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Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1152-1162
By
From the Department of Cell Biology, Amgen, Inc, Thousand Oaks, CA.
CD34 is widely used as a marker in the identification and
purification of human hematopoietic stem and progenitor cells; however, its function within hematopoiesis is largely unknown. We have investigated the contribution of cytoplasmic domain of CD34 in cytoadhesion signaling and proliferation signaling in hematopoietic cells. Engagement of particular determinants of CD34 by monoclonal antibodies leads to homotypic adhesiveness of the full-length CD34-transfected BaF3 cells. However, this homotypic adhesiveness is
abrogated in BaF3 cells transfected with the truncated CD34 lacking the
cytoplasmic domain. Cytoadhesion signaling through the cytoplasmic
domain of CD34 cannot be restored through that of erythropoietin
receptor (EPOR) or granulocyte colony-stimulating factor receptor
(G-CSFR), suggesting that the cytoplasmic domain of CD34
is required for its signal transduction of cellular adhesion. In
constrast, we show that replacing the cytoplasmic domain of EPOR or
G-CSFR with that of CD34 abolished growth signal transduction in
response to EPO or G-CSF in the chimeric receptor-transfected BaF3,
32D, and FDCP1 cells, whereas the wild-type EPOR- or G-CSFR-transfected cells responded to EPO or G-CSF growth signaling well. These results suggest that the cytoplasmic portion of CD34 may not contain the elements necessary to transduce a proliferative signal in hematopoietic cells. Thus, the function of CD34 in hematopoiesis is primarily on
hematopoietic cell adhesion.
EXPRESSION OF THE stem cell antigen CD34
is a defining hallmark of human hematopoietic stem and progenitor
cells; thus, CD34 is one of the most commonly used markers for
isolation, purification, and manipulation of these
cells.1-4 The CD34 molecule is an approximately 115-kD type
I transmembrane glycoprotein with a protein backbone of approximately
40 kD that shows no significant sequence homology to any other known
protein. The extracellular domains of the human and mouse CD34 share
approximately 63% amino acid identity and both contain an extensively
O-glycosylated mucin-like portion and a cysteine-rich region. The
N-terminal regions (1-130 amino acids) of the extracellular domains are
the least well-conserved portions of the whole molecule (~44% amino
acid identity), whereas the entire cytoplasmic domains of both species
are highly conserved (~93% amino acid identity), predicting an
essential functional importance for this domain.5,6 Studies
in humans and baboons have shown that the CD34+ bone marrow
(BM) and peripherally mobilized progenitor cells contain stem cells
that can reconstitute all of the blood cells after
transplantation.7,8 Similarly, the mouse CD34+
BM and fetal liver cells also contain stem cells that can repopulate all of the blood lineages in lethally irradiated mice. 9 In
addition to its expression on hematopoietic progenitor/stem cells, CD34
is also expressed on all vascular endothelial cells of both adults and
embryos.10,11 It has been suggested that the
CD34+ endothelial cells lining the yolk sac blood islands
interact with the CD34+ hematopoietic progenitor cells of
the yolk sac to induce differentiation, proliferation, and, possibly,
self-renewal of the stem cells.12 Similar interactions
between hematopoietic progenitor/stem cells and endothelial cells have
been proposed in the aortic-gonadal-mesonephros region of the embryo as
well.13,14 Despite the importance of CD34 as a marker of
early hematopoietic progenitor/stem cells in clinical and developmental
hematopoiesis, the function and regulation of this stem cell antigen is
still unclear.
Studies on the function of CD34 suggest that it may play a role in
adhesion and signal transduction on hematopoietic progenitor/stem cells. Ectopic expression of human CD34 in the thymocytes of transgenic mice indicates that CD34 augments the adhesive interactions of CD34+ hematopoietic cells with BM stroma and this
CD34-dependent adhesion is enhanced by the engagement of anti-CD34
monoclonal antibodies (MoAbs).15 Similarly, engagement of
certain epitopes on CD34 by anti-CD34 MoAbs triggers homotypic adhesion
of CD34-expressing KG1a cells, implicating that CD34 has signal
transducing capacity to induce cytoadhesiveness.16
Furthermore, CD34 may also be involved in the maintenance of the
hematopoietic progenitor/stem cell phenotype, ie, downregulation of
CD34 may be necessary for differentiation of hematopoietic
progenitor/stem cells. For example, the inappropriate or dysregulated
expression of the full-length CD34 in leukemic cells may contribute to
their undifferentiated phenotype.17 This notion has been
supported by an intriguing report that constitutive overexpression of
recombinant full-length CD34 protein in murine M1 myeloid leukemia
cells blocks differentiation of these cells.18 However, the
forced overexpression of the wild-type truncated form of CD34, which
lacks a major portion of its cytoplasmic domain, fails to inhibit the
cell differentiation of M1 leukemia cells, implying that the
cytoplasmic domain of CD34 is required for the negative regulatory role
for full-length CD34 in hematopoietic differentiation.18
Emerging evidence suggests that the cytoplasmic domain of CD34 may be
involved in transducing a proliferation or differentiation signal in
hematopoietic cells. The serine residues within the cytoplasmic domain
of CD34 have been found to be phosphorylated upon treatment of
hematopoietic cells with protein kinase C (PKC) activators.19 Because PKC is known to be involved in
hematopoietic cell proliferation and differentiation,20,21
it is possible that the cytoplasmic domain of CD34 may have the
capability to transduce a growth or differentiation signal in
hematopoietic cells. Furthermore, one recent study of CD34-deficient
mice shows that hematopoietic progenitor/stem cells are probably
decreased at certain developmental stages in the knockout mice, and
this hematopoietic defect can be reversed by ectopic expression of the
full-length CD34 in the CD34-deficient embryoid bodies; therefore, it
has been proposed that CD34 may be involved in proliferation, survival,
or retention of progenitor/stem cells in the hematopoietic compartment.22 However, surprisingly, a wild-type truncated form of CD34 lacking the majority of the cytoplasmic domain can also
rescue all hematopoietic phenotypes as effectively as the full-length
CD34, suggesting that the crucial functional portion of CD34 is
confined to the extracellular domain, and the cytoplasmic domain of
CD34 appears to be dispensible in hematopoietic
development.22 Thus, the normal functional role of the
cytoplasmic domain of CD34 within hematopoiesis has remained elusive.
In this report, we have examined the potential functional importance of
the cytoplasmic domain of CD34 in cytoadhesion signaling and
proliferation signaling in hematopoietic cells. By comparing the
full-length CD34 with the recombinant truncated and chimeric CD34
molecules expressing in the factor-dependent hematopoietic cell lines,
we have established that the cytoplasmic domain of CD34 is required for
its signal transduction of cellular adhesion. In constrast, we show
here for the first time, to our best knowledge, that the cytoplasmic
domain of CD34 may not contain the elements necessary to transduce a
proliferative signal in hematopoietic cells.
Reagents.
Chemical reagents, including actinomycin D, cycloheximide, cytochalasin
B, EDTA, herbimycin A, H7, sodium azide, sodium fluoride, sodium
othovanadate, staurosporine, and other chemicals for buffers, were
purchased from Sigma (St Louis, MO). Protease inhibitors, including aprotinin, leupeptin, and Pefabloc, were obtained from Boehringer Mannheim (Indianapolis, IN). Alamar Blue
reagent was purchased from Biosource International (Camarillo,
CA). The following antihuman CD34 MoAbs were used in this
study: QBEND 10 (Immunotech, Inc, Westbrook, ME), ICH-3
and BI-3C5 (Accurate Antibodies, Westbury, NY), HPCA-1
(MY10) and HPCA-2 (8G12) (Becton Dickinson, Mountain View,
CA), and VMA27 (Pharmingen, San Diego, CA).
The isotype-matched control MoAbs mouse IgG1, IgG2a, and rat IgG2a were
purchased from Pharmingen. For homotypic adhesion blocking experiments, the following MoAbs directed against mouse adhesion molecules were
used: anti-CD11a M17/4 and 2D7, anti-CD11b M1/70, anti-CD11c HL3,
anti-CD18 C71/16 and M18/2, anti-CD29 9EG7, anti-CD31 MEC13.3, anti-CD44 1M7, anti-CD45 30-F11, anti-CD45R RA3-6B2, anti-CD49d R1-2,
anti-CD54 3E2, 3E2B and KAT-1, anti-CD62L MEL-14, anti-CD71 C2, and
anti-integrin cDNA constructions.
Full-length human CD34 cDNA was obtained from a human thymus cDNA
library (Clontech, Inc, Palo Alto, CA) by the polymerase chain reaction (PCR) technique using Vent DNA polymerase (New England
Biolabs, Beverley, MA) with the 5
Cell culture and transfections.
The factor (IL-3)-dependent murine hematopoietic cell lines
BaF3,25 32D,26 and FDCP127 used in
this study were obtained from Drs Naoki Nakayama and Ian McNiece
(Amgen, Inc) and were cultured in RPMI 1640 medium (GIBCO Life
Technologies, Inc, Grand Island, NY) supplemented with
10% fetal bovine serum (FBS; HyClone Laboratories, Inc, Logan,
UT), 5 ng/mL of murine IL-3 (Amgen, Inc),
penicillin/streptomycin, and L-glutamine (P/S/G) in a CO2 (5%) incubator at 37°C. Similarly, the human hematopoietic cell line KG1a was obtained from Geraldine Trail (Amgen, Inc) and was cultured in Iscove's modified Dulbecco's medium (GIBCO Life
Technologies, Inc) supplemented with 20% FBS and P/S/G. Cells to be
transfected were washed once with HEPES-buffered saline (HeBS) (21 mmol/L HEPES, pH 7.0, 137 mmol/L NaCl, 5 mmol/L KCl, 0.7 mmol/L
Na2HPO4, 5.5 mmol/L dextrose) and resuspended
in ice-cold HeBS at a density of 5 × 106 cells/mL.
Cells were stably transfected with each expression plasmid (20 µg
linearized DNA), which contains the neo gene (G418 resistance), or
cotransfected each expression plasmid with pNeo3 (2 µg linearized
DNA) by electroporation at 300 V, 960 µF with Gene Pulser (BioRad,
Inc, Hercules, CA), and cultured in IL-3-containing medium for 48 hours. Subsequently, the transfected cells were diluted
into the selection medium containing G418 (final concentration, 1 mg/mL) at a density of 1 × 103 or 1 × 104 cells/mL and distributed into flat-bottomed 96-well
microtiter plates (100 µL/well). G418-resistant colonies were
isolated and expanded after 10 to 15 days. Transfectants expressing the
exogenous receptors were identified by fluorescence-activated cell
sorting (FACS) analyses.
Western blot analysis.
The BaF3 cell transfectants were lysed in WCE lysis buffer (20 mmol/L
HEPES, pH 7.4, 2 mmol/L EGTA, 50 mmol/L Homotypic adhesion assay.
The semiquantitative homotypic aggregation assays were performed as
described by Rothlein and Springer28 and Majdic et
al.16 The individual cell clones of BaF3 transfectants were
placed into flat-bottomed 96-well microtiter plates (Falcon, Franklin
Lakes, NJ) at a density of 1.3 × 105
cells per well in 90 µL of basic medium, and 10 µL of anti-CD34 MoAb QBEND 10 was added into each well (final concentration, 5 µg/mL)
and mixed with the cell suspension. The cells were incubated for 90 minutes at 37°C, with mild shaking, and then the degree of cell
aggregation was scored under a microscope. Scores ranged from 0+ to 4+.
0+ represented that less than 10% of the cells were in homoaggregates;
1+, 10% to 40%; 2+, 40% to 70%; 3+, 70% to 100%; and 4+ indicated
100% of the cells were in very large homoaggregates. For antibody
blocking assays, BaF3 cells were preincubated with inhibitor MoAbs
(final concentration, 50 µg/mL) for 30 minutes at 0°C before
initiation of the homotypic aggregation assay by adding anti-CD34 MoAb
QBEND 10 (final concentration, 5 µg/mL).
Cell proliferation assay.
The individual cell clones of BaF3, 32D, and FDCP1 transfectants were
washed extensively with phosphate-buffered saline to remove IL-3 and
seeded onto flat-bottomed 96-well microtiter plates at a density of 5 × 103 cells per well in basic medium supplemented
with the indicated growth factors (final concentration, 5 µg/mL),
including IL-3, EPO, and G-CSF. The cells were incubated for 36 to 48 hours at 37°C in 5% CO2. Subsequently, cell
proliferation was measured by adding 10 µL of Alamar Blue reagent
into each well and returning the 96-well plates to CO2
incubator. After 6 hours of incubation, plates were read
with quantitation of fluorescence by excitation at 530 nm and emission
at 590 nm by CytoFlor II fluorescence plate reader (PerSeptive
Biosystems, Bedford, MA).
The cytoplasmic domain of CD34 is essential for its signal transduction
of cellular adhesion.
To investigate the functional role of the cytoplasmic domain of CD34 in
cytoadhesion signaling, we constructed mammalian cell expression
vectors to carry the recombinant truncated human CD34 without the
cytoplasmic domain (designated as CD34/T) and two chimeric CD34
receptors consisting of the entire extracellular domain of human CD34
and the transmembrane and cytoplasmic regions of either human EPOR or
human G-CSFR, designated as CD34/EPOR and CD34/G-CSFR, respectively
(Fig 1). We note that the recombinant truncated CD34 protein (CD34/T)
generated in this study was truncated after the first amino acid within
the intracellular domain (after the N) and is not the same as wild-type
truncated CD34 protein that has the intracellular domain:
NRRSWSPTGERLELEP (the underlined amino acids are shared with
the full-length CD34). We also constructed an expression vector to
express the full-length human CD34. We transfected each of these
expression constructs and an empty vector alone into the
IL-3-dependent murine lymphoid precursor cell line BaF3, which does
not express endogenous CD34. The transfected G418-resistant cell clones
were analyzed for expression of the introduced receptors by FACS
analysis (Fig 2A), and positive cell clones
from each transfection were selected and expanded, and their expression
of the introduced receptors was confirmed by Western blot analysis (Fig
2B). Analysis of BaF3 cells transfected with vector alone confirmed the
lack of any endogenous CD34. Because the majority of molecular mass of
sialomucin CD34 is contributed by extensive glycosylation and heavily
sialylated glycan chains, there is no significant difference in
molecular mass (115 kD) between the full-length and the truncated CD34
molecules.
CD34 antibody-induced homotypic adhesion requires cellular adenosine
triphosphate (ATP), divalent cations, a functional cytoskeleton, and
possibly protein tyrosine kinases.
To define the cellular components crucial for cytoadhesion induction,
we next examined the effect of inhibitors on CD34 MoAb-induced homotypic aggregation. BaF3-CD34 cells were preincubated with inhibitors for 30 minutes at 37°C before initiation of the
homotypic cell adhesion assay by adding anti-CD34 MoAb QBEND 10. As
shown in Table 1, homotypic adhesion
induced by MoAb QBEND 10 was significantly inhibited by metabolic
depletion of cellular ATP by prior incubation with sodium fluoride and
was completely abrogated by chelation of divalent cations with EDTA or
inhibition of the cytoskeleton by cytochalasin B. The data suggest that
CD34 MoAb-induced homotypic adhesion requires cellular ATP, divalent
cations, and a functional cytoskeleton. However, CD34 MoAb-induced
homotypic adhesion was not affected by prior incubation with
actinomycin D or cycloheximide, suggesting that de novo protein
synthesis was not necessary for cytoadhesion induction. Moreover,
catalases were not involved in this homotypic adhesion because it was
not affected by prior incubation with sodium azide.
Lymphocyte function-associated antigen-1 (LFA-1) and intercellular
adhesion molecule-1 (ICAM-1) may be involved in CD34 antibody-induced
homotypic aggregation.
It has been previously suggested that a concomitant activation of the
integrin
The cytoplasmic domain of CD34 is not sufficient to transduce growth
signal in hematopoietic cells.
It has been postulated that CD34 may be involved in the maintenance or
proliferation of the hematopoietic progenitor/stem cells22;
therefore, we wish to use chimeric receptor constructs to investigate whether the cytoplasmic domain of CD34 contains the elements necessary to transduce a proliferative signal in hematopoietic cells. We generated two mammalian cell expression vectors to produce chimeric receptors consisting of the extracellular domain of either human EPOR
or human G-CSFR and the cytoplasmic region of human CD34, designated as
EPOR/CD34 and G-CSFR/CD34, respectively
(Fig 4). We also constructed two expression
vectors to express the full-length EPOR or G-CSFR. We transfected each
of these four receptor-expressing constructs and an empty vector alone
into the IL-3-dependent BaF3 cells, which do not express endogenous
EPOR or G-CSFR or CD34. The transfected G418-resistant cell clones were
analyzed for expression of the introduced receptors by FACS analysis
(Fig 5a through d), and positive cell
clones from each transfection were selected and expanded. In general,
the expression level of the chimeric receptor EPOR/CD34 or G-CSFR/CD34
was greater than that of the full-length EPOR or G-CSFR. Although we
could only obtain low expressing clones of BaF3-EPOR and BaF3-G-CSFR
after several times of transfection, these cell clones could respond to
the corresponding growth factors well. Subsequently, we have assayed
the positive cell clones for proliferation or survival activity in the
presence of EPO or G-CSF specifically, using IL-3 as positive control
and no factor as negative control. Our results indicated that none of
the chimeric receptors (EPOR/CD34, G-CSFR/CD34) could support cell
growth or survival, whereas the full-length EPOR or G-CSFR could
stimulate cell proliferation in response to EPO or G-CSF, respectively
(Fig 6A). Furthermore, to generalize this
investigation, we repeated the same DNA transfection and proliferation
experiments with IL-3-dependent murine myeloid cell line 32D and
hematopoietic precursor cell line FDCP1, which do not express
endogenous EPOR or G-CSFR or CD34. Analysis of BaF3 or 32D or FDCP1
cells tranfected with vector alone confirmed the lack of any endogenous
EPOR or G-CSFR or CD34. Receptor-expressing clones of 32D and FDCP1
cells (Fig 5e through l) were selected and assayed for proliferation activity in the presence of IL-3 or EPO or G-CSF as described above.
Similar results were obtained with 32D and FDCP1 cells (Fig 6B and C),
indicating that the observed phenomena are not due to a particular cell
type. Taken together, these results imply that the cytoplasmic domain
of CD34 may not contain the elements necessary to transmit a
growth-stimulatory signal in hematopoietic cells. However, it has not
been determined whether the intracellular "NRRSWSPTGERL" domain
in wild-type truncated CD34 can signal in this proliferation study.
Interestingly, overexpression of the cytoplasmic domain of full-length
CD34 through the chimeric receptors (EPOR/CD34, G-CSFR/CD34) diminished
approximately 25% of the IL-3 responsiveness of Baf3, 32D, and FDCP1
cells (Fig 6), suggesting that the cytoplasmic domain of CD34 may
either have a negative effect on cytokine-mediated signaling or compete
with the cytoplasmic domain of IL-3 receptor for vital signaling
components in these cells.
In the present study, using the truncated and chimeric receptor
approaches, we found that the cytoplasmic domain of CD34 is required
for its signal transduction of cellular adhesion in hematopoietic cells. This is consistent with the notion that the cytoplasmic domain
of CD34 is indispensable for the negative regulatory function of
full-length CD34 in hematopoietic differentiation.18 In
agreement with the previous findings,16 we showed that CD34
indeed has cytoadhesive signal transducing capability. Although
homotypic adhesion of BaF3-CD34 cells depends on molecular engagement
of certain epitopes on CD34 molecule, evidence argues against the possibility that the observed homotypic aggregation is merely a passive
agglutination of these cells by certain CD34 MoAbs. Firstly,
cytoadhesion induction of BaF3-CD34 cells by MoAb QBEND 10 was
temperature-dependent and no homoaggregate formation could be induced
at 0°C. Secondly, unlike KG1a cells, none of the anti-CD34 MoAbs
can induce homotypic aggregation of KG1 cells that also express a high
level of CD34 but a very low level of ICAM-1.16 Thirdly,
concomitant activation of the LFA-1/ICAM-1 cytoadhesion pathway may be
involved in the CD34-dependent cytoadhesion formation.16 Fourthly, ectopic expression of the human CD34 molecule in murine hematopoietic cells confers elevated binding of these cells to human BM
stromal cells or cell lines but not to their murine counterparts, implying that the cytoadhesive function of CD34 requires specific recognition of counter-receptor or ligand on stromal
cells.15 Perhaps the stimulatory anti-CD34 MoAbs act as
surrogate ligands mimicking the binding of natural ligands or
counter-receptors by interacting with the ligand binding domains,
presumably glycosylated regions, of the CD34 molecule.
Submitted September 23, 1997;
accepted November 25, 1997.
The authors thank S. Nagata for providing the full-length human G-CSFR
cDNA and pEF-BOS plasmid; S. Elliott for providing the full-length
human EPOR cDNA; R. Nelson for providing L-selectin-IgG/Fc fusion
protein; L. Antonio for DNA sequencing; T. Boone for mouse IL-3; L. Souza for human G-CSF; V. Gottmer for technical illustration; and W. Boyle, R. Bosselman, and L. Souza for their support.
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1 |
Abstract
Introduction
Abstract
7. These MoAbs were purchased either from Pharmingen
or Serotec Ltd (Indianapolis, IN). L-selectin-IgG/Fc fusion protein was kindly provided by Dr Richard Nelson (Amgen, Inc,
Thousand Oaks, CA). Recombinant murine interleukin-3 (IL-3) produced in
Escherichia coli, human erythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF) produced in Chinese hamster ovary
cells were prepared at Amgen, Inc.
primer
containing the translational start (underlined)
5
24-neo, which contains the simian virus 40 early
promoter and the R-U5 segment of human T-cell leukemia virus type I
long terminal repeat, and designated pSR
