1 Department of Dermatology, Klinikum der J. W. Goethe-Universität,
Frankfurt am Main, Germany
2 Department of Molecular Biology, Max-Planck-Institut für Physiologische
und Klinische Forschung, Bad Nauheim, Germany
* Author for correspondence (e-mail: Gille{at}em.uni-frankfurt.de)
Accepted 24 October 2002
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Summary |
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Key words: Neovascularization, Endothelial growth factors, HGF/SF, VEGF/VPF, Transcription factors, Trans-activation, Signal transduction, Promoter regions
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Introduction |
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HGF/SF is known for its pro-angiogenic properties
(Rosen and Goldberg, 1997) and
is secreted by mesenchyme-derived cells, including fibroblasts and smooth
muscle cells. HGF/SF mediates its effects through binding to the Met receptor
tyrosine kinase, which is predominantly expressed on cells of epithelial
origin, suggesting a key role for HGF/SF as a paracrine mediator of
mesenchyme-epithelial interactions (reviewed by
Birchmeier and Birchmeier,
1994
; Rosen et al.,
1994
). On the basis of its ability to induce VEGF/VPF expression
in different cell types (Clifford et al.,
1998
; Moriyama et al.,
1998
), and to potentiate its angiogenic effect via upregulation of
VEGF/VPF in vivo (Van Belle et al.,
1998
), a model of an indirect paracrine amplification loop of
angiogenesis has been proposed.
We have previously shown that HGF/SF-mediated VEGF/VPF expression by
primary and immortalized keratinocytes, as well as epithelial-derived tumor
cells, is primarily dependent on transcriptional activation, since VEGF/VPF
mRNA stability as a major determinant of mRNA abundance remains unchanged
after HGF/SF stimulation (Gille et al.,
1998). The HGF/SF-responsive region has been mapped to a
GC-box-containing region between bp -88 and -65 of the VEGF/VPF gene promoter,
which is critical for both constitutive and HGF/SF-induced transcriptional
activity. Sp1-like factors constitutively bind to this element, although the
VEGF/VPF promoter is transactivated by HGF/SF in the absence of induced
binding activity. As HGF/SF functions as a potent inducer of VEGF/VPF
expression in different cell types (Dong
et al., 2001
; Van Belle et
al., 1998
) and probably contributes to paracrine amplification of
angiogenesis via VEGF/VPF induction, we set out studies to elucidate molecular
mechanisms underlying HGF/SF-induced VEGF/VPF gene transcription.
Binding of Sp family members to the cluster of Sp1 sites in close proximity
to the transcription start of the VEGF/VPF promoter has been implicated
previously in constitutive (Shi et al.,
2001) and growth-factor-induced VEGF/VPF gene expression
(Finkenzeller et al., 1997
;
Gille et al., 1998
). In
addition, evidence has been provided that transrepression of VEGF/VPF promoter
activity by both von Hippel-Lindau and p53 tumor suppressor gene product is
mediated through Sp1 interaction via the indicated GC-rich response region
(Mukhopadhyay et al., 1997
;
Zhang et al., 2000
).
Regulation of Sp1-dependent transcription may be conveyed by changes in DNA
binding activity, by association with other transcription factors, by changes
in Sp1 abundance or in transactivation activity owing to biochemical
modification, such as phosphorylation (reviewed by
Black et al., 2001
;
Philipsen and Suske, 1999
). In
addition, Sp1-dependent transcription could be effected by changes in relative
binding activity of different Sp family members, as the respective
transactivation activity of Sp1 and Sp3 may vary in a promoter- and
cell-type-specific fashion. Pertinent to these findings, basal and Ras-induced
Sp1-dependent VEGF/VPF promoter activity has been experimentally linked to
interaction with and phosphorylation by the atypical protein kinase C (PKC)
isoform PKC-
in renal cell carcinoma and fibrosarcoma cell lines
(Pal et al., 1998
;
Pal et al., 2001
). The
significance of PKC-
as an intermediate signaling protein was
substantiated in a recent study on stretch-induced VEGF/VPF mRNA expression by
retinal pericytes and endothelial cells
(Suzuma et al., 2002
).
To determine mechanisms that mediate HGF/SF-induced Sp1 site-dependent VEGF/VPF gene transcription, we analyzed nuclear expression levels, binding activities, transactivating properties and phosphorylation states of Sp family members in HGF/SF-stimulated immortalized HaCaT keratinocytes. These studies did not reveal effects on either nuclear expression levels or on relative changes of Sp1 and Sp3 DNA-binding activity in response to HGF/SF. Since the family members Sp1 and Sp3 exert comparable functional effects on transcriptional activation of the VEGF/VPF promoter, induction of VEGF/VPF gene transcription may be conveyed via increased Sp1 transactivation activity, as HGF/SF is herein found to increase intracellular amounts of phosphorylated Sp1 in immunoprecipitation experiments.
Upon binding to its Met receptor tyrosine kinase, HGF/SF has been shown to
concomitantly activate a number of distinct protein modules, including PI
3-kinase and the mitogen-activated protein kinase/extracellular signal-related
kinase kinase (MEK) signaling cassette, which direct a genetic program that
has been shown to facilitate cell proliferation and invasive growth (reviewed
by Comoglio and Boccaccio,
2001; Stuart et al.,
2000
). By the use of pharmacological inhibition, of expression of
kinase-deficient signaling molecules and by the use of antisense
oligonucleotides targeting expression of protein signal transducers, we
provide evidence that both MEK1/2 and PI 3-kinase contribute to HGF/SF-induced
VEGF/VPF promoter activation and subsequent protein expression by HaCaT
keratinocytes. We herein show that HGF/SF increases the intracellular amount
of serine-phosphorylated Sp1, most probably by engaging PKC-
as an
intermediate signaling protein. This novel observation may provide a rational
mechanism of paracrine HGF/SF-induced upregulation of VEGF/VPF expression that
significantly adds to the understanding of a pathway implicated in indirect
amplification of angiogenesis.
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Materials and Methods |
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Cell lines and culture conditions
HaCaT cells (utilized between passages 45 and 65; provided by N. E.
Fusenig; German Cancer Research Center, Division of Carcinogenesis and
Differentiation, Heidelberg, Germany)
(Boukamp et al., 1988) and
A431 cells (American Type Culture Collection, Rockville, MD) were cultured at
37°C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM;
Gibco), supplemented with 10% fetal bovine serum (Gibco), 2 mM L-glutamine
(Gibco), 100 U/ml penicillin, 100 µg/ml streptomycin and 250 µg/ml
amphotericin B (all from Sigma). The embryonic Drosophila cell line
SL2 (provided by G. Suske, Institut für Molekularbiologie und
Tumorforschung, Philipps-Universität Marburg, Germany) was maintained at
25°C in Schneider's medium (Gibco), containing supplements as mentioned
above.
VEGF/VPF protein quantification
For quantitative determination of human VEGF/VPF protein concentrations in
cell culture supernatants, the Quantikine Human VEGF Immunoassay from R&D
Systems was utilized. HaCaT cells were grown in 12-well plates to confluence
and rendered quiescent by changing to serum-free DMEM media for 24 hours.
Cells were incubated with respective pharmacological inhibitors and were
subsequently stimulated by HGF/SF for indicated time periods. Experiments were
performed in triplicate. Total cellular protein was harvested by addition of 5
M sodium hydroxide followed by a freeze/thaw cycle. Subsequently, protein
concentration was determined using the DC Protein Standard Assay (Bio-Rad,
Munich, Germany).
Plasmids and antisense oligonucleotides
The -88/+54 bp VEGF/VPF CAT construct has been described previously
(Gille et al., 1997). Briefly,
the indicated promoter fragment with flanking 5'-HindIII and
3'-XhoI enzyme restriction sites to facilitate directional
cloning into the pCAT basic vector (Promega, Mannheim, Germany) was
synthesized by PCR technique. The construct carrying a two nucleotide mutation
within the Sp1 consensus sites (Sp1 mut) was generated identically, except
that a primer was used that included the two nucleotide mutations. The
constructs were confirmed by sequencing. The -88/+54 bp VEGF/VPF luciferase
construct (Milanini et al.,
1998
), the CMV-promoter-driven expression vectors containing cDNAs
encoding wild-type and dominant-negative (a lysine 281 to tryptophan point
mutation in the catalytic site) forms of PKC-
(Bandyopadhyay et al., 1997
),
the Sp1 (pPacUSp1), Sp3 (pPacUSp3; under the control of the
Drosophila actin 5C promoter) expression, and the corresponding
parent (pPacUbx) plasmids (Hagen et al.,
1994
) have been described earlier. The utilized antisense
oligonucleotides targeted against expression of PKC-
and PKC-
have
been reported previously (Pal et al.,
2001
; Shih et al.,
1999
), and were synthesized as phosphorothioate-modified derivates
(PKC-
: GGTCCTGCTGGGCAT; PKC-
: ATGCCCAGCAGGACC; from MWG Biotech AG,
Ebersberg, Germany).
Transient transfection and analysis of reporter gene expression
HaCaT cells (1.5x106, seeded in 60 mm dishes) were
transfected with 5 µg of appropriate -88/+54 bp VEGF/VPF CAT reporter
construct (wt/mut) using the SuperFect Transfection Reagent (Qiagen). 24 hours
after transfection, control transfectants were left untreated (media change)
and test transfectants were exposed to HGF/SF in the absence or presence of
chemical inhibitors. 16 hours later, lysates were obtained by rapid
freeze/thaw cycles. Quantitative determination of chloramphenicol
acetyltransferase (CAT) expression was performed by colorimetric enzyme
immunoassay (CAT ELISA, Roche Molecular Biochemicals, Mannheim, Germany). CAT
expression was normalized to the activity of the co-transfected
pSV-ß-galactosidase control vector (1 µg, Promega).
ß-galactosidase activity was determined by ELISA using the Enzyme Assay
System from Promega. Internal plasmid controls were not influenced by
treatment with the different protein signaling inhibitors. SL2 cells
(1.5x106, seeded in 60 mm dishes) were transfected with 5
µg -88/+54 bp VEGF/VPF CAT construct along with different combinations of
expression vectors (Sp1, Sp3) or parent vector
(Suske, 2000). After
transfection, cells were propagated for 48 hours in medium only. Cells were
lysed by rapid freeze/thaw cycles, and CAT activity was analyzed as described
above. For co-expression experiments with wild-type and dominant-negative
PKC-
expression plasmids, transfections were performed by electroporation
(Gene Pulser II, Bio-Rad) using 1x106 HaCaT cells in the
presence of 2.5 µg of the -88/+54 bp VEGF/VPF luciferase construct and 500
ng of expression vector. 24 hours after transfection, cells were stimulated
with HGF/SF or were left untreated. Reporter gene activity was measured after
16 hours by the Lumat LB 9507 (Berthold Technologies, Bad Wildbad, Germany),
utilizing Promega's Luciferase Reporter Assay System. For inhibition of PKC
isoform expression by antisense oligonucleotides, HaCaT cells
(2x105, seeded in 12 well dishes) were transfected by
Lipofectamine PLUS (Gibco) following the supplier's instructions with 1 µg
of -88/+54 bp VEGF/VPF luciferase construct and 0.01 µM or 0.1 µM (final
concentration) of the respective antisense oligonucleotides. 24 hours after
transfection, cells were stimulated with HGF/SF for 16 hours or were left
untreated. The activity of firefly luciferase was analyzed as described
above.
Preparation of nuclear extracts and gel mobility shift analysis
HaCaT cells were treated with HGF/SF for 60 minutes. Nuclear proteins were
extracted as described previously (Dignam
et al., 1983). The oligonucleotides were synthesized to span the
region between -88 bp and -65 bp of the human VEGF/VPF promoter:
5'-TTTCCGGGGCGGGCCGGGGGCGGGGGTAT-3'.
The underlined sequence served as a template for the synthesis of the second
strand (random non-wild-type flanking sequences are shown in italic letters).
Radiolabeled double-stranded DNA was synthesized by annealing an
oligonucleotide complementary to the underlined sequence listed above
(5'-ATACCCCGCCCC-3') and by extension of the second strand with
Klenow fragment, 50 µCi of [
-32P]-dCTP, unlabeled dATP,
dTTP and dGTP. Unincorporated nucleotides were removed by column
chromatography. Cold unlabeled double-stranded DNA was made in the same way
except that unlabeled dCTP was substituted for labeled dCTP. The DNA-binding
reaction was performed for 30 minutes at room temperature in a volume of 20
µl, containing 5 µg of nuclear protein extract, 2.5 mg/ml bovine serum
albumin, 105 cpm [
-32P]-labeled probe
(
0.5-1.0 ng), 0.1 mg/ml poly[dI:dC] (Sigma), 5 µl of 4x binding
buffer [1x buffer: 10 mM Tris-Cl, pH 7.8, 100 mM KCl, 5 mM
MgCl2, 1 mM EDTA, 10% (v/v) glycerol, 1 mM DTT] with or without
excess of unlabeled competitor, Sp1 consensus-oligonucleotide (Promega), Sp1
or Sp3 antibody. Samples were subjected to electrophoresis on a native 4%
polyacrylamide gel (PAGE) for 2.5 hours at 120 V.
Western blot analysis
Cells were grown to confluence and rendered quiescent by changing to
serum-free media for 24 hours. Prior to HGF/SF exposure for 10 minutes, cells
were treated with chemical inhibitors for 1 hour or were left untreated. Cells
were washed with cold phosphate-buffered saline (PBS), lysed in sample buffer
containing 187.5 mM Tris-HCl, pH 6.8, 6% sodium dodecyl sulphate (SDS) (w/v),
30% glycerol (v/v), 0.3 mM DTT, 0.3% bromophenol blue sodium salt (w/v),
sonicated and boiled at 95°C for 5 minutes. Lysates were separated in 10%
SDS-PAGE and transferred to polyvinylidene fluoride (PVDF)-membrane
(Millipore, Eschborn, Germany) at 50 V, subsequently blocked in Tris-buffered
saline-Tween 20 (TBS-T) containing 5% BSA or non-fat milk. The membranes were
incubated with primary antibodies followed by incubation with
horseradish-peroxidase-conjugated secondary antibodies (anti-mouse or
anti-rabbit, Amersham Biosciences). The blots were then developed using the
enhanced chemiluminescence detection system (ECL) according to the
instructions of the manufacturer (Amersham). For time-dependent analysis of
Sp1 and Sp3 protein, extracts were obtained by the procedure outlined for
nuclear extracts.
Immunoprecipitation
For Sp1 immunoprecipitation, cells were lysed in cold buffer containing 20
mM Tris-HCl, 300 mM NaCl, 2 mM EDTA, 2 mM ethylenglycol-bis-(ß-aminoethyl
ether)-tetraacetic acid, 2% Triton (v/v), 1% NP-40 (v/v), supplemented with
proteinase/phosphatase inhibitors, for 30 minutes on ice. Protein
concentrations were determined with the DC Protein Standard Assay (Bio-Rad).
Immunoprecipitations were carried out at antibody excess, incubating 2.5 mg of
total lysate with of Sp1 antibody (Sigma) on a rotator at 4°C overnight.
Immunocomplexes were then captured with Protein G Sepharose 4 Fast Flow
(Amersham Biosciences). After three washes with lysis buffer, the
immunoprecipitates were resuspended in electrophoresis sample buffer and
subjected to western blot analysis. For PKC- immunoprecipitation
experiments, the protocol previously reported by Standaert et al. was used
with minor modifications (Standaert et
al., 1997
).
Statistics
Normality (after Kolmogorov-Smirnov) of the data was confirmed by SigmaSTAT
(SPSS Inc. Chicago). For statistical analysis, a student's t-test was
performed using the Excel software from Microsoft (Redmond, WA). A
P<0.05 on the basis of at least three independent sets of
experiments was considered to be statistically significant.
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Results |
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To clarify whether HGF/SF induced differences in relative DNA-binding
activity of Sp family members to the cluster of Sp1 sites despite the absence
of changes in nuclear expression levels, super-shift analyses were carried out
using the -88/-65 bp promoter sequence as a specific DNA probe
(Fig. 2). These electromobility
shift assays (EMSAs) revealed multiple constitutive binding activities that
were almost entirely Sp dependent, as shown by competition with excess Sp1
consensus oligonucleotides (lanes 3 and 4). Addition of antibodies directed
against either Sp1 or Sp3 induced a supershift and/or a significant reduction
of Sp1-dependent binding activities (lanes 5 to 8). Simultaneous addition of
both antibodies leads to a nearly complete supershift (lanes 9 and 10). These
data indicate that Sp1-dependent binding activity mostly comprises Sp1 and Sp3
protein, which is in line with previous reports on NIH3T3 fibroblasts
(Finkenzeller et al., 1997)
and pancreatic adenocarcinoma cell lines
(Shi et al., 2001
). However,
both competition and super-shift analyses revealed no discernible differences
in binding activities between extracts of untreated and HGF/SF-stimulated
cells (Fig. 2). Thus our
results are in opposition to the assumption that an induced change in relative
DNA binding activity of Sp1 and/or Sp3 protein may operate as a mechanism by
which HGF/SF activates VEGF/VPF gene transcription in the absence of inducible
DNA-binding activity to the functional response element.
|
Both Sp1 and Sp3 transcription factor induce Sp1 site-dependent
VEGF/VPF promoter activation in a similar fashion to Drosophila SL2
cells
Extending our DNA-binding experiments, we subsequently determined whether
the HGF/SF effect could be mediated through differing transactivating
properties of the respective Sp family members as a function of subtle binding
variations to the GC-boxes. We therefore explored whether Sp1 and Sp3 exerted
different effects on VEGF/VPF promoter activity and whether their individual
and/or combined effects were preferentially mediated via the Sp1 consensus
binding sites. To eliminate the impact of constitutive expression of Sp family
members in HaCaT keratinocytes, these experiments were performed in
Drosophila Schneider SL2 cells. As the HGF/SF-response has been
functionally mapped to a VEGF/VPF promoter region between bp -88 and -70
(Gille et al., 1998), the
expression vectors encoding Sp1 and Sp3 were cotransfected with the -88/+54 bp
VEGF/VPF reporter plasmid. Initially, the dosage of maximal reporter gene
induction by the respective expression plasmids was determined. The results
were similar with both plasmids, and maximal induction was seen with 250 ng to
500 ng of co-transfected DNA, with no further increase observed with higher
amounts (data not shown). Co-expression of either Sp1 or Sp3 revealed marked
activation of VEGF/VPF promoter activity compared with cells co-transfected
with backbone vector only (Fig.
3). Sp1 expression only tended to act as a slightly more effective
inducer of transcriptional activation than Sp3; however, the differences were
not seen to be statistically significant. In addition, simultaneous expression
of Sp3 and Sp1 did not attenuate Sp1-driven reporter gene activity, as has
been observed previously with different promoters
(Hagen et al., 1994
). In order
to demonstrate that induced expression by Sp1 and Sp3 was Sp1 site dependent,
a -88/+54 bp VEGF/VPF reporter plasmid was utilized that carried two critical
nucleotide mutations within the adjacent Sp1 sites. Indeed, co-expression
experiments with this mutant construct showed marked inhibition compared to
co-transfections with the respective wild-type plasmid. Together, these
results indicate that HGF/SF-induced Sp1 site-dependent VEGF/VPF transcription
may not be mediated by relative changes of Sp1 and/or Sp3 DNA binding or by
differing transactivating properties of the respective Sp family members,
taking into account that subtle binding variations to the GC-boxes may not
have been detected by EMSA.
|
HGF/SF mediates serine phosphorylation of Sp1 transcription
factor
In the absence of both apparent relative changes in Sp1 and Sp3 DNA-binding
activity and significant functional differences in Sp family members, we
proposed that HGF/SF increases transactivation activity of Sp1 as a molecular
mechanism to induce VEGF/VPF gene transcription. As phosphorylation has been
implicated in changes of Sp1 transcriptional activity
(Alroy et al., 1999;
Chun et al., 1998
;
Fojas et al., 2001
), we next
determined whether HGF/SF increased the amount of phosphorylated Sp1
(Fig. 4). For this purpose,
whole protein extracts of untreated and HGF/SF-stimulated cells were
immunoprecipitated by Sp1 antibody (Sigma) and were subsequently subjected to
western blot analyses utilizing a panel of antibodies, specific for
phospho-threonine-containing proteins (binding to threonine-phosphorylated
sites in a manner largely independent of the surrounding amino-acid sequence)
or recognizing a distinct pattern of serine-phosphorylated proteins
(preferring positively charged amino acids adjacent to phospho-serine). Among
the different clones directed against phospho-serine-containing proteins (1C8,
4A3, 4A9, 4H4, 16B4, 7F12), the antibody designated 4A3 reproducibly detected
a strongly enhanced signal in extracts of HGF/SF-treated cells compared with
unstimulated controls (Fig.
4A). This effect was not HaCaT-cell-specific, as an
HGF/SF-mediated increase in serine phosphorylated Sp1 was also observed in
A431 carcinoma cells (data not shown). To monitor the correct level of
migration, the blots were also probed with Sp1 antibody
(Fig. 4A,B; upper panel). The
remaining antibodies, including the one that recognizes
phospho-threonine-containing proteins (Fig.
4B), did not display enhanced signal strength after HGF/SF
treatment relative to untreated controls. These data indicate that HGF/SF is
capable of increasing relative amounts of serine-phosphorylated Sp1
transcription factor in HaCaT keratinocytes. Nevertheless, phosphorylation
events could involve threonine residues of Sp1 protein as well, which may have
not been detected by the antibodies used. Together, in the absence of any
changes regarding nuclear expression and DNA binding, the HGF/SF-induced
increase in phosphorylated Sp1 may represent a rational mechanism to enhance
Sp1's transcriptional activity, conveying HGF/SF-induced VEGF/VPF
transcription.
|
HGF/SF-mediated transactivation of the VEGF/VPF promoter is repressed
by atypical PKC- antisense oligonucleotides and overexpression of
dominant-negative PKC-
mutant, whereas inhibition of conventional/novel
PKC isoforms fails to block induced VEGF/VPF transcription
HGF/SF has been demonstrated to transduce its biological activities through
the Met receptor tyrosine kinase, activating a number of intracellular
pathways to integrate the HGF/SF signal to the cytosol and to the nucleus
(reviewed by Comoglio and Boccaccio,
2001; Stuart et al.,
2000
). Responses to HGF/SF binding have been shown to involve
activation of different functional protein kinase C (PKC) subspecies that are
associated with enhanced cell migration and growth
(Cai et al., 2000
;
Chandrasekher et al., 2001
)
and are linked in part to increased invasive potential of cancer cells owing
to induction of protease expression
(Kermorgant et al., 2001
). As
classical and novel PKC activation contributes to induced VEGF/VPF expression
in response to certain stimuli (Hossain et
al., 2000
; Kim et al.,
2000
), we sought to determine the potential contribution of PKC
activation to the HGF/SF-induced VEGF/VPF gene expression. To verify whether
HGF/SF activates conventional/novel PKC isoforms in HaCaT keratinocytes,
western blot analyses of extracts from treated and unstimulated cells were
performed (Fig. 5A), utilizing
an antibody that detects the phosphorylated PKC-
isoform. HGF/SF was
seen to increase PKC phosphorylation, which was substantially diminished by
preincubation with known conventional/novel PKC inhibitors
(bisindolylmaleimide I, calphostin C). Consequently, we clarified whether
pharmacological PKC inhibition blocked HGF/SF-induced VEGF/VPF promoter
activation (Fig. 5B). Both
bisindolylmaleimide I and calphostin C (data not shown) failed to abrogate
VEGF/VPF-promoter-based reporter gene expression, however
(Fig. 5B). These observations
are in line with effects observed on induced VEGF/VPF protein expression by
HaCaT keratinocytes, as chemical PKC inhibition failed to block protein
synthesis as well (Fig. 5C,
equivalent data were obtained from studies with bisindolylmaleimide I). This
data thus indicate that HGF/SF-induced VEGF/VPF expression by HaCaT
keratinocytes is independent of conventional and novel PKC isoforms.
|
Besides conventional and novel PKC isoforms, atypical PKC- has
recently been closely linked to transcriptional activation of the VEGF/VPF
gene (Pal et al., 1998
;
Shih et al., 1999
). In our
studies on HaCaT keratinocytes, PKC-
was promptly phosphorylated in
response to HGF/SF (Fig. 6A).
In the current absence of isotype-specific PKC-
inhibitors, explicit
antisense oligonucleotides (AS) were utilized in transcriptional activation
studies that have been previously shown to effectively block PKC isoform
expression (Pal et al., 2001
;
Shih et al., 1999
)
(Fig. 6B). Whereas both
PKC-
and PKC-
antisense oligonucleotides at 0.01 µM did not
effect transcription (data not shown), at a concentration of 0.1 µM
AS-PKC-
, but not AS-PKC-
, inhibited reporter gene expression.
These observations are in agreement with our chemical inhibition studies
(Fig. 5B), revealing a lack of
inhibitory efficacy on VEGF/VPF promoter activity by conventional/novel PKC
inhibitor bisindolylmaleimide I. The potential contribution of PKC-
was
substantiated in experiments in which expression of wild-type and
dominant-negative PKC-
protein on VEGF/VPF gene transcription was
analyzed (Fig. 6C).
Overexpression of deficient PKC-
protein significantly inhibited basal
and HGF/SF-induced transactivation. In addition, broad-range PKC inhibitor RO
31-8220, which also affects atypical PKC-
(Standaert et al., 1997
),
blocks HGF/SF-induced VEGF/VPF transcription in a concentration-dependent
fashion (data not shown). Together, these findings suggest that PKC-
is
engaged as an intermediate signaling protein in HGF/SF-induced VEGF/VPF
expression.
|
HGF/SF-induced VEGF/VPF gene expression is mediated via activation of
PI 3-kinase and MEK1/2 signaling modules
Among the protein modules activated via HGF/SF binding to the Met receptor,
PI 3-kinase and the MEK1/2 signaling cascades appear to act as transducers of
central importance (Stuart et al.,
2000). To determine whether additional signaling proteins are
implicated in HGF/SF-mediated VEGF/VPF transcription and protein expression by
HaCaT cells, we evaluated the contribution of the indicated pathways largely
by use of selective chemical inhibitors. HGF/SF activates Akt by
phosphorylation at both the threonine 308 and the serine 473 residues
(Fig. 7A). Abrogation of Akt
activation in response to HGF/SF by the PI 3-kinase inhibitors wortmannin and
LY 294002 clearly suggests that Akt phosphorylation in HaCaT keratinocytes is
PI 3-kinase dependent. Moreover, both PI 3-kinase inhibitors effectively
inhibit HGF/SF-induced VEGF/VPF-promoter-based reporter gene expression and
subsequent protein expression (Fig.
7B,C), strongly implicating PI 3-kinase in the signaling pathway
of HGF/SF-mediated VEGF/VPF expression.
|
In addition, selective inhibition of MEK1 by compound PD 98059 completely blocked HGF/SF-induced phosphorylation of downstream ERK1/2 (Fig. 8A), attenuated baseline VEGF/VPF expression and entirely prevented upregulation of VEGF/VPF gene transcriptional activation and protein expression (Fig. 8B,C). Equivalent results were obtained by utilizing the MEK1/2 inhibitor U0126 (data not shown), establishing a key contribution of the MEK1/2 signaling protein to HGF/SF-induced gene expression by HaCaT keratinocytes. As both PI 3-kinase and MEK1/2 inhibition resulted in abrogation of induced VEGF/VPF expression, the question arose of whether crosstalk between the indicated signaling pathways exists (Fig. 9A). As shown above, both PD 98059 and LY 294002 as explicit inhibitors blocked phosphorylation of their appropriate downstream targets in HaCaT cells; however, they failed to inhibit HGF/SF-induced activation of the respective mutual downstream signaling proteins. Whereas LY 294002 inhibited HGF/SF-induced Akt activation, it did not abolish ERK1/2 phosphorylation. Similarly, PD 98059 inhibited HGF/SF-induced MEK1/2 activation but revealed no effect on Akt activation (Fig. 9A). From a hierarchical point of view, PI 3-kinase and MEK1/2 thus appear to mediate the HGF/SF signal partly in parallel in HaCaT keratinocytes, as HGF/SF-induced activation of immediate PI 3-kinase and MEK1/2 downstream targets is mutually independent.
|
|
HGF/SF-induced phosphorylation of PKC- and Sp1 is blocked by PI
3-kinase and MEK1/2 inhibition
Both pharmacological inhibitors of PI 3-kinase and MEK1 seem to jointly
block shared downstream signaling proteins, as each inhibitor by itself almost
completely prevents HGF/SF-mediated VEGF/VPF transcription and protein
expression (Figs 7 and
8). Induced phosphorylation of
respective downstream targets of PI 3-kinase and MEK1/2 occurs despite the
presence of overexpressed kinase-deficient PKC-
(Fig. 9B), indicating that
PKC-
may serve as a downstream effector of MEK1/2 and PI 3-kinase upon
HGF/SF stimulation. This assumption is further supported by experimental
evidence demonstrating that HGF/SF-induced activation of PKC-
is
efficiently blocked by both PI 3-kinase and MEK1/2 inhibition
(Fig. 10A,B). These data
strongly imply that PI 3-kinase and MEK1/2 act as upstream signaling proteins
in HGF/SF-induced PKC-
phosphorylation. To substantiate the importance
of Sp1 phosphorylation in HGF/SF-induced VEGF/VPF gene expression, we next
determined the effect of PI 3-kinase and MEK1/2 inhibition on HGF/SF-induced
Sp1 phosphorylation. In agreement with our hypothesized model, induced
phosphorylation of Sp1 is prevented by pharmacological inhibition of PI
3-kinase and MEK1 (Fig. 11).
In addition, broad range PKC inhibition by compound RO 31-8220 effectively
blocked HGF/SF-induced Sp1 phosphorylation, suggesting that Sp1 may function
as a downstream target for PI 3-kinase, MEK1/2 and PKC-
. Together, these
data indicate that HGF/SF induces serine-phosphorylation of Sp1 transcription
factor via PI 3-kinase, MEK1/2 and PKC-
signaling.
|
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![]() |
Discussion |
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Here, we provide evidence that HGF/SF-mediated VEGF/VPF gene transcription
and subsequent protein expression may be conveyed via increased Sp1
phosphorylation as a way to enhance Sp1 transactivating activity. Previously,
we showed that HGF/SF-induced VEGF/VPF expression by keratinocytes and
epithelial-derived tumor cells is primarily dependent on transcriptional
activation, localizing the HGF/SF-responsive region to a Sp1-site-containing
region between bp -88 and -65 of the VEGF/VPF gene
(Gille et al., 1998). This
region is critical for both basal and HGF/SF-induced transcriptional activity.
The Sp family members Sp1 and Sp3 bind to this element constitutively
(Fig. 2); however the VEGF/VPF
promoter is transactivated by HGF/SF in the absence of induced binding
activity. Though less frequently reported, regulated Sp1-site-dependent
transcription may be mediated also in the absence of changes in factor binding
to different genes (Alliston et al.,
1997
; Black et al.,
1999
; Borroni et al.,
1997
). As our supershift EMSAs indicate that bound complexes
comprise either Sp1 or Sp3 protein (Fig.
2), variations in DNA binding and/or association of additional
transcription factors with Sp proteins may represent unlikely mechanisms of
HGF/SF-induced VEGF/VPF transcription.
Sp1 and Sp3 are ubiquitously expressed transcription factors that recognize
GC-rich sequences (Azizkhan et al.,
1993) present in regulatory sequences of numerous housekeeping
genes and genes involved in growth regulation and cancer (reviewed by
Black et al., 2001
;
Philipsen and Suske, 1999
).
Whereas Sp1 has mostly been shown to act in a positive manner, Sp3 may
function either as repressor or activator of Sp1-mediated activation,
depending on both the promoter-dependent and cellular context. Our
co-expression experiments with Sp1 and Sp3 in Sp-deficient Schneider cells
indicate that both factors are equally capable of activating the VEGF/VPF
promoter through the HGF/SF response region
(Fig. 3). The effects may be
slightly different in pancreatic carcinoma cells, in which co-expression of
Sp3 was recently reported to induce VEGF/VPF transactivation to a lesser
extent than did Sp1 expression (Shi et
al., 2001
). The differences in our observation may be related to
the different length of the utilized VEGF/VPF-promoter-based reporter gene
construct or may be due to cell-dependent basal expression levels of the
respective Sp factors. In addition, our studies reveal that Sp3 lacks
repressive effects on Sp1-induced VEGF/VPF transcription. Hence,
HGF/SF-induced Sp1 site-dependent VEGF/VPF transcription in HaCaT
keratinocytes may not be conferred by relative changes of Sp1 and/or Sp3 DNA
binding or by differing transactivating properties of the respective Sp family
members on VEGF/VPF transactivation.
As a result, we favored the hypothesis that HGF/SF increased
transactivation activity of constitutively expressed Sp family members as a
molecular mechanism to induce VEGF/VPF gene transcription. Previously, it has
been demonstrated that Sp1 is subject to several post-transcriptional
modifications including glycosylation and phosphorylation (reviewed by
Black et al., 2001;
Philipsen and Suske, 1999
).
Sufficient evidence suggests that Sp1 phosphorylation may represent a means
for regulating transcriptional initiation. In the current study, Sp1
immunoprecipitates were assayed by western blotting for the presence of
serine- and threonine-phosphorylated proteins, revealing increased
intracellular amounts of serine-phosphorylated Sp1 in response to HGF/SF
(Fig. 4A). Therefore, of the
potential parameters tested, neither alterations in expression and DNA-binding
activity nor distinct transactivating properties of the respective Sp family
members accounted for HGF/SF-induced Sp1 site-dependent VEGF/VPF
transcription; only changes in Sp1 phosphorylation could account for it.
Alhough the evidence presented is largely indirect, along with our
supplementary findings as well as previously established mechanisms of
Sp1-site-dependent transcription, an increase in Sp1's transactivation
potential owing to changes in its phosphorylation state may provide a rational
and likely conclusion by which HGF/SF conveys induced VEGF/VPF
transcription.
Whereas Sp1 is ubiquitously expressed, activation and recruitment of
distinct transduction pathways and signaling molecules may serve as mechanisms
to promote transactivation of selected responsive genes. Sp1 is phosphorylated
by a number of cellular kinases, including DNA-dependent protein kinase,
protein kinase A (PKA) and different members of the PKC family (reviewed by
Black et al., 2001). Among the
PKC isoforms, atypical PKC-
has been implicated in Sp1-dependent VEGF/VPF
gene expression (Pal et al.,
1998
; Pal et al.,
2001
). It has been indicated that Sp1 can bind to PKC-
and may
act as a direct substrate for PKC-
. In addition, overexpression of
PKC-
induced VEGF/VPF transcription in a Sp1-site-dependent fashion,
whereas co-transfection of a dominant-negative PKC-
mutant repressed
constitutive VEGF/VPF promoter activity concentration dependently. The
importance of PKC-
was substantiated in a recent study on stretch-induced
VEGF/VPF mRNA expression (Suzuma et al.,
2002
). Overexpression of kinase-deficient PKC-
by adenoviral
transfer almost entirely blocked stretched-induced VEGF/VPF mRNA levels by
bovine retinal pericytes and endothelial cells. However, expression of
wild-type PKC-
did not induce VEGF/VPF transcript levels in these cell
types. To determine the putative role of PKC-
in HGF/SF-induced VEGF/VPF
expression, we studied the impact of antisense oligonucleotide transfection as
well as wild-type and mutant PKC-
overexpression
(Fig. 6B,C). These experiments
suggest that PKC-
is critically involved in HGF/SF-induced VEGF/VPF
expression by HaCaT keratinocytes. Inhibition of PKC-
significantly blocks
induced VEGF/VPF transcriptional activity, whereas antisense oligonucleotides
directed against conventional PKC-
fails to exert inhibitory effects in
our transfection experiments. The contribution of PKC-
is underscored by
findings showing prompt phosphorylation of PKC-
in response to HGF/SF and
inhibition of induced VEGF/VPF expression by overexpression of mutant PKC-
(Fig. 6A). To further
strengthen the significance of Sp1 phosphorylation in HGF/SF-induced VEGF/VPF
gene expression, we determined the effect of broad range PKC inhibition by
compound RO 31-8220 on HGF/SF-induced Sp1 phosphorylation. In agreement with
our hypothesis, induced phosphorylation of Sp1 is prevented by broad range PKC
inhibition (Fig. 11). As
PKC-
has been previously shown to interact with and to phosphorylate Sp1
(Pal et al., 1998
), PKC-
may be engaged as an essential downstream target of HGF/SF signaling in HaCaT
cells.
To identify critical upstream signaling molecules of HGF/SF-induced
VEGF/VPF promoter activation, the effects of specific pharmacological
inhibitors and overexpression of kinase-deficient signaling proteins were
studied. HGF/SF have been demonstrated to mediate its biological activities
through binding to the Met receptor, activating a number of intracellular
pathways (reviewed by Comoglio and
Boccaccio, 2001; Stuart et
al., 2000
). Also, basal and induced VEGF/VPF gene expression has
been shown to require distinct signaling modules, depending on the stimulus
applied as well as on the cellular context chosen. MEK1/2, PI 3-kinase and
different PKC isoforms belong to the major signaling molecules implicated in
both HGF/SF-mediated responses and regulated VEGF/VPF expression. The PKC
family currently consists of 12 isozymes, which are grouped in different
functional classes, the conventional (cPKC-
, -ßI,
-ßII, -
), novel (nPKC-
, -
, -
,
-
), atypical (aPKC-
, -
) and recently described PKC-µ and
-
on the basis of the structural differences that foster different
requirements for activation by diacylglycerol and calcium
(Dempsey et al., 2000
;
Newton, 1997
). Earlier, HGF/SF
treatment was reported to activate and translocate PKC-
, -
and
-
in neocortical cells (Machide et
al., 1998
) and PKC-
, -ßII and -
in a
lung adenocarcinoma cell line (Awasthi and
King, 2000
). In addition, chemical inhibition of
conventional/novel PKC isozymes was previously found to abrogate induced
VEGF/VPF expression in different cell types
(Hossain et al., 2000
;
Kim et al., 2000
). In our
studies, HGF/SF was shown to increase phosphorylation of novel PKC-
in
immortalized HaCaT keratinocytes, which was inhibited by preincubation with
broad-range and isotype-specific conventional/novel PKC inhibitors
(Fig. 5A). Though different PKC
isozymes may be activated by HGF/SF, induced VEGF/VPF transcription and
protein expression were not affected by chemical PKC inhibition at
concentrations that effectively blocked PKC phosphorylation
(Fig. 5B,C). Whereas
HGF/SF-mediated activation of different functional PKC isozymes has been
associated with enhanced cell migration and growth
(Cai et al., 2000
;
Chandrasekher et al., 2001
)
and has been linked to increased invasive potential of cancer cells
(Kermorgant et al., 2001
),
conventional/novel PKC seems not to contribute to angiogenesis via
HGF/SF-induced VEGF/VPF expression in keratinocyte-derived HaCaT cells.
The key signaling proteins MEK1/2 and PI 3-kinase have been clearly linked
to both HGF/SF-mediated responses and induced VEGF/VPF expression. We assessed
whether these kinases are activated by HGF/SF in HaCaT keratinocytes and
whether their explicit inhibition affected HGF/SF-induced VEGF/VPF expression.
Our data demonstrate that ERK1/2 and Akt as the respective downstream targets
of MEK1/2 and PI 3-kinase are phosphorylated in response to HGF/SF (Figs
7 and
8). Specific pharmacological
inhibition of MEK1/2 and PI 3-kinase blocked activation of their substrates
and also abrogated HGF/SF-induced VEGF/VPF promoter activity and protein
expression, indicating a critical contribution of both signaling modules.
These findings are in line with a most recent report on HGF/SF-induced
VEGF/VPF mRNA and protein expression in head and neck squamous carcinoma
(Dong et al., 2001),
indicating that the HGF/SF induction pathway may be not be restricted to
keratinocytes only but may be functional in different epithelial cells. As
chemical inhibition of either PI 3-kinase or MEK1/2 resulted in abrogation of
induced VEGF/VPF expression, the question arose as to whether there is
crosstalk between the two signaling pathways or whether they are connected
sequentially to induce VEGF/VPF expression. Previously, it has been reported
that MEK1/2 inhibition can diminish HGF/SF-induced Akt phosphorylation, as a
substrate of PI 3-kinase (Delehedde et
al., 2001
; Yu et al.,
2001
), or vice versa. PI 3-kinase inhibition may also block
HGF/SF-induced ERK1/2 activation, as a substrate of MEK1/2
(Delehedde et al., 2001
;
Sipeki et al., 2000
). In our
studies, however, neither MEK1/2 inhibition blocked Akt phosphorylation nor PI
3-kinase inhibition reduced ERK1/2 activation
(Fig. 9), whereas
phosphorylation of the corresponding substrates was abrogated.
Mechanistically, PI 3-kinase and MEK1/2 thus appear to mediate the HGF/SF
signal partly in parallel in HaCaT keratinocytes without crosstalk, as
HGF/SF-induced activation of immediate PI 3-kinase and MEK1/2 downstream
targets is mutually independent. Nonetheless, both signaling modules appear to
jointly block shared downstream signaling protein(s), since pharmacological
inhibition of each pathway almost completely blocked HGF/SF-mediated VEGF/VPF
transcription and protein expression. A comparable signaling scenario seems to
be in place to mediate anti-apoptotic HGF/SF effects in different cell lines
(Xiao et al., 2001
). PKC-
may serve as a downstream effector of MEK1 and PI 3-kinase upon
HGF/SF-stimulation, since phosphorylation of respective downstream targets
occurs despite the presence of overexpressed kinase-deficient PKC-
(Fig. 9). This assumption is
also supported by experimental evidence demonstrating that HGF/SF-induced
activation of PKC-
is efficiently blocked by both PI 3-kinase and MEK1/2
inhibition (Fig. 10A,B). These
results suggest that signaling pathways employed to direct HGF/SF biological
effects are both stimulus and cell context dependent.
Pertinent to this conclusion, regulated VEGF/VPF gene expression has also
been shown to be conferred via activation of distinct signaling molecules,
depending on the cellular context and on the inducing stimulus. Since the
first description of the significance of the Sp1 site cluster in close
proximity to the transcription start
(Gille et al., 1997;
Ryuto et al., 1996
), this
region has been implicated in the regulation of VEGF/VPF gene transcription in
response to several important stimuli
(Finkenzeller et al., 1997
;
Gille et al., 1998
;
Tanaka et al., 2000
).
Moreover, transrepression of VEGF/VPF promoter activity by von Hippel-Lindau
and p53 tumor suppressor gene product was shown to be mediated via interaction
with this GC-rich region (Mukhopadhyay et
al., 1997
; Zhang et al.,
2000
). Alhough many different stimuli for VEGF/VPF induction have
been identified in the past, studies on comprehensive evaluation of involved
upstream signaling molecules have only recently been reported. Activation of
VEGF/VPF promoter activity by MEK1 overexpression was shown to depend on the
GC-rich -88/-66 bp promoter element in lung fibroblasts. By contrast,
overexpression of c-Jun N-terminal kinase (JNK) or p38 MAPK as well as their
activating kinases had no effect on VEGF/VPF promoter activity in this
experimental set-up (Milanini et al.,
1998
). Additionally, overexpression of Ras has been shown to
mediate Sp1-dependent transcriptional activation of VEGF/VPF, involving PI
3-kinase and PKC-
as important intermediary signaling molecules
(Pal et al., 2001
). The
contribution of MEK1/2 as a potential target protein of Ras signaling was not
investigated in these studies, however. PI 3-kinase may also be involved in
hypoxia-induced VEGF/VPF gene expression, as PI 3-kinase inhibition partially
blocked VEGF/VPF in epithelial cells
(Blancher et al., 2001
). In
Ha-Ras-transformed fibroblasts, inhibition of PI 3-kinase even completely
prevented hypoxia-mediated VEGF/VPF expression
(Mazure et al., 1997
).
Notably, the hypoxia-response element is located far more upstream in the
VEGF/VPF promoter at bp -985/-939, requiring the interaction with
hypoxia-inducible factor-1 (Forsythe et
al., 1996
; Liu et al.,
1995
). VEGF/VPF activation via different response elements may
thus be channeled through similar signaling pathways. The molecular mechanisms
involved in induced VEGF/VPF expression by a given stimulus are therefore not
predictable, as they may involve PI 3-kinase/PKC-
but not MEK1/2 (e.g.
stretch) (Suzuma et al., 2002
)
or they may depend on p38 MAPK but not on MEK1/2 and PI 3-kinase (e.g.
heregulin-ß1) (Xiong et al.,
2001
). Consequently, the cellular context and the induction
pathways have to be considered to define appropriate targets in order to
intersect with the regulated VEGF/VPF expression as a denominator of
angiogenesis.
Together, we demonstrate that both MEK1/2 and PI 3-kinase contribute to
HGF/SF-induced VEGF/VPF promoter activation and protein expression by
keratinocytes. We herein provide thorough evidence that HGF/SF increases the
intracellular amount of serine-phosphorylated Sp1, most likely by engaging
PKC- as a downstream intermediate signaling protein. Our findings
characterize a novel potential mechanism of paracrine HGF/SF-induced
upregulation of VEGF/VPF expression that permits a more detailed understanding
of a pathway implicated in indirect amplification of angiogenesis.
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References |
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