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Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1152-1162
The Cytoplasmic Domain of Stem Cell Antigen CD34 Is Essential for
Cytoadhesion Signaling But Not Sufficient for Proliferation Signaling
By
Mickey C.-T. Hu and
Shu L. Chien
From the Department of Cell Biology, Amgen, Inc, Thousand Oaks, CA.
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ABSTRACT |
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.
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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  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.
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 primer
containing the translational start (underlined)
5-TATGGTACCAAGCTTGCCACCATGCCGCGGGGCTGGACCGCGCTTTGC-3 and the 3 primer containing the end of cytoplasmic domain and two stop codons (underlined)
5-TATGTCGACATCGATTCATCACAATTCGGTATCAGCCACCACGTGTTG-3. The PCR-generated product was cloned into the expression vector pEF-BOS23 between the Kpn I and Sal I sites
and designated pEF-CD34. The recombinant truncated form of CD34 cDNA,
which was truncated after the first amino acid within the intracellular
domain (after the N, amino acid 301), was constructed by the same PCR
technique with the 5 primer containing the translational start
(underlined) 5-TATGGTACCGAATTCGCCACCATGCCGCGGGGCTGGACCGCGCTTTGC-3
and the truncated 3 primer containing the end of transmembrane
domain and the first amino acid of the intracellular domain and two
stop codons (underlined)
5-TATGTCGACATCGATTCATCATTCATCAGGAAATAGCCAGTGATGCCC-3. The PCR product was cloned into the expression vector pEF-BOS between
the Kpn I and Sal I sites and designated pEF-CD34/T.
The full-length human EPOR and G-CSFR cDNAs were obtained from Drs Steven Elliott (Amgen, Inc) and Shigekazu Nagata (Osaka,
Japan), respectively, and were subcloned into the expression vector
pSR 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-EPOR and pSR-G-CSFR,
respectively. The cDNA of chimeric receptor CD34/EPOR or CD34/G-CSFR
was generated by using a two-step PCR to combine the extracellular
domain of CD34 with the transmembrane and cytoplasmic domains of either EPOR or G-CSFR, and the cDNAs were subcloned into the expression vector
pEF-BOS between Kpn I and Sal I and designated
pEF-CD34/EPOR and pEF-CD34/G-CSFR, respectively. Thus, all of the cDNAs
listed in Fig 1 are in the expression
vector pEF-BOS that uses a powerful elongation factor promoter to drive
transcription of the inserted cDNAs. Similarly, the cDNA of chimeric
receptor EPOR/CD34 or G-CSFR/CD34 was generated by using a two-step PCR
to combine the extracellular domain of either EPOR or G-CSFR with the
transmembrane and cytoplasmic domains of CD34, and the cDNAs were
subcloned into the expression vector pSR-neo between the
EcoRI and Cla I, designated pSR-EPOR/CD34 and
pSR-G-CSFR/CD34, respectively. Therefore, all of the cDNAs listed in
Fig 4 are in the expression vector pSR-neo that uses a strong SR a
promoter to drive transcription of the inserted cDNAs. The sequences of
all cDNA constructs were confirmed by DNA sequencing on both strands
using a PCR procedure employing fluorescent dideoxynucleotides and a
model 373A automated sequencer (Applied Biosystems, Foster City,
CA).
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| Fig 1.
Schematic representation of the truncated CD34 and CD34
chimeric receptors. The sequences are aligned at their transmembrane domains (shown by a solid vertical bar). The sequence of CD34 is
represented by an open box, EPOR is shown by a shaded box, and G-CSFR
is depicted by a hatched box.
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| Fig 4.
Schematic representation of EPOR/CD34 and G-CSFR/CD34
chimeric receptors. The sequences are aligned at their transmembrane domains, which are depicted by a solid vertical bar. The sequence of
EPOR is shown by a shaded box, G-CSFR is denoted by a hatched box, and
CD34 is represented by an open box.
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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 -glycerophosphate, 1%
Triton X-100, 10% glycerol, 1 mmol/L dithiothreitol, 2 µg/mL leupeptin, 5 µg/mL aprotinin, 1 mmol/L Pefabloc [Boehringer
Mannheim], and 1 mmol/L sodium othovanadate). Soluble lysates were
prepared by centrifugation at 10,000g for 30 minutes at
4°C, electrophoresed through an 8% sodium dodecyl sulfate
(SDS)-polyacrylamide gel, and electroblotted onto polyvinylidene
difluoride (PVDF) membranes (Novex, Inc, San Diego,
CA). The blot was probed with anti-CD34 MoAb QBEND 10 and
visualized by enhanced chemiluminescence (ECL) detection (Amersham,
Arlington Heights, IL) using goat antimouse IgG
conjugated to horseradish peroxidase as a secondary antibody (Pierce,
Rockford, IL).
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).
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RESULTS |
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.
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| Fig 2.
Expression of the full-length or truncated CD34 and CD34
chimeric receptors on the BaF3 cell transfectants. BaF3 cells were stably transfected with each receptor expression construct as indicated, and individual positive cell clones were selected. (A) Flow
cytometry analyses. The vector-transfected BaF3 cells (negative
controls, open histograms) or the receptor-transfected BaF3 cells
(solid histograms) were stained with the fluorescein isothiocyanate
(FITC)-labeled anti-CD34 MoAb HPCA-2. The staining profiles of the
BaF3-transfected cells with an isotype-matched control MoAb are the
same as those of negative controls (shown as open histograms). (B)
Western blot analyses. Cell lysates from the BaF3 cell transfectants
were electrophoresed through an 8% SDS-polyacrylamide gel and
transferred to a PVDF membrane. The blot was probed with anti-CD34 MoAb
QBEND 10 and visualized by ECL detection using goat antimouse IgG
conjugated to horseradish peroxidase as a secondary antibody.
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It has been reported that certain MoAbs directed against
O-sialoglycoprotease-sensitive epitopes of human CD34 can trigger homotypic aggregation of CD34+ KG1a cells.16 To
examine whether these MoAbs can induce homotypic adhesion of
BaF3-transfected cells, we incubated BaF3-CD34 cells with several
different anti-CD34 MoAbs and examined their effects on homotypic
adhesion of these cells at different time points. We found that
anti-CD34 MoAb QBEND 10 induced marked homoaggregate formation of
BaF3-CD34 cells and MoAb HPCA-1 had a less profound but definite
homoaggregation-inducing effect, whereas four other anti-CD34 MoAbs
(ICH3, BI-3C5, HPCA-2, and VMA27) were ineffective in this regard. In
accordance with the previous findings,16 adhesion induction
of BaF3-CD34 cells by MoAb QBEND 10 was temperature-dependent and no
homoaggregate formation could be induced at 0°C. The data suggest
that the observed homotypic adhesion was not due to antibody-mediated passive aggregation. The homotypic aggregation occurred after 30 minutes and reached maximal at 90 minutes at 37°C. Titration experiments indicated that the optimal concentration of QBEND 10 to
induce homoaggregate formation of BaF3-CD34 cells was around 5 µg/mL.
A typical example of the observed homoaggregation of BaF3-CD34 cells is
shown in Fig 3B.
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| Fig 3.
Homotypic aggregation of the BaF3 cell transfectants upon
addition of anti-CD34 MoAb QBEND 10. BaF3-CD34 cells were incubated with the negative control MoAb (A) or anti-CD34 MoAb QBEND 10 (B),
which represents the typical morphology of homoaggregate formation of
BaF3-CD34 cells (score, 3+). Similarly, BaF3-CD34/T cells (C),
BaF3-CD34/EPOR cells (D), and BaF3-CD34/G-CSFR cells (E) were treated
with MoAb QBEND 10. As a positive control, KG1a cells were also
incubated with MoAb QBEND 10 (F). The final concentration of MoAb is 5 µg/mL. This experiment has been repeated five times with similar
results.
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Subsequently, we investigated the contribution of the cytoplasmic
domain of CD34 in cytoadhesion signaling based on this induction of
homotypic adhesion assay. As expected, treatment of the empty vector-transfected BaF3 cells with MoAb QBEND 10 did not induce any
detectable homoaggregate formation, and a similar result was obtained
from the untransfected BaF3 cells. Whereas incubation of BaF3-CD34
cells with the control MoAb did not induce any detectable homoaggregate
formation (Fig 3A), incubation of these cells with MoAb QBEND 10 caused
strong homotypic aggregation (Fig 3B). Strikingly, incubation of
BaF3-CD34/T cells with MoAb QBEND 10 failed to induce any detectable
homotypic aggregation (Fig 3C), suggesting that the cytoplasmic domain
of CD34 is essential for induction of homotypic adhesion via the CD34
molecule. Furthermore, incubation of BaF3-CD34/EPOR and
BaF3-CD34/G-CSFR cells with MoAb QBEND 10 also failed to cause any
detectable homoaggregate formation (Fig 3D and E), implicating that
cytoadhesion signaling through the cytoplasmic domain of CD34 cannot be
restored through those domains of EPOR and G-CSFR. Similar results were
obtained with two independent BaF3 cell clones from each transfection
(Fig 3A through E) in three independent assays. As a positive control,
CD34+ KG1a cells were incubated with MoAb QBEND 10 and
showed marked homotypic aggregation (Fig 3F). Taken together, these
results strongly suggest that the cytoplasmic domain of CD34 is
required for its signal transduction of cellular adhesion.
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.
To further test whether protein tyrosine kinase or PKC activity was
involved in cytoadhesion signaling of CD34 MoAb-induced homotypic
adhesion, we pretreated BaF3-CD34 cells with the protein tyrosine
kinase inhibitor herbimycin A29,30 or the PKC inhibitors
staurosporine31 and H732 and analyzed the
influence of these agents on CD34 MoAb-induced homoaggregate formation.
Herbimycin A significantly reduced homotypic aggregation of BaF3-CD34
cells upon the binding of MoAb QBEND 10. However, staurosporine and H7
only slightly inhibited CD34 MoAb-induced homotypic adhesion. These
results suggest that certain protein tyrosine kinases may be involved
in CD34 cytoadhesion signaling. All of the substances used at the
indicated concentrations did not affect cell viability during the
experiments as determined by trypan blue staining.
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 2 adhesion pathway may be involved in CD34 MoAb-induced homotypic adhesion.16 To confirm their
interesting findings, we performed MoAb inhibition experiments by
preincubation of BaF3-CD34 cells at 0°C with blocking MoAbs against
integrin 1 (CD49d, CD29), integrin 2
(CD11a, CD18), ICAM-1 (CD54), CD11b, CD11c, CD31, CD44, CD45, and other
adhesion molecules before the addition of MoAb QBEND 10. As listed in
Table 2, one of the anti-CD18 and anti-CD54
MoAbs could indeed significantly but not completely inhibit the CD34
MoAb-induced homotypic adhesion of BaF3-CD34 cells; however, other
anti-CD18 and anti-CD54 MoAbs used here only partially reduced the
homotypic adhesion of BaF3-CD34 cells. In addition, anti-CD11a MoAbs
did not significantly block the homotypic adhesion of BaF3-CD34 cells.
Perhaps the observed incomplete inhibition of some MoAbs was due to
ineffective blocking activities of those MoAbs. Nevertheless,
anti-CD11a, anti-CD18, and anti-CD54 MoAbs were the only MoAbs tested
so far that could impede the CD34 MoAb-induced homotypic adhesion of
BaF3-CD34 cells. These data agree well with the previous
results16 and suggest that LFA-1 (CD11a/CD18) and ICAM-1
(CD54) may be involved in CD34 MoAb-induced homotypic adhesion.
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.
View larger version (31K):
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| Fig 5.
Expression of EPOR, G-CSFR, and their chimeric receptors
EPOR/CD34 and G-CSFR/CD34 on the BaF3 or 32D or FDCP1 cell
transfectants. BaF3 (a through d), 32D (e through h), or FDCP1 (i
through l) cells were stably transfected with each receptor expression
construct as indicated, and individual positive cell clones were
selected by flow cytometry analyses. The vector-transfected cells
(negative controls, open histograms) or the receptor-transfected cells
(solid histograms) were stained with either anti-EPOR MoAb or the
FITC-labeled G-CSF. The staining profiles of the transfected cells with
an isotype-matched control MoAb are the same as those of negative controls, which are depicted as open histograms.
|
|
View larger version (43K):
[in this window]
[in a new window]
| Fig 6.
Proliferation of the BaF3, 32D, and FDCP1 cell
transfectants in response to IL-3, EPO, or G-CSF. The individual cell
clones as indicated were incubated in the absence of any growth factor (strongly shaded box), as a negative control, or in the presence of
IL-3 (medianly shaded box) or EPO (lightly shaded box) or G-CSF (unshaded box) for 36 to 48 hours. Proliferation was measured by
reduction of Alamar Blue, with quantitation of fluorescence by
excitation at 530 nm and emission at 590 nm. Error bars indicate the
mean and standard deviation for triplicate assay values.
|
|
| |
DISCUSSION |
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.
Recently, one study of CD34-knockout mice shows that hematopoiesis is
delayed in developing embryoid bodies; that hematopoietic progenitor
cells from yolk sac, fetal liver, and adult BM are reduced twofold to
threefold; and that the BM progenitor cells are retarded in their
ability to expand ex vivo in response to various hematopoietic growth
factors.22 In addition, ectopic expression of the
full-length CD34 in the CD34-deficient embryoid bodies results in a
reversal of this hematopoietic deficiency and suggests that CD34 may be
involved in the proliferation or maintenance of the hematopoietic
progenitor/stem cells. However, a wild-type truncated form of CD34,
which lacks the majority of the cytoplasmic domain, can also rescue all
hematopoietic phenotypes to a similar degree as the full-length CD34,
implying that the cytoplasmic domain of CD34 is not necessary for its
function in hematopoietic development.22 Evidently, our
present results did not accord with their implications. Several
possible reasons may explain this difference. Firstly, Cheng et
al22 have pointed out that the high level overexpression of
either the full-length or the wild-type truncated form of CD34 caused
losses of normal CD34 regulation and tissue-specific expression in
their transfection system so that the transfected cells may have
overcome any need for either PKC-mediated or other intracellular
signaling phenomena. Secondly, it is possible that the intracellular
"NRRSWSPTGERLELEP" domain of the wild-type truncated CD34 may be
able to transduce signals and play an important role in rescuing the
phenotype in the CD34-knockout embryoid bodies. Thirdly, other cellular
receptors such as the transmembrane glycoprotein CD43 (leukosialin and
sialophorin),5 which structurally resemble CD34 and have
the characteristic features of cell-associated mucins, may share
similar functions as CD34 in hematopoietic development. The expression
of these CD43-like redundant receptors could be affected in their
CD34-knockout mice. When they rescue the CD34-deficient phenotype with
the wild-type truncated form of CD34, the expression of those CD43-like
receptors may be restored also and they can compensate the defect of
the wild-type truncated form of CD34 in those transfected cells.
Fourthly, the human CD34 gene locus has been mapped to chromosome 1q32, a region that contains genes encoding many hematopoietic regulatory and
signaling molecules.33 Perhaps some important regulatory or
signaling molecules, which are downstream of CD34 signaling, are also
turned on by forcibly expressing the wild-type truncated form of CD34
in the transfected cells and achieve the CD34+ functional
phenotypes. Finally, another study of CD34-deficient mice indicates
decreased eosinophil accumulation after allergen exposure but rather
normal hematopoietic development,34 suggesting that the
hematopoietic defective phenotypes observed in the former CD34-knockout
study may not be a generalized case. Perhaps the expression of other
redundant receptors such as CD43 or some vital regulatory and signaling
molecules as described above is not affected in the latter
CD34-knockout experiment. Therefore, they do not observe the same
phenotypes as described by the former study.22 Nevertheless, the discrepancy between these two CD34-knockout studies
has not been clearly resolved yet.
Using the EPOR/CD34 and G-CSFR/CD34 chimeric receptors, we showed that
the cytoplasmic domain of CD34 failed to stimulate cell growth in
response to EPO or G-CSF, respectively, implying that the cytoplasmic
portion of CD34 may not contain the elements necessary to transduce a
proliferative signal in hematopoietic cells. This is consistent with
the recent findings obtained by Dr Toshio Suda in Japan (personal
communication, November 24, 1996), where they generated
the c-Kit/CD34 chimeric receptors to test the proliferative signaling
potential of the cytoplasmic region of CD34. In addition, the observed
CD34 MoAb-induced adhesion formation could not be significantly blocked
by staurosporine or H7, which are potent PKC inhibitors, suggesting
that PKC activation may not be involved in this cytoadhesive signaling
pathway. In contrast, CD34 MoAb-induced cytoadhesion could be
significantly inhibited by herbimycin A, which is a selective inhibitor
of several protein tyrosine kinases,16 implicating that
some tyrosine phosphorylation event might be involved in the
CD34-mediated signal transduction of cellular adhesion. These results
agree well with the previous data16 and the new findings
observed by Dr Toshio Suda (personal communication, August 26, 1997). However, the cytoadhesive signaling pathways via
the cytoplasmic domain of CD34 are not understood yet and remain to be
elucidated.
In accordance with the previous findings,16 we found that
CD34 MoAb-induced cytoadhesion could be suppressed, but not completely abrogated, by blocking MoAbs directed against some epitopes of LFA-1
(CD11a/CD18) and its counter-receptor ICAM-1 (CD54). This suggests, but
does not prove, that a concomitant activation of the LFA-1/ICAM-1
cytoadhesion pathway may play a role in the CD34-mediated cellular
adhesion. MoAbs against other adhesion molecules or receptors listed in
Table 2 did not indicate any inhibitory effects on the CD34
MoAb-induced homotypic aggregation. However, certain unidentified
cellular adhesion molecules may also participate in the CD34-mediated
cytoadhesion. The mechanism by which LFA-1/ICAM-1 cooperates with CD34
in cellular adhesion is unknown. Moreover, the natural ligands for CD34
on hematopoietic precursor cells are still enigmatic. It has been found
that L-selectin, the lymphocyte homing receptor,35,36 can
bind to CD34 expressed on high endothelial venule (HEV) cells in the
lymph nodes and that this binding is sialic acid specific and
Ca2+-dependent.10,37 However, we were unable to
detect any binding of L-selectin-IgG/Fc fusion protein with BaF3-CD34
or KG1a cells (data not shown), both of which express CD34 highly. In
fact, thus far, the L-selectin-IgG/Fc chimeric protein has not been reported to bind to CD34 expressed on hematopoietic progenitor cells.4 This finding suggests that the glycosylation
patterns of CD34 on hematopoietic cells may be different from those on endothelial cells of HEV so that the adherence of CD34 to L-selectin is
abolished. Alternatively, CD34 on hematopoietic cells may primarily interact with other unidentified L-selectin-like ligands on
endothelial or stromal cells in the BM compartment.
Cellular adhesion and migration of hematopoietic progenitor/stem cells
is likely to be crucial in embryonic or fetal hematopoietic development
and the dynamic recapitulation of hematopoiesis that occurs in the
adult BM. It has been postulated that CD34+ hematopoietic
cells administered intravenously are able to migrate to the BM
compartment for their development after BM transplantation. Such a
homing mechanism may mimic the multistep process identified for
leukocyte-endothelial cell interactions.38,39 When the CD34+ hematopoietic progenitor/stem cells are halted in the
blood stream by the interaction of CD34 with L-selectin or certain
L-selectin-like adhesion molecules on the endothelial cells of blood
vessels in the BM, they may migrate through the endothelial layer, bind
to the extracellular matrix of BM stroma, and undergo differentiation or proliferation in the BM. Although this hypothesis requires further
investigation, our findings about the essential role of cytoplasmic
domain of CD34 in its cytoadhesive signaling open an avenue to
delineate the mechanism by which CD34 mediates the cellular adhesion of
hematopoietic progenitor/stem cells to BM stroma.
| |
FOOTNOTES |
Submitted September 23, 1997;
accepted November 25, 1997.
Address reprint requests to Mickey C.-T. Hu, PhD, Amgen,
Inc, 14-1-D, Thousand Oaks, CA 91320.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
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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Abstract
Introduction
Abstract