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INTRODUCTION |
The regulation of vascular endothelial growth factor
(VEGF)1 expression by human
tumor cells has been implicated as a key factor in human tumorigenic
and metastatic potential (1, 2). VEGF expression has been shown to be
controlled by environmental factors such as the limited availability of
oxygen or glucose (2). Alternatively, growth factors like
platelet-derived growth factor and acidic/basic fibroblast growth
factor or cytokines like tumor necrosis factor-
and transforming
growth factor family members also have been shown to stimulate VEGF
synthesis (2). It is clear that VEGF expression is tightly regulated by
both transcriptional and post-transcriptional mechanisms (2, 3);
however, the signal transduction pathways that regulate these
mechanisms remain largely unknown.
The protein kinase C (PKC) family consists of 12 phospholipid-dependent serine/threonine kinases that
mediate signals from the cell surface to the nucleus and play key roles
in cellular signaling pathways (4, 5). These isoenzymes have been
grouped into three functional classes based on the structures of their regulatory domains and their requirements for activation by
phosphatidylserine, calcium, and diacylglycerol (DAG) (4, 5). The first
class, the conventional PKCs (cPKCs, isoforms
,
I,
II, and
), require Ca2+ and DAG to be activated. The second
class, the novel PKCs (nPKCs, isoforms
,
,
, and
), are
Ca2+-independent. The third class, the atypical PKCs
(aPKCs, isoforms
,
,
, and µ), are dependent on
phosphatidylserine but not Ca2+ or DAG. PKC isoenzymes
exhibit distinct tissue distribution patterns, and most cell types
express PKC-
, -
, and -
, whereas other isoforms are restricted
to some specific tissues (4, 5). Several reports have indicated that
activation of PKC by the tumor promoter, phorbol 12-myristate
13-acetate (PMA), up-regulates VEGF expression in a variety of cell
types including astrocytes, fibroblasts, umbilical vascular endothelial
cells, and smooth muscle cells (6-9). However, the relative importance
of individual PKC isoforms to transcriptional and post-transcriptional
regulation of VEGF expression remains unclear.
Human glioblastomas are one of the most rapidly growing angiogenic
tumors corresponding to high mortality and morbidity rates. Here we
show that when U373 glioblastoma cells were treated with PMA, VEGF
mRNA expression was up-regulated through a post-transcriptional mRNA stabilization mechanism but not transcriptional activation. We
show that PKC-
and -
are involved in the induction of VEGF expression as defined by antisense oligonucleotide inhibition and
overexpression studies. This study indicates that PKC isoform activation, especially PKC-
and -
, are important upstream events resulting in increased VEGF mRNA stabilization.
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MATERIALS AND METHODS |
Antibodies, Oligonucleotides, and Kinase
Inhibitors--
Monoclonal antibodies to different PKC isoforms were
purchased from Transduction Laboratories (Lexington, KY). The chicken anti-hVEGF polyclonal antibody was raised to human
baculovirus-expressed human VEGF165. The mouse anti-hVEGF monoclonal
antibody was purchased from R & D Systems (Minneapolis, MN). All of the
antisense PKC oligonucleotides to PKC-
, -
, -
, -
, -
, and
-
were synthesized as phosphorothioate derivatives from Genemed
Synthesis (San Francisco, CA). Seven different kinase inhibitors were
used and purchased from Calbiochem as follows: protein kinase C
inhibitors staurosporine (highly specific inhibitor of PKC) (10),
bisindolemaleimide I (high selectivity for PKC-
, -
, -
, -
and -
) (11), Go6976 (high selectivity for PKC-
and -
) (12),
and calphostin C (highly specific inhibitor of PKC) (13); protein
kinase A inhibitor H89 (14); serine/threonine kinase inhibitor, H7
(15); and cyclic nucleotide-dependent protein kinase
inhibitor, H8 (16).
Cell Lines and Culture Conditions--
Human glioblastoma cell
line, U373-MG, was routinely grown in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, 10 units/ml penicillin, and 10 µg/ml
streptomycin. Cells were cultured under either normoxic conditions (5%
CO2, 21% O2, 74% N2) or hypoxic
conditions (5% CO2, 2% O2, 93%
N2) in a humidified triple gas incubator (Heraeus model
6060, Hanau, Germany) at 37 °C.
Western Blot and Luciferase Assays--
Cytoplasmic extracts
were obtained as described (17). Briefly, U373 cells were washed three
times in ice-cold PBS before lysis with buffer containing 50 mM Hepes, pH 7.5, 1% Triton X-100, 10 mM
sodium pyrophosphate, 150 mM NaCl, 100 mM NaF,
0.2 mM NaVO4, 1 mM EGTA, 1.5 mM MgCl2, 10% glycerol, and 5 mM
4-(2-aminoethyl)benzenesulfonyl fluoride (Sigma). The cytoplasmic
extract was collected after centrifugation at 14,000 × g for 15 min, denatured under reducing conditions, and
proteins separated in 8% SDS-PAGE. Western blots were performed using
a 1:1000 dilution of anti-PKC monoclonal antibodies followed by a
1:10,000 dilution of horseradish peroxidase-conjugated goat anti-mouse
IgG (Amersham Pharmacia Biotech) in a blocking buffer containing 1%
bovine serum albumin and 0.1% Tween 20 in Tris-buffered saline, pH
7.4. The blots were then developed with ECL system (Amersham Pharmacia
Biotech). For luciferase reporter assays, stably transfected U373 cells
containing the VEGF promoter construct (
1226 to +298 base pairs
upstream of luciferase reporter) (18) were lysed with passive lysis
buffer, and luciferase activity was measured with a Luciferase Assay
kit following the supplier's instructions (Promega, Madison, WI).
Enzyme-linked Immunosorbent Assay--
In a 96-well plate
(Dynatech Laboratories, Chantilly, VA), 100 µl of chicken anti-hVEGF
polyclonal antibody (0.5 µg/ml in PBS) was coated overnight at
4 °C. The plate was then blocked with blocking buffer (0.25% bovine
serum albumin, 0.05% Tween 20, and 1 mM EDTA in PBS) for
1 h at 37 °C. Conditioned media (100 µl) or recombinant hVEGF
standard (R & D System) was added and incubation continued for another
2 h at 37 °C. Mouse anti-hVEGF monoclonal antibody (R & D
System) was added (100 µl of 0.5 ng/ml) to each well after washing
the plate with PBS, 0.05% Tween 20 solution and then incubated at
37 °C for 2 h. Detection antibody, a peroxidase-labeled goat
anti-mouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD), was
incubated at 37 °C for 2 h, and the plates were washed again
with PBS, 0.05% Tween 20. MBT substrate (Kirkegaard & Perry
Laboratories) was used to develop color and quantitated with a
Thermomax microplate reader (Molecular Devices, Sunnyvale, CA).
Transcription Run-off--
Transcription run-off assay were
performed as described earlier (17). Briefly, U373 cell nuclei were
collected in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.35% sucrose, and 0.5% Nonidet P-40) and centrifuged for 3 min at 500 × g. Nuclei were then suspended in reaction buffer (5 mM Tris-HCl, pH 8.0, 2.5 mM MgCl2,
150 mM KCl, 0.5 mM rATP/CTP/GTP, 0.7 µM rUTP, 2.5 mM dithiothreitol, and 5 units
RNasin) in the presence of 100 µCi of [
-32P]UTP and
incubated at 30 °C for 23 min. Total RNA was extracted by the
addition of 40 µg of glycogen carrier and 0.8 ml of Trizol reagent.
The radiolabeled RNA pellets were collected after phenol/chloroform extraction and isopropyl alcohol precipitation (0.5 volume) and dissolved in 10 mM
-mercaptoethanol solution. Slot blots
used for RNA hybridizations were prepared with VEGF, 36B4 and
glyceraldehyde-3-phosphate dehydrogenase cDNAs (5 µg/slot). The
cDNAs were denatured with 100 mM NaOH at 100 °C for
10 min, neutralized with NH4OAc to 1 M final
concentration, and directly blotted onto nylon membranes (ICN, Costa
Mesa, CA) using a slot blot apparatus (Schleicher & Schuell). Membranes
were subsequently washed twice with 2× SSC, baked at 80 °C for 15 min, and prehybridized for 6 h at 65 °C in hybridization buffer
([50 mM PIPES, pH 6.5, 50 mM sodium phosphate, pH 7.0, 20 mM NaCl, 5% SDS, 2.5 mM EDTA, and
50 µg/ml denatured salmon sperm DNA). Hybridizations were performed
with equal amounts of labeled nuclear RNA (1 × 106
cpm/ml) in hybridization buffer for 20 h at 65 °C. Blots were then washed once at room temperature for 20 min and twice at 55 °C
for 20 min in 1% SDS, 1× SSC. Blots were then exposed to Kodak MR
film. VEGF mRNA was normalized to 36B4 expression using
PhosphorImager analysis (Molecular Dynamics).
Transient Transfection, mRNA Extraction, and Northern
Analysis--
Antisense oligonucleotides to PKC-
, -
, -
, -
,
-
, and -
(0.1, 0.5, or 1 µM) or FLAG epitope-tagged
PKC-
overexpression vector (19) were transfected into U373 cells
using LipofectAMINE (Life Technologies, Inc.) following the supplier's
instructions. Total RNA was extracted by using RNeasy RNA extraction
kit (Qiagen, Chatsworth, CA). Northern blot was performed as described
previously (17). Briefly, equal amounts of total RNA (10 µg/lane)
were electrophoresed in 1% agarose gels containing 2.2 M
formaldehyde. RNA was blotted by capillary transfer to a nylon membrane
(NEN Life Science Products) in 10× SSC. Blots were cross-linked with UV Stratalinker (Stratagene), baked at 80 °C for 15 min, and
prehybridized for 2-4 h at 65 °C. Hybridization was carried out
overnight at 65 °C with [
-32P]dCTP-labeled human
VEGF165 AccI/NcoI fragment (823 base pairs) encompassing the coding region and 330 base pairs of 3'-untranslated region. A ribosome-associated protein cDNA, 36B4, was used as a
control. Probes were prepared by the random-primed synthesis method
using the Multiprime Kit (Amersham Pharmacia Biotech). Blots were
washed at high stringency (1% SDS, 1× SSC at 60 °C) and exposed to
Kodak MR film. VEGF mRNA was normalized to 36B4 expression using
PhosphorImager analysis.
Determination of VEGF mRNA Half-life--
The transcription
inhibitor actinomycin D (5 µg/ml) was added to U373 cells and total
RNA isolated at various time intervals (30 min and 1, 2, and 4 h).
Total RNA was analyzed by Northern blot, and VEGF signal was quantified
by PhosphorImager analysis. The VEGF signal was normalized to ribosomal
associated protein 36B4 signal. Least squares regression analysis of
the resulting line was performed (Kaleidagraph, Synergy Software,
Reading, PA), and half-life values were determined from the regression curves.
In Vitro PKC Activity Assay--
The in vitro PKC
activity assays were performed as described (20). Briefly, U373 cells
were washed three times in ice-cold PBS followed by lysis in 1% Triton
X-100 lysis buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 0.2 mM NaVO4, and 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride. The lysate was incubated with
rabbit anti-human PKC-
, -
, or -
polyclonal antibody (Life
Technologies, Inc.) for 2 h at 4 °C followed by another 2-h
incubation with 20 µl of protein A-Sepharose beads (Amersham
Pharmacia Biotech) at 4 °C. The immunoprecipitates were washed six
times with the lysis buffer containing 0.5 M NaCl. The
immunocomplexes were suspended in 20 µl of kinase buffer (35 mM Tris-HCl, pH 7.5, 10 mM MgCl2,
0.5 mM EGTA, 0.1 mM CaCl2, and 1 mM phenylphosphate) containing 2 µg of myelin basic
protein (MBP, Sigma) and 5 µCi of [
-32P]ATP and
incubated for 30 min at 30 °C. Reactions were stopped by the
addition of SDS-PAGE sample buffer. Samples were denatured and
separated by 12% SDS-PAGE followed by exposure to Kodak MR film. MBP
and 32P-MBP signals were quantified by densitometer and
PhosphorImager analysis, respectively.
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RESULTS |
Protein Kinase C Activation Potently Induces VEGF
Expression--
To determine the relative contribution of protein
kinase C (PKC) activation to the induction of VEGF mRNA expression
in human glioblastoma U373 cells, phorbol 12-myristate 13-acetate (PMA) was used to activate PKCs, and steady-state VEGF mRNA levels were determined by Northern blot. As shown in Fig.
1A, VEGF mRNA levels were
increased 5.8-fold with 4 h PMA treatment when compared with U373
cells without PMA activation. The 4-h PMA-induced VEGF mRNA expression was approximately half of the 18-h hypoxia-induced signal
(Fig. 1A). In a time course study of PMA treatment (100 nM), induction of VEGF mRNA can be observed at 1 h
which increased to a maximum level at 8 h (Fig. 1B) and
then slowly decreased afterward (data not shown). Furthermore, this
response was dependent upon cell confluence, showing a PMA induction of
VEGF mRNA expression that varied from 3- to 10-fold at 4 h
(data not shown). Taken together, these results suggest that PMA
strongly up-regulates VEGF mRNA expression in U373 cells, and the
activation requires prolonged PMA treatment with maximum VEGF mRNA
levels reached at 8 h.

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Fig. 1.
PMA up-regulates VEGF mRNA
expression. A, VEGF mRNA expression in U373 cells.
U373 cells were cultured alone (C), treated with PMA (100 nM) for 4 h (P), or cultured under hypoxic
conditions (3% O2) for 18 h (H). The fold
induction of VEGF mRNA over control (C) was calculated
after normalization to the ribosome-associated protein, 36B4, from each
lane (Fold). B, time course studies of PMA
induction of VEGF mRNA expression. U373 cells were treated with PMA
(100 nM) for various times as indicated. Total mRNA was
extracted and analyzed for VEGF mRNA by Northern blot. The
ribosome-associated protein, 36B4, was used as mRNA control. Fold
induction of VEGF mRNA without PMA treatment (0) was
calculated after normalization to the 36B4 mRNA from each lane
(Fold).
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To study whether PMA up-regulation of VEGF mRNA expression
correlates to VEGF protein synthesis and secretion, we performed enzyme-linked immunosorbent assay analysis of U373-conditioned media
with and without the addition of PMA for 4 h. The conditioned media of control cells contained 0.313 ± 0.076 ng/ml VEGF, and PMA-treated cells contained 0.79 ± 0.124 ng/ml VEGF. This is a 2.5-fold induction of VEGF secretion in only 4 h and is
statistically significant in a Student's t test
(p
0.0047). Thus, increases observed in VEGF
mRNA levels with PMA treatment correlate well with increases in
VEGF protein synthesis and secretion.
The PKC isoforms
,
, and
, representing cPKC, nPKC, and aPKC
classes respectively, are universally expressed (4). Studies have shown
that prolonged PMA treatment can repress cPKCs and nPKCs but not aPKCs
synthesis (4, 21). Thus, it was of interest to determine the effect of
PMA on the three universally expressed PKC isoforms in U373 cells. Fig.
2A is a representative Western blot showing PMA regulation of PKC synthesis. Both PKC-
and -
protein levels decreased over time with PMA treatment, and PKC-
reached almost undetectable levels after 4 h of PMA exposure. In a
similar but less effective manner, PKC-
decreased by 50% with
8 h PMA treatment. In contrast, PKC-
protein showed a
substantial increase and almost doubled after 30 min and remained
elevated up to at least 8 h (Fig. 2A) and then slowly
decreased afterward (data not shown). By using an in vitro
PKC activity assay (Fig. 2, B and C), specific
immunoprecipitation followed by phosphorylation of myelin basic protein
(MBP), PKC-
activity reached a maximum of 141% over control at 15 min PMA and then decreased sharply over 4 h PMA treatment to 53%
of control. PKC-
activity showed a slower induction than PKC-
and
reached maximum of 121% over control at 30 min PMA and then slowly
decreased over the time of PMA treatment to 91% of control at 4 h
PMA. In contrast, PKC-
activity gradually increased during PMA
treatment and remained elevated up to 4 h PMA with a maximum of
158% over control at 4 h PMA exposure. These results suggest that
PMA differentially regulates not only PKC isoform synthesis but also
selectively modulates their activity in U373 cells. A transient, rapid
activation of PKC-
activity, followed by stable up-regulation of
PKC-
activity over the 4-h PMA treatment, suggests that a constant
total PKC activity may be necessary to functionally up-regulate VEGF
mRNA expression in U373 cells.

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Fig. 2.
Differential regulation of PKC expression and
PKC activity by PMA. A, effect of PMA on PKC isoform
synthesis. U373 cells were treated with PMA (100 nM) for
various times as indicated. Total cellular proteins were extracted, and
PKC- , - , and - protein levels were detected by Western blot.
S indicates standard PKCs from Jurkat cell lysate.
B, representative in vitro PKC activity after PMA
treatment. Total cellular proteins were prepared after different time
intervals of PMA (100 nM) treatment, PKC- , - or -
immunoprecipitated, and in vitro phosphorylation activity
tested with 21-kDa myelin basic protein (MBP) as a substrate
and analyzed 32P-MBP after separation in 12% SDS-PAGE.
M indicates 22-kDa protein marker. C, percent PKC
activity over control ( ) was calculated after normalization to
Coomassie Blue-stained MBP from each lane. MBP and 32P-MBP
signals were quantified by densitometer and PhosphorImager analysis,
respectively. Results are presented as the means ± S.D. of two
independent experiments.
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Protein Kinase C Inhibitors Suppress Phorbol Ester-induced VEGF
mRNA Expression--
In order to study further the involvement of
PKC isoforms to the phorbol ester-induced VEGF mRNA expression, we
employed several kinase inhibitors at active concentrations and studied
PMA-induced VEGF mRNA levels. A representative result is shown in
Fig. 3; the broad PKC inhibitors,
staurosporine and calphostin C, strongly decreased PMA-induced VEGF
mRNA expression. However, PKC inhibitors selective to cPKCs
(Go6967) or to both cPKCs and nPKCs (bisindolemaleimide I) were less
effective in repressing PMA-induced VEGF mRNA. This suggests that
the aPKC isoforms may also play a role in the PMA-induced VEGF mRNA
expression. Application of a broad serine/threonine kinase inhibitor H7
showed significant reduction of PMA-induced VEGF mRNA expression,
whereas the protein kinase A inhibitor H89 did not show any
suppression. This indicates that protein kinase A is not involved in
the PMA activation of VEGF mRNA expression, and inhibition of VEGF
mRNA accumulation by the serine/threonine kinase inhibitor H7
is likely mediated through PKC. The cyclic nucleotide-dependent protein kinase inhibitor, H8,
interestingly showed an increase in VEGF mRNA levels, indicating
that blocking of cyclic nucleotide-dependent protein kinase
may favor PMA activation pathways and VEGF mRNA accumulation. Taken
together, these results suggest that the induction of VEGF mRNA
expression by PMA is mainly mediated through a cascade of PKC isoform
activation, including aPKCs.

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Fig. 3.
Inhibition of PMA-induced VEGF mRNA
expression by kinase inhibitors. U373 cells were cultured alone
(C) or treated with PMA for 4 h in the absence or
presence of kinase inhibitors; staurosporine (100 nM;
Sto), H7 (25 µM), H8 (25 µM),
bisindolemaleimide I (100 nM; Bis), Go6976 (10 nM; Go), calphostin C (100 nM;
Cal), and H89 (1 µM). Total RNA was extracted,
and VEGF mRNA was detected by Northern blot analysis. The
ribosome-associated protein probe, 36B4, was used as control. Fold
induction of VEGF mRNA over control (C) was calculated
after normalization to the ribosome-associated protein mRNA, 36B4,
from each lane (Fold).
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PKC Activation Induces VEGF mRNA Steady-state Levels That Are
Independent of Transcriptional Activation--
To determine whether
increased VEGF mRNA expression by PMA is specifically due to
transcriptional activation, two studies were performed. First, VEGF
mRNA transcription in U373 cells was measured by run-off
transcription assay. As shown in Fig.
4A, only hypoxic conditions
showed measurable activation of VEGF mRNA transcription, whereas
4 h of PMA treatment did not when compared with control. This
experiment has been repeated three times with identical results.
Second, U373 cells were transfected and selected for a stable
integrated human VEGF promoter construct (
1226 to +298 base pairs)
upstream of the luciferase reporter gene. These cells were then treated
with PMA and analyzed for luciferase activity. As shown in Fig.
4B, there was little or no increase in luciferase activity
from 1 to 12 h PMA treatment, whereas 12 h of hypoxia showed
a 2.5-fold transcriptional activation. Thus, the VEGF promoter/reporter construct is functional and responsive to hypoxia, and PMA does not
activate VEGF promoter transcription. Taken together, these results
suggest that PMA-induced VEGF mRNA accumulation is not mediated
through transcriptional activation as is the case for hypoxic
stress.

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Fig. 4.
PMA-induced VEGF mRNA expression is not
transcriptionally activated. A,
transcription run-off assays. U373 cells were cultured alone
(C) or treated with PMA for 4 h (P) or
hypoxia (3% O2) for 8 h (H). Total
radiolabeled RNA (106 cpm/ml) obtained from nuclear run-off
reactions was hybridized to denatured cDNA templates encoding
GADPH, VEGF, and 36B4. B, luciferase reporter assays. The
stably transfected U373 cells containing the VEGF promoter ( 1226 to
+298 base pairs upstream of luciferase) were either treated with PMA
(100 nM) for various times as indicated or cultured under
hypoxia for 12 h (H) in triplicate. Luciferase
activities (mean ± S.D.) were measured as described under
"Materials and Methods."
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Phorbol Ester Induces VEGF mRNA Stability That Is Reversible
with Staurosporine Treatment--
To determine whether
post-transcriptional regulation plays a crucial role in the PMA
induction of VEGF mRNA expression, we treated U373 cells with PMA
for 3 h prior to the addition of the transcription inhibitor
actinomycin D, and we determined the VEGF mRNA half-life with
quantitative Northern blot analysis. As shown in Fig.
5, VEGF mRNA half-life increased from
0.8 to 3.6 h after treatment of U373 cells with PMA. Since the
broad PKC inhibitor staurosporine showed a strong inhibition of PMA
up-regulation of VEGF mRNA expression (Fig. 3), the inhibitor was
also added during the course of PMA treatment, and VEGF mRNA
stability was measured. As shown in Fig. 5, staurosporine limited the
PMA-induced VEGF mRNA half-life to 1.3 h (Fig. 5). This result
suggests that increased VEGF mRNA expression by PMA is regulated
predominantly through a post-transcriptional mechanism, and the
activation of PKC plays an important role in this mechanism.

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Fig. 5.
Staurosporine suppresses PMA-induced VEGF
mRNA stability. U373 cells were cultured alone
(control) or treated with PMA for 3 h (PMA)
in the absence or presence of staurosporine (PMA+ST) in a
triplicate experiment. The cells were then treated with actinomycin D
(5 µg/ml) for various time intervals (0, 1, 2, and 4 h), and
VEGF mRNA levels were detected by Northern blot analysis. VEGF
mRNA signal was quantified by PhosphorImager analysis and
normalized with the ribosome-associated protein, 36B4 mRNA. Values
are expressed as mean ± S.D. VEGF mRNA signal without
actinomycin D treatment was defined as 100%, and VEGF mRNA signals
after actinomycin D treatment were calculated as percent VEGF mRNA
remaining.
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PKC-
and -
Antisense Oligonucleotides Block the PMA Induction
of VEGF mRNA Expression--
Of the 12 PKC isoforms identified to
date, 8 PKC isoforms were detected in the U373 cells by Western blot
analysis, and the PKC-
I, -
II, -
, and -
isoforms were
excluded (data not shown). To delineate the PKC isoforms contributing
to PMA-induced VEGF mRNA stability, six antisense (AS)
oligonucleotides were selected from the three subclasses of PKC
isoforms to access their individual functions. The AS oligonucleotides
were designed against the translation start site of each PKC isoform
(Fig. 6A). To improve
intracellular stability and effectiveness, the AS oligonucleotides were
synthesized as phosphorothioate derivatives. To ensure the AS
oligonucleotides were effective in repressing their respective PKC
isoform expression, we performed Western blot analysis on transfected
cells. A representative result is shown in Fig. 6B, all six
of the AS oligonucleotides (0.5 µM) substantially reduced
their specific PKC protein levels. PKC-
and -
AS oligonucleotides
decreased their respective protein levels approximately 80-90%,
whereas PKC-
, -
and -
AS oligonucleotides repressed their
isoforms by about 40-50%.

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Fig. 6.
Effect of antisense oligonucleotides on PKC
synthesis. A, antisense oligonucleotide
sequences to translation start sites of individual PKC isoforms.
B, Western blot detection of PKC isoform synthesis. U373
cells were transiently transfected with Lipofectin reagent in the
absence (C) or presence of 0.5 µM antisense
(AS) oligonucleotides to PKC- , - , - , - , - , or
- . After 18 h recovery, cytoplasmic extracts were obtained, and
the respective PKC proteins were detected by Western blot.
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Given the effective reduction of PKC-
and -
isoforms by their
specific AS oligonucleotides, we examined whether down-regulation of
either of these PKC isoforms would interfere with PMA-induced VEGF
mRNA expression by quantitative Northern blot. As a control, cells
were transfected with AS oligonucleotide to PKC-
, which is not
expressed by U373 cells. As shown in Fig.
7A, cells transfected in
triplicate with PKC-
or -
AS oligonucleotide showed a
dose-dependent reduction of PMA-induced VEGF mRNA
level, whereas the control PKC-
AS oligonucleotide was ineffective.
With the exception of the lowest concentration (0.1 µM),
both 0.5 and 1 µM AS oligonucleotide transfection to
PKC-
or -
showed a statistically significant reduction in
PMA-induced VEGF mRNA expression (Fig. 7A,
p < 0.05). Fig. 7B is a representative
Northern blot showing a dose-dependent repression of
PMA-induced VEGF mRNA level by PKC-
or -
AS oligonucleotide transfection. The U373 cells transfected with PKC-
or -
AS
oligonucleotides did not demonstrate any significant repression of
PMA-induced VEGF mRNA expression when performed in triplicate (data
not shown). Taken together, these results suggest that both PKC-
and
-
isoforms are crucial mediators in the PMA activation of VEGF
mRNA expression in U373 cells.

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Fig. 7.
PKC- and
- antisense oligonucleotides block PMA-induced
VEGF mRNA expression. A, effect of PMA-induced VEGF
mRNA expression by antisense (AS) oligonucleotide to
PKC- , - , or - . U373 cells were mock-transfected or transfected
with AS oligonucleotide (0.1, 0.5, or 1 µM) to PKC- ,
- , or - . The cells were recovered overnight and treated with 100 nM PMA for 4 h in triplicate. Total mRNA was
extracted, and VEGF mRNA level was detected by Northern blot. VEGF
mRNA signal was quantified by PhosphorImager analysis and
normalized to the 36B4 ribosome-associated mRNA from each lane and
calculated as mean ± S.D. The VEGF mRNA from PMA-treated
cells were shown as 100% VEGF mRNA levels and compared the
mRNA with the control or the AS oligonucleotide-transfected cells.
*, significantly different from PMA control (p 0.05)
by Student's t test. B, representative Northern
blot of PKC- or - AS oligonucleotide suppression of PMA-induced
VEGF mRNA expression. The mock-transfected cells without PMA
treatment (C) and with PMA treatment (P) were
used as controls. Fold induction of VEGF mRNA over mock-transfected
control (C) was calculated after normalization to the
ribosome-associated protein, 36B4, from each lane
(Fold).
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Studies have suggested that PKC isoforms may regulate their activity
through direct or indirect cross-talk (22). Given the effectiveness of
transient transfection of either PKC-
or -
AS oligonucleotides
for limiting the PMA induction of VEGF mRNA expression and the
overlap in PMA-induced activity between the two isoforms, it was of
interest to detect a possible cross-talk between the two isoforms in
U373 cells. A representative Western blot is shown in Fig.
8A, identifying that the
transfection of U373 cells with PKC-
AS oligonucleotide not only
blocked its own protein synthesis but also significantly reduced
PKC-
protein levels. Conversely, AS oligonucleotide to PKC-
also
strongly inhibited PKC-
protein levels after the transfection. In
contrast, the control AS oligonucleotide to PKC-
did not show any
significant effect on the synthesis of either PKC-
or -
. By using
an in vitro PKC activity assay to access PKC isoform
activities with AS oligonucleotide transfection (Fig. 8B),
the reduction of PKC-
protein level by both PKC-
and -
AS-oligonucleotide also showed concomitant decreases in PKC-
activity. PKC-
specific activity was 42 and 58% over
mock-transfected control with PKC-
and -
AS oligonucleotide
transfection, respectively. In a similar manner, PKC-
activity
levels were 36 and 47% of mock-transfected control with PKC-
and
-
AS oligonucleotide transfection, respectively. Although the
specific isoform oligonucleotides were most effective, the alternative
isoform oligonucleotides were still fairly efficient. These results
suggest that PKC-
and -
AS oligonucleotides cross-inhibit their
protein synthesis, indicating that these PKC isoforms may be under
strict coordinate control in vivo. Since the AS
oligonucleotide sequences of the PKC-
and -
do not show
significant homology, the cross-inhibition of their protein synthesis
is likely to be dependent on isoform cross-talk as opposed to
nonspecific interactions.

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Fig. 8.
Cross-inhibition of PKC isoform synthesis and
activity by antisense oligonucleotide to PKC-
or PKC- . A,
representative Western blot of PKC- and - synthesis after
PKC- , - , or - AS oligonucleotide transfection. U373 cells were
either mock-transfected ( ) or transfected with 0.5 µM
PKC- , - , or - AS-oligonucleotide (AS). Total
cellular proteins were extracted and detected PKC- or - protein
levels by Western blot. S indicates standard PKC- or -
from Jurkat cell lysate. B, representative in
vitro PKC- and - activity after PKC- , - , or - AS
oligonucleotide transfection. PKC- or - was immunoprecipitated
from mock-transfected ( ) or 0.5 µM PKC- , - , or
- AS oligonucleotide-transfected cell lysate. The in
vitro PKC- or - activity was measured by phosphorylation of
the 21-kDa myelin basic protein (MBP), and
32P-MBP was measured after separation in 12% SDS-PAGE. MBP
and 32P-MBP signals were quantified by densitometer and
PhosphorImager analysis, respectively. Percent PKC activity over mock
transfection was calculated after normalization to the Coomassie
Blue-stained MBP from each lane.
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To measure whether blocking of PKC-
and -
synthesis through AS
oligonucleotides will also affect PMA induction of VEGF mRNA stability, we transfected U373 cells with AS oligonucleotides and
analyzed the PMA induction of VEGF mRNA half-life. As expected, a
prolonged VEGF mRNA half-life of 3 h was demonstrated with
PMA-treated/mock-transfected U373 cells (Fig.
9). In contrast, PKC-
AS
oligonucleotide-transfected cells completely prevented the PMA
induction of VEGF mRNA stability with an observed VEGF mRNA
half-life of 0.7 h, which is similar to control VEGF mRNA
half-life without PMA treatment (see Fig. 5). Transfection with PKC-
AS oligonucleotide also showed strong suppression of PMA-induced VEGF
mRNA stability (data not shown). Taken together, these results
suggest that repression of PKC-
or -
protein synthesis through AS
oligonucleotide transfection can effectively block PMA induction of
VEGF mRNA stability.

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Fig. 9.
PKC- antisense
oligonucleotide blocks PMA-induced VEGF mRNA stability. U373
cells were either mock-transfected or transfected with 0.5 µM PKC- AS-oligonucleotide. The cells were then
treated with PMA for 3 h prior to addition of actinomycin D (5 µg/ml) for the indicated times. Total mRNA was then extracted,
and VEGF mRNA level was detected by Northern blot. VEGF mRNA
signal was normalized with the ribosome-associated mRNA, 36B4, from
each lane. VEGF mRNA signal with 3 h of PMA treatment was
defined as 100% and calculated % VEGF mRNA remaining after
actinomycin D treatment.
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PKC-
Overexpression Up-regulates VEGF mRNA
Expression--
To determine whether PKC-
itself can up-regulate
VEGF mRNA expression, we overexpressed PKC-
in U373 cells using
a cytolomegalovirus enhancer driven FLAG-epitope-tagged PKC-
expression vector (18). To ensure that the FLAG-PKC-
protein was
being expressed after transfection, we used Western blot to detect
cytoplasmic PKC-
protein levels. As shown in Fig.
10A, transfection with the
FLAG-PKC-
vector resulted in the detection of a higher molecular
weight PKC-
protein than the standard PKC-
, and a substantial
increase in FLAG-PKC-
protein when compared with the control vector.
To evaluate PKC-
activity after FLAG-PKC-
transfection, we
employed an in vitro kinase assay (Fig. 10B). As
expected, PKC-
activity correlated to FLAG-PKC-
expression when
compared with the control cells (2.9-fold increase). In Northern blot
analysis, the PKC-
overexpressing cells demonstrated very high
levels of VEGF mRNA expression without requiring PMA activation
(Fig. 10C). This result further provides evidence that
PKC-
is a major mediator of VEGF mRNA expression in U373 cells.
To address whether overexpression of PKC-
was affecting VEGF
mRNA transcription, we performed co-transfection of the PKC-
vector with the VEGF promoter-luciferase reporter construct. We
observed no transcriptional up-regulation of luciferase activity from 4 to 36 h after transfection (data not shown). Thus, the high level
of steady-state VEGF mRNA expression produced in PKC-
-expressing
cells was not mediated through transcriptional mechanisms.

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Fig. 10.
PKC- overexpression
up-regulates VEGF mRNA expression. U373 cells were either
transiently transfected with empty vector alone ( ) or with
FLAG-PKC- overexpression vector ( ). A, Western blot
detection of PKC- protein expression level in transfected cells.
B, PKC- activity in empty vector ( ) and PKC-
overexpression in vector-transfected cells. C, Northern blot
detection of VEGF mRNA expression in transfected cells.
M indicates 22-kDa protein marker. S indicates
standard PKC- or  from Jurkat cell lysate.
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DISCUSSION |
The tumor promoter, phorbol 12-myristate 13-acetate
(PMA), has been shown to induce angiogenesis in mouse skin
in vivo (23) and up-regulates VEGF expression in many cell
types in vitro (2, 6-8). In this study, we show that
induction of VEGF expression by PMA treatment and PKC activation in
human U373 glioblastoma cells is mediated through mRNA
stabilization as opposed to transcriptional activation. Antisense
oligonucleotide transfection and PKC-
overexpression studies
directly demonstrated that both PKC-
and -
are involved in the
regulation of VEGF mRNA stability. Given the fact that most cells
respond to hypoxic stimuli by activating both transcriptional and
post-transcriptional mechanisms to increase VEGF mRNA expression, this study identifies for the first time that the PKC pathway is a
direct activator of VEGF mRNA stabilization independent of transcriptional events.
Diacylglycerol (DAG) binding to a specific site on cPKC and nPKC
isoforms induces translocation of PKCs from the cytosol to the cellular
membrane, where the enzyme interacts with membrane phospholipids that
are required for kinase activity (4, 5). As DAG analogues, phorbol
esters are potent activators of cPKC and nPKC isoforms (4, 5). However,
chronic treatment with PMA results in the down-regulation of cytosolic
and membrane levels of cPKCs and nPKCs (4, 21). On the other hand,
aPKCs are not directly activated or depleted by DAG or phorbol esters
(21, 24, 25) but can be activated by other lipid intracellular mediators (26-28). There is evidence that PKC-
is directly
activated by ceramide in vivo by treating NIH-3T3 cells with
sphingomyelinase C (26). In addition, phosphatidylcholine-derived DAG
activates sphingomyelinase C (27). These studies indicate that although DAG is not able to activate PKC-
directly, production of ceramide through sphingomyelinase activation will ultimately affect PKC-
activity. This indirect activation of PKC-
by DAG is further supported by studies that demonstrate that
phosphatidylcholine-phospholipase C-derived DAG can activate the
mitogen-activated protein kinase pathway through PKC-
but not
phorbol ester-sensitive PKCs (28). Furthermore, PMA-mediated activation
of cPKCs and nPKCs leads to phospholipase A2 activation which, by
releasing arachidonic acid from phospholipids, can also activate
PKC-
(29). Taken together, these studies indicate that although PMA
and DAG do not activate PKC-
directly, the lipid second messengers
produced in PMA/DAG downstream pathways will likely play an important
role in PKC-
activation which in turn involves post-transcriptional regulation of VEGF mRNA expression.
Depending on PMA dose and treatment time, PKC isoforms, including
aPKCs, have shown variable translocation rates to the plasma membrane
compartment (29, 30). Recent studies showed that phosphatidylinositol
3-kinase is required for PMA-induced AP-1 activation and cell
transformation in JB6 cells (31), and PMA stimulates PIP3
production in 3T3-L1 adipocytes (32). Accordingly, PIP3, a
phosphorylation product of phosphatidylinositol 3-kinase, potently and
selectively activates PKC-
(33). Thus, it is possible that
PMA-induced PIP3 will function to recruit PKC-
to the
cell membrane and activate it. These observations support the view that
PMA is modulating aPKCs through lipid second messengers.
Studies have shown that chronic exposure to PMA (24 h), which depletes
PKC activity, does not reduce the hypoxia-induced VEGF expression in
astrocytes (6). These data provide supporting evidence that VEGF
induction can proceed through cPKC- and nPKC-independent pathways. Our
data also support this hypothesis since strong and persistent induction
of VEGF mRNA expression (4-8 h PMA treatment) was observed despite
PMA reduction of both cPKC and nPKC proteins, thus implicating a role
for the aPKCs in VEGF mRNA expression. Furthermore, chemical
inhibitors selective to cPKC and nPKC isoforms did not abolish VEGF
mRNA expression, and overexpression of PKC-
itself strongly
up-regulated steady-state VEGF mRNA levels. Combined, these studies
indicate that aPKCs, particularly PKC-
, are critical mediators
involved in VEGF post-transcriptional regulation. Since VEGF mRNA
stabilization has been demonstrated to be one of the key mechanisms
required for maximal induction of VEGF mRNA in both hypoxic and
hypoglycemic conditions, it is interesting to postulate that activation
of PKCs, especially PKC-
, may be occurring under these conditions.
Hypoxic and/or metabolic stress may adversely affect cell membranes
resulting in the release of lipid by-products which in turn trigger
PKC-
activation and ultimately increase VEGF mRNA stability
under these conditions. Since VEGF mRNA stabilization occurs after
several hours of hypoxic stress (3), involvement of lipid by-products
and PKC-
activation are even more likely, an interesting possibility
we are currently investigating.
Recent studies showed that overexpression of PKC-
potentiated
PMA-induced PKC activation and suggested a cross-talk between different
PKCs (22). Our studies of AS oligonucleotides also suggested that PKCs
may cross-regulate their expression and activity through cross-talk. We
showed that suppression of PKC-
synthesis by AS oligonucleotides to
PKC-
also affected PKC-
protein level, and vice versa PKC-
synthesis was affected by PKC-
AS oligonucleotides. Thus, it is
possible that PKC protein synthesis and total PKC activity are under
strict control in vivo, and blocking of one PKC isoform
synthesis might negatively regulate other PKCs, especially between
PKC-
and -
isoforms, an interesting hypothesis that is currently
under investigation. This cross-talk hypothesis also helps to explain
why PKC-
protein levels started to decrease after 8 h of PMA
treatment in U373 cells (data not shown), a time when PKC-
is
decreased to very low levels and VEGF mRNA expression is no longer maintained.
The identification of PKC isoforms as specific modulators of VEGF
mRNA stabilization will allow extended investigations into the
downstream molecular events that control mRNAbinding proteins or alterations in the mRNA degradation pathways. Thus, defining PKCs as modulators of VEGF mRNA stabilization is a significant advance in understanding the role of signaling pathways that control mRNA metabolism, especially that which potentiates the survival of
cells exposed to metabolic stresses or prolonged cytokine activation. Given the data presented here, human tumor cells that are metabolically stressed likely impact the expression of VEGF through intracellular activation of the PKC proteins and PKC-
and -
, in particular, which function to increase VEGF mRNA stabilization. Thus,
alterations in lipid by-products and PKC-
activation is likely an
additive mechanism that, when combined with transcriptional increases
induced by hypoxia, results in prolonged VEGF expression in tumor cells under stress. These activation pathways will likely proceed until the
metabolic stress is relieved by increased vascular permeability and/or
neovascularization, or, alternatively, the cells do not survive; thus
representing a critical balance in tumor cell survival and progression.