(Received for publication, December 14, 1994; and in revised form, June 7, 1995)
From the
Stimulation of NIH 3T3 cells with platelet-derived growth factor
(PDGF)-BB and 12-O-tetradecanoylphorbol-13-acetate (TPA)
enhances vascular endothelial growth factor (VEGF) gene expression. To
address the question of whether Ras and Raf are involved in the
induction of VEGF gene expression by PDGF and TPA, we examined the
effects of both factors on NIH 3T3 cells stably transfected with
v-Ha-ras or v-raf. In serum-starved NIH 3T3 cells,
only low levels of mRNA expression can be detected, whereas both ras and raf transformed cell lines express enhanced
levels of a 4.3-kilobase VEGF transcript. Stimulation with PDGF or TPA
resulted in increased VEGF mRNA in all cell lines, with highest levels
found in the transformed cells. Immunofluorescence studies confirmed
that the elevated VEGF mRNA expression correlated with enhanced protein
levels. Positive immunofluorescence signals could be detected in
v-Ha-ras or v-raf transformed cell lines but not in
unstimulated NIH 3T3 cells. VEGF from conditioned medium of v-raf transformed NIH 3T3 cells was partially purified by chromatography
on heparin-Sepharose. Biological activity of this VEGF protein was
demonstrated by competition with binding of recombinant I-VEGF
to human umbilical vein endothelial
cells and by its ability to stimulate proliferation of these cells.
Vascular endothelial growth factor (VEGF) ()has
several features as a mediator of both normal and pathological
angiogenesis. It specifically stimulates growth of endothelial cells
and induces angiogenesis in
vivo(1, 2, 3) . VEGF is produced by
several differentiated cell
types(1, 4, 5, 6) , and VEGF mRNA is
also expressed in various transformed cell
lines(7, 8) . The VEGF gene is transcribed into at
least four mRNA types by alternative splicing, giving rise to four
distinct VEGF proteins of 121, 165, 189, and 206 amino acids after
cleavage of the 26-amino acid signal peptide(9, 10) .
The two smaller proteins are efficiently secreted, whereas the larger
forms apparently remain associated with heparin-containing
proteoglycans on the cell surface(10) . Four AP-1 binding sites
have been identified within the promoter sequence of the VEGF
gene(9) . Cells either treated with phorbol ester, leading to
activation of protein kinase C (PKC), or stimulated with PDGF-BB induce
VEGF gene expression, suggesting the participation of AP-1 binding
sites(9, 11) . Therefore, PKC is one essential
component in signal transduction leading to VEGF expression (11) .
Several lines of evidence strongly implicate Ras as an essential component of growth-regulating signal transduction by growth factor receptor protein-tyrosine kinases(12) . The c-ras proto-oncogene encodes a 21,000-Da guanine nucleotide-binding protein (13, 14) , which is thought to be involved in the regulation of cell growth, differentiation, and carcinogenesis(15, 16, 17) . When microinjected into quiescent fibroblasts, the Ras protein has been shown to be a potent mitogen(15) . In addition, microinjection into pheochromocytoma PC 12 cells has been shown to induce neurite outgrowth (18) . Ras also plays an important role in the pathogenesis of several malignancies including tumor growth. Mutations in the ras gene that increase the proportion of Ras in the GTP-bound state relative to Ras-GDP activate the biological function of Ras and have been found in a large number of human tumors(19, 20) .
The raf proto-oncogene
products are cytoplasmic serine/ threonine-specific protein kinases,
which are activated by growth factors or phorbol esters (21, 22, 23) . ()They transmit the
incoming signals to the transcriptional machinery in the nucleus.
Expression of constitutively activated Raf-1 and A-Raf trans-activate transcription from the oncogene-responsive
element in the polyomavirus enhancer, which contains an AP-1/Ets
binding site(24) , in a Ras-independent
manner(25, 26) . In contrast, mitogen- or Ras-induced
expression from the oncogene-responsive element was completely
dependent on functional Raf(25) . Therefore, Raf has been
positioned downstream of Ras in the signal transduction pathway leading
to proliferation and transformation of NIH 3T3 cells. Recently, direct
interaction of normal and oncogenic Ras with Raf has been
reported(27, 28) . Ras-mediated activation of Raf thus
initiates a protein kinase cascade leading to mitogen-activated protein
kinase activation and finally to increased AP-1
activity(29, 30) .
A pronounced synergism between
PKC and mutant p53 in the induction of VEGF expression was previously
reported(31) . We therefore addressed the question of whether a
functional link exists between oncogenic transformation of cells and
the expression of the tumor angiogenesis factor, VEGF. This would
generate a connection between genetic alterations, which are thought to
play a role in the process of tumor initiation, and the process of
tumor progression, mediated by enhanced expression of VEGF. For this
purpose, we have used NIH 3T3 cell lines transformed by v-Ha-ras or v-raf, the viral forms of the respective cellular
oncogenes, which have been found to be frequently activated by mutation
or overexpressed in certain human
tumors(19, 20, 32, 33, 34) . ()We show that v-Ha-Ras or v-Raf are sufficient to enhance
VEGF expression in NIH 3T3 cells without further stimulation.
Furthermore, NIH 3T3 cells transformed by v-raf are able to
secrete VEGF, which shows biological activity in proliferation assays
with HUVE cells. Our findings suggest that Ras and Raf provide
potential tumor angiogenesis inducers when constitutively activated.
Figure 1:
Basal VEGF mRNA expression in
v-Ha-ras or v-raf transformed NIH 3T3 cells. NIH 3T3
cells transformed by v-Ha-ras or v-raf and control
NIH 3T3 cells were serum starved for 24 h. Total RNA was isolated, and
10 µg were analyzed by Northern blotting using a VEGF cDNA probe(39) . The filter was exposed for 48 h at
-80 °C with intensifying screen. The rRNA 28 S band is
indicated on the left. Equal loading of the gel was confirmed
by ethidium bromide staining.
Figure 2:
Time course of VEGF expression of
v-Ha-ras transformed and control NIH 3T3 cells. The
v-Ha-ras transformed cells (NIH 3T3/v-Ha-ras) and control NIH
3T3 cells (NIH 3T3) were serum starved for 24 h to achieve quiescence
and then stimulated by the addition of TPA (100 ng/ml) and PDGF-BB (50
ng/ml). RNA was extracted at the indicated time (h) after stimulation.
10 µg of total RNA were loaded in each lane. The filters were
probed with the 600-base pair VEGF complementary DNA
(cDNA) probe (39) and exposed for 48 h at -80 °C with
intensifying screen. The rRNA 28 S band is indicated on the left. Equal loading of the gel was confirmed by ethidium
bromide staining.
It was previously reported that at least in some
cell types tumorigenic progression induced by oncogenic ras is
associated with increased PKC gene transcription and PKC
expression(42, 43, 44) . Therefore, we have
addressed the question of whether the enhanced TPA inducibility of VEGF
expression in NIH 3T3 cells transformed by v-Ha-ras may be due
to increased PKC levels in the transfected cell line. Western blot
analysis using a polyclonal antibody that recognizes PKC ,
I/
II,
, and
did not show any difference in PKC
expression between the two cell lines (data not shown), indicating that
the increased TPA inducibility of VEGF expression in v-Ha-ras transformed cells is not due to increased PKC levels.
Figure 3:
Time course of VEGF expression in
v-raf transformed NIH 3T3 cells. NIH 3T3 cells transformed by
v-raf (NIH 3T3/v-raf) as well as control cells (NIH 3T3) were
serum starved for 24 h. Cells were stimulated with PDGF-BB (40 ng/ml)
and TPA (100 ng/ml) for the indicated periods (h). Total RNA was
isolated, and 10 µg were analyzed by Northern blotting using a
VEGF cDNA probe(39) . The filters were exposed
for 48 h at -80 °C with intensifying screens. The rRNA 28 S
band is indicated on the left. Equal loading was assured by
staining the RNA gels with ethidium
bromide.
These results suggest that the Raf kinase is involved in the signal transduction leading to VEGF gene expression. Furthermore, it is strongly indicated that constitutive activation of Raf-1 releases the cell for the necessity of external stimuli to induce VEGF expression.
Figure 4: Basal VEGF protein expression in v-Ha-ras or v-raf transformed NIH 3T3 cells. NIH 3T3 cells transformed by v-Ha-ras (NIH 3T3/v-Ha-ras) or v-raf (NIH 3T3/v-raf) and NIH 3T3 control cells (NIH 3T3) were maintained for 24 h in medium supplemented with 0.05% serum. Immunofluorescence with a VEGF-specific antiserum was performed as described under ``Experimental Procedures.''
By
expressing VEGF transiently in mammalian cells, it could be shown that
only VEGF and VEGF
are efficiently secreted
into the medium, whereas the longer forms VEGF
and
VEGF
are rather cell associated(10) . Based on
this observation, together with the strong VEGF immunofluorescence in
v-Ha-ras and v-raf transformed cells, we partially
purified VEGF from medium conditioned by the v-raf transformed
cell line. VEGF could be identified in fractions eluted from
heparin-Sepharose columns by Western blot analysis (Fig. 5A).
Figure 5:
Partial purification and characterization
of VEGF protein from conditioned medium of v-raf transformed
NIH 3T3 cells. A, cells were maintained for 72 h in medium
supplemented with 0.05% serum. 100 ml of conditioned medium were run
through a heparin-Sepharose column, and proteins were eluted with 1 M NaCl and fractionated in 1-ml aliquots. 15 µl of
fractions (1-6) were mixed with non-reducing sample
buffer and run on a 10% SDS polyacrylamide gel. Western blot analysis
was performed with a VEGF-specific polyclonal rabbit antiserum. L, load; FT, flow through; C, control
recombinant VEGF expressed in Sf9 cells; M,
molecular weight marker. B, competition of VEGF binding.
Confluent HUVE cells were incubated with 1.5 ng/ml
I-VEGF
and increasing amounts of either
unlabeled recombinant VEGF
(open circles) or
VEGF from conditioned medium from v-raf transformed NIH 3T3
cells (closed circles). As negative control, a 100-fold excess
of unlabeled PDGF-BB (open triangle) or bFGF (closed
triangle) was used. The experiment was done three times with
essentially identical results. C, proliferation of HUVE cells.
HUVE cells were seeded into 24-well plates and treated with either
recombinant VEGF
(open circles) or VEGF protein
from conditioned medium (closed circles). After 7 days, cells
were counted with a coulter counter. The experiment was done three
times with essentially identical results. In B and C,
the amount of purified VEGF was estimated by Western blot
analysis.
The fraction containing the highest
amount of VEGF protein was tested for competition with I-labeled rVEGF
in a binding assay (Fig. 5B). The competition was similar to the positive
control with rVEGF
, whereas PDGF-BB and bFGF even at a
concentration of 100 ng/ml showed no competition at all. Furthermore,
VEGF protein isolated from v-raf transformed cells promoted
proliferation of HUVE cells similar to rVEGF
(Fig. 5C), suggesting that VEGF expressed in NIH 3T3
cells is biologically active.
The identification of mitogen-activated protein kinase kinase as a substrate of Raf (45) together with the demonstration of direct phosphorylation of c-Jun (46) by mitogen-activated protein kinase extended the signal transduction pathway (receptor kinase, Ras, Raf, mitogen-activated protein kinase kinase, mitogen-activated protein kinase (Ras/Raf pathway)) into the nucleus. A transforming mutant of raf leads to trans-activation of promoters containing AP1 binding sites(25) . In addition, it could be shown that various natural promoters containing AP1/Ets binding sites respond to Raf activation(47, 48, 49, 50, 51) . It was also shown that expression of oncogenic Ras leads to an increase in the expression of c-Jun and c-Fos and therefore to an increased AP1 activity(29) . The four AP1 binding sites in the promoter region of the VEGF gene (9) render this gene a potential downstream effector in Ras and Raf activation.
Finkenzeller et
al.(11) could show that VEGF mRNA expression in NIH 3T3
cells is intensified after PDGF-BB and TPA stimulation. Furthermore,
they postulate an essential role of PKC in the signal transduction
leading to VEGF induction. Together, with the demonstration of direct
phosphorylation of Raf-1 by PKC(52) , the Raf kinases are
placed in a central position in the signal transduction pathway of VEGF
expression.
The enhanced basal expression of VEGF mRNA in serum-starved NIH 3T3 cells transformed by v-Ha-ras or v-raf (Fig. 1) also indicates that the Ras/Raf signal transduction pathway plays an essential role in the regulation of VEGF gene expression. Despite the increased expression in ras- or raf-transformed cell lines, VEGF expression was still further enhanced by treatment of cells with PDGF and TPA ( Fig. 2and Fig. 3). This increase of VEGF indicates that a second, Ras- and Raf-independent, signal transduction pathway may exist or, with regard to Raf, that the already constitutively activated Raf kinase becomes additionally positively regulated by phosphorylation of specific sites in the kinase domain after PDGF or TPA stimulation.
The enhanced
basal VEGF mRNA expression also resulted in higher VEGF protein
expression, as was shown by indirect immunofluorescence (Fig. 4). Furthermore, VEGF protein could be isolated from
conditioned medium of v-raf transformed cells (Fig. 5A). Characterization of the secreted VEGF
unveiled a specific competition to I-labeled
VEGF
(Fig. 5B), and it was shown to be
biologically active for HUVE cells (Fig. 5C).
Taken together, these results clearly demonstrate the essential role of the Ras/Raf signal transduction pathway in the regulation of VEGF expression in NIH 3T3 cells. This indicates that Ras as well as Raf are not only involved in cell proliferation and differentiation but also in tumor progression, mediated by increased expression of the tumor angiogenesis factor VEGF.