From the Service of Endocrinology and the
§ Department of Physiology and Biophysics, Faculty of
Medicine, University of Sherbrooke,
Sherbrooke, Quebec J1H 5N4, Canada
Received for publication, March 13, 2002, and in revised form, November 11, 2002
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ABSTRACT |
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The angiotensin II (Ang II) type 2 (AT2) receptor is an atypical seven-transmembrane
domain receptor. Controversy surrounding this receptor concerns both
the nature of the second messengers produced as well as its associated
signaling mechanisms. Using the neuronal cell line NG108-15, we have
reported previously that activation of the AT2 receptor
induced morphological differentiation in a
p21ras-independent, but
p42/p44mapk-dependent mechanism.
The activation of p42/p44mapk was delayed,
sustained, and had been shown to be essential for neurite elongation.
In the present report, we demonstrate that activation of the
AT2 receptor rapidly, but transiently, activated the
Rap1/B-Raf complex of signaling proteins. In RapN17- and
Rap1GAP-transfected cells, the effects induced by Ang II were
abolished, demonstrating that activation of these proteins was
responsible for the observed p42/p44mapk
phosphorylation and for morphological differentiation. To assess whether cAMP was involved in the activation of Rap1/B-Raf and neuronal
differentiation induced by Ang II, NG108-15 cells were treated with
stimulators or inhibitors of the cAMP pathway. We found that dibutyryl
cAMP and forskolin did not stimulate Rap1 or
p42/p44mapk activity. Furthermore, adding H-89,
an inhibitor of protein kinase A, or Rp-8-Br-cAMP-S, an inactive cAMP
analog, failed to impair p42/p44mapk activity
and neurite outgrowth induced by Ang II. The present observations
clearly indicate that cAMP, a well known stimulus of neuronal
differentiation, did not participate in the AT2 receptor signaling pathways in the NG108-15 cells. Therefore, the
AT2 receptor of Ang II activates the signaling modules of
Rap1/B-Raf and p42/p44mapk via a
cAMP-independent pathway to induce morphological differentiation of
NG108-15 cells.
The angiotensin II (Ang
II)1 octapeptide hormone
binds two major receptor subtypes, type 1 (AT1) and
type 2 (AT2), both of which are expressed in several
tissues. One of the most remarkable features of the AT2
receptor is its high level of expression in most fetal tissues (1-3)
including the brain (4, 5). Some neuronal cell lines such as NG108-15
(6-8), PC12W (9, 10), and N1E 115 cells (11, 12) also express the
AT2 receptor at high levels. The
AT1:AT2 receptor ratio increases dramatically after birth (5, 13), suggesting an involvement of the AT2 receptor in fetal development. In the adult, the AT2
receptor expression is limited to some tissues, such as the adrenal
gland and specific areas of the brain. Several recent studies have
indicated that ligand-independent activation (14) or Ang II stimulation of the AT2 receptor is associated with antiproliferative
effects (15, 16), apoptosis (17, 18), and differentiation. Indeed, involvement of the AT2 receptor has been documented in
different models of differentiation such as steroidogenesis in gonads
or the adrenal gland (19-21), contractility in smooth muscle cells (22, 23), or neurite outgrowth in neuronal cell types (9, 24, 25) (for
review, see Refs. 26-29).
The precise nature of the signaling pathways activated by the
AT2 receptor is still controversial (for review, see Refs.
26-29). This seven-transmembrane domain receptor is not coupled to any of the classical, well established, second messengers, such as cAMP or
inositol phosphates, and its coupling to a G To study the effects of Ang II on differentiation, we have used
NG108-15 cells. In their undifferentiated state, neuroblastoma × glioma hybrid NG108-15 cells have a rounded shape and divide actively.
It is now well documented that chronic exposure to dibutyryl cAMP
induces a process resulting in both morphological and functional differentiation (42, 43). We have shown previously that these cells
express only the AT2 receptor of Ang II (6, 24) and that a
3-day treatment with Ang II or CGP42112 induced neurite outgrowth (24),
through a mechanism involving p21ras inhibition,
and a sustained increase in p42/p44mapk activity
(41). Because inhibition of p21ras using the
dominant negative mutant RasN17 failed to impair the ability of Ang II
to stimulate p42/p44mapk activity, an
alternative pathway for the activation of the
p42/p44mapk cascade must be considered. We have
demonstrated recently that nitric oxide and cGMP are involved in the
AT2 receptor effects on neurite outgrowth. However, this
pathway appeared to be a parallel, complementary, rather than an
intermediary step of the AT2 signaling cascade directed to
p42/p44mapk activation (33).
In PC12 cells stimulated with NGF, initiation of neurite outgrowth was
accompanied by a sustained increase in
p42/p44mapk activities (44, 45), mediated by
Rap1, a small guanine nucleotide-binding protein of the Ras family, and
B-Raf, a neuron-specific member of the Raf family of kinases (46-48).
The active GTP-bound form of Rap1 is known to bind, in
vitro, to most p21ras effectors of the Raf
family of kinases, in particular B-Raf and the Ral guanine nucleotide
exchange factors (RalGEFs), RalGDS (49, 50). Moreover, NGF stimulated
Rap1 activity via the exchange factor Crk/C3G and was also shown to
activate this pathway through a cAMP-dependent mechanism,
involving (51) or not involving protein kinase A (52). Similar
mechanisms could also be considered for NG108-15 cells because chronic
treatment with cAMP analogs is a well known differentiating factor for
these cells (42, 43). The aim of the present study was to investigate
whether the Rap1/B-Raf pathway could be involved in the Ang II-induced sustained increase in p42/p44mapk activity
participating in the morphological differentiation of the NG108-15
cells and to verify whether cAMP could be involved in the signaling
pathway of the AT2 receptor.
Materials--
Dulbecco's modified Eagle's medium (DMEM),
fetal bovine serum (FBS), HAT supplement (hypoxanthine, aminopterin,
thymidine), gentamycin, and LipofectAMINE were from Invitrogen.
[Val5]Ang II was from Bachem (Marina Delphen, CA, USA).
CGP42112 was obtained from CIBA-GEIGY (Basel, Switzerland).
Antiphosphorylated p42/p44mapk (1:1,000),
anti-p42/p44mapk (1:1,000), and anti-MEK1/2
(1:1,000) antibodies were from New England Biolabs (Beverly, MA).
GST-RalGDS fusion protein was a gift from Dr. J. L. Bos, Utrecht
University, Utrecht, The Netherlands. pCMVRapN17 and
pcDNA3.1/ Rap1GAP were gifts from Dr. P. J. Stork, Oregon Health
Sciences University, Portland, OR. Monoclonal anti-Rap1 (1:200)
antibody was from Transduction Laboratories (Mississauga, ON, Canada).
Antiphosphotyrosine (1:500), anti-Rap1/Krev (1:200), anti-B-Raf
(1:500), anti-Raf-1, and recombinant MEK1 were from Santa Cruz
Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were from Amersham Biosciences. CompleteTM protease inhibitor, polyvinylidene difluoride membranes, and the enhanced chemiluminescence (ECL) detection system were from
Roche Molecular Biochemicals. [ Cell Culture--
NG108-15 cells (provided by Drs. M. Emerit and
M. Hamon; INSERM, Unité 238, Paris, France) were cultured
(passages 12-21) in DMEM with 10% FBS, HAT supplement, and 50 mg/liter gentamycin at 37 °C in 75-cm2 Nunclon Delta
flasks in a humidified atmosphere of 93% air and 7% CO2
(43). Subcultures were performed at subconfluence. Under these
conditions, cells express only the Ang II AT2 receptor
subtype (24, 53). According to the experiments, cells were stimulated directly in culture medium for periods ranging from 1 min to 3 days (treated every day beginning 24 h after plating, for a 3-day treatment). Cells were treated without (control cells) or with 100 nM Ang II in the absence or presence of the inhibitors to be tested: H-89 (10 µM for short time experiments or 0.5 µM for 3-day treatment) or Rp-8-Br-cAMP-S (50 µM for all treatments).
Cell Transfection--
NG108-15 cells were transfected with
pCMVRapN17, pcDNA3.1/Rap1GAP, or with pcDNA3.1. Plasmid DNA (3 µg/ml) was mixed with 40 µg/ml LipofectAMINE and incubated at room
temperature for 30 min. For transfection, NG108-15 cells were grown to
subconfluence (70-80%) in 24-well Petri dishes and incubated for
3 h at 37 °C with the DNA-lipid complex. Transfection medium
was then replaced with complete, fresh medium and, 24 h
post-transfection, cells were harvested for quantification of
p42/p44mapk activity as described elsewhere
(33). Morphological studies were done on stably transfected cells,
grown in a Geneticin (G-418, 200 µg/ml)-containing medium.
B-Raf and Raf-1 Kinase Activity Measurements--
NG108-15 cells
were plated at a density of 2 × 105 cells in 100-mm
Petri dishes and used for experiments at 80% confluence. Cells were
incubated from 0 to 120 min with 100 nM Ang II, then washed
with Hanks' balanced solution and lysed in 1 ml of ice-cold buffer (70 mM Rap1 Activity Measurements--
The activated form of Rap1 was
pulled down with glutathione S-transferase (GST)-RalGDS
fusion protein from cell lysates as described elsewhere (56-58). Wild
type NG108-15 cells or cells stably transfected with pcDNA3.1,
RapN17, or Rap1GAP were cultured in 35- or 100-mm Petri dishes and, for
wild type cells, incubated from 0 to 60 min with 100 nM Ang
II or 10-100 nM CGP42112 (an AT2 receptor
agonist). Cells were washed with Hanks' balanced solution and
lysed in 1 ml of ice-cold lysis buffer containing 2,5% CHAPS in 50 mM Tris-HCl (pH 7.6), 140 mM NaCl, 5 mM MgCl2, 1 mM
Na3VO4 and CompleteTM mixture of inhibitors.
Lysates were centrifuged to eliminate insoluble material, and 150 µg
of total protein was incubated with 15 µl of a 50% slurry of
glutathione-Sepharose beads coupled with 5 µg of GST-RalGDS for
1 h at 4 °C. Beads were washed four times with lysis buffer,
and activated Rap1 protein was eluted with Laemmli buffer and resolved
by Western blotting (15% SDS-PAGE transferred on polyvinylidene
difluoride membrane and revealed with a Rap1 polyclonal antibody,
1:200).
Determination of Cells with Neurites--
NG108-15 cells were
plated at a density of 5 × 104 cells in 35-mm Petri
dishes and incubated for 3 days in FBS-containing medium without
(control) or in the presence of 100 nM Ang II, Ang II + 0.5 µM H-89, or Ang II + 50 µM Rp-8-Br-cAMP-S
(each introduced daily with the inhibitors applied 30 min prior to Ang
II) and were then examined under a phase contrast microscope. Cells
with at least one neurite longer than a cell body were counted as
positive for neurite outgrowth.
Western Blot Analysis--
Samples were loaded on 10 or 15%
SDS-polyacrylamide gels. Kaleidoscope prestained standards were loaded
to evaluate molecular weights of separated proteins. Gels were
transferred onto polyvinylidene difluoride membranes. Membranes were
blocked with 1% gelatin and probed with primary antibodies at the
indicated dilutions, followed by the appropriate secondary antibody
conjugated to horseradish peroxidase. Detection was performed by
chemiluminescence with ECL system on Kodak XK-1 films.
Measurement of cAMP Accumulation--
The ability of cells to
activate cAMP production and accumulation were measured by two
different methods. The capacity of cells to stimulate adenylyl cyclase
activity was determined by measuring the conversion of
[3H]ATP to [3H] cAMP, as described
previously (59). In short, cultured cells were incubated at 37 °C in
the DMEM (with 10% FBS) containing 1.5 µCi/ml
[3H]adenine. After 1 h, the cultures were washed and
incubated with 100 nM Ang II, 10 nM CGP42112,
or 20 µM forskolin in a Hanks' balanced solution
buffer containing 1 mM isobutylmethylxanthine (an inhibitor
of phosphodiesterase activity). After 15 min at 37 °C, cells were
collected, solubilized, and chromatographed on Dowex and alumina columns.
The total amount of cAMP contained in control or stimulated cells was
assayed using an enzyme immunoassay kit, as proposed by the
manufacturer (Amersham Biosciences). Briefly, NG108-15 cells were
cultured in DMEM + 10% FBS in 35-mm Petri dishes. Cells were
stimulated for various times with 100 nM Ang II, and for the last 15 min of stimulation, a final concentration of 1 mM isobutylmethylxanthine was added. At the end of the
experimental period, cells were incubated for 10 min with the lysis
reagent (0.5% dodecyltrimethylammonium bromide). Cellular extracts
were then transferred to 96-well plates, and antiserum against cAMP was
added to each well at 4 °C for 2 h. The competition procedure was initiated by adding cAMP conjugated to horseradish peroxidase to
the reaction for 60 min and terminated by extensive washes with 0.01 M phosphate buffer (pH 7.5) containing 0.05% (v/v) Tween 20. Enzyme substrate was added to each well where blue color was allowed to develop for 30 min, and the plate was then read at 630 nm on
a µQuant microplate reader (Bio-Tek Instruments, Inc.). Results are
presented as fmol of cAMP produced/µg of proteins. The protein
content of each sample was determined using Bio-Rad DC method.
Data Analysis--
The data are presented as mean ± S.E.
of the number of experiments indicated in the text. Homogeneity of
variance was assessed by Bartlett's test, and p values were
obtained from Dunnett's tables.
Ang II Stimulates B-Raf Kinase Activity--
NG108-15 cells were
treated with 100 nM Ang II for periods ranging from 5 to
120 min. Total B-Raf was immunoprecipitated from cell lysates using an
anti-B-Raf antiserum, and the kinase activity, using MEK1 as a
substrate, was determined as described under "Experimental Procedures." Quantification of MEK1 phosphorylation, expressed as a
ratio of MEK1 detected by autoradiography (Fig.
1A) over the total MEK1
determined by Western blotting (not shown), showed that Ang II induced
a rapid and transient increase in B-Raf kinase activity. The effect was
observed within 5 min of stimulation, reached a peak at 10 min, and
then decreased to control levels by 20 min (Fig. 1B). Raf-1
kinase activity was also measured but did not show any significant
differences over control level (data not shown). Western blot analyses
of cell homogenates indicated that Rap1 (left panel) and
B-Raf (right panel) proteins are indeed present at high
levels in the NG108-15 cells (Fig. 1C). Because Rap1 is
found ubiquitously and B-Raf has been demonstrated to be expressed
predominantly in neuronal tissues, the expression of both proteins was
compared using newborn rat brain homogenate as a positive control.
Interestingly, we observed that, like in the rat brain, NG108-15 cells
expressed the p95B-Raf, but the 68-kDa isoform
was undetectable.
Ang II Stimulates Rap1 Activity--
In vitro,
GTP-bound Rap1, the active form of this G protein, associates tightly
with RalGDS (50). Because B-Raf kinase activity usually arose from an
upstream stimulation of Rap1, we used this property to measure the
amount of activated Rap1, as described by Franke et al.
(58). Cell lysates were incubated with GST-RalGDS fusion protein
precoupled to glutathione beads. The activated Rap1 that bound to
RalGDS was then separated by SDS-PAGE and analyzed by Western blot
using anti-Rap1 antibodies. As shown in Fig.
2A, Ang II stimulation
significantly increased the total amount of Rap1-GTP associated with
GST-RalGDS within 30 s, persisted no longer than 5 min, to then
decrease under the basal level. The maximum response was observed after
1 min of stimulation with Ang II (1.35 ± 0.08-fold increase,
n = 9) (Fig. 2B, n = 4). As shown in Fig. 2C, increasing concentrations of the
AT2 receptor agonist CGP42112 also increased Rap1 activity
after a 1-min incubation, indicating that activation of Rap1 occurred
specifically through the AT2 receptor.
Rap1 Inhibition Prevents AT2-induced
p42/p44mapk Activation--
As described
previously, activation of Rap1 and B-Raf was shown to lead to sustained
p42/p44mapk activity (46-48). To investigate
further the involvement of Rap1 in the AT2 receptor
signaling, we transfected NG108-15 cells with RapN17 or Rap1GAP
constructs. RapN17 is a dominant negative mutant form of Rap1, and
Rap1GAP increases the GTPase activity of Rap1, thus favoring the
GDP-bound form (inactive form) of the protein (60, 61). As shown in
Fig. 3A, in transfected
NG108-15 cells (pcDNA3.1/Rap1GAP or pCMVRapN17), the active form of
Rap1 (the GTP-bound form) was decreased substantially, compared with
the native or pcDNA3.1-transfected NG108-15 cells. In
pcDNA3.1-transfected cells used as control, Ang II induced a
delayed phosphorylation of p42/p44mapk (as
determined with an antibody directed against the phosphorylated form of
p42/p44mapk). This effect is observed within 30 min of Ang II application (2.2 ± 0.4-fold increase over control),
sustained for 60 min (1.8 ± 0.3-fold increase over control), and
then decreased to basal level at 120 min (Fig. 3, B and
E). However, in RapN17- or Rap1GAP-transfected cells, Ang II
failed to activate p42/p44mapk (Fig. 3,
C-E). These results demonstrated that Rap1 activation is
necessary for Ang II to activate
p42/p44mapk.
Rap1 Activation Is Involved in AT2-induced
Morphological Differentiation--
We have shown previously that a
sustained activation of the p42/p44mapk pathway
was an essential event to promote neurite outgrowth in NG108-15 cells
treated with Ang II (41). Control pcDNA3.1-transfected cells had
similar rounded morphology, with only very short neurites, in some
cells, as in nontransfected NG108-15 cells (Fig. 4,
A and C). After a
3-day treatment with 100 nM Ang II, cells possessed many
neurites as native cells (Fig. 4, B and D).
Similar treatment conducted in cells transfected with pCMVRapN17 (Fig.
4, E and F) or pcDNA3.1/Rap1GAP (Fig. 4,
G and H) was unable to induce neurite outgrowth
and elongation. Quantification of these results demonstrated that a
3-day treatment with Ang II had no effect on neurite outgrowth in cells
expressing the dominant negative form of Rap1 (RapN17) or the specific
inhibitor for Rap1 (Rap1GAP) (Fig. 5).
Indeed, in control and pcDNA3.1-transfected NG108-15 cells, Ang II
induced a substantial increase in the number of cells with neurites
(from 6.3 ± 2.3% to 29.9 ± 3.5% in native NG108-15 cells
and from 6.1 ± 2.1% to 27.1 ± 7.6% in
pcDNA3.1-transfected cells). This effect was abolished in cells
expressing RapN17 or Rap1GAP. These observations indicated that Rap1
activation is required for the AT2-induced neurite
outgrowth.
Cyclic AMP and PKA Are Not Involved in AT2-induced
p42/p44mapk Activation--
Several studies
have documented the requirement of PKA for such a
Rap1-dependent activation of the
p42/p44mapk. To determine whether PKA could be
involved in the observed Ang II-induced
p42/p44mapk phosphorylation, we stimulated
NG108-15 cells with Ang II in the presence or in the absence of 10 µM H-89, an inhibitor of PKA. As shown in Fig.
6, A and B,
pretreatment of the cells with H-89 did not alter the activation of
p42/p44mapk induced by Ang II. Because it has
been shown that cAMP is able to activate
p42/p44mapk in a PKA-independent mechanism (52),
we verified such possibilities using cells stimulated with forskolin
(an activator of adenylyl cyclase). In NG108-15 cells, 10 nM forskolin decreased the basal level of phosphorylated
p42/p44mapk, an effect reversed when cells were
preincubated with Rp-8-Br-cAMP-S, an inactive analog of cAMP (Fig.
6C). This inhibition of p42/p44mapk
was observed within 5-10 min after forskolin application and persisted
for at least 2 h. The same results were also observed with a
cell-permeable cAMP analog, dibutyryl cAMP (1 mM) (data not
shown). These results indicated that cAMP was not involved in the
signaling pathway of the AT2 receptor which leads to a sustained, but delayed, p42/p44mapk
activation.
To support further our observations that cAMP is not involved in the
activation of p42/p44mapk in our model, Rap1
activity was measured in NG108-15 cells incubated in the presence of
forskolin at concentrations as high as 20 µM. This
cAMP-elevating agent failed to increase significantly the association
of Rap1 with GST-RalGDS (1.1 ± 0.1-fold increase over control)
(Fig. 6, D and E), at a time (5 min) where
p42/p44mapk was strongly inhibited with any
concentrations of forskolin tested (from 10 nM to 20 µM) (not shown). In comparison, Ang II induced a
significant increase in Rap1 activity of 1.35 ± 0.08-fold over control. These results suggest that in NG108-15 cells, cAMP was not
sufficient to increase Rap1 activity, reinforcing its absence of effect
in the AT2 receptor signaling mechanisms.
Cyclic AMP and PKA Are Not Involved in AT2-induced
Neurite Elongation--
To document further the absence of any
involvement of PKA and/or cAMP in the AT2-induced neurite
elongation, cells were left untreated (Figs.
7A and 8A) or
treated for 3 days with 100 nM Ang II (Figs. 7B
and 8B), in the absence or in
the presence of 0.5 µM H-89 (Fig. 7, C and
D) or 50 µM Rp-8-Br-cAMP-S (Fig. 8, C and D) (to block all cAMP-dependent
mechanisms). Both inhibitors were added, daily, 30 min before the
application of Ang II. Phase contrast micrographs indicated that cells
stimulated with Ang II alone or in the presence of H-89 (Fig. 7) or
Rp-8-Br-cAMP-S (Fig. 8) exhibit similar neurite elongations.
Quantification of these results indicated that cells stimulated with
Ang II alone or in the presence of H-89 exhibited numerous well
developed neurites (29.0 ± 3.9% and 30.6 ± 2.3%,
respectively), compared with control cells (8.6 ± 1.7%) (Fig.
9A). Similar results were
found for cells stimulated in the presence of Rp-8-Br-cAMP-S (Fig.
9B). These observations confirmed that inhibition of PKA did
not impair the ability of Ang II to induce neurite outgrowth in
NG108-15 cells.
The AT2 Receptor Is Not Coupled to cAMP
Production--
As shown in Fig.
10A, incubation of cells
with 100 nM Ang II or 10 nM CGP42112 (an
AT2 receptor agonist at that concentration), for period
ranging from 0 to 120 min, had no effect on cAMP accumulation. In
contrast, 20 µM forskolin induced a 110-fold increase in
cAMP accumulation, indicating that adenylyl cyclase is functional in the NG108-15 cells. Because cAMP could also be produced much later after hormonal stimulation, we also measured the intracellular content
of cAMP in NG108-15 cells after longer period of Ang II treatment. As
shown in Fig. 10B, the same kinds of result were obtained
using an enzyme immunoassay to determine the cAMP content in cells.
Together, these results clearly established that the AT2
receptor signaling did not involved cAMP as a second messenger for Rap1
and p42/p44mapk activation as well as for
neurite elongation.
In the present study, we have demonstrated that the binding of Ang
II to the AT2 receptor, in NG108-15 cells, increased
Rap1/B-Raf activities, two events accompanied by a concomitant decrease
in p21ras and a sustained increase in
p42/p44mapk activities. However, in contrast to
many G protein-coupled receptors that activate the Rap1/B-Raf cascade,
the action of Ang II is independent of cAMP or PKA, again confirming
the atypical nature of this seven-transmembrane domain receptor.
Application of Ang II, on NG108-15 cells, led to a transient
stimulation of B-Raf kinase activity, as measured by an in
vitro kinase assay using MEK1 as a substrate, but it did not
affect the basal level of Raf-1 activity. One of the better described activators of B-Raf is the small G protein Rap1 (62). Using RalGDS as
affinity probe, we found that Ang II treatment of NG108-15 cells
significantly increased the active, GTP-bound form, of Rap1 within 1 min and B-Raf within 5 min, whereas Raf-1 activity was not modified.
Members of the Raf family of proteins include Raf-1, A-Raf, and B-Raf.
Neurons are known to express the ubiquitous isoform Raf-1 and the
specific neuronal isoform B-Raf (63). In neurons stimulated with NGF,
B-Raf is the major isoform activated, with only 5% of the total
activity attributed to Raf-1 (64, 65). The findings that both proteins,
Rap1 and B-Raf, are activated after Ang II treatment are thus
compatible with the observation that activation of
p42/p44mapk is stimulated in a
p21ras-independent mechanism by the
AT2 receptor (41). Moreover, our results demonstrated that
Rap1 activation is responsible for the AT2-induced
p42/p44mapk phosphorylation as well as for the
Ang II-induced neurite outgrowth in NG108-15 cells. In fact, we find
that in cells transfected with RapN17 or Rap1GAP constructs (in which
the endogenous Rap1 activity is substantially decreased), Ang
II-induced p42/p44mapk activation is abolished.
In addition, these transfected cells exposed to Ang II have the same
morphology as the native, untreated NG108-15 cells. As we have shown
previously that a sustained activation of the
p42/p44mapk pathway was an essential event to
promote neurite outgrowth in NG108-15 cells treated with Ang II (41),
these results indicate that Rap1 activation is necessary for Ang II to
induce p42/p44mapk activation and that Rap1 is
required for the Ang II-induced neurite outgrowth.
How Rap1 could be activated in our model remain unknown. In several
models, cAMP and cAMP-activated proteins appeared to be of great
importance, and many studies have reported the requirement of PKA for
NGF activation of Rap1 as well as for
p42/p44mapk (47). Indeed, several studies
indicate that treatment of cells with cAMP or analogs induced neurite
outgrowth, both in PC12 (44) and in NG108-15 cells (24, 42, 43).
Activation of Rap1 by cAMP favored its association with Raf-1,
consequently inhibiting the transient
p21ras-dependent activation of
p42/p44mapk (66). In parallel, the action of
Rap1 on B-Raf accounted for the sustained stimulatory effect on
p42/p44mapk activity (51, 55, 66). In PC12 cells
stimulated with NGF, cAMP is required for a sustained increase of
p42/p44mapk activity (51), even if in these
cells, the NGF TrkA receptor is coupled directly to Rap1 through the
adaptor protein CrkII (47).
However, in the NG108-15 cells stimulated with Ang II, the present
results indicate that cAMP (or analogs) and PKA are not involved in the
increased activities of Rap1 and B-Raf. We report that the PKA
inhibitor H-89 or an inactive cAMP analog, Rp-8-Br-cAMP-S, neither
impaired the ability of Ang II to induce
p42/p44mapk activation nor did either affect
morphological differentiation. Furthermore, forskolin-induced cAMP
production in NG108-15 cells inhibited
p42/p44mapk phosphorylation but did not modify
Rap1 activity. These results corroborate our previous observations that
coincubation of Ang II with dibutyryl cAMP inhibits differentiation
induced by each stimulus alone (24). Furthermore, Ang II did not
stimulate cAMP production or accumulation, demonstrating that the
cAMP/PKA pathway is not involved in the AT2 receptor
signaling mechanisms leading to p42/p44mapk
activation and neuronal differentiation of NG108-15 cells. Those results corroborate the observations of Sanchez et al. (67) which indicate that in the human neuroblastoma cell line SH-SY5Y, cAMP-induced neurite outgrowth was independent of
p42/p44mapk activation.
Fig. 11 shows, in the same time scale,
the sequential activation of Rap1/B-Raf and
p42/p44mapk. The delay between B-Raf and
p42/p44mapk could be explained by the presence
of an intermediate type of reaction, such as activation of MEK. In
NGF-stimulated cells, York et al. (56) have shown a
sustained Rap1 activation mediated by TrkA internalization and
phosphoinositide 3-kinase activation. In this system, the
authors proposed that phosphoinositide 3-kinase favors
clathrin-dependent TrkA internalization into the
endocytotic compartment where Rap1 is localized. However, such a
mechanism is incompatible with the known behavior of the
AT2 receptor. Indeed, in contrast to many G protein-coupled
receptors, including the AT1 receptor, the AT2
receptor does not internalize (68, 69). In addition, the differences in
activation kinetics could be also the result of variations in the
molecular composition and the stability of protein complexes recruited
by the different receptors (65).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i
protein, reported by several authors (30-34), is not a
consensus. However, various mediators, which could individually exert
opposite effects, such as cGMP, tyrosine or serine/threonine
phosphatases, and the extracellular signal-regulated kinases ERK1/ERK2
(p42/p44mapk) have been associated with
activation of the AT2 receptor, depending on cell types and
experimental conditions used. More precisely, AT2 receptor
activation was shown to decrease p42/p44mapk
activities (35, 36), a process often associated with programmed cell
death (36-39), or stimulate a sustained increase in
p42/p44mapk activity (32), a process more
often associated with cellular differentiation (40, 41).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was from
PerkinElmer Life Sciences. All other chemicals were of grade A purity.
-glycerophosphate buffer (pH 7.2)
containing 0.5% Triton X-100, 100 µM
Na3VO4, 2 mM MgCl2, 1 mM EGTA, 5 µg/ml leupeptin, 20 µg/ml aprotinin, 1 mM dithiothreitol for B-Raf or 10 mM Tris (pH
7.4) containing 1% Triton X-100, 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 0.1% bovine serum albumin,
20 µM aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 2 mM Na3VO4 for
Raf-1). Briefly, total B-Raf and Raf-1 were, respectively,
immunoprecipitated from 2-mg cell extracts after Ang II treatment. The
precipitate was washed several times. Immune complex kinase assays were
performed as described, using recombinant MEK1 as substrate and
[
-32P]ATP as a phosphate donor for 30 min at 30 °C,
and the reaction was stopped by adding Laemmli buffer (54-56). The
kinase assay products were separated by electrophoresis on 10%
SDS-polyacrylamide gels, revealed with Kodak Biomax MS films and
analyzed with Image Quant software (Molecular Dynamics).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of Ang II on B-Raf kinase activity in
NG108-15 cells. A, NG108-15 cells (8 × 106 cells in 100-mm Petri dishes) were stimulated for the
indicated time periods in culture medium (DMEM containing 10% FBS).
B-Raf kinase activity induced by 100 nM Ang II was analyzed
as described under "Experimental Procedures" by immune complex
kinase assay using anti-B-Raf antiserum, recombinant MEK1 as a
substrate, and [ -32P]ATP as the phosphate donor. The
reaction product was resolved by SDS-PAGE and detected by
autoradiography. Numbers on the left indicate the
molecular mass of proteins in kDa. B, densitometric
analysis of the mean ± S.E. of three different experiments.
C, total extracts of NG108-15 cells (NG) were
submitted to SDS-PAGE followed by Western blot analysis to verify the
presence of Rap1 and B-Raf proteins as described under "Experimental
Procedures." A 2-day-old rat brain homogenate (B) was used
as a positive control. Western blots are representative of three
individual experiments. The lower band that appeared on the
Western blot analysis of p21rap1 has been
described by the manufacturer (Santa Cruz Biotechnology) to be a
nonspecific cross-reaction of this antibody.
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Fig. 2.
Activation of Rap1 by the AT2
receptor of Ang II in NG108-15 cells. NG108-15 cells (8 × 106 cells in 100-mm Petri dishes) were stimulated in
culture medium (DMEM containing 10% FBS). A and
B, time course effect of 100 nM Ang II and
C, dose-dependent effect of CGP42112 (after 1 min of incubation) on Rap1 activity. Rap1 activity was measured as
described under "Experimental Procedures." Cell lysates were
incubated with GST-RalGDS fusion protein precoupled to glutathione
beads. The activated Rap1 that bound to RalGDS was separated by
SDS-PAGE and analyzed by Western blot analysis using anti-Rap1
antibodies. The densitometric analysis of the mean ± S.E. of four
different experiments is shown.
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Fig. 3.
Effect of RapN17 and Rap1GAP expression on
Rap1 and p42/p44mapk activities in
NG108-15 cells. A, basal level of Rap1 activity in
pcDNA3.1-, RapN17-, or Rap1GAP-transfected NG108-15 cells compared
with wild type NG108-15 cells. Western blot analysis of GTP-bound Rap1
is representative of three different experiments. NG108-15 cells were
transiently transfected with pcDNA3.1 (B), pCMVRapN17
(C), or pcDNA3.1/Rap1GAP (D) as described
under "Experimental Procedures." 24 h post-transfection, cells
were incubated with 100 nM Ang II for the indicated times
and then harvested as described (33). Western blot analyses of
phosphorylated p42/p44mapk
(pp42mapk and pp44mapk)
are representative of three independent transfection experiments each
performed in duplicate. The total amount of
p42/p44mapk (shown in the lower part
of each panel) was determined after reprobing the membrane
presented in the upper panel with an anti-total
p42/p44mapk antibody. E,
densitometric analysis of the mean ± S.E. of three different
experiments, each in duplicate. *, p < 0.05, difference compared with basal level of
p42/p44mapk phosphorylation over total
p42/p44mapk.
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Fig. 4.
Effect of RapN17 and Rap1GAP on the Ang
II-induced neurite outgrowth in the NG108-15 cells. Nontransfected
NG108-15 cells (A and B) and NG108-15 cells
stably transfected with pcDNA3.1 (C and D), with
pCMVRapN17 (E and F), or with
pcDNA3.1/Rap1GAP (G and H) as described under
"Experimental Procedures." Control and transfected cells were left
untreated (A, C, E, and G)
or were treated with 100 nM Ang II for 3 days
(B, D, F, and H). Cells
were examined by phase contrast microscopy. All panels are
seen at the same magnification of ×1,000.
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Fig. 5.
Quantification of neurite outgrowth induced
by Ang II in transfected NG108-15 cells. Native NG108-15 cells
and NG108-15 cells stably transfected with pcDNA3.1, with
pCMVRapN17, or with pcDNA3.1/Rap1GAP were grown as described under
"Experimental Procedures" with or without 100 nM Ang
II. Cells with at least one neurite longer than a cell body were
counted as positive for neurite outgrowth. Results are expressed as
percentage of cells with neurites over the total amount of cells in the
micrographs. Results are the mean ± S.E. of at least 250 cells
from three independent transfection experiments, each performed in
triplicate. *, p < 0.05 and **, p < 0.02, difference compared with control, untreated cells.
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Fig. 6.
Effect of cAMP and PKA pathway on
p42/p44mapk activity in NG108-15
cells. NG108-15 cells (8 × 106 cells in 100-mm
Petri dishes) were incubated in the culture medium as described under
"Experimental Procedures," in the presence of 100 nM
Ang II alone (A) or with 10 µM H-89, a PKA
inhibitor (preincubated for 30 min; B). Cells were also
incubated in the presence of 10 nM forskolin with or
without 50 µM Rp-8-Br-cAMP-S (C). Western blot
analyses of phosphorylated p42/p44mapk
(pp42mapk and pp44mapk)
are representative of at least three different experiments. The total
amount of p42/p44mapk (shown in the lower
part of each panel) was determined after reprobing the
membrane presented in the upper panel with an anti-total
p42/p44mapk antibody. D, effect of 20 µM forskolin (5 min) (FSK) and 100 nM Ang II (1 min) on Rap1 activation. C
indicates control. E, densitometric analysis of the
mean ± S.E. of nine different experiments for Ang II and three
different experiments for forskolin. *, p < 0.05, difference compared with basal level of Rap1 activity.
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Fig. 7.
Effect of a PKA inhibitor (H-89) on Ang
II-induced neurite outgrowth in NG108-15 cells. NG108-15 cells
(plated at 5 × 104 cells in 35-mm Petri dishes) were
cultured for 3 days in DMEM containing 10% FBS alone (control cells,
A) or were stimulated daily with 100 nM Ang II
(B), 0.5 µM H-89, a PKA inhibitor
(C), or Ang II and H-89 (D). Cells were then
examined by phase contrast microscopy. All panels are seen
at the same magnification of ×1,000 and are representative of four
different experiments.
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Fig. 8.
Effect of Rp-8-Br-cAMP-S on Ang II-induced
neurite outgrowth in the NG108-15 cells. NG108-15 cells (plated at
5 × 104 cells in 35-mm Petri dishes) were cultured
for 3 days in DMEM containing 10% FBS alone (control cells,
A) or were stimulated daily with 100 nM Ang II
(B), 50 µM Rp-8-Br-cAMP-S, a cAMP antagonist
(C), or Ang II and Rp-8-Br-cAMP-S (D). Cells were
then examined by phase contrast microscopy. All panels are
seen at the same magnification of ×1,000 and are representative of at
least three different experiments.
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Fig. 9.
Quantification of neurite outgrowth induced
by Ang II. NG108-15 cells were grown as described under
"Experimental Procedures" and treated with 100 nM Ang
II in the absence (control, C) or in the presence of 0.5 µM H-89 (A) or 50 µM
Rp-8-Br-cAMP-S, an inactive analog of cAMP (B). Cells with
at least one neurite longer than a cell body were counted as positive
for neurite outgrowth. Results are expressed as percentage of cells
with neurites over the total amount of cells in the micrographs.
Results are the mean ± S.E. of three different experiments, each
performed in triplicate. *, p < 0.01, difference
compared with control cells.
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Fig. 10.
Effect of Ang II and CGP42112 on adenylyl
cyclase activity and cAMP accumulation. NG108-15 cells were grown
as described under "Experimental Procedures" and treated without
(control, C) or with 100 nM Ang II, 10 nM CGP42112, or 20 µM forskolin
(FSK) for the indicated time periods. The effects of Ang II
and CGP42112 on adenylyl cyclase activity (A) and of Ang II
on the intracellular cAMP level (B) were quantified as
described under "Experimental Procedures." Results are the mean of
two different experiments, each performed in triplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 11.
Activation kinetics of Rap1, B-Raf, and
p42/p44mapk after Ang II activation in
NG108-15 cells. The kinetics data for Rap1 and B-Raf are from
Figs. 1 and 2. The kinetic data for p42/p44mapk
activities are from Figs. 3 and 6 combined with data published
previously (33, 41). Results are shown as protein activity (-fold
increase over basal level) over the duration of the stimulation with
100 nM Ang II.
Our recent observations on the AT2 receptor signaling
mechanisms in NG108-15 cells are summarized in Fig.
12. After binding of Ang II, the
activated AT2 receptor rapidly inactivates
p21ras (5-120 min) (41) and enhances the level
of the GTP-bound form of Rap1 (1-5 min). Activated Rap1 then enhances
the activity of B-Raf (5-15 min) which in turn stimulates
p42/p44mapk phosphorylation (30-60 min).
Finally, the return of p42/p44mapk
phosphorylation to basal level, occurring much later (seen after 120 min of Ang II treatment), may be under a phosphotyrosine phosphatase activity such as SHP-1, shown to be activated after the AT2
receptor stimulation (39).
|
Ang II also stimulates the nitric oxide/soluble guanylyl cyclase/cGMP cascade of signaling (33). Our studies have shown that this cascade, together with MAPK activation, is involved in neurite outgrowth, through a mechanism independent of Ras activation. All of these pathways may have as final targets regulation of gene expression and modulation of the phosphorylation states of different microtubule-associated proteins such as MAP2, tau, and MAP1b, as we have shown previously in NG108-15 cells (24) as well as in granule cells from rat cerebellum (25).
Three important conclusions can be drawn from the present work. First, Ang II, in NG108-15 cells, induced a rapid activation of Rap1 and B-Raf, two proteins that account for the sustained activation of p42/p44mapk induced by the AT2 receptor. Second, AT2 signaling mechanisms leading to Rap1/B-Raf-dependent p42/p44mapk activation and neurite elongation are clearly cAMP-independent. Third, the capability of cAMP to induce neurite outgrowth and neuronal differentiation in the NG108-15 cells occurs independently of the sustained increase in p42/p44mapk activity. Altogether, these observations suggest that when present in the cell type studied, such as neurons, the Rap1/B-Raf signaling cascade could play a pivotal role in transduction (63). Thus, the presence of Rap1/B-Raf may explain the controversial results surrounding the AT2 receptor action (in particular apoptosis versus differentiation). In addition, as many G protein-coupled receptors, the AT2 receptor may probably recruit some not yet identified partners, which altogether, contribute to the complex regulation of morphological neuronal differentiation.
Lessons from knockout mice or from neurological disorders reinforce the
idea that the AT2 receptor may be important for neuronal development. Indeed, perturbations in exploratory behavior and locomotor activity were observed (70, 71) as well as an anxiety-like behavior (72). In addition, a decrease in the expression of the
AT2 receptor is observed in areas of the adult brain
implicated in the development of neurological disorders such as
Alzheimer's disease, Huntington's disease, or Parkinson's disease
(caudate nucleus, putamen and substantia nigra, temporal cortex) (73). Finally, recent observations indicate that AGTR2 mutations
are correlated with mental retardation (74). Together, such
observations indicate inappropriate neuronal differentiation and
plasticity (in adult) when AT2 is absent or genetically modified.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. J. L. Bos (Utrecht University, Utrecht, The Netherlands) for the GST-RalGDS fusion protein and Dr. P. J. Stork (Oregon Health Sciences University, Portland, OR) for the pCMVRapN17 and pcDNA3.1/Rap1GAP. We also thank Lucie Chouinard for experimental assistance.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Canadian Institute for Heath Research (to N. G.-P. and M. D. P.).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.
¶ Recipient of a studentship from the Fonds de la recherche en santé du Québec.
These authors contributed equally to this work.
** To whom correspondence should be addressed: Service of Endocrinology, Faculty of Medicine, University of Sherbrooke, 3001 12th Ave., Sherbrooke, Quebec J1H 5N4, Canada. Tel.: 819-564-5243; Fax: 819-564-5292; E-mail: Nicole.Gallo_Payet@USherbrooke.ca.
¶¶ Holder of the Canadian Research Chair in endocrinology of the adrenal gland.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M202446200
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ABBREVIATIONS |
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The abbreviations used are: Ang II, angiotensin II; AT1 and AT2, type 1 and 2 Ang II receptors, respectively; 8-Br-cAMP-S, 8-bromo-adenosine cyclic 3':5'-phosphorothioate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GST, glutathione S-transferase; mapk, mitogen-activated protein kinase gene; MEK, mitogen-activated protein kinase kinase; NGF, nerve growth factor; PKA, protein kinase A.
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