled quickly t o 4°C and stained with the
ACK-2 antibody as above or with isotype control. Results show (A)
level of surface expression with no growth factor (compared with
isotype control); (B) downmodulation of surface expression induced
by rrSCF; and (C) downmodulation induced by rhSCF. Experiments
performed at 4°C demonstrated that the presence of the ligand had
no effect on detection of surface expression of receptor (data not
shown). Results shown are of one of three independent experiments
demonstrating similar results.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
TRANSGENIC HUMAN SCF MIMICS C-KIT MUTATIONS
"
c-kit+ rSCF hSCF MBP
cells
alone
Fig 6. Activation of MAP kinase activity b y mSCF versus hSCF.
Representative experiment showing activation of MAP kinase in ckit' mouse bone marrow cells stimulated with either rSCF or hSCF
for 15 minutes. Mean f SD phosphate incorporation into substrate
is shown for unstimulated primary bone marrow cells, rSCF-treated
cells, hSCF-treated cells, or substrate alone. The difference between
values of rSCF and hSCF induced activity is significant t o a level of
P < .001.
physical interaction between human SCF and murine receptor. The results of receptor down-modulation studies support
the possibility that hSCF interacts with mc-kit at concentrations found to be insufficient to stimulate cell proliferation.
Uncoupling of receptor binding and signal transduction has
been previously reported by Langley et aI3' with truncated
forms of human SCF expressed in Escherichia coli. Recombinant forms of hSCF truncated at aa 127, 130, and 137 all
demonstrated receptor binding, but induced significantly less
cell proliferation of UT-7 human megakaryoblastic leukemia
cell line. Our data suggest that transgenic expression of analogous truncated murine forms of SCF in vivo should yield
phenotypically abnormal mice.
The phenotypic abnormalities of the transgenic mice reported here may be contributed to by the membrane localization of the human growth factor in murine cells. It has been
postulated that the phenotypic abnormalities associated with
the SZd mutant, which lacks the membrane-spanning portion
of the protein, implicates membrane-associated SCF in normal biologic processes involving c-kit." Our laboratory has
previously identified both the primary and secondary proteolytic cleavage sites present in SCF protein and demonstrated
that the human and mouse sequence are divergent around
the secondary site." Expression of human SCF2" in murine
cells in vitro leads to inefficient cleavage at the second site,
with diminished secretion of protein from the hSCF2" isoform. In the studies reported here, hSCF**' also appears to
be restricted to the membrane in vivo. The low affinity of
human SCF for murine c-kit4." and the results of immunoprecipitation from cells derived from the transgenic mice
suggest that the local concentration of hSCF at the cell surface in vivo in TgN(PGKhSCFZ2') must be high. Delay in
c-kit receptor internalization in response to membrane-asso-
3209
ciated versus soluble SCF has been previously demonstrated
with human SCF and c-kit positive human cell lines2' and
with murine SCF and c-kit positive murine primary bone
marrow cells (R. Cooper and D.A.W., unpublished results).
By extrapolation, membrane-associated SCF may play two
important roles in physiologic c-kit functioning, both by increasing the local concentration of the ligand and increasing
the time the receptor is occupied and activated to induce
signaling.
On the other hand the mechanism of suboptimal mc-kit
activation could involve interference with the local presentation of mSCF by the transgene. Although we were unable
to detect differences in steady-state mRNA levels of mSCF
in stromal cell lines generated from transgenic mice, protein
analysis of these same cell lines demonstrated a substantial
decrease in the surface presentation of mSCF. These data
imply a posttranslational modulation of mSCF expression
may contribute to the abnormalities seen in these transgenic
mice. Therefore, phenotypic abnormalities seen in the
transgenic mice reported here may be due both to hSCF
T
60
40
20
Ofi
.
....
0
...
.*..*.a
0
7
L
Stromal Cells
Fig 7. Effect of expression of h S C F in transgenic-derived stromal cells on survival of HPP-CFC derived from murine c-kit' bone
marrow cells. Call lines are described in the Materials and Methods
and Fig 2 legend. c-kit' bone marrow cells were plated onto stromal
cells for 3 days, then all cells were harvested and plated in HPP-CFC
assays. Cell lines include +/lo, a murine fatal liver stromal cell line
generated from a heterozygous (SI/+)'; U2, a murine stromal cell line
generated from the bone marrow of a normal mousez3;hm220-5 and
hm220-2, both stromal cell lines generated from adult transgenic
mice expressing the h S C f2o transgene (and endogenous mSCF); SI4/
h220, a transfected stromal cell line derived from SIISI' expressing
only human membrane-associated SCF13; Sl'lm220. a transfected
stromal cell line derived from SI/S14 expressing only murine membrane-associated SCF"; SIISI', a stromal cell line expressing no SCF;
and no stroma. Results show one of three experiments with similar
results; mean f SD, n = 4. Differences between hm220-5 and hm2202 versus U2 or Sl'lm220, P < .005.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
MAJUMDAR ET AL
3210
::j
1o4
interference with c-kit signaling and to diminished expression of normal MA endogenous in SCF.
SI and W mutations have been widely studied by cell and
developmental biologists and a
x easily recognized due to distinctive coat color abnormalities. Uncoupling receptor binding and
proliferative responses may provide novel approaches to manipulating c-kit positive cell populations both in vitro and in vivo,
as has been used with other receptorAigand pairs." The data
provided by the transgenic mouse line reported here suggest. that
melanocytes may have increased sensitivity to subtle changes in
c-kit signaling in vivo.
ACKNOWLEDGMENT
We thank Drs Larry Bennett, Frank Martin, and Kris Zsebo for
providing reagents; members of our laboratory for many helpful
discussions; and Dorothy Giarla and Karen Roller for preparation
of the manuscript.
0
REFERENCES
A
-
/'//:
hm220-2 lootype
hm220-2 mSCF
Fluorescence
Intensity
Fig 8. Cell surface expression of hSCF and endogenous mSCF in
stromal cell lines derived from transgenic mice. Stromal cells were
stained as described in the Materials and Methods. (A) SIISI', U2,
hm220-2 ~ r o m acell
l lines were stained with rabbit antihuman SCF
polyclonal antibody and an FlTC conjugated goat F(ab'), antirabbit
IgG secondary. (6)U2 cells were Jtained with either isotype control
or rat antimouse SCF MoAb and an FlTC conjugated goat Flab')*
antirat IgG secondary. (C) hm220-2 cells were stained with either
isotype control or rat antimouse SCF MoAb and an FlTC conjugated
goat Flab'), antirat IgG secondary.
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1996 87: 3203-3211
Xenogeneic expression of human stem cell factor in transgenic mice
mimics codominant c-kit mutations
MK Majumdar, ET Everett, X Xiao, R Cooper, K Langley, R Kapur, T Vik and DA Williams
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