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INTRODUCTION |
A pivotal process in a healing wound as well as in a growing tumor
is the development of new blood vessels, or angiogenesis. Some of the
most potent angiogenic stimulators are the fibroblast growth factors,
including the classical angiogenic activators of this family,
FGF-11 and FGF-2 (1). High
concentrations of biologically active FGF-1 and FGF-2 are found in
extracts of normal human tissues that are not necessarily undergoing
active new blood vessel growth (1). This is due to the storage of FGF-1
and FGF-2 in the extracellular matrix, where they are found tightly
bound to membrane-attached heparan sulfate proteoglycans (2), which
quenches their biological activity. One mechanism by which FGF-1 and
FGF-2 are released from the extracellular matrix is through the
secretion of the carrier protein FGF-BP that binds to FGF-1 and FGF-2
in a noncovalent and reversible manner (3). FGF-BP is actively secreted
from cells and, once bound, prevents degradation of FGF-1 and FGF-2 (3,
4). The importance of FGF-BP secretion in promoting tumor growth was
shown in studies using the nontumorigenic adrenal carcinoma cell line
SW-13, which has high expression of FGF-2 but is negative for FGF-BP.
Stable overexpression of FGF-BP in SW-13 cells led to a dramatic
increase in FGF-2-dependent colony formation and formation
of highly vascularized tumors in nude mice (4). Additional studies were
carried out to characterize the biological role of FGF-BP in highly
tumorigenic cell lines such as ME-180 (human cervical squamous cell
carcinoma) and LS174T (colon adenocarcinoma), which express high levels
of endogenous FGF-BP (4, 5). Reduction of FGF-BP mRNA levels in
these cell lines using ribozyme targeting significantly inhibited tumor development and angiogenesis (6). These studies demonstrated that
FGF-BP serves as a rate-limiting angiogenic modulator for some tumor
types (6, 7).
FGF-BP is highly expressed in squamous cell carcinoma (SCC) cell lines
from lung, bladder, skin, and cervix and is positive in primary SCC
tumor samples (4). Furthermore, its expression has been shown to be
up-regulated during mouse embryonic development of the skin, lung, and
intestine and is low in most adult tissues (8). A potential role for
FGF-BP during skin carcinogenesis was described in studies showing
dramatic FGF-BP up-regulation in
human2 and mouse skin treated
topically with DMBA and TPA (8). These observations suggested several
mechanisms that might be involved in the direct regulation of the
FGF-BP gene. First, DMBA treatment has been shown to cause a specific
point mutation in the ras oncogene (9), suggesting that the
Ras signal transduction pathway might regulate FGF-BP expression. This
is also indicated by the observation that FGF-BP is induced in
ras-transformed keratinocytes (8). Second, the role of TPA
as a direct regulator of FGF-BP gene expression was confirmed upon TPA
treatment of several SCC cell lines, including ME-180, which caused
rapid transcriptional induction of the FGF-BP gene (10). We further
identified that Sp1, AP-1, and C/EBP sites within the proximal FGF-BP
promoter are all required for TPA regulation of FGF-BP (10).
SCC cell lines and tumors, including the ME-180 cell line, typically
express high levels of EGF receptors (EGFRs) (11, 12), and
overexpression of EGFR in SCC has been shown to confer greater tumorigenicity (13), which led us to investigate the possible role and
mechanisms of EGF regulation of FGF-BP. EGF signaling occurs
predominantly through binding to its receptor EGFR (HER1) and its
dimerization partner ErbB2 (HER2/Neu). Autophosphorylation of activated
EGF receptors stimulates a number of signal transduction pathways,
including the classical Ras/Raf/MAP kinase kinase (MEK)/MAP kinase
(ERK) pathway, which is known to phosphorylate and activate AP-1 (14).
MEK/ERK activation occurs either through phosphorylated EGFR
recruitment of the Shc-Grb2-SOS complex and subsequent Ras activation
or through recruitment of phospholipase C
and subsequent PKC
activation. PKC can in turn modulate Raf through both
Ras-dependent and -independent mechanisms (15, 16). Other
signaling pathways induced by EGF include the stress-activated protein
kinases such as JNK and p38 (17), the PI 3-kinase pathway (18), and the Janus kinase/signal transducers and activators of transcription pathway
(19).
Here we show a possible link between EGF signaling and angiogenic
activation through the regulation of the FGF-BP gene in SCC. We found
that EGF treatment induces rapid up-regulation of FGF-BP transcription
occurring through the AP-1 and C/EBP sites in the FGF-BP promoter.
Furthermore, inhibition of either the MEK/ERK pathway or the p38
pathway abrogates induction by EGF, implicating dual activation of
these MAP kinases as an important step in FGF-BP regulation.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Reagents--
The ME-180 cervical squamous cell
carcinoma cell line was obtained from American Type Culture Collection
(ATCC; Manassas, VA). Cells were cultured in improved minimum essential
medium (IMEM) (Biofluids Inc., Rockville, MD) with 10% fetal bovine
serum (Life Technologies, Inc.). Actinomycin D, calphostin C, and
wortmannin were purchased from Sigma. Anisomycin and tyrphostin AG1478
were from Alexis Corp. SB203580, SB202190, and SB202474 were from
Calbiochem, and U0126 was purchased from RBI (Natick, MA). All
compounds were dissolved in Me2SO.
Northern Analysis--
ME-180 cells were grown to 80%
confluence in 10-cm dishes, washed twice in serum-free IMEM, and
incubated for 16 h in serum-free IMEM prior to treatment. Cells
were pretreated for 1 h with the indicated drug or with vehicle
alone (Me2SO; final concentration 0.1%). EGF or anisomycin
treatment was for 6 h unless otherwise indicated. Total RNA was
isolated with RNA STAT-60TM (Tel-Test Inc.), and Northern
analysis was carried out as described previously (10). Hybridization
probes were prepared by random-primed DNA labeling (Amersham Pharmacia
Biotech) of purified insert fragments from human FGF-BP (4) and human
GAPDH (CLONTECH). Quantitation of mRNA levels
was performed using a PhosphorImager (Molecular Dynamics, Inc.).
Plasmids--
Human FGF-BP promoter fragments were cloned into
the pXP1 promoterless luciferase reporter vector and have been
described previously (10). The mutant AP-1 FGF-BP promoter construct
was generated by PCR as described previously (10), introducing point mutations that convert the AP-1 site from GTGAGTAA (
66 to 59) to
TGGAGCAA. The MEK2 (K101A) dominant negative
was provided by Dr. J. Holt (Vanderbilt University) (20). The dominant
negative of ERK2 (K52R) and the empty vector pCEP4 were provided by Dr. M. Cobb (University of Texas Southwestern) (21). The expression plasmids containing dominant negative JNK1 (APF), dominant negative p38
(AGF), wild-type JNK (pCDNA3-Flag-JNK1), wild-type p38
(pCDNA3-Flag-p38), JIP (pCDNA3-Flag-JIP1), and constitutively
active MKK6 (pCDNA3-Flag-MKK6(Glu)) were provided by Dr. R. Davis
(University of Massachusetts) (22-25). All effects of dominant
negatives were compared with their empty vector control or with the
empty vector pCDNA3 (Invitrogen).
Transient Transfections and Reporter Gene Assays--
24 h
before transfection, ME-180 cells were plated in six-well plates at a
density of 750,000 cells/well. pRL-CMV Renilla luciferase
reporter vector (Promega) was included as a control for transfection
efficiency. For each transfection, 1.0 µg of FGF-BP
promoter-luciferase construct, 0.1 ng of pRL-CMV (transfection efficiency control), and 8 µl of LipofectAMINE (Life Technologies, Inc.) were combined and added to cells for 3 h in serum-free
conditions as described previously (10). For co-transfections, 1.0 µg
of 118/+62Luc FGF-BP promoter construct, 500 ng of expression vector, 0.1 ng of pRL-CMV, and 8 µl of LipofectAMINE were added to cells. Transfected cells were treated for 18 h with EGF (5 ng/ml) in serum-free IMEM before cell lysis in 150 µl of passive lysis buffer (Promega). 20 µl of extract was assayed for both firefly and
Renilla luciferase activity using the
Dual-LuciferaseTM reporter assay system (Promega). Due to a
small background induction (1.5-2 fold) of the pRL-CMV plasmid by EGF,
all luciferase values were normalized for protein content. There were
no significant differences, however, in the transfection efficiencies
between plasmid constructs in untreated controls as determined by
Renilla luciferase assay. Protein content of cell extracts
was determined by Bradford assay (Bio-Rad).
Gel Shift Assays--
ME-180 cells were grown to 80% confluency
on 150-mm dishes, serum-starved in IMEM for 16 h, and treated with
or without 5 ng/ml EGF for 1 h. Nuclear extracts were prepared as
described previously (10). Binding reactions with 70/51 and
80/63 probes were carried out as described previously (10) with 5 or 1 µg, respectively, of ME-180 nuclear extracts, binding buffer (20 mM Tris, pH 7.5, 60 mM KCl, 5% glycerol, 0.5 mM dithiothreitol, 2.0 mM EDTA), and 250 ng of
poly(dI-dC). Binding reaction with 55/30 probe was carried out with
5 µg of ME-180 nuclear extracts and 500 ng of poly(dI-dC). Supershift
antibodies (2 µg) were added to the binding reaction for 10 min on
ice before adding 20 fmol of labeled probe. Reactions were carried out
45 min on ice and analyzed by 6% polyacrylamide gel electrophoresis.
Supershift antibodies purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA) were the following: Fos-specific antibodies c-Fos
(K-25), c-Fos (4), Fos B (102), Fra-1 (R-20), and Fra-2 (Q-20);
Jun-specific antibodies c-Jun/AP-1 (D), c-Jun/AP-1 (N), JunB (N-17),
and JunD (329); Sp1-specific antibody Sp1 (PEP 2); and C/EBP-specific
antibodies C/EBP (14AA), C/EBP
(C-19), C/EBP (
198),
C/EBP (C-20), C/EBP
(M-17), and C/EBP
(C-22). For
oligonucleotide competition, a 50-fold molar excess of unlabeled
double-stranded competitor was added to the binding reaction and
incubated for 10 min on ice before the addition of the labeled probe.
Sequence of the C/EBP consensus fragment was 5'-TGCAGATTGCGCAATCTGCA-3'
(Santa Cruz Biotechnology).
Immunocomplex Akt Kinase Assay--
ME-180 cells were grown in
150-mm dishes and pretreated as described above for Northern analysis.
Cells were treated with EGF (5 ng/ml) for 5 min. Cell lysates were
prepared by scraping the cells into immunoprecipitation buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.5%
deoxycholic acid, 1% Nonidet P-40, 10% glycerol, 1 mM
sodium orthovanadate, 1 µM okadaic acid, 50 mM sodium fluoride, 2 µg/ml aprotinin, 1 µg/ml
pepstatin A) and incubating for 15 min at 4 °C in a rotating rack.
The lysates were cleared by centrifugation, and protein content was
measured with the Bio-Rad protein assay kit. Lysates were precleared
with protein G-Sepharose and incubated for 4 h at 4 °C with 3 µg of sheep anti-Akt1 antibody (Upstate Biotechnology, Inc., Lake
Placid, NY). Immunocomplexes were captured with protein G-Sepharose at
4 °C for 1 h. The beads were then washed with 50 mM
Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM
dithiothreitol. The Akt kinase assay was carried out as described
previously (26), using the K9 peptide as a substrate.
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RESULTS |
EGF Treatment Increases FGF-BP mRNA in SCC Cells--
We have
shown previously that phorbol ester (TPA) treatment of the human
cervical SCC cell line ME-180 results in a time- and
dose-dependent increase of FGF-BP mRNA, which is
mediated via the PKC signal transduction pathway (10). Similarly,
treatment of ME-180 cells with EGF resulted in a rapid increase in the
steady-state levels of FGF-BP mRNA with no effect on GAPDH mRNA
levels (Fig. 1). FGF-BP mRNA
induction was detectable after 1 h of treatment and was maximal
after 6 h with an average increase of 4.5-fold. The rapid and
transient nature of EGF induction of FGF-BP mRNA is identical to
that seen after TPA treatment (10), suggesting that these agents might
induce FGF-BP through a similar transcriptional mechanism.
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Fig. 1.
Induction of FGF-BP mRNA by EGF in ME-180
cells. Shown is Northern analysis of FGF-BP mRNA levels in
ME-180 cells that were either untreated or treated with 5 ng/ml EGF for
the indicated amounts of time. Northern blot signal intensities of
FGF-BP mRNA were quantitated by PhosphorImager and normalized to
GAPDH. Open circles represent control (untreated)
levels, and closed circles represent EGF treatment. Values represent
the mean and S.D. of at least two separate experiments.
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To determine whether FGF-BP is up-regulated by EGF at the
transcriptional level, we tested the effect of the transcription inhibitor actinomycin D on the EGF induction of FGF-BP mRNA.
Pretreatment with actinomycin D completely blocked the induction of
FGF-BP by EGF (see Fig. 4). Combined treatment with EGF and
cycloheximide had no effect on induction of FGF-BP mRNA (data not
shown), indicating that de novo protein synthesis is not
required for the EGF response. Furthermore, transient transfection of
the full-length FGF-BP promoter from 1060 to +62 into ME-180 cells
resulted in a 4.5-fold increase in luciferase activity upon EGF
treatment (Fig. 2). These data
demonstrate that EGF, like TPA, can directly increase the rate of
FGF-BP gene transcription.
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Fig. 2.
Effect of FGF-BP promoter mutations on the
transcriptional induction by EGF. The histogram on the
left shows the impact of each promoter deletion on the basal
(uninduced) luciferase activity of each construct. The basal activity
of the 118/+62 construct was set at 100%. The right
histogram shows the transcriptional activity in the presence
of EGF and is expressed as -fold induction of EGF-treated over
untreated for each construct. ME-180 cells were transiently transfected
with the indicated FGF-BP promoter luciferase constructs and were
either untreated or treated with 5 ng/ml of EGF for 18 h. Promoter
constructs are described under "Experimental Procedures" and in
Ref. 10. Values represent the mean and S.E. from at least three
separate experiments, each done in triplicate wells. Statistically
significant differences relative to the 118/+62 promoter construct
are indicated (*, p < 0.05; **, p < 0.01, t test).
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Identification of EGF Response Elements within the FGF-BP
Promoter--
Functional analysis of the FGF-BP promoter has shown
that TPA-induced transcription involves a combination of both positive and negative regulatory elements located within the first 118 base
pairs of the proximal promoter (10). Full TPA induction was mediated by
a C/EBP consensus site between 48 and 40, as well as through a
juxtaposed Sp1/AP-1 element between 76 and 59. The Sp1(b) site
between 76 and 68 was described previously as an Ets element based
on its homology to other Ets consensus binding sites (10). However, we
have now determined through supershift analysis that this site is bound
by the Sp1 transcription factor (Fig.
3D). To investigate whether
these same regulatory elements play a role in EGF induction of the
FGF-BP promoter, we transiently transfected a series of mutated
promoter constructs into ME-180 cells and tested their ability to be
induced after treatment with EGF. Deletion of promoter sequences from
1060 to 118 had no effect on the level of promoter activity and
resulted in a similar 5-fold EGF induction (Fig. 2), demonstrating that the EGF regulatory region of the promoter is located within the first
118 base pairs of the promoter. Although the upstream Sp1 site (Sp1(a))
drives a significant portion of basal promoter activity (10), deletion
of this site had no effect on EGF induction (data not shown). Deletion
of the Sp1(b) site within the context of the 118/+62 promoter
fragment also had no effect on EGF induction (Fig. 2). Basal activity
of the promoter, however, did drop significantly in the absence of the
Sp1(b) element (Fig. 2 and Ref. 10). In contrast, mutation of the AP-1
site resulted in a significant 40% decrease in EGF induction as
compared with the wild-type 118/+62 promoter. Similarly, complete
deletion of the juxtaposed Sp1(b)/AP-1 site resulted in a 46% decrease
in EGF response. Mutation of the AP-1 site had no effect on basal
promoter activity, demonstrating that changes in EGF responsiveness can
occur independently of changes in the basal rate of transcription.
These results show that unlike TPA induction, which requires the entire
Sp1(b)/AP-1 element, EGF induction is driven mainly through the AP-1
site in the promoter, reflecting possible differences in the mechanisms by which each of these agents regulate the transcription of FGF-BP.
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Fig. 3.
Characterization of transcription factor
binding to FGF-BP promoter elements. A, double-stranded
oligonucleotide sequences of FGF-BP promoter elements used for gel
shift analysis. Supershift analysis of transcription factor binding to
the AP-1 site (B), C/EBP site (C), or Sp1(b) site
(D) of the FGF-BP promoter. Gel shift assay with the labeled
FGF-BP promoter sequences as indicated were incubated in the presence
of nuclear extracts from untreated or EGF-treated ME-180 cells. Binding
reactions were incubated in the presence of supershift antibodies as
indicated in each figure. Specific binding of AP-1, C/EBP, and Sp1 are
indicated by an arrow at the left of each
panel. Supershift complexes are labeled by an
asterisk on either side of the gel. Competition for C/EBP
binding was carried out in the presence of a 50-fold molar excess of
unlabeled oligonucleotides as indicated.
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To test the contribution of the C/EBP element to EGF induction, we
introduced an internal deletion of this site within the context of the
118/+62 promoter. Removal of the C/EBP site significantly reduced the
EGF effect by 46% but had no influence on basal promoter activity
(Fig. 2). Together, these data demonstrate the requirement for an
intact C/EBP site and AP-1 site in the EGF induction of the FGF-BP promoter.
In addition to these positive regulatory elements, we recently showed
that TPA regulation of the FGF-BP promoter involves a negative
regulatory element, lying just downstream of the AP-1 site, which shows
similarity to an E-box factor binding site (10). We wanted to determine
whether this E-box repressor site also played a role in the regulation
by EGF. Point mutation of this site at position 58, or deletion of
this site from 57 to 47 resulted in a dramatic increase in EGF
induction of the promoter from about 5-fold in the wild-type promoter
to about 8-10-fold in the repressor mutants (Fig. 2), indicating loss
of repressor activity. Therefore, the repressor element between 58
and 47 also plays a regulatory role in EGF induction by limiting the transcriptional response to growth factor stimulation.
AP-1, C/EBP, and Sp1 Binding to the FGF-BP Promoter--
Because
the AP-1 site in the FGF-BP promoter appears to be important during TPA
and growth factor regulation of FGF-BP, we determined which members of
the Fos and Jun family might be binding to the FGF-BP AP-1 site. To
identify transcription factor binding to the AP-1 site, we carried out
gel shift analysis using labeled promoter sequence fragment from 70
to 51 (Fig. 3A), which was incubated in the presence of
nuclear extracts from control or EGF-treated ME-180 cells. Protein
binding in the uppermost complex, which has previously been shown to
represent AP-1 (10), is highly induced by EGF treatment (Fig.
3B, lanes 1 and 2). To
determine the composition of the AP-1 complex, we used Fos and
Jun-specific antibodies for supershift analysis. As shown in Fig.
3B, the addition of a Fos antibody that recognizes all Fos
family members (lane 3) or that specifically
recognizes c-Fos (lane 4) resulted in a
supershifted complex. Antibodies against FosB, Fra-1, or Fra-2 had no
effect. Furthermore, incubation with a general Jun family antibody
(Fig. 3B, lane 8) or with a
JunD-specific antibody (lane 11) either blocked
or supershifted the AP-1 complex, respectively. The c-Jun- or
JunB-specific antibodies had no effect on AP-1 binding. These results
demonstrate that c-Fos and JunD are the major components of AP-1
binding to the FGF-BP promoter.
The binding of C/EBP to the FGF-BP promoter was investigated using a
promoter fragment spanning the C/EBP site from 55 to 30 (Fig.
3A). Gel shift analysis with this fragment in the presence of nuclear extracts from untreated or EGF-treated ME-180 cells showed
no significant change in the binding of C/EBP (Fig. 3C, lanes 1 and 2). This finding is
consistent with previously published observations that PKC activation
phosphorylates and enhances C/EBP transcriptional activity with no
effect on DNA-binding activity (27). The specificity of C/EBP binding
in the upper complex of the gel shift (Fig. 3C,
arrow) is demonstrated through competition with a molar
excess of unlabeled 55/30 fragment (lane 9)
as well as with a C/EBP consensus element (lane
10). Incubation with an antibody against C/EBP that also
cross-reacts with other members of the C/EBP family resulted in one
prominently supershifted band (Fig. 3C, lane
3). In addition, antibodies specific for C/EBP (lane 4) and C/EBP (lane
7) also supershifted the complex, whereas antibodies for
C/EBP (lane 5), C/EBP (lane
6), and C/EBP (lane 8) had little
effect on C/EBP binding. Because the C/EBP site is required for full
EGF induction of the FGF-BP promoter (Fig. 2), these results suggest
that C/EBP and - are involved in mediating the EGF effect. The
mechanism by which these factors are activated in response to EGF,
however, remains to be determined.
As mentioned previously, the promoter element between 80 and 63
shows some homology to an Ets factor binding site. Closer analysis of
this site also revealed homology to a variant Sp1 site, which was
previously shown to respond to EGF treatment (28). In fact, supershift
analysis of the 80 to 63 element showed specific binding of Sp1
(Fig. 3D). This element has been denoted Sp1(b) in order to
distinguish it from the Sp1 site further upstream (Sp1(a)).
Interestingly, the binding of Sp1 to the Sp1(b) site increased in the
presence of nuclear extracts from EGF-treated cells (Fig.
3D, lanes 1 and 2) despite
the fact the removal of the Sp1(b) site alone has no impact on the
overall EGF induction of the FGF-BP promoter (Fig. 2).
Role of PKC and MEK1/2 Signal Transduction Pathways in FGF-BP
Regulation--
In order to differentiate between the possible
signaling pathways involved in EGF induction of FGF-BP, we tested
pharmacological inhibitors of signal transduction components for their
effect on FGF-BP regulation. We found that treatment with the EGFR
tyrosine kinase inhibitor tyrphostin AG1478 reduced EGF induction of
FGF-BP to 30% (Fig. 4), which was not
significantly different from the basal level of expression (without EGF
or drug treatment) of approximately 25% (data not shown). As expected,
EGFR tyrosine kinase activity is essential for the EGF effect on the
FGF-BP gene. In addition, we have shown previously that TPA induction
of FGF-BP transcription was mediated through a
PKC-dependent pathway (10). To establish whether PKC
activation was also required for the EGF effects on FGF-BP, we treated
ME-180 cells with the specific PKC inhibitor calphostin C (29) and
found that this completely blocked EGF induction of FGF-BP mRNA
(Fig. 4). Therefore, PKC activation is central in mediating FGF-BP
transcriptional activation upon either EGF or TPA stimulation.
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Fig. 4.
Effect of signal transduction inhibitors on
EGF induction of FGF-BP mRNA. FGF-BP mRNA levels from
ME-180 cells treated with 5 ng/ml EGF for 6 h. Cells were
pretreated for 1 h with vehicle alone, 5 µg/ml actinomycin D
(transcription inhibitor), 100 µM tyrphostin AG1478 (EGFR
tyrosine kinase inhibitor), 100 nM calphostin C (PKC
inhibitor), or 10 µM U0126 (MEK1/2 inhibitor). Northern
blot signal intensities of FGF-BP mRNA were quantitated, normalized
to GAPDH, and expressed relative to mRNA levels after EGF treatment
alone (without inhibitor), which was set to 100%. Basal FGF-BP level
(without EGF or inhibitor) was approximately 25%. Values represent the
mean and S.D. of at least two separate experiments.
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Since the MEK/ERK pathway is known to be stimulated by EGF, we
investigated the contribution of the MAP kinase kinases MEK1 or MEK2 to
FGF-BP using pharmacological inhibitors of MEK1/2. Treatment with the
drug U0126, which is a potent inhibitor of both MEK1 and MEK2,
abrogated EGF induction of FGF-BP mRNA (Fig. 4). Consistent with
the role of MEK in FGF-BP induction, treatment with a less potent MEK
inhibitor PD98059 also blocked the EGF effect on FGF-BP, albeit at
higher concentrations (data not shown). Therefore, activation of the
MEK pathway is a necessary step in EGF regulation of FGF-BP.
EGF is also known to stimulate intracellular signaling via the PI
3-kinase pathway (18). In order to test the contribution of PI 3-kinase
to FGF-BP regulation, we used the drug wortmannin to specifically
inhibit PI 3-kinase activity. Pretreatment with wortmannin at two
different concentrations had no effect on EGF induction or FGF-BP
mRNA (Fig. 5A). Endogenous
PI 3-kinase activity in ME-180 cells, as measured by immunoprecipitated
Akt kinase activity, was induced approximately 2-fold by EGF and was
effectively blocked by the same concentrations of wortmannin and under
the same experimental conditions used for the analysis of FGF-BP (Fig. 5B). Therefore, we conclude that activation of PI 3-kinase
upon EGF treatment does not play an essential role in the regulation of
FGF-BP expression. Furthermore, the lack of effect by wortmannin served
as a negative control and demonstrates specificity of the inhibitions
observed in Fig. 4.
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Fig. 5.
Effect of PI 3-kinase inhibition on
EGF-induced FGF-BP mRNA. A, FGF-BP mRNA levels
after pretreatment with vehicle alone or with the indicated
concentration of wortmannin (PI 3-kinase inhibitor) for 1 h,
followed by 6 h of treatment with 5 ng/ml EGF. FGF-BP mRNA
levels were analyzed by Northern blot, normalized for GAPDH, and
expressed relative to untreated control. B, inhibition of PI 3-kinase
activity by wortmannin. ME-180 cells were pretreated for 1 h with
or without wortmannin, stimulated for 5 min with 5 ng/ml EGF, and
assayed for Akt kinase activity (downstream target of PI 3-kinase) as
described under "Experimental Procedures."
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Induction of FGF-BP through p38 Kinase--
Stimulation of the
JNK/p38 MAP kinase pathway has also been shown to regulate AP-1
activity in response to mitogens and stress (17). We therefore tested
whether JNK or p38 activation could induce FGF-BP gene expression by
treating with the antibiotic anisomycin. Anisomycin treatment at
concentrations below 200 nM is known to be an effective
stimulator of both JNK and p38 kinase activity (30). Treatment of
ME-180 cells with anisomycin alone resulted in a significant and
dose-dependent increase of FGF-BP mRNA levels up to
2.3-fold (Fig. 6A), suggesting
that activation of the JNK/p38 pathway might be involved in the
regulation of FGF-BP.
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Fig. 6.
Involvement of p38 MAP kinase in the
anisomycin and EGF induction of FGF-BP mRNA. A,
anisomycin induction of FGF-BP mRNA. ME-180 cells were treated for
6 h with the indicated concentrations of anisomycin. The mean and
S.E. of at least three separate experiments are given. Statistically
significant differences relative to the control (untreated) group are
indicated (*, p < 0.05; **, p < 0.01, t test). B, p38 inhibition blocks EGF and
anisomycin (inset) induction of FGF-BP mRNA. ME-180
cells were pretreated for 1 h with the indicated concentration of
p38 inhibitors SB203580 (closed circles) and SB202190
(open circles) or with the control compound SB202474
(open triangles), followed by 6-h treatment with EGF (5 ng/ml) or anisomycin (200 nM). Northern blot signal
intensities of FGF-BP mRNA were quantitated, normalized to GAPDH,
and expressed relative to mRNA levels after EGF or anisomycin
treatment alone (without inhibitor), which was set to 100%. Basal
FGF-BP level (without EGF or inhibitor) was approximately 25% of
EGF-treated. Values represent the mean and S.D. of at least two
separate experiments.
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To further investigate the involvement of p38 kinase in FGF-BP
regulation, we tested the contribution of p38 to FGF-BP induction using
the p38-specific inhibitors SB203580 and SB202190, which have no
inhibitory activity for JNK or ERK1/2 (31-33). Treatment with SB203580
or SB202190 significantly reduced EGF induction of FGF-BP mRNA by
50% to 75% in a dose-dependent manner (Fig. 6B). In contrast, there was no reduction after treatment
with SB202474, a drug with a similar structure as SB203580 and SB202190 but with no inhibitory activity for p38. Additionally, p38 inhibition completely blocked anisomycin induction of FGF-BP (Fig. 6B,
inset), demonstrating that both anisomycin and EGF induction
of FGF-BP mRNA require p38 activation.
To investigate a possible additive or synergistic interaction between
ERK and p38 pathways in EGF-induced FGF-BP expression, we treated cells
simultaneously with the MEK inhibitor (U0126) and the p38 inhibitor
(SB202190) and examined the effect on FGF-BP mRNA. As shown in Fig.
7, treatment with suboptimal doses of
U0126 alone or with SB202190 alone resulted in approximately 20%
decrease in EGF-induced FGF-BP. Simultaneous treatment with both
inhibitors resulted in a 40% reduction of FGF-BP mRNA levels,
indicating that the contribution of each of these pathways is additive
and not synergistic.
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Fig. 7.
Additive contribution of ERK and p38
pathways. FGF-BP mRNA levels from ME-180 cells treated with 5 ng/ml EGF for 6 h. Cells were pretreated for 1 h with vehicle
alone or with suboptimal concentrations of U0126 (MEK1/2 inhibitor)
and/or SB202190 (p38 inhibitor). Northern blot signal intensities of
FGF-BP mRNA were quantitated, normalized to GAPDH, and expressed
relative to mRNA levels after EGF treatment alone (without
inhibitor), which was set to 100%. Basal FGF-BP level (without EGF or
inhibitor) was approximately 25%. Values represent the mean and S.D.
of at least two separate experiments.
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Contribution of MEK/ERK and p38 Pathways to FGF-BP Promoter
Activity--
Inhibition of MEK and p38 with pharmacological agents
demonstrated the importance of these kinases in mediating the induction of FGF-BP by EGF. To confirm the role of each of these pathways in the
regulation of FGF-BP transcription, we expressed dominant-negative mutants that specifically suppress the enzymatic activity of their endogenous counterparts. Co-transfection of a dominant-negative form of
MEK2 with the FGF-BP promoter luciferase construct (118/+62) into
ME-180 cells resulted in a significant decrease in the amount of EGF
induction (Fig. 8A). In
addition, inhibition of ERK, the MAP kinase target of MEK, by
co-transfection of a kinase-deficient dominant negative ERK2 mutant,
also resulted in a significant decrease in EGF induction (Fig.
8A). Inhibition of the MEK/ERK signaling pathway with these
dominant negative mutants led to nearly a 50% decrease in EGF
induction of the FGF-BP promoter. These results support the hypothesis
that activation of MEK and ERK by EGF are necessary for induction of
the FGF-BP gene.
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Fig. 8.
Effect of dominant negatives on EGF induction
of FGF-BP promoter activity. A, ME-180 cells were
transiently co-transfected with the 118/+62 FGF-BP promoter construct
along with either empty vector or with dominant negative mutant
constructs for MEK2, ERK2, JNK, or p38. Transfected cells were either
untreated or treated with 5 ng/ml EGF for 18 h. EGF induction is
determined by the -fold increase in luciferase activity and expressed
relative to the empty vector control, which is set at 100%. Basal
(uninduced) promoter activity was approximately 18% of EGF-treated.
Values represent the mean and S.E. from at least three separate
experiments, each done in triplicate wells. Statistically significant
differences relative to the empty vector control are indicated (*,
p < 0.05; **, p < 0.01; ***,
p < 0.001, t test). B, ME-180
cells co-transfected with 118/+62 FGF-BP promoter construct and
either empty vector or expression vectors for JNK, p38, MKK6 (Glu), or
JIP. Cells were treated as in A, and the FGF-BP promoter
activity is shown relative to the untreated empty vector control.
Statistical analysis was as described in A, and differences
in the absence or presence of EGF are compared with the empty vector
control in the absence or presence of EGF, respectively.
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To test the contribution of p38 and JNK to EGF induction of FGF-BP, we
co-transfected dominant negative mutants of JNK and p38 along with the
FGF-BP promoter into ME-180 cells (Fig. 8A). Each dominant
negative mutant contains an alanine and phenylalanine in place of the
activating threonine or tyrosine phosphorylation sites (22, 23).
Expression of the dominant-negative JNK mutant had no significant
effect on EGF induction compared with the empty vector. On the other
hand, expression of the dominant-negative p38 mutant consistently
reduced EGF induction of FGF-BP transcription. Although inhibition of
p38 reduced promoter activity by only 23%, this difference was
statistically significant from empty vector-transfected cells.
Additionally, we tested whether overexpression of wild-type JNK or p38
constructs could affect EGF induction of the FGF-BP promoter. In
agreement with the dominant-negative data, expression of JNK had no
significant effect on promoter activity, whereas expression of p38
consistently resulted in a higher level of EGF induction of FGF-BP
transcription, which was significantly higher than the empty vector
control (Fig. 8B). Furthermore, expression of a
constitutively active mutant of MKK6, MKK6(Glu), a MAP kinase kinase
that specifically phosphorylates p38 (25), was also tested for its
effect on FGF-BP induction. Overexpression of MKK6(Glu) caused a
significant increase in both basal and EGF-induced FGF-BP promoter
activity (Fig. 8B). Overall, these data implicate ERK and
p38 as being necessary for the full induction of FGF-BP by EGF. The
lack of effect on FGF-BP promoter activity by either JNK overexpression
or by inhibition of JNK through expression of a dominant-negative JNK
mutant or expression of JIP, which prevents nuclear translocation of
activated JNK (24) (Fig. 8B), suggests that JNK activation
does not play a role in the regulation of FGF-BP in these cells.
| |
DISCUSSION |
This study shows for the first time that transcription of FGF-BP,
an important mediator of FGF activation in SCC, is directly induced by
EGF. The EGF family of growth factors, which include EGF, transforming
growth factor-, and other structurally related peptides, cause
cellular signaling through the EGFR pathway, regulating proliferation
and differentiation of many tissue types. Deregulation of the
EGF-induced signaling network is known to play an important role in the
tumorigenesis of several human cancers, including neoplasms of the
brain, lung, breast, ovary, pancreas, prostate, and colon, as well as
in SCC of the skin and cervix (11, 12, 34). Here, we have shown that
EGF up-regulates FGF-BP gene expression, suggesting a link between
activated EGF signaling in a cell and subsequent FGF activation,
ultimately leading to activation of FGF-mediated processes, such as
angiogenesis, during development and tumor growth.
The up-regulation of FGF-BP by EGF was found to show characteristics
similar to TPA induction of this gene, including a rapid and transient
increase in FGF-BP mRNA levels, and the requirement of
transcriptional elements within the TPA regulatory region (118 to
+62) of the promoter. Transcriptional regulation of FGF-BP by EGF
required the AP-1 site between 65 and 61, which is bound by c-Fos
and JunD family members (Figs. 2 and 3B). The finding that
c-Fos is associated with FGF-BP up-regulation is significant, since an
important role for c-Fos during SCC formation has been observed.
Studies with c-fos knockout mice demonstrated that
c-fos is required for malignant transformation of skin
papillomas into malignant carcinomas, since c-fos(/)
papillomas became desiccated and hyperkeratinized, lacked
vascularization, and remained benign (35). Since the FGF-BP gene was
previously found to be highly induced during skin carcinogenesis (8),
it seems possible that this gene could be a target of c-Fos activation
during skin SCC tumor formation, providing at least one mechanism for
the recruitment of new blood vessels to the tumor.
The other required EGF response element in the FGF-BP promoter is the
C/EBP site between 47 and 33, which is predominantly bound by the
C/EBP and C/EBP family members (Figs. 2 and 3C). C/EBP
proteins are a family of leucine zipper transcription factors that play
a central role in the acute phase response and in a number of cell
differentiation pathways (reviewed in Ref. 36). Regulation of C/EBP
activity occurs at multiple levels, including increased gene expression
(37, 38), nuclear localization (39), enhanced DNA binding (40), and
post-transcriptional modification by protein kinases (27, 41). Because
we observed no increase in C/EBP binding to the FGF-BP promoter after
EGF treatment, it seems likely that stimulation of C/EBP
transcriptional activity occurs through a post-translational
modification. This conclusion is consistent with other studies
demonstrating that TPA stimulation of the PKC pathway in hepatoma cells
results in increased phosphorylation of C/EBP, enhanced
transcriptional efficacy, and no obvious changes in DNA binding (27,
42).
C/EBP family members recognize similar DNA elements in their target
genes, where they bind either as homodimers or heterodimers with other
C/EBP family members or with other leucine zipper factors (43). Whereas
C/EBP is generally associated with growth arrest and cellular
differentiation, C/EBP and C/EBP are often correlated with gene
activation during cellular proliferation, inflammation, and
tumorigenesis (44-46). Analysis of different C/EBP target gene promoters has demonstrated a finely tuned regulation of gene expression through the interplay of different C/EBP factors. During the acute phase response, for example, the amount of C/EBP homodimers or heterodimers is reduced and replaced by C/EBP and C/EBP complexes (47-49). Interestingly, study of cyclooxygenase-2 promoter regulation by C/EBP during skin carcinogenesis showed a change in C/EBP complexes from C/EBP·C/EBP in normal skin to predominantly
C/EBP·C/EBP complexes in skin SCC (50). The presence of
C/EBP·C/EBP complexes on the FGF-BP promoter in ME-180 SCC
cells raises the possibility that FGF-BP up-regulation during tumor
formation may be in part due to an interplay between the different
C/EBP family members.
In trying to delineate the signal transduction pathway regulating
FGF-BP gene expression, we found that activation of PKC plays a central
role, since inhibition of PKC with calphostin C blocks both the EGF
(Fig. 4) and TPA (10) induction of this gene. PKC activation in
response to growth factor stimulus can lead to stimulation of the
classical MAP kinase pathway Raf/MEK/ERK and subsequent activation of
AP-1. PKC-mediated signaling through this pathway can be achieved
through either Ras-dependent (15) or Ras-independent (16)
mechanisms. The involvement of Ras in FGF-BP regulation is suspected,
since FGF-BP expression is increased after DMBA treatment and in
ras-transformed keratinocytes (8). A direct role for Ras in
the activation of FGF-BP expression, however, has yet to be determined.
In addition to PKC, the EGF-activated MEK/ERK pathway plays a
significant role in the regulation of FGF-BP gene expression, since
pharmacological and dominant-negative inhibition of MEK or ERK
abrogates EGF induction. Although the exact target of ERK activation
was not examined in this study, ERK has been shown to phosphorylate and
activate both AP-1 (14) and C/EBP (41) family members.
p38 is a JNK-related MAP kinase that is activated in response to a
variety of stimuli including growth factors, phorbol esters, cytokines,
and environmental stress (17). Using a number of different approaches,
we show in this study that in addition to ERKs, p38 contributes to the
EGF induction of FGF-BP. We demonstrated the inhibition of EGF-induced
FGF-BP by pyridinyl imidazole compounds (SB202190 and SB203580), which
selectively inhibit p38 and p382 isoforms but have no effect on
the activity on other p38 isoforms, JNK, or ERK (31-33). In addition,
we have shown that FGF-BP promoter activity is inhibited by expression
of a dominant-negative p38 mutant but is activated by overexpression of
wild-type p38. p38 is phosphorylated and activated by the dual
specificity protein kinases MKK3, MKK4, and MKK6 (23, 25, 51-54).
Although overexpression of the p38-specific kinase MKK6 can stimulate
FGF-BP transcription, the signal transduction cascades that connect
EGFR activation to phosphorylation of p38 in ME-180 cells remain
unknown. One possible pathway that is currently under investigation is
the involvement of two members of the Rho family of GTPases, Rac and CDC42, which are known to regulate the activity of both JNK and p38
(55-58). Furthermore, Rac and CDC42 can be activated downstream of Ras
(55), thereby connecting p38 and JNK activation to growth factor
effects on cell growth and proliferation. Transcription factor targets
of p38 include CREB and ATF1 (59, 60), ATF2 (25, 61), MEF-2C (62), and
the C/EBP family members CHOP (63) and C/EBP (64, 65), suggesting
that C/EBP and/or C/EBP could be targets of p38 activation on the
FGF-BP promoter.
In general, EGF regulation of FGF-BP gene expression seemed to be more
dependent on the MEK/ERK pathway, since inhibition of MEK1/2 with U0126
completely abrogated induction, and expression of dominant negative MEK
or ERK reduced induction by 50%. p38, on the other hand, appears to
play a somewhat lesser role, since pharmacological inhibition of p38
caused a maximum 50% reduction of FGF-BP, and expression of dominant
negative p38 reduced induction by only 23%. Although stimulation of
the p38 pathway plays a less prominent role, regulation of FGF-BP by
p38 may be independent of MEK/ERK activation, since they function in an
additive rather than synergistic manner (Fig. 7). While the mechanism
for these differences in activity of each pathway remains unclear, one
explanation could be the differences in their transcription target
specificities at the level of the FGF-BP promoter.
This study also examined the possible role of JNK in the regulation of
the FGF-BP gene. Based on several lines of evidence, we have concluded
that JNK is unlikely to be involved in FGF-BP regulation. Inhibition of
JNK activity, either through expression of a dominant-negative JNK
mutant or expression of JIP, had no effect on EGF induction of the
FGF-BP promoter. Overexpression of wild-type JNK also had no effect on
FGF-BP expression. In addition to this, we examined the activation of
Elk1 by MEKK, a potent activator of JNK (66), and found no effect on
this pathway by dominant negative JNK or JIP expression (data not
shown). Furthermore, we found no activation of FGF-BP gene expression
in the presence of UV light (data not shown), which is another known
stimulator of the JNK pathway (17). Together, these findings indicated that there may be very little JNK activity in ME-180 cells and that JNK
activation does not significantly contribute to FGF-BP gene expression
in response to EGF.
In conclusion, this study demonstrates that the growth factor EGF
induces FGF-BP gene transcription and characterizes the mechanisms by
which this effect is accomplished. The EGF-mediated pathways leading to
FGF-BP transcription and subsequent angiogenic activation include the
selective activation of MEK/ERK and p38 MAP kinase pathways. This study
highlights several targets for potential anti-angiogenic therapy of
human cancers, which utilize FGF-BP's angiogenic properties for
tumor growth.
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: The Research Building,
Rm. E307, Georgetown University, 3970 Reservoir Rd., N.W., Washington,
D.C. 20007. Tel.: 202-687-1479; Fax: 202-687-4821; E-mail:
ariege01@gunet.georgetown.edu.
