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Next Article 
Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1101-1134
REVIEW ARTICLE
c-kit Ligand and Flt3 Ligand: Stem/Progenitor Cell Factors
With Overlapping Yet Distinct Activities
By
Stewart D. Lyman and
Sten Eirik W. Jacobsen
From the Department of Molecular Genetics, Immunex Corp, Seattle, WA;
and the Stem Cell Laboratory, Department of Internal Medicine,
University Hospital of Lund, Lund, Sweden.
 |
INTRODUCTION |
HEMATOPOIESIS IS A life-long process
responsible for replenishing both hematopoietic progenitor cells and
mature blood cells from a pool of pluripotent, long-term reconstituting
stem cells.1 The daily turnover in a normal adult of
approximately 1012 blood cells is tightly regulated,
involving, in part, a complex interaction between soluble and
membrane-bound stimulatory and inhibitory cytokines and their
corresponding receptors.2-4 The molecular cloning of these
hematopoietic growth factors (HGFs) and their receptors has been
instrumental in delineating the pathways that lead from a single
hematopoietic stem cell to the various terminally differentiated cells
in the hematopoietic system.
Although a number of cytokines have effects on progenitor and stem
cells in vitro or in vivo, two cytokines discovered in the early 1990s,
c-kit ligand and flt3 ligand, appear to have unique and
nonredundant activities on primitive progenitor/stem cells.
Because of the broad range of hematopoietic activities mediated through
interaction of c-kit ligand (KL) and flt3 ligand (FL) with
their receptors, it is beyond the scope of this report to review the
effects of these proteins outside of the hematopoietic system. Rather,
we will focus on the discovery, structure, function, expression, and
biological roles of these two ligand-receptor pairs. Special attention
will be directed towards hematopoietic activities in which KL and FL
show either distinct or synergistic effects. For a more detailed
overview of other hematologic and immunologic effects of KL and FL,
other reviews can be recommended.5-8 Two subjects have been
deliberately left out of this report, because they are deserving of
their own separate reviews (signal transduction pathways involving
c-kit and flt3 and activities of KL and FL outside of the
hematopoietic system).
| |
DISCOVERY OF THE DOMINANT WHITE SPOTTING (W)
LOCUS AND ITS RELATIONSHIP TO THE c-kit TYROSINE KINASE
RECEPTOR |
The W (dominant White spotting) locus in mice was first
described in the early 1900s.9,10 Mice afflicted with
mutations at the W locus were originally identified, as the
name implies, by the presence of a white spot on the bellies of
pigmented mice. Detailed examination of these mice showed that the
mutation was pleiotropic. The mice suffer from defects in germ cell
development (manifested as reproductive difficulties) and in
hematopoiesis (characterized by a macrocytic anemia). Over the years,
at least 20 allelic variants of the W locus have been
described; most have a similar, although not identical,
phenotype.9,10 The W locus is on chromosome 5 and
is one of the most mutable loci in mice.9,10
A central question that remained was what kind of protein the W
locus encoded, and how did it affect so many different tissues. A
breakthrough came in 1988 when it was shown that the W locus encoded a tyrosine kinase receptor known as
c-kit.11,12 The c-kit protein has the same
general structure as four other tyrosine kinase receptors:
c-fms, the receptor for macrophage colony-stimulating factor
(M-CSF)13-15; flt316-19; and both of the
receptors for platelet-derived growth factor (PDGF;
designated as A and B).20-23 Each of these
receptors is approximately 1,000 amino acids in length, has five
Ig-like domains in the extracellular region, and contains a split
catalytic domain in the cytoplasmic region that phosphorylates tyrosine
residues in specific target proteins after activation of the receptor
by ligand. The exact defect in the c-kit receptor has been
identified at the molecular level for a number of alleles of the
W locus24-28 (see section on genetic alterations in
c-kit and KL genes).
| |
THE STEEL (Sl) LOCUS AND ITS RELATIONSHIP TO
W |
Many years after the discovery of the W locus, a mutation in
mice that had a phenotype virtually identical to W mice was
identified.29 Despite the similarities in phenotype, this
new mutation, designated Steel (Sl), was localized to mouse
chromosome 10, so it was clearly not allelic with the W locus
on chromosome 5.10,30 Because mutations on two different
chromosomes had the same complex phenotype that affects pigmentation,
germ cells, and hematopoiesis, researchers hypothesized that there
would be some relationship between the proteins encoded at these two
loci. Elizabeth Russell, who did much of the pioneering research on
both of these mutations, suggested (years before the discovery that the
W locus encoded c-kit and that c-kit was a
receptor) that the W and Sl loci might encode a
receptor and its cognate ligand.10
| |
CLONING OF THE STEEL FACTOR (THE c-kit LIGAND, KL) |
With the recognition that the W locus encoded
c-kit,11,12 the search for the c-kit ligand
began in earnest. A number of approaches were undertaken to identify
the protein encoded at the Sl locus, including chromosome
walking31 and expression cloning. However, the successful
approach turned out to be the purification of the Steel factor protein.
The cloning of a cDNA encoding the Steel factor was reported
simultaneously by three different groups, each of which discovered a
different source of the factor.32-34 All three groups used
a similar approach; they first purified the protein from medium conditioned by a cell line, obtained N-terminal amino acid sequence, and then made degenerate oligonucleotide primers based on the protein
sequence to isolate cDNA clones by polymerase chain reaction (PCR). The
three groups named this protein mast cell growth factor, stem cell
factor, and c-kit ligand (see below). In this review, we will
use the name c-kit ligand (KL) for the protein that binds to
the c-kit receptor and is encoded at the Sl locus on
mouse chromosome 10 (see below).32,35,36
Once the murine and rat KL cDNAs had been cloned, cross-species
hybridization was used to clone KL cDNAs from a number of other
species.33,37-40 The mouse and human proteins are 82%
identical at the amino acid level.
| |
DISCOVERY OF THE Flt3 TYROSINE KINASE RECEPTOR |
In contrast to the discovery of c-kit, analysis of mouse
mutations did not play a role in the discovery of the flt3 receptor. This receptor was isolated independently by two groups using distinct cloning strategies.18,19,41 One group used low stringency hybridization with a DNA probe from the M-CSF receptor (c-fms) to isolate a portion of a related DNA sequence that was named flt3
(fms-like tyrosine kinase 3).41 The partial clone was then used to isolate a full-length receptor clone.18
A second group used degenerate oligonucleotides (based on conserved
regions within the kinase domain of tyrosine kinase receptors) in a
PCR-based strategy to isolate a novel receptor fragment from highly
purified murine fetal liver stem cells.19 This fragment was
used to isolate a full-length receptor clone given the name flk-2
(fetal liver kinase 2). The flt3/flk-2 receptor has also been referred
to as Stk-1 (stem cell kinase-1),17 but this name is not
widely used, perhaps because it has been previously designated to
denote a gene regulating stem cell kinetics42 as well as a
different receptor tyrosine kinase of the met/sea/ron
family.43
Comparison of the murine flt3 and flk-2 receptor sequences showed that
these sequences differ by only two amino acids in their extracellular
domains.44 In contrast, a large number of amino acid
differences were seen in a region near their C-terminal ends. The
murine flt3 receptor sequence has been independently confirmed by
several groups,44-46 and the human receptor sequence is
directly homologous to the murine flt3, but not the murine flk-2
sequence.16,17 No independent confirmation of the sequence
of flk-2 has been reported. Differences between flt3 and flk-2
sequences are not a result of tissue-specific expression of distinct
isoforms.46 The differences in the murine flt3 and flk-2
sequences have never been fully explained, and the validity of the
sequence reported as flk-2 is still unclear.47 As a result
of this, we refer to the receptor as flt3 and to its ligand as flt3
ligand (FL).
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CLONING OF THE LIGAND (FL) FOR THE Flt3 RECEPTOR |
A soluble form of the flt3 receptor was the key reagent used by two
groups to clone FL. Lyman et al48 screened a variety of
cell lines to look for one that expressed a ligand on the cell surface
that was capable of binding the soluble receptor. A murine T-cell line
was identified that specifically bound the soluble flt3 receptor. The
ligand was then cloned from a cDNA expression library made from mRNA
isolated from these cells.
An alternative approach employed by Hannum et al49 used an
affinity column made with the mouse flt3 receptor extracellular domain
to purify FL from medium conditioned by a murine thymic stromal cell
line. N-terminal sequencing of the purified protein generated a short
amino acid sequence, which was then used to design degenerate
oligonucleotide primers to amplify a portion of the FL gene by PCR.
Isolation of this FL gene fragment led to the cloning of a full-length
murine cDNA.
Once the murine FL cDNA had been isolated, it was used to isolate cDNAs
encoding the human gene.49,50 The mouse and human FL
proteins are 72% identical at the amino acid level; homology is
greater in the extracellular region (73%) than in the cytoplasmic domain (57%).
| |
SPECIES SPECIFICITY OF KL AND FL |
No restriction in species specificity has been observed with regard to
FL binding or biological activity. Both the mouse and human ligand
proteins are fully active on cells bearing either the mouse or human
receptors.51 The human FL protein has been found to
stimulate mouse, cat (Janis Abkowitz, University of Washington, Seattle, WA, unpublished data), rabbit,
nonhuman primate, and human cells. This lack of species specificity of
FL is in marked contrast to KL, where the mouse protein is active on
human cells but the human protein has limited activity on murine
cells.33 Analysis of chimeric mouse/human KL proteins has
helped define regions of the protein that regulate its species-specific
action.52
| |
STRUCTURE OF THE c-kit AND Flt3 RECEPTORS |
The murine and human c-kit receptors are each 976 amino acids
in length, have nine potential sites for N-linked glycosylation in
their extracellular domains,53,54 and are glycosylated at one or more of these sites.54,55 Immunoprecipitation shows two proteins of approximately 140 kD and 155 kD54; the
predicted size of the protein backbone alone is approximately 108 kD.
Pulse-chase analysis has shown that the larger 155-kD protein arises
from the smaller protein,56 presumably due to glycosylational processing of the protein from one containing high
mannose carbohydrates to one containing complex carbohydrates. Furthermore, cell surface iodination of c-kit-expressing cells radiolabels only the larger protein.54 The size of the
c-kit protein varies between tissues,55 although
whether this is due to differential glycosylation or expression of
different isoforms is unclear (see below).
The murine (1,000 amino acids) and human (993 amino acids) flt3
receptors have 9 and 10 potential sites for N-linked glycosylation, respectively, in their extracellular domains16-19 and are
also glycosylated at one or more of these sites.44
Immunoprecipitation shows two proteins of 130-143 kD and 155-160 kD44,57,58; the predicted size of the protein backbone
alone is approximately 110 kD. As with c-kit, pulse-chase
analysis has shown that the larger protein arises from the smaller
protein44; again, this most likely results from
glycosylational processing. Consistent with this interpretation is the
finding that only the 158-kD species is found on the cell
surface.44 There do not appear to be any O-linked sugars on
the protein.59
| |
BINDING OF KL AND FL TO THEIR RECEPTORS |
A number of studies have measured the binding affinity of KL to the
c-kit receptor60-64 and that of FL to the flt3
receptor.65 Both high (kd, 16 to 310 pmol/L) and low (kd, 11 to 65 nmol/L) affinity binding of KL to its
receptor have been reported.60,61,63 Some primary cells and
cell lines have only high- affinity sites, whereas others have
both.61,63 Neither the number of receptors per cell nor the
finding of one or two classes of receptors can be correlated with the
ability of cells to proliferate in response to KL.60
The binding affinity of human FL for the flt3 receptor on human myeloid
leukemia cells has been estimated to be 200 to 500 pmol/L,65 and only high-affinity binding is seen. The high
binding affinity of FL for the flt3 receptor is therefore in the same range of affinities as the binding of KL to c-kit.
The c-kit and flt3 receptors each have five Ig-like domains in
their extracellular regions. Mutagenesis studies on c-kit have shown that the first three domains are both necessary and sufficient for binding of ligand66 and that the fourth Ig-like domain
is required for dimerization of the receptor,66 although
this has recently been called into question.67 Several
models have been proposed for binding of KL to
c-kit,66-71 but it is beyond our scope to review
these studies. Whatever the mechanism responsible for the formation of
the complex, the ultimate result is that a dimeric form of the ligand
is associated with a dimeric form of the receptor, which results in
signal transduction. Although similar studies have not been performed
with FL and flt3 receptors, a similar process most likely occurs with
this ligand-receptor pair.
| |
ISOFORMS OF THE c-kit AND Flt3 RECEPTORS |
Analysis of independently derived cDNA clones has shown that there are
two isoforms of both the murine and human c-kit-encoded protein.72 These c-kit receptor isoforms differ by
four amino acids (glycine-asparagine-asparagine-lysine, abbreviated
GNNK) that are either present or absent just upstream of the
transmembrane domain. The different isoforms result from alternative
splicing of c-kit mRNAs at a cryptic splice donor site located
at the 3 end of exon 9.73 Although it is not clear if
physiologic differences occur because of ligand signaling via one
c-kit isoform versus another, ligand-independent constitutive
phosphorylation of the receptor occurs only in the isoform missing
these four amino acids.72
Crosier et al74 examined expression of the two c-kit
isoforms in both leukemic cell lines and in primary acute myeloid
leukemias; both isoforms appeared to be expressed in all of the cells
examined, with the ratio of GNNK to GNNK+
isoforms ranging from 10:1 to 15:1. A second study confirmed the
expression of both isoforms in a series of acute myeloid
leukemias.75
In addition to the isoforms discussed above, other variants have been
seen in the c-kit receptor. Alternative splicing of mRNAs has
been shown to insert an extra serine residue in the cytoplasmic domain
at position 715; a survey of human cell lines and acute myeloid
leukemia samples shows that both of these isoforms are normally
expressed.74
Finally, soluble c-kit receptors are produced by some
hematopoietic cell lines in culture,64 and a soluble
version of c-kit has been found in human serum at high levels
(324 ± 105 ng/mL).76 How this soluble c-kit
receptor is generated is unknown, although it does appear capable of
binding KL.60,64 In each of the cases described above, the
physiologic significance, if any, of the receptor variant is unknown.
Fewer isoforms of the flt3 receptor have been reported than have been
seen with c-kit. One isoform of the murine flt3 receptor is
missing the fifth of the five Ig-like regions in the extracellular domain as a result of the skipping of two exons during
transcription.77 This alternative isoform is present at
lower levels than the wild-type receptor, although it is able to bind
ligand and is phosphorylated as a result of this binding. Thus, the
fifth Ig domain of flt3 is not required for either ligand binding or
receptor phosphorylation. Similarly, the c-kit receptor
requires only the first three Ig-like domains for ligand
binding.66 The physiologic significance of this flt3
receptor isoform is presently unknown, and a soluble version has not
yet been identified in human serum.
| |
STRUCTURES OF THE KL AND FL PROTEINS |
The KL and FL proteins are structurally similar to each other (as
described below)48-50 and to M-CSF.78 The
primary translation product of the KL gene is a type 1 transmembrane
protein, ie, the N-terminus of the protein is located outside of the
cell. This protein is biologically active on the cell
surface.79 The murine and human KL proteins are each 273 amino acids in length, with a 25 amino acid leader, a 185 amino acid
extracellular domain, a 27 amino acid transmembrane domain, and a 36 amino acid cytoplasmic tail.
The murine32,79 KL protein has four potential sites for
N-linked sugar addition; the human protein has five. KL made by Buffalo
rat liver cells is N-glycosylated in a heterogeneous fashion and
probably contains O-linked sugars. Analysis of human KL produced by
Chinese hamster ovary (CHO) cells shows that it is glycosylated in a
somewhat different manner than the rat protein and that it also
contains O-linked sugars.80
Circular dichroism spectra of KL shows that it has considerable
secondary structure, including both  helical and  sheets.80 There are four cysteine residues that are
conserved between KL, FL, and M-CSF. In the case of KL, these form
two intramolecular disulfide bonds that establish the
three-dimensional structure of the protein.81 Although KL
forms homodimers in solution, they are not covalently
linked.80 KL is thus different from M-CSF, which contains
three intramolecular disulfide bonds and an unpaired cysteine residue
that forms an intermolecular disulfide bond.82 Preliminary
data suggest that FL also contains three intramolecular disulfide bonds
and exists as a noncovalently linked homodimer (Rick Remmele, Immunex,
Seattle, WA; unpublished observation).
Mutagenesis studies of mouse and human KL have identified a core region
that is required for biological activity; this region constitutes the
major portion of the extracellular domain and encompasses all four of
the cysteine residues conserved between KL, FL, and
M-CSF.83,84 Neither the cytoplasmic, transmembrane, spacer,
nor tether regions of KL (Fig 1) is
required for biological activity. Similar studies on FL have yielded
essentially identical results.85
View larger version (25K):
[in this window]
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| Fig 1.
Sequence alignment of human FL and KL proteins. The
figure illustrates that both colony-stimulating factors are type I
transmembrane proteins with short cytoplasmic domains; both are likely
to be four helix bundle proteins (based on x-ray crystallography data in the case of M-CSF82). The approximate positions of the
four helices are shown. The vertical red lines show the locations of
introns (to the nearest amino acid) within the
genes33,93,95,104 and illustrate their common genomic
structure and ancestral origin. Conserved cysteine residues are shaded
in color to reflect the formation of proposed intramolecular disulfide
bonds (3 in the case of FL and 2 in the case of KL). Possible sites for
N-linked glycosylation are boxed. The alignment is based on the one
originally proposed by Bazan78 for KL and M-CSF.
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The primary translation product of the FL gene is also a type 1 transmembrane protein. The mouse and human proteins contain 231 and 235 amino acids, respectively. The first 27 (mouse) or 26 (human) amino
acids constitute a signal peptide that is absent from the mature
protein, followed by a 161 (mouse) or 156 (human) amino acid
extracellular domain, a 22 (mouse) or 23 (human) amino acid
transmembrane domain, and a 21 (mouse) or 30 (human) amino acid
cytoplasmic tail. The cytoplasmic domains of murine and human FL are
only 52% identical and are much more divergent than the cytoplasmic
domains of murine and human KL (92% identical). Why the cytoplasmic
domains of mouse and human FL are so much more divergent in sequence
than the cytoplasmic domains of mouse and human KL is unknown. The
mouse and human FL proteins each contain two potential sites for
N-linked glycosylation. The human FL protein contains N-linked sugars
(Claudia Jochheim, Immunex; unpublished observation).
| |
KL AND FL ISOFORMS |
The mature mouse and human KL proteins (from which the amino acid
signal sequence has been cleaved) undergo proteolytic cleavage to
generate a soluble, biologically active, 164-165 amino acid protein.32,33,79,86 The primary site for proteolytic
cleavage is encoded within exon six33; however, mutagenesis
experiments have shown that there is a secondary proteolytic cleavage
site just upstream of the transmembrane region within exon
7.87 This secondary site is used only if the primary site
is missing, which can occur by splicing out the sixth
exon.79,88,89
Splicing has been suggested to be a method of regulating the generation
of soluble versus membrane-bound forms of the protein. Alternative
splicing of the sixth exon of the KL gene has been reported in both
mouse and human cells.40,79,88,90,91 The cell-bound form of
KL appears to be required for normal development in mice since a
mutation (Sld) that eliminates the membrane-bound
form of the factor, but still makes a biologically active soluble form,
results in developmental abnormalities.88,92 Huang et
al90 showed that there is tissue-specific expression of the
different isoforms. The physiologic significance of these altered
isoform ratios is unknown but presumably reflects the capacity of each
tissue to produce a form of KL that is capable of interacting with
specific c-kit-expressing cells.
It is unclear what regulates the proteolytic cleavage of KL, and what,
if any, the physiologic effects of this process are. The protease
responsible for cleavage of KL has not been identified, and it is
unknown if it is the same protease that generates soluble, biologically
active forms of M-CSF and FL.48,49,93
Multiple isoforms of both mouse and human FL have been identified by
analysis of multiple cDNA clones and PCR.48-50,94 The biological significance of these isoforms is presently unknown. The
predominant isoform of human FL is the transmembrane protein that is
biologically active on the cell surface.48-50 This isoform is also found in the mouse, although it is not the most abundant isoform in that species (see below). The transmembrane FL protein can
be proteolytically cleaved to generate a soluble form of the protein
that is also biologically active.48 Neither the protease responsible for this cleavage nor the exact site in the FL amino acid
sequence where cleavage occurs has been identified.
The most abundant isoform of murine FL95 is an alternative,
220 amino acid form that is membrane bound, but is not a transmembrane protein.49,94 This form arises due to a failure to splice
an intron from the mRNA. This leads to a change in the reading frame, which terminates in a stretch of hydrophobic amino acids that serve to
anchor the protein in the membrane.50 This isoform is
missing the spacer and tether regions that contain the proteolytic cleavage site seen in the transmembrane isoform. As a result, this
membrane-associated isoform is resistant to proteolytic
cleavage,94 although it is biologically active on the cell
surface. This isoform has not been identified in any human FL cDNAs
examined.
A third FL isoform identified in mouse94 and
human95 tissues arises because of an alternatively spliced
sixth exon. This exon introduces a stop codon near the end of the
extracellular domain and thereby generates a soluble, biologically
active protein that appears to be relatively rare compared with other
isoforms.95 Another method of generating soluble FL in the
human is to splice out the transmembrane domain,50 but the
relative abundance of this isoform has not been quantitated.
There is a difference between KL and FL in regard to their
alternatively spliced sixth exons. The amino acids in exon 6 of mouse
and human KL are nearly identical, whereas those of mouse and human FL
have virtually no homology.95 In the case of KL, the sixth
exon is normally part of the transmembrane protein and contains the
proteolytic cleavage site. In the case of FL, it is not a part of the
transmembrane protein; introduction of the sixth exon results in the
generation of a soluble protein due to a shift in the reading frame.
Thus, evolution has made two different uses of the sixth exon of KL and
FL, allowing the generation of a soluble protein by different
mechanisms.
| |
STRUCTURE OF THE GENOMIC LOCI ENCODING THE c-kit AND Flt3
RECEPTORS |
The genomic loci encoding the c-kit, flt3, and
c-fms receptors share overall conservation of exon size,
number, sequence, and exon/intron boundary positions,96 and
these genes have likely arisen from a common ancestral gene. The
genomic loci encoding the mouse97 and
human98-100 c-kit receptors show clear evidence of
evolutionary conservation. The coding region of the c-kit
receptor encompasses 21 exons, and both the mouse and human loci span
more than 70 kb of genomic sequence.
The human flt3 receptor genomic locus is approximately 100 kb in
size.101 The exon:intron structure of the entire receptor has been reported to contain 24 exons,102 but only the
portion of the gene encoding the C-terminal domain has been published.
| |
STRUCTURE OF KL AND FL GENOMIC LOCI |
The genomic locus encoding KL has been cloned from the
human,33 rat,33 and mouse.103 The
human KL locus is more than 50 kb in length (Vann Parker, Amgen,
Thousand Oaks, CA; personal communication) and consists
of eight exons that contain the entire coding region of the protein.
The intron:exon boundaries identified within the rat, human, and murine
genes occur at identical positions. In the case of the mouse protein, a
ninth exon is present and encodes the C-terminal end of the cytoplasmic
domain.103
The genomic loci encompassing the coding regions of mouse and human FL
are approximately 4.0 kb and 5.9 kb, respectively; the coding region
comprises 8 exons.95 The human FL locus is thus
significantly smaller than the human KL locus. The sizes of the
individual FL exons are well conserved between species,95 although the intron sizes are much more variable.
The genomic locus encoding M-CSF also contains eight
exons.104 A comparison of exon sizes between FL, KL, and
M-CSF shows that identically numbered exons are similar in size in all
three proteins.95 If the sizes of the exons are taken as a
measure of overall relatedness, then M-CSF and KL are more closely
related to each other than they are to FL. For example, the sizes of
exons 3 and 4 are identical between M-CSF and KL, but are not the same as the corresponding exons in FL. The location of the introns in the
three genes are also fairly well conserved, indicating that these
proteins are probably ancestrally related.
| |
CHROMOSOMAL LOCATION OF c-kit AND Flt3 RECEPTORS |
The murine c-kit locus is located in the D-E region of mouse
chromosome 511,12 near two other tyrosine kinase receptors
(PDGF A and flk-1/KDR). The murine flt3 receptor gene is also on
chromosome 5, but at the G region.41 The flt3
receptor105 is located less than 350 kb from the murine flt
tyrosine kinase receptor106 but is separated from the
clustered c-kit, PDGF A, and flk-1/KDR receptors.
The human c-kit locus is on the centromeric region of
chromosome 4, in the area of 4q31-34,53
4q11-21,54 and 4q11-12.107 The gene encoding
the human flt3 receptor maps to chromosome 13q12,41 again
near the flt receptor locus. The flt3 and flt genes are linked105 in a head to tail fashion and are separated by
about 150 kb.101
| |
CHROMOSOMAL LOCATION OF KL AND FL GENES |
The KL gene is, as expected, encoded on mouse chromosome 10 and is
deleted in some, but not all, Sl alleles.32,35,36
The FL gene maps to the proximal portion of mouse chromosome
7.94
The gene encoding human KL has been mapped to chromosome
12q22-2440 and 12q14.3-qter108 in a region that
is syntenic with mouse chromosome 10. The human FL gene maps to
chromosome 19q13.3-13.4,94,109 which is syntenic with mouse
chromosome 7. The chromosomal locations of KL, FL, M-CSF, and their
receptors are summarized in Table 1.
| |
GENETIC ALTERATIONS IN c-kit AND KL GENES |
The exact defect in the c-kit receptor has now been identified
at the molecular level for a number of alleles of the W
locus.24-28 Most of the alleles result from point mutations
in the cytoplasmic domain of the receptor; these changes decrease the
tyrosine-phosphorylating activity of the protein. However, in several
cases, the mutations appear to be of a regulatory instead of a
structural nature and result in reduced expression of the c-kit
receptor.
There is a rare, autosomal dominant genetic disease in humans known as
piebald trait. Affected individuals have a white forelock and large,
nonpigmented patches on the chest and/or other areas. All cases
of piebald trait that have been molecularly analyzed result from
missense or frameshift mutations in the c-kit tyrosine kinase
receptor (Ezoe110 and references therein). Affected
individuals are heterozygous for defects in the c-kit protein;
the dominant nature of the trait reflects the dominant-negative effects
of the mutant c-kit allele. The dominant-negative effects of
these mutations are thought to result because receptor dimerization is
required for proper biological function.
Because pigmentation defects in W and Sl mice are often
indistinguishable, it would be reasonable to expect that at least some
cases of piebald trait in humans would arise from mutations in the KL
gene, ie, from a defect in the ligand instead of the receptor. However,
no defects in the KL gene have been reported in piebald humans. Piebald
trait thus represents the human homologue of the W mutation in
mice.
Mutations at the Steel locus35 have occurred spontaneously
or have been induced by chemical mutagenesis, x-ray irradiation, or
transgene insertion.111 In addition to the
Sld mutation (see above), the molecular defect
responsible for three other Sl mutations has been identified.
In the Sl17H mutation,103 the
cytoplasmic tail of KL is altered as a result of a splicing defect; in
contrast, the Slcon and Slpan
mutations are of a regulatory nature and result in altered,
tissue-specific expression of mRNAs encoding KL.112
| |
GENETIC ALTERATIONS IN Flt3 RECEPTOR AND FL GENES |
In contrast to the well-described mutations in the c-kit
receptor and its ligand (see above), there are no reports of any genetic defects associated with either the flt3 receptor or its ligand.
As described above, FL maps to human chromosome 19q13.3. Trisomy 19 is
strongly associated with myeloid malignancies.113 However,
whether overexpression of FL plays a role in the increased incidence of
leukemia in trisomy 19 remains to be determined.
| |
EXPRESSION OF KL AND FL IN MOUSE AND HUMAN HEMATOPOIETIC TISSUES |
The expression of the c-kit and flt3 receptors, and not their
ligands, is the key to understanding the function of these growth factors. Numerous studies have shown that both KL and FL are widely expressed in different tissues, in contrast to their receptors, which
are expressed on a more limited number of cells, especially in the case
of flt3. KL is widely expressed during
embryogenesis,114-116 suggesting that KL may affect the
growth, survival, and/or differentiation of cells in addition
to the three lineages (hematopoietic cells, germ cells, and
melanocytes) shown to be affected in both W and Sl
mutant mice. Cells expressing KL are frequently contiguous with cells
expressing c-kit, ie, ligand and receptor expression are
complementary. KL is expressed on stromal cells,117,118
fibroblast,26,79,119 endothelial cells,117
visceral yolk sac,115 and other places.
FL, like KL, is widely expressed in both murine and human
tissues.49,50,94 Highest levels of FL mRNA on human tissue
Northern blots are in peripheral blood mononuclear cells, but the
ligand is also expressed in almost every tissue that has been
examined.48-50 Mouse developmental in situ hybridization
studies have not yet been performed with FL, although it would be
interesting to see how the distribution of FL would compare with flt3
receptor.120
| |
EXPRESSION OF c-kit AND Flt3 RECEPTORS ON HEMATOPOIETIC CELL
LINES |
Expression of the c-kit receptor has been extensively surveyed
on mouse and human hematopoietic cell lines
(Table 2). It is seen on only a small
percentage of myeloid and myeloblastic cell lines.121-124
In contrast, the majority of erythroid and erythroleukemia cell lines
express c-kit,121-123,125 as do virtually all
megakaryocytic cell lines.121,123,125 Mast cell lines
generally express c-kit.51,126-128 In contrast,
expression of c-kit is generally not seen on lymphoid leukemia
cell lines (including pre-B, B, and T cells),121,123,125 on
B-cell or T-cell lymphoma cell lines,121,122,125 or on
myeloma cell lines.121
Flt3 receptor expression on mouse and human cell lines is quite
different from that of c-kit. No flt3 expression is seen on any
of the mouse myeloid, macrophage, erythroid, megakaryocyte, or mast
cell lines examined46,129 or most early mouse B-cell lines,
but it has been reported on several mature B-cell lines.129 This lack of expression is different from what is seen on most human
pre-B-cell lines, which do express flt3 receptor.123,130 In
addition, flt3 expression has been seen on only one mouse pro-T cell
line, but not on any T-cell lines.46,129
A number of studies have been published that show expression of flt3
receptor on a limited range of human cell lines. The flt3 receptor is
found on a high percentage of human myeloid and monocytic cell
lines,123,129,130 in contrast to mouse cell
lines.46,129 No flt3 expression is seen on myeloma cell
lines,129,130 and only a few megakaryocytic cell lines are
positive.123,129,130 All erythroid and erythroblastic cell
lines are flt3 negative as well.129,130
Among lymphoid cell lines, pro-B as well as pre-B lines are flt3
receptor positive,129,130 whereas natural killer (NK) cell lines and Hodgkin's cell lines are negative,130 as are all
T-cell lines.123,129,130
| |
EXPRESSION OF c-kit AND Flt3 RECEPTORS ON PRIMARY HUMAN
LEUKEMIAS |
Both the c-kit and flt3 receptors are frequently seen on acute
myelogenous leukemia (AML) blasts. The c-kit protein is
expressed on blast cells obtained from a high percentage of patients
with AML from all French-American-British (FAB)
subtypes.61,124,131-139 Receptor levels on AML blast cells
are variable, but in general are similar to or less than c-kit
levels on normal stem and progenitor cells.140
Expression of the flt3 receptor in primary leukemias has also been
investigated and recently reviewed.141 As with
c-kit, the majority of adult AML samples from all FAB classes
are positive for flt3 receptor expression.57,142-146
Among lymphoid leukemias, little or no expression of c-kit is
observed on blast cells in acute lymphoblastic leukemia
(ALL).133,143 c-kit is expressed on Reed-Sternberg
cells in about half of Hodgkin's disease patients as well as on some
anaplastic large-cell lymphoma samples.147
All B-lineage ALL samples examined are flt3 receptor
positive,142-144 as are most hybrid (also known as mixed or
biphenotypic) leukemia samples.144 The greatest variability
reported in flt3 receptor expression is on T-lineage ALL, which have
been reported to be all negative,142 have a small
percentage that are positive,143 or have about half of the
samples positive.144 In contrast, both T-cell and B-cell
lymphomas are negative for flt3 receptor expression.144 Tandem in-frame duplications in the juxtamembrane region of the human
flt3 receptor have been reported to be associated with both leukocytosis148 and leukemic transformation.149
The c-kit receptor is expressed on a majority of samples from
chronic myelogenous leukemia (CML) patients in blast
crisis134,150 and at least some samples of chronic phase
CML138 and CML in blast transition.151 In
contrast, almost all chronic-phase or accelerated-phase CML samples are
negative for flt3 receptor expression.143,144 However,
about two thirds of the samples from CML patients in blast crisis are
flt3 receptor positive.143,144
| |
RESPONSIVENESS OF PRIMARY LEUKEMIA CELLS TO KL AND FL |
AML.
Numerous studies have been performed on human leukemia samples to
determine whether the cells proliferate in response to KL, FL, or other
growth factors, although a lack of proliferation should not necessarily
be considered negative expression. For example, a growth factor could
drive differentiation or inhibit apoptosis; in fact, both
KL152 and FL153 have been shown to have this
latter effect. In the case of nonproliferative cells, the cells may be
truly nonresponsive or may be producing endogenous ligand, and thus are
refractory to exogenously added growth factor.
c-kit receptor expression is variable among AML FAB subtypes
and does not predict responsiveness to KL.145 The
majority of AML samples proliferate in response to
KL.61,131,137,154,155 Many of these studies show that
KL synergizes with other cytokines to enhance the proliferation of
leukemic blast cells. Some AML cell lines express KL in addition to
c-kit,140,156 suggesting that an autocrine loop may
play a role in the transformation of these cells. However, the low
level of KL expression on some AML cells has led one group to conclude
that a c-kit and KL autocrine cycle is not common in
AML.140
Whether flt3 receptor or its ligand play a causal role in the
development of human leukemias has not been determined. A large percentage of AML cells from children142 and
adults145,146 proliferate (as measured by both
[3H]-thymidine incorporation or colony formation) in
response to FL. Within age groups (children or adults), some FAB
subtypes show a greater response compared with
others.142,146 It is unclear whether there is a difference
in the FL responsiveness of flt3 receptor-positive AML samples of
different FAB subtypes from children and adults because not enough
samples of each FAB subtype have been analyzed.
Primary AML samples that proliferate in response to FL also frequently
proliferate in response to granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and KL, and additive or synergistic responses are observed. Some AML cells are therefore similar to normal hematopoietic progenitor cells in that both show
synergistic responses to FL in combination with other cytokines. Many
of the AML samples that do not proliferate in response to FL do
proliferate in response to other cytokines,142 indicating that the cells do not lack a general capacity to proliferate. In
summary, flt3 receptor expression on AML samples is not predictive of
FL responsiveness, just as c-kit expression is not predictive of KL responsiveness.
CML.
KL can weakly stimulate the proliferation of CML blast cells on
its own and strongly stimulate them in the presence of IL-3 and/or GM-CSF.138 Culturing of bone marrow (BM)
cells from CML patients in the presence of KL favors the growth of
malignant progenitor cells.157 In contrast, preliminary
results suggest that FL favors the outgrowth of benign progenitors from
5-FU-treated CD34+ CML BM cells at the expense of malignant
cells158 and that FL generates a significantly greater
percentage of normal progenitors (Philadelphia chromosome-negative
cells) compared with KL.
ALL.
Because c-kit is not generally expressed on ALL
cells,124,133,134,139 the capacity of these cells to
proliferate in response to KL has not been examined. As mentioned
above, all B-lineage ALL and some T-lineage ALL samples express flt3
receptor. However, only a small percentage of B-lineage ALL samples
proliferate in response to FL.142
In one study, pediatric T-lineage ALL samples did not proliferate in
response to FL, but none of these samples was positive for flt3
expression.142 In a separate study on a variety of ALLs, several flt3 receptor-positive samples proliferated in
FL.159 However, the majority of samples failed to
proliferate in FL, even though they were flt3 receptor
positive.159 Flt3 receptor expression is therefore not
predictive for proliferation of ALL cells to FL in vitro.
| |
EXPRESSION AND FUNCTION OF c-kit AND Flt3 IN THE
HEMATOPOIETIC HIERARCHY |
Studies of cytokine receptor expression have proven valuable in
pinpointing where specific ligand-receptor pairs have biological activities. Not only can such studies identify cell types in which a
specific receptor might be important, they also allow functional characterization of distinct cell populations separated based on
various levels of receptor expression. The expression of c-kit and flt3 in the hematopoietic system has been studied in detail, and in
the following sections we review the findings of flt3 and c-kit
expression on various cell types (summarized in
Fig 2), followed by the in
vitro biological effects (summarized in
Table 3) of FL and KL on the same cell
types. It is important to emphasize that the extensive c-kit
and flt3 expression studies to be described have inherent limitations.
Most expression studies have been performed by flow cytometric
evaluation of cell-surface c-kit and flt3 expression. Because
flow cytometry has a rather high detection limit (~500 molecules/cell), so- called c-kit and
flt3 populations might prove to express low levels of
c-kit and flt3, respectively. On the other hand, reverse
transcriptase-PCR (RT-PCR) detection of c-kit
and flt3 mRNA has much greater sensitivity, but unless performed at the
single-cell level does not provide a quantitative measurement of
c-kit+ and flt3+ cells. Thus, a minor
contaminating (nonrelevant) cell type might account for detected
expression (particularly relevant for heterogenous primary cell
populations).
View larger version (43K):
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| Fig 2.
c-kit and Flt3 expression in the hematopoietic
hierarchy. The figure indicates expression of c-kit (red, upper
symbol on side of each cell) and flt3 (green, lower symbol on side of
each cell) on various classes of hematopoietic stem and progenitor
cells as well as mature blood cells, as described in the text. Because most hematopoietic cell populations are heterogeneous and hard to
purify, it is not possible to exclude c-kit and/or flt3
expression on a minority of cells in the different cell populations.
Therefore, the figure illustrates the c-kit and flt3 receptor
status on the majority of cells within a specific population, based on
studies of receptor expression and/or functional studies. As
discussed in the text, the proposed hierarchy of pluripotent stem cells is based solely on different levels of c-kit and flt3
expression and does not take into account other stem cell
antigens/characteristics, which are likely to uncover additional
heterogeneity. Symbols: ( ) most/all cells appear to lack
c-kit or flt3 expression; (+) most/all cells appear to
express c-kit or flt3; (+/) the cell type appears to
consist of significant receptor-positive as well as receptor-negative
populations; (?) sufficient expression or functional data not
available; (high and low) cell populations have been separated based on
high and low levels of c-kit expression. Abbreviations:
BFU, burst-forming units; CFU, colony-forming units; E, erythroid; Mk,
mega karyocyte; G, neutrophilic progenitor; M, monocyte/macrophage; DC,
dendritic cell; Baso, basophil; RBC, red blood cell; NK, natural killer
cell.
|
|
| |
EXPRESSION OF c-kit AND Flt3 ON MATURE BLOOD CELLS |
c-kit and flt3 expression in the hematopoietic system appear
predominantly restricted to the progenitor/stem cell compartment (outlined in the following sections). However, some differentiated blood cells also express these receptors (Fig 2).
c-kit is expressed on primary mast cells as well as mast cell
lines and primary neoplastic mast cells.160 In addition,
c-kit is constitutively activated in a number of mast cell
tumor lines (mastocytomas),127,161 but mast cells do not
express flt3.128
There are other differentiated hematopoietic cells that express
c-kit and/or flt3, although the functional significance
is less clear. In mouse BM, very low levels of
c-kit can be detected on promyelocytes and myelocytes, but not
on neutrophils.162 Approximately 50% of murine BM
eosinophils and monocytes express low levels of
c-kit.162 Seven percent of lymphocytes in murine BM
express high levels of c-kit.162 However, still
other studies suggest that mature B and T cells do not express
c-kit; therefore, this small fraction of
c-kit+ cells might represent B- and T-cell
precursors/progenitors.163-165
Similar studies have revealed that flt3 expression in murine BM is
restricted to blast cells, monocytes, and a small fraction of
lymphocytes.166 Nucleated murine erythroid cells lack both c-kit and flt3 expression.162,166 Early murine
megakaryocytes (stage I and II) express c-kit, whereas the most
mature (stage III) megakaryocytes appear to be
c-kit.167 Also, human
megakaryocytes express c-kit,61,168 but not flt3.169 In addition, activated but not resting platelets
express c-kit.170
Initial studies indicated that flt3 mRNA is expressed by murine B and T
cells from thymus, spleen, and peripheral blood.18 However,
several later studies of mature murine B and T cells suggest that these
do not express flt3.166,171 Thus, the initial findings
potentially were due to a small fraction of contaminating flt3+ cells, such as more primitive B- and T-cell
progenitors.
Peripheral human blood cells contain less than 0.1%
c-kit+ cells, suggesting that very few mature human
blood cells express c-kit.172-174 c-kit is
constitutively expressed on a small subset of resting human NK cells in
peripheral blood that are characterized by high CD56 expression,
whereas c-kit is not expressed on the larger fraction of more
differentiated NK cells with low CD56 expression.175 These
c-kit+ NK cells appear to be the only mature,
resting lymphocytes that constitutively express c-kit.
No expression of flt3 mRNA has been reported on mature
lympohematopoietic cells fractionated from human peripheral
blood17 or B cells, T cells, monocytes, or
granulocytes.144 However, in other studies, monocytes and
granulocytes have been shown as weakly positive at the mRNA and
cell-surface level.16,176
| |
RESPONSE OF MAST CELLS TO KL, BUT NOT FL |
The effects of KL on mast cell populations have been extensively
reviewed6 and will be only briefly summarized here. KL regulates the migration, maturation, proliferation, and activation of
mast cells in vivo.6 Injection of recombinant KL into
rodents,86,177 primates,178 or
humans179 results in an increase in mast cells at both the
site of injection and at distant sites. Treatment of rats with KL
generates both connective tissue mast cells and mucosal mast
cells.177 Animals treated with KL generally do not appear
to suffer from serious adverse events despite the large-scale expansion
of mast cells in vivo.178 However, at least one study has
shown that KL administration to mice leads to degranulation of mast
cells in the lungs, which leads to acute respiratory
distress.180 The effects of KL on mast cells may have a
significant impact on the clinical potential of this molecule for
humans.179,181,182
In contrast to c-kit, flt3 is not expressed on primary mast
cells or mast cell lines, and these cells, not surprisingly, do not
respond to FL.51,128 This lack of flt3 expression on mast cells is one of the key differences between KL and FL.
| |
COMMITTED MYELOID PROGENITOR CELLS ARE
c-kit+Flt3+ OR
c-kit+Flt3, WHEREAS EARLY ERYTHROID
PROGENITOR CELLS APPEAR TO BE ONLY
c-kit+Flt3 |
Half of c-kit+ murine BM cells coexpress
lineage-specific cell surface antigens such as GR-1 and MAC-1
(Lin+), characteristic of cells committed to the myeloid
lineage, whereas the remaining half express higher levels of
c-kit and are Lin |
Introduction
References