Signals from the AT2 (Angiotensin Type 2) Receptor of Angiotensin II Inhibit p21ras and Activate MAPK (Mitogen-Activated Protein Kinase) to Induce Morphological Neuronal Differentiation in NG10815 Cells
Louis Gendron*,
Liette Laflamme*,
Nathalie Rivard,
Claude Asselin,
Marcel D. Payet and
Nicole Gallo-Payet
Service of Endocrinology (L.G., L.L., N.G-P.) Department
of Anatomy and Cell Biology (L.G., L.L., N.R., C.A., N.G-P.)
Department of Physiology and Biophysics (M.D.P, N.G-P.)
Faculty of Medicine University of Sherbrooke Sherbrooke J1H
5N4, Quebec, Canada
 |
ABSTRACT
|
---|
In a previous study, we had shown that activation
of the AT2 (angiotensin type 2) receptor
of angiotensin II (Ang II) induced morphological differentiation of the
neuronal cell line NG10815. In the present study, we
investigated the nature of the possible intracellular mediators
involved in the AT2 effect. We found that
stimulation of AT2 receptors in NG10815 cells
resulted in time-dependent modulation of tyrosine phosphorylation of a
number of cytoplasmic proteins. Stimulation of NG10815 cells with Ang
II induced a decrease in GTP-bound p21ras but a
sustained increase in the activity of p42mapk
and p44mapk as well as neurite outgrowth.
Similarly, neurite elongation, increased polymerized tubulin levels,
and increased mitogen-activated protein kinase (MAPK) activity
were also observed in a stably transfected NG10815 cell line
expressing the dominant-negative mutant of
p21ras, RasN17. These results support the
observation that inhibition of p21ras did not
impair the effect of Ang II on its ability to stimulate MAPK activity.
While 10 µM of the MEK inhibitor, PD98059,
only moderately affected elongation, 50 µM
PD98059 completely blocked the Ang II- and the RasN17-mediated
induction of neurite outgrowth. These results demonstrate that some of
the events associated with the AT2
receptor-induced neuronal morphological differentiation of NG10815
cells not only include inhibition of p21ras but
an increase in MAPK activity as well, which is essential for neurite
outgrowth.
 |
INTRODUCTION
|
---|
The two main types of angiotensin II (Ang II) receptors, namely
AT1 and AT2, are expressed in high levels in
the brain (1, 2). While the AT1 receptor is closely
associated with the regulation of blood pressure, hydromineral balance,
and thirst, the role of the AT2 receptor is not clearly
established. The AT2 receptor is highly expressed in many
fetal tissues (3, 4, 5), including brain (6, 7) and cell lines of neuronal
origin (8, 9, 10). Interestingly, with a few exceptions (limbic system,
several thalamic nuclei, adrenal medulla, adrenal zona glomerulosa),
the AT1/AT2 receptor ratio changes dramatically
after birth (7, 11), suggesting that the AT2 receptor may
be involved in fetal development. From the large number of studies
conducted during the past 5 yr, three main functions appear to be
associated with the activation of this receptor. The first clearly
identified function is the inhibition of proliferation induced by
growth factors (12, 13), including Ang II, via the AT1
receptor (14, 15). Second, the activation of the AT2
receptor has an antagonistic action of the effects mediated by the
AT1 receptor at the central level, such as vasopressin
release (16), control of thirst, and cognitive effects (17, 18, 19). The
third action appears to be a role in development, such as morphological
neuronal differentiation (10, 20) and control of programmed cell death
(3, 21).
The signaling pathway involved in the mechanism of action of the
AT2 receptor is still a matter of controversy (22, 23).
This receptor belongs to the seven-transmembrane domain receptor family
(24, 25). However, it does not fulfill the criteria that characterize G
protein-coupled receptors. The binding of Ang II to the AT2
receptor lacks sensitivity to GTP analogs (26, 27), even when
overexpressed in COS cells (24, 25). Furthermore, the AT2
receptor is not associated with activation or inhibition of adenylyl
cyclase or phospholipid hydrolysis, characteristic archetype pathways
for the family of G protein-linked membrane receptors (7, 28).
Nevertheless, some studies have shown the involvement of a G protein
that was either sensitive (24, 29) or insensitive (27, 30) to pertussis
toxin (PTX) treatment. However, other studies have failed to show any G
protein interaction with the AT2 receptor (25, 31).
AT2 receptor activation was found to decrease (32, 33) or
increase (34, 35) intracellular cGMP, through a PTX-sensitive
Gi-dependent mechanism (33), by selective inhibition of
particulate guanylate cyclase activity, through a
phosphotyrosinephosphatase (PTPase) activity (30, 32), or by an
increase in nitric oxide (NO) production (35). Signal transduction
events after AT2 receptor activation seem to involve
protein phosphatases acting either on tyrosine or serine/threonine
residues. Some controversy persists regarding stimulation (12, 21, 30, 32) or inhibition (24, 36) of tyrosine phosphorylation (Tyr-P). The
AT2 receptor also modulates ion channel activity. For
instance, in NG10815 cells, a decrease in a T-type Ca2+
channel activity by a PTX-insensitive mechanism involving a PTPase
activity has been described (27). However in cultured hypothalamic
neurons, AT2 receptors stimulate a K+ channel
activity occurring via a PTX-sensitive G protein and activation of a
Ser/Thr phosphatase (29).
In their nondifferentiated state, neuroblastoma x glioma hybrid
NG10815 cells, which express only the AT2 receptors, have
a spherical shape and divide actively. We have previously shown that a
3-day treatment with Ang II or CGP 42112 induced neurite outgrowth
characterized by an increase in the level of polymerized tubulin and an
increase of the level of the microtubule-associated protein, MAP2c,
associated with microtubules (10). In rat pheochromocytoma PC12W cells,
a similar effect was observed and was associated with an increase in
another microtubule-associated protein, MAP 1B (20), and a decrease in
the neurofilament middle molecular weight subunit, NF-M (37).
In the present study, we investigated whether the
p21ras-MAP kinase [mitogen-activated protein kinase
(MAPK)] cascade could be involved in the Ang II effect mediated
by the AT2 receptor on the induction of neurite outgrowth.
We found that stimulation of AT2 receptors in NG10815
cells resulted in a time-dependent modulation of Tyr-P of several
proteins. However, stimulation of AT2 receptors decreased
GTP-bound p21ras but induced a sustained increase in the
activities of p42 and p44 MAPK (p42mapk and
p44mapk). Similarly, neurite outgrowth, increased
polymerized tubulin levels, and increased MAPK activity were also
observed in a stably transfected NG10815 cell line expressing the
dominant-negative mutant of p21ras, RasN17. These results
demonstrate that some of the events associated with the AT2
receptor-induced neuronal morphological differentiation of NG10815
cells include not only inhibition of p21ras but an increase
in MAPK activity, which is essential for neurite outgrowth.
 |
RESULTS
|
---|
Effect of Ang II on Protein Tyrosine Phosphorylation
Figure 1A
shows that stimulation of
NG10815 cells with 100 nM Ang II induced a time-dependent
increase in the level of Tyr-P of proteins of about 35 and 45 kDa,
reaching a plateau between 60 and 240 min. Other proteins between 125
and 210 kDa exhibited a time-dependent increase in Tyr-P for up to 30
min followed by a strong decrease, while the phosphorylation state of
other proteins of approximately 50 kDa did not change over the course
of the experimental period. The specificity of the AT2
receptor-mediated effects was assessed by coincubating the cells with
various Ang II analogs. As shown in Fig. 1B
, incubation with 10
nM CGP 42112, an AT2 receptor agonist, induced
a more pronounced effect than that of Ang II, while coincubation with 1
µM DUP 753, an AT1 receptor antagonist,
slightly increased the effect seen with Ang II alone, suggesting that
the cells contain a small quantity of AT1 receptors.

View larger version (62K):
[in this window]
[in a new window]
|
Figure 1. Western Blot Analysis of the Effect of Ang II on
Tyrosine Phosphorylation in NG10815 Cells
Cells were cultured in 35-mm Petri dishes (1 x 106
cells per dish), as described in Materials and Methods
and were stimulated with 100 nM Ang II under different
experimental conditions. A, Time course study. B, Pharmacological
study. Cells were incubated for 15 min with buffer alone (Control), 100
nM Ang II, 100 nM Ang II + 1 µM
DUP 753 (AT1 receptor antagonist), or 10 nM CGP
42112 (AT2 receptor agonist). For all these experiments, 30
µg of proteins from whole-cell lysates were analyzed by Western
blotting, and phosphotyrosinated proteins were revealed with an
antiphosphotyrosine antibody as detailed in Materials and
Methods. Numbers on the right indicate position
of molecular mass markers (kDa). The results are representative of six
different experiments.
|
|
p21ras Activity
We have previously shown that Ang II activates a GTPase activity.
However, the G protein involved is not of the classical type, since
GTP
S does not affect the binding of Ang II to its receptor (27). A
potential candidate for a protein downstream from the AT2
receptor displaying a GTPase activity bearing the above properties
could be p21ras. We thus analyzed the activity of
p21ras in NG10815 cells. Time-course studies indicated
that the maximal decrease in p21ras activity was observed
after a 10-min stimulation with Ang II and was maintained below control
levels for the duration of the 2-h treatment (Fig. 2
, A and B). Pharmacological studies
measuring p21ras activity indicated that Ang II decreased
the Ras-GTP-bound form by 48.5 ± 7.9% (n = 3) (Fig. 2
, C
and D). CGP 42112 also induced a decrease of 35.5 ± 8.5% (n
= 3), while coincubation of Ang II with PD123319 reversed these
inhibitory effects (Fig. 2
, C and D). A novel assay to detect
p21ras activity based on the Ras-binding domain of Raf
(RBD) as a specific trap to selectively precipitate p21ras
only in its GTP-bound state was used. Ang II and CGP42112 both
decreased, by 58% and 48%, respectively, the level of Ras-GTP
bound to GST-RBD compared with control cells while coincubation with 1
µM PD123319 (AT2 receptor antagonist)
prevented this effect (Fig. 2E
).

View larger version (43K):
[in this window]
[in a new window]
|
Figure 2. Effect of Ang II on p21ras Activity in
NG10815
NG10815 cells were cultured for 3 days in 100-mm Petri dishes
(1.0 x 107 cells per dish), labeled for 18 h
with [32P] orthophosphate, and stimulated with Ang II
and/or analogs as described in Materials and Methods.
After incubation, cells were solubilized and p21ras
immunoprecipitated with antisera OP21 or OP40 recognizing
p21ras. p21ras-[32P]GDP and
p21ras-[32P]GTP were separated by TLC and
autoradiographed as detailed in Materials and Methods.
A, Representative autoradiograph of one time course experiment. B,
Densitometric analysis of the experiment shown in panel A. C, D, and E,
Pharmacological analysis of the effect of Ang II on p21ras
bound to GTP. C, Pharmacological autoradiogram of the effect of Ang II
and Ang II receptor analogs on p21ras activity, using the
OP21 antibody. D, Densitometric analysis of three different
experiments. E, Pharmacological analysis of the effect of Ang II on
p21ras activity using GST-RBD fusion protein. Cells were
stimulated during 10 min with 100 nM Ang II, 10
nM CGP 42112 (AT2 receptor agonist), or with
100 nM Ang II + 1 µM PD 123319
(AT2 receptor antagonist), as described in Materials
and Methods. *, P < 0.01, difference
compared with the control value.
|
|
MAP Kinase Activity
The increase in Tyr-P observed after AT2 receptor
stimulation, as well as modulation of p21ras, may be
involved in the regulation of different kinases such as the
mitogen-activated protein kinases (MAPK), which are modulated by
phosphorylation on both Thr and Tyr residues by the dual specificity
MAPKK, MEK1, and MEK2 (38). The effect of Ang II on MAPK activity was
first estimated by in-gel kinase assays (data not shown) and further
assessed using a specific antibody directed against the phosphorylated
form of MAPK (Fig. 3
). A time-course
study indicated that the increase in MAPK activity persisted for more
than 2 h with maximal 4.7- and 2.3-fold increased activity over
control for p42mapk and p44mapk, respectively,
after 120 min of stimulation with Ang II (Fig. 3
, A and C). This effect
was specific for MAPK activity since coincubation with 10
µM PD98059, a specific inhibitor of MEK1 (39), abolished
this increase (Fig. 3B
). To further emphasize the specificity of
AT2 stimulation on MAPK, the effect of the AT1
receptor stimulation was analyzed on (Bu)2cAMP (1
mM) differentiated cells (10). In these conditions, the
cells express both types of receptors, i.e. AT1
and AT2. Figure 3D
shows that after blockade of the
AT2 receptor by PD123319, Ang II induced a rapid and
transient increase in MAPK phosphorylation.

View larger version (34K):
[in this window]
[in a new window]
|
Figure 3. Effect of Ang II on MAPK Activity in NG10815
Cells
NG10815 cells were cultured for 3 days in 35-mm Petri dishes (1
x 106 cells per dish) and then stimulated without
(control) or with 100 nM Ang II for the indicated times,
followed by extraction of cells and resolution of 30 µg of proteins
from whole-cell extracts on 10% SDS-polyacrylamide gels, as detailed
in Materials and Methods. Time course study of MAPK
activity revealed by Western blot experiments using an
antiphosphorylated MAPK antibody. Effect of Ang II alone (A) or after a
30-min preincubation with 10 µM of the MEK1 inhibitor,
PD98059 (B). C, Comparative histogram analysis of three different
experiments, conducted as in A. D, MAPK analysis in 3-day cultured
cells in the presence of 1 mM (Bu)2cAMP. Cells
were stimulated with Ang II (100 nM) plus PD 123319 (1
µM). Data are mean ± SE. *,
P < 0.02; **, P < 0.01,
difference compared with the control value. Numbers on the
right indicate position of p42 and p44mapk (kDa).
|
|
Effect of PD98059 on the Ang II-Induced Outgrowth of Neurites
Numerous studies using PC12 cells have shown that a sustained
activation of MAPK was necessary, and even sufficient, to induce
neuronal differentiation. However, inhibition of MAPK using a specific
MEK1 inhibitor, PD98059, did not interfere with the outgrowth of
neurites from sensory and sympathetic neurons (40) as well as PC12D
cells (41). Since we observed a prolonged increase in MAPK activity in
NG10815 cells after Ang II treatment, we aimed at verifying whether
MAPK activation was necessary for the differentiation of NG10815
cells. As previously published (10), a 3-day treatment with Ang II
caused an increase in neurite formation (Fig. 4B
vs. 4A). Coincubation of
Ang II with 10 µM PD98059 for 3 days, a dose at which
MAPK is inhibited (Fig. 3B
), caused a decrease in the length and number
of neurites (Fig. 4C
), an effect further amplified using 50
µM of PD98059 (Fig. 4D
), where cells exhibited rounded
bodies with even fewer neurite-like processes than control cells. The
level of polymerized tubulin was increased in cells treated for 3 days
with 100 nM Ang II, comparatively to control cells (Fig. 4E
). Decreases of 44 ± 12% and 76 ± 15% (n = 3) in
the amount of polymerized tubulin were observed when Ang II was
incubated with 10 µM and 50 µM PD98059,
respectively, reflecting the observed decrease in neurite length and
number (Fig. 4E
). In addition, while 10 µM PD98059 alone
or in the presence of Ang II only caused a modest decrease in cell
number (12 ± 4% and 19 ± 2%, respectively; n = 3), a
concentration of 50 µM PD98059, applied for 3 days,
strongly affected cell division and probably cell viability, since
cells showed a 80 ± 5% (n = 3) decrease in the number of
attached cells (Fig. 4F
).

View larger version (105K):
[in this window]
[in a new window]
|
Figure 4. Effect of PD98059 on Neurite Outgrowth in NG10815
Cells
Cells were plated at a density of 5 x 104 cells per
dish in 35-mm Petri dishes and either cultured for 3 days in the
absence (A) or in the presence of 100 nM Ang II (B), 100
nM Ang II and 10 µM PD98059 (C), or 100
nM Ang II and 50 µM PD98059 (D). All panels
are shown at the same magnification of x680. E, Densitometric analysis
of the amount of polymerized tubulin from cells treated as described
above (n = 3). F, Measurements of cell number after the various
treatments (n = 3). *, P < 0.05; **, P < 0.02,
difference compared with the control value.
|
|
Properties of NG10815 Cells Transfected with RasN17
To further investigate the involvement of p21ras on
Ang II-induced neurite outgrowth, we produced a stably transfected
NG10815 cell line expressing an hemagglutinin (HA)-tagged
dominant-negative mutant of p21ras, Asn-17 Ras (RasN17
cells), using the Lac Switch-inducible expression system to control the
expression of inhibitory RasN17. Northern blot analysis (Fig. 5A
) indicated that daily application of
isopropyl-1-thio-ß-D-galactopyranoside (IPTG) induced a
strong level of mRNA induction of p21ras after 3 days
(2.94-fold increase), compared with that observed in control
transfected cells (without IPTG induction). The mRNA induction was
followed by an IPTG-regulated expression of a higher molecular form of
Ras protein (representing the HA epitope-tagged HA-Ras mutant) (Fig. 5B
), which was present 24 h after induction. Moreover, the
presence of an unique band in the transfected cells (line 0) indicated
the absence of basal expression of HA-Ras without IPTG induction (no
leakiness). The functional consequences of expressing RasN17 is shown
in Fig. 5C
, where the basal level of GTP-bound Ras was decreased by
47% in RasN17 cells, compared with control transfected cells (Fig. 5C
). Figure 6
shows the morphological
consequences of a 3-day expression of RasN17 in NG10815 cells. The
control transfected cells were polygonal, actively dividing and
extended one or two small processes, much like untransfected NG10815
cells (compare Fig. 6A
with 4A). After 3 days of IPTG induction, the
RasN17-transfected cells exhibited several long processes (Fig. 6B
).
The neurites appeared longer and more branched than that of
untransfected NG10815 cells treated for 3 days with Ang II (Fig. 6B
vs. Fig. 4B
). Immunofluorescence for ß-tubulin exemplified
the morphological appearance of neurites. One or two thin processes
were observed in control transfected cells (Fig. 6C
), while long
neurites with distinct growth cones at each tip were observed in
IPTG-induced RasN17 cells (Fig. 6D
). These morphological changes were
correlated with an increase in the level of polymerized tubulin (Fig. 6E
).

View larger version (28K):
[in this window]
[in a new window]
|
Figure 5. Characterization of the RasN17-Transfected Cells
A, Northern blot analysis of mRNA levels of control RasN17-transfected
cells (0) and after 3 days of daily induction of RasN17 with 5
mM IPTG (72 ). Total cellular RNA (30 µg) was processed
according to the procedure described in Materials and
Methods, using 32P-RasN17 cDNA as a probe.
Expression levels were compared with hybridization of the same blot
with -tubulin as probe. B, Western blot analysis of
p21ras expression in control RasN17-transfected cells (0)
and after 24, 48, or 72 h of IPTG induction. Endogenous
p21ras in brain (B) and in NG10815 cells (N) was compared
with the hemagglutinin-tagged Ras. C, Analysis of p21ras
activity in control RasN17-transfected cells (C) and after 72 h of
IPTG induction (IPTG) was measured with GST-RBD fusion protein as
described in Materials and Methods.
|
|

View larger version (131K):
[in this window]
[in a new window]
|
Figure 6. Morphology of RasN17-Transfected NG10815 Cells
Stably RasN17-transfected cells were cultured for 3 days in normal
culture medium (A and C) or in medium containing 5 mM IPTG
for induction of RasN17 expression (B and D). A and B, Phase contrast
microscopy; C and D, after formaldehyde fixation and permeabilization
with 0.1% Triton X-100, cells were processed for immunofluorescence
labeling using anti-ß-tubulin antibody and fluorescein isothiocyanate
as described in Materials and Methods. All panels are
shown at the same magnification of x680. E, Neurite quantification:
extraction of microtubules and associated proteins were done as
detailed in Materials and Methods and aliquots
equivalent to 3 x 104 cells were resolved on 8%
polyacrylamide gels. Proteins were transferred to PVDF membranes, and
detection of tubulin was performed using an anti-ß tubulin antibody.
|
|
Finally, MAPK activity was measured. Addition of Ang II in control
transfected cells (Fig. 7A
) or in
IPTG-induced RasN17 cells (Fig. 7B
) induced the same time-dependent
increase in MAPK activity, as described in NG10815 cells (Fig. 3
). As
for NG10815 cells, the maximal increase was 7.92-fold over control
for p42mapk and 10.5 over control for p44mapk
in control transfected cells (Fig. 7C
) and 3.92- and 7.15-fold over
control for p42mapk and p44mapk, respectively,
in IPTG-induced RasN17 cells (Fig. 7D
). Moreover, IPTG induction of
RasN17 increased the basal activity of
p42mapk/p44mapk compared with control
transfected cells or NG10815 cells (Fig. 7E
, c vs. a).

View larger version (43K):
[in this window]
[in a new window]
|
Figure 7. Effect of Ang II on MAPK Activity in Stably
RasN17-Transfected NG10815 Cells
Three-day induction of RasN17 expression was done by addition of 5
mM IPTG to the culture medium as detailed in
Materials and Methods. Control RasN17-transfected cells
(A) or IPTG-induced RasN17-expressing cells (B) were grown in 35-mm
Petri dishes (1 x 106 cells per dish) and stimulated
without (control) or with 100 nM Ang II for the indicated
times, followed by extraction of cells and separation of 30 µg of
proteins from whole-cell extracts on 10% SDS-polyacrylamide gels, as
detailed in Materials and Methods. Time course study of
MAPK activity was revealed by Western blot experiment using an
antiphosphorylated MAPK antibody. C and D, Comparative histogram
analysis of three different time course studies of MAPK activity
performed in control cells (C) and in IPTG-induced RasN17-expressing
cells (D). E, Comparative effect of Ang II on MAPK activity in native
NG10815 cells (a), control RasN17-transfected cells (b), and 72-h
IPTG-induced RasN17-transfected cells (c). *, P <
0.05; **, P < 0.01, difference compared with the
control value. Numbers on the right indicate position of
p42 and p44mapk (kDa).
|
|
Effect of PD98059 on Neurite Outgrowth of RasN17-Expressing
Cells
We further verified whether the induction of neurite outgrowth
from the RasN17-transfected cells was dependent on MAPK activation.
Daily application of 10 µM PD98059 to RasN17-transfected
cells for 3 days caused a reduction in the number, length, and
branching of neurites (Fig. 8C
vs. Fig. 8B
), adopting morphology similar to control
cells (Fig. 8C
vs. 8A). Application of 50 µM
PD98059 had a major inhibitory effect on cell morphology (Fig. 8D
).
They lost their neurite processes, exhibited a rounded cell body, and
detached easily from the substratum. The effects of PD98059 on neurite
extension were again quantified using polymerized tubulin as an index
of neurite length (Fig. 8E
). The results showed a 76 ± 5%
(n = 3) decrease in the amount of polymerized tubulin with
application of 10 µM PD98059 down to undetectable levels
at 50 µM PD98059 (Fig. 8E
). The MEK inhibitor also had a
major effect on cell proliferation, cell adhesion, and cellular
viability. In the presence of 10 µM PD98059, cell number
was decreased by 23 ± 8% (n = 3) and by 90 ± 0.5%
(n = 3) in the presence of 50 µM PD98059 (Fig.
8F).

View larger version (117K):
[in this window]
[in a new window]
|
Figure 8. Effect of PD98059 on Neurite Outgrowth in
RasN17-Transfected NG10815 Cells
RasN17-transfected cells were plated at a density of 5 x
104 cells in 35-mm Petri dishes and cultured for 3 days in
normal culture medium (A) or in medium containing 5 mM IPTG
(B) or 5 mM IPTG and 10 µM PD98059 (C) or 5
mM IPTG and 50 µM PD98059 (D). All panels are
shown at the same magnification of x680. E, Densitometric analysis of
the amount of polymerized tubulin from cells treated as described above
(n = 3). F, Measurement of cell number (n = 3). **,
P < 0.001, difference compared with the control
value.
|
|
 |
DISCUSSION
|
---|
In the present study, we describe intracellular events initiated
by the activation of the AT2 receptor, which may be
responsible for the induction of neurite outgrowth in NG10815 cells.
Using RasN17-transfected cells, we were able to demonstrate that
p21ras inhibition did not impair the effect of Ang II on
its ability to stimulate MAPK activity, which is essential for neurite
outgrowth.
The AT2 receptor signaling events remain a subject of
controversy, in particular with regard to Tyr phosphorylation of
proteins and G protein coupling. In contrast to previous studies
describing an inhibitory (9, 12, 21, 30, 32) or stimulatory (24, 36)
effect on Tyr phosphorylation, we found a strong modulatory effect of
Ang II. Indeed, using nondifferentiated cells that expressed only
AT2 receptors, we observed three different patterns in
protein phosphorylation of tyrosine residues: 1) a progressive increase
until 30 min followed by a decrease; 2) a monotonic time-dependent
increase; and finally 3) no change over the 2-h incubation. These
observations suggest that AT2 receptor signaling is
associated with several proteins that could be modulated by tyrosine
phosphorylation. Among these proteins are those at around 44 kDa, final
targets of the p21ras/Raf/MAPK pathway.
The role of p21ras/MAPK cascade has been extensively
studied for the neurotrophic nerve growth factor (NGF)-induced cell
differentiation of the sympathetic-like PC12 cells, but, even in this
model, results are contradictory. It has been shown that activation of
p21ras in PC12 cells is necessary for neuronal
differentiation. In fact, induction of p21ras was shown to
mimic the effect of NGF on neurite outgrowth (42), while expression of
inactive p21ras, RasN17, blocked this action (43).
Sustained activation of MAPK by NGF was necessary and sufficient for
neurite outgrowth (44, 45), and blockade of this activation by the MEK
inhibitor PD98059 impeded NGF-induced differentiation (45). It was
previously shown that Ang II, through its seven- transmembrane
AT2 receptor, also induced neurite outgrowth, not only in
NG10815 cells (10), but also in PC12 cells, where it potentiated
NGF-induced differentiation (20). However, our results clearly
demonstrate that the mechanism of differentiation induced by the
AT2 receptor is different. We found that Ang II stimulation
caused an inhibition of p21ras. This effect was mimicked by
the AT2 agonist CGP 42112 and blocked by the antagonist
PD123319, demonstrating the specificity of the AT2
receptor-mediated inhibition. Even if Ang II potentiates NGF-induced
neurite outgrowth, it also decreased the NGF-activation of
p21ras (data not shown). These observations indicate that
the initial intracellular events involved in the morphological neuronal
differentiation in these two cell lines are different. Moreover, our
results indicate that despite the reduction of the p21ras
activity, a sustained increase in MAPK activity, essential to neurite
outgrowth, is still observed.
To further investigate whether p21ras inhibition was
involved in the initiation of neurite outgrowth, we used cells
transfected with the inactive form of the Ras protein, RasN17.
Induction of RasN17 expression increased the basal state of MAPK
activation, which remains sensitive to Ang II stimulation. These
results indicate that p21ras inhibition alone activates
MAPK and that Ang II stimulation further increased this activity by a
p21ras-independent mechanism. The role of sustained
increase in MAPK in the induction of neuronal differentiation is now
well established (46, 47). What is novel in the present study is that
inhibition of p21ras did not impair the ability of Ang II
to stimulate MAPK activity. In addition, this inhibition initiated
neurite outgrowth, further stimulated by Ang II. These results clearly
indicate that MAPK could be activated by p21ras-independent
pathways. Indeed, recent studies indicate that the sustained phase of
MAPK activation leading to neurite outgrowth could be mediated by Rap1
(48, 49, 50). Vossler et al. (46) demonstrated that activation
of Rap1/B-Raf was accompanied by a concomitant decrease in the
Ras/Raf-1 pathway. These effects seem to be mediated by PKA, through a
cAMP-dependent or -independent manner (47). Similar mechanisms could
also be considered in NG10815 cells, where chronic treatment with
cAMP analogs is the usual method to differentiate these cells (51, 52).
Alternatively, Rap1 could also be activated by phospholipase C, by
intracellular calcium (50, 53), or protein kinase G (54). The sustained
increase in MAPK shown in NG10815 cells may be also linked to the
observation that AT2 receptor activation did not undergo
desensitization as do most G protein-coupled receptors (55).
The transient vs. sustained increases in MAPK appear to
control the switch between proliferation and differentiation (56, 57, 58).
This observation may apply to the effect of Ang II. Indeed, several
studies indicate that the growth-promoting effect of the
AT1 receptor, both in vascular smooth muscle cells and in
adrenal cells, is associated with a rapid and transient increase in
MAPK activity (59, 60, 61, 62). Accordingly, our results show the same time
course, in conditions in which NG10815 cells express AT1
receptor (i.e, cell treatment with (Bu)2cAMP,
10), and stimulation with Ang II plus PD123319 induced a transient
increase in MAPK activity. In vascular smooth muscle cells, inhibitors
of Tyr kinases completely inhibited p21ras stimulation
mediated by activation of the AT1 receptor, without
affecting MAPK (63), indicating that the exact mechanism in which
AT1 receptor stimulates MAPK is not known, although the
involvement of a protein kinase C (64) or Raf-1 (65) are described.
These results, including ours, demonstrate the possibility of
stimulating MAPK pathway without the involvement of
p21ras.
To verify whether MAPK activation was essential for Ang II
differentiation of NG10815- or RasN17-expressing cells, we used the
specific MEK1 inhibitor, PD98059. At a concentration of 10
µM, PD98059 blocked the Ang II-induced MAPK activity and
decreased neurite outgrowth in native NG10815 cells but had a much
stronger effect on the number and length of neurites in
RasN17-transfected cells. At this concentration, neurite outgrowth was
also inhibited in PC12 cells, while even higher doses failed to inhibit
NGF-induced neurite outgrowth in PC12D and in sensory or sympathetic
neurons (40, 41). A concentration of 50 µM blocked
neurite outgrowth both in Ang II-treated NG10815 cells and in RasN17
cells. PD98059 is a MEK1 inhibitor with a reported IC50
value of 10 µM, but it also inhibits MEK2 at an
IC50 of 50 µM (66). While p21ras
inhibition had a negligible effect on cell proliferation, both
p21ras and MAPK inhibitions completely blocked cell
proliferation and neurite outgrowth and eventually led to cell death,
at high concentrations of the MEK inhibitor.
What is the initial event and link between AT2 receptor
activation and sustained MAPK activity? Recent observations, including
ours (67), have shown that the AT2 receptor induces an
increase in NO synthase activity, which is required for initiation of
neurite outgrowth, and trigger the switch between growth arrest and
differentiation (68). Because NO is known to increase cGMP and because
this increase is followed by inactivation of phosphodiesterase activity
(69), which consequently increases cAMP, NO could be one or the first
second messenger in the AT2 receptor-signaling pathway
through cAMP to MAPK activation.
Taken together, our results indicate that the morphological neuronal
differentiation of NG10815 cells after Ang II treatment involves a
decrease in p21ras activity, but also an increase in MAPK
activity, an absolute mediator of neurite outgrowth.
 |
MATERIALS AND METHODS
|
---|
Materials
The chemicals used in the present study were obtained from the
following sources: DMEM, FBS, HAT supplement (hypoxanthine,
aminopterin, thymidine), gentamycin, lipofectamine, geneticin,
hygromycin B, and IPTG from Life Technologies, Inc.
(Burlington, Ontario, Canada); [32P]orthophosphate (10
mCi/ml), [
-32P]dCTP (3000 Ci/mmol), horseradish
peroxidase-conjugated antimouse antibody and enhanced
chemiluminescence (ECL) detection system from Amersham Pharmacia Biotech (Oakville, Ontario, Canada);
antiphosphotyrosine antibody and monoclonal antibody to
p21ras (pan-ras (Ab3), OP40 and
pan-ras (Ab1), OP21) from Oncogene Science, Inc. (Scarborough, Ontario, Canada); anti-ß-tubulin and
antimouse Ig-fluorescein and Complete protease inhibitor from
Roche/Boehringer Mannheim (Montreal, Quebec, Canada), and
antiphosphorylated MAPK from New England Biolabs, Inc.
(Missisauga, Ontario, Canada). Plastic coverslips were from Sarstedt
(St-Laurent, Quebec, Canada) and Vectashield mounting medium was from
Vector Laboratories, Inc. (Burlingame, CA). PD98059 was a
gift from Dr. David Dudley (Parke-Davis, Pharmaceutical
Research Division, Ann Arbor, MI). CGP 42112 was provided by Dr.
Marc de Gasparo (Ciba-Geigy, Basel, Switzerland); PD
123319 from RBI (Natick, MA) [Val5]-Ang II was from
Bachem California, Inc. (Torrance, CA). All other
chemicals were of A grade purity. The dominant negative mutant of
p21ras (RasN17) was kindly provided by Dr. Gary Johnson
(division of Basic Sciences, National Jewish Center for Immunology and
Respiratory Medicine and Department of Pharmacology, University of
Colorado Medical School, Denver, CO).
Cell Culture
NG10815 cells (provided by Drs M. Emerit and M. Hamon; INSERM,
U. 238, Paris, France) were cultured (passage 1221) in DMEM with 10%
FBS, HAT supplement, and 50 µg/liter gentamycin at 37 C in 80
cm2 Nunclon Delta flasks (Life Technologies, Gaithersburg,
MD) in a humidified atmosphere of 93% air and 7%
CO2, as suggested by Hamprecht et al. (51). The
medium was replaced every 2 days. Subcultures were performed at
subconfluency. Under these conditions, cells express only the
AT2 receptor subtype of Ang II (10, 30, 32).
Western Blot Analysis of Tyr-Phosphorylated Proteins
Analysis of protein tyrosine phosphorylation was performed using
whole- cell lysates essentially as described by Ohmichi et
al. (70). NG10815 cells (1 x 106 cells) were
incubated for various time intervals at 37 C in Hanks buffered saline
(HBS: NaCl, 130 mM; KCl, 3.5 mM;
CaCl2, 1.8 mM; MgCl2, 0.5
mM; NaHCO3, 2.5 mM; HEPES, 5
mM, supplemented with 1 g/liter glucose and 0.1% BSA) in
the presence of various drugs incubated 10 min before the addition of
Ang II. The reaction was stopped by aspiration of the medium and
immediate washing with ice-cold HBS containing 1 mM sodium
orthovanadate (Na3VO4) and 0.1 µM
staurosporine. Cells were homogenized in 125 mM Tris buffer
(pH 6.8) containing 1% Nonidet P-40, 1 mM
Na3VO4, 0.1 µM staurosporine, 2
mM phenylmethylsulfoxide (PMSF), 0.04 trypsin inhibitory
unit (TIU)/ml aprotinin, and 1 mM benzamidin.
Homogenates were centrifuged for 15 min at 8,000 x g,
and the supernatants were collected and stored at -20 C. Equal amounts
of protein (30 µg) were separated on 8% SDS-polyacrylamide gels.
Proteins were transferred electrophoretically to polyvinylidene
difluoride (PVDF) membranes (Immobilon P, Millipore Corp.,
Bedford, MA). Membranes were blocked with 1% gelatin, 0.05%
Tween 20 in TBS buffer (pH 7.5). After four washes with TBS-Tween 20
(0.05%), membranes were incubated with antiphosphotyrosine antibody
(dilution 1:650) for 2 h at room temperature, followed by four
washes with TBS-Tween 20. Detection was performed using horseradish
peroxidase-conjugated antimouse antibody (1:2,000) and ECL detection
system.
Western Blot Analysis of Phosphorylated MAPK
NG10815 cells (1 x 106 cells) were incubated
for various time intervals at 37 C in culture medium in the presence of
Ang II and antagonists and incubated 10 min before the addition of Ang
II. The reaction was stopped by aspiration of the medium. Cell lysis
was done at 4 C in 50 mM HEPES, pH 7.8, 1% Triton X-100,
0.1 µM staurosporine, 1 mM sodium
Na3Vo4, 0.04 TIU/ml aprotinin, 2 mM
PMSF, 1 mM benzamidin. Cell extracts were centrifuged at
8,000 x g for 15 min at 4 C, and the supernatants were
stored at -20 C. Equal amounts of protein (30 µg) were separated on
10% SDS-polyacrylamide gels. Western blot procedure was performed as
stated above but with the use of an antiphosphorylated MAPK antibody
(dilution 1:1,000). Detection was accomplished using horseradish
peroxidase-conjugated antirabbit antibody (1:2,000) and ECL detection
system.
Western Blot Analysis of Microtubule Proteins
Extractions of microtubules were done as previously described
(10). Extracts equivalent to 3 x 104 cells were
separated on 8% polyacrylamide gels. Western blot procedure was
performed as stated above. Tubulin was detected with an
anti-ß-tubulin antibody (1:500) and visualized using horseradish
peroxidase-conjugated antimouse antibody (1:2,000) and ECL detection
system.
Analysis of p21ras-Bound
GDP/GTP
NG10815 cells were incubated in serum-free, phosphate-free
DMEM and labeled with [32P]orthophosphate (0.5 mCi/100-mm
Petri dish) for 18 h, as described by Satoh et al.
(71), with slight modifications. Cells were then stimulated for 10 min
at 37 C with or without Ang II and/or analogs. After hormonal
treatment, cells were washed, scraped from the substratum, centrifuged,
and homogenized in lysis buffer (50 mM Tris-HCl, pH 7.5, 15
mM NaCl, 20 mM MgCl2, 5
mM EGTA, Complete protease inhibitor, 1% Triton X-100, 1%
N-octylglucoside) for 15 min at 4 C. Insoluble material was
removed by centrifugation at 12,000 x g for 2 min at 4
C. Cell lysates were incubated with antisera recognizing
p21ras (OP21 in Fig. 2C
; OP40 in panels A and E) (1
µg/sample) for 2 h at room temperature. The immune complexes
were collected with protein A-sepharose and washed twice with lysis
buffer and twice with PBS. p21ras was recovered by
incubation at 95100 C for 3 min in solubilization buffer (1
M KH2PO4, pH 3.4).
p21ras-bound nucleotides were separated on a
phosphoethyleneimine-cellulose plate eluted with 1 M
potassium dihydrogen phosphate (KH2PO4)
solution, pH 3.4, and subjected to autoradiography. 32P
incorporation was quantified using a PhosphoImager analysis system
(Molecular Dynamics, Inc., Sunnyvale, CA).
p21 Ras Activation Assays
The assay to measure the activity status of p21ras
was as described previously (72). Briefly, NG10815 cells were
harvested at 80% confluence, 100% confluence (day 0), and 4, 8, and
14 days postconfluence in lysis buffer A (50 mM Tris-HCl,
pH 7.5, 15 mM NaCl, 20 mM MgCl2, 5
mM EGTA, Complete protease inhibitor, 1% Triton X-100, 1%
N-octylglucoside) for 15 min at 4 C. Insoluble material was
removed by centrifugation at 12,000 x g for 2 min at 4
C. Proteins from lysates (1000 µg) were incubated with 30 µg of
GST-RBD fusion protein [where RBD is amino acids 81131 of Raf-1 and
is the minimal domain required for binding of Ras-GTP (73)]
preadsorbed to glutathione-sepharose beads for 2 h at 4 C.
Precipitates were washed three times with buffer A. The presence of
p21ras was detected by resuspending the final pellet in 30
µl of Laemmli buffer, followed by protein separation on 12%
polyacrylamide gels, and Western blotting with antisera OP40 (1:100)
recognizing p21ras.
Cell Transfection
The plasmid pOPNRas contains a cDNA encoding an
hemagglutin-tagged dominant negative Asn-17 mutant of
p21ras, RasN17 (74), cloned into the LacI repressible
vector (a gift from Dr. Gary Johnson, University of Colorado Medical
School, Denver, CO). Cells were cotransfected with pOPNRas and the
plasmid p3'SS coding for the LACI repressor. Plasmid DNA used for
transfection was prepared by cesium chloride extraction. pOPNRas (1.5
µg) and p3'SS DNA (1.5 µg) were mixed with 40 µg Lipofectamine
(Life Technologies, Inc.) in 500 µl serum-free DMEM and
incubated for 30 min at room temperature. For transfection, NG10815
cells grown to subconfluency in 24-well Petri dishes were incubated
with the lipid-DNA complex for 5 h at 37 C in a humidified
atmosphere of 93% air and 7% CO2. Transfection medium was
then replaced with complete medium. Selection of stably transfected
clones expressing both RasN17 and the Lac repressor was initiated
36 h after transfection with 400 µg/ml geneticin (G 418) and 200
µg/ml hygromycin B. Stable clones were isolated and were routinely
maintained in normal complete NG10815 cell medium containing 200
µg/ml G418 and 100 µg/ml hygromycin B. The expression of the
dominant-negative RasN17 was induced with 5 mM IPTG,
applied daily for 3 days, and the induction of RasN17 transcription was
verified by Northern blot analysis (75), by using
32P-RasN17 cDNA as a probe.
Immunofluorescence Microscopy Studies
Immunofluorescence microscopy studies were performed as
described previously (10) with slight modifications. Untransfected or
transfected NG10815 cells were cultured for 4 days on plastic
coverslips according to the different experimental conditions. Cells
were washed twice with cold PBS and then fixed for 1 min with 3%
(vol/vol) formaldehyde in PEM buffer (80 mM
piperazine-N,N'-bis-[2-ethanesulfonic acid], 5
mM EDTA, 2 mM MgCl2, pH 6.5) at
room temperature, and 8 min in 3% (vol/vol) formaldehyde in 100
mM sodium borate (pH 11). Cells were washed twice (5 min)
with PBS and were incubated twice (15 min) with 0.1% (wt/vol) sodium
borohydride/PBS, pH 8.0. Cells were then permeabilized twice (5 min)
with 0.2% Triton X-100/PBS and incubated three times for 10 min at
room temperature in blocking solution (0.5% BSA/PBS) followed by two
5-min washes with PBS. Cells were then incubated with an
anti-ß-tubulin antibody (1:50/0.5% BSA/PBS) overnight at 4 C. On the
next day, cells were incubated in 0.5% BSA/PBS and washed three times
for 10 min with PBS before a 60-min incubation with an antimouse
Ig-fluorescein antibody (1:30/0.5% BSA/PBS) at room temperature
and washed three times for 10 min with PBS. Cells were then postfixed
in 3% formaldehyde/PBS for 30 min at room temperature and treated with
50 mM ammonium chloride/PBS solution for 15 min at room
temperature. After three 10-min washes with PBS, slides were mounted in
Vectashield mounting medium and examined on a DM 400 microscope
equipped for epifluorescence using B-1E fluorescein
isothiocyanate filter (Nikon, Melville, NY).
Data Analysis
The data are presented as means ± SE.
Statistical analyses of the data were performed using the Student
t test.
 |
ACKNOWLEDGMENTS
|
---|
The authors would like to thank Dr. David Dudley
(Parke-Davis, Pharmaceutical Research Division, Ann
Harbor, MI) for PD98059, the MEK1 inhibitor, and Dr. Gary Johnson
(division of Basic Sciences, National Jewish Center for Immunology and
Respiratory Medicine and Department of Pharmacology, University of
Colorado Medical School, Denver, CO) for the gift of the dominant
negative mutant of p21ras (RasN17).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Nicole Gallo-Payet, Service of Endocrinology, Faculty of Medicine, University of Sherbrooke, Sherbrooke J1H 5N4, Quebec, Canada.
This work was supported by Medical Research Council and FCAR
(Fonds pour la Formation des Chercheurs et Aide à la Recherche)
grants to N.G.-P. and M.D.P.
Received for publication October 7, 1998.
Revision received May 13, 1999.
Accepted for publication June 3, 1999.
 |
REFERENCES
|
---|
-
Lenkei Z, Palkovits M, Corvol P, Llorens-Cortes C 1997 Expression of angiotensin type-1 (AT1) and type-2 (AT2) receptor mRNAs
in the adult rat brain: a functional neuroanatomical review. Front
Neuroendocrinol 18:383439[CrossRef][Medline]
-
Griendling K, Lassègue B, Alexander R 1996 Angiotensin
receptors and their therapeutic implications. Annu Rev Pharmacol
Toxicol 36:281306[CrossRef][Medline]
-
Tanaka M, Ohnishi J, Ozawa Y, Sugimoto M, Usuki S, Naruse M,
Murakami K, Miyazaki H 1995 Characterization of angiotensin II receptor
type 2 during differentiation and apoptosis of rat ovarian cultured
granulosa cells. Biochem Biophys Res Commun 207:593598[CrossRef][Medline]
-
Breault L, Lehoux J-G, Gallo-Payet N 1996 The angiotensin
receptor AT2 is present throughout the human fetal adrenal
gland of the second trimester gestation. J Clin Endocrinol Metab 81:39143922[Abstract]
-
Schütz S, Le Moullec J-M, Corvol P, Gasc J-M 1996 Early
expression of all the components of the renin-angiotensin-system in
human development. Am J Pathol 149:20672079[Abstract]
-
Millan MA, Jacobowitz DM, Aguilera G, Catt KJ 1991 Differential distribution of AT1 and AT2
angiotensin II receptor subtypes in the rat brain during development.
Proc Natl Acad Sci USA 88:1144011444[Abstract]
-
Tsutsumi K, Strömberg C, Viswanathan M, Saavedra JM 1991 Angiotensin-II receptor subtypes in fetal tissues of the rat:
autoradiography, guanine nucleotide sensitivity and association with
phosphoinositide hydrolysis. Endocrinology 129:10751082[Abstract]
-
Webb ML, Liu EC-K, Cohen RB, Hedberg A, Bogosian EA,
Monshizadegan H, Moloy C, Serafino R, Moreland S, Murphy TJ, Dickinson
KEJ 1992 Molecular characterization of angiotensin II type 2 receptors
in rat pheochromocytoma cells. Peptides 13:499508[CrossRef][Medline]
-
Nahmias C, Cazaubon SM, Briend-Sutren MM, Lazard D,
Villageois P, Strosberg AD 1995 Angiotensin II AT2
receptors are functionally coupled to protein tyrosine
dephosphorylation in N1E-115 neuroblastoma cells. Biochem J 306:8792[Medline]
-
Laflamme L, de Gasparo M, Gallo J-M, Payet MD, Gallo-Payet N 1996 Angiotensin II induction of neurite outgrowth by AT2
receptors in NG10815 cells. Effect counteracted by the AT1 receptors.
J Biol Chem 271:2272922735[Abstract/Free Full Text]
-
Grady EF, Sechi L, Griffin C, Schambelan M, Kalinyak J 1991 Expression of AT2 receptors in the developing rat fetus.
J Clin Invest 88:921933[Medline]
-
Tsuzuki S, Eguchi S, Inagami T 1996 Inhibition of cell
proliferation and activation of protein tyrosine phosphatase mediated
by angiotensin II type 2 (AT2) receptor in R3T3 cells.
Biochem Biophys Res Commun 228:825830[CrossRef][Medline]
-
Bedecs K, Elbaz N, Sutren M, Masson M, Susini C, Strosberg AD,
Nahmias C 1997 Angiotensin II type 2 receptors mediate inhibition of
mitogen-activated protein kinase cascade and functional activation of
SHP-1 tyrosine phosphatase. Biochem J 325:449454[Medline]
-
Stoll M, Steckelings UM, Paul M, Bottari SP, Metzger R, Unger
T 1995 The angiotensin AT2-receptor mediates inhibition of
cell proliferation in coronary endothelial cells. J Clin Invest 95:651657[Medline]
-
Munzenmaier DH, Greene AS 1996 Opposing actions of angiotensin
II on microvascular growth and arterial blood pressure. Hypertension 27:760765[Abstract/Free Full Text]
-
Hohle S, Culman J, Boser M, Qadri F, Unger T 1996 Effect of angiotensin AT2 and muscarinic receptor blockade
on osmotically induced vasopressin release. Eur J Pharmacol 300:119123[CrossRef][Medline]
-
Hein L, Barsh GS, Pratt RE, Dzau VJ, Kobilka BK 1995 Overexpression of angiotensin AT1 receptor transgene in the
mouse myocardium produces a lethal phenotype associated with myocyte
hyperplasia and heart block. Nature 377:744747[CrossRef][Medline]
-
Ichiki T, Labosky PA, Shiota C, Okuyama S, Imagawa Y, Fogo A,
Niimura F, Ichikawa I, Hogan BL, Inagami T 1995 Effects on blood
pressure and exploratory behaviour of mice lacking angiotensin II
type-2 receptor. Nature 377:748750[CrossRef][Medline]
-
Braszko J 1996 The contribution of AT1 and
AT2 angiotensin receptors to its cognitive effects. Acta
Neurobiol Exp 56:4954[Medline]
-
Meffert S, Stoll M, Steckelings UM, Bottari SP, Unger T 1996 The angiotensin II AT2 receptor inhibits proliferation and
promotes differentiation in PC12W cells. Mol Cell Endocrinol 122:5967[CrossRef][Medline]
-
Yamada T, Horiuchi M, Pratt R, Dzau V 1996 Angiotensin II type
2 receptor mediates programmed cell death. Proc Natl Acad Sci USA 93:156160[Abstract/Free Full Text]
-
Unger T, Chung O, Csikos T, Culman J, Gallinat S, Gohlke P,
Hohle S, Meffert S, Stoll M, Stroth U, Zhu YZ 1996 Angiotensin
receptors. J Hypertens Suppl 14:S95S103
-
Helin K, Stoll M, Meffert S, Stroth U, Unger T 1997 The role
of angiotensin receptors in cardiovascular diseases. Ann Med 29:2329[Medline]
-
Kambayashi Y, Bardhan S, Takahashi K, Tsuzuki S, Inui H,
Hamakubo T, Inagami T 1993 Molecular cloning of a novel angiotensin II
receptor isoform involved in phosphotyrosine phosphatase inhibition.
J Biol Chem 268:2454324546[Abstract/Free Full Text]
-
Mukoyama M, Nakajima M, Horiuchi M, Sasamura H, Pratt RE, Dzau
VJ 1993 Expression cloning of type 2 angiotensin II receptor reveals a
unique class of seven-transmembrane receptors. J Biol Chem 268:2453924542[Abstract/Free Full Text]
-
Bottari SP, Taylor V, King IN, Bogdal Y, Whitebread S, De
Gasparo M 1991 Angiotensin II AT2 receptors do not interact
with guanine nucleotide binding proteins. Eur J Pharmacol 207:157163[CrossRef][Medline]
-
Buisson B, Laflamme L, Bottari SP, de Gasparo M, Gallo-Payet
N, Payet MD 1995 A G protein is involved in the angiotensin
AT2 receptor inhibition of the T-type calcium current in
non-differentiated NG10815 cells. J Biol Chem 270:16701674[Abstract/Free Full Text]
-
Bottari SP, De Gasparo M, Steckelings UM, Levens NR 1993 Angiotensin II receptor subtypes: characterization, signaling
mechanisms, and possible physiological implications. Front
Neuroendocrinol 14:123171[CrossRef][Medline]
-
Kang J, Posner P, Sumners C 1994 Angiotensin II type 2
receptor stimulation of neuronal K+ currents involves an
inhibitory GTP binding protein. Am J Physiol 267:C1389C1397
-
Brechler V, Reichlin S, De Gasparo M, Bottari SP 1994 Angiotensin II stimulates protein tyrosine phosphatase activity through
a G-protein independent mechanism. Receptors and Channels 2:8997[Medline]
-
Dudley DT, Hubbell SE, Summerfelt RM 1991 Characterization of
angiotensin II (AT2) binding sites in R3T3 cells. Mol
Pharmacol 40:360367[Abstract]
-
Bottari SP, King IN, Reichlin S, Dahlstroem I, Lydon N, de
Gasparo M 1992 The angiotensin AT2 receptor stimulates
protein tyrosine phosphatase activity and mediates inhibition of
particulate guanylate cyclase. Biochem Biophys Res Commun 183:206211[Medline]
-
Sumners C, Tang W, Zelezna B, Raizada MK 1991 Angiotensin II
receptor subtypes are coupled with distinct signal-transduction
mechanisms in neurons and astrocytes from rat brain. Proc Natl Acad Sci
USA 88:75677571[Abstract]
-
Siragy HM, Carey RM 1996 The subtype-2 (AT2)
angiotensin receptor regulates renal cyclic guanosine 3',
5'-monophosphate and AT1 receptor-mediated prostaglandin E2 production
in conscious rats. J Clin Invest 97:19781982[Abstract/Free Full Text]
-
Siragy HM, Carey RM 1997 The subtype-2 (AT2)
angiotensin receptor mediates renal production of nitric oxide in
conscious rats. J Clin Invest 100:264269[Abstract/Free Full Text]
-
Takahashi K, Bardhan S, Kambayashi Y, Shirai H, Inagami T 1994 Protein tyrosine phosphatase inhibition by angiotensin II in rat
pheochromocytoma cells through type 2 receptor, AT2.
Biochem Biophys Res Commun 198:6066[CrossRef][Medline]
-
Gallinat S, Csikos T, Meffert S, Herdegen T, Stoll M, Unger T 1997 The angiotensin AT2 receptor down-regulates
neurofilament M in PC12W cells. Neurosci Lett 9:2932
-
Brunet A, Pouyssegur J 1997 Mammalian MAP kinase modules: how
to transduce specific signals. Essays Biochem 32:116[Medline]
-
Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel A 1995 A
synthetic inhibitor of the mitogen-activated protein kinase cascade.
Proc Natl Acad Sci USA 92:76867689[Abstract]
-
Klinz FJ, Wolff R, Heumann R 1996 Nerve growth
factor-stimulated mitogen activated kinase activity is not necessary
for neurite outgrowth of chick dorsal root ganglion sensory and
sympathetic neurons. J Neurosci Res 46:720726[CrossRef][Medline]
-
Sano M, Kitajima S 1998 Activation of mitogen-activated
protein kinases is not required for the extension of neurites from
PC12D cells triggered by nerve growth factor. Brain Res 785:299308[CrossRef][Medline]
-
Kojima H, Hara K, Mineta-Kitajima R, Taguchi F, Matsutani S,
Yamamoto N, Kodate S, Shirataka M, Tamai Y 1993 Isolation of a
subclonal cell line of PC12 transfected with dexamethasone-regulated
ras oncogene: morphological differentiation, biochemical
properties and tumorigenicity. J Biochem 114:194202[Abstract]
-
Szeberenyi J, Cai H, Cooper GM 1990 Effect of a dominant
inhibitory Ha-Ras mutation on neuronal differentiation of PC12 cells.
Mol Cell Biol 10:53245332[Medline]
-
Cowley S, Paterson H, Kemp P, Marshall CJ 1994 Activation of
MAP kinase kinase is necessary and sufficient for PC12 differentiation
and for transformation of NIH 3T3 cells. Cell 77:841852[Medline]
-
Pang L, Sawada T, Decker S, Saltiel A 1995 Inhibition of MAP
kinase kinase blocks the differentiation of PC-12 cells induced by
nerve growth factor. J Biol Chem 270:1358513588[Abstract/Free Full Text]
-
Vossler M, Yao H, York R, Pan M, Rim C, Stork P 1997 cAMP
activates MAP kinase and Elk-1 through a B-Raf- and Rap1-dependent
pathway. Cell 89:7382[Medline]
-
Yao H, York R, Misra-Press A, Carr D, Stork P 1998 The cyclic
adenosine monophosphate-dependent protein kinase (PKA) is required for
the sustained activation of mitogen-activated kinases and gene
expression by nerve growth factor. J Biol Chem 273:82408247[Abstract/Free Full Text]
-
Bos J 1998 All in the family? New insights and questions
regarding interconnectivity of Ras, Rap1 and Ral. EMBO J 17:67766782[Abstract/Free Full Text]
-
York RD, Yao H, Dillon T, Ellig CL, Eckert SP, McCleskey EW,
Stork PJ 1998 Rap1 mediates sustained MAP kinase activation induced by
nerve growth factor. Nature 392:622626[CrossRef][Medline]
-
Zwartkruis F, Wolthuis R, Nabben N, Franke B, Bos J 1998 Extracellular signal-regulated activation of Rap1 fails to interfere in
Ras effector signalling. EMBO J 17:59055912[Abstract/Free Full Text]
-
Hamprecht B, Glaser T, Reiser G, Bayer E, Propst F 1985 Culture and characteristics of hormone-responsive neuroblastoma X
glioma hybrid cells. Methods Enzymol 109:316341[Medline]
-
Beaman-Hall CM, Vallano ML 1993 Distinct mode of
microtubule-associated proteins expression in the neuroblastoma, glioma
cell line 108CC15/NG10815. J Neurobiol 24:15001516[Medline]
-
Posern G, Weber C, Rapp U, Feller S 1998 Activity of Rap1 is
regulated by bombesin, cell adhesion, and cell density in NIH3T3
fibroblasts. J Biol Chem 273:2429724300[Abstract/Free Full Text]
-
Hood J, Granger H 1998 Protein kinase G mediates vascular
endothelial growth factor-induced Raf-1 activation and proliferation in
human endothelial cells. J Biol Chem 273:2350423508[Abstract/Free Full Text]
-
Hein L, Meinel L, Pratt R, Dzau V, Kobilka B 1997 Intracellular trafficking of angiotensin II and its AT1 and
AT2 receptors: evidence for selective sorting of receptor
and ligand. Mol Endocrinol 11:12661277[Abstract/Free Full Text]
-
Greene L, Tischler A 1976 Establishment of a noradrenergic
clonal line of rat adrenal pheochromocytoma cells which respond to
nerve growth factor. Proc Natl Acad Sci USA 73:24242428[Abstract]
-
Peeper D, Upton T, Ladha M, Neuman E, Zalvide J, Bernards R,
DeCaprio J, Ewen M 1997 Ras signalling linked to the cell-cycle
machinery by the retinoblastoma protein. Nature 386:177181[CrossRef][Medline]
-
Pneuova N, Enikolopov G 1995 Nitric oxide triggers a switch to
growth arrest during differentiation of neural cells. Nature 375:6873[CrossRef][Medline]
-
Marrero M, Paxton W, Schieffer B, Ling B, Bernstein K 1996 Angiotensin II signalling events mediated by tyrosine phosphorylation.
Cell Signal 8:2126[CrossRef][Medline]
-
Schieffer B, Paxton WG, Chai Q, Marrero MB, Bernstein KE 1996 Angiotensin II controls p21ras activity via
pp60c-src. J Biol Chem 271:1032910333[Abstract/Free Full Text]
-
Chabre O, Cornillon F, Bottari S, Chambaz E, Vilgrain I 1995 Hormonal regulation of mitogen-activated protein kinase activity in
bovine adrenocortical cells: cross-talk between phosphoinositides,
adenosine 3', 5'-monophosphate, and tyrosine kinase receptor pathways.
Endocrinology 136:956964[Abstract]
-
Tian Y, Smith R, Balla T, Catt K 1998 Angiotensin II activates
mitogen-activated protein kinase via protein kinase C and Ras/Raf-1
kinase in bovine adrenal glomerulosa cells. Endocrinology 139:18011809[Abstract/Free Full Text]
-
Takahashi T, Kawahara Y, Okuda M, Ueno H, Takeshita A,
Yokoyama M 1997 Angiotensin II stimulates mitogen-activated protein
kinases and protein synthesis by a Ras-independent pathway in vascular
smooth muscle cells. J Biol Chem 272:1601816022[Abstract/Free Full Text]
-
Ueda Y, Hirai S, Osada S, Suzuki A, Mizuno K, Ohno S 1996 Protein kinase C activates the MEK-ERK pathway in a manner independent
of Ras and dependent on Raf. J Biol Chem 271:2351223519[Abstract/Free Full Text]
-
Arai H, Escobedo J 1996 Angiotensin II type 1 receptor signals
through Raf-1 by a protein kinase C-dependent, Ras-independent
mechanism. Mol Pharmacol 50:522528[Abstract]
-
Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR 1995 PD
098059 is a specific inhibitor of the activation of mitogen-activated
protein kinase kinase in vitro and in vivo.
J Biol Chem 270:2748927494[Abstract/Free Full Text]
-
Côté F, Laflamme L, Payet MD, Gallo-Payet N 1998 Nitric oxide, a new second messenger involved in the action of
angiotensin II on neuronal differentiation of NG 10815 cells. Endocr
Res 24:403407[Medline]
-
Poluha W, Schonhoff C, Harrington K, Lachyankar M, Crosbie N,
Bulseco D, Ross A 1997 A novel, nerve growth factor-activated pathway
involving nitric oxide, p53, and p21WAF1 regulates neuronal
differentiation of PC12 cells. J Biol Chem 272:2400224007[Abstract/Free Full Text]
-
Beavo J, Conti M, Heaslip R 1994 Multiple cyclic nucleotide
phosphodiesterase. Mol Pharmacol 46:399405[Abstract]
-
Ohmichi M, Decker SJ, Saltiel AR 1992 Activation of
phosphatidylinositol-3 kinase by nerve growth factor involves indirect
coupling of the trk proto-oncogene with src homology 2 domains. Neuron 9:769777[Medline]
-
Satoh T, Endo M, Nakafuku M, Akiyama T, Yamamoto T, Kaziro Y 1990 Accumulation of p21ras-GTP in response to stimulation
with epidermal growth factor and oncogene products with tyrosine kinase
activity. Proc Natl Acad Sci USA 87:79267929[Abstract]
-
Herrmann C, Martin G, Wittinghoffer A 1995 Quantitative
analysis of the complex between p21Ras and the Ras-binding domain of
the human Raf-1 protein kinase. J Biol Chem 269:29012905[CrossRef]
-
Warne PH, Viciana P, Downward J 1993 Direct interaction of Ras
and the amino-terminal region of Raf-1 in vitro. Nature 364 364:10311034
-
Albanese C, Johnson J, Watanabe G, Eklund N, Vu D, Arnold A,
Pestell RG 1995 Transforming p21ras mutants and c-Ets-2
activate the cyclin D1 promoter through distinguishable regions. J
Biol Chem 270:2358923597[Abstract/Free Full Text]
-
Boudreau F, Blais S, Asselin C 1996 Regulation of
CCAAT/enhancer binding protein isoforms by serum and glucocorticoids in
the rat intestinal epithelial crypt cell line IEC-6. Exp Cell Res 222:19[CrossRef][Medline]