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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2301-2309
Neuropilin-1 Is Expressed on Bone Marrow Stromal Cells: A Novel
Interaction With Hematopoietic Cells?
By
Rafaèle Tordjman,
Nathalie Ortéga,
Laure Coulombel,
Jean Plouët,
Paul-Henri Roméo, and
Valérie Lemarchandel
From INSERM U474, Hopital Henri Mondor, Créteil, France;
Laboratoire de Biologie Moléculaire Eucaryote, CNRS UPR 9006, Toulouse, France; and INSERM U363, IGR, Villejuif, France.
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ABSTRACT |
In adult bone marrow, hematopoietic stem cells are found in close
association with distinctive stromal cell elements. This association is
necessary for maintenance of hematopoiesis, but the precise mechanisms
underlying the cross-talk between stromal cells and hematopoietic stem
cells are poorly understood. In this study, we used a bone marrow
stromal cell line (MS-5) that is able to support human long-term
hematopoiesis. This hematopoietic-promoting activity cannot be related
to expression of known cytokines and is abolished by addition of
hydrocortisone. Using a gene trap strategy that selects genes encoding
transmembrane or secreted proteins expressed by MS-5 cells, we obtained
several insertions that produced fusion proteins. In one clone, fusion
protein activity was downregulated in the presence of hydrocortisone,
and we show that insertion of the trap vector has occurred into the
neuropilin-1 gene. Neuropilin-1 is expressed in MS-5 cells, in other
hematopoietic-supporting cell lines, and in primary stromal cells but
not in primitive hematopoietic cells. We show that neuropilin-1 acts as
a functional cell-surface receptor in MS-5 cells. Two neuropilin-1
ligands, semaphorin III and VEGF 165, can bind to these cells, and the addition of VEGF 165 to MS-5 cells increases expression of 2 cytokines known to regulate early hematopoiesis, Tpo and Flt3-L. Finally, we show
that stromal cells and immature hematopoietic cells express different
neuropilin-1 ligands. We propose that neuropilin-1 may act as a novel
receptor on stromal cells by mediating interactions between stroma and
primitive hematopoietic cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DURING ADULT HEMATOPOIESIS, proliferation
and differentiation of hematopoietic progenitors occur within the bone
marrow. Stromal cells, which form the backbone of the bone marrow
microenvironment, consist of myofibroblasts, endothelial cells,
adipocytes, osteoclasts, and macrophages. These cells provide a complex
array of extracellular matrix proteins that facilitate cell-cell
interactions. In addition, stromal cells are instrumental in providing
various soluble or resident cytokines for the controlled
differentiation and proliferation of early hematopoietic progenitors.
Molecules mediating interactions between hematopoietic cells and
stromal cells (adhesion molecules, cytokines, and their receptors) are
either transmembrane or secreted proteins.
Long-term bone marrow cultures have been developed to reproduce the
bone marrow microenvironment in vitro. In these cultures, maintenance
of hematopoiesis requires the prior formation of an adherent layer
whose cellular complexity prevents the understanding of the
relationship between stromal cells and hematopoietic
cells.1,2 Some murine stromal cell lines have been shown to
support growth and differentiation of human immature cells. One of
them, the murine MS-5 cell line, was derived from the irradiated
adherent layer of a Dexter-type long-term culture.3 MS-5
promotes the expansion of human hematopoietic cells selected for their
high expression of CD34 antigen and lack of expression of CD38 antigen (CD34+/CD38 ) for 5 to 10 weeks and
without the addition of human growth factors.4 Futhermore,
MS-5 cells also support the B-lymphoid potential of human
CD34+/CD38 cells.5 No
combination of murine cytokines, which are active on human cells, was
able to mimic the hematopoietic-promoting activity of
MS-5.6 Finally, addition of hydrocortisone in the coculture
inhibits the hematopoietic-promoting activity of MS-5 cells,7 and exogenous supply of growth factors cannot
reverse this effect. Altogether, these data strongly suggest that
hydrocortisone may downregulate an MS-5-derived activity which is
necessary for proliferation or differentiation of
CD34+/CD38 cells or, alternatively,
upregulate an MS-5-derived inhibitory activity.
In this study, we used a gene-trap strategy designed to clone secreted
and transmembrane molecules expressed by MS-5 cells, and we have
selected insertions that produced fusion proteins whose expression was
modulated by hydrocortisone. We obtained 10 independent clones in
which fusion protein activity was downregulated by hydrocortisone. In
one of these clones, vector insertion has occurred into the
neuropilin-1 gene. We show that neuropilin-1 acts as a functional
receptor on stromal cells and that neuropilin-1 ligands are
differentially expressed in stromal cells and in immature hematopoietic cells.
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MATERIALS AND METHODS |
Cell Lines
Stromal MS-5, 30E, 30W, and S17 cell lines.
These murine bone marrow-derived cell lines were maintained in  Modified Eagle's Medium (MEM) supplemented with 10%
fetal calf serum (FCS; purchased from Stem Cell Technologies,
Vancouver, Canada). Hydrocortisone (106 mol/L,
hydrocortisone 21-hemisuccinate; Sigma, St Louis, MO) was
added when indicated. 30E and 30W cells were kindly provided by Dr D. Rennick (DNAX Institute, Palo Alto, CA), MS-5 cells by Dr K. Mori
(Niigata University, Niigata, Japan), and S17 cells by Dr
A. Cumano (Pasteur Institute, Paris, France).
Chinese hamster ovary (CHO) cells.
Xylosyltransferase-deficient pgsA-745 CHO cells were cultured in
DMEM-F12 medium supplemented with 10% FCS. Cells were transfected with
10 µg of expression vector carrying either the rat NP-1
cDNA8 or the human KDR cDNA and 1 µg of pSV2 expression
vector carrying the neomycin resistance gene with the lipofectin
reagent (GIBCO, Ceigy Pontoise, France). The KDR cDNA we
used consists of coding sequences from position 1 to 24779
and encodes a receptor that lacks the kinase domain. Stable integrants were selected using 500 µg/mL G418 (GIBCO). Cloning was performed by
colony isolation using a Pasteur pipette and clones were further screened for their ability to specifically bind iodinated
VEGF.10
293 cells.
The semaphorin III-AP secreting cell line was previously
described.8
Human umbilical vein endothelial cells (HUVEC).
HUVEC were cultured as described.11 For proliferation
assays, 10,000 HUVEC were seeded on gelatin-coated 12 multiwell plates in SFM medium (GIBCO) supplemented with 10% fetal calf serum.
Bone Marrow-Derived Stromal Cells and Hematopoietic Progenitors
Human bone marrow samples were obtained from patients undergoing hip
surgery. Cells were extracted from the bone fragments as previously
described.7
Culture of human marrow adherent cells.
Long-term bone marrow cultures were established in a mixture of FCS and
horse serum and 106 mol/L
hydrocortisone.7 After 4 to 6 weeks in culture at 33°C, nonadherent cells were discarded and adherent cells were detached with trypsin.
Selective culture of human fibroblasts.
To grow selectively fibroblastic cells, bone marrow cells were
incubated in MEM with 10% FCS (but without horse serum) and hydrocortisone in 25-cm2 flasks at a concentration of
106 cells/mL. After 24 hours, nonadherent
cells were removed and fresh medium was added. After 3 to 4 weeks of
culture at 37°C, cells were subcultured and collected after 3 passages as previously described.12 At this time, adherent
cells were devoid of endothelial cells because no expression of von
Willebrand factor could be detected by reverse transcription-polymerase
chain reaction (RT-PCR) (data not shown).
Isolation of CD34+/CD38 cells.
Low-density mononuclear cells were subjected to a standard CD34
immunomagnetic bead separation using the miniMACS system following the
manufacturer's guidelines (Miltenyi Biotec, Auburn, CA).
CD34+ cells obtained after bead separation were further
purified by cell sorting either immediately or after overnight
incubation at 4°C in high serum concentration. Before sorting,
CD34+ cells were incubated with a 1/5 dilution of a
Cy-5-phycoerythrin (PE)-anti-CD34 monoclonal antibody (MoAb)
(Immunotech, Marseille, France) and PE-anti CD38 MoAb (Becton
Dickinson, San Jose, CA). Sorting of
CD34+CD38low fractions was performed using a
FACS-Vantage equipped with an argon ion laser (Innova 70-4-Coherent
radiation; Innova, Palo Alto, CA) tuned to 488 nm and
operating at 500 mW. A morphological gate including all the
CD34+ cells was determined on 2-parameter histogram side
scatter (SSC) versus forward scatter (FSC). Limit for
CD38low cells was defined using control cells labeled with
PE-CD34 (HPCA2; Becton Dickinson) and an irrelevant IgG1 MoAb.
CD38low represented 10% to 15% of total
CD34high cells. Compensation was set up as described above.
Culture of murine marrow adherent cells.
Bone marrow from C56Bl mice was obtained by flushing cells from 1 tibia
and 1 femur. Undissociated cells were grown in a 25-cm2
flask in LTC medium (purchased from Stem Cell Technologies) with 106 mol/L hydrocortisone. At weekly intervals, half
of the medium containing nonadherent cells was collected and replaced
by fresh medium. After 5 weeks, adherent cells were detached with trypsin.
Gene Trap Strategy
Transfection and selection conditions.
The gene-trap vector pGT1.8TM, provided by Dr William Skarnes
(University of California, Berkeley), was introduced into MS-5 cells
using Lipofectamine reagent (GIBCO) following the manufacturer's instructions. A total of 108 cells divided in 50 100-mm
Petri dishes were transfected with 600 µg of linearized plasmid DNA.
After 3 days, the content of each Petri dish was split up into 5 dishes
and cells were selected for resistance to G418 at a concentration of
250 µg/mL for 15 days. After G418 selection, 750 clones were tested
for  -galactosidase activity.
For in situ staining, cells were washed in phosphate-buffered saline
(PBS), fixed in 4% paraformaldehyde for 10 minutes, and stained with
X-gal solution (4 mmol/L K4 [Fe (CN)6]; 4 mmol/L K3 [Fe(CN)6]; 4 mmol/L
MgCl2; 0.4 mg/mL of Xgal [Amersham, Pharmacia Biotech, Orsay, France] in PBS). For quantitative assays,
MS-5 cells grown in 100-mm Petri dishes were collected, washed, and lysed in 100 µL of 250 mmol/L Tris pH 7.8, 0.2% TritonX-100, 5 mmol/L dithiothreitol (DTT), 10% glycerol. Thirty
microliters of cell lysate was assayed with
o-nitrophenyl--D-galactopyranoside (Pharmacia) as previously
described.13
5' RACE.
Total RNA from MS-5 clones was prepared using Trizol reagent (GIBCO).
Cloning of 5' cDNA ends was performed using the 5' RACE system (GIBCO). First-strand cDNA was reverse transcribed in a volume
of 25 µL where 1 µg of total RNA was annealed to 2.5 pmol of
oligonucleotide 1 (5' CCA GAA CCA GCA AAC TGA AGG G 3', CD4 sequences located 364 bp from the splice site), as
previously described.14 PCR of dC-tailed cDNA was performed
using the Abridged Anchor Primer (AAP) from the kit and oligonucleotide
2 (5' AGT AGA CTT CTG CAC AGA CAC C 3', CD4 sequences
located 281 bp from the splice site). This primary PCR product was
diluted 500-fold following the manufacturer's instructions and used as
template in a nested amplification using the Abridged Universal
Amplification Primer (AUAP) and oligonucleotide 3 (5' TGC TCT GTC
AGG TAC CTG TTG G 3', engrailed-2 sequences located 130 bp from
the splice site). PCR products were cloned in pCRII vector (Invitrogen,
Carlsbad, CA) for sequencing.
RT-PCR Analysis
First-strand cDNA was reverse-transcribed in a volume of 30 µL. One
microgram of total RNA was annealed to 300 ng of desoxyhexanucleosides [pd(N)6; Pharmacia] and incubated for 1 hour at 37°C
with Superscript II (GIBCO) in buffer supplied by the manufacturer. One
microliter of RT product was used as template in a 50-µL PCR reaction
containing 100 ng of each oligonucleotide and 2.5 U of AmpliTaqGold
(Perkin Elmer). Reactions were performed in a 2400 Gene Amp PCR System (Perkin Elmer) under the following conditions: 10 minutes at 94°C, 30 to 40 cycles composed of 10 seconds at 94°C, 30 seconds at annealing temperature, and 30 seconds at 72°C, and, finally, 7 minutes at 72°C.
Primer sequences are the following. Mouse neuropilin-1 forward
primer 5'GAA GTT TAT GGC TGC AAG 3' and reverse primer
5' CTT CTC TGT GGC CAG GAC 3' (35 cycles, annealing at
50°C). Human neuropilin-1 forward primer 5' ACG ATG
AAT GTG GCG ATA CT 3' and reverse primer 5' AGT GCA TTC AAG
GCT GTT GG 3' (35 cycles, annealing at 50°C). S14
forward primer 5' GGC AGA CCG AGA TGA ATC CTC A 3' and
reverse primer 5' CAG GTC CAG GGG TCT TGG TCC 3' (30 cycles, annealing at 64°C). HPRT forward primer 5'
GCT GGT GAA AAG GAC CTC T 3' and reverse primer 5' CAC AGG
ACT AGA ACA CCT GC 3' (30 cycles, annealing at 50°C).
Human semaphorin III forward primer 5' ACT CAC TGT TCA
GAC TTA C 3' and reverse primer 5' GAG CTG CAT GAA GTC TCT
3' (35 cycles, annealing at 50°C). Human semaphorin
IV forward primer 5' GCG CAT GAA GTT GAT CAC 3' and
reverse primer 5' ACC AGT GGA TGC CCT TCT 3' (40 cycles,
annealing at 54°C). Human semaphorin V forward primer
5' CAA CTG GGC AGG GAA GGA CAT 3' and reverse primer
5' CGT CTG GGT TCT CGC TCT CCG 3' (35 cycles, annealing at
60°C). Human and murine VEGF165 isoform forward primer 5' TGG TCC CAG GCT GCA CCC A 3' and reverse 5' AAC
AAA TGC TTT CTC CGC TCT G 3' (40 cycles annealing at 60°C).
Human and murine semaphorin E forward primer 5' GCA AAA
TGG CTG GCA AAG ATC C 3' and reverse primer 5' CCC ATG AAA
TCT ATA TAC ATT CC 3' (35 cycles, annealing at 60°C).
Murine semaphorin III forward primer 5' GGT CCC AAC TAT
CAG TGG 3' and reverse primer 5' GGC ACA GTA AGG GTC CCG
3'. Murine semaphorin IV forward primer 5' TGT GGG
CAG CAA GGA CTA CG 3' and reverse primer 5' GGT AGA AGA TGT
AAT CCT GTG C 3' (35 cycles, annealing at 60°C). Murine
semaphorin V forward primer 5' GCA ACT GGG CAG GGA AGG A
3' and reverse primer 5' CGC ACA TCG TTC ATG CTG TAC
3' (35 cycles, annealing at 60°C). Murine Tpo forward
primer 5' ACT TTA GCC TGG GAG AAT GGA AA 3' and reverse primer 5' AGG AGT AAT CTT GAC TGT GAA TC 3' (32 cycles,
annealing at 50°C). Murine Flt3-ligand forward primer
5' AGT TGA CTG ACC ACC TGC TT 3' and reverse primer
5' AGG AGT AAT CTT GAC TGT GAA TC 3' (36 cycles, annealing
at 58°C).
Binding Experiments
Semaphorin III-AP.
MS-5 cells were cultured in 6-well plates. Subconfluent cells (5 × 105) were treated for 90 minutes with
concentrated conditioned medium containing semaphorin III-AP.
After incubation, cells were processed for AP
chemistry.8 The cell content of 1 well was assayed in 1 mL.
For competition assays, cells were incubated with conditionned medium
containing 5 ng/mL of semaphorin III-AP and various dilutions of
purified VEGF 165.
VEGF165.
Recombinant human VEGF (VEGF 165) was synthesized in Sf9 cells infected
with a recombinant baculovirus containing the VEGF 165 cDNA. Native
VEGF 165 was further cleaved by plasmin as already described10 and further purified by heparin affinity
chromatography.15 The 110-amino acid (aa) fragment (V110)
corresponding to the N-terminus was contained in the
flow-through and the 55-aa carboxy-terminal fragment
(V55) was eluted with 0.5 mol/L NaCl, whereas uncleaved VEGF was
collected with 0.65 mol/L NaCl.
Four micrograms of VEGF was iodinated by the iodogen procedure
according to Jonca et al.10 The specific activity averaged 200,000 cpm/ng. Cell cultures (106 cells per dish) were
washed twice in ice-cold binding medium (DMEM-20 mmol/L HEPES, 1 mg/mL
pH 7.3-gelatin, 0.5 µg/mL heparin) and incubated in the same medium
with 0.5 ng/mL 125I-VEGF and various dilutions of VEGF 165, V110 and V55. The binding was performed for 2 hours at 4°C with
gentle agitation. After 2 washes in PBS, the cell monolayers were
dissolved with 0.2 mol/L NaOH and the radioactivity counted in a gamma counter.
For cross-linking experiments, the procedure was similar except that
concentration of 125I-VEGF was 5 ng/mL and concentration of
unlabeled VEGF 165, V55, and semaphorin III-AP was 1 µg/mL.
Cross-linking was performed with 0.15 mmol/L disuccinimidyl suberate
(DSS; Pierce, Rockford, IL) in PBS for 15 minutes at room
temperature and stopped with 0.2 mol/L glycin pH 7.4. MS-5 cell pellets
were resuspended in (0.1% sodium dodecyl sulfate [SDS], 0.3 mol/L
NaCl, 10 mmol/L EDTA, 0.02 mol/L Tris-HCl pH 7.4, 1% Triton-X100,
0.05% Tween-20), centrifuged, and further incubated with 2 µg/mL of
an antibody directed against the cytoplasmic domain of neuropilin-1 (a
gift of Drs Zhigang He and Marc Tessier-Lavigne, University of
California, San Francisco). The immunocomplexes were
collected using protein A-Sepharose beads (Amersham, Pharmacia
Biotech), washed, and dissolved in 1X sample buffer
(Tris-HCl pH 6.9, 0.1 mol/L; SDS 0.2%; glycerol 10%).
The CHO pgs A-745 cell pellets were directly dissolved in 1X sample
buffer. Proteins were resolved on a 7% SDS-polyacrylamide gel
electrophoresis (SDS-PAGE). Gels were stained with Coomassie blue,
dried, and analyzed on a PhosphorImager (Amersham, Pharmacia Biotech).
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RESULTS |
A Gene Trap Approach to Identify Genes Encoding Transmembrane or
Secreted Proteins Expressed by MS-5 Cells
To identify membrane or secreted proteins involved in MS-5 function, we
used an original gene trap strategy called "secretory trap."16 The pGT1.8TM gene trap vector
(Fig 1) contains the CD4 transmembrane
domain fused to geo, a chimeric protein that possesses both
-galactosidase (-gal) and neomycin phosphotransferase (neoR) activities. This coding sequence is preceded by a
splice acceptor cassette; insertion into an intron is predicted to
generate a fusion mRNA which may, in some cases, code for a fusion
protein containing geo at its C-terminus. In protein fusions that
lack a signal peptide, the CD4 transmembrane domain acts as a signal anchor sequence that exposes geo to the lumen of the endoplasmic reticulum where -galactosidase activity, but not neomycin
phosphotransferase activity, is lost. In fusions that contain a signal
peptide, -galactosidase activity can be detected, presumably because
geo does not enter the endoplasmic reticulum.16
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| Fig 1.
Structure of the secretory trap vector, pGT1.8TM. En-2,
murine engrailed-2 sequences; SA, splice acceptor site; TM,
transmembrane domain; geo, fusion between LacZ and neomycin
phosphotransferase sequences; polyA, polyadenylation signal.
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We have transfected the pGT1.8TM plasmid into MS-5 cells, selected
G418-resistant clones for 10 to 15 days, and stained purified clones
for -galactosidase activity. Thirty-two of a total of 750 clones
selected for G418 resistance were stained blue with X-gal.
-Galactosidase activity was then measured in the absence and in the
presence of hydrocortisone. In these conditions, 20 clones showed a
constitutive activity of -galactosidase, 2 clones an upregulated
activity, and 10 clones a downregulated activity.
Gene Trap Insertion Into the Neuropilin-1 Gene
We focused our attention on the clones for which -galactosidase was
downregulated by hydrocortisone. Endogenous trapped sequences were
cloned from the fusion transcripts using the 5' RACE method. cDNA
fragments derived from the fusion transcripts were cloned and
sequenced. In the 15D3 clone, for which -galactosidase activity was
downregulated 2-fold in the presence of hydrocortisone
(Fig 2), the sequence of the 5' RACE product
revealed an insertion into the coding region of the neuropilin-1 gene
that codes for a type I cell-surface glycoprotein.17
Sequence analysis showed a fusion to the neuropilin-1 coding sequence
after the codon 642 (Fig 3A). The structure
of the 15D3 cDNA was surprising. Fifty base pairs of vector DNA were
found between the En-2 sequences and the neuropilin-1 sequences. These
3 sequences formed an in-frame fusion transcript (Fig 3B). Because only
1 band could be detected in Northern blot analysis using a LacZ probe
(data not shown), neuropilin-1-geo must be the unique fusion
transcript produced by the 15D3 clone.
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| Fig 2.
-Galactosidase activity is downregulated by
hydrocortisone in 15D3 cells. Confluent 15D3 cells were grown in the
absence (top panel) or in the presence (bottom panel) of
106 mol/L hydrocortisone. After 48 hours, cells were
fixed with paraformaldehyde and stained for -galactosidase
activity.
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| Fig 3.
Characterization of the fusion cDNA produced by 15D3
cells. (A) Predicted structure of the mutant fusion protein. The
neuropilin-1 protein and the mutant protein are shown. SS, signal
sequence; TM, transmembrane domain. a1, a2, b1, b2, and MAM are
previously described neuropilin-1 sub-domains.42 (B)
Portion of the sequence obtained by 5' RACE, displaying the
fusion between neuropilin-1 and gene trap vector sequences. 642 is the
last codon of neuropilin-1 sequences and 220 is the first codon of En-2
sequences present in the trap vector.
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The Neuropilin-1 Gene Is Expressed in Bone Marrow Adherent Cells But
Not in Immature Hematopoietic Cells
Reports of neuropilin-1 expression have been limited predominantly to
the nervous system of the developing embryo and, more recently, to some
adult tissues.18 Because no data about its expression in
hematopoietic and stromal cells have been reported so far, we performed
RT-PCR assays. In addition to MS-5, neuropilin-1 transcripts were found
in the murine 30E, 30W, and S17 stromal cell lines
(Fig 4), which are derived from long-term
bone marrow cultures19 but barely detectable in NIH3T3
cells (not shown). Neuropilin-1 was also expressed in both human and
murine marrow adherent cells (Fig 4, lanes marked Hu Adh and Mo Adh),
and also in cultures of human bone marrow-derived fibroblasts that are devoid of endothelial cells (Fig 4, lane marked Fibro). Interestingly, neuropilin-1 expression could not be detected in human primitive hematopoietic cells sorted on the basis of their high expression of
CD34 and low expression of CD38 (Fig 4, lane
34+/38).
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| Fig 4.
Expression pattern of the neuropilin-1 gene. RT-PCRs were
performed using total RNA isolated from stromal cell lines (MS-5, 30E,
30W, and S17), adherent layers of long-term murine and human cultures
(Mo Adh and Hu Adh), human fibroblasts (Fibro), and human
CD34+/CD38 (34+/38 ) hematopoietic
primitive cells. PCR products are 450 bp for human neuropilin-1 and 635 bp for murine neuropilin-1. As a control, amplification of RT products
with either S14 primers (human samples) or HPRT primers (murine
samples) is shown.
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Semaphorin III Binding to MS-5 Cells Is Displaced by VEGF 165
Neuropilin-1 was first described as a semaphorin III
receptor.8,20 To ensure that neuropilin-1 acts as a
cell-surface receptor in MS-5 cells, we first tested the binding of
semaphorin III to MS-5 cells. The coding region of human semaphorin III
was fused to that of alcaline phosphatase (AP), and the resulting chimeric protein (sema III-AP) was expressed in human embryonic kidney
293 cells.8 Conditioned medium from these cells was applied
to MS-5 cells and AP activity was measured. The binding affinity of
sema III-AP to MS-5 cells was measured in equilibrium binding
experiments, based on the relative amounts of AP activity in the
supernatant and bound to cells. Sema III-AP binding to MS-5 cells
increased in a dose-dependent manner and reached saturation at
approximately 100 ng/mL (data not shown). Scatchard analysis of sema
III-AP binding showed a single class of semaphorin III binding sites
with a kd of 125 pmol/L and 2,500 binding sites per cell
(Fig 5A). The value for the dissociation
constant for the interaction of semaphorin III with neuropilin-1 has
been previously estimated at 325 pmol/L8 or 1,500 pmol/L.20
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| Fig 5.
Semaphorin III binding to MS-5 cells. Sub-confluent MS-5
cells were treated for 90 minutes with different concentrations of
semaIII-AP. Then, AP activity from bound semaIII-AP was measured
colorimetrically (see Materials and Methods for details). Specific
binding was determined by substraction of values obtained from binding
to MS-5 cells and to COS cells. COS cells do not express neuropilin-1
and are unable to bind semaIII-AP.8 (A) Scatchard's
analysis. The values shown are an average of 3 different assays. Linear
regression analysis of values showed that MS-5 cells express 2,500 binding sites per cell and bind Semaphorin III with a kd of 1.2 × 1010 mol/L. (B) MS-5 cells were incubated
with concentrated conditioned medium containing 5 ng/mL of semaIII-AP
in the absence or in the presence of indicated concentrations of VEGF
165. Values shown are an average of 2 different assays. Reaction time
for AP activity detection was 3 hours.
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As VEGF 165 was recently described as a neuropilin-1 ligand, we tested
whether VEGF 165 was able to displace sema III-AP binding to MS-5
cells. MS-5 cells were incubated with conditioned medium containing 5 ng/mL of sema III-AP in the absence or in the presence of purified VEGF
165. As shown in Fig 5B, sema III-AP binding was progressively
displaced in the presence of increasing concentrations of VEGF 165. These results indicate that semaphorin III can bind to MS-5 cells and
that this binding can be competed by VEGF 165.
Neuropilin-1 Is a VEGF 165 Receptor on MS-5 Cells
The active form of VEGF is a homodimer whose activities are mediated on
endothelial cells by 2 specific tyrosine kinases receptors, the
fms-like tyrosine kinase Flt-1 and the kinase insert domain-containing receptor KDR/Flk-1. Cell-surface receptor cross-linking experiments with 125I-VEGF have shown that additional VEGF receptors
may exist that are neither KDR or Flt-1. Recently, a 130- to
135-kD VEGF receptor was identified as
neuropilin-1.18 A striking feature of neuropilin-1 is that
it binds VEGF 165 but not VEGF 121.
The structural difference between VEGF 165 and VEGF 121 is that VEGF
165 contains 44 additional aa encoded by exon 7 of the VEGF gene. A
fusion protein containing the exon 7-encoded domain of VEGF 165 binds
to neuropilin-1 directly and is able to compete for VEGF 165 binding.21 Thus, VEGF 165 binding to neuropilin-1 occurs
through the VEGF 165 exon 7-encoded domain. By contrast, KDR and Flt-1
interact with domains of VEGF 165 that are encoded by exons 4 and 3, respectively.
MS-5 cells bound 125I-VEGF 165 in a saturable fashion.
Scatchard's analysis showed the presence of a single class of binding sites and a dissociation constant of 120 pmol/L (data not shown). To
test the specificity of VEGF 165 binding, 125I-VEGF 165 was
bound to MS-5 cells in the presence of an increasing excess of
nonlabeled VEGF 165 proteolytic fragments. Both native VEGF 165 and
V55, which consists of a peptide encoded by exons 7 and 8, competed
VEGF 165 binding to MS-5 cells while V110, which consists of a peptide
encoded by exons 1 to 5, was unable to displace VEGF 165 binding (Fig
6A). Therefore, VEGF 165 binding by MS-5 cells is
unlikely to be mediated by KDR or Flt-1.
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| Fig 6.
VEGF 165 binding to MS-5 cells. (A) Left: Structure of
native VEGF 165 (V165) and its proteolytic fragments (V110 and V55).
Right: Confluent MS-5 cells were incubated with 0.5 ng/mL of
125I VEGF 165 and various amounts of V165, V110 (domain
encoded by exons 1 to 5 that do not bind neuropilin-1), and V55 (domain
encoded by exons 7 and 8 that bind neuropilin-1). Nonspecific binding
was measured in the presence of 2 µg/mL of unlabeled V165. The
competitive displacement is expressed as the average of iodinated V165
specific binding in triplicate assays. Standard errors were less than
10%. (B) Subconfluent cells were washed twice in ice-cold binding
medium and incubated with 5 ng/mL 125I-VEGF for 2 hours in
the absence (lane 0) or in the presence of 1 µg/mL of V165 (lane
V165), V55 (lane V55), or sema III-AP (lane sema III). For MS-5 cells,
cross-linked complexes were incubated in the presence of 2 µg/mL of
anti-neuropilin-1 antibodies. Proteins were resolved on a 7% SDS
polyacrylamide gel, stained with Coomassie Blue, and autoradiographed.
Position of molecular-weight markers is indicated on the right.
pgs-A745: xylosyltransferase deficient pgs-745 CHO cells
constitutively expressing either neuropilin-1 (pgs-A745 NP-1) or KDR
(pgs-A745 KDR). The upper band corresponds to species that did not
enter the gel. Arrowheads indicate cross-linked complexes.
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Next we estimated the molecular weight of VEGF 165 cell-surface
receptors on MS-5 cells using cross-linking assays. As size controls,
we transfected CHO cells with either human KDR or rat neuropilin-1.
125I VEGF 165 was bound to transfected CHO cells and
cross-linked (Fig
6B). The
radiolabeled complexes formed in cells transfected with KDR (CHO-KDR
cells) had an apparent molecular weight of 180 kD (Fig 6B, bottom
panel). Subtraction of the molecular weight of the VEGF
165 dimer (45 kD) from these cross-linked complexes gave us 135 kD, the
molecular weight of this form. In CHO cells transfected with
neuropilin-1 (CHO-Np-1 cells), labeled complexes displayed a 165-kD
molecular weight (Fig 6B, middle panel). This molecular weight was
consistent with the binding of a VEGF 165 dimer (45 kD) to receptors of
120 kD. This value is in accordance with the molecular weight of
neuropilin-1 detected on endothelial cells (130 to 135 kD).18 When 125I VEGF 165 was cross-linked to
MS-5 cells in the same conditions, a faint complex was detected (data
not shown). This suggested that neuropilin-1 was weakly expressed on
the MS-5 cell surface. To confirm that VEGF 165 binds on MS-5 cells
through neuropilin-1, cross-linked complexes were immunoprecipitated
with anti-neuropilin-1 antibodies. As shown in Fig 6B (top panel), a
165-kD radiolabeled complex was detected. As in CHO-Np-1 cells, this
molecular weight was consistent with the binding of a VEGF 165 dimer
(45 kD) to receptors of 120 kD. As expected, the labeled complex was
displaced in the presence of VEGF 165, V55, or sema III-AP.
We conclude that VEGF 165 can bind on MS-5 cells to receptors of 120 kD, which are specifically recognized by anti-neuropilin-1 antibodies.
This binding was competed by V55 or sema III-AP but not by V110.
Because sema III-AP binding was displaced by VEGF 165, we conclude that
both VEGF 165 and semaphorin III compete for binding to 120-kD
receptors that we identified as neuropilin-1.
VEGF 165 Has No Mitogenic Activity on MS-5 Cells but
Increases Expression of Thrombopoietin (Tpo) and Flt-3 Ligand
(Flt3-L)
To understand the role of neuropilin-1 on stromal cells, we first
investigated whether growth of MS-5 cells could be influenced by VEGF
165. MS-5 cells were incubated in the absence or in the presence of 5 ng/mL VEGF 165 for 5 days. As shown in
Table 1, VEGF 165 has no effect on MS-5
cell proliferation, although it is able to stimulate the growth of
HUVEC. Then, we investigated whether VEGF 165 could modulate the
expression of cytokines by MS-5 cells. MS-5 cells were incubated for 72 hours in the absence or in the presence of 50 ng/mL or 100 ng/mL VEGF
165. Expression of several cytokines known to influence the
proliferation and differentiation of hematopoietic progenitors was
determined by RT-PCR. Expression of stem cell factor (SCF), granulocyte
colony-stimulating factor (G-CSF), hepatocyte growth factor
(HGF),22 macrophage inhibitory factor-1 (MIP-1),
nerve growth factor (NGF),23 transforming growth
factor-1 (TGF-1), tumor necrosis factor- (TNF-), and
stromal cell-derived factor-1 (SDF-1)24 was
unaffected by the presence of VEGF 165 (not shown), but expression of
Flt3-L and Tpo was increased 5-fold and 3-fold, respectively, in the presence of VEGF 165 (Fig 7).
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[in a new window]
| Fig 7.
VEGF 165 stimulates stromal Tpo and Flt-3 ligand mRNAs.
Confluent MS-5 cells (7.5 × 105) were
incubated for 72 hours in the absence (lane VEGF 0) or the the presence
of either 50 ng/mL (lane VEGF 50) or 100 ng/mL (lane VEGF100) VEGF 165. RT-PCRs were performed using 1 µg of total RNA. Expected PCR products
are 270 bp for Flt3-L and 460 bp for Tpo. Amplification of the HPRT
mRNA is shown as a control. The intensity of ethidium bromide-stained
bands was quantified with a CCD camera and Image Quant
v1.11 software (Amersham, Pharmacia Biotech).
|
|
Different Neuropilin-1 Ligands Are Expressed in Stromal Cells and in
Immature Hematopoietic Cells
In vitro, neuropilin-1 is also able to bind secreted semaphorins
related to semaphorin III, ie, semaphorins V and E. Neuropilin-1 is
also able to bind semaphorin IV, but with a lower
affinity.25
We investigated whether stromal cells and immature hematopoietic cells
expressed known ligands for neuropilin-1 using RT-PCR experiments. As
shown in Fig 8, high expression of
semaphorin IV and weak expression of semaphorin III and semaphorin V
were detected in MS-5 cells. Neither VEGF 165 nor semaphorin E
expression was detected. Expression profile in murine marrow-derived
adherent cells was similar (data not shown). Then, we analyzed
expression of neuropilin-1 ligands in
CD34+/CD38 hematopoietic progenitors.
These cells express high levels of semaphorins IV, V , E, and VEGF 165. No semaphorin III expression could be detected.
View larger version (39K):
[in this window]
[in a new window]
| Fig 8.
Differential expression of secreted semaphorins and VEGF
165. RT-PCRs were performed using total RNA from human
CD34+/CD38 cells and from murine MS-5
cells (see Materials and Methods). Expected PCR products are 130 bp for
human semaphorin IV, 430 bp for human semaphorin III, 410 bp for human
semaphorin V, 255 bp for human and murine semaphorin E, 260 bp for VEGF
165, 260 bp for murine semaphorin IV, 720 bp for murine semaphorin V,
and 530 bp for murine semaphorin III. Controls consist of amplification
with either S14 primers (human samples) or HPRT primers (murine
samples).
|
|
Therefore, we conclude that although neuropilin-1 ligands are expressed
in both stromal cells and hematopoietic cells, their expression
profiles are clearly different.
| |
DISCUSSION |
Bone marrow stroma is made of several cell types and an extracellular
matrix, which together form a suitable microenvironment for growth and
differentiation of hematopoietic stem cells. So far, the precise
mechanisms by which stromal cells influence the fate of hematopoietic
cells have not been elucidated. To overcome the complexity of stromal
cells, primary marrow-derived cells have been substituted by
established cell lines that exhibit similar supportive functions. One
of the most studied, the murine MS-5 cell line, allows expansion of
human primitive CD34+/CD38 cells in the
absence of exogenous growth factors. The hematopoietic promoting
activity of MS-5 cells cannot be related to expression of the major
hematopoietic cytokines.6 Furthermore, addition of
hydrocortisone dramatically decreases the ability of MS-5 cells to
support human hematopoiesis.7 This inhibition cannot be reversed by addition of exogenous growth factors. This suggests that
hydrocortisone may either downregulate an activity which promotes
differentiation of CD34+/CD38 cells or
may upregulate an inhibitory activity.
The action of hydrocortisone is mediated by the glucocorticoid
receptor, a transcription factor that regulates transcription through
DNA-binding-dependent and -independent mechanisms.26 Howewer, examples have been reported that suggest a role for the glucocorticoid receptor in posttranscriptional
regulation.27,28 Direct binding of the glucocorticoid
receptor to RNA, especially transfer RNAs,29 has been
reported. This RNA binding activity may reflect regulation of gene
expression through posttranscriptional mechanisms, such as alterations
in translational efficiency. Therefore, it is likely that the effects
of hydrocortisone on MS-5 are due to both transcriptional and to
posttranscriptional events.
A number of approaches have been used to clone genes with differential
expression. These include differential cDNA library construction,30 differential
hybridization,31,32 and differential display
RT.33 Identification of a candidate gene using these methods relies on its differential mRNA levels, and genes that are
posttranscriptionally regulated are not taken into account. Gene
trapping is an efficient way to target active genes and allows monitoring of the expression of the endogeneous gene by a
LacZ reporter gene. This method has been used
successfully to clone retinoid acid induced genes in embryonal
carcinoma P19 cells34 or in embryonic stem (ES)
cells.35 We chose the gene trap strategy because it may
allow cloning of transcriptionally and
posttranscriptionally regulated genes. In addition, the vector we used,
pGT1.8TM, allows the screening of stable clones for insertional
mutations in genes encoding secreted and membrane-spanning proteins.
This particular gene trap relies on capturing the amino-terminal signal
sequence of a gene to produce an active -galactosidase fusion
protein. In our screen, 4% of the clones selected for G418 resistance
displayed -galactosidase activity, a percentage similar to that
obtained in experiments performed in ES cells.16
In the 15D3 clone, insertion has occurred within the neuropilin-1 gene.
Sequence of the 15D3 fusion transcript showed an insertion of bacterial
DNA between the En-2 sequences and the neuropilin-1 sequences. This
event may be due to multiple insertions. In this case, splicing may
have occurred within vector tandems. Insertion of multiple and
rearranged copies of the vector was confirmed by Southern blot
experiments (data not shown). However, because only 1 band was detected
in Northern blot experiments, only 1 fusion transcript is produced in
15D3 cells.
In 15D3 cells, -galactosidase activity was downregulated in the
presence of hydrocortisone. To ensure that this modulation reflected
neuropilin-1 activity, we studied VEGF 165 binding to MS-5 cells in the
presence of hydrocortisone. We showed that it was downregulated 2-fold
in the presence of hydrocortisone. Regulation of neuropilin-1 by
glucocorticoids may be a posttranscriptional event, since we observed
no difference in mRNA levels (data not shown).
Neuropilin-1 is expressed in adult endothelial cells, in
some tumor-derived cells, and in a variety of tissues including
placenta, heart, lung, liver, skeletal muscle, kidney, and
pancreas.18 During embryonic development, neuropilin-1 is
expressed in particular neuronal circuits, in the cardiovascular
system, and in limb buds. Neuropilin-1 is a receptor for the axonal
repellent semaphorin III. When semaphorin III binds to the receptor on
the growing tips of neurons, its repels the cells, keeping them from
getting off-track. Recently, neuropilin-1 has been described as a new receptor for the VEGF 165 isoform and may stimulate blood vessel growth. The role of neuropilin-1 in angiogenesis seems to be different from its role in axonal guidance. In developing blood vessels, neuropilin-1 seems to work in concert with KDR to stimulate vessel growth. Cells in which both receptors are expressed are 4-fold more
effective at binding VEGF 165 and 3-fold more effective at migrating
than cells carrying KDR alone.18
In our study, semaphorin III-AP and VEGF 165 could bind to MS-5 cells,
both a similar kd of approximately 120 pmol/L. This value
is comparable to the estimated kd for the interaction of semaphorin III
with neuropilin-1 (325 to 1,500 pmol/L) and for interaction of VEGF 165 with neuropilin-1 (333 pmol/L). We have performed binding experiments
in the presence of neuropilin-1 blocking antibodies (provided by Drs Z. He and M. Tessier-Lavigne). Although these antibodies block the ability
of semaphorin III to repel axons and to induce collapse of their growth
cones,8 they were unable to block semaphorin III binding in
our assays. They were actually raised against a portion of the
neuropilin-1 ectodomain that is dispensable for semaphorin III
binding.36,37 Therefore, the antibodies we used are
expected to block biological activity but not semaphorin III binding
(data not shown). Semaphorin III-AP binding to MS-5 cells is displaced
by VEGF 165 while VEGF 165 binding is competed by V55 and semaphorin
III-AP. When 125I-VEGF was cross-linked to MS-5 cells, a
165-kD complex was detected and recognized by anti-neuropilin-1
antibodies. These results indicated that both VEGF 165 and semaphorin
III bound to MS-5 cells through the neuropilin-1 receptor.
To elucidate the role of neuropilin-1 on stromal cells, we first
investigated whether genes encoding secreted semaphorins or VEGF 165 are expressed in stromal cells. In MS-5 cells, as in bone
marrow-derived adherent cells, we detected no VEGF 165 mRNA
expression, a high expression of semaphorin IV, and a weak expression
of semaphorins III and V. These results indicate that neuropilin-1 and
secreted semaphorins may act as mediators of a cross-talk between
stromal cells to organize or maintain the stromal structure of the bone marrow.
Another possibility is that neuropilin-1 may bind ligands produced by
primitive hematopoietic cells. Therefore, we looked for neuropilin-1
ligands in primitive hematopoietic cells and we found that semaphorin
IV, V, E, and VEGF 165 mRNAs were expressed in
CD34+/38 progenitors. These results
suggest that semaphorins and/or VEGF 165 secreted by hematopoietic
cells bind to neuropilin-1 on stromal cells. We showed that although
VEGF 165 was unable to stimulate the growth of MS-5 cells, it was able
to increase both Tpo and Flt3-L mRNA expression. Interestingly, these 2 cytokines have been shown to regulate early hematopoiesis by increasing
numbers of primitive hematopoietic progenitors in vitro.38
,39
It was shown previously that stromal cultures are a source of early
acting cytokines, especially Tpo, SCF, and Flt3-L,40 and
that the ability of MS-5 cells to enhance the hematopoietic potential
of ES cells is, in part, due to their secretion of Tpo.41 These data and ours suggest that interaction between stromal cells and
hematopoietic cells through neuropilin-1 may induce cytokine production
by the former and proliferation of the latter.
| |
ACKNOWLEDGMENT |
We are very grateful to Drs Marc Tessier-Lavigne and Zhigang He for the
gift of the semaphorin III-AP secreting cell line and
anti-neuropilin-1 antibodies, and for their advice and encouragement; to Dr William Skarnes for the gift of pGT1.8TM and for his advice; to
Drs Alain Chédotal, Isabelle Godin, and Patrick Mayeux for helpful discussions; and to Prof André Baruchel and Dr Vincent Mignotte for their encouragement.
| |
FOOTNOTES |
Submitted December 1, 1998; accepted May 27, 1999.
Supported by the Institut National de la Santé et de la Recherche
Médicale (INSERM), the Centre National de la Recherche Scientifique, the Fondation de France, and the Ligue Nationale contre
le Cancer. R.T. was supported by the INSERM. N.O. was supported by the
Ligue Nationale contre le Cancer.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Valérie Lemarchandel,
PhD, INSERM U474, Hopital Henri Mondor, 94010 Créteil, France; e-mail: valerie{at}im3.inserm.fr.
| |
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TOP
ABSTRACT
INTRODUCTION
