From the Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0575
Received for publication, August 15, 2002, and in revised form, January 23, 2003
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ABSTRACT |
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Vascular endothelial growth factor
(VEGF) stimulates angiogenesis during development and in disease.
In pheochromocytoma (PC12) cells, VEGF expression is regulated by
A2A adenosine receptor (A2AAR)
activation. The present work examines the underlying signaling pathway.
The adenylyl cyclase-protein kinase A cascade has no role in the
down-regulation of VEGF mRNA induced by the A2AAR agonist,
2-[4-[(2-carboxyethyl)phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680). Conversely, 6-h exposure of cells to either phorbol 12-myristate 13-acetate (PMA) or protein kinase C (PKC) inhibitors mimicked the CGS21680-induced down-regulation. PMA activated PKC Vascular endothelial growth factor
(VEGF)1 was described
initially as a vascular permeability factor (1), and was
characterized subsequently as an endothelial cell mitogen (2). VEGF is
involved primarily in angiogenesis, the pruning and reorganization of
pre-existing vasculature to create new vasculature, and it has a
critical role in embryonic development (3). In the adult, VEGF is
required for the development and maintenance of the female reproductive cycle (4) and may be cardioprotective during ischemia (5). However,
elevated levels of VEGF have been associated with pathologies, such as
diabetic retinopathy, endometriosis, rheumatoid arthritis, and
tumorigenesis (3, 6-8). Many tumors demonstrate elevated levels of
VEGF, which can be correlated to disease progression (9-12). This
correlation reflects the requirement of an expanding vasculature for
tumor growth, and disruption of VEGF signaling retards cancer
progression (13-15).
Several factors, including hypoxia (16-19), various growth factors
(20-22), and oncogenic mutations (23-25), up-regulate VEGF and the
underlying mechanisms have been extensively examined. Less is known
about the factors that down-regulate VEGF: natriuretic peptides (26),
N-acetylcysteine (27), somatostatin (28), and certain
anti-inflammatory drugs (29, 30). The pathways mediating the
down-regulation of VEGF have not been elucidated.
Rat pheochromocytoma (PC12) cells are a frequently employed model for
hypoxia-initiated responses and have been used to study VEGF gene
regulation, as hypoxia is a potent stimulant of VEGF expression
(16-19). Additionally, PC12 cells express A2A and
A2B adenosine receptors (AR) (31) and have been employed to
study AR signal transduction and physiologic activity. This laboratory has shown previously that activation of the A2AAR in PC12
cells results in a substantial reduction of VEGF, which is observed at
both the mRNA and protein levels (32). Furthermore, this down-regulation of VEGF mRNA occurs because of an inhibition of VEGF gene transcription (32). The nonselective AR agonist,
5'-(N-ethylcarboxamido)adenosine, was also reported to
down-regulate VEGF expression in PC12 cells (33). Other cell types have
been shown to respond to AR agonists with either increases or decreases
in VEGF expression (34-36). This differential regulation may exist
because of the subtype specificity of various AR ligands, and because
of cell-specific variations in the signal transduction cascade to which
a distinct AR subtype may be linked.
The A2AAR is typically coupled via the Gs
protein to the stimulation of adenylyl cyclase (AC) and activation of
protein kinase A (PKA) (31, 37, 38). However, certain effects mediated by the A2AAR have been linked to protein kinase C (PKC)
activation (39-42). Based on their requirements for activation, three
PKC classes are defined: conventional ( The goal of the present study was to elucidate the signal transduction
cascade responsible for the down-regulation of VEGF mRNA that is
induced by
2-[4-[(2-carboxyethyl)phenyl]-ethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680), a selective agonist for the A2AAR. Our results
indicate that stimulation of PKC activity by either CGS21680 or phorbol 12-myristate 13-acetate (PMA) produces an initial up-regulation of VEGF
mRNA that is rapidly followed by a marked reduction in VEGF
expression. The latter response appears to result from a decrease in
PKC activity with specifically PKC Cell Culture--
Rat pheochromocytoma (PC12) cells were grown
in complete RPMI medium (RPMI medium 1640 supplemented with 10% fetal
bovine serum, 10% equine serum, 1× penicillin-streptomycin-glutamine, and 0.25 µg/ml fungizone), and were maintained in a 5%
CO2-humidified incubator at 37 °C as previously
described (32). Cells were subcultured into collagen-coated six-well
dishes, 100-mm dishes, or T-75 flasks for experiments 24 h prior
to treatment. Culture medium was replaced with fresh complete RPMI
medium or with RPMI 1640 medium, when noted, 30-60 min prior to
treatment. The times of treatment with various agonists and inhibitors
are provided under "Results." Control cells were treated with
appropriate volumes of dimethyl sulfoxide when appropriate.
Radioimmunoassay of cAMP--
Intracellular cAMP levels in PC12
cells were determined with a cAMP 125I-Radioimmunoassay Kit
(PerkinElmer Life Sciences). Cells in six-well dishes were
treated for the indicated amount of time and washed twice with
phosphate-buffered saline (PBS) (1.36 M NaCl, 27 mM KCl, 80.5 mM
Na2HPO4, 14.7 mM
KH2PO4), scraped, and suspended in 1 ml of
EtOH. A 250-µl aliquot of the lysate was then dried at 60 °C for
3 h in a SpeedVac (Savant), and lysates were resuspended in 250 µl of sterile H2O. These solutions were diluted 1:50 in assay buffer, and 100-µl aliquots of the diluted solutions were used
for the assay in duplicate. Samples were incubated overnight with
125I-labeled cAMP and antiserum complex prior to cAMP
precipitation and centrifugation the following day. Gamma counts of the
precipitated cAMP pellets were determined, and intracellular cAMP
concentrations determined from a set of freshly prepared standards as
instructed by the manufacturer.
Northern Blot Analysis--
Northern blot analysis was performed
to determine VEGF mRNA content as previously described (32), with
minor modifications. Total RNA was isolated from PC12 cells with TRIzol
reagent (Invitrogen) and RNA samples were run on 1% agarose
gels containing 2.2 M formaldehyde. RNA was then
transferred to Zeta-Probe nylon membranes (Bio-Rad) and UV cross-linked
with a Strata-linker (Stratagene, La Jolla, CA) prior to
prehybridization at 42 °C for 2-5 h. Hybridization was conducted
overnight with a 600-bp fragment of murine VEGF165 cDNA
random prime-labeled with [32P]dCTP. The membrane was
sequentially washed and subjected to autoradiography. To normalize
total RNA levels, membranes were additionally hybridized with a
1,100-bp fragment of human glyceraldehyde-3-phosphate dehydrogenase
(Clontech, Palo Alto, CA) random prime labeled with [32P]dCTP. Autoradiographic signals were quantitated by
an AlphaImager 2000 (Alpha Innotech Corp.).
Western Blot Analysis--
Total protein from PC12 cells was
isolated and analyzed to determine PKC protein levels. Cells were
treated for the indicated amounts of time and then scraped in 250 µl
of lysis buffer (125 mM Tris-HCl, pH 6.8, 2% SDS, 5%
glycerol, 0.1 M dithiothreitol), boiled for 5 min, and
microcentrifuged at 13,000 × g for 15 min at 4 °C.
Total protein concentration of the resulting supernatant was determined
with Bio-Rad Protein Assay, and equal amounts of protein were
electrophoresed on 8% polyacrylamide gels. Protein was then
transferred to nitrocellulose membranes and blocked for 1 h with
Blotto (5% nonfat dry milk, 0.2% Triton X-100, 0.05% thiomerosal, in
PBS), prior to being incubated overnight at 4 °C with the
appropriate primary antibody at a 1:1000 dilution in Blotto. The
following antibodies were employed: cPKC Nuclear, Cytosolic, and Membrane PKC Analysis--
PC12 cells
were treated with the appropriate agonists for 5 min. After treatment,
subcellular fractions were isolated as previously described (49), with
minor modifications. Briefly, cells from 100-mm dishes were scraped in
100 µl of extraction buffer (20 mM Tris, pH 7.6, 2 mM EDTA, 5 mM EGTA, 10 mM
Adenoviral Vector Expression of PKC Data Analysis--
All experiments were performed a minimum of
three times, and in duplicate when noted. Results are expressed as
mean ± S.E. Statistical analysis was performed by one-way
analysis of variance followed by a Newman-Keuls post-test. A
p value < 0.05 was considered significant.
Reagents--
The following compounds were purchased from
Calbiochem (La Jolla, CA): forskolin,
N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89), protein kinase A inhibitor 14-22 amide (PKI), PMA, MG-132, bisindolylmaleimide IX (Ro-31-8220), and bisindolylmaleimide I (GFX).
Epidermal growth factor (EGF) and all cell culture reagents were
obtained from Invitrogen. The following compounds were purchased from
Sigma: 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), CGS21680, and phorbol 12, 13-diacetate (PDA).
Examination of the Involvement of PKA in the
A2AAR-mediated Down-regulation of VEGF--
As stimulation
of the A2AAR is linked to AC activation (31, 37, 38), the
signaling pathway mediating the down-regulation of VEGF mRNA
induced by CGS21680, a selective agonist for the A2AAR, was
initially explored using activators and inhibitors of PKA (Fig.
1A). As previously described
(32), treatment of PC12 cells for 6 h with 1 µM
CGS21680 reduced the VEGF mRNA level to 27.0 ± 8.1% of that
in control cells. Forskolin (5 µM), an activator of AC
and 8-Br-cAMP (1 mM), a cell-permeable cAMP analogue, did
not alter VEGF mRNA levels (Fig. 1A). The PKA
inhibitors, H-89 (5 µM) and PKI (10 µM),
had no effect on VEGF mRNA and were unable to reverse the
CGS21680-induced down-regulation of VEGF mRNA (Fig. 1A).
To further examine the role of cAMP in CGS21680-induced down-regulation
of VEGF mRNA, whole cell accumulation of cAMP was assessed in
response to forskolin (5 µM) or CGS21680 (1 µM) (Fig. 1B). Radioimmunoassay of cAMP
content showed that forskolin elicited a greater increase in cAMP
levels than CGS21680 at all time points examined during a 6-h time
course. As forskolin increased cAMP levels significantly more than
CGS21680, but had no effect on VEGF mRNA levels, the data strongly
suggest that cAMP is not involved in the A2AAR-mediated
down-regulation of VEGF. Overall, the sum of these data indicates that
pathways involving cAMP and/or PKA are not involved in the observed
down-regulation of VEGF following the stimulation of the
A2AAR in PC12 cells.
PKC Activation in Response to Stimulation of the
A2AAR--
As activation of the A2AAR has been
linked to PKC stimulation in certain systems (39-41), including PC12
cells (42), the role of PKC in the regulation of VEGF mRNA was
explored. For the initial analysis, cells were serum-starved for
14 h prior to a 6-h agonist treatment, as growth factors found in
serum can stimulate PKC activity and/or up-regulate VEGF expression
(21, 32). Under serum-free conditions, CGS21680 (1 µM),
PMA (100 nM), and EGF (10 ng/ml), a growth factor known to
activate PKC (21, 53), similarly regulated VEGF mRNA in a biphasic
manner over a 6-h time period (Fig.
2A). At 1 h of treatment,
all three compounds induced an initial increase in VEGF mRNA
levels, followed by a down-regulation of VEGF mRNA by 6 h. The
similarity of the PMA-, EGF-, and CGS21680-induced responses indicated
that PKC may be involved in the A2AAR-mediated regulation
of VEGF. To explore this possibility, the up-regulation of VEGF
mRNA induced at 1 h by CGS21680 and PMA was examined for
sensitivity to chemical inhibitors of PKC. As shown in Fig.
2B, administration of GFX (5 µM) and
Ro-31-8220 (5 µM) blocked the increase in VEGF expression that was induced by CGS21680 and PMA. Under these conditions, the PKC
inhibitors alone had no effect on VEGF mRNA.
Based on the above findings, the ability of CGS21680 and PMA to
activate various PKC isoforms was assessed by examining the translocation of PKC isoforms in the nuclear, cytosolic, and membrane fractions as translocation to the membrane or the nucleus is an indicator of enzyme activation (54-56). PKC Role of PKC Inhibition in the Down-regulation of VEGF
mRNA--
To further explore the role of PKC activity in the
down-regulation of VEGF, modulation of VEGF mRNA expression by a
6-h treatment with CGS21680 (1 µM) or PMA (100 nM) was examined for sensitivity to GFX (5 µM) and Ro-31-8220 (5 µM). These
experiments were conducted in cells maintained in complete growth
medium. As shown in Fig. 4A,
the CGS21680- or PMA-induced down-regulation of VEGF mRNA was
unaltered in the presence of GFX and Ro-31-8220. Interestingly, however, when these PKC inhibitors were administered alone, GFX and
Ro-31-8220 lowered VEGF mRNA to 44.8 ± 8.7% and 40.7 ± 7.8% of control, respectively. To characterize this decrease in VEGF mRNA elicited by GFX and Ro-31-8220, time-course studies for these PKC inhibitors were performed. As shown in Fig. 4B, both
compounds significantly reduced VEGF mRNA levels at 30 min of
treatment (to 55.0 ± 17.2% by Ro-31-8220 and to 48.3 ± 0.7% by GFX) and this down-regulation remained throughout 6 h.
The above data suggest that prolonged exposure of PC12 cells to
CGS21680 or PMA may decrease VEGF mRNA content via an inhibition of
PKC activity that may result from a down-regulation of PKC levels. Such
a regulation of novel and conventional PKC isoforms is well documented
in cells exposed for extended periods of time to PMA (57, 58). To
examine this possibility, PC12 cells were treated with CGS21680 (1 µM) or PMA (100 nM) for 6 h and whole cell lysates were analyzed for PKC content (Fig.
5). Relative to control cells, PMA
reduced PKC Identification of PKC Isoforms Involved in the CGS21680- and
PMA-induced Down-regulation of VEGF mRNA--
To further define a
role for PKC and to identify the specific PKC isoform(s) that may be
involved in the CGS21680- or PMA-induced down-regulation of VEGF
mRNA, PC12 cells were treated with 1 µM PMA for
24 h to remove conventional and novel PKCs. Cells were then
treated with cobalt chloride (50 µM) with or without
CGS21680 (1 µM) or PMA (100 nM) for 6 h.
Application of cobalt chloride mimics hypoxia and has been demonstrated
to elevate VEGF mRNA expression (16). As shown in Fig.
6, in control cells and cells treated for
24 h with PMA, cobalt chloride induced VEGF expression by 3.1 ± 0.5- and 4.9 ± 1.3-fold, respectively. In control cells, this
cobalt chloride-induced up-regulation of VEGF mRNA was inhibited by
71.0 ± 9.5% by CGS21680 and 124.9 ± 13.8% by PMA,
i.e. PMA reduced VEGF mRNA to a level lower than that
observed in cells not treated with cobalt chloride. In cells treated
for 24 h with PMA, CGS21680 similarly produced a 66.7 ± 7.5% inhibition of the cobalt chloride-induced response. Conversely,
the ability of PMA to block the cobalt chloride-induced up-regulation
of VEGF mRNA was nearly abolished, with an observed inhibition of
20.3 ± 9.1%. These data indicate that CGS21680-induced
down-regulation of VEGF mRNA is not dependent on conventional
and/or novel PKC isoforms, but that it may depend on PKC Use of a Proteasomal Inhibitor and a Selective Phorbol Ester to
Identify the Specific PKC Isoform That Is Involved in the PMA-induced
Response--
The present findings suggest that the PMA-induced
reduction in VEGF mRNA occurs as a result of the ability of this
phorbol ester to down-regulate susceptible PKC isoforms. The
down-regulation of PKC isoforms frequently results from targeting of
activated PKCs for degradation through proteasome pathways (58-60).
Thus, PMA- and CGS21680-induced down-regulation of VEGF mRNA was
analyzed for sensitivity to MG-132, a chemical inhibitor of proteasomal degradative activity. As shown in Fig. 7,
treatment with MG-132 (500 µM) alone increased VEGF
mRNA levels to 215.0 ± 29.0% of control. It may be
speculated that MG-132 inhibits the degradation of HIF-1
To further support the role of PKC down-regulation as the mechanism by
which PMA reduces VEGF mRNA, PDA was explored for its ability to
regulate PKC isoform expression and VEGF mRNA levels. PDA has been
reported to activate but not promote the degradation of PKC Application of Adenoviral Vectors Expressing Either Wild-type or
Dominant Negative PKC Research examining the signal transduction cascade linking
A2AAR activation to physiologic responses has typically
demonstrated a critical role for the AC-PKA pathway (31, 37, 38).
Therefore, the initial focus of this study was the role of AC and PKA
in the A2AAR-mediated down-regulation of VEGF mRNA.
Forskolin and 8-Br-cAMP did not decrease VEGF mRNA, and PKA
inhibitors did not modulate the CGS21680-induced down-regulation of
VEGF. As there is evidence for PKA-independent, but
cAMP-dependent events in signal transduction (63), changes
in cellular cAMP were evaluated. Forskolin elevated cAMP levels to a
greater degree than CGS21680 throughout a 6-h time course, indicating
that CGS21680-induced VEGF down-regulation is not
cAMP-dependent. Thus, multiple results strongly imply that
AC and PKA are not involved in the A2AAR-mediated down-regulation of VEGF mRNA.
Subsequent focus was placed on the PKC pathway, as there have been
reports linking A2AAR stimulation to PKC activation
(39-42). Under serum-free conditions, CGS21680, PMA, and EGF initially up-regulated VEGF mRNA, and this was followed by a down-regulation at 6 h. The ability of two well described activators of PKC, PMA and EGF, to mimic the CGS21680-induced response suggested that the
A2AAR may be linked to PKC stimulation. Indeed, the
up-regulation of VEGF mRNA produced by CGS21680, PMA, and EGF was
blocked by two different chemical inhibitors of PKC. Moreover, CGS21680
induced the translocation of PKC The findings with the PKC inhibitors suggested that down-regulation of
VEGF mRNA in response to CGS21680 and PMA over a prolonged period
may occur as a result of a decrease in PKC activity, and several
approaches were taken to examine this possibility. These studies
demonstrated the differential regulation and roles of distinct PKC
isoforms in the CGS21680- and PMA-induced responses. Treatment of PC12
cells with PMA for 6 h significantly decreased PKC The proteasome inhibitor MG-132 was employed to determine whether PKC
degradation was central to the mechanism by which PMA down-regulates
VEGF mRNA. MG-132 blocked PMA-induced down-regulation of VEGF
mRNA, but had no effect on the CGS21680-induced response. PDA, a
phorbol ester reported not to promote the degradation of PKC To further explore the putative role of PKC Our findings implicating PKC Our findings also raise questions regarding the mechanisms of
differential feedback regulation of PKC isoforms. It is clear that PMA
ultimately promotes the proteasomal degradation of PKC, PKC
, and PKC
, and CGS21680 activated PKC
and PKC
as
assessed by cellular translocation. By 6 h, PMA but not CGS21680
decreased PKC
and PKC
expression. Neither compound affected
PKC
levels. Following prolonged PMA treatment to down-regulate
susceptible PKC isoforms, CGS21680 but not PMA inhibited the cobalt
chloride induction of VEGF mRNA. The proteasome inhibitor, MG-132,
abolished PMA- but not CGS21680-induced down-regulation of VEGF mRNA.
Phorbol 12,13-diacetate reduced VEGF mRNA levels while
down-regulating PKC
but not PKC
expression. In cells
expressing a dominant negative PKC
construct, CGS21680 was
unable to reduce VEGF mRNA. Together, the findings suggest that phorbol
ester-induced down-regulation of VEGF mRNA occurs as a result of
a reduction of PKC
activity, whereas that mediated by the
A2AAR occurs following deactivation of PKC
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
I,
II,
), which
are activated by Ca2+ and diacylglycerol (DAG); novel (
,
,
,
), which are Ca2+-independent; and atypical
(
,
,
), which are activated independently of Ca2+
or DAG. PC12 cells have been reported to express PKC isoforms
,
I,
II,
,
,
,
, and
(43-45). Two specific PKC
isoforms, PKC
and PKC
, have been demonstrated to regulate VEGF
expression. For example, increases in PKC
activity up-regulate VEGF
expression in glioblastoma U373 cells (46), and in HT1080 fibrosarcoma and 786-0 renal carcinoma cells (25). Activation of PKC
has also
been implicated in stretch-induced up-regulation of VEGF in retinal
capillary pericytes (47). In addition, it has been reported that
ischemic preconditioning induces translocation of PKC
to the nucleus
in cardiomyocytes, which causes up-regulation of VEGF expression
(48).
and PKC
mediating the
PMA- and CGS21680-induced response, respectively.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(C-20), nPKC
(C-15), and
aPKC
(C-20)-G (Santa Cruz Biotechnology, Santa Cruz, CA). Membranes
were washed three times for 5 min with Blotto and incubated with the
appropriate horseradish peroxidase-conjugated secondary antibody at
1:10,000 dilution for 1 h at room temperature. Membranes were then
washed three times for 5 min in Blotto and two times for 5 min with PBS
prior to being developed with ECL Western blotting detection reagents
(Amersham Biosciences) and being exposed to x-ray film. Signals were
analyzed with an AlphaImager 2000.
-mercaptoethanol (
-ME), and protease inhibitors including 0.1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 1 µg/ml pepstatin, and 0.5 µg/ml aprotinin). Cells were then Dounce
homogenized with 10 strokes, and lysates were microcentrifuged at
500 × g for 10 min. The supernatant (plasma membrane
and cytosolic fractions) was removed and centrifuged at 100,000 × g for 60 min, and the resulting pellet (plasma membrane) was
resuspended in 50 µl of suspension buffer (10 mM Tris, pH 7.6, 5 mM MgCl2, 5 mM
-ME, and
protease inhibitors as described above) by sonication. The supernatant
was collected as the cytosolic fraction. The pellet from the initial
500 × g spin (nuclear fraction) was resuspended in 100 µl of nuclei buffer (5 mM Tris, pH 7.6, 10.5 mM MgCl2, 10 mM
-ME, and
protease inhibitors described above) supplemented with 0.1% Triton
X-100 and then layered over 100 µl of nuclei buffer + 0.5 M sucrose and microcentrifuged for 10 min at 500 × g. The pellet (nuclear fraction) was resuspended in 25 µl
of suspension buffer by sonication. All fractions were then resuspended
in lysis buffer, boiled for 5 min, and microcentrifuged for 15 min at
13,000 × g prior to protein concentration
determination by Bio-Rad protein assay. Equal amounts of protein were
run on 8% polyacrylamide gels as described above. Nuclear sample
purity was demonstrated with the histone H1 (AE-4) antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
--
PC12 cells were
infected with previously characterized replication-deficient adenoviral
vectors containing either wild-type or kinase-deficient mutant
(dominant negative) forms of PKC
, or a vector containing
-galactosidase as a control (50, 51). Each recombinant adenovirus
was prepared as previously described (52). The vectors were expanded in
HEK293 cells, and the amount of plaque-forming units for each was
determined to infect at an equal multiplicity of infection (m.o.i.).
The wild-type PKC
adenoviral vector was infected at an m.o.i. of
600, whereas the dominant negative PKC
adenoviral vector was
infected at an m.o.i. of 800. PC12 cells were infected for 8 h in
4 ml of RPMI 1640 medium supplemented with 2% fetal bovine serum with
gentle shaking in a 5% CO2-humidified 37 °C cell
incubator. Complete RPMI medium was then added to the cells overnight,
and cells were either treated for 24 h with 1 µM PMA
prior to a 6-h treatment with the appropriate agonist in fresh complete
medium, or cells were directly given fresh complete RPMI medium and
treated for 6 h with the appropriate agonist. Cells were then
scraped in 1 ml of PBS, and a 150-µl aliquot was microcentrifuged at
13,000 × g, and the resulting pellet was lysed in 100 µl of lysis buffer. PKC protein levels were assessed by Western blot
as described above. The remaining cell suspension was spun at 200 × g, and the resulting pellet was used for RNA isolation.
VEGF mRNA levels were assessed by Northern blot analysis as
described above.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
CGS21680-induced down-regulation of VEGF
mRNA in PC12 cells is not mediated by the adenylyl cyclase-PKA
pathway. A, effect of elevated intracellular cAMP and
PKA inhibitors on VEGF mRNA in control and CGS21680-treated cells.
PC12 cells were treated for 6 h with 5 µM forskolin,
1 mM 8-Br-cAMP, 5 µM H-89, 10 µM PKI, or 1 µM CGS21680 (CGS),
or cells were pretreated for 1 h with a PKA inhibitor prior to
6 h of treatment with 1 µM CGS21680. Total RNA was
collected, and Northern blot analysis was performed. Significant
difference from control denoted by ** (p < 0.001). There was no significant difference between CGS21680-treated
cells and cells treated with PKA inhibitors in combination with
CGS21680 (p > 0.05). B, forskolin elevated
intracellular cAMP levels to a greater degree than CGS21680. PC12 cells
were treated with CGS21680 (1 µM) or forskolin (5 µM) for the indicated amount of time, and intracellular
cAMP levels were quantitated by radioimmunoassay. All experiments were
conducted in duplicate. Data are presented as percentage of cAMP in
untreated cells. Significant difference between CGS21680- and
forskolin-treated cells is denoted by * (p < 0.05), # (p < 0.01), or ** (p < 0.001).
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Fig. 2.
PKC regulation of VEGF expression in
serum-starved PC12 cells. A, biphasic regulation of
VEGF mRNA by CGS21680, PMA, and EGF. PC12 cells were serum-starved
for 14 h prior to treatment for the indicated times with CGS21680
(1 µM), PMA (100 nM), or EGF (10 ng/ml).
Total RNA was collected, and Northern blot analysis was performed.
Significant difference from control denoted by *
(p < 0.05), # (p < 0.01),
or ** (p < 0.001). B, PKC
inhibitors Ro-31-8220 and GFX blocked CGS21680- or PMA-induced
up-regulation of VEGF mRNA in serum-starved PC12 cells. PC12 cells
were serum-starved for 14 h prior to treatment with 1 µM CGS21680 (CGS) or 100 nM PMA, either alone
or in combination with 5 µM Ro-31-8220 or 5 µM GFX. Total RNA was collected, and Northern blot
analysis was performed. Significant difference from control is denoted
by * (p < 0.05) or # (p < 0.01). Significant difference between CGS21680 or PMA treatments and
CGS21680 or PMA treatments in combination with a PKC inhibitor is
denoted by + (p < 0.05).
, PKC
, and PKC
isoforms were studied, as each represents one of the three PKC classes: conventional, novel, and atypical, respectively. As shown in Fig. 3, PMA promoted the nuclear translocation
of PKC
, PKC
, and PKC
by 710.8 ± 181.8%, 520.5 ± 107.2%, and 397.7 ± 98.9%, respectively. CGS21680 induced the
nuclear translocation of PKC
and PKC
by 358.7 ± 86.3% and
416.0 ± 146.1%, respectively. This correlates with previous data
indicating activation of the PKC pathway following stimulation of the
A2AAR (39-42).
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Fig. 3.
PMA activated PKC ,
PKC
, and PKC
, whereas
CGS21680 activated PKC
and
PKC
in PC12 cells. Cells were treated for
5 min with either 1 µM CGS21680 (CGS) or 100 nM PMA and nuclear, cytosolic, and membrane protein
fractions were collected as described under "Experimental
Procedures." Protein fractions were analyzed by Western blot.
Significant difference from control is denoted by *
(p < 0.05) or ** (p < 0.001).
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Fig. 4.
PKC inhibitors Ro-31-8220 and GFX did not
reverse the CGS21680-induced down-regulation of VEGF mRNA, but
alone these inhibitors decreased basal VEGF mRNA levels in PC12
cells. A, cells were treated for 6 h with 1 µM CGS21680 (CGS), 100 nM PMA, 5 µM Ro-31-8220 (Ro), or 5 µM GFX;
or cells were treated simultaneously with 5 µM Ro-31-8220
or 5 µM GFX and 1 µM CGS21680 or 100 nM PMA for 6 h. Total RNA was collected, and Northern
blot analysis was performed. Significant difference from control is
denoted by * (p < 0.001). There is no
significant difference between CGS21680- or PMA-treated cells and cells
treated with PKC inhibitors in combination with CGS21680 or PMA
(p > 0.05). B, Ro-31-8220 and GFX promoted
a rapid and sustained down-regulation of VEGF mRNA in PC12 cells.
Cells were treated for the indicated time with either Ro-31-8220 (5 µM) or GFX (5 µM). Total RNA was collected,
and Northern blot analysis was performed. Significant difference from
control is denoted by * (p < 0.05),
# (p < 0.01), or **
(p < 0.001).
and PKC
expression by 55.7 ± 4.5% and
91.2 ± 1.7%, respectively. PMA did not significantly alter
PKC
levels. Treatment of PC12 cells with CGS21680 for 6 h
caused no significant change in the expression of any examined PKC
isoform.
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[in a new window]
Fig. 5.
Regulation of PKC isoform expression by 6-h
treatment of PC12 cells with either CGS21680 or PMA. Cells were
treated for 6 h with either 100 nM PMA or 1 µM CGS21680 (CGS). Total protein was collected
and analyzed by Western blot. Significant difference from control
denoted by * (p < 0.05) or **
(p < 0.001).
, as
CGS21680 can further down-regulate VEGF mRNA when PKC
remains
available.
View larger version (14K):
[in a new window]
Fig. 6.
CGS21680, but not PMA, inhibited cobalt
chloride-induced VEGF mRNA up-regulation in PC12 cells pre-treated
with PMA for 24 h. PC12 cells were treated with PMA (1 µM) for 24 h prior to a 6-h treatment with 50 µM cobalt chloride (Cobalt) alone or in
combination with either 1 µM CGS21680 (CGS) or
100 nM PMA. Control cells did not receive 24 h PMA
treatment. Total RNA was collected, and Northern blot analysis was
performed. For control group, significant difference from cobalt
chloride treatment is denoted by * (p < 0.05) or ** (p < 0.001); and for the 24-h
PMA group, significant difference from cobalt chloride treatment is
denoted by # (p < 0.01).
, a
transcription factor known to up-regulate VEGF mRNA and to be
subject to constitutive proteasomal degradation (61). MG-132 abolished
the ability of PMA to down-regulate VEGF mRNA while having no
effect on the reduction of VEGF mRNA elicited by CGS21680. To
confirm the ability of MG-132 to inhibit the PMA-induced down-regulation of PKC isoforms, whole cell lysates of PC12 cells were
analyzed for PKC expression by Western blotting following treatment
with PMA in the absence or presence of MG-132 (Fig. 7,
inset). The PMA-induced down-regulation of both PKC
and
PKC
was completely reversed by MG-132. MG-132 itself had no effect on PKC expression. These data indicate a possible role for PKC
and/or PKC
in PMA down-regulation of VEGF mRNA, while also
supporting the above described findings (Fig. 6) that indicate these
PKC isoforms are not involved in the mechanism by which CGS21680
down-regulates VEGF mRNA.
View larger version (27K):
[in a new window]
Fig. 7.
MG-132 blocked PMA-induced degradation of
PKC and PKC
and
inhibited PMA-induced, but not CGS21680-induced, down-regulation of
VEGF mRNA in PC12 cells. PC12 cells were treated with 1 µM CGS21680 (CGS), 100 nM PMA, or
500 nM MG-132, alone or in combination, for 6 h as
indicated. Total RNA was collected, and Northern blot analysis was
performed. Significant difference from control is denoted by *
(p < 0.05), whereas significant difference between
MG-132 and MG-132/CGS21680 is denoted by **
(p < 0.001). Inset, PC12 cells were treated
for 6 h with either 100 nM PMA or 500 nM
MG-132 alone or in combination as indicated. Total protein was
collected, and PKC isoform expression was analyzed by Western blot.
Significant difference from control denoted by # (p < 0.01) or ** (p < 0.001).
in rat
brain cortical slices (62). As demonstrated in Fig.
8, a 6-h treatment of PC12 cells with 10 µM PDA promoted a down-regulation of VEGF mRNA nearly
identical to that observed with 100 nM PMA. Although
producing an 85.2 ± 4.3% reduction in PKC
expression, 10 µM PDA had no significant effect on PKC
levels relative to untreated cells. As observed with PMA, PDA did not regulate
PKC
expression. The sum of these findings indicates a role for
PKC
in the phorbol ester-mediated down-regulation of VEGF
mRNA.
View larger version (19K):
[in a new window]
Fig. 8.
PDA promoted specific degradation of
PKC and down-regulated VEGF mRNA in PC12
cells. Cells were treated for 6 h with the indicated
concentration of PDA or 100 nM PMA. Total RNA was
collected, and Northern blot analysis was performed. Significant
difference from control denoted by * (p < 0.05).
Inset, in parallel, PC12 cells were treated under the same
conditions, total protein was collected, and expression of PKC isoforms
was analyzed by Western blot. Significant difference from control is
denoted by * (p < 0.05) or **
(p < 0.001).
to Alter the CGS21680-induced
Response--
To study the putative role of PKC
in constitutive
VEGF expression in PC12 cells and more specifically the
A2AAR-mediated down-regulation of VEGF mRNA, we
employed adenoviral vectors that directed expression of either
wild-type PKC
or a dominant negative form of PKC
(DNPKC
).
Control cells were infected at the same m.o.i. with a
-galactosidase
adenoviral construct. 24 h after infection, cells were treated
with 1 µM PMA for 24 h prior to treatment with
CGS21680. The addition of long term PMA allowed for the specific
analysis of PKC
activity, as novel and conventional PKC isoforms
were removed. As shown in Fig.
9A, 1 µM
CGS21680 reduced VEGF mRNA to 45.0 ± 12.1% of that observed
in untreated cells expressing
-galactosidase. In cells in which
there was a 3.9 ± 0.9-fold overexpression of wild-type PKC
(Fig. 9A, inset), CGS21680 was unable to induce a
significant decrease in VEGF mRNA. In a separate set of
experiments, cells were infected with the
-galactosidase or DNPKC
adenoviral constructs at the same m.o.i. (Fig. 9B). The
addition of CGS21680 to cells expressing the
-galactosidase construct reduced the VEGF mRNA level to 40.7 ± 6.4% of that
in untreated cells. In cells expressing DNPKC
(Fig. 9B,
inset), VEGF mRNA levels were 43.7 ± 1.3% of that
observed in
-galactosidase-expressing cells. However, 1 µM CGS21680 did not further down-regulate VEGF mRNA
in cells expressing DNPKC
.
View larger version (22K):
[in a new window]
Fig. 9.
Involvement of PKC in the
CGS21680-induced down-regulation of VEGF mRNA in PC12 cells.
A, overexpression of wild-type PKC
. Cells were infected
for 8 h with adenovirus directing either the expression of
-galactosidase or wild-type PKC
prior to exposure for 24 h
to 1 µM PMA. Cells were then treated for 6 h with 1 µM CGS21680 (CGS) as indicated. Total RNA was
collected, and Northern blot analysis was performed. Significant
difference from control is denoted by * (p < 0.05). Inset, protein samples from cells treated as
described above were analyzed for PKC
expression by Western blot.
Representative blot is shown. B, expression of DNPKC
.
Cells were infected for 8 h with adenovirus directing either the
expression of
-galactosidase or DNPKC
. After 24 h, cells
were treated with 1 µM CGS21680 (CGS) as
indicated. Total mRNA was collected, and Northern blot analysis was
performed. Significant difference from control is denoted by
** (p < 0.001). Inset, protein
samples from cells treated as described above were analyzed by Western
blot with PKC
antibody. Representative blot is shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and PKC
to the nucleus whereas
PMA directed nuclear translocation of PKC
, PKC
, and PKC
.
Recently, Huang and co-workers (42) demonstrated the rapid nuclear
translocation of PKC
following A2AAR activation in PC12
cells and associated this response with the anti-apoptotic effects of
CGS21680. It should be noted that, although insensitivity to direct
activation by DAG has served to define atypical PKC isoforms,
stimulation of PKC
by PMA in various cell types including PC12 cells
has been observed (64, 65). Several studies have demonstrated that
translocation of PKC to the nucleus is concurrent with activation (42,
44, 48, 55). This facet of PKC activity is particularly relevant to
VEGF mRNA regulation, as PKC-mediated activation of nuclear
transcription factors such as Sp-1 has a critical role in VEGF gene
transcription (66, 67). The importance of PKC activity in VEGF mRNA
regulation was further demonstrated, as GFX and Ro-31-8220 were found
to rapidly down-regulate VEGF mRNA in PC12 cells maintained in
complete medium. This suggests that VEGF mRNA is maintained at a
high constitutive level in PC12 cells most likely because of high basal
PKC activity resulting from growth factors present in the
serum-supplemented medium. Indeed, VEGF mRNA and protein expression
are markedly lower in serum-starved PC12 cells relative to cells
maintained in complete medium (32).
levels, and
PKC
expression was nearly abolished. It is well documented that long
term (
24 h) treatment with PMA down-regulates conventional and novel
PKC (59), but it was surprising to observe such a dramatic decrease at
6 h. In cells depleted of conventional and novel PKC isoforms by
prolonged exposure to PMA, CGS21680, but not PMA, maintained the
ability to inhibit the cobalt chloride-induced up-regulation of VEGF
mRNA. This finding strongly implies a role for conventional/novel
PKC isoforms in the PMA-induced VEGF down-regulation whereas PKC
may
mediate the response observed with A2AAR activation.
(62),
was administered to determine which PKC isoform may mediate the
PMA-induced down-regulation of VEGF mRNA. PDA promoted the
degradation of specifically PKC
, and in a consistent temporal fashion down-regulated VEGF mRNA. These findings indicate that PMA-induced down-regulation of VEGF mRNA occurs because of the degradation of specifically PKC
that follows the activation of this
PKC isoform.
in the
A2AAR-mediated down-regulation of VEGF mRNA,
replication-deficient adenoviruses containing either wild-type PKC
or a dominant negative PKC
construct were employed. In cells
overexpressing wild-type PKC
and treated for 24 h with PMA,
CGS21680 did not down-regulate VEGF mRNA. One possibility for this
lack of responsiveness is that the mechanism by which prolonged
stimulation of the A2AAR abrogates PKC
signaling is
overwhelmed in the presence of excess PKC
levels. We did not observe
up-regulation of VEGF mRNA levels upon overexpression of PKC
,
and this was similarly reported for retinal capillary pericytes (47).
Conversely, in other cell lines, an up-regulation of VEGF mRNA
occurs upon overexpression of PKC
(25, 46). It is possible that, in
PC12 cells, a relatively high basal activity of endogenous PKC
prevents any activity of experimentally introduced PKC
to be
apparent. When a dominant negative PKC
isoform was expressed, VEGF
mRNA levels were reduced relative to those in
-galactosidase-expressing cells, an observation in agreement with
the hypothesis regarding high constitutive activity of endogenous PKC
in PC12 cells. In these cells, CGS21680 did not down-regulate VEGF mRNA. Thus, the reduction in PKC
function induced via
expression of the dominant negative construct may result in the loss of
the signaling activity that is typically targeted for inhibition upon prolonged A2AAR stimulation.
and PKC
in VEGF regulation in PC12
cells are consistent with observations in other cell lines. For
example, Kawata et al. (48) found that the specific
translocation of PKC
to the nucleus of cardiomyocytes 10 min after
an ischemia/reperfusion protocol in rats up-regulated VEGF mRNA at
3 h. A PKC inhibitor blocked both the translocation of PKC
and
the up-regulation of VEGF mRNA. PKC
has also been shown to
regulate VEGF mRNA expression in several cell types. In both human
glioblastoma (46) and fibrosarcoma (25) cells, overexpression of PKC
resulted in constitutive up-regulation of VEGF mRNA. Similarly,
expression of a dominant negative PKC
has been shown to abrogate
VEGF up-regulation induced by various stimulants (25, 47). Particularly
intriguing is the present finding that the reduction, and not the
direct activation, of PKC signaling by a physiologically relevant
stimulus, adenosine, underlies the VEGF mRNA down-regulation. The
cellular effects occurring in response to acute PKC activation have
been extensively explored. However, there have been reports of PKC
deactivation/degradation upon prolonged agonist exposure as the
mechanism underlying cellular response. For example, in an examination
of the tumor-promoting effects of PMA in rat fibroblasts overexpressing
c-Src, Lu and co-workers (68) found that phenotypic transformation of
these cells corresponded temporally with phorbol ester-induced
depletion of PKC
. Similar to results we describe, inhibitors of PKC
and expression of a dominant negative PKC
also promoted phenotypic changes similar to those observed with prolonged PMA exposure (68).
Additionally, Shizukuda et al. (69) reported that the VEGF-induced migration and proliferation of endothelial cells was
mediated by a decrease in PKC
activity although PKC
expression remained unchanged. Overexpression of wild-type PKC
blocked the ability of VEGF to induce cell response (69), and this finding is
similar to our observation that CGS21680 did not regulate VEGF in cells
overexpressing PKC
. It is apparent that deactivation of PKCs, either
through reduction in kinase activity or protein expression, can be as
important or more important than their activation.
, but it is
not known how A2AAR activation apparently decreases PKC
activity without modifying protein levels. Indeed, relatively little is
known about the regulation of atypical PKC isoforms although it has
been reported that PKC
may be processed by ubiquitinylation followed
by proteasomal degradation (60). Additionally, of special interest in
light of the lack of CGS21680-induced PKC
degradation is the
prostate apoptosis response-4 (Par-4) gene product. Par-4 has been
shown to directly and specifically associate with PKC
with a
resulting dramatic reduction in PKC
kinase activity (70). Our PC12
cells express the Par-4 gene product (data not shown), and it is
currently being examined whether A2AAR activation modulates Par-4 expression or promotes its complex formation with PKC
.
![]() |
ACKNOWLEDGEMENTS |
---|
We are very grateful to Dr. Jeff Molkentin
(Children's Hospital Medical Center, Cincinnati, OH) for the generous
gift of the PKC adenoviral constructs. We thank Dr. Ron Millard
(University of Cincinnati, Cincinnati, OH) for advice with statistical
analysis. We very much appreciate the excellent technical assistance of John Meinken.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health NCI Grant R01 CA79531 (to M. E. O.).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.
Supported by National Institutes of Health Training Grant 5T32 HL07382.
§ To whom correspondence should be addressed. Tel.: 513-558-2361; Fax: 513-558-1169; E-mail: mark.olah@uc.edu.
Published, JBC Papers in Press, February 17, 2003, DOI 10.1074/jbc.M208366200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
VEGF, vascular
endothelial growth factor;
PC12, pheochromocytoma;
AR, adenosine
receptor;
AC, adenylyl cyclase;
PKA, protein kinase A;
PKC, protein
kinase C;
DAG, diacylglycerol;
CGS21680, 2-[4-[(2-carboxyethyl)phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine;
PMA, phorbol 12-myristate 13-acetate;
-ME,
-mercaptoethanol;
DNPKC
, dominant negative PKC
;
m.o.i., multiplicity of infection;
H-89, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide;
PKI, protein kinase A inhibitor 14-22 amide;
Ro-31-8220, bisindolylmaleimide IX;
GFX, bisindolylmaleimide I;
EGF, epidermal
growth factor;
8-Br-cAMP, 8-bromoadenosine 3',5'-cyclic monophosphate;
PDA, phorbol 12, 13-diacetate;
Par-4, prostate apoptosis response-4;
PBS, phosphate-buffered saline.
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REFERENCES |
---|
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---|
1. | Senger, D. R., Galli, S. J., Dvorak, A. M., Perruzzi, C. A., Harvey, V. S., and Dvorak, H. F. (1983) Science 219, 983-985[Medline] [Order article via Infotrieve] |
2. | Ferrara, N., and Henzel, W. J. (1989) Biochem. Biophys. Res. Commun 161, 851-858[Medline] [Order article via Infotrieve] |
3. |
Ferrara, N.,
and Davis-Smyth, T.
(1997)
Endocr. Rev.
18,
4-25 |
4. | Shweiki, D., Itin, A., Neufeld, G., Gitay-Goren, H., and Keshet, E. (1993) J. Clin. Invest. 91, 2235-2243[Medline] [Order article via Infotrieve] |
5. | Deindl, E., and Schaper, W. (1998) Mol. Cell Biochem 186, 43-51[CrossRef][Medline] [Order article via Infotrieve] |
6. | Wilkinson-Berka, J. L., Kelly, D. J., and Gilbert, R. E. (2001) J. Vasc. Res. 38, 527-535[CrossRef][Medline] [Order article via Infotrieve] |
7. | Folkman, J. (1995) Nat. Med. 1, 27-31[Medline] [Order article via Infotrieve] |
8. | Smith, S. K. (2001) Trends Endocrinol. Metab. 12, 147-151[CrossRef][Medline] [Order article via Infotrieve] |
9. | Volm, M., Koomagi, R., Mattern, J., and Stammler, G. (1997) Anticancer Res. 17, 99-103[Medline] [Order article via Infotrieve] |
10. | Brown, L. F., Berse, B., Jackman, R. W., Tognazzi, K., Guidi, A. J., Dvorak, H. F., Senger, D. R., Connolly, J. L., and Schnitt, S. J. (1995) Hum. Pathol. 26, 86-91[Medline] [Order article via Infotrieve] |
11. | Brown, L. F., Berse, B., Jackman, R. W., Tognazzi, K., Manseau, E. J., Dvorak, H. F., and Senger, D. R. (1993) Am. J. Pathol. 143, 1255-1262[Abstract] |
12. | Sowter, H. M., Corps, A. N., Evans, A. L., Clark, D. E., Charnock-Jones, D. S., and Smith, S. K. (1997) Lab. Invest. 77, 607-614[Medline] [Order article via Infotrieve] |
13. | Millauer, B., Shawver, L. K., Plate, K. H., Risau, W., and Ullrich, A. (1994) Nature 367, 576-579[CrossRef][Medline] [Order article via Infotrieve] |
14. | Warren, R. S., Yuan, H., Matli, M. R., Gillett, N. A., and Ferrara, N. (1995) J. Clin. Invest. 95, 1789-1797[Medline] [Order article via Infotrieve] |
15. |
Cheng, S. Y.,
Huang, H. J.,
Nagane, M.,
Ji, X. D.,
Wang, D.,
Shih, C. C.,
Arap, W.,
Huang, C. M.,
and Cavenee, W. K.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
8502-8507 |
16. | Minchenko, A., Bauer, T., Salceda, S., and Caro, J. (1994) Lab. Invest. 71, 374-379[Medline] [Order article via Infotrieve] |
17. | Shweiki, D., Itin, A., Soffer, D., and Keshet, E. (1992) Nature 359, 843-845[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Levy, A. P.,
Levy, N. S.,
Loscalzo, J.,
Calderone, A.,
Takahashi, N.,
Yeo, K. T.,
Koren, G.,
Colucci, W. S.,
and Goldberg, M. A.
(1995)
Circ. Res.
76,
758-766 |
19. |
Levy, A. P.,
Levy, N. S.,
Wegner, S.,
and Goldberg, M. A.
(1995)
J. Biol. Chem.
270,
13333-13340 |
20. | Goldman, C. K., Kim, J., Wong, W. L., King, V., Brock, T., and Gillespie, G. Y. (1993) Mol. Biol. Cell 4, 121-133[Abstract] |
21. |
Heasley, L. E.,
and Johnson, G. L.
(1989)
J. Biol. Chem.
264,
8646-8652 |
22. | Lohrer, P., Gloddek, J., Hopfner, U., Losa, M., Uhl, E., Pagotto, U., Stalla, G. K., and Renner, U. (2001) Neuroendocrinology 74, 95-105[CrossRef][Medline] [Order article via Infotrieve] |
23. | Rak, J., Mitsuhashi, Y., Bayko, L., Filmus, J., Shirasawa, S., Sasazuki, T., and Kerbel, R. S. (1995) Cancer Res. 55, 4575-4580[Abstract] |
24. | Rak, J., Yu, J. L., Klement, G., and Kerbel, R. S. (2000) J. Invest. Dermatol. Symp. Proc. 5, 24-33[CrossRef] |
25. |
Pal, S.,
Datta, K.,
Khosravi-Far, R.,
and Mukhopadhyay, D.
(2001)
J. Biol. Chem.
276,
2395-2403 |
26. |
Pedram, A.,
Razandi, M.,
and Levin, E. R.
(2001)
Endocrinology
142,
1578-1586 |
27. | Redondo, P., Jimenez, E., Perez, A., and Garcia-Foncillas, J. (2000) Arch. Dermatol. Res. 292, 621-628[CrossRef][Medline] [Order article via Infotrieve] |
28. | Mentlein, R., Eichler, O., Forstreuter, F., and Held-Feindt, J. (2001) Int. J. Cancer 92, 545-550[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Nagashima, M.,
Wauke, K.,
Hirano, D.,
Ishigami, S.,
Aono, H.,
Takai, M.,
Sasano, M.,
and Yoshino, S.
(2000)
Rheumatology
39,
1255-1262 |
30. |
Gille, J.,
Reisinger, K.,
Westphal-Varghese, B.,
and Kaufmann, R.
(2001)
J. Invest. Dermatol.
117,
1581-1587 |
31. | Arslan, G., Kull, B., and Fredholm, B. B. (1999) Naunyn Schmiedebergs Arch. Pharmacol. 359, 28-32[Medline] [Order article via Infotrieve] |
32. |
Olah, M. E.,
and Roudabush, F. L.
(2000)
J. Pharmacol. Exp. Ther.
293,
779-787 |
33. |
Kobayashi, S.,
and Millhorn, D. E.
(1999)
J. Biol. Chem.
274,
20358-20365 |
34. |
Feoktistov, I.,
Goldstein, A. E.,
Ryzhov, S.,
Zeng, D.,
Belardinelli, L.,
Voyno-Yasenetskaya, T.,
and Biaggioni, I.
(2002)
Circ. Res.
90,
531-538 |
35. |
Grant, M. B.,
Tarnuzzer, R. W.,
Caballero, S.,
Ozeck, M. J.,
Davis, M. I.,
Spoerri, P. E.,
Feoktistov, I.,
Biaggioni, I.,
Shryock, J. C.,
and Belardinelli, L.
(1999)
Circ. Res.
85,
699-706 |
36. | Wakai, A., Wang, J. H., Winter, D. C., Street, J. T., O'Sullivan, R. G., and Redmond, H. P. (2001) Shock 15, 297-301[Medline] [Order article via Infotrieve] |
37. | Olah, M. E., and Stiles, G. L. (1992) Annu. Rev. Physiol. 54, 211-225[CrossRef][Medline] [Order article via Infotrieve] |
38. | Linden, J. (2001) Annu. Rev. Pharmacol. Toxicol. 41, 775-787[CrossRef][Medline] [Order article via Infotrieve] |
39. | Gubitz, A. K., Widdowson, L., Kurokawa, M., Kirkpatrick, K. A., and Richardson, P. J. (1996) J. Neurochem. 67, 374-381[Medline] [Order article via Infotrieve] |
40. | Cunha, R. A., and Ribeiro, J. A. (2000) Neurosci. Lett. 289, 127-130[CrossRef][Medline] [Order article via Infotrieve] |
41. | Cunha, R. A., and Ribeiro, J. A. (2000) Neuropharmacology 39, 1156-1167[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Huang, N. K.,
Lin, Y. W.,
Huang, C. L.,
Messing, R. O.,
and Chern, Y.
(2001)
J. Biol. Chem.
276,
13838-13846 |
43. | Ohmichi, M., Zhu, G., and Saltiel, A. R. (1993) Biochem. J. 295, 767-772[Medline] [Order article via Infotrieve] |
44. | Borgatti, P., Mazzoni, M., Carini, C., Neri, L. M., Marchisio, M., Bertolaso, L., Previati, M., Zauli, G., and Capitani, S. (1996) Exp. Cell Res. 224, 72-78[CrossRef][Medline] [Order article via Infotrieve] |
45. | Sparatore, B., Patrone, M., Passalacqua, M., Pedrazzi, M., Pontremoli, S., and Melloni, E. (2000) Biochem. Biophys. Res. Commun. 275, 149-153[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Shih, S. C.,
Mullen, A.,
Abrams, K.,
Mukhopadhyay, D.,
and Claffey, K. P.
(1999)
J. Biol. Chem.
274,
15407-15414 |
47. |
Suzuma, I.,
Suzuma, K.,
Ueki, K.,
Hata, Y.,
Feener, E. P.,
King, G. L.,
and Aiello, L. P.
(2002)
J. Biol. Chem.
277,
1047-1057 |
48. |
Kawata, H.,
Yoshida, K.,
Kawamoto, A.,
Kurioka, H.,
Takase, E.,
Sasaki, Y.,
Hatanaka, K.,
Kobayashi, M.,
Ueyama, T.,
Hashimoto, T.,
and Dohi, K.
(2001)
Circ. Res
88,
696-704 |
49. | Huang, H. M., Weng, C. H., Ou, S. C., and Hwang, T. (1999) J. Neurosci. Res. 56, 668-678[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Ohba, M.,
Ishino, K.,
Kashiwagi, M.,
Kawabe, S.,
Chida, K.,
Huh, N. H.,
and Kuroki, T.
(1998)
Mol. Cell. Biol
18,
5199-5207 |
51. |
Liu, Y. F.,
Paz, K.,
Herschkovitz, A.,
Alt, A.,
Tennenbaum, T.,
Sampson, S. R.,
Ohba, M.,
Kuroki, T.,
LeRoith, D.,
and Zick, Y.
(2001)
J. Biol. Chem.
276,
14459-14465 |
52. |
Braz, J. C.,
Bueno, O. F.,
De Windt, L. J.,
and Molkentin, J. D.
(2002)
J. Cell Biol.
156,
905-919 |
53. | Altin, J. G., and Bradshaw, R. A. (1990) J. Neurochem. 54, 1666-1676[Medline] [Order article via Infotrieve] |
54. | Toker, A. (1998) Front. Biosci. 3, D1134-D1147[Medline] [Order article via Infotrieve] |
55. | Martelli, A. M., Sang, N., Borgatti, P., Capitani, S., and Neri, L. M. (1999) J. Cell. Biochem. 74, 499-521[CrossRef][Medline] [Order article via Infotrieve] |
56. | Dempsey, E. C., Newton, A. C., Mochly-Rosen, D., Fields, A. P., Reyland, M. E., Insel, P. A., and Messing, R. O. (2000) Am. J. Physiol. 279, L429-L438 |
57. |
Lee, H. W.,
Smith, L.,
Pettit, G. R.,
and Smith, J. B.
(1997)
Mol. Pharmacol.
51,
439-447 |
58. |
Lu, Z.,
Liu, D.,
Hornia, A.,
Devonish, W.,
Pagano, M.,
and Foster, D. A.
(1998)
Mol. Cell. Biol.
18,
839-845 |
59. | Parker, P. J., Bosca, L., Dekker, L., Goode, N. T., Hajibagheri, N., and Hansra, G. (1995) Biochem. Soc. Trans. 23, 153-155[Medline] [Order article via Infotrieve] |
60. |
Smith, L.,
Chen, L.,
Reyland, M. E.,
DeVries, T. A.,
Talanian, R. V.,
Omura, S.,
and Smith, J. B.
(2000)
J. Biol. Chem.
275,
40620-40627 |
61. | Semenza, G. L. (2001) Cell 107, 1-3[Medline] [Order article via Infotrieve] |
62. |
Kotsonis, P.,
Funk, L.,
Prountzos, C.,
Iannazzo, L.,
and Majewski, H.
(2001)
Br. J. Pharmacol.
132,
489-499 |
63. |
Kawasaki, H.,
Springett, G. M.,
Mochizuki, N.,
Toki, S.,
Nakaya, M.,
Matsuda, M.,
Housman, D. E.,
and Graybiel, A. M.
(1998)
Science
282,
2275-2279 |
64. | Wooten, M. W., Zhou, G., Seibenhener, M. L., and Coleman, E. S. (1994) Cell Growth Differ 5, 395-403[Abstract] |
65. | Kim, M. S., Lim, W. K., Cha, J. G., An, N. H., Yoo, S. J., Park, J. H., Kim, H. M., and Lee, Y. M. (2001) Cancer Lett. 171, 79-85[CrossRef][Medline] [Order article via Infotrieve] |
66. |
Pal, S.,
Claffey, K. P.,
Cohen, H. T.,
and Mukhopadhyay, D.
(1998)
J. Biol. Chem.
273,
26277-26280 |
67. |
Ryuto, M.,
Ono, M.,
Izumi, H.,
Yoshida, S.,
Weich, H. A.,
Kohno, K.,
and Kuwano, M.
(1996)
J. Biol. Chem.
271,
28220-28228 |
68. | Lu, Z., Hornia, A., Jiang, Y. W., Zang, Q., Ohno, S., and Foster, D. A. (1997) Mol. Cell. Biol. 17, 3418-3428[Abstract] |
69. |
Shizukuda, Y.,
Tang, S.,
Yokota, R.,
and Ware, J. A.
(1999)
Circ. Res.
85,
247-256 |
70. | Diaz-Meco, M. T., Municio, M. M., Frutos, S., Sanchez, P., Lozano, J., Sanz, L., and Moscat, J. (1996) Cell 86, 777-786[Medline] [Order article via Infotrieve] |