© 1999 American Heart Association, Inc.
Original Contribution |
Identification and Cloning of a Secreted Protein Related to the Cysteine-Rich Domain of Frizzled
Evidence for a Role in Endothelial Cell Growth Control
From INSERM U441, Pessac, France (C.D., B.J., C.M.), and Shepens Eye Research Institute and Department of Ophthalmology and Pathology (P.A.D'A.), Harvard Medical School, Boston, Mass.
Correspondence to Cécile Duplàa, INSERM U441, Av du Haut-Lévêque, 33600 Pessac, France. E-mail cecile.duplaa{at}bordeaux.inserm.fr
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Abstract |
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Abstract—We report the isolation of a cDNA, FrzA (frizzled in aorta; GenBank accession No. U85945), from bovine aortic endothelium. It is the bovine counterpart of the mouse sFRP1, which encodes for a secreted protein that is homologous to the cysteine-rich domain of frizzled. Members of the frizzled family of genes have been shown to be required for tissue polarity and to act as receptors for Wnt. The predicted protein product of this gene includes the cysteine-rich extracellular domain, but not the 7 putative transmembrane domains that are highly conserved among members of the frizzled family. Visualization of FrzA mRNA and protein revealed that it was widely distributed among adult tissues. FrzA is expressed by highly differentiated or polarized cells, eg, neurons, cardiocytes, or various epithelia. Analysis of its expression in endothelium revealed that FrzA mRNA levels were high in endothelial cells scraped from freshly obtained bovine aortas, decreased when cells were placed in culture and began to proliferate, but increased at confluence. Transient transfection assays and an assay using addition of purified protein indicate that FrzA reduces the proliferation of endothelial cells. These data demonstrate the existence of a secreted protein homologous to the extracellular domain of the fz receptor, which we speculate plays a role in controlling cell growth and differentiation, possibly by regulating accessibility to Wnt family members.
Key Words: cloning • bovine-secreted frizzled-related protein • tissue expression • endothelial differentiation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Increasing evidence indicates a role for cell-cell interactions in the regulation of cell growth and differentiation, as well as in the formation of organ and tissue structures. Genetic studies in Drosophila melanogaster have revealed that the frizzled (fz) locus participates in such intercellular communication. The product of the fz gene functions in Drosophila to transmit information regarding tissue polarity,1 organizing hair and bristle orientation throughout the cuticle. Genetic mosaic experiments have demonstrated fz function in the transduction of the polarity signal.2 A large family of putative transmembrane receptors homologous to fz has been described in mammals.3 4 These proteins are conserved from flies to mammals, which suggests functional parallels between Drosophila fz and its homologues or related genes in other species. Additional families of highly conserved genes are involved in the determination of cell and tissue polarity, including Notch5 and Wnt/wingless.6 7
All fz sequences share the same overall domain structure with a signal peptide, a unique and highly conserved cysteine-rich domain (CRD), and 7 putative transmembrane domains. Frizzled family members are expressed in diverse organisms, show specific tissue distribution, and are regulated during embryonic development. The role of fz proteins as transmembrane proteins with potential receptor function has attracted much attention.8 A recent in vitro study demonstrated that frizzled proteins could function as receptors for Wnt (Wingless).9 10 The Wnt family includes numerous genes isolated from diverse species, which encode secretory glycoproteins that appear to influence multiple biological responses, including cell growth, differentiation, and survival.6 11 12 The importance of the fz-Wnt interaction was highlighted by the fact that cells transfected with soluble Wg protein bind both Drosophila and mammalian fz proteins and initiate signal.9 10 Furthermore, and relevant to our observations, the extracellular CRD of fz was shown to be required for Wnt ligand binding.
We report the isolation and characterization of the bovine orthologue of mouse sFRP1, FrzA (frizzled in aorta; GenBank accession No. U85945), which encodes a deduced protein that contains a frizzled CRD, but no transmembrane domains. It belongs to a novel family of secreted mammalian proteins related to the frizzled family, sFRP, but encoding only for the CRD.9 10 13 14 15 16 We document that in adult tissue the transcripts and the protein were widely distributed but had specific cellular localization. The expression of FrzA in bovine aortic endothelial cells (BAECs) was examined, and FrzA mRNA was found to be expressed in cells in situ, diminished with increasing passage of the cultured cells, and inducible by serum-free culture conditions. Moreover, results of transient transfection studies and an assay using exogenously added purified protein assays implicate FrzA in the control of endothelial and smooth muscle proliferation. These data indicate the existence of a secreted protein, a member of the secreted frizzled-related protein (sFRP) family14 consisting of a fz-like CRD, which we hypothesize plays a role in regulating cell growth and differentiation in adult organisms, perhaps by controlling accessibility to Wnt family members.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cDNA Library Construction
RNA was isolated by a CsCl gradient from endothelium scraped directly from bovine aortas. Poly(A)+ RNA was isolated by chromatography on an oligo(dT)-cellulose column (Stratagene). A cDNA library was constructed in the Uni-ZAP XR vector using the ZAP-cDNA synthesis kit (Stratagene) and size fractionated on a column following the manufacturer's instructions. The yield was 500 000 primary recombinants with an average size of 2 kb.
Approximately 600 000 phages plaques were absorbed onto nylon membrane
(New England Nuclear Research Products) and screened. Four
successive hybridization rounds were carried out to obtain a complete
cDNA sequence. The cDNA tag (A1), which was initially isolated from
vessel endothelium by differential
display,17 was used as a probe for the first round of
hybridization. A probe, corresponding to 300 bp of the 5' end of the
clone obtained in the first round of screening, was used in the second
screening, and the same strategy was applied in 3 subsequent screens.
The probes were random-prime labeled (Rediprime, Amersham), and
hybridization was carried out in a mixture containing 1% SDS, 2x SSC,
10% dextran sulfate, 50% formamide, 5x Denhardt's solution, and 50
ng/mL ssDNA. The filters were washed successively in 2x SSC-0.1% SDS
at room temperature for 20 minutes and then twice in 0.5x SSC-0.1%
SDS at 50°C and were then exposed to x-ray film. Positive clones were
isolated and rescreened until they were plaque purified. The inserts in
ZAP vector were excised using the protocol recommended by the
manufacturer (Stratagene). The structure of the double-stranded
phagemid cDNA insert was determined by the cycle-sequencing method
using fluorescent dideoxy terminator nucleotides
with an Applied Biosystems 373A automated DNA sequencer (Dana-Farber
Cancer Institute Core Facility); the sequence was determined for 100%
of both strands.
Protein and Nucleic Sequence Analysis
Sequence analysis was carried out using the GCG package
(Genetic Computing Group). Homology searches against a protein databank
were accomplished with the Blast algorithm.18 Alignment of
2 sequences was accomplished with Bestfit and of multiple sequences
with Pileup19 algorithms.
Cell Culture
BAECs were isolated as previously described20 by
scraping the intimas of bovine aortas with a scalpel blade. Bovine
smooth muscle cells (BSMCs) were explanted from bovine aortas as
previously described21 and used at passages 2 to 10. All
cells were grown in DMEM (Sigma) with 10% FCS (Hyclone Laboratories,
Inc), L-glutamine (29.2 mg/mL), and penicillin-streptomycin
(10 000 U/mL) (Irvine Scientific) (DMEM/10% FCS). For each
experiment, endothelial cells (ECs) scraped from
several aortas were pooled and were either processed for RNA isolation
or cultured in DMEM/10% FCS. Once cells had reached confluence, they
were switched for 24 hours to medium with serum or serum-free medium
containing BSA (1%), insulin (1 µmol/L), transferrin (200
µg/mL), ascorbate (0.2 mmol/L), and sodium selenite (6.25 ng/mL)
(all from Sigma).
Northern Blot Analysis
Tissue obtained directly from the slaughterhouse was frozen
immediately in liquid nitrogen. Total RNA was isolated by
homogenizing the tissues in RNAzol B (Tel-Test, Inc)
following the manufacturer's instructions. Total RNA (20 µg) was
electrophoresed in a 1% agarose/formaldehyde gel and transferred by
capillary technique to a GeneScreen membrane (NEN Research
Products). An EcoRI/SmaI fragment of FrzA
cDNA was used as a probe, and hybridization was carried out with
random-labeled probe.
RNase Protection
Total RNA was prepared from adult bovine brain, retina, spleen,
heart, kidney, liver, muscle, and aorta (adventitia, media, and
endothelia were extracted separately) in RNAzol as described above.
Total RNA was extracted from cultured cells by a CsCl step gradient.
Ten micrograms of total RNA from the aortic adventitia, media,
endothelia, and cultured BAECs or 50 µg of each of the other tissues
(or tRNA as a control) was assayed by RNase protection. The cRNA probes
for GAPDH and FrzA were prepared as follows. A GAPDH cDNA, amplified by
polymerase chain reaction (PCR) using 5'-TGAAGGTCGGAGTCAACGGA as sense
primer and 5'-CATGTGGGCCATGAGGTCCA as antisense primer, was cloned into
the pCRII using a T/A cloning kit (Invitrogen). The primer sequences
were chosen on the basis of the human sequence (Clontech). The
construct was digested with EcoRI and AccI,
subcloned into the pBluescript II KS with or without plasmid in
EcoRI/AccI sites, and transcribed. A riboprobe
for FrzA was synthesized from the A1-tag PCR-amplified product
cloned into the pCR1000 plasmid (Invitrogen). Transcription was carried
out with bacteriophage T3 RNA polymerase for 5' end-linearized FrzA
vector and GAPDH vector using the Maxiscript kit (Ambion). Reactions
were incubated for 1 hour at 37°C, and full-length radiolabeled
probes were purified by electrophoresis through a denaturing
polyacrylamide gel. Transcripts were located in the gel by
autoradiography and eluted from gel slices. Reagents
were obtained from Ambion, and the hybridization and digestion
conditions were as recommended by the manufacturer.
In Situ Hybridization
Adult bovine tissues obtained directly from the slaughterhouse
were fixed in 4% formaldehyde in PBS for 24 hours and embedded in
paraffin. In situ hybridization was carried out using the Genius
Labeling and Detection Kit (Boehringer Mannheim), as described
elsewhere.22 Serial 5-µm sections were fixed in 4%
paraformaldehyde for 30 minutes, rehydrated, treated
with proteinase K (20 µg/mL) for 15 minutes, and then
acetylated with triethanolamine, dehydrated, and air dried.
Labeled single-strand RNA probes were prepared using digoxigenin-11-UTP
according to the manufacturer's instructions. For a FrzA probe, a 2-kb
cDNA fragment containing nearly the entire 3'-untranslated region of
FrzA cDNA was used. The cDNA, subcloned into the polylinker region of
pBluescript KS, was linearized by digestion with the appropriate
enzymes to allow synthesis of a digoxigenin-labeled RNA in either the
sense or the antisense orientation (using T3 or T7 RNA polymerase,
respectively). To obtain small RNA fragments of 
100 bp, the RNA
transcript was partially hydrolyzed in 60 mmol/L
Na2CO3/40 mmol/L
NaHCO3 at 60°C for 1 hour and ethanol
precipitated. Hybridization was carried out at 50°C for 16 hours with
200 ng/mL of probe in a hybridization buffer containing 50% deionized
formamide, 1x Denhardt's solution, 4x SSC, 10% dextran sulfate, and
0.4 mg/mL ssDNA. Sections were washed twice for 15 minutes in 2x SSC
at room temperature, treated with RNase A (100 µg/mL) in 2x SSC at
37°C for 30 minutes, and then washed twice for 20 minutes in 2x SSC
at 42°C and then at room temperature. The alkaline
phosphatase–conjugated anti-digoxigenin antibody (1:500 dilution) was
incubated for 2 hours in 10% horse serum at room temperature, and the
sections were rinsed overnight. The sections were incubated for 8 hours
with the substrate nitroblue tetrazolium salt X-phosphate and then
counterstained with the methyl green nuclear stain (Vector
Laboratories).
Production of Polyclonal Antisera and
Immunohistochemistry Analysis
Oligopeptides corresponding to C-terminal amino acids 206 to 218
and 269 to 296 were synthesized by Research Genetics, Inc. The peptides
were conjugated to keyhole-limpet hemocyanin, emulsified with an equal
volume of Freund's complete adjuvant, and used to immunize New Zealand
White rabbits. Injections were repeated at 4-week intervals, and the
sera were obtained 10 weeks after the initial injection.
Immunoperoxidase staining was performed on serially cut 5-µm sections. Endogenous peroxidase was neutralized with 3% hydrogen peroxide, and the sections were preincubated in blocking serum (10% goat serum in PBS) for 20 minutes at room temperature. The sections were incubated in primary antibody (1/500) overnight at 4°C in PBS, 5% goat serum, 2% BSA, and 0.2% Tween and were then incubated with biotinylated rabbit secondary antibody (Amersham) (1/100) for 30 minutes at room temperature in PBS with 5% goat serum, 2% BSA, and 0.2% Tween followed by streptavidin–horseradish-peroxidase complex (Amersham) (1/100) for 30 minutes at room temperature in PBS with 5% goat serum, 2% BSA, and 0.2% Tween. The sections were rinsed with PBS and visualized by incubation in 3,3'-diaminobenzidine tetrahydrochloride dihydrate (Vector Laboratories). Sections were counterstained with 10% Gill hematoxylin. For each tissue section labeled with FrzA antibody, a negative control was prepared by staining a serially cut section with a nonspecific rabbit polyclonal antibody.
Protein Cellular Distribution and Purification
COS-7 cells were stably transfected with the pcDNA3 mammalian
expression vector (Invitrogen) containing FrzA fused in the C terminus
with a myc::his epitope using an activated-deutrimer reagent
(Superfect, Qiagen) according to the manufacturer's protocol. Stable
transfectants were selected, and expression of the gene was confirmed
by PCR and Western blot analysis. Clones were incubated for 72
hours in serum-free medium (Opti-MEM, Gibco-BRL), the last 4 hours in
the presence or absence of 50 µg/mL heparin (bovine lung, Sigma), and
processed for secretion studies or purification as described below.
Secretion Studies
Conditioned medium (CM) was collected and concentrated 20 times
by using Microsept-10 concentrators (Filtron, Polylabo). Cells were
scraped, resuspended in lysis buffer consisting of 10 mmol/L Tris
and 5 mmol/L EDTA, and lysed using a syringe fitted with a
25-gauge needle. Large debris, nuclei, and nonlysed cells were removed
by centrifugation for 10 minutes at 3000g.
The supernatant was centrifuged for 30 minutes at
100 000g. The pellet was extracted with 1% NP-40, 0.5%
sodium deoxycholate, and 0.1% SDS in PBS. Cytosolic and
membrane/particulate fractions and concentrated CM were
analyzed by SDS-PAGE on a 10% gel under reducing conditions
and transferred to nitrocellulose membrane (Amersham). The membranes
were blocked in 10% nonfat dry milk in Dulbecco-PBS (PBS) plus
0.1% Tween 20 for 1 hour at room temperature and then incubated with
anti-myc monoclonal antibody (1/5000) (Invitrogen) for 90 minutes in
PBS+5% milk. The membrane was washed 3 times for 10 minutes each in
PBS+0.1% Tween and incubated with anti-mouse IgG-peroxidase conjugate
for 1 hour at room temperature. After 3 10-minute washes in PBS+0.5%
Tween, the membrane was stained with a luminescence detection system
(enhanced chemiluminescence, Amersham) according to the manufacturer's
instructions.
Purification
Recombinant FrzA protein was purified using a
Ni2+-charged agarose resin (ProBond resin,
Invitrogen) under native conditions according to the manufacturer's
protocol. Medium conditioned by heparin-treated cells was collected by
using Centriprep-10 concentrators (Amicon) and equilibrated in native
binding buffer (20 mmol/L sodium phosphate buffer [pH 7.8] and
500 mmol/L NaCl). The concentrated supernatant was then incubated
with resuspended resin at 4°C in a rotating device. After
centrifugation at 800g for 2 minutes, the
column was washed 3 times with 5 mL of native binding buffer by
resuspending the resin, rocking it for 2 minutes, and then separating
the resin by centrifugation. The resin was then washed
3 times with 5 mL of phosphate buffer (20 mmol/L sodium phosphate
buffer [pH 6] and 500 mmol/L NaCl) with the same procedure. The
retained protein was eluted stepwise by application of increasing
imidazole concentrations (50, 200, 350, and 500 mmol/L) in elution
buffer (20 mmol/L sodium phosphate buffer [pH 6] and 500
mmol/L NaCl). All of the fractions obtained were blotted on a
nitrocellulose membrane using a dot blot apparatus. For
detection of FrzA, an immunoassay using myc monoclonal antibody
(Invitrogen) was conducted as previously described in secretion
studies. FrzA protein was detected in the fractions of elution buffer
containing 200 mmol/L imidazole. These fractions were pooled and
dialyzed overnight against PBS at 4°C, and the protein concentration
was calculated with the micro-BCA method (Pierce).
Effect of FrzA on Cell Proliferation
Transient Transfection
BAECs (passage 6) were plated at
2x104 cells/cm2 in 6-well
plastic dishes (Costar) in serum-containing medium and allowed to
attach overnight. The cells were then incubated for 3 hours with 1 µg
of plasmid and 3 µL of Superfect reagent (Qiagen) in DMEM/10% CS.
After the transfection, the medium was removed and replaced with fresh
DMEM/10% CS. Cell counts were determined 3 and 5 days later using a
Coulter counter. The culture medium was not replaced during the time
course. The transient transfections were conducted either with a
control plasmid (pZeoSV coding for the ß-galactosidase; Invitrogen)
or with the same plasmid containing the FrzA coding sequence in its
polylinker region. The efficiency of each transfection was determined
by immunostaining the cells for the Sh Ble
resistance gene and counting the number of positively stained cells.
Briefly, this involved cell fixation in paraformaldehyde,
preincubation in blocking serum, and application of the polyclonal
antibody against Sh Ble protein (1/1000) (Cayla) for 90
minutes in 5% goat serum, 2% BSA, and 0.2% Tween. A biotinylated
anti-rabbit secondary antibody (Amersham) was then applied for 30
minutes at room temperature followed by
streptavidin–horseradish-peroxidase complex (Amersham). After 30
minutes at room temperature, sections were rinsed with PBS and
visualized by incubation with 0.05% (wt/vol) 3,3'-diaminobenzidine
tetrahydrochloride dihydrate. A counterstain of 10% Gill hematoxylin
was applied.
Preparation of BAEC CM
Media conditioned for 5 days by cells transiently transfected
with the control plasmid (CM-C) or with the plasmid containing the
insert (CM-pFrzA) were assayed for their effects on the proliferation
of BAECs. BAECs plated at 20 000/cm2 on 6-well
plates (Falcon) were grown in the presence of the CM for 5 days before
being harvested and counted. All experiments were repeated at least 3
times.
Effect of Purified FrzA
BAECs were plated at 20 000/cm2 and BSMCs
at 10 000/cm2 on 12-well plastic dishes
(Falcon). After cell attachment, the medium was replaced with 1.2 mL of
the mixture of 10% CS plus DMEM with or without purified FrzA. Cells
were grown for 2 days in the presence of increasing concentrations of
the FrzA-purified fraction before being harvested and counted. All
experiments were repeated at least 3 times in duplicate dishes with 3
different preparations of FrzA. Controls consisted of media conditioned
by native COS-7 cells concentrated and eluted with the same procedure,
as well as the fraction of eluent obtained before and after the peak of
FrzA elution. Neither of these control eluents affected cell
proliferation.
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Results |
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cDNA Cloning and Sequence Analysis
Differential display was initially used to identify genes differentially expressed between endothelium in vivo and ECs in culture.17 A PCR-amplified cDNA fragment (called A1) was isolated from endothelium in vivo, cloned, and sequenced. To isolate the full-length cDNA, a cDNA library constructed from RNA isolated from endothelium directly scraped from adult bovine aortas was screened with the A1 cDNA probe. Five independent overlapping cDNA clones were isolated. Together, the clones were found to cover a cDNA sequence of 4.240 bp (Figure 1
). The complete cDNA sequence obtained
from different overlapping clones contains a single, complete long open
reading frame (nucleotides 198 to 1124) and encodes a
predicted protein product of 308 amino acids (Figure 1). On
the basis of the hydrophobicity profile (data not shown), there is a
predicted signal sequence of 29 amino acids at the N terminus with a
characteristic composition. Close to the N terminus are 3 polar
residues (Gly2–4), and within the leader is a hydrophobic core
consisting largely of hydrophobic amino acids (Trp6–Ala22). A polar
C-terminal (Gly23–Tyr30) region is also present. Two putative
N-glycosylation sites were identified at positions 167 and
257.
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While this work was in progress, reports were published defining a
novel family of secreted protein related to the frizzled transmembrane
protein.15 Bovine FrzA gene was the bovine
counterpart of the cloned mouse sFRP1 encoding for proteins
presenting 93% of identity. It seemed very unlikely that
FrzA is an alternatively spliced gene. A single transcript
of 4.3 kb was detected when a Northern blot of bovine brain and
liver total RNA was probed with different cDNA probes that had been
used to screen the library. Levels of the transcript were high in the
brain and quite low in the liver (Figure 2), observations that were later
confirmed by both RNase protection (Figure 4) and in situ
hybridization (Figure 5a, 5b, 5k, and 5l). A human orthologue
was subsequently cloned presenting 94% identity with the bovine
amino acid sequence.16 23 24
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Tissue Distribution of FrzA
RNase Protection and In Situ Hybridization
In an attempt to gain some insight into the potential function of
FrzA gene product, we studied the expression pattern of
FrzA transcripts in various adult bovine tissues by RNase
protection and by in situ hybridization. RNase protection revealed that
FrzA mRNA levels were highest in aortic
endothelium, heart, spleen, and eye. Lower but
significant levels were found in the lung, brain, and kidney. Faint
signals were detected in liver, skeletal muscle, and the medial layer
of the aorta, and no signal was detected in the aortic adventitia
(Figure 3).
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To determine which cells were producing FrzA, various adult
bovine tissues were examined by in situ hybridization. Our results
indicate that FrzA synthesis is highly cell specific. The cortical part
of the brain was labeled, with neuronal cells and small vessels
strongly positive (Figure 4a
and 4b). ECs and SMCs were labeled. FrzA mRNA was
expressed in several layers of the retina, with labeling strongest in
the inner nuclear layer, where it was localized to astrocytes and
ganglion cells (Figure 4c and 4d). In the sclera of the eye, the
endothelium and smooth muscle of blood vessels
expressed FrzA (Figure 4e and 4f), whereas the
connective tissue was negative. Neural cells in the optic disc were not
labeled, and FrzA expression was confined to the
endothelium of the central artery (data not
shown). Expression of FrzA in the heart was restricted to
myocytes (Figure 4g and 4h). The skeletal muscle (Figure 4i and 4j) and the liver (Figure 4k and 4l) were both
negative for FrzA transcript. In the lung, a high level of
labeling was seen in terminal bronchioles, where FrzA
transcripts were detected in the epithelium as well as in cells that we
suspect are connective tissue macrophages (Figure 4m and 4n). It was notable that not all of the epithelium was labeled;
ciliated cells strongly expressed FrzA, whereas Clara cells,
which have been described as undifferentiated stem
cells,25 were negative. Interestingly, transcripts
were not detected in the large vessels in the lung. In the kidney,
the FrzA mRNA was localized in the epithelium of collecting
ducts in the medulla (Figure 4o and 4p), whereas the cortical
kidney was negative, with no labeling in the glomeruli. The
endothelium and the media of the major arteries were
mildly positive. In the spleen, FrzA expression was
restricted to the red pulp in cells associated with the sinuses (Figure 4q and 4r); these cells could be vascular or lymphatic ECs or
macrophages. In the white pulp, the central arteries, capsula
rich in smooth muscle cells, and connective tissue were all
negative. In general, the labeling pattern observed by in situ
hybridization was in agreement with that noted by RNase protection
(Figure 3) and indicates a cell-specific pattern of
FrzA expression.
Immunohistochemistry
Immunohistochemical analysis was used to examine
FrzA protein localization in adult bovine tissues. Serially
cut, hematoxylin-stained sections of brain, retina, sclera, aorta,
lung, and kidney were examined (Figure 5). In general, protein localization was
similar to that observed for mRNA by in situ hybridization. In the
cortical brain, staining was localized to the neurons and small blood
vessels (Figure 5a and 5b). FrzA was localized in the retina to
the inner and the outer nuclear layers, with strong labeling of the
neuronal cell bodies (Figure 5c and 5d), and displayed a strong
immunolabeling in vessels in the choroid and the sclera (data not
shown). In the aorta, there was strong immunoreactivity in the
endothelium, and a few SMCs in the medial layer were
moderately labeled (Figure 5e and 5f). Epithelial cells of the
terminal bronchioles in the lung, which showed a strong signal by in
situ hybridization, were also intensely immunopositive. Interestingly,
the blood vessels in the lung were unstained by the FrzA antisera
(Figure 5g and 5h). Immunolocalization in the kidney displayed a
pattern of FrzA protein expression similar to that seen for mRNA by in
situ hybridization, with staining in the epithelium of the collecting
ducts (data not shown) and the media and the
endothelium of the arteries (Figures 5i and 5j).
The 2 antisera yielded similar staining patterns on different bovine
tissues; control preimmune serum showed no signal.
Expression of FrzA by ECs In Vitro
Because FrzA was originally identified as
differentially expressed between ECs in culture and
endothelium in vivo, we examined its expression by ECs
in vitro. RNase protection was used to investigate the effect of growth
state on endothelial expression of FrzA.
ECs, collected by scraping the intimas from freshly obtained bovine
aortas, were pooled and plated at low density. FrzA levels
were examined after 72 hours, when cells were sparse and proliferating,
and after 10 days, when cells were confluent (Figure 6). Sparse BAECs expressed barely
detectable levels of FrzA, and expression increased when
cells were confluent. There was significant FrzA
expression in first-passage ECs, but after 2 additional passages
FrzA mRNA levels were undetectable. Culture in serum-free
conditions resulted in a strong induction of FrzA in primary
BAECs. Serum-free culture conditions, however, did not reinduce
FrzA levels in BAECs that had been passaged.
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Protein Expression, Secretion, and Purification
To determine whether FrzA was secreted, different protein
fractions were examined by immunoblot analysis.
FrzA was associated with the plasma membrane but released in the medium
by heparin treatment. After 3 days in culture under serum-free
conditions, cells were treated for 4 hours with heparin. Then the CM
was collected, and the cells were extracted. The majority of the tagged
protein was detected in the medium, and the intensity of the band
increased after heparin treatment. The protein was not detected in the
cytosolic fractions and was barely detectable in the lysates. There was
no evidence of partial hydrolysis (Figure 7A). The presence of 6 histidines in the
C terminus of FrzA allowed us to purify it from the supernatant of
stably transfected COS-7 cells. CM was collected and fractionated on a
Ni2+-charged agarose resin column. The major
fractions containing FrzA protein were identified by dot blot by
probing with myc antibody. FrzA was detected in the 200 mmol/L
imidazole fractions (Figure 7B). The purified protein was
visualized as a major band by silver-staining
SDS-polyacrylamide gel. The identity of the protein was
confirmed by immunoblotting with a myc antibody (Figure 7C).
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Characterization of FrzA
To investigate its role, studies were conducted to determine
whether FrzA would influence EC proliferation in vitro. BAECs were
transiently transfected with a plasmid coding either ß-galactosidase
(control) or FrzA and allowed to grow for 3 or 5 days without a change
in media, and cell number was determined. The efficiency of each
transfection was determined by immunostaining the cells
for the Sh Ble resistance gene and counting the number of
positively stained cells. The level of transfection was equivalent
between cells transfected with the control plasmid and cells
transfected with the plasmid containing the FrzA insert
(data not shown), allowing comparison of the effect of FrzA expression
on cell proliferation.
Cells transfected with FrzA were growth inhibited when compared with
the cells transfected with the control plasmid (Figure 8A). There was a 50% decrease in
cell number 5 days after the transfection. Parallel studies revealed a
similar decrease in [3H]thymidine incorporation
(data not shown). Because our studies confirmed that FrzA is a secreted
protein, medium conditioned by the FrzA-transfected cells was also
assessed for its effect on the proliferation of untransfected BAECs
(Figure 8B). Media conditioned for 5 days by cells transfected
with the control plasmid (CM-C) or cells transfected with the plasmid
containing the insert (CM-FrzA) were added at a final concentration of
25%, 50%, or 75% to BAECs plated at a sparse density. The cells were
allowed to grow for 4 days without a medium change, and cell number was
determined. CM-FrzA inhibited endothelial growth
relative to CM-C. Cell growth was inhibited by 27%, 30%, and 33% at
25%, 50%, and 75% CM, respectively. Parallel studies revealed a
similar decrease in [3H]thymidine incorporation
(data not shown).
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Next, we examined the effect of highly enriched fractions of
recombinant FrzA on BAEC growth. BAECs were grown for 48 hours in the
presence of FrzA, and cell number was determined. FrzA inhibited the
proliferation in a dose-dependent fashion. At estimated doses of 1.4 to
14 nmol/L, EC proliferation was inhibited 10% to 60%, respectively
(Figure 8C). We also tested the effect of FrzA on BSMCs and
showed a significant proliferation inhibition at the same doses (data
not shown). As a control, we tested the effect of (1) fractions
from CM from heparin-treated native COS-7 cells purified on a
Ni2+-charged agarose resin column and (2)
fractions obtained before and after the FrzA peak elution fractions on
cell proliferation. These fractions did not affect EC growth (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This work was initially motivated by an interest in identifying genes involved in the induction and maintenance of the quiescent, differentiated phenotype of vascular endothelium. Previous studies have demonstrated phenotypic differences between quiescent endothelium in vivo and their in vitro counterparts. The phenotype of EC in culture is more consistent with cells that have been injured or otherwise activated than cells that are quiescent.26 27 Differential display was used to compare gene expression of quiescent ECs isolated directly from bovine aorta with that of cultured, proliferating ECs. Several cDNA tags expressed predominantly by bovine aortic endothelium in vivo were isolated, but sequence information did not reveal any clue to their possible functions.17
To isolate the complete cDNA of one of these tags (called A1), we constructed a cDNA library from ECs directly scraped from bovine aortas and used the gene tag obtained by differential display to isolate an additional sequence. Sequence homology analyses of this gene revealed that it encodes a predicted protein that contains the characteristic CRD of fz proteins but lacks both the transmembrane and cytoplasmic domains that characterize members of the fz family. Sequence analysis indicated that the protein was secreted. We therefore hypothesized that this protein was a form of the extracellular ligand binding portion of the fz receptor. Western blot analysis of FrzA protein production as a result of transient translation has confirmed that FrzA is secreted.
This gene does not appear to be a splice variant of the longer fz
receptor. In the 4 successive library screens, the same sequence was
obtained by restriction profile comparison. In a fifth screen, 5
independent clones yielded the identical sequence. Moreover, Northern
blot analysis of bovine tissues using probes spanning different
regions of the cDNA revealed a single transcript of 4.3 kb. PCR
analysis using different sets of primers spanning the entire
cDNA sequence yielded a single band for each primer set (data not
shown).
Recently, there have been several reports describing proteins in mouse
and human homologous to the Fz CRD called secreted frizzled-related
proteins (sFRPs)15 or secreted apoptosis-related
proteins (SARPs).16 SDF5, now referred to as sFRP2 or
SARP1, was cloned by signal sequence trapping from a murine bone marrow
stromal cell library and is expressed at high levels in lung and
kidney.28 Frzb, an orthologue of the mouse sFRP3, was
identified in bovine articular chondrocytes.13 Fritz, a
CRD-related protein initially cloned from hamster fibroblasts, is
highly homologous to Frzb.29 FrzA is the bovine orthologue
of the mouse sFRP1 or SARP2. sFRP, the human orthologue, was purified
with hepatocyte growth factor by its heparin-binding
capacity from CM by a human embryonic lung fibroblast
line.23 Interestingly, hSARP2 was isolated as a result of
an attempt to clone genes expressed in quiescent but not in
proliferating cells. Experiments with SARP2 demonstrated an increased
sensitivity of 10T1/2 cells to proapoptotic
agent.16 Screening for apoptosis-related genes led
to the cloning of hsFRP gene, which was suggested to be
involved in growth inhibition and in breast tumor
development.24 The sequence of FrzA is most similar
to that of mouse, rat, and Drosophila fz CRD, with 38%
identity at the amino acid level and conservation of all 10 cysteine
residues.1 3 4 13 The CRD motif is also found in a
variant of type XVIII collagen.30
To gain some insight into the potential role of FrzA, we studied the expression of FrzA mRNA and protein distribution. FrzA mRNA and protein were expressed widely in adult bovine tissues, yet the cellular localization was quite specific. Immunohistochemical analysis of FrzA protein revealed a pattern of localization that was consistent with the mRNA distribution seen by in situ hybridization. We demonstrated that FrzA is expressed at high levels by epithelium of lung and kidney, by neural elements of the brain and the retina, and by cardiocytes. There was a striking lack of signal in skeletal muscle and liver. In general, a majority of vessels were labeled. In large blood vessels, there was a significant expression in the endothelium, whereas labeling in medial smooth muscle cells was scattered. Expression in microvessel was variable; small vessels in skeletal muscle were not labeled; vessels in all other organs were strongly labeled both in the intima and media. Detection of the protein in the cytoplasm is consistent with its localization in vesicles, as it has been reported for other secreted proteins such as platelet-derived growth factor.31 Our description of FrzA transcript distribution at the tissue level is in agreement with that described by RNase assay by Rattner et al15 for sFRP1 in mouse adult tissues. Our study, furthermore, has described the expression of FrzA at the cellular level. These data at the level of sequence taken together indicate a high level of conservation and expression of protein, which suggests a functional conservation among the species.
The expression of FrzA was confined to cells that are highly polarized, nonproliferating, and differentiated. A possible role for FrzA in the regulation of growth and differentiation is supported by our in vitro observations. The endothelium of both large and small adult blood vessels does not proliferate and has been documented to have a labeling index of <0.01%.32 BAECs scraped directly from fresh bovine aortas had the highest level of FrzA mRNA. Placement of ECs into culture in the presence of serum, which is known to stimulate proliferation and induce dedifferentiation of EC,27 led to reduced FrzA mRNA levels. Repeated passage of the BAECs led to a further reduction in FrzA mRNA until, by passage 5, the transcript levels were nondetectable by RNase protection and could not be reinduced by withdrawal of serum. In addition, BAEC proliferation was reduced by transient expression of FrzA and by culture either in medium conditioned by cells overexpressing FrzA or in the presence of highly enriched FrzA fractions. This effect was not specific to BAECs, because BSMCs were also inhibited by FrzA.
Indirect evidence suggests that FrzA binds heparin. Indeed, we demonstrated that heparin releases FrzA in the CM. The highly human homologue of FrzA, hFRP, was purified on a heparin-Sepharose column.23 As we obtained enriched FrzA fractions from stably transfected COS-7 cells treated with soluble heparin, one could argue that heparin could inhibit the ability of FrzA to suppress BAEC proliferation. Three kinds of experiments were conducted to demonstrate that FrzA influences the growth of vascular cells: transfection experiment, CM assays, and the addition of recombinant purified protein. In the last experiment only, cells were treated with heparin to enhance the free fraction of FrzA protein in the CM, which was purified on a Ni2+-charged agarose resin column. Recombinant protein was collected and, during the concentration step, was extensively washed with NaCl before being applied to the column. To remove heparin, different concentrations of NaCl ranging from 0.5 to 1.5 mol/L were used in the washing step in preliminary experiments. Each time, we obtained the same growth-inhibition level (data not shown). In addition, the fact that the transfection experiment (without added heparin in the CM) led to growth inhibition is strong evidence that this effect is due to FrzA. The silver stain of the purified preparation fractions reports the presence of additional bands. Also, we cannot rule out the possibility that the antiproliferative activity present in the FrzA preparation is due to the presence of contaminating cytokine. This is not probable, however, given that fractions from CM from heparin-treated native COS-7 cells and fractions obtained before and after FrzA peak elution fractions did not affect cell growth.
Interestingly, hsFRP, the human orthologue, was found to be regulated during the cell cycle24 ; there was an increase in hsFRP expression in serum-deprived, growth-arrested cells and a decrease in proliferating cells. Moreover, in human breast tumors and in proliferative lesions, hsFRP expression was downregulated. Similarly, although it did not affect cell growth, the overexpression of SARP2 in epithelial tumoral MCF7 cells sensitized the cells to proapoptotic events.16 Our study reports for the first time that FrzA influences the growth of vascular cells. The mechanism of the EC and SMC growth inhibition is currently under investigation. The expression of Wnt family members in the adult endothelium has not been reported.
The extracellular CRD motif of Fz is thought to serve as a site of ligand binding interaction.9 10 The Drosophila fz protein, Dfz2, and other frizzled mammalian members have been shown to bind Wg, a Wnt-1 homologue. The 7 transmembrane domains are theoretically involved in cellular signal transduction. Some insight into the possible role played by FrzA and related proteins is provided by the fact that FrzA,33 sFRP2, and sFRP314 15 34 have been shown to bind and inhibit the effects of members of the Wnt family. Our identification of a secreted truncated form of frizzled receptor protein in the vasculature is intriguing in terms of a potential biological role of this protein in vivo. Other secreted forms of growth factor receptors have been described, including the interleukin 4 receptor,35 the interleukin 7 receptor,36 and the fibroblast growth factor receptor.37 Similarly, secreted forms of adhesion molecule have been described, including the neuronal cell adhesion molecule,38 the vascular cell adhesion molecule39 and endothelial leukocyte adhesion molecule.40 As for these other secreted proteins, FrzA may well act as a competitive inhibitor and therefore a naturally occurring antagonist for the ligand.
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Acknowledgments |
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This work was supported by grants from the Fulbright Foundation and from the Association pour la Recherche sur le Cancer (to C.D.) and by NIH Grants EY05985 and CA45548 (to P.A.D'A.). B.J. is a recipient of a fellowship from the Groupe de Réflexion sur la Recherche Cardiovasculaire.
Received February 4, 1999; accepted April 7, 1999.
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