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Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2569-2576
HEMATOPOIESIS
Cloning of PRV-1, a novel member of the uPAR receptor superfamily,
which is overexpressed in polycythemia rubra vera
Sne ana Temerinac,
Steffen Klippel,
Elisabeth Strunck,
Sabine Röder,
Michael Lübbert,
Winand Lange,
Marc Azemar,
Gerold Meinhardt,
Hans-Eckart Schaefer, and
Heike
L. Pahl
From the Department of Experimental Anaesthesiology, University
Hospital Freiburg, Center for Tumor Biology, Freiburg, Germany; the
Department of Hematology and Oncology, University Hospital Freiburg,
Freiburg, Germany; the Center for Tumor Biology, Freiburg, Germany; the
Department of Hematology/Oncology, University Hospital Munich, Munich,
Germany; and the Department of Pathology, University Hospital Freiburg,
Freiburg, Germany.
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Abstract |
Polycythemia vera (PV) is a clonal stem cell disorder characterized
by hyperproliferation of the erythroid, myeloid, and megakaryocytic lineages. Although it has been shown that progenitor cells of patients
with PV are hypersensitive to several growth factors, the molecular
pathogenesis of this disease remains unknown. To investigate the
molecular defects underlying PV, we used subtractive hybridization to
isolate complementary DNAs (cDNAs) differentially expressed in patients
with PV versus normal controls. We isolated a novel gene, subsequently
named PRV-1, which is highly expressed in granulocytes from patients
with PV (n = 19), but not detectable in normal control granulocytes
(n = 21). Moreover, PRV-1 is not expressed in mononuclear cells from
patients with chronic myelogenous leukemia (n = 4) or acute
myelogenous leukemia (n = 5) or in granulocytes from patients with
essential thrombocythemia (n = 4) or secondary erythrocytosis
(n = 4). Northern blot analysis showed that PRV-1 is highly expressed
in normal human bone marrow and to a much lesser degree in fetal liver.
It is not expressed in a variety of other tissues tested. Although
PRV-1 is not expressed in resting granulocytes from normal controls,
stimulation of these cells with granulocyte
colony-stimulating factor induces PRV-1 expression. The PRV-1 cDNA
encodes an open reading frame of 437 amino acids, which contains a
signal peptide at the N-terminus and a hydrophobic segment at the
C-terminus. In addition, PRV-1 contains 2 cysteine-rich domains
homologous to those found in the uPAR/Ly6/CD59/snake
toxin-receptor superfamily. We therefore propose that PRV-1
represents a novel hematopoietic receptor.
(Blood. 2000;95:2569-2576)
© 2000 by The American Society of Hematology.
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Introduction |
Polycythemia vera (PV) is 1 of 4 diseases termed the
myeloproliferative disorders (MPDs).1 Besides PV, this
group includes essential thrombocythemia (ET), idiopathic myelofibrosis
(IMF), and chronic myelogenous leukemia (CML). All MPDs result from the clonal expansion of a mutant pluripotent hematopoietic stem
cell.2-5 PV is characterized by an increased proliferation
of all 3 myeloid lineages, which results in an excess production of
mature red cells, granulocytes, and platelets.6 Because the
disease results from the hyperproliferation of a single aberrant stem
cell, the peripheral red blood cells, granulocytes, monocytes, and
platelets are clonal in these patients.2,7-9 Although the
molecular etiology of PV remains unknown, progress has recently been
made in characterizing the malignant cells.
The PV cells are hypersensitive to several hematopoietic growth factors
including interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), and thrombopoietin (TPO).10-13 However, regarding
erythropoietin (EPO), a long debate had ensued whether PV cells are
hypersensitive to or in fact independent of this growth factor. In
elegant experiments using a novel serum-free medium devoid of any
burst-promoting activity, Correa et al have recently shown that PV
erythroid progenitor cells are independent of EPO.14 In
addition, these cells are exquisitely hypersensitive to insulin-like
growth factor-1 (IGF-1).14 Interestingly, patients with PV
also show 4-fold elevated plasma levels of insulin-like growth factor
binding protein-1 (IGFBP-1), which, together with IGF-1, stimulates
erythroid burst formation in vitro.15
The observation that PV cells are hypersensitive to a large variety of
growth factors suggests that signal transduction pathways may be
altered in these cells. Moliterno and colleagues have recently reported
that platelets from patients with PV displayed impaired tyrosine
phosphorylation in response to TPO stimulation, whereas the response to
thrombin remained intact.16 The TPO response was also
deficient in patients with IMF, but not in patients with a variety of
other hematopoietic diseases. The inability to transduce the TPO signal
was due to a dramatic reduction or a complete absence of the TPO
receptor, c-mpl, in 34 of 34 PV and 13 of 14 IMF
patients.16 However, it is at present not clear how a loss
of c-mpl expression could contribute either to the growth factor
hypersensitivity of PV cells, or, more generally, to the molecular
pathology of the disease.
An intriguing observation about PV cells was recently reported. Silva
et al showed that PV erythroid precursor cells express the
anti-apoptotic protein bcl-xL to a much higher
proportion than normal precursor cells (21.8% versus
6.6%).17 In addition, in PV, more mature cells, which
normally show no bcl-xL expression, still
express high levels of the protein. Hematopoietic growth factors act in
part by suppressing apoptosis. IGF-1, in particular, has been shown to
suppress apoptosis of erythroid progenitors and myeloid
cells.18,19 Thus, perhaps the observed growth factor hypersensitivity of PV cells results from an intrinsic protection from
apoptosis, thereby requiring less protection through growth factor stimulation.
Despite these recent advances in characterizing the malignant PV clone,
the molecular defect leading to the development of this disease remains
unclear. In an attempt to find such a defect, we wished to define
differences in gene expression between PV and normal cells. We used
subtractive hybridization to clone complementary DNAs (cDNAs) that are
either over- or underexpressed in PV cells. We report here the cloning
of a novel hematopoietic cell surface receptor, which is strongly
overexpressed in cells from 19 of 19 patients with PV and not expressed
in 21 of 21 normal controls. We have named this novel receptor
polycythemia rubra vera-1 (PRV-1).
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Materials and methods |
Patients
Peripheral blood samples were obtained from 19 patients with PV as
well as 6 patients with ET, 4 patients with CML in chronic phase, 1 patient with IMF, 5 patients with acute myelogenous leukemia (AML), 4 patients with secondary erythrocytosis, and 21 healthy volunteers. The
diagnosis of PV and ET was made according to the clinical and
laboratory criteria established by the Polycythemia Vera Study
Group.20 Patient characteristics are listed in Table 1. The study protocol was approved by the
local ethics committee and informed consent was obtained from all
patients.
Separation of cells
Heparinized blood from patients and healthy controls was used as a
source of granulocytes and mononuclear cells. Granulocytes were
purified by dextran sedimentation followed by Ficoll-Paque (Pharmacia
Biotech, Uppsala, Sweden) separation.21
Erythrocytes were eliminated by hypotonic lysis (0.2% NaCl for 30 seconds). This method consistently yielded granulocyte preparations
with more than 98% purity as judged by visual inspection of
Wright-Giemsa-stained slide preparations. Peripheral blood mononuclear
cells from patients with AML or CML were isolated by Ficoll-Paque separation.
RNA isolation and Northern blots
Total cellular RNA was harvested using an acidic phenol extraction
(Trizol, GIBCO/BRL, Rockville, MD). Either 5 µg or 10 µg of RNA was
analyzed in a Northern blot. Alternatively, Multiple Tissue Northern
Blots (Clontech, Palo Alto, CA) were used. The blots were hybridized in
ExpressHyb Hybridization Solution (Clontech) at 68°C. PRV-1 and
actin cDNAs were labeled using the Prime-It-II labeling kit
(Stratagene, La Jolla, CA) and  -32P-dCTP (Amersham,
Uppsala, Sweden). The blots were washed 3 times for 10 minutes in
2× sodium chloride sodium citrate (SSC), 0.05% sodium dodecyl
sulfate (SDS) at room temperature and twice at 50°C for 20 minutes
in 0.1 × SSC, 0.1% SDS.
After the first hybridization, membranes were reprobed with a 1.2-kb
Pstl-fragment of the human beta-actin gene to control for equal loading
of the RNA.
Isolation of poly(A)+RNA and subtractive hybridization
Poly(A)+RNA was isolated using the Fast Track 2.0 Kit (Invitrogen,
Carlsbad, CA). Double-stranded cDNA was synthesized using the
PCR-Select cDNA Subtraction Kit (Clontech) at the manufacturer's recommendation. Subtractive hybridization was performed with cDNA from
PV granulocytes pooled from 5 patients (MT, WZ, IP, FB, and DJ, Table
1) and cDNA from normal control granulocytes using the PCR-Select cDNA
Subtraction Kit (Clontech) according to the manufacturer's
recommendation. The subtracted cDNA was cloned directly into the pCR
2.1 vector (TA Cloning Kit, Invitrogen).
Western blots
Total cell extracts were prepared using a high-salt detergent buffer
(Totex) as previously described.22 Cell extracts (30 µg)
were boiled in Laemmli sample buffer and subjected to
SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting as
described.22 Primary polyclonal antibodies were raised by
injection of 2 hapten coupled synthetic peptides into rabbits. These
peptides encode amino acids 13 to 25 and 368 to 383 of the predicted
mature PRV-1. The purified rabbit serum was used at a dilution of
1:500. Bound antibody was decorated with peroxidase conjugated
secondary antibody (goat antirabbit IgG, Amersham). The immunocomplexes
were detected using ECL Western blotting reagents (Amersham). Exposure
to Kodak XAR-5 films was performed for 5 to 10 seconds.
Immunohistochemistry
The bone marrow biopsy was decalcified, paraffin embedded, and
stained as previously described.23-25 Serial sections were
stained as follows: (1) with a mouse polyclonal antiserum raised
against native PRV-1 by DNA vaccination (Genovac GmbH, Freiburg,
Germany); for vaccination, the 1.4-kb PRV-1 coding region cloned into
pCDNA3.1 (Invitrogen) was used; (2) enzymatically for
naphthol-AS-D-chloroacetate esterase; or (3) with an antibody against
hemoglobin (DAKO, Hamburg, Germany).
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Results |
Cloning of PRV-1
Messenger RNA (mRNA) from granulocytes of 5 PV patients (MT,
WZ, IP, FB, and DJ, Table 1) was compared to normal granulocyte mRNA in a subtractive hybridization. Five clones were obtained, which
encoded different overlapping fragments of the same cDNA. Northern blot
hybridization to granulocyte RNA from 5 patients with PV and 5 healthy
volunteers showed that this cDNA hybridized to 2 distinct RNAs, 2.1 kb
and 3.1 kb in size, in all 5 PV patients, but showed no hybridization
to normal granulocyte RNA (Figure 1). RNA
from a total of 19 patients with PV and 21 normal controls has been
analyzed to date. In Northern blots, peripheral blood granulocytes from
all 19 PV patients displayed strong hybridization to the cDNA probe,
whereas none of the controls showed a detectable signal (data not
shown).
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| Fig 1.
Expression of PRV-1 in peripheral blood granulocytes from
patients with PV and normal controls.
Granulocytes were isolated from peripheral blood to more than 98%
purity. Total RNA was extracted and a Northern blot prepared using 10 µg RNA. Lanes 1-5 healthy volunteer donors, lanes 6-10 patients with
PV. (Top) The membrane was probed with a 1.1-kb fragment of the PRV-1
cDNA. The positions of the 18S and 28S ribosomal RNAs are indicated.
(Bottom) The membrane was stripped and reprobed with a 1.2-kb fragment
of the human  -actin cDNA.
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Structure of the PRV-1 protein
From 3 clones, a complete cDNA was constructed. (The nucleotide
sequence has been submitted to the GenBank/EBI Data Bank with the
accession number AF 146747.) Homology searches with several databases
revealed that the sequence represents a novel previously uncharacterized cDNA, which we subsequently named polycythemia rubra
vera-1 (PRV-1). This sequence encodes an open reading frame of 437 amino acids (Figure 2), which contains the
following features: (1) an N-terminal signal sequence of 21 amino
acids, (2) 2 highly homologous cysteine-rich domains of 188 amino
acids, and (3) a highly hydrophobic C-terminal sequence.
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| Fig 2.
The protein sequence of PRV-1.
The 2 homologous cysteine-rich regions are shown aligned to each other.
Identical amino acids are shown in red, conserved cysteine residues in
bold red. Similar amino acids are depicted in blue. The position of the
leader peptide, the carboxy-terminal hydrophobic region as well as the
 -sites, potential sites of GPI-anchor attachment, are shown. The
postulated cysteine-disulfide bridges are indicated by brackets.
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The presence of a signal peptide suggests that the protein is imported
into the endoplasmic reticulum and destined either for insertion into
the plasma membrane or for secretion from the cell.
The 2 cysteine-rich domains show homology to the previously described
uPAR-domains, found in cell surface receptors of the uPAR/Ly6/CD59/snake toxin-family of proteins (Figure
3).26 These domains contain 8 to 10 cysteine residues spaced at conserved distances from each
other.28 PRV-1 contains 6 of these cysteines with the
conserved spacing (Figure 3).
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| Fig 3.
Alignment of PRV-1 to members of the uPAR/Ly6/CD59/snake
toxin family.
The protein sequences were retrieved from the Swissprot database and
aligned to PRV-1 according to the model of Kieffer et
al.27 Identical amino acids are shown in red,
conserved cysteine residues in bold red. Similarities shared only
between PRV-1 and the snake toxin family are shown in blue. Numbers
preceding the sequences indicate the first amino acid aligned.
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The C-terminal hydrophobic sequence could encode a transmembrane
domain. However, the 12 amino acids predicted to form the transmembrane
domain are too short for spanning the membrane and would leave a
cytoplasmic tail of only 7 amino acids. All members of the
uPAR/Ly6/CD59 family of proteins described so far are attached to the
membrane via a glycosyl phosphatidylinositol anchor
(GPI-anchor).28-31 It seems likely, therefore, that the
hydrophobic sequence at the C-terminus of PRV-1 encodes a signal for
the addition of a GPI-link.
To verify that the polypeptide predicted from the amino acid
translation of the cDNA was indeed synthesized in PV cells, we raised
antibodies against 2 peptides in the predicted primary sequence. These
peptides encode amino acids 13 to 25 and 368 to 383 of the predicted
mature PRV-1. Total cell extracts were prepared from purified
granulocytes of a PV patient (MR). The extracts were separated by
SDS-PAGE and subjected to Western blotting with antibodies raised
against the 2 peptides. Both antibodies recognized a single polypeptide
of approximately 60 kd (Figure 4, lanes 2 and 3). The apparent molecular weight of the detected polypeptide is 14 kd larger than the weight calculated from the amino acid sequence. This
observation can be explained by the existence of 3 potential
glycosylation sites in the PRV-1 sequence (N-46, N-189, and N-382). The
addition of sugar residues could account for the additional weight
observed. Many GPI-linked proteins including uPAR are modified by
glycosylation.28
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| Fig 4.
Expression of the PRV-1 protein in granulocytes from a
patient with PV.
Granulocytes were isolated from peripheral blood to more than 98%
purity. Total cell extracts were prepared and subjected to SDS-PAGE.
The proteins were blotted onto a nylon membrane and decorated with
either the antibody raised against the C-terminal peptide (amino acids
368-383, lane 2) or the antibody raised against the N-terminal peptide
(amino acids 13-25, lane 3). Immune complexes were detected by
decoration with peroxidase-conjugated secondary antibody and visualized
by chemoluminescence. Lane 1 shows a molecular size marker. The film
was exposed for 5 seconds.
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Because the structure of PRV-1 suggests that it is expressed on the
cell surface, we co-stained granulocytes of a PV patient with an
antibody against native PRV-1 and an antibody against CD13, a myeloid
marker. Fluorescence-activated cell sorter analysis revealed that all
PV granulocytes express PRV-1 on their surface (data not shown).
PRV-1 expression in MPDs
Because PV is 1 of 4 diseases collectively termed the MPDs, we
investigated whether overexpression of PRV-1 is unique to PV or whether
it also occurs in the other MPDs. Peripheral blood mononuclear cells
were purified from 4 patients with chronic phase CML. Morphologically,
these cells represent various stages of granulocyte precursors. RNA was
prepared and hybridized in a Northern blot with a 1.1-kb fragment of
the PRV-1 cDNA (Figure 5A). There was no
PRV-1 expression in CML mononuclear cells from 4 different patients
(Figure 5A). Hybridization to a -actin cDNA confirmed the presence
of similar amounts of RNA in all samples.
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| Fig 5.
Expression of PRV-1 in other MPDs.
Mononuclear cells were isolated from peripheral blood of patients with
CML (A) or AML (D). Granulocytes were isolated from peripheral blood of
patients with ET (B), IMF (C), secondary erythrocytosis (E), PV (A, B,
D, and E) or healthy volunteers (A and D). Total RNA was extracted and
Northern blots prepared using 10 µg RNA. (Top) The membrane was
probed with a 1.1-kb fragment of the PRV-1 cDNA. (Bottom) The membrane
was stripped and reprobed with a 1.2-kb fragment of the human -actin
cDNA.
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Likewise, granulocytes were purified from 6 patients with ET and the
RNA hybridized to the PRV-1 cDNA (Figure 5B). Four of the
patients showed no PRV-1 expression (Figure 5B, lanes 1, 2, 3, and 5). Two patients (FW and RW, Figure 5B, lanes 4 and 6) showed PRV-1 expression, 1 (RW) weakly, the other (FW) strongly.
Peripheral granulocytes were isolated from 1 patient with IMF. This
patient showed weak PRV-1 expression in a Northern blot (Figure 5C).
White blood cells from patients with PV frequently display a "left
shift," morphologically less mature cells appear in the peripheral
blood. If PRV-1 is normally expressed on immature granulocytes, but its
expression is down-regulated during maturation, its expression on PV
cells could simply result from the immaturity of the cells. The
observation that PRV-1 is not found on CML-mononuclear cells, which
represent all stages of granulocytic differentiation, argues against
this hypothesis. Nevertheless, we wished to investigate additional
immature myeloid cells. We therefore isolated peripheral blast cells
from 5 patients with AML. RNA was prepared and analyzed for PRV-1
expression in a Northern blot (Figure 5D). None of the 5 AML patients
showed any PRV-1 expression.
Polycythemic states are classified as either primary conditions, which
include PV, or as secondary conditions. The latter category is also
called secondary erythrocytosis and describes reactive processes. We
investigated whether PRV-1 expression discriminates between PV and
secondary erythrocytosis by analyzing PRV-1 expression in granulocytes
from 4 patients with secondary erythrocytosis. None of these patients
showed PRV-1 expression (Figure 5E). In addition, we investigated
whether PRV-1 expression correlates with an elevated leukocyte alkaline
phosphatase (LAP) score. Patients with PV frequently display elevated
LAP scores, whereas the LAP is decreased in patients with CML. We
observed no correlation between LAP score and PRV-1 expression. Three
patients who express PRV-1 (HH, RW, and EH; Figure 1, lane 10, Figure
5B, lane 6, and Table 1) have LAP scores in the normal range (10-100).
Conversely, 1 patient who does not express PRV-1 (MS, Figure 5B, lane
5) has an elevated LAP score. In fact, her LAP score is higher than the score of patient DJ, who does express PRV-1. We therefore conclude that
PRV-1 expression does not correlate with an elevated LAP score.
PRV-1 expression in normal tissues
We wished to determine the expression pattern of PRV-1 in normal
tissues. A Northern blot containing 2 µg mRNA from various hematopoietic tissues was probed with the PRV-1 cDNA (Figure
6). PRV-1 expression was very strong in
bone marrow and a slight expression was detected in fetal liver (Figure
6, lanes 5 and 6). The other tissues including spleen, lymph node,
thymus, and peripheral blood leukocytes did not express PRV-1 (Figure
6, lanes 1-4). In addition, several other tissues including heart,
brain, kidney, testis, ovary, small intestine, and skeletal muscle did
not express PRV-1 (data not shown). These data indicate that PRV-1 is
selectively expressed in human bone marrow.
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| Fig 6.
Expression of PRV-1 in hematopoietic tissues.
(Top) A Northern blot containing 2 µg of polyA+RNA from the indicated
tissues (Clontech) was probed with a 1.1-kb fragment of the PRV-1 cDNA.
(Bottom) The membrane was stripped and reprobed with a 1.2-kb fragment
of the human -actin cDNA.
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PRV-1 expression in bone marrow
To specify which cell types in the bone marrow express PRV-1, we
performed immunohistochemistry (Figure 7). Paraffin sections of a bone
marrow biopsy from a patient with PV were stained with a polyclonal
antibody directed against the native PRV-1 protein (Figure 7, panel A).
In addition, serial sections were stained for
naphthol-AS-D-chloroacetate esterase (see Figure 7, NACE, panel B) and
with an antibody against hemoglobin (Figure 7, panel C). Comparison of
these differently stained serial sections allows identification of the
PRV-1-positive cells. The anti-PRV-1 antibody stains early
erythroblasts, which have a basophilic cytoplasm, as well as
megakaryocytes, promyelocytes, and myelocytes. In early erythrocytes,
the anti-PRV antibody stains a solitary paranuclear apparently globular
structure, which is located at a site similar to the Golgi zone (large
arrowhead, Figure 7, panel A).
PRV-1 expression is stimulated by granulocyte colony-stimulating
factor and GM-CSF
In PV, bone marrow progenitor cells are hyperproliferating, thereby
producing the elevated cell counts recorded in the peripheral blood. A
hyperproliferation of the myeloid lineage, however, can also be induced
in healthy volunteers, for example, when these individuals act as
donors for peripheral stem cell transplantation. To increase the number
of circulating hematopoietic progenitor cells, these donors are
stimulated with 10 µg/kg body weight of recombinant human G-CSF twice
daily for 4 days. We investigated whether hyperproliferation of the
bone marrow in these healthy donors also leads to PRV-1 expression in
the peripheral granulocytes. Granulocytes were purified from 2 individuals treated with granulocyte colony-stimulating factor (G-CSF)
and the RNA analyzed by Northern blot (Figure
8A). Both G-CSF-treated donors showed a
very strong PRV-1 expression (Figure 8A, lanes 3 and
4). This observation prompted us to
investigate whether PRV-1 expression can be stimulated by G-CSF in
normal, mature peripheral granulocytes as well. Granulocytes from
healthy volunteer donors were purified and treated in vitro with 100 ng/mL G-CSF for various times. PRV-1 expression was analyzed by
Northern blot (Figure 8B). Stimulation with G-CSF for as little as 3 hours induced PRV-1 expression in mature granulocytes. Interestingly, the larger 3.1 kb mRNA appeared first, after 3 hours of stimulation (Figure 8B, lane 3), whereas the smaller 2.1 kb mRNA was only seen
after 5 hours of G-CSF stimulation (Figure 8B, lane 4). These 2 RNA
species arise from alternative polyadenylation (data not shown).
Stimulation of normal peripheral granulocytes with 100 ng/mL GM-CSF
also induces PRV-1 expression with kinetics similar to G-CSF (data not
shown).
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| Fig 7.
Immunohistochemical staining of paraffin sections of a
bone marrow biopsy from a patient with PV.
Serial sections were stained with a polyclonal antibody to native PRV-1
(panel A), to naphthol-AS-D-chloroacetate esterase (NACE, panel B), or
with an antibody to hemoglobin (panel C). In panel A, the large
arrowhead points to an early erythroblast, the small arrowhead points
out a megakaryocyte, and the open arrow shows a promyelocyte. A 1:400
magnification is shown.
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| Fig 8.
Expression of PRV-1 in granulocytes from healthy
volunteers treated with G-CSF in vivo and in vitro.
Granulocytes were isolated from peripheral blood of untreated and
G-CSF-treated healthy volunteers and a patient with PV. Total RNA was
extracted and a Northern blot prepared using 10 µg RNA. (A) Lane 1 patient with PV, lane 2 empty, lanes 3 and 4 healthy volunteers treated
with G-CSF in vivo, and lanes 5 and 6 untreated healthy volunteer
donors. (B) Lane 1 unstimulated granulocytes; lanes 2-8, granulocytes
stimulated in vitro for the indicated times with 100 ng/mL G-CSF. (Top)
The membrane was probed with a 1.1-kb fragment of the PRV-1 cDNA.
(Bottom) The membrane was stripped and reprobed with a 1.2 kb fragment
of the human -actin cDNA.
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Discussion |
We describe the cloning of a novel cell surface receptor,
named PRV-1 (polycythemia rubra vera-1), which under physiologic conditions is selectively expressed in human bone marrow. The amino
acid sequence of PRV-1 shows that it is a novel protein most closely
related to members of the uPAR/Ly6/CD59 family of cell surface
receptors. Overall amino acid identity between members of this family
is low, ranging between 20% and 30%.30 However, the
family is defined by the presence of cysteine-rich domains, which
contain 8 to 10 cysteine residues spaced at conserved
distances.28 PRV-1 contains 2 cysteine-rich domains that
are highly homologous to each other, showing 42% identity over 146 amino acids (Figure 2). In each of these domains, 6 cysteines are
spaced precisely like those found in the uPAR domains (Figure 3). At
first sight, the remaining cysteines found in the classical uPAR domain
appear to be missing. However, all uPAR domains described
to date contain a "signature sequence," a conserved
sequence found around the last 2 C-terminal cysteine residues of the
domain (cysteines 7 and 8, or 9 and 10, depending on the number of
cysteines in the domain).26,32 This signature sequence
consists of 7 residues with the sequence CXXDXCN. This sequence is also
found twice in PRV-1 (amino acids 104-111 and amino acids 294-300;
amino acids 104-111 contain 1 additional amino acid resulting in the
sequence CXXXDXCN). However, instead of occurring at the C-terminus of the cysteine-rich domain, where this sequence is found in all other
uPAR/Ly6/CD59 family members, in PRV-1 it is found N-terminal to the
other cysteines.
The genomic structure of several uPAR genes has been
elucidated.32-34 In all cases, the uPAR domain is encoded
by 2 separate exons, an intron occurring in the middle of the domain.
We have determined the genomic structure of PRV-1 and found that the
cysteine-rich domains are also encoded by 2 separate exons (data not
shown). When the 2 exons encoding the PRV-1 cysteine- rich
domains and the 2 exons encoding the first domain of uPAR (exons 3 and 4 of uPAR) are compared, the N-terminal PRV-1 exon is similar to
uPAR exon 4, whereas the C-terminal PRV-1 exon is similar to uPAR
exon 3. It appears therefore, that an exon switching has occurred in PRV-1, such that the cysteines surrounded by the signature sequence are
now located N-terminal to the other cysteines.
In the first, N-terminal domain of the uPAR receptor, the precise
location of the disulfide bonds has been elucidated.28 It
has been shown that the 1st and the 5th cysteines, the 2nd and the 3rd
cysteines, and the 4th and the 6th cysteines in this domain form
disulfide bridges. Furthermore, cysteine 7 and 8, which are surrounded
by the signature sequence, have also been shown to bind in the first
uPAR domain.28 Therefore, the relocation of these 2 cysteines to the N-terminus of the PRV-1 domain may not disrupt
disulfide bridge formation (Figure 2). However, it is likely that the
tertiary structure formed by the PRV-1 domain is different from that of
other uPAR/Ly6/CD59 domains.
Members of the uPAR/Ly6/CD59 family of receptors fulfill a diverse set
of functions. For example, CD59 protects cells from autologous lysis by
binding the C5-C8 complex, thereby preventing the formation of a
membrane attack complex.35,36 TSA, the thymic shared
antigen, plays a role during the positive selection of T cells and
lineage commitment to the CD4 or CD8 pathways.30 The
recently cloned RoBo-1 protein, which is expressed selectively in bone,
has been implicated in bone growth and remodeling.37 Although these receptors are tethered to the cell membrane via a lipid
anchor, they nonetheless participate actively in signal transduction.38 Several groups have shown that uPAR
interacts with and can activate protein tyrosine kinases including hck, fyn, lyn, fgr, and lck.39-41 In addition, uPAR has been
shown to activate the JAK/STAT pathway.40,42 It is
therefore likely that PRV-1 also mediates signal transduction in bone
marrow cells.
The first cysteine-rich domain of uPAR (uPAR-1), which shares the
greatest homology with the PRV-1 domains, because it also contains only
8 cysteine residues, has been shown to contain the ligand
binding activity of uPAR.43 uPAR binds its ligand, uPA, via
an epithelial growth factor-like domain (EGF domain) within uPA.44 It is therefore possible that the ligand for PRV-1
may also contain an EGF domain, perhaps also constitutes a
growth factor. We are currently conducting experiments to
identify the PRV-1 ligand.
This study demonstrated that PRV-1 is overexpressed in the peripheral
blood granulocytes of 19 of 19 patients with PV and not expressed in 21 of 21 healthy controls (Figure 1 and Table 1). Moreover, PRV-1 is not
expressed in 4 of 4 patients with CML (Figure 5A), 5 of 5 patients with
AML (Figure 5D), or 4 of 4 patients with secondary erythrocytosis
(Figure 5E). PRV-1 expression was detected in the single patient with
IMF (Figure 5C) and 2 of 6 patients with ET (Figure 5B). In a bone
marrow biopsy, the patient with IMF displayed hyperplastic
erythropoiesis and granulopoiesis as well as increased numbers of
megakaryocytes. Thus, all 3 lineages appear to be hyperproliferating in
this patient. Interestingly, both patients with ET positive for PRV-1
had slightly elevated peripheral leukocyte counts at the time of blood
sampling. Patient RW had 13 000 leukocytes/µL on the day of sampling
and had consistently shown leukocyte counts exceeding 12 000/µL for
the past 4 months. She had shown detectable levels of PRV-1 mRNA on
Northern blots in 3 different blood samples during this time. Patient
FW had 17 000 leukocytes/µL on the day of blood sampling. The other
4 patients with ET consistently showed leukocyte counts below
10 000/µL.
It has been previously reported that some patients who initially
present with thrombocytosis but normal hematocrit and are therefore
diagnosed with ET, subsequently progress to PV.45 Shih and
Lee have reported that of 30 patients presenting with idiopathic marked
thrombocytosis (platelets > 1000 × 109/L) and a
normal or reduced hemoglobin, 11 were found to fulfill the diagnostic
criteria for PV 2 to 45 months after initial evaluation.45 At the initial presentation, the bone marrow from these 11 patients had
shown endogenous erythroid colony formation in the absence of added
erythropoietin. Shih and Lee have therefore proposed that endogenous
colony formation may be used in the early identification of
PV.45 It remains to be seen whether patients PW and RW
progress to a diagnosis of PV. If this is the case, PRV-1
overexpression may likewise prove helpful in distinguishing ET from PV.
A sensitive reverse transcriptase-polymerase chain reaction assay for
PRV-1 has been established, which can be used to screen for PRV-1
overexpression. Together with clinical data, PRV-1 expression may thus
be useful in establishing a diagnosis of PV. The consistency with which this receptor is overexpressed in PV suggests that it may play a role
in the pathophysiology of this MPD.
| |
Acknowledgments |
The authors would like to thank Brigitte Schneider
and Gesa Santos for excellent technical assistance. We gratefully
acknowledge very helpful and stimulating discussions with Prof Dr J. Prchal and Dr F. Schriever. Our sincere thanks go to Prof Dr K. Geiger for his continuing support. A very special thank you to the
helpful staff of the hematology clinic and the therapy ward. Thanks
also to the Photo Center at the University Hospital Freiburg for
excellent photographic support. This article is dedicated to Irmgard
Pahl, mother of the senior author; her diagnosis with polycythemia vera sparked this research.
| |
Footnotes |
Submitted May 4, 1999; accepted December 17, 1999.
Reprints: Heike L. Pahl, Department of Experimental
Anaesthesiology, University Hospital Freiburg, Center for Tumor Biology, PO Box 1120, 79106 Freiburg, Germany; e-mail:
pahl{at}uni-freiburg.de.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction