From the Department of Pathology, Anatomy and Cell
Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, the
Division of Cell, Molecular and Developmental Biology,
American Type Culture Collection, Manassas, Virginia 20110, and the
** Cellular Biology and Signaling Program, Kimmel Cancer Center, Thomas
Jefferson University, Philadelphia, Pennsylvania 19107
Received for publication, December 20, 2000
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ABSTRACT |
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Perlecan, a widespread heparan sulfate
proteoglycan, functions as a bioactive reservoir for growth factors by
stabilizing them against misfolding or proteolysis. These factors,
chiefly members of the fibroblast growth factor (FGF) gene family, are coupled to the N-terminal heparan sulfate chains, which augment high
affinity binding and receptor activation. However, rather little is
known about biological partners of the protein core. The major goal of
this study was to identify novel proteins that interact with the
protein core of perlecan. Using the yeast two-hybrid system and domain
III of perlecan as bait, we screened ~0.5 106
cDNA clones from a keratinocyte library and identified a
strongly interactive clone. This cDNA corresponded to
FGF-binding protein (FGF-BP), a secreted protein previously shown to
bind acidic and basic FGF and to modulate their activities. Using a
panel of deletion mutants, FGF-BP binding was localized to the second
EGF repeat of domain III, a region very close to the binding site for
FGF7. FGF-BP could be coimmunoprecipitated with an antibody against perlecan and bound in solution to recombinant domain III-alkaline phosphatase fusion protein. Immunohistochemical analyses revealed colocalization of FGF-BP and perlecan in the pericellular stroma of
various squamous cell carcinomas suggesting a potential in vivo interaction. Thus, FGF-BP should be considered a novel
biological ligand for perlecan, an interaction that could influence
cancer growth and tissue remodeling.
Heparan sulfate proteoglycans are emerging as key molecules
governing crucial events in embryonic development, pattern formation, inflammation, wound repair, and cancer (1-5). Perlecan is a major heparan sulfate proteoglycan of basement membranes (6), which is
expressed in virtually all vascularized tissues (7). As demonstrated by
sequence analysis (7-11), perlecan has a complex multidomain structure
based on seven protein modules arranged in five distinct domains (12).
The modules harbor protein motifs with similarities to proteins
involved in nutrient uptake, cell growth, adhesion, and signaling (13,
14). Functional pleiotropism is inferred from its complex structure and
broad expression (15). Perlecan can self-aggregate into dimeric or
multimeric forms when incubated under neutral isotonic conditions (16)
and is involved in heterotypic interactions with various macromolecules
including laminin (17-19), nidogen (20), fibronectin (12, 21, 22), fibulin-2 (12), collagen type IV (12, 23), During mammalian development, its expression is detected quite early in
tissues of vasculogenesis, being deposited along nearly all the
endothelial-lined vascular beds (26, 27). Perlecan is present not only
in the basement membranes but also within extracellular matrices (13,
28) and in close proximity to cell surfaces (29), where its binding is
likely mediated by members of the integrin family (30-32). Targeted
disruption of the perlecan gene causes embryonic lethality at day 10.5 with widespread cephalic and skeletal abnormalities (33, 34). Notably, the basement membranes are normally formed in the homozygous null animals, but vascular and cephalic abnormalities are generated in areas
of increased pressure, suggesting that perlecan is required for
maintaining basement membrane integrity (35). The abnormal cartilage
structure and the disregulated endochondral ossification of the
perlecan null animals are suggestive of a phenotype encountered with
activating mutations of the fibroblast growth factor
(FGF)1 receptor 3, thereby
positioning perlecan as a negative regulator of this signaling pathway
(33).
Perlecan affects cell proliferation, tumor invasion, angiogenesis, and
thrombosis (13, 36-38), but the pathways by which it is able to
influence such important events are not completely understood.
Increased perlecan deposition is observed in breast and colon (13)
carcinomas as well as in metastatic melanomas (39), and these changes
correlate with enhanced metastatic potential (40). Perlecan functions
as a ligand reservoir for angiogenic growth factors that become
stabilized against misfolding or proteolysis (41, 42). For example,
perlecan binds FGF2 (43, 44) and promotes receptor activation and
mitogenesis (45). In a rabbit ear model of angiogenesis,
perlecan-FGF2 complexes induce blood vessel formation at levels
higher than those induced by heparin-FGF2 complexes (45). FGF2 binds to
the heparan sulfate chains of perlecan, and its displacement by various
proteolytic enzymes offers a plausible physiological mechanism whereby
a powerful angiogenic stimulus becomes operational at the site of
active tumor invasion (42). Thus, we hypothesized that perlecan
deposition in the newly formed tumor stroma may act as a scaffold on
which capillaries proliferate to generate new vascular anastomoses
(14). Suppression of perlecan expression blocks autocrine and paracrine activities of FGF2 in human melanoma cells (46) and halts melanoma cell
proliferation and invasion (47). In fibrosarcoma cells, however,
perlecan appears to act as a negative regulator of growth and invasion
(48), suggesting that the specific cellular context may play a cardinal
role in perlecan's biological function. We have recently discovered
(49) that antisense targeting of the perlecan gene correlates with a
reduced colon carcinoma cell growth and a markedly attenuated
responsiveness to mitogenic FGF7. FGF7 binds specifically to domains
III and V of perlecan (50), and exogenous perlecan efficiently
reconstitutes FGF7 mitogenic activity in perlecan-deficient cells
(49).
The major goal of this study was to identify novel proteins that
interact with the protein core of perlecan. Using the yeast two-hybrid
system and domain III of perlecan as bait, we identified a
strongly interactive clone that corresponded to HBp17 (51), also
known as FGF-BP (52), a protein previously shown to modulate the
activity of FGF1 and FGF2 to enhance the tumorigenicity of A431
squamous carcinoma cells (51) and to act as a potent angiogenic stimulus (52). FGF-BP bound specifically to domain III within the
second EGF repeat, a region very close to the major binding site for
FGF7 (50). FGF-BP could be coimmunoprecipitated with an antibody
against perlecan and bound in solution to recombinant domain
III-alkaline phosphatase (AP) fusion protein. Immunohistochemical studies revealed a significant up-regulation of FGF-BP in various squamous cell carcinomas with a distribution similar to that of perlecan, suggesting a potential in vivo interaction.
Therefore, FGF-BP should be considered a novel biological ligand for
perlecan, an interaction that could influence cancer growth and tissue remodeling.
Materials and Cell Cultures--
Media and fetal bovine serum
were obtained from Hyclone Laboratories (Logan, UT). 125I
and Hybond ECL membranes were purchased from Amersham. Monoclonal antibodies 7B5 against perlecan domain III (15) or monoclonal C9
against human FGF-BP (51) have been previously described.
Yeast Two-hybrid Library Screening--
The Matchmaker
two-hybrid system (CLONTECH Laboratories, Inc.,
Palo Alto, CA) was used to screen a human keratinocyte cDNA library
constructed in pGAD10 (complexity ~5 × 106
recombinants) with perlecan domain III. The full-length domain III and
the deletion constructs were cloned into pGBT9 and pGAD424 (50), and
further deletion fragments were generated by endonuclease digestion of
the constructs. Competent HF7c cells were prepared and combined with 50 µg each of the library DNA and the domain III construct. The mixture
was incubated at 30 °C for 30 min with shaking (200-250 rpm) and
then heat-shocked for 15 min at 42 °C and incubated at 30 °C for
30 min in the presence of 1 ml of Trp Yeast Two-hybrid One-on-one Interactions--
FGF-BP was cloned
in the yeast two-hybrid plasmids by PCR amplification, digestion of the
PCR product with EcoRI/SalI, and ligation into
EcoRI/SalI-digested pGBT9 and pGAD424 plasmids. Both constructs were analyzed by DNA sequencing. Transformation of the
yeast reporter strain SFY526 with all the combinations of hybrid
constructs, using FGF-BP and perlecan domain III as both prey and bait
and utilizing the various deletion constructs of domain III to
specifically map the site of interaction, was performed as described
previously (50). The transfected cells were plated in
Trp In Vitro Transcription/Translation--
The TNT T7 Quick-coupled
Transcription/Translation System (Promega) was used for in
vitro transcription and translation of FGF-BP and the positive
luciferase control. FGF-BP was subcloned from pGAD424 into pcDNA3.1
downstream from the T7 promoter by EcoRI/SalI
digestion followed by ligation into an
EcoRI/XhoI-digested pCDNA3.1. One µg of the
plasmid DNA, 40 µl of the TNT Quick Master Mix, and 20 µCi of
[35S]methionine were combined and incubated at 30 °C
for 90 min. A 5-µl aliquot was resuspended in SDS-sample buffer and
loaded on a 6-12% SDS-PAGE. The gel was incubated with fixing
solution (40% methanol, 10% glacial acetic acid) for 1 h, dried,
and exposed on Kodak X-Omat AR film for 6-15 h at room temperature.
Generation of A431 Cells Expressing Domain III-AP Fusion Protein
and Co-immunoprecipitation Studies--
Perlecan domain III was
subcloned by PCR (ExpandTM Template PCR kit, Roche
Molecular Biochemicals) using the oligonucleotides 5'-CGCAATTGCCCTGCCCTGACGGCC-3' and
5'-CGGGATCCAATTGTGGGGCTTGGTTTGTCTC-3', which introduced an
MfeI site. The purified digested fragment was ligated into
an engineered pcDNA3.1 vector linearized with EcoRI
containing the sequence of the human placental AP preceded by the BM-40
signal peptide. The construct was sequenced, and 10 µg of the
purified plasmid were used to transfect A431 cells by electroporation
(375 V, 490 microfarads, 1 ms). Cells were cultured in G418 (400 µg/ml), and clones were isolated by ring cloning and expanded.
Conditioned media from confluent cells were collected after 2 days and
assayed for AP enzymatic activity using a SEAP (secreted
alkaline phospatase) Chemiluminescence Detection Kit
(CLONTECH). The expression of the recombinant RNA
was analyzed by Northern blotting and the chimeric protein detected in
Western immunoblotting employing a specific antibody directed against domain III. For immunoprecipitation, aliquots of the media containing domain III-AP fusion proteins were brought to 50 mM HEPES,
pH 7.4, 150 mM NaCl, 5 mM EGTA, 10% glycerol,
1% Triton X-100, 100 mM NaF, 200 µM
Na3VO4, 10 mM sodium pyrophosphate,
and a mixture of protease inhibitors (CompleteTM, Roche
Molecular Biochemicals). Following a 4-h incubation at 4 °C,
1 µg of monoclonal antibodies against domain III or FGF-BP was added
and incubated overnight at 4 °C. Sepharose-A/G beads (1:1 v/v) were
added and incubated for 1 h with shaking, centrifuged at
10,000 × g for 10 min, and washed three times with 20 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Triton
X-100, 10% glycerol, 200 µM
Na3VO4, 10 mM NaF, and protease
inhibitors. The final pellet was boiled in Laemli buffer and analyzed
by SDS-PAGE. Additional details are provided in the text and and the
figure legends.
In Vivo Interaction of Human Perlecan Domain III and FGF-BP in
Transiently Transfected COS-1 Cells--
Subconfluent cultures
of COS-1 cells were transfected using LipofectAMINE Plus reagent (Life
Technologies, Inc.) with 4 µg of perlecan domain III/AP and FGF-BP,
both cloned into pcDNA3.1 (Invitrogen) containing the sequence of
the BM-40 signal peptide. After 72 h of incubation, the serum-free
conditioned medium was collected and filtered, and various protease
inhibitors were added (see above). Two µg of either anti-domain III
or anti-FGF-BP antibody were added to 1 ml of medium of transfected and
untransfected COS-1 cells, and the proteins were immunoprecipitated at
4 °C for 18 h with continuous rocking. The immunocomplexes were
captured with protein A/G-agarose beads (Pierce). Samples were analyzed in a 3-15% SDS-PAGE gradient gel under nonreducing conditions and
transferred into nitrocellulose. The membranes were incubated in 5%
nonfat dry milk at room temperature for 1 h. The lower portion of
the membrane (proteins <40 kDa) was then incubated with the anti-FGF-BP antibody and the upper portion with the anti-domain III
antibody for 1 h. Membranes were washed three times in
Tris-buffered saline containing 0.1% Tween 20 and incubated with
horseradish peroxidase-conjugated secondary antibody for 1 h.
After three washes, the immunocomplexes were visualized by enhanced
chemiluminescence (Pierce).
Immunohistochemical Studies--
A survey of normal and
neoplastic frozen human tissues, obtained from the Tumor Bank of Thomas
Jefferson University, was investigated using monoclonal antibodies C9
against FGF-BP (51) or 7B5 against perlecan domain III (15). A total of
38 samples was investigated including normal samples of skin, colon,
kidneys, brain, tongue, pharynx, and lung. In addition we stained
several samples of squamous cell carcinomas from skin, esophagus,
tongue, lung and cervix, as well adenocarcinomas of the urinary
bladder, lung, and kidney. Immunohistochemistry was done essentially as
previously described (15) with minor variations. Routinely, we used
primary antibodies at 1:1000 dilution and secondary antibodies
conjugated with either AP or horseradish peroxidase at 1:2000 dilution.
In all cases, the frozen sections were blocked with either lovamisole
or H2O2 before the immunohistochemical
reaction. Images were captured with a Pixera digital camera and
assembled using Adobe Photoshop version 5.0.
Discovery of FGF-BP as a Binding Partner for Perlecan Protein
Core--
The yeast two-hybrid system (53) was used to identify
candidate proteins that interact with perlecan domain III in
vivo. Before proceeding with the screening, the bait plasmid
harboring domain III was tested for its inability to activate the
prototrophic reporter gene HIS3. As a control for possible
interactions, all constructs were also tested as either bait or prey.
In addition, all of the constructs were assayed for growth in double
minus (Trp
The full-length FGF-BP cDNA is 1163 base pairs, and the primary
structure of the encoded protein consists of 234 amino acids, including
a 21-residue signal peptide sequence and a single putative N-linked glycosylation site. Computer searches revealed that
homologues of human FGF-BP have been cloned previously from bovine,
rat, and mouse tissues (54-56) and that FGF-BP is well conserved among species (Fig. 1B). Human FGF-BP showed 84, 57, and 49%
identical amino acid sequence to the bovine, rat, and mouse species,
respectively. Ten Cys residues are fully conserved among species, and
there are two partial heparin-binding consensus sequences (57) (Fig. 1B) in addition to a highly basic region (residues 110-143)
proposed to be the principal heparin binding domain in FGF-BP (58).
To establish the nature of FGF-BP cDNA, we cloned FGF-BP into the
expression vector pcDNA3.1 and used in vitro
transcription/translation to determine its molecular mass. The results
showed a single band of the predicted size of ~23 kDa (Fig.
1C, lane 2) in contrast to the empty vector (Fig.
1C, lane 1), which showed no detectable bands.
The positive control was provided by the luciferase cDNA, which
produced the expected ~66 kDa protein (Fig. 1C, lane
3).
Nature of FGF-BP--
FGF-BP is a secreted, heparin-binding
protein originally purified from media conditioned by A431 human
squamous carcinoma cells (51). Notably, FGF-BP is localized to squamous
epithelia (51) and to squamous cell carcinomas (59). FGF-BP binds FGF1 and FGF2 in a noncovalent reversible manner (51) and potentiates the
activity of FGF7 (59), similar to the action of perlecan (49). In
addition, A431 squamous carcinoma cells transfected with FGF-BP become
more tumorigenic than wild type cells, and nontumorigenic A431-4
subclones, which do not express FGF-BP, become tumorigenic upon
de novo expression of FGF-BP (51). Ectopic expression of
FGF-BP in adrenal adenocarcinoma cells causes a release of FGF2 and
formation of highly vascularized tumors in nude mice (52). FGF-BP
expression is down-regulated by retinoic acid (56, 60) and induced by
EGF (61), and it is often enhanced in squamous cell carcinomas and
in a few colon carcinoma cell lines (54). Notably, FGF-BP can
serve as an angiogenic switch in vivo because reduction of
FGF-BP expression by targeting FGF-BP with specific ribozymes causes a
reduction in the growth and angiogenesis of tumor xenografts (62).
FGF-BP Binds Specifically to Perlecan's Domain III--
To
further verify that FGF-BP was a true interactive protein, we cloned
the full-length FGF-BP cDNA into plasmids carrying both the binding
(pGB) and activating (pGAD) domains and adopted them as either prey or
bait with perlecan domain III using a different yeast host strain
(SFY526). As positive and negative controls, we utilized the pTD1/pVA3
and pTD1/pLAM5' plasmids, respectively (CLONTECH).
The results showed a robust growth in triple minus media of
clones coexpressing FGF-BP/perlecan domain III proteins, expressed as
either bait or prey, with growth rates comparable with the positive
control (Fig. 2A). To further
prove this interaction, we performed FGF-BP Binds to the Second EGF Motif of Domain III--
Next, we
sought to establish the precise location of the FGF-BP-binding site
within domain III of perlecan, a1172-residue domain that is encoded by
27 exons (65) and shares homology with the short arm of laminin
Coimmunoprecipitation of FGF-BP and Domain III--
To verify that
the interaction detected using the yeast two-hybrid system could occur
outside the yeast, and to show its relevance within the context of
secreted proteins from mammalian cells, we studied the interaction of
secreted FGF-BP and recombinant domain III. As mentioned above, FGF-BP
was originally isolated from media conditioned by A431 squamous
carcinoma cells (51). To investigate the potential interaction in
solution between perlecan and FGF-BP, we generated stable transfectant
clones of A431 secreting the full-length domain III fused to the human
placental AP to serve as a marker. The generation of domain III-AP
fusion protein would avoid the potential interference of the heparan
sulfate chains, because this module has been shown to be synthesized
without glycosaminoglycan substitution (66), and would facilitate a direct protein-protein interaction. Several clones were isolated, and
two (clones 1 and 11) were synthesized and released into the medium of
the A431 cells, the fusion protein of the correctly predicted mass of
~190 kDa. This protein was recognized by anti-domain III monoclonal
antibodies (Fig. 4A, top
panel) and by an antibody against alkaline phosphatase (not
shown). Interestingly, FGF-BP was detected at high levels in these
cells (Fig. 4A, bottom panel) and migrated as a ~23 kDa
protein under reducing conditions, to a position identical to that
obtained with in vitro transcription/translation (cf. Fig. 1C). Proper folding of domain
III-AP fusion protein was verified by detection of strong AP activity
in clones 1 and 11 (Fig. 4B). This assumption is based on
the fact that the placental AP is positioned C-terminally, and thus we
presume that domain III is also properly folded because the fusion
protein expressed high enzymatic activity. In addition, domain III
folds into an individual entity, as determined by rotary shadowed
electron microscopy and biophysical studies (66, 67).
Immunoprecipitation studies using anti-FGF-BP or anti-domain III
monoclonal antibodies followed by Western immunoblotting showed the
coimmunoprecipitation of domain III and FGF-BP (Fig.
4C).
We further confirmed the interaction identified in the two-hybrid
screen system by coimmunoprecipitation of domain III and FGF-BP in
transiently transfected kidney COS-1 (African green monkey) cells,
which do not express FGF-BP. The media conditioned by COS-1
cells cotransfected with domain III and FGF-BP cDNAs showed that
both proteins interacted in solution and could be identified by their
respective antibodies (Fig. 5).
Collectively, these data indicate that FGF-BP is a binding partner for
perlecan domain III and that FGF-BP can bind perlecan protein core in
solution.
FGF-BP Is Increased in Squamous Cell Carcinomas and Codistributes
with Perlecan--
To elucidate FGF-BP distribution in human tissues,
we tested a number of normal and neoplastic frozen human tissues with
the monoclonal antibody directed toward FGF-BP. In adult normal skin, FGF-BP was expressed at relatively low levels in the suprabasal region
of the epidermis and focally in hair follicles (Fig.
6B). Interestingly, in a few
cases of squamous epithelia, FGF-BP epitopes were clearly present along
the basement membrane at the dermo-epidermal junction (Fig.
6C), occasionally extending into the basement membrane of
dermal blood vessels (Fig. 6D). This is similar to the
distribution of human perlecan using anti-domain III antibodies (15).
All of the other normal tissues, including lung, brain cortex, kidney, uterus, breast, colon, and various fibro-adipose tissues, were essentially negative. In contrast, invasive squamous cell carcinoma showed a marked induction of FGF-BP, especially around the most undifferentiated cells (Fig. 6E). Notably, even at
relatively high concentrations of the primary antibody (1:200) there
was no detectable staining in the normal tissues and the nonsquamous cell carcinomas tested (see below), in contrast to skin and squamous cell carcinomas where the antibody had to be diluted significantly (1:1000) to achieve optimal staining. Overall, these data are in good
agreement with in situ hybridization studies in the mouse, which have shown that FGF-BP expression starts at embryonic day 9 reaches its peak perinatally and is subsequently down-regulated during
adult life (54). In concert with our findings, FGF-BP mRNA
expression is dramatically increased upon induction of mouse skin
papillomas and carcinomas (54).
We then investigated the expression of FGF-BP vis á
vis that of perlecan in dysplastic skin and various tumors,
including carcinomas of the lung, uterus, bladder, kidney, and squamous cell carcinomas of the esophagus, skin, tongue, pharynx, lung, and
penis. A total of 38 samples were investigated. In dysplastic skin
overlaying an infiltrating squamous cell carcinoma of the tongue,
FGF-BP staining was markedly induced (Fig.
7B). Perlecan distribution was
primarily along the basement membrane and blood vessels of the upper
dermis (Fig. 7, C and F) in agreement with previous studies (15, 68). Again, there was a marked expression of
FGF-BP in the invasive squamous cell carcinoma cells of the esophagus(Fig. 7E), and consecutive sections showed a
significant codistribution of FGF-BP (Fig. 7G) and perlecan
(Fig. 7, H and I). Keratin pearls, considered to
represent a sign of cellular differentiation, did not contain either
FGF-BP or perlecan epitopes (Figs. 6E and 7I). No
significant FGF-BP immunoreactivity was observed in all the other
nonsquamous carcinoma tumors (not shown) with the exception of focal
positivity in colon and lung carcinomas. The latter finding is
in agreement with the reports that FGF-BP is expressed by some colon
carcinoma cell lines (52) and that FGF-BP transcript can be detected in
the developing mouse intestine and lung (54).
Overall, our data indicate that under normal conditions, the
distributions of FGF-BP and perlecan overlap only focally. The former
is located in the suprabasal layer (stratum spinosus) of the epidermis,
whereas the latter is located primarily in the dermis, along the
basement membrane zone at the dermal-epidermal junction and along the
vasculature. When the squamous epithelium becomes transformed,
regardless of its site of origin, FGF-BP is deposited in the
pericellular space in close proximity to the cell surface of the most
aggressive squamous carcinoma cells. This distribution clearly overlaps
with that of perlecan, suggesting a potential in vivo
interaction between these two proteins.
Conclusions--
Because of its eukaryotic nature, the yeast
two-hybrid system has been widely used to study protein-protein
interactions, primarily those occurring among intracellular proteins
(69). Only recently, however, has it been successfully used to
investigate interactions among extracellular proteins such as those
involving collagen types VI and IV (70), thrombospondin (71), EMILIN (72), or matrix metalloproteinase 2 (73). Binding sites for FGF7 and
platelet-derived growth factor-AA and -BB have been identified in
subdomains III-1 (50) and III-2 (74), indicating that there are unique
binding specificities for perlecan modules containing highly repetitive
sequences. The results of this study indicate that the second EGF-like
repeat (LE2) of domain III-1 interacts specifically with FGF-BP and
that this interaction could modulate the ability of squamous epithelia
to respond to FGF-mediated signaling. Binding of FGF-BP to subdomain
III-1 is the second extracellular ligand identified so far for this
particular perlecan region. Coimmunoprecipitation and codistribution in
cancer tissues suggest that these interactions may take place in
vivo. Perlecan may function as an extracellular sink for FGF7 and
FGF-BP, acting as a reservoir for these growth factors. FGF-BP appears
to be located within the keratinocyte layer of squamous epithelia,
primarily in the stratum spinosus. During cancer growth and invasion,
FGF-BP could be released from the transformed epithelial cells and
interact with perlecan, which is highly expressed in the stroma and
perivascular microenvironment. It is plausible that FGF-BP, FGF7, and
perlecan form a trimolecular complex that potentiates the activity of
FGF7. It is seemingly possible that perlecan may modulate the targeting of FGF7 and FGF-BP to the epithelium and may function as an important molecular entity in the signaling of these proteins. Upon displacement by partial proteolysis of the protein core, FGF7 and FGF-BP would become available to the surrounding cellular environment and could behave as a promoter of growth and differentiation. An alternate pathway would involve an FGF-BP-mediated displacement of FGF2 and FGF7
stored in the cell surface heparan sulfate proteoglycans. This pathway
does not require the need of active proteolysis or glycolytic enzymes
and could be operational at the site of tumor growth. According to this
working model (Fig. 8), overproduction of
FGF-BP in malignant cells or induction by EGF would liberate FGF-BP in
the microenvironment where it would displace FGF2 or FGF7 bound to
heparan sulfate chains of syndecan, glypican, or perlecan
proteoglycans. In addition, FGF-BP could interact and displace FGF7
bound to perlecan's domain III, without the need of proteolytic
activity. Various FGFs could then be released into the microenvironment
where they would stimulate angiogenesis and tumorigenesis. Hence,
perlecan would play a central role not only as molecular storage of
growth factors but also as repository for FGF-BP, and possibly related
proteins, that would act as angiogenic modulators.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-dystroglycan (24), and
lipoproteins (25).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/Leu
medium with constant shaking. The transfected cells were plated in
medium lacking Trp and Leu to obtain an indication of the primary transformation efficiency and on plates lacking Trp, Leu, and His to
select for colonies expressing interacting hybrid proteins. The plates
were incubated at 30 °C for 8 days. The His+ colonies
were isolated and screened by PCR for the inserts in the activation
domain vector using primers specific for the GAL4 activation domain plasmid.
/Leu
and
Trp
/Leu
/His
agar plates to
check for interactions, analyzed initially by growth in the triple
minus media compared visually with that of the positive control.
For qualitative
-galactosidase assays, cells grown in
Trp
/Leu
plates were transferred onto
Whatman No. 3MM paper filters and 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-3indolyl-
-D-galactopyranoside). Yeast cells were frozen in liquid nitrogen for 10 s, thawed,
transferred onto a filter, and incubated at 30 °C for ~8 h to
visualize the appearance of blue colonies. In addition, the two-hybrid
interactions were verified and quantified by liquid assays of
-galactosidase activity using the Luminescent
-galactosidase
detection kit II (CLONTECH). This system uses a
chemiluminescent substrate (Galacton-StarTM), and the light
emission can be used as a quantitative measure of
-galactosidase
activity. Five-ml overnight cultures in
Trp
/Leu
medium were transferred to 8 ml of
YPD medium and incubated at 30 °C for 3 h with shaking
(250 rpm). The cells were harvested by centrifugation at 10,000 × g for 30 s, and the pellets were resuspended in 300 µl of buffer and subjected to two consecutive freeze/thaw cycles.
25-µl aliquots of each cell lysate were incubated with 200 µl of
Galacton-StarTM reaction mixture at room temperature for 60 min and centrifuged at 10,000 × g for 1 min, and the
supernatants were transferred to luminometer tubes. The light emission
(measured in relative light units) was recorded at 5-s
intervals, and the
-galactosidase activity was normalized on the
relative OD, and expressed as relative light units
/A600 unit of cell culture.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/Leu
) media as a control
for transfection efficiency. The screening of ~0.5 × 106 cDNAs from a human keratinocyte library resulted in
the isolation of 50 independent clones identified as large colonies
growing in triple minus
(Trp
/Leu
/His
) plates. Of
these, 40 clones grew back and 38 clones showed inserts ranging between
0.4 and 1.6 kilobase pair. To minimize the number of false positives
and to increase the likelihood of obtaining pure clones, the initial
isolates were regrown in triple minus media and subjected to
direct PCR screening using flanking primers specific for the pGAD
plasmid. All of the bands were isolated and sequenced, and DNA homology
searches using the NCBI BLAST program of the Genetics Computing
Group package led to the elimination of many false positives, the
majority of which were nuclear proteins that could themselves
activate the transcription of the HIS3 reporter gene.
A highly interacting clone contained a 1.2-kilobase pair insert
(Fig. 1A) that encoded a small
protein, named HBp17 (Fig. 1B) because it binds
heparin-binding growth factors 1 and 2 (51) (also called FGF-BP, for
FGF-binding protein (52)). The cDNA was in frame with the
activating domain of the plasmid and coded for the full-length FGF-BP,
further supporting the concept of a real protein-protein
interaction.
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Fig. 1.
Discovery of FGF-BP as a biological
partner for perlecan protein core. A, PCR amplification
of a positive clone (lane 3) interacting with perlecan
domain III by the yeast two-hybrid system. Lanes 1 and
2 correspond to the Mr markers and a
negative control, respectively. B, alignment of human
(NP005121), bovine (AAF75792), rat (AAF23079), and mouse (NP032035)
sequences encoding FGF-BP (the respective GenBankTM
accession numbers are given in parentheses). Fully conserved residues
are shown in red, partially conserved residues are in
green, and weakly conserved residues are in blue.
The 10 conserved Cys are represented by an asterisk, while
the 2 heparin-binding partial consensus sequences are represented by a
horizontal line. C, in vitro
transcription/translation of empty plasmid (lane 1),
FGF-BP-containing plasmid (lane 2), and
luciferase-containing plasmid (lane 3). Samples were
incubated with [35S]methionine in a rabbit reticulocyte
lysate, separated in a 6-12% SDS-PAGE, and subjected to
autoradiography.
-galactosidase assays on the
cotransfectants. In addition to growth in triple minus media,
transcription of lacZ containing the upstream binding
sites of GAL4 and the subsequent ability of cotransformant yeast
strains to express functional
-galactosidase are additional strong
proofs for a true protein-protein interaction (63, 64). Domain III,
present as either bait or prey, showed a robust production of blue
colonies (Fig. 2B), comparable in intensity with the
positive control pTD1/pVA3 harboring the p53 and the SV40 T antigen
(Fig. 2C). This suggests that the affinity between domain
III and FGF-BP is relatively high. Preliminary solid phase binding
experiments using purified FGF-BP and soluble domain III-AP
demonstrated a saturable and high affinity (Kd ~ 18 nM) binding (not shown).
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Fig. 2.
Specific interaction between FGF-BP and
perlecan protein core. A, growth in triple minus
media of FGF-BP cDNA cloned into plasmids carrying both the
binding (pGB) and activating (pGAD) domains, used as either prey or
bait with perlecan domain III and employing the yeast host strain
SFY526. As positive and negative controls, pTD1/pVA3 and pTD1/pLAM5'
plasmids were used, respectively. B, -galactosidase
assays of the cotransfectant clones. C,
-galactosidase assays of the
positive and negative controls.
1-chain (8-10). Domain III consists of alternating globular domains
(L4 modules, characteristically devoid of cysteine residues) and short
connecting rod-like segments of laminin-type EGF-like repeats (LE
modules). We generated five deletion fragments of domain III,
1-
-5 (Fig. 3A).
Growth was observed in cells cotransformed with full-length domain III
and the first two deletion constructs, but it was markedly attenuated using
3-5 constructs (Fig. 3A). These results were
corroborated by qualitative (Fig. 3B) and quantitative (Fig.
3C)
-galactosidase assays. In the latter, the two-hybrid
interactions were quantified by liquid assays of
-galactosidase
activity using a very sensitive luminescent detection system. For
testing each interaction, five separate transformant colonies were
assayed and each assay was performed in triplicate. Notably,
-galactosidase activity of domain III-FGF-BP cotransformants was
about 50% of that observed by the positive control, further suggesting
that the affinity of FGF-BP for perlecan protein core is relatively
strong. The results indicate that the second EGF-like repeat (LE2) of
domain III-1 interacts specifically with FGF-BP.
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Fig. 3.
FGF-BP interacts specifically with the second
EGF-like repeat of domain III-1 of perlecan. A,
schematic representation of domain III and various deletion mutants.
Domain III has been subdivided as indicated at the top
according to a recently proposed nomenclature (67). Green
ovals indicate globular domains (L4 modules), and red
rectangles indicate the laminin-type EGF-like repeats (LE
modules). The numbers within parentheses
designate the amino acid position based on the mature protein core.
Growth is indicated by semiquantitative assessment, with maximal growth
at +++. B, representative -galactosidase assays of all
the clones carrying domain III or its deletion mutants. C,
liquid assays quantization of
-galactosidase activity using a
sensitive luminescent detection system (CLONTECH).
The values are the mean ± S.D. (n = 5) and are
expressed as relative light units (RLU) normalized on cell
content (OD600).
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Fig. 4.
FGF-BP can be coimmunoprecipitated with
perlecan's domain III-AP. A, generation of A431 clones
stably expressing domain III-AP fusion protein. Aliquots of serum-free
media conditioned for 24-36 h by A431 squamous carcinoma cells
were precipitated with 6 volumes of ethanol-0.1% potassium acetate at
20 °C, separated on a 8.5% SDS-PAGE, and analyzed by Western
immunoblotting with either anti-domain III (
DIII) or anti-FGF-BP
(
FGF-BP). Notice, in the top panel,
the presence of a ~190-kDa immunoreactive protein in clone 1 (lanes 1 and 2) or clone 11 (lanes 5 and 6), in contrast to clone 4 or the wild type cells
(lanes 3 and 4 and 7 and 8,
respectively). The bottom panel, reacted with
FGF-BP,
shows a significant amount of FGF-BP migrating with an average mass of
~23 kDa. B, alkaline phosphatase assays of various clones
as indicated. C, immunoprecipitation (ip)
followed by Western immunoblotting (wb) as indicated. Notice
the presence of domain III-AP fusion protein and FGF-BP in both
immunoprecipitates. The reactive ~85 kDa band (lanes 1 and
2, top panel) corresponds to IgG homo- or heterodimers since
the gel was run under nonreducing conditions because the C9 antibody
does not work well following reduction. Lane 3 shows
immunoblotting of the total medium alone without any
immunoprecipitation.
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Fig. 5.
Perlecan-FGF-BP interaction in transiently
transfected cells. To establish the in vivo interaction
of perlecan's domain III and FGF-BP, the two full-length cDNAs
were subcloned into pcDNA3.1 containing the signal peptide of BM-40
and cotransfected into COS-1 cells. The 72-h conditioned media were
immunoprecipitated with the two antibodies as indicated and subjected
to Western immunoblotting (wb). The bottom panel
represents the IgG to show equal loading. Molecular mass markers in kDa
are shown in the right margin. Notice the presence of the
predicted ~190- and ~23-kDa proteins corresponding to perlecan's
domain III-AP and -FGF-BP, respectively.
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Fig. 6.
FGF-BP is expressed at low levels in normal
skin but is up-regulated in squamous cell carcinoma. A,
skin negative control (no primary antibody). B, skin reacted
with anti-FGF-BP monoclonal antibody. Notice the presence of
immunoreactive protein in the suprabasal layer (stratum spinosus) of
the epidermis (arrows) and focally in a hair follicle
(asterisk). C and D, two areas of
human skin showing immunoreactive deposits along the basement membrane
(BM) of the dermo-epidermal junction and of a blood vessel
(Bw). E, squamous cell carcinoma of the skin.
Notice the increased deposits of FGF-BP in a pericellular and/or cell
surface distribution, particularly around less differentiated squamous
carcinoma cells (arrows). A keratin pearl (Kp)
within the tumor is totally negative. After blockade of endogenous AP
with lovamisole, immunohistochemistry was performed using
primary C9 antibody (1:1000) followed by AP-linked secondary antibody
(1:2000) and fast red chromogen (Dako Laboratories).
Bar = 50 µm.
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Fig. 7.
FGF-BP expression is elevated in dysplastic
skin and codistributes with perlecan in the pericellular environment of
squamous cell carcinomas. A, dysplastic skin, negative
control (no primary antibody). B, dysplastic skin overlaying
an invasive squamous cell carcinoma of the tongue. Notice the marked
staining with anti-FGF-BP antibody of the expanded stratum spinosus.
C, skin stained with anti-perlecan antibody. Notice the
marked staining of the basement membrane (arrows) and of the
upper dermis and blood vessels. D, squamous cell carcinoma
of the esophagus, negative control (no primary antibody). E,
parallel section stained with anti-FGF-BP. Notice the intense staining
of the invasive cancer cells (Ca). F, dysplastic
skin stained with anti-perlecan antibody. Notice the marked staining of
the basement membrane (arrows) and of the upper dermis and
blood vessels. G, invasive squamous cell carcinoma of the
esophagus reacted with anti-FGF-BP antibody. H, parallel
section stained with anti-perlecan antibody. Notice a distribution
within the invasive cancer (Ca) nearly identical to that of
FGF-BP. I, a different case of squamous cell carcinoma
stained with anti-perlecan antibody showing similar pericellular
distribution. A keratin pearl (Kp) is negative. Following
blockade of endogenous peroxidase activity with 3%
H2O2, immunohistochemistry was
performed using primary C9 or 7B5 monoclonal antibodies (both at
1:1000) followed by AP-linked secondary antibody (1:2000) visualized
with horseradish peroxidase-labeled secondary antibody (1:200) and
diaminobenzidine as a substrate (DAKO Laboratories).
Bar = 60 µm.
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Fig. 8.
Proposed model showing how secreted FGF-BP
would facilitate the release of FGFs from the cell surface and basement
membrane heparan sulfate proteoglycans. FGF-BP expression is
blocked by retinoic acid but highly induced by transformation and EGF.
Released FGF-BP mobilizes FGF2 and FGF7 bound, in a presumably inactive
status, to the heparan sulfate chains of various proteoglycans. In
addition, FGF7 and FGF-BP can interact with the protein core of
perlecan thereby providing an additional level of control. The
liberated (active) FGFs can thus mediate various functions
such as induction of angiogenesis and tumorigenesis. The model is
modified from the work of Rak and Kerbel (75).
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ACKNOWLEDGEMENTS |
---|
We thank I. Eichstetter for excellent technical assistance, Chris Munnery for help with the immunohistochemistry, and Jim San Antonio for helpful advice.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants RO1 CA39481 and RO1 CA47282 (to R. V. I).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.
§ Supported in part by a fellowship from the American-Italian Cancer Foundation.
¶ These two authors contributed equally to 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, January 8, 2001, DOI 10.1074/jbc.M011493200
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ABBREVIATIONS |
---|
The abbreviations used are: FGF, fibroblast growth factor; FGF-BP, FGF-binding protein (also known as HBp17); FGF7, fibroblast growth factor-7 (also known as keratinocyte growth factor); EGF, epidermal growth factor; AP, alkaline phosphatase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.
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