From the Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215
Received for publication, August 28, 2000, and in revised form, October 13, 2000
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ABSTRACT |
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Vascular permeability factor/vascular endothelial
growth factor (VPF/VEGF), a multifunctional cytokine, is regulated by
different factors including degree of cell differentiation, hypoxia,
and certain oncogenes namely, ras and
src. The up-regulation of VPF/VEGF expression by Ras has
been found to be through both transcription and mRNA stability. The
present study investigates a novel pathway whereby Ras promotes the
transcription of VPF/VEGF by activating protein kinase C The growth and metastasis of tumors depend on the development of
an adequate blood supply via angiogenesis which is attributed in large
part to the production of angiogenesis promoting growth factors by
tumor (1, 2). Although many angiogenic factors have been described, one
in particular stands out for its potency and specificity, namely
vascular permeability factor/vascular endothelial growth factor
(VPF1/VEGF) (3, 4). VPF/VEGF
is overexpressed by a wide variety of human tumors and plays a critical
role in tumorigenesis. Although constitutively expressed by many tumor
cells, VPF/VEGF expression is substantially up-regulated by hypoxia,
cytokines, hormones, and certain oncogenes including activated forms of
src and ras (5-7). We have recently shown that
protein kinase signaling pathways also play an important role in tumor
angiogenesis (8, 9). Among the protein kinase C (PKC) family, the
isoform PKC PKC For the past few years, research has focused on the role of the
oncogene in the signaling pathways controlling cell growth and
differentiation (15, 16). Ras has also been found to be involved both
in transcriptional and post-transcriptional up-regulation of VPF/VEGF
expression, and thus angiogenesis (17). The persistent activation of
signaling pathways induced by Ras accounts for overexpression of
VPF/VEGF in a significant fraction of human tumors (7, 17). PKC Some recent findings have shown that the enzyme
phosphoinositide-dependent protein kinase-1 (PDK-1) is at
the hub of many signaling pathways, activating PKB and PKC isoenzymes
as well as p70-S6 kinase and perhaps PKA (13, 34-36). It has been
shown that PDK-1 induces PKC There are several reports that PKC In previous studies, we have shown that PKC Cell Culture--
Human fibrosarcoma (HT1080) and renal cell
carcinoma (786-0) cell lines were maintained in Dulbecco's modified
Eagle's medium with 10% fetal bovine serum (Hyclone Laboratories).
Plasmids--
All the VPF/VEGF reporter constructs used in
transient transfection assays contain sequences derived from the human
VPF/VEGF promoter driving expression of firefly luciferase. The 0.35- and 0.07-kb deletion mutant constructs were made by polymerase chain reaction from the 2.6-kb VPF/VEGF promoter fragment and subcloned into
pGL-2 Basic vector (Promega) as described earlier (48). The
overexpressed PKC Antiserum and Oligonucleotides--
A polyclonal anti-rabbit
antibody directed against the phosphorylated activation loop
Thr410 of PKC Northern Blot Analysis--
RNA, isolated by the single-step
acid-phenol extraction method (8, 49), was separated on a
formaldehyde-agarose gel, transferred to a GeneScreen membrane by using
10 × SSC, and probed with random primer-labeled cDNAs in a
solution containing 0.5 M sodium phosphate (pH 7.2), 7%
SDS, 1% bovine serum albumin, 1 mM EDTA, and sonicated
herring sperm DNA (50 µg/ml) at 68 °C. Blots were washed three
times with a solution containing 40 mM sodium phosphate (pH
7.2), 0.5% SDS, 0.5% bovine serum albumin, and 1 mM EDTA
at 68 °C and quantitated by laser densitometry.
Immunoprecipitations--
As described earlier, cells were
washed twice with cold phosphate-buffered saline, lysed with ice-cold
lysis buffer (50 mM Tris, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 1 mM Na3VO4, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 0.5% aprotinin, and 2 mM pepstatin A),
incubated for 10 min on ice, and centrifuged for 10 min at 4 °C (8,
48). Immunoprecipitations were carried at antibody excess, using 0.5 mg
of total protein either with a mouse monoclonal antibody (1 µg)
directed against Ras (Transduction Laboratories) or a rabbit polyclonal
antibody (1 µg) directed against PKC Western Blot Analysis--
Protein samples were mixed with
2 × sample buffer (125 mM Tris-HCl, pH 6.8, 20%
glycerol, 10% Transfection Assays--
Cells were plated at 2-3 × 105 cells/60-mm dish 1 day before transfection with
VPF/VEGF promoter-luciferase construct and expression plasmids using
the calcium-phosphate precipitation method (50). The expression was
normalized with a control empty expression vector. Cells were harvested
for luciferase assay 40 h after transfection. Luciferase activity
was measured using the luciferase assay kit (Promega). In all
co-transfection experiments, transfection efficiency was normalized by
assaying Nuclear Extract Preparation and Electrophoretic Mobility Shift
Assays (EMSAs)--
Nuclear extracts were prepared from HT1080 cells
following a standard protocol (48), with modifications. Cells were
washed in cold phosphate-buffered saline, suspended in buffer A (10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM
MgCl2, 0.1 mM EDTA, 10 µg/ml aprotinin, 3 mM dithiothreitol, and 0.1 mM
phenylmethylsulfonyl fluoride) and incubated for 15 min on ice. Cells
were then lysed with 0.5% Nonidet P-40 and the pellets were
resuspended in buffer C (50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA,
10% glycerol, 3 mM dithiothreitol, and 0.1 mM
phenylmethylsulfonyl fluoride). Following incubation on a rotating rack
for 25 min, samples were centrifuged at 14,000 rpm for 10 min. Clear
supernatants, containing the nuclear proteins were collected and stored
at
EMSAs were performed as described previously (48). Briefly, EMSA
binding reaction mixtures (25 µl) contained 20 mM HEPES (pH 8.4), 100 mM KCl, 20% glycerol, 0.1 mM
EDTA, 0.2 mM ZnSO4, 0.05% Nonidet P-40, and 1 µg of bovine serum albumin. Extract protein and 200 ng of
poly(dA-dT)·poly(dA-dT) were added at room temperature 10 min prior
to addition of ~0.1 ng of radiolabeled oligonucleotide probe. After
20 min incubation at 4 °C, samples were run on 7% acrylamide gel in
1 × TAE (40 mM Tris acetate, 1 mM EDTA) buffer.
The radiolabeled oligonucleotide used in EMSA studies was a 188-base
pair polymerase chain reaction-generated fragment (base pair PKC
To elucidate the role of PKC Ras and PKC Ras Activates PKC
We also observed by EMSA, that in presence of Ras, the Sp1-mediated
transcription of VPF/VEGF through PKC
Observations from different laboratories clearly indicate that
oncogenic Ras-mediated transformation needs different downstream targets, one of which involves Raf-1/MEK/MAPK pathways (23, 24, 52).
From our observations, it appears that PKC Both Raf-dependent and Raf-independent Pathways Are
Involved in Channeling Ras Signals for PKC
Next, we co-transfected the HT1080 cells with 0.35-kb VPF/VEGF
promoter-luciferase construct and either the Ras mutants alone or in
combination with PKC PDK-1 Plays a Crucial Role in Activation of PKC
We next sought to determine whether the combination of both PDK-1 and
Ras can activate PKC PI 3-Kinase Acts as Major Upstream Effector to Activate
PKC
To elucidate the involvement of PI 3-kinase in Ras, PDK-1, and
PKC VPF/VEGF, a multifunctional cytokine, was originally discovered
because of its ability to increase the permeability of microvessels, primarily post-capillary venules and small veins, to circulating macromolecules (4, 44-47). It plays an important role in both pathological and physiological angiogenesis (3, 4). Although constitutively expressed by many tumor cells and transformed cell lines, VPF/VEGF expression is also subject to regulation by both PKC
and cAMP-dependent kinase pathways (58) and by mechanisms involving alterations in both mRNA transcription and stability (59,
60). Other factors that can regulate VPF/VEGF expression include the
degree of cell differentiation; local concentrations of oxygen,
glucose, and serum; cytokines; hormones; prostaglandins; modulators of
PKC; calcium influx; the electron transport chain; depolarizing agents;
angiotensin II; stimulators of adenylate cyclase; nitric oxide; and
expression levels of certain oncogenes, like src or
ras (5-7, 17, 61, 62).
The data presented here investigate a novel mechanism by which
transforming human Ras regulated the transcription of VPF/VEGF through
PKC Ras is a point of convergence for many signaling pathways and plays a
significant role in expression of VPF/VEGF (17). Here, we demonstrate
that Ras promotes VPF/VEGF transcription in a Sp1-dependent manner (Fig. 1). It has been reported that Ras triggers activation of
series of kinases known as the MAPK cascade (Ras > MEK > MAPK) (16, 20-22). Raf-1 serine/threonine kinase has been reported to
be one of the downstream effectors of Ras (23, 24, 52). Raf-1 in turn
activates MAPK kinases (MEK1 and MEK2), which in turn activate p42 and
p44 MAPKs/extracellular signal-regulated kinases (19, 63, 64).
Activated MAPKs then translocate into the nucleus, where they
phosphorylate and activate nuclear transcription factors (65, 66),
resulting in immediate early gene induction. As from the previous
reports, PKC We next set out to dissect the role of Raf-independent downstream
effectors that are involved in mediating Ras signals. Mutations in the
Ras effector domain (residues 32-40) can impair Ras transforming activity and interaction with effector proteins without causing alterations in intrinsic GDP and GTP regulation (31, 67). We made use
of two different effector loop mutants of Ha-Ras(G12V). Ha-Ras(G12V,T35S) retained the full-length Raf-1 binding activity, while Ha-Ras(G12V,E37G) was impaired in its ability to bind to Raf-1.
Consistent with its impaired ability to up-regulate Raf-1 kinase
activity, it has been demonstrated that cells expressing the later
mutant show no significant increase in p42 and p44 MAPK activities (31,
55). In contrast, both Ha-Ras(G12V)- and Ha-Ras(G12V,T35S)-expressing cells show elevated MAPK activities. Recent studies have demonstrated that, like Rho family proteins, Ha-Ras(G12V,E37G) can cause activation of the stress-activated protein kinase (SAPK/JNK) (16, 24, 68). It has
also been shown to mediate its functions through Rho family of
proteins, mainly, RhoA, Rac1, and CDC42 (24, 31). In this study, we
observed that both Ha-Ras(G12V,T35S) and Ha-Ras(G12V,E37G) caused
significant activation of VPF/VEGF transcription through PKC Recent works have identified PDK-1, constitutively active in mammalian
cells, as a major upstream activating kinase for PKC PKC In summary, our results indicate that Ras-mediated VPF/VEGF
transcription occurs mainly through PKC (PKC
).
The Ras-mediated overexpression of VPF/VEGF was also found to be
inhibited by using the antisense or the dominant-negative mutant of
PKC
. In co-transfection assays, by overexpressing oncogenic Ha-Ras
(12 V) and PKC
, there was an additive effect up to 4-fold in
activation of Sp1-mediated VPF/VEGF transcription. It has been shown
through electrophoretic mobility shift assay that Ras promoted the PKC
-induced binding of Sp1 to the VPF/VEGF promoter. In the presence of PDK-1, a major activating kinase for PKC, the Ras-mediated activation of VPF/VEGF promoter through PKC
was further increased, suggesting that PKC
can serve as an effector for both Ras and PDK-1.
In other experiments, with the use of a dominant-negative mutant of
phosphatidylinositol 3-kinase, the activation of VPF/VEGF promoter
through Ras, PDK-1, and PKC
was completely repressed, indicating
phosphatidylinositol 3-kinase as an important component of this
pathway. Taken together, these data elucidate the signaling mechanism
of Ras-mediated VPF/VEGF transcriptional activation through PKC
and
also provide insight into PKC
and Sp1-dependent transcriptional regulation of VPF/VEGF.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
plays a critical role in regulating VPF/VEGF overexpression.
represents an atypical PKC isoform in that: 1) it lacks the C2
domain making its kinase activity Ca2+ independent, and 2)
it possesses only one zinc finger region in its regulatory domain (10).
Consequently, PKC
does not bind Ca2+ and cannot be
activated by diacylglycerol or phorbol esters (11). In addition,
prolonged treatment with phorbol esters does not down-regulate PKC
(10), and most PKC inhibitors do not decrease PKC
activity (11).
PKC
is found to be involved in a wide range of physiological
processes including mitogenesis, protein synthesis, cell survival, and
transcriptional regulation (12, 13). Like many other protein kinases,
PKC
requires phosphorylation within its activation loops to express
full catalytic potential (13, 14).
could have an important cross-talk with Ras (18). Several reports have
shown that cell stimulation activates Ras which triggers a number of
important serine/threonine kinases that have MAP kinase kinase (MEK) as
substrate, such as MEK kinase, c-Raf-1, and B-Raf, culminating in the
activation of MAP kinase (MAPK) (19-22). The kinase-deficient mutants
of Raf-1, MEKs, and MAPKs have been shown to block Ras-mediated
signaling events and transformation (23, 24). It has been suggested
that Ras may function primarily to promote the translocation of Raf-1
from the cytosol to the plasma membrane, where subsequent
Ras-independent events trigger Raf-1 kinase activation (25). In some
studies, it has clearly been shown that PKC
may also serve as a
downstream target of Ras (26). For example, both proteins have been
found to be critically involved in the activation of NF-
B (27, 28).
It appears that the mechanism whereby PKC
controls cell signaling
could, at least in part, implicate the channeling of Ras signals in the
activation of MAPK (26). However, despite the evidence that Raf-1 is a
critical downstream effector of Ras function, there is increasing
evidence that Ras may mediate its action through the activation of
multiple downstream effector-mediated pathways (29). For example, the existence of Raf-independent Ras signaling pathways is suggested by the
expanding roster of candidate Ras effectors that have been identified
(30-33). Like Raf-1, these functionally diverse proteins, including
Rho family members, show preferential binding to the active GTP-bound
form of Ras and this interaction requires an active Ras effector domain
(amino acids 32 to 40).
phosphorylation in vivo,
leading to its activation (13). Furthermore, PDK-1 directly
phosphorylates PKC
at the activation-loop Thr410 residue
in vitro (13, 14, 37). As PKC
phosphorylation/activation is almost completely blocked by coexpression of dominant-negative PDK-1
or by mutation of Thr410, it is likely that a pre-requisite
for PKC
activation is phosphorylation of Thr410 (13,
38). PKC
is found to be associated with PDK-1 in the same complex,
and also identified as its in vivo substrate along with
Akt/PKB and p70 S6 kinase (39). It has also been shown that PDK-1 is
recruited in the signaling pathways through PI 3-kinase and serves as a
multifunctional downstream effector (13, 37, 39).
is activated by important lipid
intracellular mediators like phosphatidic acid (40), phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3) (11), and ceramide (41). It has been shown that insulin and insulin-like growth factor-1 leads to the activation of PKC
which can be
inhibited by chemical inhibitors of PI 3-kinase (42). Similarly,
activation of PI 3-kinase by lipopolysaccharide leads to the activation
of PKC
which is sensitive to PI 3-kinase inhibitors and a
dominant-negative PI 3-kinase mutation (43). Taken together, these
observations pinpoint PKC
as a target of important lipid second
messengers and support its role in cell signaling.
plays a significant role
in promotion of tumor angiogenesis by stimulating the expression of
VPF/VEGF (8, 9). In addition to rendering microvessels hyperpermeable,
VPF/VEGF stimulates endothelial cells to migrate and divide and
profoundly alters their pattern of gene expression (3, 4, 44-47). We
have shown that PKC
interacts with and phosphorylates the
transcription factor Sp1 and increases the VPF/VEGF promoter activity
in human fibrosarcoma (HT1080) and renal cell carcinoma (786-0) cell
lines, where basal level of VPF/VEGF is also very high (9). In the
present study, we dissect the upstream signaling pathways, with special
emphasis on Ras, that is required for activation of PKC
to promote
Sp1-mediated VPF/VEGF transcription. Our observations clearly indicate
that coordinated signaling through Ras, PDK-1, and PI 3-kinase may be
required to mediate PKC
-induced activation of VPF/VEGF promoter.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and a kinase inactive PKC
cDNA (PKC
KW; LYS-275 to tryptophan substitution), both subcloned into pCMV2FLAG vector were generous gifts from Alex Toker (13). All Ras
expression constructs encode mutant versions of the transforming human
Ha-Ras(G12V). The pDCR-ras(G12V),
pDCR-ras(G12V,T35S), and pDCR-ras(G12V,E37G) mammalian constructs encode effector domain mutants of Ha-Ras(G12V) in
which expression is under the control of the cytomegalovirus promoter
(31). The Ras(Q61L,C186S) is the dominant inhibitory mutant of Ras
(31). The Myc-PDK-1, Myc-PDK-1.K/N, and GST-
p85 were generous gifts
from Alex Toker (18). The kinase-inactive variant Myc-PDK-1.K/N was
made by mutating the conserved critical Lys110 residue to Asn.
was received from Alex Toker as a generous
gift (13). All PKC oligonucleotides were synthesized as
phosphorothioate derivatives from Genemed Synthesis (San Francisco, CA)
(49).
(Chemicon International Inc).
Immunocomplexes were captured with protein A-agarose beads (Amersham
Pharmacia Biotech). After three washes with cell lysis buffer,
bead-bound proteins were subjected to Western blot analysis.
-mercaptoethanol, 4% sodium dodecyl sulfate (SDS),
and 0.0025% bromphenol blue), boiled, and run on 7.5-10%
polyacrylamide gels with Tris glycine-SDS running buffer (Bio-Rad).
Agarose beads with bound proteins were handled in the same manner and
directly loaded on the gel. Size-separated proteins were transferred to
a polyvinylidene difluoride membrane (PerkinElmer Life Sciences) at 70 volts. For immunodetection, the membranes were blocked with 5% milk or
2% bovine serum albumin in phosphate-buffered saline-Tween 20 (PBST)
and then coated with primary antibody. After washings, the membranes
were incubated with peroxidase-linked secondary antibody and the
reactive bands were detected by chemiluminescent substrate.
-galactosidase activity using the
-galactosidase gene
under control of cytomegalovirus immediate early promoter as internal
control. 786-0 cells were transfected using Effectene transfection
reagent (Qiagen), following manufacturer's protocol. 1:25 ratio of DNA
to Effectene was used for all the experiments. For all the transfection
assays, average results from three independent experiments were plotted.
70 °C.
195 to
7, relative to the transcription start site) of the VPF/VEGF promoter
containing the four putative Sp1-binding sites (48).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, an Intermediary Molecule for Ras-mediated Overexpression of
VPF/VEGF--
In the present study we demonstrate that oncogene
Ha-Ras(G12V) promoted the Sp1-mediated VPF/VEGF transcriptional
activation in human fibrosarcoma (HT1080) and renal cell carcinoma
(786-0) cell lines (Fig. 1). 786-0 and
HT1080 cells were co-transfected with a 2.6-kb VPF/VEGF
promoter-luciferase construct and plasmid containing Ha-Ras(G12V).
VPF/VEGF reporter activity was increased up to 2-fold in comparison
with cells transfected with expression vector alone. To define the
region of the VPF/VEGF promoter that is responsive to Ras, we utilized
two different 5' deletions of the 2.6-kb promoter-reporter vector and
co-transfected these deletions with a plasmid containing Ha-Ras(G12V).
Ras increased the reporter activity by 2-fold in the 0.35-kb segment of
the VPF/VEGF promoter that contains the Sp1-binding site, although
there was no change of reporter activity in the 0.07-kb VPF/VEGF
promoter having the deleted Sp1-binding site (Fig. 1) (48). These
results suggest that transforming human Ras itself activates VPF/VEGF
transcription in a Sp1-dependent manner.
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Fig. 1.
Effect of oncogenic Ras on VPF/VEGF promoter
activity. Human renal cell carcinoma (786-0) and fibrosarcoma
(HT1080) cell lines were co-transfected with 2.6-, 0.35-, or 0.07-kb
VPF/VEGF promoter-luciferase constructs (1.0 µg) and Ha-Ras(G12V)
(1.2 µg) expression vectors. Cells were harvested for luciferase
assays 40 h after transfection, and fold activation was calculated
as relative to the activity of same reporter construct co-transfected
with an empty expression vector (pDCR). The raw values of luciferase
activities of 2.6-, 0.35-, or 0.07-kb VPF/VEGF promoters are
32,194 ± 180, 31,534 ± 177, and 19,532 ± 140, respectively, in 786-0 cells. In case of HT1080 cells, these values are
8,606 ± 92, 8,230 ± 90, and 4,115 ± 64, respectively.
Open bars represent the empty expression vector, while the
black bars represents Ha-Ras(G12V) expression vector.
in Ras-mediated VPF/VEGF
overexpression, we studied the effect of the dominant-negative mutant (kw) and the antisense (AS) oligonucleotide of PKC
on the expression of VPF/VEGF mRNA in HT1080 and 786-0 cells. In these two cell lines, the oncogenic Ras is already activated and as a result the basal
level of VPF/VEGF is very high. Through Northern blot analysis, we
demonstrate that the PKC
(kw) and PKC
(AS) clearly reduced the
VPF/VEGF mRNA expression level by 50 and 70%, respectively (at
their highest doses) in HT1080 cells (Fig.
2). We also found the same pattern of
inhibition of VPF/VEGF expression in 786-0 cells (data not shown).
Previously, we have shown that this oligonucleotide could effectively
reduce the PKC
protein level, whereas the other PKC isoforms
remained unchanged (49). There was no change in VPF/VEGF expression
with the antisense oligonucleotide of PKC
(data not shown). This
result suggests that PKC
plays a critical role as an intermediary
molecule in Ras-mediated overexpression of VPF/VEGF. We recently showed
that PKC
can also promote the Sp1-dependent
transcription of VPF/VEGF in HT1080 and 786-0 cell lines (9). We
demonstrated that co-transfection of HT1080 and 786-0 cells with a
plasmid overexpressing PKC
(at a concentration of 0.6 µg) and
different deletion mutants of VPF/VEGF promoter luciferase constructs
results in activation of Sp1-mediated transcription, whereas expression
of a dominant-negative mutant of PKC
represses this activation (9).
Interestingly, with the increase in the dose of PKC
overexpressing
plasmid beyond 1.2 µg concentration, the VPF/VEGF transcription was
decreased and gradually came down to the basal level (Fig.
3). These results indicate that
PKC
-mediated VPF/VEGF transcription might be a rate-limiting step
and required activation through upstream signaling pathways.
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Fig. 2.
Effect of dominant-negative mutant and
antisense oligonucleotide of PKC on VPF/VEGF
mRNA expression. Total RNA (5 µg) was extracted from HT1080
cells that had been transfected with different concentrations of the
dominant-negative mutant and the antisense oligonucleotide of PKC
and subjected to Northern blot analysis. The blot was probed with
32P-labeled VPF/VEGF cDNA. Fold expression was
calculated by densitometry using 36B4 ribosome-associated mRNA
expression as a normalization control. The lower panel shows
ethidium staining of the RNA samples prior to transfer.
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Fig. 3.
Dose-dependent effect of
overexpressed PKC on VPF/VEGF
transcription. HT1080 cells were co-transfected with 0.35-kb
VPF/VEGF promoter-luciferase construct (1.0 µg) and increasing
concentrations (0.2-2.0 µg) of wt-PKC
cDNA. Cells were
harvested for luciferase assay 40 h after transfection, and fold
activation was calculated as relative to the activity of the same
reporter construct co-transfected with an empty expression vector
(pCMV-FLAG). In all the doses of wt-PKC
, the amount of total DNA was
balanced by the empty expression vector.
Are Present in the Same Complex--
Since several
reports clearly indicated PKC
as a critical step downstream of Ras
(12, 18, 26, 51) and as Ras was also found to be involved in VPF/VEGF
transcriptional regulation (17), here we set out to dissect whether
there was any association between PKC
and Ras. To this end, we
prepared the lysates of HT1080 and 786-0 cells and immunoprecipitated
with anti-Ha-Ras antibody followed by immunoblotting with antibody
directed against PKC
. We found a strong band corresponding to PKC
in the immunoprecipitates prepared from both the cell lines (Fig.
4A). This experiment
demonstrated that PKC
and Ras were present in the same complex, but
did not elucidate whether these proteins might interact directly with each other. To test for this possibility, we performed in
vitro association experiment using GST-Ras fusion protein and
recombinant PKC
isoform. Bacterially expressed GST protein alone or
GST protein fused to activated form (GTP-bound) of Ras was bound to
glutathione-Sepharose beads, and these were mixed with purified
recombinant PKC
in a buffer designed to approximate intracellular
ionic concentrations. After suitable incubation and extensive washing
with the same buffer, the bound proteins were separated by SDS-PAGE and
subjected to Western blotting with antibodies to PKC
. But we did not
observe any significant association of PKC
with immobilized
activated Ras (data not shown), which reflected that, although PKC
and Ras were present in the same complex, they may not interact
directly. We next sought to determine whether association of Ras could
activate PKC
. As it has been shown previously that phosphorylation
at the activation loop Thr410 residue is an important step
for activation of PKC
(13), we made use of a phospho-specific
antibody raised against the phosphorylated activation loop sequence.
This antibody can specifically recognize PKC
when phosphorylated at
Thr410. We transfected the human HT1080 cells either with
an expression plasmid for PKC
alone or in combination with a plasmid
containing human Ha-Ras(G12V). Lysates were prepared from the
transfected cells, immunoprecipitated with an antibody specific to
PKC
and then subjected to Western blot analysis with
phospho-specific PKC
antibody. A band of phosphorylated PKC
was
detected in case of cells transfected with the combination of PKC
and Ha-Ras(G12V) (Fig. 4B). This result reveals that Ras
plays a significant role in regulating phosphorylation of the
activation loop Thr410 of PKC
.
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Fig. 4.
Ras associates with and also phosphorylates
PKC . A, lanes 2 and
3, extracts were prepared from both 786-0 and HT1080 cells
and immunoprecipitated with a monoclonal antibody directed against
Ha-Ras. In lanes 1 and 4, 786-0 cell extracts
were immunoprecipitated with antibody against PKC
and c-Src,
respectively. B, HT1080 cells were transfected with
different combinations of wt-PKC
(0.6 µg), Ha-Ras(G12V) (1.2 µg), PDK-1 (2.0 µg), and PI 3-kinase dominant-negative mutant
(p85DN) (2.0 µg). Cells were lysed 40 h after transfection and
the cellular extracts were immunoprecipitated using PKC
polyclonal
antibody. All the immunoprecipitates (IP) were then captured
by protein A-Sepharose beads. After thorough washings, the Sepharose
beads were boiled in SDS buffer and separated by SDS-PAGE. Western
blottings (Blot) were performed by using (A)
PKC
polyclonal antibody and (B) a phospho-specific
antibody, raised against the phosphorylated (T410) activation loop
sequence of PKC
. In the lower panel of B, the
protein extracts from the cells transfected with different combinations
of PKC
, Ha-Ras(G12V), PDK-1 and p85DN were separated by SDS-PAGE and
blotted with PKC
polyclonal antibody.
for Sp1-mediated VPF/VEGF
Transcription--
We attempted to explore the involvement of Ras in
PKC
-mediated activation of VPF/VEGF transcription. To this end,
HT1080 cells were co-transfected with a 0.35-kb VPF/VEGF
promoter-luciferase construct and the plasmid containing either
overexpressed PKC
or transforming human Ha-Ras(G12V). Both PKC
and Ha-Ras(G12V) increased the VPF/VEGF reporter activity by 2- and
2.5-fold, respectively, in comparison to the cells transfected with
expression vector alone (Fig.
5A). Interestingly, when we
transfected the cells with a combination of PKC
and Ha-Ras(G12V),
the VPF/VEGF reporter activity increased up to 4-fold. A dominant
negative mutant of transforming human Ras (Ras(Q61L,C186S), which
prevents downstream signaling) decreased the PKC
-mediated reporter
activation almost to the control level (Fig. 5A). In 786-0 cells, we also observed a similar type of activation of VPF/VEGF
promoter activity through PKC
and Ha-Ras(G12V) (Fig. 5B).
Expression of both PKC
and Ras in the transfected cells were
confirmed through Western blot analysis (Fig. 5C). Together
these results indicate that PKC
is a key activator of Sp1-mediated
VPF/VEGF transcription and oncogenic ras plays a
significant role in activation of PKC
for such transcriptional regulation. Interestingly, a dominant-negative mutant of PKC
completely blocked the Ras-mediated transcriptional activation of
VPF/VEGF (Fig. 5A). Moreover, the combination of PKC
kw
and Ras(Q61L,C186S) reduced the VPF/VEGF transcriptional activation to
the same extent when these two mutants were used individually which
suggests that PKC
and Ras are in the same signaling pathway (Fig.
5A).
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Fig. 5.
Ras promotes transcription of VPF/VEGF
through PKC . A, HT1080; and
B, 786-0 cells were co-transfected with 0.35-kb VPF/VEGF
promoter-luciferase construct (1.0 µg) and different combinations of
wt-PKC
(0.6 µg), Ha-Ras(G12V) (1.2 µg), Ras(Q61L,C186S) (2.0 µg) or PKC
(KW) (2.0 µg) cDNAs. In all the transfection
experiments, cells were harvested for luciferase assay 40 h after
transfection, and expression in each experiment was normalized to
respective empty expression vector. Fold activation was calculated as
relative to the activity of the same reporter construct, co-transfected
with the control vector. C, expression of PKC
and Ras in
the transfected samples of HT1080 cells were confirmed by Western blot
analysis, using PKC
(polyclonal) and Ras (monoclonal)
antibodies.
was further increased. We made
use of a 188-base pair VPF/VEGF promoter fragment that contained all
the four Sp1-binding sites and performed EMSA with the nuclear extracts
of HT1080 cells, transfected with overexpressed PKC
in the presence
or absence of Ha-Ras(G12V). As shown in Fig. 6A, overexpression of PKC
promoted the binding of Sp1 to the VPF/VEGF promoter and in presence of
Ras, this binding was further increased. This specific protein-DNA
complex formation was competed away with 10-fold molar excess of Sp1
consensus oligonucleotide. Interestingly, the dominant-negative mutant
of PKC
(PKC
kw) significantly reduced the Ras-induced binding of
Sp1 to the VPF/VEGF promoter (Fig. 6B). These results again
clearly indicate that PKC
induces VPF/VEGF transcription through
Sp1, and for which it needs activation through Ras.
View larger version (54K):
[in a new window]
Fig. 6.
Ras promotes the
PKC -induced binding of Sp1 to the VPF/VEGF
promoter. A-C, by using a 188-base pair VPF/VEGF
promoter fragment (having all the four putative Sp1-binding sites) as
the probe, EMSA were performed with partially purified nuclear extracts
of HT1080 cells transfected with different combinations of PKC
(0.6 µg), Ha-Ras(G12V) (1.2 µg), PDK-1 (2.0 µg), and the
dominant-negative (DN) mutants of PKC
and PI 3-kinase (2.0 µg).
Nuclear extracts were prepared from the transfected cells 40 h
after transfection. A, unradiolabeled Sp1 consensus
oligonucleotide (oligo) (10-fold molar excess) was added to
the binding reaction mixture of the control sample (without any
transfection) run in lane 6, to show that it can compete
away the specific protein-DNA complexes.
could be an intermediary
member of this Raf/MEK/MAPK signaling cascade, which is downstream of
Ras. Although Raf-1 is a critical downstream of Ras, it has also been
demonstrated that oncogenic Ras-mediated transformation occurs through
both Raf-dependent and Raf-independent pathways (29, 30,
32, 33, 53, 54). Here we set out to determine which particular pathway
of Ras signaling is involved in VPF/VEGF transcriptional activation.
-mediated Activation of
VPF/VEGF Transcription--
To define the role of
Raf-dependent and Raf-independent pathways in promoting Ras
activity, we selected two different effector loop mutants of Ha-Ras.
The mutant Ha-Ras(G12V,T35S) retained full-length Raf-1 binding
activity while the other mutant, Ha-Ras(G12V,E37G) failed to bind and
activate Raf-1 but could effectively activate some of the other Ras
effector-mediated pathways, like the Rho pathway (31, 55).
. In the presence of Ha-Ras(G12V,T35S) or
Ha-Ras(G12V,E37G) alone, there was an almost 1.7-fold activation of
VPF/VEGF reporter activity (Fig. 7). When
the cells were transfected with these two Ras effector mutants in
presence of PKC
, the VPF/VEGF reporter activity was increased up to
3-fold (Fig. 7). These results are consistent with the hypothesis that
Ras plays a critical role in PKC
-mediated activation of VPF/VEGF
transcription. The above experiments also clearly suggest the
involvement of both Raf-dependent and Raf-independent
pathways in channeling of Ras signals in the activation of PKC
.
Earlier studies have also shown that the Ha-Ras(G12V,E37G) mutant could
complement the transforming activity of the Ha-Ras(G12V,T35S) mutant,
indicating that Raf-independent pathways activated by Ras can
contribute to transformation (31, 55). The effector loop mutant
Ha-Ras(G12V,E37G) mainly involves the RhoA, Rac1, or CDC42 among the
Rho family member of proteins.
View larger version (21K):
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Fig. 7.
Activation of
PKC -mediated VPF/VEGF transcription through
different effector loop mutants of Ha-Ras(G12V). HT1080 cells were
co-transfected with 0.35-kb VPF/VEGF promoter-luciferase
construct (1.0 µg) and different combinations of wt-PKC
(0.6 µg)
and two effector loop mutants of Ha-Ras(G12V), Ha-Ras(G12V,T35S)
and Ha-Ras(G12V,E37G) (each 1.2 µg) expression vectors. The cells
were harvested for luciferase assay 40 h after transfection and
expression in each experiment was normalized with respective empty
expression vector. Fold activation was calculated as relative to the
activity of the same reporter construct, co-transfected with the
control vector.
for VPF/VEGF
Transcription--
Over the last few years, PDK-1 was identified as
the first known upstream activating kinase for PKC (36, 37, 46). It has
been shown that PDK-1 could phosphorylate and activate PKC
in
vivo, and this activation was due to phosphorylation of threonine 410 in PKC
activation loop (13). Based on this observation, we
explored the role of PDK-1 in PKC
-mediated transcriptional activation of VPF/VEGF promoter. The HT1080 cells were co-transfected with 0.35-kb VPF/VEGF promoter and different combinations of PKC
and
PDK-1. As shown in Fig. 8A,
overexpression of PDK-1 increased the PKC
-mediated activation of
VPF/VEGF promoter about 3.3-fold, suggesting PDK-1 was capable of
activating PKC
. When we used a dominant-negative mutant of either
PKC
or PDK-1, the activation of VPF/VEGF promoter was reduced almost
to control level (Fig. 8A). From the above results, it is
likely that PKC
needs activation through PDK-1, as the
kinase-inactive variant of PDK-1 in which the critical
Lys110 residue was mutated to an Asn (PDK-1 Lys/Asn), could
effectively block the activation of VPF/VEGF transcription through
PKC
.
View larger version (19K):
[in a new window]
Fig. 8.
Role of PDK-1 in PKC
and Ras-induced activation of VPF/VEGF transcription.
A, HT1080; and B, 786-0 cells were co-transfected
with 0.35-kb VPF/VEGF promoter-luciferase construct (1.0 µg) and
different combinations of wt-PKC
(0.6 µg), Ha-Ras(G12V) (1.2 µg), wt-PDK-1 (Myc-tagged) (2.0 µg), PDK-1(KN) (Myc-tagged) (2.0 µg), and PKC
(KW) (2.0 µg) expression vectors. The cells were
harvested for luciferase assay 40 h after transfection, and
expression in each experiment was normalized with a respective empty
expression vector. Fold activation was calculated as relative to the
activity of the same reporter construct, co-transfected with control
vector. C, expression of PKC
, Ras, and PDK-1 in the
transfected samples of HT1080 cells were confirmed by Western blot
analysis, using PKC
(polyclonal), Ras (monoclonal), and Myc
(monoclonal) antibodies.
. Fig. 8A indeed shows that in presence of both PDK-1 and transforming Ha-Ras(G12V) the
PKC
-mediated activation of VPF/VEGF promoter activity was increased
~4.5-fold. In 786-0 cells, we also observed a similar type of
activation of VPF/VEGF promoter through PDK-1, Ras, and PKC
(Fig.
8B). In a separate experiment, it has been shown that the
Ras-mediated phosphorylation of PKC
was further increased in
presence of PDK-1 (Fig. 4B). We also found through EMSA that
in presence of PDK-1, the binding of Sp1 to the VPF/VEGF promoter
through Ras and PKC
was further increased (Fig. 6A).
Together, these results clearly indicate that PKC
needs upstream
signaling through both PDK-1 and Ras for VPF/VEGF transcription.
Interestingly, when we used the dominant-negative mutant of PDK-1, the
activation of VPF/VEGF promoter mediated by the combination of Ras and
PKC
was not lowered significantly (Fig. 8A). This
suggests that although both PDK-1 and Ras activate PKC
, they mediate
their action through two different signaling pathways.
--
PI 3-kinases and their lipid products play a crucial role
in various aspects of cell function (37, 43, 53). Interestingly, PI
3-kinase may regulate PKC
by generation of activating molecules (e.g. phosphatidylinositol 1,4,5-trisphosphate) and/or by
acting as a "linker" protein to bring PKC
in contact with other
activating molecules (11, 40, 56). PI 3-kinase consists of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit (57). It has been
shown previously that PI 3-kinase interacts directly with Ras through
its catalytic subunit and the effector site of Ras in a
GTP-dependent manner (33). Recent reports have shown that
PDK-1 also binds with high affinity to the PI 3-kinase lipid product
phosphatidylinositol 3,4,5-triphosphate (PtdIns-3,4,5-P3) (18). As PDK-1 also interacts with PKC
, their association reveals extensive cross-talk between the enzymes in the PI 3-kinase signaling pathway.
-mediated up-regulation of VPF/VEGF transcription, HT1080 cells
were co-transfected with 0.35-kb VPF/VEGF promoter and different combinations of Ha-Ras(G12V), PDK-1, and PKC
in the presence or
absence of a dominant-negative mutant (
p85) of PI 3-kinase. As shown
in Fig. 9,
p85 reduced the PDK-1 and
PKC
-mediated activation of VPF/VEGF promoter to the basal level. In
the presence of
p85 alone, the VPF/VEGF promoter activation was
almost completely shut down. This observation clearly demonstrates that
PKC
and PDK-1 need signaling through PI 3-kinase, as shown by
another group (13). Through EMSA, it has also been shown that
p85
significantly inhibited the PKC
-induced binding of Sp1 to the
VPF/VEGF promoter sequence (Fig. 6C). Interestingly,
p85
could not significantly decrease the individual effect of Ras or the
additive effect of Ras and PKC
in mediating activation of VPF/VEGF
promoter (Fig. 9). But in the presence of
p85, the Ras-mediated
phosphorylation of PKC
was significantly reduced (Fig.
4B). These data reflect that, although Ras-mediated
activation of PKC
is dependent on PI 3-kinase, the Ras-induced
transcription of VPF/VEGF through PKC
is partially dependent upon
the PI 3-kinase pathways. It appears that for VPF/VEGF transcription,
PI 3-kinase activates PKC
mainly through PDK-1.
View larger version (20K):
[in a new window]
Fig. 9.
Role of PI 3-kinase in
PKC , Ras, and PDK-1-induced transcriptional
activation of VPF/VEGF. HT1080 cells were co-transfected with
0.35-kb VPF/VEGF promoter-luciferase construct (1.0 µg) and different
combinations of wt-PKC
(0.6 µg), Ha-Ras(G12V) (1.2 µg), wt-PDK-1
(Myc-tagged) (2.0 µg), and PI 3-kinase dominant negative mutant
(p85DN) (2.0 µg) expression vectors. The cells were harvested for
luciferase assay 40 h after transfection, and expression in each
experiment was normalized with respective empty expression vector. Fold
activation was calculated as relative to the activity of the same
reporter construct, co-transfected with a control vector.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, finally resulting in tumor angiogenesis. By blocking PKC
with the use of its dominant-negative mutant or antisense oligonucleotide, we demonstrate that PKC
acts as an important intermediary molecule in Ras-mediated overexpression of VPF/VEGF (Fig.
2). In accord with earlier work, we also found that PKC
promoted the
Sp1-mediated transcription of VPF/VEGF in human HT1080 and 786-0 cells
where the oncogene ras was already activated. With the
increase in the dose of PKC
, there was gradual activation of
Sp1-mediated VPF/VEGF transcription, but from a certain dose (1.2 µg), this activation was lowered and then gradually came down to the
basal level (Fig. 3). These findings clearly suggest that
PKC
-mediated activation of VPF/VEGF transcription is a rate-limiting step and may need activation through its upstream signaling pathways. Thus, if we overexpress PKC
beyond a certain concentration, it acts
like a dominant-negative factor since it is no longer activated. Here
we attempt to dissect the role of Ras, along with PDK-1 and PI-3 kinase
in activating PKC
and show that they play a significant role in
PKC
-mediated activation of VPF/VEGF transcription.
appears to be located downstream of Ras (12, 18, 28,
51), it seems conceivable that PKC
could be critically involved in
channeling Ras signals for activation of MAPK resulting in
up-regulation of VPF/VEGF transcription. In the current study, we
observe that PKC
or Ras alone increased the VPF/VEGF transcription
in human HT1080 or 786-0 cells up to 2- and 2.5-fold, respectively
(Fig. 5, A and B). But when the cells were
transfected in combination with PKC
and Ha-Ras(G12V), the
transcriptional activation was increased up to 4-fold (Fig. 5,
A and B). A dominant-negative mutant of Ha-Ras
blocked the PKC
-mediated activation of VPF/VEGF transcription (Fig.
5A). The dominant-negative mutant of PKC
reduced the
VPF/VEGF transcription below the control level (Fig. 5A).
These results clearly indicate that PKC
needs signaling through Ras
for activation. It may also play an important role in channeling a part
of Ras signal to its downstream targets to promote tumor angiogenesis.
Observations from others have prompted suggestions that PKC
is a
required step of Ras-mediated mitogenic signaling and that Ras directly interacts in vitro with the regulatory domain of PKC
as
well as that the association of PKC
with Ras in vivo is
triggered by platelet-derived growth factor (51). In our studies,
although we were unable to detect any direct interaction between PKC
and Ras, these two proteins were found to be present in the same
immunocomplex (Fig.-4A). Therefore, it is quite clear that
Ras-mediated signaling is an important step for PKC
activation and
thus increasing VPF/VEGF promoter activity. We have also confirmed this
through EMSA, where Ras promoted the PKC
-induced binding of Sp1 to
the VPF/VEGF promoter (Fig. 6A). Moreover, it is also
evident that even the activated form of Ras cannot bypass PKC
in
regulating VPF/VEGF transcription, as the dominant-negative mutant of
PKC
clearly inhibited the Ras-induced binding of Sp1 to the VPF/VEGF
promoter (Fig. 6B).
(Fig.
7). This result clearly suggests that Ras-induced activation of PKC
for VPF/VEGF transcription is mediated through both
Raf-dependent and Raf-independent pathways.
(13, 34-36).
Like Akt/PKB and p70 S6K, PKC
has also been shown to be one of the
in vivo substrates of PDK-1 (13, 39). Phosphorylation of
PKC
Thr410 by PDK-1 leads to activation of the enzyme,
both in vivo and in vitro (38). In the present
study, we observe that PDK-1 plays a significant role in promoting
VPF/VEGF transcription induced by PKC
. PDK-1 increased
PKC
-mediated activation of VPF/VEGF transcription up to 3.3-fold in
human HT1080 and 786-0 cells (Fig. 8, A and B). A
dominant-negative mutant of either PDK-1 or PKC
completely blocked
the transcription of VPF/VEGF (Fig. 8A). The mutant PKC
reduced the VPF/VEGF transcription well below the control level (Fig.
8A). All of these observations clearly present PDK-1 as an
activator of PKC
. Interestingly, when we transfected the HT1080 or
786-0 cells with combinations of both Ras and PDK-1, there was an
additive effect up to 4.5-fold in PKC
-mediated activation of
VPF/VEGF transcription (Fig. 8, A and B). This
suggests that upstream signaling from both Ras and PDK-1 is essential
for the activation of PKC
. In EMSA, we have shown that PDK-1
promoted the Ras and PKC
-induced binding of Sp1 to the VPF/VEGF
promoter (Fig. 6A). We have also demonstrated that the
signaling through Ras and PDK-1 are mediated by two distinctly
different pathways, as the dominant-negative mutant of PDK-1 could not
effectively block PKC
and Ras-induced transcriptional activation of
VPF/VEGF promoter (Fig. 8A). Through the use of a
phospho-specific antibody, it has been shown that both Ras and PDK-1
stimulate phosphorylation of the PKC
activation loop
Thr410 (Fig. 4B). Observations from other
laboratories (13, 38) have demonstrated that PKC
phosphorylation/activation is almost completely blocked by coexpression
of dominant-negative PDK-1 or by mutation of Thr410. Thus,
it is likely that a prerequisite for PKC
activation is phosphorylation of Thr410. Taken together, these
observations indicate that Ras and PDK-1 constitute two distinct
pathways, both of which are required for PKC
-mediated VPF/VEGF
transcriptional activation.
has received considerable attention in recent years as it has
been implicated as a downstream target of PI 3-kinase (11, 40, 42, 43).
PDK-1 also serves as an important member of PI 3-kinase pathways (13,
37, 39). The observation that PKC
associates with PDK-1 in
vivo suggests considerable cross-talk between effector molecules
in the PI 3-kinase signaling pathway (13, 37). On the other hand, PI
3-kinase also interacts with Ras·GTP but not with Ras·GDP and is
activated both in vitro and in vivo as a result
of this interaction (53, 69). We have found that a dominant-negative
mutant of PI 3-kinase significantly reduces the activation of PKC
through Ras (Fig. 4B). In the present study, we have shown
that PI 3-kinase acts as a major activator for PDK-1 and
PKC
-mediated pathway, regulating VPF/VEGF transcription. The
dominant-negative mutant of PI 3-kinase significantly reduced the
PKC
-induced binding of Sp1 to the VPF/VEGF promoter sequence as well
as inhibited the PDK-1 and PKC
-induced activation of VPF/VEGF
promoter almost to the basal level (Figs. 6C and 9). These
observations suggest that both PDK-1 and PKC
need activation through
PI 3-kinase. In contrast, the dominant-negative mutant of PI 3-kinase
could not lower the Ras and PKC
-mediated VPF/VEGF transcription
below the control level (Fig. 9). Moreover, the mutant PI 3-kinase
failed to reduce the VPF/VEGF transcriptional activation mediated by
Ras alone (Fig. 9). Thus, although Ras interacts with PI 3-kinase, it
may not need activation through PI 3-kinase to promote VPF/VEGF transcription.
, in a
Sp1-dependent manner. PDK-1 and PI-3 kinase also play an
important Ras-independent role in exerting an additive effect in this
signaling cascade. All of these mechanisms which up-regulate VPF/VEGF
may have important connections with tumor angiogenesis where mutant Ras
alleles contribute to solid tumor development and metastasis. It
appears that in addition to hypoxia, different growth factors or
hormones, the activation of oncogenes such as Ras, also play a
significant role in tumor angiogenesis in a stable manner by
stimulating constitutive expression of VPF/VEGF. Moreover,
possibilities of common pathways for different stimuli to promote
VPF/VEGF expression cannot be ignored. Therefore, understanding the
signaling pathways for the activation of VPF/VEGF expression and
designing inhibitory molecule(s) of these signaling cascades might have
comprehensive effects in tumor angiogenesis, progression, and metastasis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Alex Toker for PDK-1, PDK-1 DN,
p85 DN expression vectors and also for anti-phospho-PKC antibodies;
Dr. Jack Lawler for helpful comments on the manuscript; Rinku Pal and
Alexis Bywater for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant CA78383, the Massachusetts Public Health, and under terms of a contract from the National Foundation for Cancer Research (to D. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Howard Temin awardee supported by National Institutes of Health
Grant CA 78396).
§ Eugene P. Schonfeld Medical Research awardee from the National Kidney Cancer Association. To whom correspondence should be addressed: Dept. of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Ave., RN 270H, Boston, MA 02215. Tel.: 617-667-7853; Fax: 617-667-3591; E-mail; dmukhopa@caregroup.harvard.edu.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M007818200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
VPF, vascular permeability factor;
VEGF, vascular denothelial growth factor;
PKC, protein kinase C
;
MAP, mitogen-activated protein;
MAPK, mitogen activated protein kinase;
PI 3-kinase, phosphatidylinositol
3-kinase;
PI(3, 4,5)P3, phosphatidylinositol
3,4,5-triphosphate;
kb, kilobase(s);
EMSA, electrophoretic mobility
shift assay;
PAGE, polyacrylamide gel electrophoresis.
![]() |
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