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INTRODUCTION |
Complex cellular processes such as proliferation, migration,
differentiation, and apoptosis are regulated in part by a diverse group
of molecules known as polypeptide growth factors. These factors act by
binding and thereby activating specific transmembrane receptor tyrosine
kinases. The activation of cell surface receptors by polypeptide
ligands triggers downstream intracellular events, including the
stimulation of protein phosphorylation cascades and the transcriptional
activation of numerous genes (1, 2). Many mitogen-inducible genes have
been identified, and they encode a diverse group of proteins including
transcription factors, protein kinases and phosphatases, cell cycle
regulators, and cytoskeletal and extracellular matrix proteins (2, 3).
A recent study using cDNA microarray technology has demonstrated
that >500 genes are transcriptionally activated after serum
stimulation of quiescent human fibroblasts and that a subset of these
genes encode proteins implicated in the wound healing process in
vivo (3).
Our laboratory has been studying fibroblast growth factor-1
(FGF-1)1-regulated gene
expression in murine NIH 3T3 cells. FGF-1 (also referred to as acidic
FGF) is one of the most extensively characterized members of the FGF
family of heparin-binding proteins (4-6). It is a potent mitogenic,
chemotactic, angiogenic, and neurotrophic factor both in
vitro and in vivo. These cellular responses are mediated via high affinity binding to a family of related
membrane-spanning tyrosine kinase receptors (4-6). We have shown by
Northern blot hybridization analysis that FGF-1 stimulation of
quiescent NIH 3T3 cells induces the expression of several previously
described serum-regulated genes; e.g. c-fos,
c-jun, c-myc, thrombospondin-1, and ornithine
decarboxylase (7, 8). In addition, we have used a differential display
approach to isolate several cDNA clones representing previously
unreported mitogen-inducible genes (9). Genes identified to date using
this strategy include those encoding an aldose reductase-related
protein (10, 11), a member of the polo family of serine/threonine
protein kinases (12, 13), and a member of the transcriptional enhancer
factor-1 family of DNA-binding proteins (14).
In this paper, we report that the murine Fn14 gene is a growth factor-
and phorbol ester-regulated immediate-early response gene encoding a
novel type Ia transmembrane protein, with an amino-terminal signal
peptide, a 53-aa ectodomain, a single hydrophobic transmembrane domain,
and a 28-aa carboxyl-terminal cytoplasmic domain. Analysis of
transfected NIH 3T3 cell lines that constitutively express the Fn14
protein indicate that it may be involved in the regulation of cellular
adhesion, growth, and migration.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Murine NIH 3T3 fibroblasts (American Type
Culture Collection) were grown, expanded, and serum-starved as
described (15). Serum-starved cells were then either left untreated or
treated with one of the following: 10 ng/ml human recombinant FGF-1
(kind gift of W. Burgess, Holland Laboratory) in combination with 5 units/ml heparin (Upjohn), 10 ng/ml human recombinant FGF-2 (Bachem), 10 ng/ml human recombinant PDGF-BB (Genzyme), 2 ng/ml human recombinant TGF-
1 (R&D Systems), 20 ng/ml human recombinant IGF-1 (Bachem), 10%
calf serum (HyClone), or 30 ng/ml PMA (Sigma). In some experiments, NIH
3T3 cells were treated with 2 µg/ml actinomycin D (Calbiochem) or 10 µg/ml cycloheximide (Sigma).
RNA Isolation and Differential Display--
Cells were harvested
by trypsin/EDTA treatment, and total RNA was isolated using RNA Stat-60
(Tel-Test) per the manufacturer's instructions. Tissues from newborn
or adult FVB/N mice (Taconic Farms) were obtained, and RNA was isolated
as above after the samples were initially homogenized using a
Tissumizer (Tekmar). RNA concentrations were calculated by measuring UV
light absorbance at 260 nm. RNA (1 µg) from quiescent or
FGF-1/cycloheximide-treated NIH 3T3 cells was converted to cDNA
using Moloney murine leukemia virus reverse transcriptase (Life
Technologies, Inc.) and random hexamer primers (Roche Molecular
Biochemicals) as described (16). PCR assays were performed using a
degenerate sense protein kinase domain oligonucleotide primer (12) and
a degenerate antisense leucine zipper domain oligonucleotide primer
(17). An equivalent aliquot of each amplification mixture was subjected
to electrophoresis in a 2% agarose gel, and DNA was visualized by
ethidium bromide staining. An ~230-bp DNA fragment was excised,
recovered using the freeze-squeeze method (18), reamplified, and
ligated into the vector pCRII using a T/A cloning kit (Invitrogen).
cDNA Library Screening--
A mouse Balb/c 3T3 cell cDNA
library (kind gift of T. Lanahan, Johns Hopkins University School of
Medicine, Baltimore, MD) was screened with the PCR-derived Fn14
cDNA fragment to obtain longer cDNA clones. The DNA fragment
was labeled with [
-32P]dCTP (3000 Ci/mmol, Amersham
Pharmacia Biotech) using a random primer DNA labeling kit (Roche
Molecular Biochemicals). Approximately 2 × 105 phage
were plated at a density of 2 × 104 plaque-forming
units/150-mm dish using Escherichia coli C600 Hfl as the
host. Duplicate plaque lifts (Colony/Plaque screen, DuPont) were
pre-hybridized, hybridized, washed and exposed to Kodak X-Omat AR film
as described (14). Five positive phage were plaque-purified and
amplified on E. coli C600 Hfl cells. Plasmids were excised
from the phage clones using XL-1 Blue cells and R408 helper phage
(Stratagene) as described (19) and designated pBluescript/Fn14.
5'-RACE Assays--
A cDNA fragment representing the 5'
region of Fn14 mRNA was identified by PCR using mouse heart
5'-RACE-Ready cDNA (CLONTECH) per the
manufacturer's instructions. The Fn14 antisense oligonucleotide primer
used in the PCR amplification step was complementary to nucleotides
361-381 of the Fn14 cDNA sequence. Amplification products were
subjected to electrophoresis in a 1.2% agarose gel and visualized by
ethidium bromide staining. One DNA fragment was recovered and cloned
into the vector pCRII using a T/A cloning kit (Invitrogen).
cDNA Sequence Analysis--
Plasmid DNA was purified using a
Magic Miniprep kit (Promega), and both strands of the entire ~950-bp
Fn14 cDNA clone were sequenced by the dideoxynucleotide chain
termination method. Both strands of the Fn14 cDNA fragment isolated
by the 5'-RACE technique were also completely sequenced. Sequencing was
done either automatically using an Applied Biosystems model 373A DNA
sequencer and a Dye Terminator Cycle Sequencing kit (Perkin-Elmer) or
manually using a Sequenase 2.0 kit (U.S. Biochemical Corp.) and
[-35S]dATP (1000 Ci/mmol, Amersham Pharmacia Biotech).
The nucleic acid and deduced protein sequences were compared with
sequences in the GenBank sequence data base using BLAST search programs accessed through the National Center for Biotechnology Information web
site. The predicted Fn14 protein sequence was analyzed using several
programs (SignalP, ScanProsite, PSORT II, TMpred, etc.) accessed
through the ExPASy Molecular Biology Server.
Interspecific Mouse Backcross Mapping--
Interspecific
backcross progeny were generated by mating (C57BL/6J × Mus
spretus)F1 females and C57BL/6J males as described (20). A total of 205 N2 mice were used to map the
Fn14 locus (see text for details). DNA isolation,
restriction enzyme digestion, agarose gel electrophoresis, Southern
blot transfer, and hybridization were performed essentially as
described (21). All blots were prepared with Hybond-N+
nylon membranes (Amersham Pharmacia Biotech). The probe, an ~1.0-kb EcoRI/XhoI cDNA insert from the
pBluescript/Fn14 plasmid, was labeled with [-32P]dCTP
(3000 Ci/mmol; Amersham Pharmacia Biotech) using a random primer
labeling kit (Stratagene). Blots were washed to a final stringency of
0.8× SSCP, 0.1% SDS, 65 °C. A fragment of ~8.0 kb was detected
in EcoRI-digested C57BL/6J DNA, and a fragment of ~6.0 kb
was detected in EcoRI-digested M. spretus DNA.
The presence or absence of the ~6.0-kb EcoRI M. spretus-specific fragment was followed in backcross mice. A
description of the probes and RFLPs for the loci linked to
Fn14 including Mas1, E4f1, and Pim1
has been reported previously (22, 23). Recombination distances were
calculated using Map Manager, version 2.6.5. Gene order was determined
by minimizing the number of recombinant events required to explain the
allele distribution patterns.
Northern Blot Analysis--
RNA samples (10 µg) were denatured
and subjected to electrophoresis in 1.2% agarose gels containing 2.2 M formaldehyde. The gels were stained with ethidium bromide
to verify that each lane contained similar amounts of undegraded rRNA.
RNA was transferred onto Zetabind nylon membranes (Cuno Inc.) by
electroblotting and cross-linked to the membrane by UV light
irradiation using a Stratalinker (Stratagene). A Northern blot
containing 2 µg of poly(A)+ RNA isolated from mouse
embryos at different developmental stages was purchased from
CLONTECH. Membrane pre-hybridization,
hybridization, and washing conditions were as described (14). The two
cDNA hybridization probes were: (a) mouse Fn14,
~1.0-kb EcoRI/XhoI fragment of
pBluescript/Fn14, and (b) mouse -actin, ~1.1-kb
EcoRI fragment of pVAA (kind gift of G. Liau, Genetic
Therapy Inc., Gaithersburg MD). The probes were radiolabeled with
[-32P]dCTP as described above under cDNA library screening.
In Situ Hybridization--
A pBluescript/Fn14 plasmid containing
the Fn14 cDNA sequence without the 3'-untranslated region was
constructed and linearized using the appropriate enzymes, and sense or
antisense riboprobes were transcribed in vitro using T3 or
T7 RNA Polymerase (Roche Molecular Biochemicals),
[-35S]UTP (1000 Ci/mmol, NEN Life Science Products)
and reagents included in the SureSite II in situ
hybridization kit (Novagen). Riboprobes were subjected to alkaline
hydrolysis to yield an average length of ~100 nt, as verified by
denaturing PAGE. Embryo sections (Novagen) were dewaxed,
rehydrated, and deproteinized according to the hybridization kit
instructions. Pre-hybridization and hybridization conditions were also
per the manufacturer's instructions except that the hybridization step
was performed at 65 °C. Post-hybridization washes were once with 2×
SSC for 5 min at room temperature, once with 2× SSC for 30 min at
50 °C, once with 2× SSC containing 20 µg/ml RNase A for 30 min at
37 °C, once with 2× SSC containing 50% formamide for 30 min at
50 °C, twice with 1× SSC for 30 min at 50 °C, and once with
0.1× SSC for 30 min at 65 °C. Slides were dipped in Kodak NTB-2
emulsion, counterstained with hematoxylin and eosin, and mounted.
Expression of Recombinant Fn14 in Bacterial Cells and Generation
of Fn14 Antiserum--
Recombinant Fn14 was produced by expression of
the cDNA in E. coli as a GST fusion protein. The
expression plasmid was constructed by ligation of an
EcoRI-XhoI restriction fragment of
pBluescript/Fn14 into the same sites located in the polylinker of
pGEX-KG (kind gift of R. Friesel, Maine Medical Center Research
Institute, Portland ME). DNA sequence analysis was performed to confirm
the construct. E. coli HB101 cells (Life Technologies, Inc.)
that had been transformed with this plasmid were cultured overnight at
room temperature in Luria broth containing 100 µg/ml ampicillin
(Sigma). Cells were diluted 1:10 in Luria broth, grown for 5 h at
room temperature, induced with 0.1 mM
isopropyl--D-thiogalactopyranoside (Life Technologies,
Inc.) for 3 h, and pelleted by centrifugation. Cells were
resuspended by repeated pipetting in STE buffer (10 mM
Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA)
containing 1 mg/ml lysozyme (Sigma) and incubated for 20 min on ice.
Dithiothreitol (Sigma) was added to a final concentration of 5 mM, the lysate was vortexed, N-laurylsarcosine (Sigma) was added to a final concentration of 1.5%, and again the
lysate was vortexed. After mild sonication, the lysate was clarified by
centrifugation, and Triton X-100 was added to the supernatant at a 2%
final concentration. The supernatant was then incubated with hydrated
glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) for 30 min
at 4 °C with end-over-end mixing. The beads were collected by a
brief centrifugation and washed five times with PBS containing 1%
Triton X-100. To prepare antigen for rabbit immunization, GST-Fn14 was
released from glutathione-Sepharose beads by the addition of 100 mM Tris/HCl, pH 6.8, 4% SDS, 20% glycerol, 10%
2-mercaptoethanol and dialyzed against PBS. A New Zealand White rabbit
was injected with ~0.5 mg of the antigen in complete Freund's
adjuvant (Calbiochem) and boosted five times with ~0.3 mg of the
antigen in incomplete adjuvant (Calbiochem). Crude serum was used for
the Western blot experiments.
Construction of the Fn14-GFP and Fn14-HA Eukaryotic Expression
Plasmids--
The plasmid pEGFP-N3/Fn14, which encodes Fn14 with a
carboxyl-terminal EGFP tag (24), was constructed as follows. PCR was performed using pBluescript/Fn14 as the template, a sense primer containing a 5' BamHI restriction site followed by Fn14
nucleotides 
12 to +12, an antisense primer representing Fn14
nucleotides 380-399 and Taq polymerase (Life Technologies,
Inc.). The DNA product was isolated and then ligated into pCR2.1 using
a T/A cloning kit (Invitrogen). A BamHI/EcoRI
fragment representing the Fn14 coding region was isolated (18) and
subcloned into the BglII and EcoRI cloning sites
of the expression vector pEGFP-N3 (CLONTECH). The
plasmid pcDNAIneo/Fn14-HA, which encodes Fn14 with a
carboxyl-terminal influenza HA epitope tag (25), was constructed by
first subcloning an EcoRI/XhoI fragment of
pBluescript/Fn14 into the EcoRI and HpaI cloning
sites of the expression vector pMEXneo (26). DNA sequence encoding the
HA epitope tag was added to the Fn14 cDNA immediately upstream of
the stop codon using the PCR overlap extension method with
Taq polymerase (27). A NotI/ApaI
fragment containing the Fn14-HA coding sequence was filled-in using T4
DNA polymerase and then cloned into the EcoRV site of the
expression vector pcDNAIneo (Invitrogen). DNA sequence analysis was
performed to confirm the identity of the two expression constructs
described above.
Transfection Experiments--
NIH 3T3 cells were grown to
~60% confluence and transfected with pEGFP-N3, pEGFP-N3/Fn14,
pcDNAIneo, or pcDNAIneo/Fn14-HA using the LipofectAMINE PLUS
reagent (Life Technologies, Inc.). The first two plasmids were used for
transient transfection experiments. To generate stable cell lines,
cells were transfected with either pcDNAIneo or
pcDNAIneo/Fn14-HA, cultured for 24 h in standard growth
medium, and split 1:10. After 24 h, cells were cultured in medium
containing 400 µg/ml G418 (Life Technologies, Inc.). Individual
G418-resistant colonies were visible approximately 10 days later, and
they were recovered with glass cloning cylinders. Clones were screened
for Fn14-HA expression by Western blot analysis.
Western Blot Analysis--
Cells were washed with PBS, collected
by scraping with a rubber policeman, and pelleted by centrifugation at
500 × g. TNEN buffer (50 mM Tris/HCl, pH
7.5, 150 mM NaCl, 2.0 mM EDTA, 1% Nonidet P-40, 1× protease inhibitor mixture; PharMingen) was added, and lysis
was performed for 10 min at 4 °C. Lysates were clarified by
centrifugation at 15,000 × g for 10 min at 4 °C.
Protein concentrations were determined using the BCA assay kit
(Pierce). Equivalent amounts of each protein sample were mixed with 2×
gel loading buffer (100 mM Tris/HCl, pH 6.8, 4% SDS, 20%
glycerol, 10% 2-mercaptoethanol, 0.2% bromphenol blue), heated at
95 °C for 10 min, and subjected to SDS-PAGE using either a 15% or
4-15% gradient acrylamide gel. For the FGF-2 time course experiment,
proteins were transferred to an Immobilon-PSQ membrane
(Millipore) by electroblotting. The membrane was stained with Ponceau S
(Sigma) to verify that equivalent amounts of protein were present in
each gel lane and then blocked overnight at 4 °C in TBST (25 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.1% Tween
20) containing 5% nonfat dry milk. The membrane was incubated for 1 h at room temperature in TBST containing 5% BSA and a 1:1000 dilution of anti-Fn14 polyclonal serum, washed twice in TBST, and
incubated for 1 h in TBST containing 5% nonfat dry milk and a
1:20,000 dilution of goat anti-rabbit Ig-HRP (Bio-Rad). For analysis of
transfected cells, proteins were transferred to Protran nitrocellulose
membranes (Schleicher & Schuell) by electroblotting. The membrane was
blocked overnight at 4 °C in TBST containing 5% nonfat dry milk and
1% BSA and incubated for 1 h at room temperature in TBST
containing 1% BSA and a 1:1000 dilution of rat anti-HA monoclonal
antibody clone 3F10 (Roche Molecular Biochemicals). The membrane was
then washed three times in TBST and incubated for 1 h in TBST
containing 5% nonfat dry milk, 1% BSA, and a 1:2000 dilution of sheep
anti-rat Ig-HRP (Amersham Pharmacia Biotech). For all Western blots,
bound secondary antibodies were detected using the ECL system (Amersham
Pharmacia Biotech). Autoradiographic signals were quantified by
densitometry (VISAGE 4.6I software, BioImage Products).
Fluorescence Microscopy--
Cells were plated on
fibronectin-coated coverslips and transfected with either pEGFP-N3 or
pEGFP-N3/Fn14. One day later, the cells were washed in PBS and fixed to
the coverslips with 3% paraformaldehyde in PBS (pH 7.2) for 30 min.
The coverslips were washed three times in PBS and mounted on glass
slides in 50% glycerol in PBS. The cells were viewed with an Olympus
BH-2 fluorescence microscope.
Cell Surface Biotinylation/Immunoprecipitation
Analysis--
Surface biotinylation of the pcDNAIneo V5 and
pcDNAIneo/Fn14-HA 10 cell lines was carried out using EZ-Link
Sulfo-NHS-LC-Biotin (Pierce) per the manufacturer's instructions. The
cells were lysed in modified 1× RIPA buffer (50 mM
Tris/HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride,
1× protease inhibitor mixture; PharMingen) for 30 min at 4 °C and
clarified by centrifugation. The cellular lysate was precleared with a
50% slurry of GammaBind Plus-Sepharose (Amersham Pharmacia Biotech) in
RIPA buffer by mixing on an orbital rocker for 10 min at 4 °C. The
anti-HA antibody (described above) and control rat IgG (Sigma) were
immobilized by mixing 50 µl of a 50% Sepharose bead slurry with 0.5 µg of anti-HA antibody or rat IgG for 2 h at 4 °C on an
orbital rocker. The conjugated beads were washed twice with RIPA
buffer, equivalent amounts of cellular lysate were added in a total
volume of 1 ml of RIPA, and the beads were incubated overnight at
4 °C on an orbital rocker. Immunoprecipitates were washed three
times with RIPA buffer, mixed with 2× gel loading buffer (see above),
and heated at 95 °C for 10 min. Samples were subjected to SDS-PAGE
and transferred to a Protran membrane by electroblotting. The membrane
was blocked for 1 h at room temperature in TBST containing 3% BSA
and incubated in TBST containing 3% BSA and 200 ng/ml streptavidin-HRP
(Pierce) for 30 min. Biotinylated proteins were detected using the
Amersham Pharmacia Biotech ECL system.
Cell Adhesion Assays--
Adhesion assays were performed as
described (28, 29). After detachment using 5 mM EDTA in
PBS, cells were plated in DMEM at 5 × 104 cells/well
on 96-well collagen I-, collagen IV-, fibronectin-, laminin- or
vitronectin-coated CytoMatrix cell adhesion strips (Chemicon). The
cells were incubated for 1 h at 37 °C, rinsed in PBS, fixed by
treatment with 3% paraformaldehyde in PBS for 20 min, rinsed again in
PBS, stained with 0.2% crystal violet in 80% methanol for 30 min, and
finally rinsed three times in H2O. The relative number of
adherent cells in each well was evaluated by measuring the absorbance
at 590 nm using a Perkin-Elmer HTS 7000 BioAssay plate reader.
Additional vitronectin adhesion assays were conducted as above except
for the following two modifications. First, the cells were plated on
wells that had been coated with various concentrations of vitronectin
(Chemicon) for 1 h at 37 °C. After washing the coated wells
with PBS, nonspecific binding sites were blocked using 1% BSA in PBS.
The wells were washed again with PBS before the cells were plated.
Second, the crystal violet stain was eluted with 0.1 M
sodium citrate (pH 4.2) in 50% ethanol prior to measuring absorbance.
Cell Growth Assays--
Cells were plated in standard growth
medium at 103 cells/well in a 96-well cell culture plate.
Cell growth was monitored using the cell proliferation reagent WST-1
(Roche Molecular Biochemicals) per the manufacturer's instructions by
measuring absorbance at 450 nm with a 650-nm reference wavelength subtracted.
Cell Migration Assays--
Migration assays were performed using
24-well Transwell inserts (Costar) with 8.0-µm pore polycarbonate
membranes per the manufacturer's instructions. After detachment using
5 mM EDTA in PBS, cells were plated in DMEM in the upper
compartment of the Transwell insert at 2 × 104
cells/insert, and either DMEM alone or DMEM supplemented with 10% calf
serum was placed in the lower compartment. The cells were incubated for
18 h at 37 °C, and those cells remaining on the upper surface
of the filter were removed with a cotton swab. The membranes were
rinsed once with PBS, and the cells that had migrated to the lower
surface of the membrane were fixed in 25% acetic acid/75% methanol
and stained with crystal violet. The stain was eluted, and the relative
number of cells on each membrane was determined by measuring absorbance
as described above for the vitronectin adhesion assays.
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RESULTS |
Identification of an FGF-1-inducible Gene by mRNA Differential
Display--
We have described previously a differential display
approach that employs random hexamer-primed cDNA templates, PCR
oligonucleotide primers designed to anneal with DNA sequences encoding
motifs found in particular protein structural domains, and agarose gel electrophoresis (9). In the present experiments, RNA was isolated from
either quiescent NIH 3T3 cells or cells that had been treated with both
FGF-1 and cycloheximide for 2 h. This RNA was converted to
cDNA using the enzyme reverse transcriptase, and PCR assays were
performed using sense protein kinase domain and antisense leucine
zipper domain oligonucleotide primers. Amplification products were
displayed using agarose gel electrophoresis and ethidium bromide
staining. An ~230-bp DNA fragment was amplified to a greater degree
when cDNA representing the RNA isolated from cells treated with
FGF-1 and cycloheximide was used as template (data not shown). This DNA
fragment was isolated, cloned, and used as a probe in a preliminary
Northern blot hybridization experiment to confirm that it did indeed
represent an FGF-1-inducible gene. This gene was named the
FGF-inducible 14 (Fn14) gene,
because it was predicted to encode an ~14-kDa protein (see below).
Fn14 cDNA Sequence Analysis--
A mouse fibroblast cDNA
library was then screened with the differential display-derived
cDNA fragment in order to isolate longer cDNA clones. Both
strands of the longest cDNA insert (~950 bp) were sequenced by
the dideoxynucleotide chain termination method. The nucleotide sequence
contained a 12-nt 5'-untranslated region, a 387-nt open-reading frame,
and a 550-nt 3'-untranslated region with a consensus polyadenylation
signal and one copy of an AT-rich sequence motif implicated in rapid
mRNA decay (30) (Fig. 1). The
presumed initiating ATG codon is flanked by a favorable sequence for
translation initiation (31); nevertheless, we used the 5'-RACE method
to screen for cDNAs containing additional 5' sequence information.
The longest cDNA we identified had an additional 12 nucleotides
that were not present in the original cDNA clone.
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Fig. 1.
Murine Fn14 nucleotide sequence and deduced
amino acid sequence. Numbers to the left
refer to the first amino acids on the lines, and the numbers
to the right refer to the last nucleotides on the lines. The
nucleotide sequence obtained from a 5'-RACE-derived cDNA clone is
in boldface type. The solid
line below amino acids 1-27 indicates the predicted signal
peptide sequence, and the boxed amino acid stretch indicates
the predicted transmembrane domain. In the 3'-untranslated region, the
stop codon is denoted by an asterisk, a putative mRNA
destabilization sequence motif is underlined, and the
polyadenylation signal is boxed.
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The Fn14 gene is predicted to encode a protein of 129 aa
with a molecular mass of 13,637 daltons and an isoelectric point of 8.18. The protein is proline-rich (9.3%) and contains eight cysteines but is devoid of asparagine or tyrosine residues. Computer analysis of the predicted Fn14 amino acid sequence using several sequence analysis programs indicated that Fn14 contains two relatively hydrophobic regions: one at the amino terminus that terminates with a
signal peptidase cleavage site and another that is predicted to
function as a membrane-spanning domain. This predicted structure indicates that Fn14 is a type Ia transmembrane protein (32) containing
a 27-aa signal peptide, a 53-aa ectodomain, a 21-aa transmembrane
domain, and a 28-aa cytoplasmic tail. After signal peptidase cleavage,
the mature Fn14 polypeptide would be 102 aa in length and have a
predicted molecular mass of 10,832 daltons. The possibility that Fn14
is a type Ia integral membrane protein is supported by the presence of
a cluster of charged amino acids immediately following the second
hydrophobic region, which may function as a stop-transfer signal. The
predicted Fn14 protein has no other characteristic sequence motifs that
may suggest cellular location or function except for several putative
serine or threonine phosphorylation sites and a L-I motif in the
cytoplasmic domain that could serve as an endocytosis signal
(33-35).
Fn14 Amino Acid Sequence Comparisons--
A search of the
available sequence data bases using the Fn14 deduced amino acid
sequence revealed no significant degree of sequence identity between
Fn14 and other known proteins. However, we have noted that the Fn14
transmembrane domain has some sequence similarity to the transmembrane
region found in the syndecan family of integral plasma membrane
proteins. Specifically, alignment of the Fn14 transmembrane sequence to
the transmembrane sequences of the mouse, rat, and human syndecan
proteins revealed that four amino acids are conserved between Fn14 and
all the syndecans, while five more amino acids are conserved between
Fn14 and one or more of the syndecans. These nine conserved Fn14 amino
acids are shown in parentheses in the following Fn14 transmembrane
domain sequence:
(I)-L-(G-G)-A-L-S-(L)-V-L-V-L-A-(L-V)-S-S-(F-L-V)-W. Of special
interest is conservation of the double glycine, since the transmembrane
region of the syndecans contains this motif and in general, glycine
residues are found infrequently in transmembrane sequences (36).
Furthermore, the Fn14 pattern of bulky and small side chain residues in
this region is similar to the conserved pattern found in the syndecans
(37).
Fn14 Chromosomal Location--
The chromosomal location of Fn14
was determined by interspecific backcross analysis using progeny
derived from matings of (C57BL/6J × M. spretus)F1 × C57BL/6J mice. This interspecific backcross mapping panel has been
typed for over 2800 loci that are well distributed among all the
autosomes as well as the X chromosome (20). C57BL/6J and M. spretus DNAs were digested with several restriction enzymes and
analyzed by Southern blot hybridization for informative RFLPs using a
Fn14 cDNA probe. The ~6.0-kb EcoRI M. spretus
RFLP (see "Experimental Procedures") was used to follow the
segregation of the Fn14 locus in backcross mice. The mapping results
indicated that Fn14 is located in the proximal region of mouse
chromosome 17 linked to Mas1, E4fl, and Pim1 (Fig.
2). Although 127 mice were analyzed for
every marker and are shown in the segregation analysis, up to 189 mice
were typed for some pairs of markers. Each locus was analyzed in
pairwise combinations for recombination frequencies using the
additional data. The ratios of the total number of mice exhibiting
recombinant chromosomes to the total number of mice analyzed for each
pair of loci and the most likely gene order are: centromere,
Mas1-2/189-Fn14- 0/156-E4fl-1/132-Pim1. The recombination frequencies
(expressed as genetic distances in centimorgans ± the standard
error) are: Mas1-1.1 ± 0.7-(Fn14, E4fl)-0.8 ± 0.8-Pim1. No
recombinants were detected between Fn14 and E4fl in 156 animals typed
in common, suggesting that the two loci are within 1.9 centimorgans of
each other (upper 95% confidence limit).
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Fig. 2.
Chromosomal location of the murine Fn14
locus. Fn14 was placed on mouse chromosome 17 by interspecific
backcross analysis. The segregation patterns of Fn14 and flanking genes
in 127 backcross animals that were typed for all loci are shown at the
top of the figure. For individual pairs of loci, more than
127 animals were typed (see text). Each column represents the
chromosome identified in the backcross progeny that was inherited from
the (C57BL/6J × M. spretus) F1 parent. The
shaded boxes represent the presence of a C57BL/6J
allele, and white boxes represent the presence of
a M. spretus allele. The number of offspring inheriting each
type of chromosome is listed at the bottom of each column. A
partial chromosome 17 linkage map showing the location of Fn14 in
relation to linked genes is shown at the bottom of the
figure. Recombination distances between loci in centimorgans are shown
to the left of the chromosome and the positions of loci in
human chromosomes, where known, are shown to the right.
References for the human map positions of loci cited in this study can
be obtained from the Genome Data Base, a computerized data base of
human linkage information maintained by the William H. Welch Medical
Library of the Johns Hopkins University (Baltimore, MD).
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Regulation of Fn14 mRNA Expression in NIH 3T3 Cells--
We
first investigated the kinetics of Fn14 mRNA accumulation following
FGF-1 stimulation of NIH 3T3 cell growth by Northern blot hybridization
analysis. A single Fn14 transcript of ~1.2-kb in size was detected in
FGF-1-treated cells (Fig. 3A).
Increased Fn14 mRNA levels were first evident at 1 h after
FGF-1 addition, and maximal levels were present at 4 h. The effect
of the RNA synthesis inhibitor actinomycin D and the protein synthesis
inhibitor cycloheximide on FGF-1 induction of Fn14 mRNA levels was
also examined. Actinomycin D co-treatment inhibited FGF-1 induction of
Fn14 mRNA (Fig. 3B); in contrast, cycloheximide
co-treatment resulted in Fn14 mRNA superinduction (Fig.
3C). Cycloheximide treatment alone also increased Fn14
mRNA levels. Taken together, these results indicate that Fn14 is an
FGF-1-inducible, transcriptionally activated immediate-early response
gene.
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Fig. 3.
Regulation of Fn14 mRNA expression in NIH
3T3 cells. A, serum-starved cells were either left
untreated or treated with FGF-1 for the indicated time periods. RNA was
isolated, and equivalent amounts of each sample were analyzed by
Northern blot hybridization. The positions of 28 and 18 S rRNA are
noted. In the bottom panel, a photograph of the
18 S rRNA band is shown to demonstrate that equivalent amounts of RNA
were present in each gel lane. B, serum-starved cells were
either left untreated (NT, no treatment) or treated with
FGF-1, FGF-1 and actinomycin D (Act.D), or actinomycin D
alone for 4 h. RNA was isolated, and equivalent amounts of each
sample were analyzed as described above. C, serum-starved
cells were either left untreated or treated with FGF-1, FGF-1 and
cycloheximide (Chx), or cycloheximide alone for 4 h.
RNA was isolated, and equivalent amounts of each sample were analyzed
as described above. D, serum-starved cells were either left
untreated or treated with FGF-1, FGF-2, PDGF-BB, TGF-1, EGF, IGF-1,
calf serum (CS), or PMA for 4 h. RNA was isolated, and
equivalent amounts of each sample were analyzed as described
above.
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We then determined whether the FGF-1-related mitogen FGF-2 (also
referred to as basic FGF), PDGF-BB, TGF-1, EGF, IGF-1, calf serum,
or PMA could also increase Fn14 mRNA levels. FGF-1, FGF-2, PDGF-BB,
calf serum, and PMA could each induce Fn14 mRNA expression to a
similar extent (Fig. 3D). In contrast, TGF-1 or EGF
treatment had only a slight stimulatory effect, while IGF-1 had no
detectable activity.
Regulation of Fn14 Protein Expression in NIH 3T3 Cells--
We
next investigated whether mitogenic stimulation of quiescent NIH 3T3
cells resulted in elevated Fn14 expression. We first obtained Fn14
polyclonal antiserum by immunizing rabbits with recombinant GST-Fn14
fusion protein purified from bacterial cultures. Initial Western blot
experiments indicated that this antiserum specifically recognized
recombinant Fn14 protein expressed in either bacterial or insect cell
systems (data not shown). To analyze Fn14 expression levels in growth
factor-stimulated cells, serum-starved cells were either left untreated
or treated with FGF-2 for different lengths of time and Western blot
analysis was conducted using the Fn14 antiserum. A major immunoreactive
protein of ~22 kDa was detected in FGF-2-treated cells (Fig.
4). FGF-2 stimulation increased the
expression level of this protein, with maximal levels present at
12 h after mitogen addition. The apparent molecular mass of this
immunoreactive protein is approximately twice the predicted size of the
mature Fn14 protein (minus signal peptide); however, we have concluded
that this protein is Fn14 based on several experimental findings (see
"Discussion").
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Fig. 4.
Regulation of Fn14 protein expression in NIH
3T3 cells. Serum-starved cells were either left untreated
(0') or treated with FGF-2 for the indicated time periods.
Cell lysates were prepared, and equivalent amounts of protein were
subjected to SDS-PAGE and Western blot analysis using anti-Fn14
polyclonal antiserum. Molecular masses of protein size standards (in
kDa) are shown on the left.
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Fn14 mRNA Expression in Mouse Embryos and Tissues--
The
pattern of Fn14 mRNA expression during mouse development was
investigated. A blot containing RNA isolated from four different developmental time-points was obtained, and Fn14 mRNA levels were examined by Northern hybridization analysis. Fn14 mRNA expression was detected at all of the time points examined (Fig.
5A). Maximal levels of
expression were detected at 7.5 days post-coitum; however, this RNA
sample was isolated from both the developing embryo and the surrounding
extra-embryonic and maternal
tissues.2 To identify the
precise sites of Fn14 mRNA expression, we performed in
situ hybridization analysis on serial sections from 8.5-day post-coitum mouse embryos sectioned in utero. Fn14
transcripts were detected primarily in the maternal decidual tissue
nearest the ectoplacental (mesometrial) pole (Fig. 5B).
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Fig. 5.
Regulation of Fn14 mRNA expression during
mouse embryonic development and in situ hybridization
to serial sections of an embryonic day 8.5 mouse embryo.
A, a Northern blot containing RNA isolated from 7.5, 11.5, 15.5, or 17.5 days post-coitum mouse embryos was obtained, and
hybridization analysis was performed using the cDNA probes
indicated. RNA size markers (in kb) are shown on the left.
B, serial sections of embryonic day 8.5 mouse embryos
sectioned in utero were used for in situ
hybridization analysis using Fn14 antisense (panel
1) or sense (panel 2) riboprobes.
These two dark-field photographs, which reveal the hybridization signal
grains in white, were taken at the same exposure level. A
bright-field view showing the histology of another serial section is
shown in panel 3. Original magnification,
×25.
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The tissue distribution of Fn14 mRNA was evaluated by Northern blot
analysis using RNA isolated from tissues obtained from either newborn
or adult mice. In the newborn animals, Fn14 transcripts were expressed
at a relatively high level in all six tissues examined (Fig.
6A). In adult mice, Fn14
mRNA was expressed at the highest level in heart and ovary and at
an intermediate level in kidney, lung and skin (Fig. 6B).
Fn14 mRNA was expressed at a relatively low level in the other
seven adult tissues examined. Thus, the Fn14 gene is expressed in a
developmental stage- and tissue-specific manner in vivo.
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Fig. 6.
Fn14 mRNA expression levels in various
mouse tissues. RNA was isolated from the indicated newborn
(panel A) or adult (panel
B) mouse tissues, and equivalent amounts of each sample were
analyzed by Northern blot hybridization. In the bottom part
of each panel, a photograph of the 18 S rRNA band is shown to
demonstrate that equivalent amounts of RNA were present in each gel
lane.
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Fn14 Subcellular Localization Studies--
Fn14 was transiently
expressed as a EGFP fusion protein in NIH 3T3 cells in order to
determine its subcellular location. When EGFP itself is expressed in
these cells, it is primarily found in the cytoplasm (Fig.
7A). In contrast, the
Fn14-EGFP fusion protein is localized to the plasma membrane and to the
trans-Golgi network near the nuclear membrane (Fig. 7, B and
C). At the cell surface, Fn14-EGFP is especially prominent
in thin membrane extensions resembling microspikes or filopodia (Fig.
7C). In additional experiments, Fn14 containing distinct
amino- and carboxyl-terminal epitope tags was expressed in transfected
NIH 3T3 cells and localization was assayed by immunofluorescence
microscopy of permeabilized or non-permeabilized cells. We found that
Fn14 was oriented with its amino terminus located outside of the cell
(data not shown), and this result is consistent with the orientation
predicted by computer analysis of the Fn14 primary sequence.
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Fig. 7.
Localization of Fn14-EGFP fusion protein
expressed in transiently-transfected NIH 3T3 cells. Cells
transfected with either the pEGFP-N3 vector (panel
A) or the pEGFP-N3/Fn14 plasmid (panels
B and C) were processed at 24 h after
transfection and viewed using fluorescence microscopy. Original
magnification, ×600.
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We confirmed that Fn14 was present on the plasma membrane using a
biochemical approach. First, the expression plasmid
pcDNAIneo/Fn14-HA, which encodes a Fn14 protein containing a
carboxyl-terminal HA epitope tag (25), was constructed. Second, NIH 3T3
cells were transfected with either the pcDNAIneo vector or the
pcDNAIneo/Fn14-HA plasmid, stable cell lines were isolated by drug
selection with G418, and Fn14-HA expression levels were assayed by
Western blot analysis using an anti-HA antibody. One control cell line
(V5) and three experimental cell lines expressing different levels of
Fn14-HA (lines 3, 11, and 10) were chosen for subsequent biochemical and phenotypic studies. The major immunoreactive protein detected in
the Fn14-HA-expressing cells was ~23 kDa in size; however, a
~16-kDa protein was also detected in lysates prepared from the cell
line with the highest level of Fn14-HA production (Fig.
8, A and B). Third,
we harvested an equivalent number of pcDNAIneo V5 and
pcDNAIneo/Fn14-HA 10 cells and biotinylated the surface proteins
using a cell membrane-impermeable biotinylation reagent. Cell lysates
were prepared, immunoprecipitation was performed using either anti-HA
IgG or control IgG, and the biotinylated cell surface proteins were
detected by Western blot analysis using a streptavadin-HRP conjugate.
Several biotinylated proteins were detected, but a ~23-kDa protein
was immunoprecipitated specifically by anti-HA IgG (Fig.
8C). Thus, taken together, the results described above
indicate that Fn14 is a plasma membrane-anchored protein.
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Fig. 8.
Localization of Fn14-HA fusion protein
expressed in stably-transfected NIH 3T3 cells. A, cells
transfected with either the pcDNAIneo vector (line
V5) or the pcDNAIneo/Fn14-HA plasmid (lines
3, 11, and 10) were harvested, and
cell lysates were prepared. Equivalent amounts of protein were
subjected to SDS-PAGE and Western blot analysis using anti-HA
monoclonal antibodies. Molecular masses of protein size standards (in
kDa) are shown on the left. B, the Fn14-HA
expression data shown in panel A were quantified
by densitometry (only the 23-kDa species) and plotted as fold increase
over the background signal found in vector-transfected cells.
C, the pcDNAIneo V5 and pcDNAIneo/Fn14-HA 10 cell
lines were surface biotinylated, cell lysates were prepared, and
immunoprecipitation analysis was performed using either control IgG or
anti-HA IgG. Biotinylated plasma membrane proteins were visualized
using HRP-conjugated streptavidin. Molecular masses of protein size
standards (in kDa) are shown on the left.
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Biological Effects of Fn14-HA Expression in Transfected NIH 3T3
Cells--
We compared the properties of the control
(vector-transfected) or Fn14-HA-expressing clonal cell lines described
above using several different assays in order to gain insight into the
possible biological functions of the Fn14 protein. First, we determined whether constitutive Fn14 expression had an effect on NIH 3T3 cell
adhesion to various extracellular matrix proteins. Neither the control
nor the three experimental cell lines were able to adhere to collagen I
or collagen IV; furthermore, these lines displayed only weak adherence
to laminin (Fig. 9A). In
contrast, the control and Fn14-HA-expressing cell lines did adhere to
fibronectin or vitronectin. In comparison to the control V5 line, the
two experimental lines expressing the highest levels of the Fn14-HA protein (lines 11 and 10) exhibited decreased adhesion to fibronectin, while all three experimental cell lines (lines 3, 11, and 10) exhibited
decreased adhesion to vitronectin. Adhesion of the Fn14-HA 10 cell line
on fibronectin and vitronectin was reduced by ~42% and ~74%,
respectively. We subsequently performed an additional set of adhesion
assays in which the control V5 and the Fn14-HA 10 cell lines were
plated onto increasing concentrations of vitronectin. For both cell
lines, the extent of cell adhesion increased in a
dose-dependent manner; however, in comparison to the
control cell line, the Fn14-HA-expressing cell line exhibited decreased adhesion at all of the vitronectin concentrations tested (Fig. 9B). We have also observed that when the V5 and Fn14-HA 10 cells are plated on vitronectin and examined ~4 h later, the V5 cells are spread out and display a typical fibroblast morphology while the
Fn14-HA-expressing cells are more spherical in shape (data not
shown).
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Fig. 9.
Effect of constitutive Fn14-HA expression on
NIH 3T3 cell adhesion to purified extracellular matrix proteins.
A, the pcDNAIneo V5 and the pcDNAIneo/Fn14-HA 3, 11, and 10 cell lines were suspended in serum-free medium and plated on
wells coated with the indicated extracellular matrix protein. Cell
adhesion was measured after a 1-h incubation. The data shown are the
means ± S.D. of triplicate wells and are representative of three
independent experiments. The horizontal line
denotes the mean absorbance of all cell lines plated in wells coated
with BSA. *, p < 0.05; #, p < 0.005 in comparison with V5, t test. B, the V5 ( )
and 10 ( ) cell lines were suspended in serum-free medium and plated
on wells coated with the indicated vitronectin concentrations. Adhesion
was measured after a 1-h incubation. The data shown are the means ± S.D. of triplicate wells and are representative of three independent
experiments.
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Second, since cell-extracellular matrix interactions play a key role in
cellular proliferation and migration, we determined whether
constitutive Fn14 expression altered serum-stimulated NIH 3T3 cell
growth or motility. When the growth properties of the control V5 cell
line and the Fn14-HA 10 cell line were compared in serum-containing
medium using a colorimetric assay, we observed that the
Fn14-HA-expressing cell line had a statistically significant decrease
in growth rate. In comparison to the V5 cells, growth of the Fn14-HA 10 cells was reduced by ~60% after 4 days of culture (Fig.
10A). Similar results were
obtained when cellular proliferation was measured by directly counting
viable cells (data not shown). These same two cell lines were then used
in cellular migration assays. Each cell line was resuspended in
serum-free medium and plated in the upper compartment of Transwell
inserts, and their migration through a porous membrane in response to
either serum-free medium or serum-containing medium was measured. When
serum-free medium was present in both the upper and lower compartments,
no cellular migration occurred (data not shown). When serum-containing medium was placed in the lower compartment, both the control and experimental cells exhibited chemotactic migration; however, migration of the Fn14-HA-expressing cell line was ~40% of that observed with
the control cell line (Fig. 10B).
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Fig. 10.
Effect of constitutive Fn14-HA expression on
NIH 3T3 cell proliferation and migration. A, The
pcDNAIneo V5 () and the pcDNAIneo/Fn14-HA 10 () cell
lines were plated at low density and cultured in normal growth medium.
At the days indicated, cells were harvested, and cell growth was
measured using a colorimetric assay. The data shown are the means ± S.D. of quadruplicate wells and are representative of three
independent experiments. B, the two cell lines described
above were suspended in serum-free medium and plated on the
polycarbonate membranes of Transwell inserts. Serum-containing medium
was placed into the bottom compartment, and the extent of cell
migration after a 18-h incubation period was measured. The data shown
are the means ± S.D. of duplicate wells and are representative of
four independent experiments.
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DISCUSSION |
The addition of serum or polypeptide growth factors to quiescent
fibroblast cultures initiates an intracellular signal transduction cascade that promotes the transcriptional activation of numerous cellular genes (1-3). These genes are generally classified as either
immediate-early, delayed-early, or late response genes, and they encode
proteins with diverse functions (2, 3). We have used a differential
display approach to identify FGF-1-inducible genes in NIH 3T3 cells
(9), and here we describe the cloning, chromosomal location, and
expression pattern of the Fn14 gene. We also report the biological
effects of constitutive Fn14 expression in transfected cells.
The original Fn14 cDNA was isolated by differential display using
degenerate sense and antisense oligonucleotide primers designed to
anneal to DNA sequences encoding conserved amino acid motifs found in
protein kinase domains and leucine zipper domains, respectively. We
found that neither of these structural domains were present in the
predicted Fn14 sequence. This result was not unexpected since
comparison of the PCR primer sequences with the corresponding regions
of the Fn14 cDNA sequence indicated that both primers annealed to
regions of relatively low sequence identity. The Fn14 gene is predicted
to encode a type Ia transmembrane protein containing a 27-aa signal
peptide, a 53-aa extracellular domain, a 21-aa transmembrane domain,
and a 28-aa cytoplasmic domain. The mature 102-aa Fn14 polypeptide is
predicted to have a molecular mass of ~10.8 kDa. Sequence motifs
present in the Fn14 sequence include a cluster of charged amino acids
following the predicted membrane-spanning domain and a L-I dipeptide in
the cytoplasmic tail. The first motif may function as a stop-transfer
signal during membrane insertion, and the second motif could serve as
an endosomal targeting signal (33-35).
The deduced Fn14 protein sequence was compared with the GenBank
sequence data base, and no significant sequence identity was found
between Fn14 and known proteins. However, a pattern search using Fn14
transmembrane domain residues revealed some sequence similarity to the
single transmembrane domain found in the syndecan family of heparan
sulfate proteoglycans (38, 39). The highest overall amino acid sequence
identity in this domain, ~33%, was between Fn14 and murine
syndecan-1. Of particular interest is that (i) Fn14 and all four
syndecan family members contain a G-G dipeptide motif in their
transmembrane domains, even though glycine residues are not generally
found in membrane-spanning sequences (36) and (ii) Fn14 contains a
pattern of bulky and small side chain residues that is similar, but not
identical, to the pattern found in the syndecan transmembrane domains
(37). It has been demonstrated by site-specific mutagenesis that this
pattern is critical for the formation of noncovalent, SDS-resistant
syndecan-3 dimers and oligomers (37). It is presently unknown whether
the Fn14 protein, like the syndecans, can self-associate, but it does not migrate at its predicted molecular weight when analyzed by SDS-PAGE
(see below).
The Fn14 gene is located on mouse chromosome 17, in the
middle of the T-locus. We have compared our interspecific map of
chromosome 17 with a composite mouse linkage map that reports the map
location of many uncloned mouse mutations (Mouse Genome Data
Base). Fn14 mapped in a region of the composite map that
lacks mouse mutations with a phenotype that might be expected for an
alteration in this locus. The proximal region of mouse chromosome 17 shares a region of homology with human chromosomes 6 and 16 (summarized
in Fig. 2). In particular, E4fl has been assigned to human
16p13.3-p13.2. The tight linkage between E4fl and
Fn14 in mouse suggests that the human homolog of
Fn14 will map to this same chromosomal location. Consistent
with this possibility, a recent search of the GenBank data base using
the murine Fn14 nucleotide sequence revealed a significant degree of
sequence relatedness between Fn14 and human chromosome 16p13.3 genomic
DNA (GenBank accession no. AC004643).
The Fn14 gene can be classified as a growth factor-inducible,
immediate-early response gene in vitro that is expressed in a developmental stage- and tissue-specific manner in vivo.
FGF-1 stimulation of quiescent fibroblasts rapidly increases Fn14
mRNA levels, with peak expression detected at 4 h. It is
likely that this response is due, at least in part, to transcriptional
activation of the Fn14 gene since Fn14 mRNA accumulation does not
occur in the presence of an RNA synthesis inhibitor. Fn14 mRNA
levels remain elevated for a significant period of time after mitogen
addition, suggesting that Fn14 gene transcription is somewhat sustained and/or Fn14 mRNA is relatively stable. In regard to this second possibility, we have noted that the Fn14 mRNA 3'-untranslated region contains one copy of an AU-rich motif implicated in rapid mRNA decay (30), and therefore this transcript may have a short half-life. However, nuclear run-on assays and mRNA stability
measurements are required in order to determine the precise molecular
basis for the Fn14 mRNA temporal expression pattern that is
observed. FGF-1 induces Fn14 mRNA levels in the presence of a
protein synthesis inhibitor; thus, Fn14 gene activation does not
require the de novo synthesis of intermediary proteins. The
simultaneous addition of FGF-1 and cycloheximide promoted Fn14 mRNA
superinduction and, in addition, cycloheximide treatment alone induced
Fn14 mRNA levels. These cycloheximide effects are likely to occur
because the drug is preventing the synthesis of labile proteins
required for Fn14 transcriptional repression and/or Fn14 mRNA
decay. We found that, like the majority of the immediate-early genes
identified to date, the Fn14 gene can be induced by various polypeptide
growth factors as well as by PMA, a tumor-promoting phorbol ester that
activates protein kinase C. Finally, Fn14 transcripts are expressed in
mice at a relatively high level in the maternal decidual tissue of the
developing embryo, in many of the major organs of newborn animals, and
in the adult heart, kidney, lung, ovary, and skin.
We generated Fn14 polyclonal antiserum and then performed Western blot
analysis to investigate whether the mitogenic stimulation of NIH 3T3
cells increased Fn14 protein expression. FGF-2 treatment of quiescent
cell cultures induced the accumulation of a ~22-kDa immunoreactive
protein, and the temporal expression kinetics observed were consistent
with the kinetics of Fn14 mRNA expression in FGF-1-treated cells.
Although this immunoreactive protein migrated at an apparent molecular
mass twice the predicted mass of mature Fn14 (~10.8 kDa), we believe
it is likely to be Fn14 for two reasons. First, when Fn14 cDNA is
expressed in baculovirus-infected insect cells, the 129-aa protein is
processed and mature Fn14 migrates an apparent molecular mass of ~23
kDa (data not shown). Second, when an HA epitope-tagged Fn14 protein
predicted to be ~11.9 kDa in size is expressed in transfected NIH 3T3
cells, the major Fn14-HA protein species detected is ~23 kDa in size
(see Fig. 8). Fn14 may be migrating at a higher apparent molecular mass
when analyzed by SDS-PAGE due to post-translational modifications,
self-association into reducing agent/SDS-resistant dimers or an amino
acid composition (e.g. high proline content) that causes the
protein to resist denaturation or to bind SDS poorly. In regard to the
first possibility, this explanation is unlikely because (i) there are
no consensus sequence motifs for N-glycosylation or
glycosaminoglycan attachment in the predicted Fn14 protein, (ii) the
major Fn14 protein synthesized in an in vitro
transcription/translation system also migrates at ~22 kDa (data not
shown), and (iii) full-length Fn14 expressed in bacterial cells also
migrates at a higher apparent molecular mass than predicted from the
cDNA sequence when it is analyzed by SDS-PAGE (data not shown). In
regard to the second possibility, although it is possible that Fn14
could be forming dimers, especially when one considers that the Fn14
transmembrane domain has some sequence identity to the region of the
transmembrane domain implicated in syndecan-3 self-association (37), we
have never detected Fn14 migrating at a molecular mass indicative of a
monomeric molecule or of oligomeric complexes. Thus, we presently favor
the third possibility, anomalous migration due to amino acid
composition, as the likely explanation for the higher than predicted
apparent molecular mass of the Fn14 protein.
Two independent experimental approaches were used to demonstrate that
Fn14 is a plasma membrane protein. Fluorescence microscopy analysis of
Fn14-EGFP localization in transfected NIH 3T3 cells indicated that Fn14
was concentrated in the trans-Golgi network (probably due to
inefficient transport of overexpressed protein through the secretory
pathway) and in areas along the cell periphery that could represent
regions of cell-substratum attachment; however, additional
co-localization experiments are required to confirm that these are in
fact focal adhesion sites. Fn14 was also present on the cell surface in
thin membrane protrusions resembling actin microspikes or filopodia,
which are believed to act as sensory structures that play a role in the
control of cell growth and migration (40-42). Fn14 was also localized
to the plasma membrane of a Fn14-HA-expressing cell line using cell
surface biotinylation followed by immunoprecipitation and Western blot analysis.
NIH 3T3 stable cell lines expressing HA epitope-tagged Fn14 were
isolated in order to examine the biological consequences of
constitutive Fn14 expression. We are aware that a limitation of this
experimental approach is that Fn14 may exhibit abnormal behavior when
overexpressed in cells; therefore, we have analyzed several independent
cell lines with varying levels of expression. Since Fn14 was present on
the cell surface, we initially assayed cellular adhesiveness to several
immobilized extracellular matrix proteins. Cellular adhesion to the
extracellular matrix is mediated primarily by the integrin family of
heterodimeric transmembrane proteins (43, 44). Neither the vector
control nor the three Fn14-HA-expressing cell lines adhered
significantly above background levels when plated on collagen I,
collagen IV, or laminin. This result is consistent with previous
reports (45-47) and our results (data not shown) demonstrating that
the 1 and 2 integrin subunits (which
represent two of the major -integrin subunits which bind collagen
and/or laminin) are expressed at relatively low levels in NIH 3T3
fibroblasts. Both the control and the Fn14-HA-expressing cell lines
could adhere to fibronectin or vitronectin, consistent with previous
studies (45-48) and our results (data not shown) demonstrating
3/1, 5/1,
and V/1 expression in NIH 3T3
fibroblasts. In comparison to the control V5 line, the two experimental
cell lines expressing the highest Fn14-HA levels showed decreased
attachment to fibronectin while all three Fn14-HA-expressing lines
showed decreased attachment to vitronectin. In these assays, the level of Fn14-HA expression did not strictly correlate with the loss of
cellular adhesiveness. This may reflect a saturation of the inhibitory
effect at intermediate (for fibronectin binding) or low (for
vitronectin binding) levels of ectopic expression. Additional experiments using only the control cell line and the experimental cell
line expressing the highest level of Fn14-HA revealed that constitutive
Fn14 expression inhibited cellular adhesion to various concentrations
of immobilized vitronectin.
Nontransformed cells in culture are dependent on interactions with an
adhesive surface for both cellular proliferation (49-52) and migration
(42, 53, 54). Therefore, we performed serum-stimulated growth and
chemotactic migration assays using the control cell line and the
experimental cell line expressing the highest level of Fn14-HA. In
these growth and migration experiments, it is likely that serum-derived
vitronectin is the major attachment factor adsorbed to the tissue
culture plastic or Transwell nucleopore membranes, respectively (55).
We found that constitutive Fn14 expression inhibited both
serum-stimulated NIH 3T3 cell growth and motility.
In conclusion, we have identified a growth factor- and tumor
promoter-inducible immediate-early response gene on mouse chromosome 17, designated Fn14, which encodes a novel plasma membrane protein. Constitutive Fn14 expression in transfected NIH 3T3 cells reduces cellular adhesion to fibronectin and vitronectin and also inhibits serum-stimulated cell proliferation and migration. The molecular basis
for these effects is presently under investigation, but we propose that
Fn14 could be altering integrin subunit expression, ligand binding, or
signal transduction. This could occur by direct binding of Fn14 to
integrin subunits; alternatively, Fn14 could interact with other
transmembrane proteins that mediate integrin function; for example,
integrin-associated protein CD47 (56), calveolin (57), syndecan-2 or -4 (58, 59), or members of the tetraspanin family (60). Finally, it is
possible that the transient expression of this protein in growth
factor-stimulated cells may be important for the "de-adhesion"
process associated with cell division and motility (61).
§,
**
TOP
ABSTRACT
INTRODUCTION
