We isolated the cDNA encoding a novel member
(207 amino acids) of the fibroblast growth factor (FGF) family from rat
embryos. Because this protein is the 18th documented member of the FGF family, we tentatively termed it FGF-18. We have also determined mouse
and human FGF-18 with high amino acid identity (99.5 and 99.0%) to rat
FGF-18, respectively. Among FGF family members, FGF-18 is most similar
(52.7% amino acid identity) to FGF-8 and FGF-17. FGF-18 has a typical
signal sequence at its amino terminus. Recombinant rat FGF-18, which
was efficiently secreted by High Five insect cells infected with
recombinant baculovirus containing the cDNA, induced neurite
outgrowth in PC12 cells. The expression of FGF-18 mRNA
was examined in adult rat tissues and embryos by Northern blotting
analysis and in situ hybridization. FGF-18
mRNA of ~2.7 kilobases was preferentially detected in the lung
among adult rat tissues examined. In rat embryos, FGF-18
mRNA was detected in several discrete regions at embryonic days
14.5 and 19.5 but not at E10.5. The temporal and spatial patterns of
FGF-18 mRNA expression in embryos are quite different
from those of FGF-8 and FGF-17 mRNAs
reported. The present results indicate that FGF-18 is a unique secreted
signaling molecule in the adult lung and developing tissues.
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INTRODUCTION |
The prototypic fibroblast growth factors
(FGFs),1 FGF-1 (acidic FGF)
and FGF-2 (basic FGF), were originally isolated from the brain and
pituitary as mitogens for fibroblasts. FGF-1 and FGF-2 are widely
expressed in developing and adult tissues and are polypeptides with
multiple biological activities including angiogenesis, mitogenesis, cellular differentiation, and repair of tissue injury (1, 2). The FGF
family now consists of 17 members, FGF-1 to FGF-17. They have a
conserved ~120-amino acid residue core with ~30-60% amino acid
identity. FGF-3 was identified to be a common target for activation by
the mouse mammary tumor virus (3). FGF-4 to FGF-6 were identified as
oncogene products (4-6). FGF-7 to FGF-9 were identified as mitogens
for culture cells (7-9). FGF-10 was identified from the rat lung by
homology-based polymerase chain reaction (PCR) (10). FGF-11 to FGF-14
(FGF homologous factors (FHFs)-1 to -4) were identified from the human
retina by a combination of random cDNA sequencing, data base
searches, and homology-based PCR (11). FGF-15 was identified as a
downstream target of a chimeric homeodomain oncoprotein (12). FGF-16
and FGF-17 were identified from the rat heart and embryos by
homology-based PCR, respectively (13, 14). These FGFs also appear to
play important roles in both developing and adult tissues. Recently, we
isolated the cDNA encoding a novel member, the 18th documented, of
the FGF family from rat embryos by homology-based PCR. Here, we report the structure and expression of FGF-18 mRNA.
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EXPERIMENTAL PROCEDURES |
Preparation of RNA--
RNA was prepared from adult rat
tissues, rat embryos, and mouse embryos using an RNA extraction kit
(Amersham Pharmacia Biotech). Poly(A)+ RNA was prepared
using oligo(dT)-cellulose (Type 2, Collaborative Biomedical Products,
Bedford, MA).
Isolation and Analysis of Rat, Mouse, and Human FGF-18
cDNAs--
The cDNA was synthesized from rat embryo (E14.5)
poly(A)+ RNA as described (10). To amplify cDNAs, PCR
was performed for 30 cycles in 25 µl of a reaction mixture containing
an aliquot of the above cDNA solution and 5 pmol/µl of each of
the sense and antisense degenerate primers representing all possible
codons corresponding to the amino acid sequences, ETDTFG and ENNYTA, of
the core of mouse FGF-8 (8), respectively, as described (14). The
amplified DNA of expected size (approximately 150 base pairs) was
cloned into the pGEM-T DNA vector (Promega, Madison, WI). The
nucleotide sequence of the cloned DNA was determined by a DNA sequencer
(Applied Biosystems, Foster City, CA). To determine the entire coding
region, the rat embryo cDNA was analyzed by rapid amplification of
cDNA ends (15).
The human FGF-18 cDNA was amplified from the cDNA
synthesized from human poly(A)+ RNA
(CLONTECH) and analyzed essentially according to
the method described above. The mouse FGF-18 cDNA was
amplified from the mouse embryo cDNA (E13.5) by PCR with primers
corresponding to the rat FGF-18 cDNA sequence and
analyzed. The apparent evolutionary relationships of members of the FGF
family were examined by the unwaited pair-group method with arithmetric
mean method with the sequence analysis software, Genetyx (Software
Development Co., Tokyo, Japan).
Production of Recombinant Rat FGF-18 in High Five Insect
Cells--
The rat FGF-18 cDNA with a DNA fragment (75 base pairs) encoding an E tag (GAPVPYPDPLEPR) and a His6
tag (HHHHHH) at the 3' terminus of the coding region was constructed in
a transfer vector DNA, pBacPAK9
(CLONTECH). Recombinant baculovirus containing the FGF-18 cDNA with the tag sequences was obtained by
cotransfection of Sf9 cells with the recombinant
pBacPAK9 and a Bsu36I-digested expression vector,
BacPAK6 (CLONTECH). High Five insect
cells were infected with the resultant recombinant baculovirus and
incubated at 27 °C for 24 h in TC-100 insect medium (Life
Technologies, Inc.) with 10% fetal bovine serum. After the infection,
the cells were further cultured at 27 °C for 60 h in the medium
without fetal calf serum.
Detection of Recombinant FGF-18 by Western Blotting
Analysis--
The culture medium and cell lysate of High Five cells
infected with the recombinant baculovirus were separated by
SDS-polyacrylamide gel (12.5%) electrophoresis under reducing
conditions and transferred onto a nitrocellulose membrane (Hybond-ECL,
Amersham Pharmacia Biotech). The membrane was incubated with anti-E tag
antibodies (1:500) (Amersham Pharmacia Biotech). The protein with the E
tag was visualized as described (14).
Neurite Outgrowth Assay--
Cultures of PC12 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum and 5% horse serum at 37 °C in a humidified
atmosphere of 5% CO2 air. Cells were seeded in 24-well
culture plates coated with poly-L-lysine. After 48 h,
the cultures were supplemented with the culture medium of High Five
insect cells and further maintained for 72 h. Outgrowth of
neurites in PC12 cells was monitored under a phase contrast microscope.
Northern Blotting Analysis--
Aliquots of RNAs (20 µg) from
rat embryos and adult tissues were dissolved on a denaturing agarose
gel (1%) containing formaldehyde and transferred to a nitrocellulose
membrane in 20× SSC (1× SSC = 0.15 M NaCl/0.015
M sodium citrate) overnight. A 32P-labeled
FGF-18 cDNA probe (~650 base pairs) was labeled by a random primer labeling kit (Amersham Pharmacia Biotech) with
deoxycytidine 5'-[
-32P]triphosphate (~110 TBq/mmol)
(ICN Biomedicals Inc., Costa Mesa, CA). The membrane was incubated in
hybridization solution containing the labeled probe as described (14)
and analyzed with a radio-imaging analyzer (BAS 2000, Fuji Photo Film
Co., Tokyo, Japan).
In Situ Hybridization--
Wistar rat embryos (E14.5 and E19.5)
were frozen in powdered dry ice, and sagittal sections were cut at 16 µm with a cryostat, thaw-mounted onto
poly-L-lysine-coated slides, and stored at
85 °C until hybridization. A 35S-labeled rat
FGF-18 antisense cRNA probe was transcribed using SP6 RNA
polymerase (TaKaRa, Kusatsu, Japan) with uridine
5'-[
-35S]thiotriphosphate (~30 TBq/mmol) (Amersham
Pharmacia Biotech). The sections were examined by in situ
hybridization with the labeled probe as described (10).
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RESULTS AND DISCUSSION |
Isolation and Analysis of the Rat, Mouse, and Human cDNAs
Encoding FGF-18--
Members of the FGF family have a conserved
~120-amino acid residue core with ~30-70% amino acid identity. To
isolate cDNAs encoding novel FGFs, cDNAs were amplified from
cDNAs of rat adult tissues and embryos as templates by PCR using
various sets of primers corresponding to the core of the FGF family and
then cloned. We isolated a cDNA fragment encoding a novel FGF from
rat embryo (E14.5) cDNA as a template using primers corresponding
to amino acid sequences, ETDTFG (amino acids 78-83) and ENNYTA (amino
acids 124-129), of the core of FGF-8 (see Fig. 2) (8). The nucleotide sequence of the entire coding region was determined by rapid
amplification of cDNA ends using the rat embryo cDNA as a
template. The nucleotide sequence of the coding region allowed
elucidation of the complete amino acid sequence of a novel FGF (207 amino acids), which has a conserved amino acid residue core (amino
acids 45-164) (Fig. 1). Because this
protein is the 18th documented protein related to FGFs, we tentatively
term it FGF-18. We also isolated the mouse and human FGF-18
cDNAs from the mouse embryo (E13.5) and human lung cDNAs,
respectively. The nucleotide sequences allowed elucidation of the amino
acid sequences of mouse and human FGF-18, which have high identity
(99.5 and 99.0% amino acid identity) to rat FGF-18, respectively (Fig.
1). Among FGF family members, FGF-18 is most similar (52.7% amino acid
identity) to FGF-8 and FGF-17 (8, 14) (Fig.
2). The apparent evolutionary
relationships of 18 members of the FGF family are shown in Fig.
3. FGF-18 was closest to FGF-8 and
FGF-17.

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Fig. 1.
Comparison of amino acid sequences of rat,
mouse, and human FGF-18. The numbers refer to the amino
acid positions of FGF-18. Asterisks indicate identical amino
acid residues of the sequences.
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Fig. 2.
Comparison of amino acid sequences of mouse
FGF-8, FGF-17, and FGF-18. The numbers refer to the
amino acid positions of FGF-8, FGF-17, and FGF-18. Asterisks
indicate identical amino acid residues of the sequences.
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Fig. 3.
The apparent evolutionary relationships of 18 members of the FGF family. The length of each horizontal
line is proportional to the degree of amino acid sequence
divergence.
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Two cysteine residues are well conserved in the FGF family, and these
amino acids correspond to residues 48 and 127 in the FGF-18 sequence
(Fig. 1). Although a cysteine residue was found at position 127, a
serine instead of a cysteine residue was found at position 48 (Fig. 1).
The same substitution was also observed in FGF-8, FGF-10, and FGF-17
sequences (8, 10, 14). Although FGF-1, FGF-2, FGF-9, FHF-1 to FHF-4,
and FGF-16 have no typical signal sequence in their termini (2, 11),
FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-15, and FGF-17 have
typical signal sequences and are secreted proteins (3-7, 9, 12, 14).
FGF-18 also has a hydrophobic amino terminus (~27 amino acids), which
is a typical signal sequence, and appears to be a secreted protein. The
signal sequence cleavage site was predicted to lie between amino acid
positions 27 (Ala) and 28 (Glu) by the method of von Heijne (16).
Production of Recombinant Rat FGF-18 in High Five Insect
Cells--
To produce recombinant rat FGF-18, High Five insect cells
were infected with recombinant baculovirus containing the rat
FGF-18 cDNA with the 3'-terminal extension encoding E
and His6 tags. To detect recombinant FGF-18, both the
culture medium and cell lysate were examined by Western blotting
analysis with anti-E tag antibodies. A major band of approximately 28 kDa was detected only in the culture medium, indicating that FGF-18 is
efficiently secreted (Fig. 4). The
observed molecular mass was larger than the calculated molecular mass
of recombinant FGF-18 (23,731 Da). Because a possible
N-glycosylation site is found at position 137 (Asn), FGF-18
might be glycosylated.

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Fig. 4.
Detection of recombinant rat FGF-18 from the
culture medium and cell lysate of High Five cells infected with
recombinant baculovirus containing the rat FGF-18
cDNA. The culture medium and cell lysate of the
recombinant baculovirus-infected High Five cells were separated by
SDS-polyacrylamide gel (12.5%) electrophoresis. Recombinant rat FGF-18
was detected by Western blotting analysis with anti-E tag antibodies.
Prestained Protein Marker Broad Range (New England Biolabs) was used as
molecular mass standard proteins.
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Neurite Outgrowth Activity of Recombinant Rat FGF-18--
FGFs
exhibit neurotrophic properties similar to those of neurotrophins (17).
The PC12 cell line has provided a useful model for studying the actions
of neurotrophins (18). These cells respond to FGFs and neurotrophins by
the elaboration of a sympathetic neuron-like phenotype. To examine the
biological activity of FGF-18, the culture medium of High Five cells
containing FGF-18 was added to PC12 cells. The medium induced neurite
outgrowth in PC12 cells (Fig. 5). In
contrast, the control medium containing no FGF-18 did not induce
neurite outgrowth in PC12 cells. FGF-induced neurite outgrowth in PC12
cells occurs via FGF receptor (FGFR)-1 but not the other FGFRs (FGFR-3
and FGFR-4) expressed in these cells (19). These results indicate that
FGF-18 can activate at least FGFR-1.

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Fig. 5.
Effect of recombinant rat FGF-18 on neurite
outgrowth in PC12 cells. PC12 cells were plated onto
poly-L-lysine-coated dishes. After 48 h, the cells
were supplemented with the culture supernatant of High Five cells
containing FGF-18. Cells supplemented with the culture medium
containing no FGF-18 were used as a control. The morphology of the
cells was examined after 72 h of treatment.
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Expression of FGF-18 mRNA in Adult Rat Tissues and Rat
Embryos--
The expression of FGF-18 mRNA in adult rat
tissues was examined. RNA from the brain, heart, lung, liver, kidney,
and small intestine was examined by Northern blotting analysis using a
32P-labeled FGF-18 cDNA probe. The integrity
of RNA was confirmed by electrophoresis on a denaturing agarose gel
containing formaldehyde. The labeled probe hybridized to a mRNA of
approximately 2.7 kilobases in the lung (Fig.
6). However, the mRNA was not
detected in the brain, heart, liver, kidney, and small intestine. The
amino acid sequence of FGF-18 is highly homologous to that of FGF-8 and
FGF-17. Among adult tissues, the FGF-8 mRNA expression
was weak and confined to the gonadal tissue (20). FGF-17
mRNA was not detected in adult major tissues examined (14). In
contrast, FGF-18 mRNA was abundantly expressed in the
lung. The expression profile of FGF-18 mRNA is quite
different from that of FGF-8 and FGF-17
mRNAs

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Fig. 6.
Expression of FGF-18
mRNA in rat adult tissues and embryos. Aliquots of total
RNA (20 µg each) were electrophoresed on a denaturing agarose gel
(1%) containing formaldehyde and transferred onto a nitrocellulose
membrane. Hybridization was performed with a 32P-labeled
rat FGF-18 cDNA probe. The positions of 28 and 18 S RNAs
are indicated. Lanes E10.5, E14.5,
E19.5, Brain, Heart, Lung,
Liver, Kidney, and Small Intestine
indicate RNA from rat E10.5, E14.5, and E19.5 embryos and the adult
brain, heart, lung, liver, kidney, and small intestine,
respectively.
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To examine the expression of FGF-18 mRNA in
rat embryos, RNA from embryos at three different embryonic stages
(E10.5, E14.5, and E19.5) was also examined by Northern blotting
analysis. FGF-18 mRNA was detected in the embryos at
E14.5 and E19.5. but not at E10.5 (Fig. 6). The expression of
FGF-18 mRNA in the embryos at E14.5 and E19.5 was also
examined by in situ hybridization with a
35S-labeled antisense FGF-18 cRNA probe,
followed by macroautoradiography. In the embryo at E14.5, discrete
labeling was observed in various regions including the isthmus,
pituitary, spinal cord, tongue, intervertebral disc, dorsal root
ganglion, and pelvis (Fig. 7). In
contrast, discrete labeling in the embryo at E19.5 was observed in
restricted regions including the lung and anterior pituitary (Fig. 7).
FGF-8 mRNA was detected at E10.5 to E12.5 but not at E13.5 in mouse embryos (20). FGF-17 mRNA was detected at
E14.5 but not at E10.5 and E19.5 in rat embryos (14). In contrast, FGF-18 mRNA was detected at E14.5 and E19.5, but not at
E10.5 in rat embryos. The spatial expression patterns of
FGF-8 and FGF-17 mRNAs in embryos were highly
restricted (14, 20). However, FGF-18 mRNA was expressed
in various regions in rat embryos. The temporal and spatial patterns of
FGF-18 mRNA expression in embryos is also quite
different from those of FGF-8 and FGF-17
mRNAs.

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Fig. 7.
Localization of FGF-18
mRNA in sagittal sections of rat embryos (E14.5 and
E19.5). Sagittal sections of rat embryos were hybridized with an
35S-labeled antisense FGF-18 cRNA probe and
exposed to x-ray film (B and D). The sections
were also counterstained with hematoxylin and eosin (A and
C). Pi, pituitary; Is, isthmus;
Sp, spinal cord; To, tongue; ID,
intervertebral disc; DRG, dorsal root ganglion;
Pe, pelvis; Lu, lung. Scale bars, 0.5 cm.
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During developmental processes, FGFs were shown to play important roles
in the induction and patterning of developing tissues. FGF-8 was shown
to be an important signaling molecule in the midbrain development and
limb development (21, 22). FGF-17 is also thought to be a signaling
molecule in the midbrain and forebrain (14). The present results
indicate that FGF-18 is a unique secreted signaling molecule in the
developing tissues.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB004638, AB004639, and AB007422.