From the Center for Cancer Biology and Nutrition, Institute of Biosciences and Technology, Texas A&M University System Health Science Center and Department of Biochemistry and Biophysics, Texas A&M University, Houston, Texas 77030-3303
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ABSTRACT |
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Fibroblast growth factor (FGF)-10, a homologue of
FGF-7, is expressed significantly in normal rat prostate tissue, well
differentiated rat prostate tumors with an epithelial and stromal
compartment and only in derived prostate stromal cells in culture.
Similar to FGF-7, recombinant rat FGF-10 was a specific mitogen for
prostate epithelial cells. In contrast to FGF-7 which is widely
expressed among stromal cells in tissues, the expression of FGF-10
correlated with the presence of stromal cells of muscle origin.
Radioreceptor binding assays and covalent cross-linking analysis
revealed that FGF-10 binds with an affinity equal to FGF-7 to resident
epithelial cell receptor, FGFR2IIIb, but unlike FGF-7 also binds the
IIIb splice variant of FGFR1. Analysis of mRNA expression by RNase protection revealed that, similar to FGF-7, the expression of FGF-10
was responsive to androgen in stromal cells from normal prostate and
non-malignant differentiated tumors. Although FGF-10 cDNA exhibits
a signal sequence for secretion, cultured stromal cells exhibit
strictly a cell-associated FGF-10 antigen that correlates with an
alternately translated intracellular isoform. FGF-10 requires 1.4 times
higher NaCl for elution from immobilized heparin than does FGF-7 and
binds to four times the number of sites on the pericellular matrix of
epithelial cells. The results show that prostate stromal cell-derived
FGF-10, like FGF-7, exhibits the properties of an andromedin which may
indirectly mediate control of epithelial cell growth and function by
androgen. Although FGF-10 and FGF-7 bind and activate the same resident
epithelial cell receptor (FGFR2IIIb), differences in cell type of
origin, compartmentation by alternate translation, the affinity for
FGFR1IIIb, and access to FGFR by differential interaction with
pericellular matrix heparan sulfate suggest they may play both
independent and compensatory roles in prostate homeostasis.
Members of the fibroblast growth factor (FGF)1 family
which is comprised of 20 or more
homologous polypeptides and four tyrosine kinase receptor (FGFR) genes
play a role in prostate homeostasis and tumor progression (1-3).
FGF-1, FGF-2, FGF-3, FGF-5, and FGF-7 and splice variants of FGFR1 and
FGFR2 have been implicated. FGF-1, which acts on FGFR isoforms that are
both in epithelium and stroma, is expressed in both prostate
compartments and expression increases with malignant progression (4).
FGF-2, FGF-3, and FGF-5 are expressed ectopically in malignant
epithelial cells (3). FGF-7, whose expression is limited to the stromal
cells, is the androgen-responsive signal of a directionally specific paracrine communication system from stroma to epithelium in normal prostate and non-malignant differentiated tumors with a stromal compartment (3). Reception of the stromal signal appears to be mediated
by FGFR2IIIb, a mutually exclusive alternate splice variant, which
recognizes FGF-7 with high affinity (2, 3, 5). In progressively
malignant prostate tumors, a phenotypic splice switch from expression
of the FGFR2IIIb to the FGFR2IIIc isoform occurs with eventual loss of
FGFR2 gene expression altogether in some malignant clones (3, 6).
Restoration of the FGFR2 kinase to malignant tumor cells restores
limitations on growth rates by increased differentiation in response to
stroma cells expressing FGF-7 (6). Therefore, it is thought that in the epithelial cell context, FGFR2 mediates a homeostatic balance through
both positive and negative effects on population growth rates and
differentiation. This is in contrast to FGFR1, which is a resident FGFR
in stromal cells (6). In this study, we show that, similar to FGF-7,
FGF-10 is expressed only in the stroma of normal prostate and
differentiated prostate tumors, and that expression is sensitive to
androgen. FGF-10 is a mitogen for isolated prostate epithelial cells,
but not for stromal cells which express it, and binds specifically to
the resident epithelial cell receptor, FGFR2IIIb. FGF-10 differs from
FGF-7 by its more restricted expression pattern, the ability to bind
FGFR1IIIb in addition to FGFR2IIIb, and in the interaction with heparin
and pericellular matrix heparan sulfate. The two factors exhibit
potential for both compensatory and specific activities in the prostate.
Construction, Expression, and Purification of Recombinant
FGF-10--
Total RNA was isolated from Dunning R3327PAP rat prostate
stromal cells by guanidinium thiocyanate/phenol/chloroform extraction and first strand cDNA was prepared using SuperScript II RNase H
Reverse Transcriptase (Life Technologies) with random primers. The
polymerase chain reaction (PCR) was carried out for 38 cycles at
94 °C for 1 min, 55 °C for 2 min, and 72 °C for 2 min.
Full-length FGF-10 cDNA was generated from the template with sense
primer p1001 5'-CGGGATCCATGTGGAAATGGATACTG-3' which
included a BamHI restriction site (underlined) and antisense
primer p1002 5'-GGGAATTCCTATGAGTGGAC-CACCAT-3' containing
an EcoRI site (underlined). The PCR product was digested by
BamHI and EcoRI and then fractionated on a 1.5%
agarose gel, recovered by electroelution and cloned into pBluescript II
SK vector (Stratagene, La Jolla, CA) at BamHI and
EcoRI sites. The cDNA was verified by nucleotide
sequencing. After digestion with HinPI and EcoRV
and treatment with Klenow enzyme, the cDNA was then cloned in-frame
into the pGEX-2T expression vector at the SmaI site and
expressed in BL21 (DE3) cells (Novagen) resulting in an expression
product fused to glutathione S-transferase at the C terminus
to N-terminal amino acid residue alanine 69 of FGF-10. Homogenous
FGF-10 with the N terminus at residue serine 75 was generated and
recovered by a novel and general method for production of FGF fused at
the N terminus with glutathione S-transferase and generation
of the most active FGF isoform by modification with serine proteases
while immobilized on heparin-agarose (7). Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid
sequence verified the homogeneity of FGF-10. FGF-7 beginning at Ser-54
was produced by the same method (7). FGF-1 was from Asn-21 and FGF-2
from Ala-1.
Analysis of Expression of FGF-7 and FGF-10 mRNA by Reverse
Transcriptase-Polymerase Chain Reaction (RT-PCR)--
FGF-10 mRNA
was analyzed in the PCR using oligonucleotide primers p1001 and p1002
which flank the coding sequence as described above. Since mouse FGF-10
C-terminal cDNA sequence is different from that of rat, another
pair of primers, sense p1001 and antisense p1004
5'-GCCGTTGTGCTGCCAGTT-3' was used to analyze mouse FGF-10 mRNA
expression. Sense primer p701 5'-CCGCCCG-GGATGGCTTGCAATGACATGA-3' and
antisense primer p702 5'-CCAATTCTTCTCTGC-ATGCTTCTT-3' which flank a
367-base pair coding sequence of rat FGF-7 were used for analysis of
FGF-7 mRNA expression. Total RNA (4 µg) from tissues and cells
indicated in the text was converted to cDNA. FGF-10 was analyzed
using the same conditions described above for cloning the cDNA.
Optimized conditions determined by experiment for analysis of FGF-7 was
35-40 cycles at 94 °C for 1 min, 57 °C for 2 min, and 72 °C
for 2 min. The RT-PCR products were analyzed in 1.5% agarose gel.
Purified cDNA templates for FGF-7 and FGF-10 and cDNA from
total RNA of lung tissue were used for positive controls. Analysis
of Analysis of mRNA Expression by Ribonuclease (RNase)
Protection--
A fragment spanning base pairs 49 to 207 between
NciI and BstUI restriction sites in rat FGF-10
cDNA was treated with Klenow enzyme and subcloned into pBluescript
SK vector at EcoRV site. The resulting plasmid template was
linearized with EcoRI and the antisense RNA probes were
transcribed using T7 RNA polymerase and labeled to a specific activity
of about 108 cpm/mg of linearized cDNA template using
the Ambion MAXIscript kit. Rat FGF-7 mRNA was determined by
protection of a 205-base antisense BamHI-KpnI
fragment from base pairs 23 to 224 as described previously (3, 8).
About 20 µg of total RNA from tissues or cells was hybridized with
1 × 105 cpm of labeled antisense RNA probe. Hybrid
duplexes were digested with RNase A/T1 for 30 min at 37 °C.
Protection fragments were analyzed on 5% polyacrylamide gels
containing 8 M urea followed by autoradiography. Yeast tRNA
was used as a control for nonspecific hybridization. All analyses were
performed using the HybSpeed ribonuclease protection kit (Ambion). To
quantitate the relative expression of FGF-7 and FGF-10, 1 × 105 cpm each of FGF-7, FGF-10, and Expression of FGF-10 Protein in Cultured Dunning Tumor Stromal
Cells--
DT-S of DT-E cells were cultured in 1:1 RPMI medium 1640 and Dulbecco's modified Eagle's medium (RD medium) supplemented with 2% fetal bovine serum in 40 × T75 cell culture flasks until
subconfluent, and the cells were then placed in serum-free RD medium.
After culture overnight, the medium was collected, and the cells were exposed to 1.5 M sodium chloride in 1% Triton. The medium
(300 ml) from about 1.5 × 108 cells was concentrated 100 times to 12 mg/ml total protein and the cell extract was concentrated
30 times from 100 ml of cell lysate to 67 mg/ml of total protein. The
extracts were treated with rabbit anti-rat FGF-10 antibody and protein
A beads followed by analysis by SDS-PAGE and immunoblotting. Homogenous
recombinant rat FGF-10 (75-214) was prepared from bacteria as
described (7). This pure antigen was used to immunize rabbits (Cocalico
Biologicals, Inc.) which produced antiserum that reached a titer of
1:1600 in immunoblot analysis.
Expression of Recombinant FGFR Isoforms in Insect
Cells--
Expression of all isoforms except FGFR1IIIb and FGFR3IIIb
in baculovirus-infected insect cells have been described previously (9). The human FGFR1IIIb and the murine FGFR3IIIb cDNA were kindly
provided by Dr. David M. Ornitz (5, 10). FGFR1IIIb from pBluescript
KS(+) vector (Stratagene, La Jolla, CA) was digested with
ApaI followed by Klenow enzyme treatment, then digested with BamHI. The resulting 2.3-kilobase fragment was cloned into
the BamHI and SmaI sites of baculovirus transfer
vector pVL1393. FGFR3IIIb from MomFR3SV vector was first digested with
HindIII and XhoI, ligated to pBluescript-SK
vector, then digested with BamHI and SalI or
digested with KpnI (treated with Klenow enzyme) and
SalI, respectively, the two resulting fragments were ligated
into pBluescript-SK vector at BamHI and SmaI
sites. Finally, this construction was digested with EcoRV
and ligated to pVL1392 vector at the SmaI site; the sense
clone was confirmed by restriction enzyme digestion. The expression
vectors bearing FGFR1IIIb and FGFR3IIIb were then co-infected with
BaculoGold viral DNA into Spodoptera frugiperda (Sf9)
insect cells, respectively, using the Lipofectin method (9).
DNA Synthesis, Radioreceptor Assays, and Covalent Affinity
Cross-linking--
DNA synthesis monitored by thymidine incorporation
was carried out as described previously (2-4, 6). Native bovine FGF-1, recombinant FGF-2, FGF-7, and FGF-10 prepared as described above were
iodinated to specific activities of about 3 × 105
cpm/ng. The determination of specific binding of radiolabeled FGF to
either FGF receptor kinase sites or heparan sulfate sites in the
pericellular matrix was carried out as described (2-4, 6, 11).
Expression of FGF-10 in Prostate Stromal Cells--
A screen of 18 FGF mRNAs using the RT-PCR with multiple sets of paired primers
revealed the presence of FGF-10 mRNA in normal rat prostate (NP)
and the transplantable non-malignant well differentiated Dunning
R3327PAP tumor (DT) (Fig. 1A)
in addition to adult lung tissue, which was used as a positive standard
(12). The FGF-10 signal parallels that of FGF-7 in the normal prostate
tissue (NP), both primary cultures and lines of stromal cells from the
DT tissue (DT-S), non-malignant, differentiated tumors (SE)
reconstituted from isolated DT epithelial (DT-E) and stromal (DT-S)
cells (2, 3). Similar to FGF-7, the FGF-10 mRNA could not be
detected in isolated epithelial cells from the DT tumors (DT-E). As
reported previously, FGF-7 can be detected in E tumors derived from
isolated DT-E cells as they progress to malignancy, malignant Dunning
R3327AT3 tumors (AT3) and derived cells (AT3-C) (2) (Fig.
1A). However, the FGF-10 transcript was undetectable in
these samples when analyzed under the same conditions.
Since expression of FGF-10 mRNA was limited to the stromal cells of
normal prostate and differentiated prostate tumors, we compared
expression of the two mRNAs in lung, skin, and muscle and
mesenchymal cells derived from them in which FGF-7 is expressed. Although FGF-10 is expressed in adult lung tissue (12), FGF-10 could
not be detected in isolated lung fibroblasts from fetal tissue relative
to FGF-7 (Fig. 1B). Compared with FGF-7, the FGF-10 signal
was weak in mouse 3T3 cells, which are also thought to be
fibroblast-like cells. Both FGF-7 and FGF-10 were undetectable in human
umbilical vein endothelial cells. The FGF-7 mRNA was barely
detectable in subcutaneous skin samples taken from tumor implantation
sites in rat hosts and FGF-10 was undetectable. Relative to FGF-7,
FGF-10 mRNA was undetectable in normal human fibroblasts isolated
from neonatal foreskin. In contrast to the fibroblast-like cells, the
embryonic rat skeletal muscle myoblast cell line L6, and a smooth
muscle cell line from normal rat prostate (PS-1) (13), exhibited the
strongest positive signals of FGF-10 relative to FGF-7 mRNA. In
contrast to skin, both rat skeletal muscle tissue and small intestine
tissue, which are rich in smooth muscle cells, exhibited FGF-10 signals
equal to or greater than FGF-7. Both FGF-7 and FGF-10 were absent in
mouse keratinocytes, human hepatoblastoma cells (HepG2), and the human
prostate tumor epithelial cell line (PC3). These results show that,
similar to FGF-7, the expression of FGF-10 is associated with the
stromal or mesenchymal compartment of prostate and a number of other
tissues. However, the expression of FGF-10 is much more restricted than
that of FGF-7 and may partition with muscle cell lineages relative to
the general stromal or mesenchymal cell population in the adult and
derived tumors.
To confirm that the RT-PCR results reflected authentic and more than
trace levels of FGF-7 and FGF-10 mRNAs, the mRNA levels for the
two factors in prostate tissues and derived cells were further examined
by ribonuclease protection (RPA) (Fig. 2
and Table I). The results generally
confirmed the RT-PCR analysis. The more reliable quantitative analysis
suggested that the ratio of expression of FGF-7 to FGF-10 mRNA in
NP tissue was about 2.5, but that levels of FGF-10 increased near
4-fold in the DT tumors and derived DT-S cells resulting in an
expression ratio of near 1:1 for the two homologues. As tumors and
cells progress to malignancy, expression of FGF-10 is lost while FGF-7
remains detectable, although at reduced levels (Fig. 2A and
Table I). Although an FGF-7 signal could be detected by PCR in tissue
at the subcutaneous site of injection (Fig. 1B, Skin)
of the transplantable tumors in control hosts, expression of both FGF-7
and FGF-10 was below the limits of detection by the ribonuclease
protection method (Fig. 2A, Table I).
Expression of FGF-10 in Prostate Stromal Cells Is
Androgen-responsive--
To determine whether the expression of FGF-10
mRNA was responsive to androgen as previously reported for FGF-7,
primary cultured stromal cells from both normal prostate and the
androgen-responsive Dunning R3327PAP tumor were isolated and analyzed
as described previously (2, 3). Cultures were maintained in serum-free medium in the absence or presence of 10
To determine whether FGF-10 mRNA gave rise to FGF-10 secretory
product, the medium of the cultured DT tumor stromal cells (DT-S) was
harvested and analyzed by immunoprecipitation and immunoblot with
rabbit polyclonal antiserum prepared against recombinant FGF-10 from
bacteria. FGF-10 antigen from medium exposed overnight to 1.5 × 108 stromal cells from the DT tumors was below the limits
of detection. Subsequent extraction and analysis of the cell monolayers
with 1.5 M NaCl and detergent revealed a single major band
at 20.2 kDa estimated from electrophoretic standards that could not be detected in equivalent amounts of the DT epithelial cells (Fig. 2C). This species is smaller than the bacterial-derived
recombinant FGF-10 (37-214) band at apparent mass 21.4 kDa (predicted
mass = 19.8 kDa), which was engineered to begin at Ala-37. A
shorter bacterial recombinant isoform, FGF-10 (75-214), at apparent
mass 15.5 kDa (predicted = 15.4 kDa), which started at Ser-75 (7) which arises by proteolysis of heparin-bound precursor is also shown.
FGF-10 Supports Growth of and Binds to Receptors on
Specifically the Prostate Epithelial Cells--
Recombinant rat
FGF-10 was generated by a novel method for production and
recovery of FGF polypeptides in bacteria developed in our laboratory
(7). Fig. 3 shows that FGF-10 stimulates DNA synthesis at 50% of maximum at 1 ng/ml in non-malignant rat prostate tumor epithelial cells (DT-E) which was slightly less than the
potency of FGF-7. Neither FGF-10 nor FGF-7 support DNA synthesis of
stromal cells (DT-S) from the same tumors in which both factors are
expressed.
Competition binding assays using radiolabeled FGF-1 which stimulated
and bound to receptors on both DT-E and DT-S cells indicated that FGF-7
and FGF-10 compete to near equal extent with FGF-1 bound to the DT-E
epithelial cells, but both failed to compete with FGF-1 bound to
stromal DT-S cells (Fig. 4). To ensure
that the mitogenic activity and competition of FGF-10 with the binding of FGF-1 reflected authentic binding to DT-E cell surface receptors, FGF-10 was radiolabeled, covalently cross-linked with the bifunctional cross-linker, DSS, and the resultant radiolabeled complexes analyzed by
SDS-PAGE and autoradiography (Fig. 5).
Radiolabeled FGF-1, FGF-7, and FGF-10 yielded expected bands at 140 and
75 kDa from DT-E cells which represent the native FGFR and a truncated
product resulting from proteolysis (15). Neither FGF-7 nor FGF-10
cross-linked to FGFR on the stromal cells (DT-S), even at 5 times
higher radiolabeled ligand and after long exposure of the analytical
gels.
FGF-10 Binds to Complexes of Heparin and Recombinant FGFR2IIIb and
FGFR1IIIb--
FGFR1IIIb and IIIc, FGFR2IIIb and IIIc, FGFR3IIIb and
IIIc, and FGFR4 were expressed on the surface of baculoviral-infected insect cells and the ability of FGF-10 to compete with the binding of
radiolabeled FGF-1 which binds to all isoforms equally in the presence
of heparin. FGF-10 competed effectively with 125I-FGF-1
bound to Sf9 insect cells expressing recombinant FGFR1IIIb and
FGFR2IIIb with an ID50 (dose required to displace 50% of
radiolabeled FGF-1) of 33 and 1.3 ng, respectively (Fig.
6). No significant competition was
observed with FGF-1 bound to cells expressing FGFR1IIIc, FGFR2IIIc,
FGFR3IIIb, FGFR3IIIc, and FGFR4. Covalent affinity cross-linking
analysis revealed that the binding to cells expressing FGFR2IIIb and
FGFR1IIIb was to molecular species that corresponded to the recombinant
FGFR (Fig. 6, inset). Both FGF-7 and FGF-10 competed with
radiolabeled FGF-1 for binding to recombinant FGFR2IIIb expressed in
Sf9 cells at nearly identical levels (Fig. 7A) and each competed
similarly with the other when one or the other was the radiolabeled
ligand (Fig. 7, B and C). These results suggest
that when all other conditions are equal, FGF-10 and FGF-7 exhibit near
equal affinities for FGFR2IIIb.
Differential Interaction of FGF-7 and FGF-10 with Heparin and the
Pericellular Matrix--
Similar to other members of the FGF
polypeptide family, FGF-10 exhibits affinity for heparin, which can be
utilized in its isolation, stabilization, and recovery, and cellular
heparan sulfate sites. In contrast to FGF-7, which elutes from
immobilized heparin at 0.8 M NaCl, FGF-10 elutes at 1.15 M NaCl, which is similar to FGF-1 (Fig.
8A). This is still below the
1.55 M that is required to elute FGF-2 from the same
column. Comparative Scatchard analysis of the binding of FGF-7 and
FGF-10 to pericellular matrix sites, which are presumed to be heparan
sulfate sites displayed on DT3 epithelial cells, revealed that FGF-10
binds to 1 × 105 sites per cell, while FGF-7 binds to
2.3 × 104 sites per cell (Fig. 8B). Both
factors exhibit a similar affinity (Kd = about 2.2 nM) to the pericellular matrix sites.
FGF-10 Is a Second Candidate Prostate Andromedin--
Here we show
that, similar to FGF-7, expression of FGF-10 is restricted to stromal
cells of normal adult prostate and androgen-responsive differentiated
non-malignant prostate tumors. FGF-10 mRNA levels were dependent on
androgen in cells from normal prostate and responsive in stromal cells
from a non-malignant, well differentiated transplantable tumor. In
addition, recombinant FGF-10 specifically stimulates epithelial cells
and binds specifically to the resident epithelial cell FGFR splice
variant, FGFR2IIIb, with similar affinity to FGF-7. Thus FGF-10
constitutes a second FGF polypeptide that is potentially involved in a
directional paracrine signaling system from prostate stroma to
epithelium. Stromal cells exhibit no response to either FGF-7 or
FGF-10. As proposed previously for FGF-7, FGF-10 is also a candidate
involved in relay of the action of androgen in the stroma to epithelial
cells (2, 3).
The full-length FGF-10 cDNA predicts a translation product of 23.8 kDa which exhibits a candidate secretory signal sequence, the removal
of which, in the absence of post-translational modification, would
result in a 19.8-kDa product (12). In vitro translation of
the full-length cDNA yields two products (12, 16). In agreement with a report which analyzed epitope-tagged FGF-10 overexpressed by
transfection in mammalian cells (16), FGF-10 antigen could not be
detected in the medium of cultured prostate stromal cells from
differentiated non-malignant tumors which naturally express the FGF-10
mRNA. Immunoanalysis with an antibody against bacterial recombinant
antigen revealed a single band with apparent mass of about 20 kDa in
extracts of the stromal cells that could not be detected in an
equivalent amount of extract from epithelial cells. Without
compensating for losses during recovery, the analysis suggested that
about 1 µg of FGF-10 antigen is associated with 108
cells. This single product was detectably smaller than the FGF-10 (37-214), which is the mature unmodified product predicted to arise by
removal of the secretory signal sequence. Although the single FGF-10
species may arise by an efficient proteolytic modification from a
higher molecular weight glycosylated form, which has been suggested to
exist in addition to unglycosylated species (16), it also may be an
unmodified, alternately translated product beginning at Met-42 with
predicted molecular mass of 19.2 kDa by amino acid content. An
alternately translated intracellular product is consistent with the
lower molecular weight product observed from in vitro translation (12, 16), the presence of a significant portion of FGF-10
antigen in cell extracts and the slightly lower molecular mass caused
by six less amino acid residues of the major prostate stromal cell
product. FGF-3, which exhibits similar receptor specificity to FGF-10
(7), also exhibits secreted and intracellular isoforms due to alternate
translational initiation (17). These observations raise the possibility
that disposition of FGF-10 product is also regulated at the level of
alternate initiation, and production of the secretory form from
prostate stromal cells requires the in vivo environment and
signals from neighboring epithelial cells.
The Different Expression Pattern of FGF-7 and FGF-10--
Despite
the apparent similarity in function in vitro, the expression
of FGF-10 appears much more restricted than FGF-7, which is expressed
broadly in the stromal or mesenchymal compartment of most embryonic and
adult tissue (18). FGF-10 was first identified in E14 rat embryos using
PCR primers homologous to FGF-3 and FGF-7, and reported to be expressed
only in adult lung and heart tissue (19). Subsequent reports showed
that FGF-10 was expressed at low levels in specific areas of the brain
(20) and prominently in the prospective limb mesoderm in the embryo
(20). We confirmed that expression of the FGF-10 mRNA is much more
restricted than FGF-7 in tissues and cells employed in the current
study. Although the FGF-10 to FGF-7 ratio is very low in
fibroblast-like cells, expression of FGF-10 was equal to or greater
than that of FGF-7 in prostate stromal cells which are thought to
consist of predominantly smooth muscle cells. The presence of
significant quantities of FGF-10 relative to FGF-7 in rat prostate
smooth muscle cells (PS-1), myoblasts, and tissues rich in smooth
muscle cells relative to skin suggest that the FGF-10-expressing
stromal cells from the non-malignant, transplantable Dunning R3327PAP
tumors (4, 21) likely derive from the transplanted tumor stroma rather
than invading stroma from the subcutaneous environment in the host
animals (4, 6). Although we cannot exclude the possibility that FGF-10 expression is induced in surrounding host tissue by presence of the
tumor or host stroma that has invaded the tumor, FGF-10 expression was
very low or undetectable in skin samples from subcutaneous pre-implantation sites. From these results we propose that FGF-10 might
be a useful marker to distinguish smooth muscle cells in mixed stromal
cell populations.
In addition, similar to normal prostate that has a well defined
epithelial and stromal compartment, FGF-10 is detectable only in tumors
that exhibit both compartments. This includes both the parent
pre-malignant DT tumor and SE tumors which were reconstituted with
isolated epithelial and stromal cells derived from the DT tumors. SE
tumors, which are at first slow growing and predominantly differentiated epithelial cells surrounded by stroma, but also exhibit
islands of undifferentiated cells (3), express a significantly reduced
ratio of FGF-7 to FGF-10. Only FGF-7 was observed in E tumors derived
from cloned DT tumor epithelial cells which are devoid of stroma within
the tumor and the fully malignant AT3 tumors that emerge from them.
Neither E nor AT3 tumors, which exhibit FGF-7 mRNA, have a distinct
stromal compartment. The fact that isolated and cloned cells (AT3-C)
derived from the malignant AT3 tumors after many generations in culture
still exhibit FGF-7 mRNA suggests that FGF-7, but not FGF-10, may
be abnormally activated in epithelial cells during the evolution to malignancy.
Interaction of FGF-10 with FGFR1IIIb in Addition to
FGFR2IIIb--
The specificity of FGF-7 for the resident epithelial
cell FGFR2IIIb isoform remains the most specific of the 18 FGF
polypeptides tested to date. We show here that FGF-7 and FGF-10
specifically stimulate DNA synthesis and compete with the resident FGFR
isoforms displayed on prostate epithelial cells with equal efficacy,
while no effect on the counterpart prostate stromal cells which do not express FGFR2IIIb could be detected at concentrations up to 100 ng/ml.
This is in contrast to a recent report which showed that FGF-10 was
only 50% as effective as FGF-7 in stimulation of DNA synthesis in
mouse keratinocytes and that FGF-10 stimulated DNA synthesis in mouse
3T3 fibroblasts at concentrations greater than 5 nM (100 ng/ml) (22). Although FGF-10 exhibited an equal affinity for
recombinant FGFR2IIIb, in contrast to FGF-7, it bound the FGFR1IIIb
isoform, but was 25 times less effective in competing FGF-1 from
FGFR1IIIb than from FGFR2IIIb. In this respect, FGF-10 appears to be
similar to FGF-3 which elicits a mitogenic response in transfected
cells bearing the FGFR1IIIb as well as the FGFR2IIIb ectodomain (5). A
detailed analysis of expression pattern of FGFR1IIIb relative to
FGFR1IIIc in the prostate tissue and cells utilized in this study is
under investigation. Previously we have demonstrated that the reduction
of resident FGFR2 gene products and the ectopic appearance of FGFR1 in
premalignant prostate tumor epithelial cells correlate with progression
to malignancy (3, 6). If the FGFR1IIIb isoform is part of the ectopic
activation of the FGFR1 gene in epithelial cells, then stromal-derived
FGF-10, which otherwise contributes to homeostasis of the epithelial
cell population through resident FGFR2IIIb, may contribute to malignant progression. Preliminary analysis of FGFR1IIIb expression in our model
rat prostate tissues indicates that expression of the isoform is
elevated in the premalignant differentiated tumors with well defined
stromal compartments, but not detectable in fully malignant tumors with
no clear stroma.2
Identification of the cell type expressing the FGFR1IIIb variant is
under investigation.
FGF-10 Differs from FGF-7 in the Interaction with Heparin and
Pericellular Matrix--
Heparin and transmembrane or pericellular
matrix heparan sulfate proteoglycans play roles in stabilization and
control of access of FGF to FGFR (1) and an integral role as a subunit of the FGFR complex (23, 24). In contrast to FGF-1, the mitogenic activity of FGF-7 on epithelial cells is conditionally inhibited by
soluble heparin (22, 25, 26), although the binding of FGF-7 to isolated
cell-free FGFR2IIIb is dependent on heparin similar to FGF-1 (26). In
the mitogenic assays and conditions utilized in this study, we observed
no significant effect of external heparin on the mitogenic activity of
FGF-1, FGF-7, or FGF-10 for prostate epithelial cells at concentrations
up to 100 µg/ml. However, as reported previously for FGF-7 and
FGFR2IIIb (26), heparin was required for the binding of all three
ligands to recombinant FGFR expressed on the surface of insect cells or
isolated and immobilized cell-free FGFR (results not shown). However,
our results show that FGF-10 has a higher affinity for immobilized
heparin than does FGF-7 and that the pericellular matrix of epithelial cells exhibits 4 times more binding sites for FGF-10 than for FGF-7.
Although this difference is not reflected as a differential mitogenic
or binding activity under the assay conditions employed, the results
suggest that heparan sulfate may differentially regulate access or
affinity of the two FGFs for FGFR2IIIb under specific conditions
present in the tissue microenvironment. We have recently demonstrated
that the heparan sulfate subunit of the FGFR kinase-cellular heparan
sulfate duplex discriminates between FGF ligands that can bind to the
duplex in contrast to heparin, which is an artificial mimic of
pericellular matrix heparan sulfate (27).
In conclusion, although FGF-7 and FGF-10 are potentially redundant in
respect to stromal origin, responsiveness to androgen and the binding
and activation of epithelial cells through FGFR2IIIb, the differential
expression among stromal cell subtypes, the differential interaction
with pericellular heparan sulfate and the affinity of FGF-10 for
FGFR1IIIb may confer unique roles on the two FGFs in a physiological context.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin was used as an internal mRNA level control.
-actin probes (2)
were added in the same RNase protection reaction. All three protection
probes were carefully standardized to the same specific activity and the relative expression was quantitated by densitometry of protected bands.
-Actin was used as the standard to normalize total RNA loads
across lanes from the different cell and tissue sources.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Comparative analysis of expression of FGF-10
and FGF-7 mRNA by PCR. cDNA templates reverse-transcribed
from mRNA of the indicated tissues and cells were used with FGF-10
and FGF-7-specific primers in the PCR described under "Experimental
Procedures." Upper part, FGF-7; lower part,
FGF-10. All lane 1, size standards in base pairs; all
lane 2, cDNA from rat lung as a positive control; all
lane 3, H2O as a negative control (no
templates). A, expression in rat prostate tissues and cells.
Lane 4, NP; lane 5, parent DT tumor tissue (DT);
lane 6, stromal cells from DT tumor (DT-S); lane
7, epithelial cells from DT tumor (DT-E); lane
8, reconstituted tumor from DT-derived epithelial and stromal
cells (SE); lane 9, tumor derived from DT
epithelial cells (E); lane 10, malignant AT3
tumor cells (AT3-C); lane 11, malignant AT3 tumor
tissue (AT3). B, comparative expression in diverse cells and
tissues. Lane 4, human fetal lung fibroblasts cells
(HLF); lane 5, NIH 3T3 cells (3T3); lane
6, human umbilical vein endothelial cells (HUVEC); lane
7, rat skeletal muscle myoblast cells (L6); lane 8, rat
prostate smooth muscle cells (PS1); lane 9, tissue isolated
from rat skin (Skin); lane 10, human foreskin
fibroblast cells (HFF); lane 11, mouse keratinocytes
(MK); lane 12, human hepatoblastoma cells
(HepG2); lane 13, human prostate tumor cells
(PC3); lane 14, tissue isolated from rat skeletal
muscle (Sk-Mu); lane 15, mouse small intestine
(S-Intes).
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Fig. 2.
Expression and androgen responsiveness of
FGF-10 and FGF-7 in prostate cells and tissues by RNase
protection. A, comparative expression of mRNA.
mRNA levels were determined by RNase protection using 20 µg of
total RNA from the indicated source as described under "Experimental
Procedures" and Refs. 2 and 3. Samples in the first two
lanes at left were the protection probe (3000 cpm) and
50 µg of yeast tRNA. DT, Dunning R3327PAP tumor tissue;
DT-S, cultured stromal cells from the DT tumor;
DT-E, cultured epithelial cells from DT tumor;
SE, tumor tissue arising from mixtures of DT-E and DT-S
cells 12 months after implantation; E, tumor tissue arising
from DT-E cells 6 months after implantation; E-C, cultured
cells from E tumors; AT3-C, cultured cells from Dunning
R3327AT3 tumors; AT3, AT3 tumor tissue; SKIN, tissue from
the subcutaneous injection site. B, the effect of androgen
on expression of FGF-7 and FGF-10. Nuclease protection analysis was
performed on total RNA from the cultured cell types treated with
dihydrotestosterone as indicated. NP-S, stromal cells from
normal prostate; DT-SP, stromal cells from DT tumors in
primary culture; DT-SL, a long term serially cultured line
of stromal cells from DT tumors. C, analysis of FGF-10
protein from DT-S. The pericellular matrix of DT-S cells was extracted
with NaCl and analyzed with anti-FGF-10 antiserum as described under
"Experimental Procedures." FGF-7 (54-193), 200 ng of
bacterial-derived recombinant FGF-7 beginning at residue Ser-54; FGF-10
(75-214), 200 ng of recombinant FGF-10 beginning at residue Ser-75;
FGF-10 (37-214), 200 ng of recombinant FGF-10 beginning at residue
Ala-37 with both Arg-71 and Arg-74 mutated to Ala; DT-S,
cultured stromal cells from the DT tumor; DT-E, cultured
epithelial cells from the DT tumor.
The relative levels of FGF-7 and FGF-10 mRNA in prostate cells and
tissues
-actin anti-sense RNA probes labeled to the same specific
activity in the same reaction mixture. Intensity of the three bands in
each lane was determined in arbitrary units by scanning densitometry
(Alpha Innotech Corp.). To compare expression levels between lanes,
band intensities were normalized to
-actin. The average density of
the
-actin bands across lanes of samples indicated was averaged
(107 ± 8.7 units). Intensity of the FGF-7 and FGF-10 bands in
each lane was adjusted by division by the intensity of the actin band
in the individual lane divided by the mean of all lanes. The density of
the FGF-7 band from DT-S cells (FGF-7, 123 units; FGF-10, 99 units;
-actin, 112 units) was assigned a value of 100% and all other band
densities were expressed as a percent of that value. The data shown is
representative of three independent reproductions.
8 M
dihydrotestosterone for 48 h and total RNA was isolated and assayed by nuclease protection (Fig. 2B). Expression of
FGF-10 mRNA in primary cultures of stromal cells from normal rat
prostate (NP-S) was completely dependent on androgen. Densitometric
analysis of protected autoradiographic bands indicated that
dihydrotestosterone stimulated the expression of FGF-10 mRNA by
about 4-fold in both primary (DT-SP) and serially cultured lines
(DT-SL) of stromal cells from DT tumors. As reported previously for
FGF-7, tumor-derived stromal cells were characterized by an elevated
level of baseline androgen-independent expression of FGF-10, which
rises, with extent of serial culture. No effect on the
-actin
control mRNA could be detected in this time frame (14).
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Fig. 3.
Selective stimulation of DNA synthesis by
FGF-10 in prostate epithelial cells. The indicated amounts of
recombinant FGF-1, FGF-2, FGF-7, or FGF-10 were added in the presence
of 25 µg/ml heparin to cultures of DT-E and DT-S cells. DNA synthesis
was determined by thymidine incorporation. The data shown is a
representative experiment of three independent reproductions.
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Fig. 4.
FGF-10 competes with radiolabeled FGF-1
binding to specifically prostate epithelial cells. Cultured DT-E
and DT-S cells were incubated with radiolabeled FGF-1 (2 ng/ml) and the
indicated amounts of unlabeled FGF isotypes in the presence of 2 µg/ml heparin. The data is representative of three separate
experiments. 100% binding represented (7.4 ± 0.38) × 103 and (1.6 ± 0.08) × 104 cpm in DT-E
and DT-S, respectively.
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Fig. 5.
Covalent affinity cross-linking of
radiolabeled FGF-10 to epithelial cell receptors. The indicated
radiolabeled FGFs were bound to DT-E and DT-S cells followed by
addition of DSS and analysis as described under "Experimental
Procedures." The positive analyses were intentionally overexposed to
reveal potential trace levels of FGF-2 bound to DT-E cells or FGF-7 and
FGF-10 bound to DT-S cells.
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Fig. 6.
Competition by FGF-10 of FGF
binding to recombinant FGFR2IIIb and FGFR1IIIb.
Sf9 insect cells infected with baculovirus bearing the indicated
FGFR cDNAs were incubated with radiolabeled FGF-1 and the indicated
amounts of FGF-10. Except for FGFR4, the closed and
open symbols indicate cells expressing the IIIb and IIIc
isoforms of FGFR, respectively. The plotted data is one of three
independent reproductions. 100% values were 1.8 ± 0.12 × 104, 1.2 ± 0.09 × 104, 1.2 ± 0.08 × 104, 1.4 ± 0.06 × 104,
5.5 ± 0.25 × 103, 1.1 ± 0.03 × 104, and 1.0 ± 0.07 × 104 cpm for
FGFR1IIIb, FGFR1IIIc, FGFR2IIIb, FGFR2IIIc, FGFR3IIIb, FGFR3IIIc,
and FGFR4, respectively. Inset, covalent cross-linking of 2 ng/ml radiolabeled FGF-10 to (left to right) FGFR1IIIb,
FGFR1IIIc, FGFR2IIIb, FGFR2IIIc, FGFR3IIIb FGFR3IIIc, FGFR4,
and uninfected Sf9 cells.
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Fig. 7.
Mutual competition of FGF-10 and FGF-7 for
binding to FGFR2IIIb. Sf9 cells expressing FGFR2IIIb were
incubated with the indicated radiolabeled FGF in the presence of
increasing amount of unlabeled FGF-7 (closed squares) or
FGF-10 (open squares).
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Fig. 8.
Differential interaction of FGF-7 and FGF-10
with heparin and peri-cellular matrix heparan sulfate sites.
A, elution from immobilized heparin. The indicated
radiolabeled FGFs were bound to heparin-agarose beads, then eluted by a
linear gradient of increasing concentrations of NaCl. B,
Scatchard analysis of the binding of FGF-7 and FGF-10 to heparan
sulfate-like sites on the pericellular matrix of prostate epithelial
cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. David M. Ornitz (George Washington University, St. Louis, MO) for providing FGFR1IIIb and FGFR3IIIb cDNAs, Dr. David Rowley (Baylor College of Medicine, Houston, TX) for the PS1 cell line, and Makiko Kan for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grants DK40739 and DK35310 from the NIDDK, National Institutes of Health, and NCI, National Institutes of Health, Grant CA59971.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.
Contributed equally to the results of this work.
§ To whom correspondence should be addressed: Institute of Biosciences and Technology, Texas A&M University System Health Science Center, 2121 W. Holcombe Blvd., Houston, TX 77030-3303. Tel.: 713-677-7522; Fax: 713-677-7512; E-mail: wmckeeha{at}ibt.tamu.edu.
2 W. Lu, Y. Luo, M. Kan, and W. L. McKeehan, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor kinase; FGFR1-4, type 1 through 4 of the FGFR kinases; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; NIH 3T3, fibroblast-like mesenchymal cells; NP, normal prostate; DT-E, Dunning tumor epithelial cells; DT-S, Dunning tumor stromal cells.
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