From the Department of Pathology, Anatomy and Cell
Biology and the
Cellular Biology and Signaling Program, Kimmel
Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
19107 and ¶ PhenoPath Laboratories,
Seattle, Washington 98103
Received for publication, October 15, 2002, and in revised form, February 4, 2003
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ABSTRACT |
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The goal of this study was to discover novel
partners for perlecan, a major heparan sulfate proteoglycan of basement
membranes, and to examine new interactions through which perlecan may
influence cell behavior. We employed the yeast two-hybrid system and
used perlecan domain V as bait to screen a human keratinocyte cDNA library. Among the strongest interacting clones, we isolated a ~1.6-kb cDNA insert that encoded extracellular matrix protein 1 (ECM1), a secreted glycoprotein involved in bone formation and angiogenesis. The sequencing of the clone revealed the existence of a
novel splice variant that we name ECM1c. The interaction was validated
by co-immunoprecipitation studies, using both cell-free systems and
mammalian cells, and the specific binding site within each molecule was
identified employing various deletion mutants. The C terminus of ECM1
interacted specifically with the epidermal growth factor-like
modules flanking the LG2 subdomain of perlecan domain V. Perlecan and
ECM1 were also co-expressed by a variety of normal and transformed
cells, and immunohistochemical studies showed a partial expression
overlap, particularly around dermal blood vessels and adnexal
epithelia. ECM1 has been shown to regulate endochondral bone formation,
stimulate the proliferation of endothelial cells, and induce
angiogenesis. Similarly, perlecan plays an important role in
chondrogenesis and skeletal development, as well as harboring pro- and
anti-angiogenic activities. Thus, a physiological interaction could
also occur in vivo during development and in pathological events, including tissue remodeling and tumor progression.
Perlecan is a modular heparan sulfate proteoglycan harboring five
distinct domains that share homology with laminin, the neural cell
adhesion molecule and the low-density lipoprotein receptor (1-6).
Perlecan is an intrinsic constituent of basement membranes (7) and it
can self-assemble into dimers and oligomers (8). It is expressed quite
early during development being deposited at the two-cell embryo stage
(9) and during acquisition of attachment competence (10). In adulthood,
it is present in all the basement membranes and it is richly deposited
along the vessel walls where, together with other extracellular matrix
molecules, maintains structural integrity in sites of high pressure (1, 11-15). Perlecan has also been detected at the cell surface (16, 17)
where it is likely to bind through a member of the integrin family
(18-20), an adhesion modulated by glycosaminoglycans (21). Perlecan
null mice are embryonic lethal at day 10.5. Surviving mice die shortly
after birth because of vascular and cephalic abnormalities, and display
chondrodysplasia with dyssegmental ossification of the spine (22,
23), which resembles the human dyssegmental dysplasia
Silverman-Handmaker type (24, 25). Notably, perlecan null
animals exhibit a high incidence of malformations of the cardiac
outflow tract with complete transposition of the great vessels (26),
further stressing a central role for perlecan in vasculogenesis.
Perlecan is involved in controlling cell proliferation, thrombosis,
tumorigenesis, and angiogenesis (20, 27-30), but the mechanisms and
pathways regulating these effects are not completely understood. Its
expression is increased in breast and colon carcinomas (27) and
metastatic melanomas (31). The effects exerted by perlecan are thought
to be in part indirect, because of its ability to bind several
molecules via the heparan sulfate chains (32) and the protein core
(33). Perlecan heparan sulfate binds several signaling molecules
including various members of the fibroblast growth factor
(FGF)1 family and the
platelet-derived growth factor (34-36). The binding of FGF2 to
perlecan promotes receptor activation and mitogenesis (37), and
increases blood vessel formation to a higher extent than that induced
by FGF2-heparin complexes (37). Recent studies have shown that perlecan
protein core binds to several extracellular matrix and growth-promoting
proteins including: fibronectin (38), laminin (39), collagen type IV
(17, 36), fibulin-2 (36, 38), ECM1 has been shown to play an important role in endochondral bone
formation by inhibiting alkaline phosphatase activity and bone
mineralization (50), and to promote blood vessel formation in the
chicken chorioallantoic membrane assay (51). ECM1 can be detected in
close association with the embryonic endothelial cells and has been
shown to trigger the proliferation of endothelial cells in
vitro (51). More recently, ECM1 has been
associated to the lipoid proteinosis, an autosomal recessive disorder
characterized by thickening of the skin, mucosae, and certain viscera
(52). Loss of functionality of this ECM1, induced by point
mutations, leads to thickening of the basement membrane at the
dermal-epidermal junction, around blood vessels and adnexal epithelia.
In this paper we discovered a specific binding of the perlecan C
terminus to the corresponding C terminus of ECM1, we identified a novel
splice variant (ECM1c), and demonstrated co-expression and partial
co-distribution of these two molecules in both cells and tissues.
We propose that perlecan/ECM1 interactions could be
physiological and could potentially influence endochondral bone formation, angiogenesis, and tumorigenesis.
Yeast Two-hybrid Library Screening--
To lessen the number of
false positives we employed the MATCHMAKER GAL4 Two-hybrid system 3 (Clontech, Palo Alto, CA), which adopts three
independent reporter genes for the selection, His, Ade, and Analysis of the ECM1c Splice Variant by RT-PCR and Real Time
PCR--
To verify that the ECM1c variant was actually being
expressed, RNA from HT1080 and A431 cancer cell lines was extracted
employing the TRI-Reagent (Sigma). The RNA was treated with RNase-free
DNase for 10 min at 37 °C and reverse transcribed using the avian
myeloblastosis virus reverse transcriptase (Promega) and random
primers. The following oligonucleotides were used in the RT-PCR:
forward 5'-CTCTGAGGGAGGCTTCACG-3' and reverse
5'-CGGGATCCATAGTTGGGGTAAGGAGCC-3'. For the nested PCR the reverse
oligonucleotide 5'-ATCTCCTCCTGATATGGAAGAGGT-3' specific for the splice
variant was used. The PCR products were then analyzed on agarose gels.
Real time PCR was performed on the RNA samples as well. Ten serial 1:1
dilutions of the extracted RNAs were carried out and the samples were
reverse transcribed as before. The following oligonucleotides were
synthesized: forward 5'-ACCAAAGAGTTCTCACCAAGCA-3' and reverse
5'-GCAGTCATGTTGTGGATCAGC-3', common to all three variants, and forward
5'-GCACCCCAATGAACAGAAGGA-3' and reverse
5'-CGGCATGGAATGGATGATTATC-3', specific of the ECM1c variant.
Yeast Two-hybrid Constructs--
Seven deletions of perlecan
domain V were cloned into the pGBKT7 vector. For deletions Co-immunoprecipitation and Western Blotting--
To corroborate
the interaction between ECM1 and perlecan domain V we performed
co-immunoprecipitation studies. The two proteins were in
vitro transcribed and translated in the presence of
[35S]methionine (ICN Pharmaceuticals Inc., Costa Mesa,
CA) employing the TNTTM reticulocyte lysate
system (Promega). One µg of the pGBKT7 or pGADT7 constructs was
employed and the reactions were incubated for 90 min at 30 °C and
performed according to the manufacturer's instructions in a total
volume of 50 µl. An amount of 15 µl of each translated protein was
employed for the co-immunoprecipitation, the proteins were co-incubated
for 1 h at 30 °C and 1 ml of co-immunoprecipitation buffer was
added (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM dithiothreitol, 5 mg/ml aprotinin, 0.5 mM
phenylmethylsulfoniyl fluoride, and 0.1% Tween 20 (v/v)). About 2 µg
of the anti-hemagglutinin antibody (the pGADT7 vector carries the
hemagglutinin tag sequence) or the anti-c-Myc antibody (the pGBKT7
vector carries the c-Myc tag sequence) were added and the samples were
incubated for 18 h at 4 °C with constant rocking. The immune
complexes were captured with protein A/G-agarose beads (Pierce).
The beads were washed three times with HNTG buffer (10 mM
Hepes, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 10%
glycerol, 200 µM Na3VO4, 20 mM NaF), and an EDTA-free protease inhibitor mixture
(Roche Molecular Diagnostics GmbH). The bands were separated in
polyacrylamide gels and, after fixation, enhanced with
AMPLIFYTM (Amersham Biosciences AB). The gels were
then exposed to Eastman Kodak films.
To investigate the interaction between ECM1 and perlecan in mammalian
cells we stably transfected 293 EBNA cells (human embryonic kidney
cells expressing the Epstein-Barr virus nuclear antigen) with 4 µg of
pCEP-Pu vector harboring the full-length domain V as described before
(53). The EBNA cells allow high episomal replication of the transfected
pCEP-Pu vector. Stable clones were obtained by culturing cells in
puromycin (1 µg/ml) for at least 4 weeks. Following verification of
domain V expression by enzyme-linked immunosorbent assay and
microsequencing (53), three domain V-expressing clones were pooled
together and transiently transfected with a full-length ECM1-containing
pcDNA3.1 plasmid (Invitrogen), harboring the BM-40 signal peptide
and a polyhistidine tag at the C terminus, using LipofectAMINE 2000 (Invitrogen). As a further control, we transfected wild type 293-EBNA
cells with the ECM1 construct as above. After 48 h of incubation,
the serum-free conditioned medium was collected and filtered, and a
mixture of protease inhibitors (CompleteTM, Roche Molecular
Biochemicals) was added. Rabbit polyclonal anti-ECM1 antiserum (51) was
added to 1 ml of conditioned medium (1:200 dilution), and the proteins
were immunoprecipitated at 4 °C overnight with continuous rocking.
Sepharose G beads (Amersham Biosciences) were added and incubated for
another 4 h with rocking, and subsequently washed four times
with 20 mM Hepes, pH 7.4, 150 mM NaCl,
0.1% Triton X-100, 10% glycerol, 200 µM
Na3VO4, 10 mM NaF. The final pellet
was boiled in Laemmli buffer and analyzed by SDS-PAGE. Western
immunoblotting was performed using anti-His monoclonal antibodies
(Qiagen), followed by horseradish-conjugated secondary antibody and
chemiluminescence detection as described before (53).
Immunohistochemical Studies--
Frozen sections of human skin
were briefly fixed in acetone, incubated in phosphate-buffered saline
containing 0.3% H2O2 and 1% sodium azide for
10 min, blocked in 5% goat serum for 30 min and incubated with the
primary antibody, either a rabbit polyclonal anti-ECM1 antiserum (51)
or 7B5 mouse monoclonal antibody raised against domain III of human
perlecan (12). Incubations were for either 1 or 18 h and
dilutions, all in the presence of 5% goat serum, ranged between 1:200
and 1:500 for each antibody. The sections were washed again, incubated
with goat anti-rabbit antibodies conjugated with horseradish peroxidase
(Envision, DAKO), and finally developed with diaminobenzidine and
counterstained with hematoxylin. Paraffin sections of skin were also
tested after various antigen retrieval including pretreatment with 1%
protease type XXIV, citrate antigen retrieval (S30), 0.1% Pronase
(Calbiochem) for 20 min each, or without any pretreatments. Peroxidase
blocking, serum blocking, and antibody incubations were the same as
described above for the frozen sections. Images were captured with a
Pixera digital camera and assembled using Adobe Photoshop version
6.0.
ECM1 Is a Novel Protein Interacting with Perlecan Protein
Core--
Only recently the two-hybrid system has been employed to
identify interactions among extracellular matrix proteins (43, 54-56).
To identify new partners for the perlecan protein core, we employed
perlecan domain V as a bait (Fig.
1A) and screened a human
keratinocyte cDNA library. This domain is located at the C-terminal
end of the protein core and harbors a tandem array of three laminin-G
type (LG1-LG3) modules separated by four EGF-like (EG1-EG4) modules,
in an arrangement highly conserved across species (1, 57, 58).
Initially, domain V was cloned into the pGBK-T7 vector and was tested
by itself to ensure that it did not activate the reporter genes
HIS3 and ADE. Next, about 0.5 × 106 cDNAs were screened. To minimize the number of
false positives, the double transfected yeast cells growing in minimal
medium were replated in the same medium containing X- Identification of the ECM1c Splice Variant and Expression
Studies--
Two splice variants of the molecule have been described
so far, ECM1a and ECM1b, generating two transcripts of 1.8 and 1.4 kb,
respectively. The smaller isoform lacks exon 7 (Fig.
3A). Interestingly, both
isoforms are larger in the murine counterpart because of the fact that
the sequence of exon 6 was not found in the human homologue. However,
this exon appears to be conserved within the 5th intron of the human
gene (48). The sequencing of the pACT2 clone of ECM1, isolated from our
library screening, revealed the presence of an extra exon, which we
named exon 5a (Figs. 2 and 3A). The peptide encoded by the
resulting splice variant was highly homologous to the mouse sequence
encoded by the murine exon 6 (Fig. 3A). To corroborate the
presence of this new splice variant, we extracted RNA from two
different cell lines, HT1080 and A431, and amplified an ECM1 fragment
by RT-PCR. A nested PCR, employing an internal oligonucleotide was
necessary to visualize the ECM1c splice variant, indicating that the
expression of this splice form was very low, at least in these two cell
lines (Fig. 3B). As a control no cDNA, derived from the
first amplification, was added to the reaction. To support these
findings and to calculate the relative amounts of this new splice
variant, we performed real time PCR experiments. We used
glyceraldehyde-3-phosphate dehydrogenase and/or
To determine whether perlecan and ECM1 were co-expressed,
we tested by RT-PCR a series of normal and malignant cells including fibrosarcoma cells, endothelial cells, dermal fibroblasts,
keratinocytes, and two osteosarcoma cells. In every case, with the
exception of Saos2 osteosarcoma cells, we found significant
co-expression of both genes (Fig. 3, E and F).
Thus, cells with a diverse histogenetic origin express ECM1 and perlecan.
Analysis of Perlecan/ECM1c Interactions--
To confirm
that ECM1 alone did not trigger the growth of yeast cells, the
construct was subcloned into the pGADT7 vector and used with the
pGBKT7-53 construct (coding for the p53 molecule) to transfect the
AH109 yeast strain. In double minus plates all transfectants grew,
indicating that the cells were successfully transfected. On the
contrary, only the cells transfected with the positive control were
able to grow in minimal media (Fig. 4A), suggesting that the
growth observed in the previous experiment was actually induced by the
interaction of perlecan domain V with the ECM1 molecule. Next, the
interaction was re-tested in this system using the pGADT7-ECM1 and the
pGBKT7-domain V constructs in one-to-one interactions. As a further
control, the molecules were employed as either a prey or bait. Several
clones resulting from the transfection were replated in minimal media
containing X- Interaction of ECM1 and Perlecan Domain V in Cell-free Systems and
in Mammalian Cells--
To corroborate the results obtained with the
yeast two-hybrid system we performed co-immunoprecipitation
experiments. The two proteins were in vitro transcribed and
translated; as a negative control, we used an empty plasmid. The
constructs confer to perlecan domain V a Myc epitope and to ECM1 a
hemagglutinin epitope fused to their respective C termini. The
translated proteins were analyzed on SDS-PAGE and the molecular weight
of the bands corresponded to the predicted sizes of the molecules,
i.e. ~81,000 for perlecan domain V and ~61,000
for the ECM1c construct (Fig.
5A, left panel). The anti-Myc monoclonal antibody was capable of co-precipitating both
proteins (Fig. 5A, right panel). In contrast, the
anti-hemagglutinin monoclonal antibody failed to do so (not
shown), suggesting that the antibody might have interfered
with the protein/protein interaction.
Because both domain V and ECM1 are cysteine-rich glycoproteins with
several disulfide bonded residues, a modification that does not occur
in yeast, we wanted to confirm the interaction using mammalian cells.
We utilized the 293-EBNA cells, human embryonic kidney cells that
harbor the Epstein-Barr virus nuclear antigen, that have been
previously shown to allow the synthesis and proper glycosylation of a
variety of extracellular matrix proteins, including portions of
perlecan (53, 58-62). Following transfection with either ECM1 or
domain V, the predicted bands of ~61 and 81 kDa for the respective
proteins were secreted (Fig. 5B). When the cells were doubly
transfected with ECM1- and domain V-containing vectors, the two
proteins were co-immunoprecipitated by the anti-ECM1 antiserum and
detected by Western immunoblotting with anti-His monoclonal antibody
(Fig. 5C). In contrast, untransfected 293-EBNA cells showed
no reactive bands (Fig. 5D). Collectively, these data
corroborate the results obtained with the yeast two-hybrid system, and
indicate that ECM1 is a novel interacting partner for the perlecan
protein core.
ECM1 Interacts with the EGF-like Repeats of Perlecan Domain
V--
The results of the co-immunoprecipitation studies described
above indicated that the interaction identified with the yeast two-hybrid system was specific. Thus, we wanted to analyze in detail
the precise site of interaction on the perlecan molecule. To this end,
we generated eight deletions of domain V and cloned each one of them
into the pGBKT7 vector (Fig.
6A). The plasmids were
co-transfected with the ECM1 construct into AH109 cells and the cells
were plated on selective media. Four independent clones from each
transfection were replated in minimal media containing X- Perlecan Domain V Binds to the C-terminal Domain of
ECM1--
Next, we determined the specific site of perlecan
interaction within the ECM1 molecule. We used a strategy similar to
that described above. We generated five deletions of ECM1, cloned all of them into the pGADT7 vector and used them to co-transfect cells with
the full-length domain V pGBKT7 construct. All the constructs that
contained the sequence of the C-terminal fourth domain of ECM1 were
able to trigger the growth of the yeast cells and the production of
Partial Expression Overlap between ECM1 and Perlecan in
Skin--
Because both perlecan and ECM1 are widely expressed in human
skin we chose to investigate the potential expression overlap of these
two proteins in this organ. Frozen sections of human skin were reacted
with either a rabbit polyclonal anti-ECM1 antiserum (51) or 7B5 mouse
monoclonal antibody raised against domain III of human perlecan (12).
The anti-ECM1 antibody has been raised against human recombinant ECM1,
and recognizes both ECM1a and ECM1b splice variants. Thus, we can
reasonably assume that this antiserum also recognizes ECM1c, because
this splice variant harbors both ECM1a and ECM1b epitopes. ECM1 was
widely distributed throughout the dermis with minimal expression in the
epidermis (Fig. 8A). It was
diffusely present around small blood vessels (Fig. 8A,
arrows) and hair follicles (Fig. 8B). The
reaction was specific because omission of the primary antibody failed
to produce any signal (Fig. 8C). Perlecan was prominently
expressed within the dermal basement membrane, at the dermal-epidermal
junction and dermal blood vessels (Fig. 8D); however,
perlecan epitopes were also observed as delicate punctuate deposits
throughout the upper dermis. Perlecan expression was also prominent
around hair follicle (Fig. 8E) and, as in the case for the
ECM1 antiserum, omission of the primary antibody failed to generate any
detectable signal (Fig. 8F). No significant signal was
obtained with paraffin-embedded tissue, even after several attempts of
antigen retrieval. Collectively, the immunolocalization studies confirm
the secreted, extracellular nature of ECM1 and further indicate that
perlecan protein core could potentially interact in vivo
with ECM1 within restricted areas of the skin, including perivascular
and perifollicular regions of the dermis, and diffusely within
collagen-rich regions of the upper dermis.
The screening of a human keratinocyte library using perlecan
domain V as bait led to the identification of a novel molecule interacting with the perlecan protein core. The construct of ECM1 consisted of a ~1.6-kb fragment encompassing nucleotides 260-1822 of
the published sequence (48). Interestingly, a sequence within the human
intron 5, which shares high homology with murine exon 6, was not
spliced out in our cDNA. This sequence coded for an additional
19-amino acid residues thereby generating a novel splice variant, which
we termed ECM1c. We performed RT-PCR experiments on different cell
lines and found this isoform to be expressed by two transformed cell
lines, the fibrosarcoma HT1080 and the squamous cell carcinoma A431. To
determine whether perlecan and ECM1 were co-expressed, we tested by
RT-PCR a series of normal and malignant cells including fibrosarcoma
cells, endothelial cells, dermal fibroblasts, keratinocytes, and two
osteosarcoma cells. In all the cases, with the exception of Saos2
osteosarcoma cells, we found significant co-expression of both genes,
indicating that ECM1 and perlecan are co-expressed by cells with a
diverse histogenetic origin. It should be pointed out, however, that
other researchers found ECM1 to be expressed by Saos2 osteosarcoma
cells (48).
The interaction between ECM1 and perlecan domain V was validated by
co-immunoprecipitation studies, using both cell-free systems and
mammalian cells. The latter experiments assure proper disulfide bonding
and glycosylation of the secreted glycoproteins. Using a battery of
deletion mutants we then demonstrated that the major sites of
interaction within perlecan domain V reside in the EGF-like repeats
connecting the LG subdomains of the molecule, which did not directly
participate in the binding. These results clearly show that both the
EGF-like tandem repeats are directly involved in the interaction within
ECM1, and further suggest that more than one ECM1 molecule might bind
to the perlecan protein core insofar as the binding region is quite
large. It is not yet known if the binding to ECM1 can alter or
influence the binding of other molecules that interact with this domain
such as nidogen-1, fibulin-2, heparin, The binding site on the ECM1 molecule was identified in the C-terminal
cysteine-rich repeat. The arrangement of the cysteine residues in this
molecule is noteworthy following the
CC-(X7-10)-C pattern found also in the serum
albumin family of proteins (47, 49). This peculiar arrangement of
cysteine residues is responsible for the formation of double-loop
domains, which may play important roles in protein/protein interactions
(47, 63). ECM1 contains three double-loop domains, one present in each
of the two central tandem repeats and one in the C-terminal domain.
Notably, only the C-terminal domain of ECM1 was found to interact with
the perlecan molecule, indicating that the double-loop domains may have
different affinity for different ligands. The three cysteine-rich
domains present in this molecule are the most conserved among species, the human being 75% identical to the mouse, whereas the overall identity between the two species reached 67%. This indicates that this
unique pattern is fundamental in maintaining the structure of the
molecule and probably indispensable for the interaction with other
extracellular molecules and in particular with perlecan.
Interestingly, loss of function of ECM1 in humans is
responsible for lipoid proteinosis (52), an autosomal recessive disease characterized by hoarse voice in early infancy because of thickening of
the vocal cords, followed by chicken pox-like scars and thickening of
the skin and mucous membranes. Several point mutations have been
described, which disrupt the molecule. Because most of the point
mutations have been found in exon 7, leading to a truncated molecule
lacking the C-terminal repeat, it is possible that the disrupted
interaction with perlecan might be responsible for some of the
pathological changes observed in this disorder. Notably, the major
changes in lipoid proteinosis occur in the dermis and subcutis, with
basement membrane thickening at the dermal-epidermal junction, around
blood vessels and adnexal epithelia (52). These changes are in
agreement with our immunohistochemical data and further stress a
physiologically relevant interaction between ECM1 and perlecan protein core.
ECM1 is also involved in the regulation of endochondral bone formation
(50). The studies conducted so far have been performed in
vitro employing 15-day-old fetal mouse metatarsal bones consisting of undifferentiated cartilage. The explants undergo sequential steps of
endochondral bone formation in a 5-day culture period. ECM1 is
expressed in the connective tissues surrounding the developing bones
and is thought to affect mineralization by regulating the alkaline
phosphatase activity, a marker of chondrocyte differentiation. Similarly, both in vitro (64, 65) and gene targeting studies (22, 23) have shown that perlecan affects chondrogenesis and endochondral ossification. Perlecan null mice develop both exencephaly and chondrodysplasia with severe disorganization of the columnar structures of the chondrocytes and defective endochondral ossification (24, 25). Part of the effects observed in perlecan null mice have been
ascribed to extracellular matrix disorganization. Because the
disruption of these two molecules leads to similar effects, it is
possible that the interaction of perlecan with ECM1 is responsible, at
least in part, in coordinating chondrocyte differentiation and bone development.
Notably, ECM1 is associated with blood vessels similarly to perlecan,
and its expression overlaps with Flk-1, one of vascular EGF receptors
(51). Recombinant ECM1 can stimulate the proliferation of endothelial
cells and can induce the formation of vessels in chorioallantoic
membrane assays (51). Perlecan is expressed quite early during
development in tissues of vasculogenesis (11, 13), although the precise
mechanism by which perlecan influences the formation of blood vessels
is not completely understood. Some of these effects are thought to be
indirect, mediated by the ability of the heparan sulfate side chains to
bind several growth factors and cytokines (66-68). Perlecan binds
FGF2, thereby promoting receptor activation and mitogenesis (34, 35).
Experiments conducted using a rabbit ear model, showed that
perlecan-FGF2 complexes induce the formation of blood vessels in much
higher levels than heparin-FGF2 complexes (37). We have recently
discovered that perlecan domain V is a potent anti-angiogenic factor
and have named it endorepellin to signify its anti-vascular function
(53). Several proteins or degradation products of the extracellular matrix have been shown to control blood vessel formation in a positive
or negative way (69-72). Thus, one possibility is that processed forms
of matrix proteins may regulate each others' activity. The interaction
of domain V/endorepellin and ECM1 around blood vessels could lead to a
fine regulation of blood vessel formation and maintenance. Moreover,
the influence on angiogenesis exerted by these two molecules may be
important in many pathological events such as wound repair (73),
diabetic retinopathy, rheumatoid arthritis, as well as tumor growth and
metastasis (74, 75), all of which are largely dependent on the
formation of new blood vessels.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dystroglycan (40),
platelet-derived growth factor (41), FGF7 (42), and FGF-binding protein
(43). A similar distribution in a number of tissues and some aspects of
the functionality are shared by extracellular matrix protein 1 (ECM1),
a secreted glycoprotein containing six cysteine doublets, with a
CC-(X7-10)-C pattern (44). This cysteine
arrangement, which is also found in serum albumin and the sea urchin
Endo16 protein (45, 46), leads to the formation of double-loop
structures that are involved in protein/protein interactions (45, 47).
Two splice variants have been described so far: ECM1a, encoded in
humans by a 1.8-kb mRNA, and ECM1b, encoded by a 1.4-kb mRNA
lacking exon 7 (48). ECM1a is expressed by the highly vascularized
heart and placenta. It is also present in liver, ovary, kidney, lung,
pancreas, testis, muscle, and colon, while ECM1b expression is confined
to skin and tonsils (48, 49).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- or
-galactosidase. A keratinocyte library
(Clontech) constructed in the pACT2 vector was
amplified and the plasmids were extracted employing the NucleoBond AX
Giga plasmid purification kit (Clontech). Perlecan
domain V was subcloned into the pGBKT7 vector from a previous construct
available in our laboratory (42) using EcoRI and
BamHI restriction sites and the construct was used as a bait
to screen the keratinocyte library (complexity ~0.5 × 106). The transfected yeast cells were plated onto
quadruple minus plates lacking Trp and Leu to select for the presence
of both plasmids, and His and Ade that are selective for the putative interactions. The yeast cell clones growing in selective medium were
re-plated in quadruple minus plates containing X-
-gal. The plasmids from the blue colonies were extracted using the Yeast Plasmid
Isolation Kit (Bio 101, Carlsbad, CA) and employed to transfect DH5
cells. The pACT2 clones only would grow on ampicillin LB plates. The
plasmids were extracted and the inserts were analyzed on agarose gels
after restriction digestion with BglII. The inserts were
identified by automatic sequencing as described before (43). For the
-galactosidase assays, 100-µl aliquots of a X-
-gal
solution (20 mg/ml) in N,N-dimethylformamide were
spread in 10-cm quadruple minus plates. The yeast clones transfected
with putative interactive molecules would turn blue (the
-galactosidase is a secreted enzyme unlike the
-galactosidase).
For
-galactosidase assays, the grown yeast colonies were transferred
onto Whatman 3MM paper filters, soaked in Z buffer/X-gal solution (0.1 M Na2HPO4, 45 mM
NaH2PO4, 10 mM KCl, 2 mM MgSO4, 0.3%
-mercaptoethanol, and 3.3 mg/ml 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside), frozen in liquid nitrogen for 10 s and incubated at 30 °C for ~8 h to visualize the blue colonies.
-Actin or glyceraldehyde-3-phosphate dehydrogenase were used as a
control. The experiments were performed using the
QuantiTectTM SYBR Green PCR kit (Qiagen) and the PCR was
performed using an ABI PRISMTM 7700 Sequence Detector
(Applied Biosystems, Forster City, CA). The real time PCRs were run in
2% agarose gels to confirm the specificity of the fluorescent
detection system. The relative amounts of ECM1c were identified using
the comparative method (ABI PRISM 7700 sequence bulletin 2) employing
the formula 2
CT, where the
CT
was determined by subtracting the average
-actin
CT (threshold cycle) value from the average ECM1c CT value. The
CT value was finally
calculated by subtracting the
CT calibration value
(
CT of all the ECM1 splice variants). A validation
experiment was performed prior to the calculations and the absolute
value of the slope of the log input amounts of RNA used
versus
CT values was found to be <0.1. For
the comparative expression of ECM1 and perlecan in different cell lines
the following oligonucleotides were used: forward
5'-GGATACCCTTGACAAATACTGTG-3' and reverse 5'-ATTCTTCCTTGGGCTCAGA-3',
forward 5'-GCGGGTGAATGGTGGAC-3' and reverse 5'-AGGGAAGGCGAGGAAGC-3',
respectively. RNA was extracted from the different cell lines and
reverse transcribed as described previously. The amplified products
were separated onto 1% agarose gels.
1,
2,
and
3, the following forward primer was used:
5'-GGAATTCGAGATCAAGATCACCTTC-3'. The reverse oligonucleotides for these deletions were:
1
5'-CGGGATCCATGTCCAGAGCCTTGTTG-3',
2
5'-CGGGATCCTGTCACACCTTCCTCACA-3', and
3
5'-CGGGATCCGGTGGGGCAGTGGGAGAT-3'. The forward oligonucleotide for
deletion
4 was 5'-GGAATTCTGTCGGGACCGGCCCTGC-3' and the reverse
primer corresponded to the one used for deletion
2. For
deletion
5 the forward oligonucleotide was
5'-GGAATTCGTGACCACCCCCTCGCTG-3' and the reverse primer
corresponded to the one used for deletion
1. The reverse primer for
deletions
6 and
7 was 5'-CGGGATCCCGAGGGGCAGGGGCGTGT-3', and the
normal nucleotides were 5'-GGAATTCTGTGAGCGCCAGCCTTGC-3' and
5'-GGAATTCGGCATAGCAGAGTCCGAC-3', respectively. In all the forward
nucleotides an EcoRI site was introduced and a
BamHI site introduced in the reverse primers. The ECM1
construct was subcloned into the pGADT7 and pGBKT7 vectors by PCR using
the pACT2 construct as a template. The forward primer was
5'-GGAATTCCCCGAAGCCTCCCCATGGAT-3', which introduced an
EcoRI site, and the reverse primer was
5'-CGGGATCCCCAAATCCAAGAGGTGTTTAGG-3', which introduced a
BamHI site. The forward primers used to amplify the deletion
fragments of ECM1 were 5'-GGAATTCGGAACGCCAGCTCCATTT-3', 5'-GGAATTCTGGGAGGAAGCAATGAGC-3', 5'-GGAATTCTGGGAGGATACCCTTGACAAA-3', 5'-GGAATTCGACCGGGACATCTTGACCAT-3' for deletions
E1,
E2,
E3,
E4, respectively. For deletion
E5 the same oligonucleotide
used to amplify the entire ECM1 fragment was employed and the reverse primer was 5'-CGGGATCCATAGTTGGGGTAAGGAGCC-3'. For all the PCR amplifications the GC-Rich Taq polymerase (Roche Molecular
Diagnostics GmbH) was used.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-gal. The
screening led to the isolation of ~100 blue colonies. The inserts,
ranging between 0.5 and 4 kb, were analyzed either by PCR using
specific primers or by restriction digestion. All inserts were
sequenced and identified by homology searching with the NCBI BLAST
program. More than 10% of the sequenced clones encoded secreted
proteins including several extracellular matrix proteins, likely to
bind perlecan. The rest of the cDNAs were false positives, being
intracellular proteins such as keratins, highly expressed by
keratinocytes, and transcription factors, which can independently
activate the reporter genes. One of the fastest growing clones
contained a ~1.6-kb cDNA insert (Fig. 1B) spanning
between nucleotides 260 and 1822 of the ECM1 (Fig.
2). The cDNA isolated with the
screening encoded for 487 amino acid residues beginning at residue 53 to the very end of the published sequence (48). ECM1 is a secreted glycoprotein first isolated from an osteogenic mouse cell line in
concomitance with studies on bone matrix biology (49). ECM1 contains a
19-amino acid residue signal peptide and it has been divided into four
distinct domains (Figs. 1C and 2). The first is a
cysteine-free domain followed by the second and third repeat and by the
C-terminal domain that contain the CC-(X7-10)-C arrangement of cysteine residues, typically observed in members of the
albumin protein family. This pattern generates double-loop structures
that are believed to be involved in protein/protein interactions. The
ratio of acidic and basic amino acids is balanced; furthermore, the
different double loops may have different binding affinities increasing
the potential of interaction with a variety of biological ligands
(47).
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Fig. 1.
ECM1 is a novel interacting partner for the
perlecan protein core. A, schematic
representation of the C-terminal perlecan domain V used as bait in the
yeast two-hybrid screening. This domain consists of three laminin-G
type (LG1-LG3) modules (orange ovals) separated by four
EGF-like (EG1-EG4) modules (blue rectangles), in an
arrangement highly conserved across species (1, 57, 58). B,
agarose gel electrophoresis showing the isolation of the ~1.6-kb
insert (ECM1) from the pACT-2 cDNA clone that interacted with
perlecan domain V in the yeast two-hybrid screening. The construct was
digested with the restriction enzyme BglII and the bands
were separated into a 1% agarose gel. The migration of the markers in
kb is indicated in the left margin. C, schematic
representation of ECM1 and its three splice variants, with ECM1c being
the novel splice variants described in this paper (see also Fig. 2).
The different domains are color-coded and the key for each domain is
provided in the right box. The numbers indicate
the amino acid residues. Potential glycosylation sites are
indicated.
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Fig. 2.
ECM1c sequence of the pACT-2 clone.
Nucleotide and deduced amino acid sequence of human ECM1c (see
NCBI accession number NM_004425.2). Vertical blue arrows
indicate the beginning and end of the pACT-2 clone interacting with
perlecan domain V that was identified during the library screening. The
sequence of the novel exon 5a is highlighted in yellow. The
horizontal arrows indicate the sequences of the
oligonucleotides used in the real time PCR experiments (see Fig. 3).
Potential glycosylation sites are underlined.
-actin as a control.
A representative example of an amplification plot is shown in Fig.
3C. The reduced normalized fluorescence values
(
Rn) are plotted against the cycle value. The threshold
cycle (CT) value, defined as the fractional cycle number at
which the fluorescence passes the fixed threshold, is indicated in the
plot by a bold line. The amplicons were run in 2% agarose
gels and showed the bands of the expected size (Fig. 3D),
indicating that the fluorescent readings were specific. Comparative analysis of the CT (see "Experimental Procedures")
indicated that the amount of ECM1c RNA was ~15% of the total amount
of ECM1 RNA in both cell lines. These data confirm the existence of a
new ECM1c splice variant, which is expressed, albeit at low levels, in
both cell lines. The level of expression between the two cell lines was
comparable.
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Fig. 3.
ECM1c is a new splice variant.
A, schematic representation of the ECM1 genomic
organization. Boxes represent exons, in scale, and
thin lines represent introns (not in scale). The new exon,
named exon 5a, is in red, whereas exon 7, which is spliced
out in ECM1b, is in blue. The 5'- and 3'-untranslated
regions are in yellow. The lower rectangle
contains the alignment of the two peptides coded by the human exon 5a
and the corresponding murine exon 6. Identical and conserved amino acid
residues are highlighted in green and cyan,
respectively. The amino acid residues present only in the human
counterpart are highlighted in yellow. B, agarose
gel electrophoresis showing the nested PCR bands amplified using
specific ECM1c oligonucleotides for the ECM1c splice variant. An RT-PCR
was performed employing specific ECM1 oligonucleotides and RNA from the
A431 cell line (lane 2) or the HT1080 cell line (lane
3), and subsequently re-amplified. As a control, the first
amplification was not added to the sample (lane 4).
Lane 1, molecular markers. C, semi-log
amplification plot showing the change in fluorescence of the SYBR Green
I dye plotted versus cycle number; the bold line
represents the threshold from which the CT values are
deducted. The code for each curve is explained in the right
margin. D, photograph of a 2% agarose gel
showing the amplicons obtained with the real time PCR. Lane
1, molecular markers. The same cell lines were used in this
experiment, A431 (lanes 2-4) and HT1080 (lanes
6-8). The total amount of ECM1 RNA (lanes 3 and
7) was compared with the amount of ECM1c (lanes 4 and 8). -Actin was used as a positive control in each
experiment (lanes 2 and 6). As a negative
control, no cDNA was added to the reaction (lanes 5 and
9). E, agarose gel electrophoresis showing the
expression of perlecan (upper panel) and ECM1 (lower
panel) in different human cells using RT-PCR, including HT1080
fibrosarcoma cells (lane 2), umbilical vein endothelial
cells (lane 3), dermal fibroblasts (lane 4),
Saos2 osteosarcoma cells (lane 5), and MG63 osteosarcoma
cells (lane 6). Molecular weight markers, lane 1;
negative control, lane 7. F, agarose gel
electrophoresis showing the expression of perlecan (upper
panel) and ECM1 (lower panel) in keratinocytes
(lane 2), dermal fibroblasts (lane 3), and
negative control (lane 4). Molecular weight markers are
shown in lane 1. The identity of each amplified band was
confirmed by automatic sequencing.
-gal. The growth of the blue colonies and the intensity
of the blue color were comparable with the positive control (Fig. 4,
B-D).
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Fig. 4.
Specific interaction between perlecan domain
V and ECM1. A, ECM1 alone could not trigger the growth
of the transfected yeast cells in minimal media. The pGBKT7-Lam
construct (containing the lamin c cDNA) was used as a partner for
the interaction. All clones were able to grow in double minus media,
which select for the presence of both plasmids (boxes
1-3). Box 1, positive control
(pGBKT7-53/pGADT7-T); box 2, negative control
(pGBKT7-Lam/pGADT7-T); box 3, pGADT7-ECM1/pGBKT7-Lam. The
same transfectants were plated on quadruple minus media to check for
putative interactions. Growth was observed with the positive control
only (box 4); no growth was detected with the negative
control (box 5) or with the ECM1 construct (box
6). B, specificity of the interaction was corroborated using
the pGADT7 and pGBKT7 plasmids in which the ECM1c cDNA was
subcloned. Growth was observed using ECM1 both as a "bait" and
"prey" and was comparable with that of the positive control
(pGADT7/pGBKT7-53). As a negative control the lamin c construct was
used in combination with the ECM1 construct. The name of the respective
constructs is indicated in the margins. C and
D, -galactosidase assays performed on six independent
clones using ECM1 as prey or bait, respectively. The
pGBKT7-53/pGADT7-T and pGBKT7-Lam/pGADT7-T constructs were used as a
positive (+) and negative (
) control, respectively.
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Fig. 5.
Corroboration of perlecan domain V/ECM1
interaction. A, autoradiograph of a 10% SDS-PAGE
(left panel) showing the in vitro transcribed and
translated domain V of perlecan (~81 kDa) and ECM1 (~61 kDa),
respectively, as indicated on the top margin. The two
molecules were in vitro transcribed and translated using the
pGBKT7-DV and pGADT7-ECM1 constructs and [35S]methionine
as the labeled precursor. The right panel shows the
co-immunoprecipitation of equimolar amounts of domain V and ECM1 using
the anti-Myc antibody (the sequence for the Myc epitope is present in
the pGBKT7 vector). In contrast no band could be detected when the
primary antibody was omitted in the incubation medium
(control). B, Western immunoblotting
(WB) of media conditioned by 293-EBNA cells transfected with
either ECM1 or domain V as indicated. Immunodetection was performed
with anti-histidine ( His) antibody. The predicted bands
of ~61 and 81 kDa for the respective proteins were detected.
C, Western immunoblotting of media conditioned by 293-EBNA
cells doubly transfected with ECM1- and domain V-containing vectors,
following co-immunoprecipitation with the anti-ECM1 antiserum and
detection with anti-His monoclonal antibody. The two lanes include
media conditioned by the doubly transfected 293-EBNA cells in the
absence or presence of fetal bovine serum, respectively. D,
same experiment as in C but without co-transfection.
-gal. Only
three deletions (
3,
7, and
8) failed to induce growth of the
yeast cells (Fig. 6A). The other deletions induced growth
and blue color of the yeast cells in a manner comparable with the
positive control (Fig. 6B). These results clearly indicate that the sites of interaction reside within the EGF-like repeats flanking the LG2 domain, and further indicate that each individual LG
module of perlecan is dispensable for the interaction.
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Fig. 6.
The sites of interaction with the ECM1
molecule reside within the EGF-like repeats of perlecan domain V. A, schematic representation of the full-length perlecan
domain V and eight deletions, cloned into the pGBKT7 vector. The
full-length pGADT7-ECM1 construct was used as prey. The name
of the constructs, the number of the amino acid residues,
and the semi-quantitative estimation of growth in selective media are
indicated. B, -galactosidase assays conducted on the
perlecan domain V constructs. Four independent clones of each
transfection were plated on quadruple minus plates containing
X-
-gal. The pGBKT7- 53/pGADT7-T and the pGBKT7-Lam/pGADT7-T
plasmids were used as positive and negative controls, respectively. The
results clearly indicate that the ECM1 molecule has affinity for the
EGF-like tandem repeats of perlecan domain V.
-galactosidase (Fig. 7,
A and B), indicating that the interaction with
perlecan domain V resides in this region. In fact, with deletion
E5,
which lacks the terminal 135 amino acid residues (i.e. the
fourth domain of ECM1), no growth of the transfected cells was observed
(Fig. 7A). Thus, we conclude that the two C termini of
perlecan and ECM1 are specifically interacting with each other.
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Fig. 7.
Perlecan domain V binds to the C-terminal
domain of ECM1. A, schematic representation of the
full-length ECM1 and its five deletion mutants cloned into the pGBKT7
vector. The full-length pGADT7-domain V construct was used as prey. The
name of the constructs, the amino acid residue number, and the
semiquantitative estimation of growth in selective media are indicated.
B, -galactosidase assays conducted on four
independent clones for each transfectant. The pGBKT7-53/pGADT7-T and
pGBKT7-Lam/pGADT7-T plasmids were used as positive and negative
controls, respectively. For this experiment the transfected yeast cells
were plated on quadruple minus plates containing X-
-gal. No growth
or
-galactosidase expression was observed employing the ECM1
deletion
E5, indicating that the site of interaction resides in the
C-terminal ECM1 domain.
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Fig. 8.
Partial expression overlap between perlecan
and ECM1. Frozen sections of human skin were reacted with either a
rabbit polyclonal anti-ECM1 antiserum (51) (A and
B) or 7B5 mouse monoclonal antibody raised against domain
III of human perlecan (12) (D and E). Notice that
ECM1 is widely distributed throughout the dermis with minimal
expression in the epidermis, and is present around dermal blood vessels
(arrows) (A). ECM1 is also diffusely expressed
around hair follicles (B). The reaction is antibody specific
because removal of the primary antibody fails to produce any detectable
signal (C). Notice that perlecan is expressed at the dermal
epidermal junction and within dermal blood vessels (D);
however, perlecan is also present as delicate punctuate deposits
throughout the upper dermis (D), and more intensely around
hair follicles (E). Removal of the primary antibody failed
to generate any detectable signal (F). Primary antibody
dilution was 1:500 for both the anti-ECM1 and anti-perlecan.
Scale bars = 160 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-integrin, and
-dystroglycan (36).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. J. Ni, Human Genome Sciences, for providing the anti-ECM1 antibody and Shelly Campbell and Marilyn Skelly for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants RO1 CA47282 and RO1 CA39481 (to R. V. I.), United States Department of the Army Grants DAMD17-00-1-0663 and DAMD17-00-1-0425 (to R. V. I.), and a fellowship from the American-Italian Cancer Foundation, New York (to M. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to the results of this work.
** To whom correspondence should be addressed: Dept. of Pathology, Anatomy and Cell Biology, Rm. 249 Jefferson Alumni Hall, Thomas Jefferson University, 1020 Locust St., Philadelphia, PA 19107. E-mail: iozzo@lac.jci.tju.edu.
Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.M210529200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
FGF, fibroblast
growth factor;
ECM1, extracellular matrix protein 1;
LG, laminin-G like
module;
EGF, epidermal growth factor;
RT, reverse transcriptase;
X--gal, 5-bromo- 4-chloro-3-indolyl-
-D-galactopyranoside.
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REFERENCES |
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