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 NG108–15 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 NG108–15. 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 NG108–15 cells resulted in time-dependent modulation of tyrosine phosphorylation of a number of cytoplasmic proteins. Stimulation of NG108–15 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 NG108–15 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 NG108–15 cells not only include inhibition of p21ras but an increase in MAPK activity as well, which is essential for neurite outgrowth.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 NG108–15 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 NG108–15 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 NG108–15 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 NG108–15 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 NG108–15 cells include not only inhibition of p21ras but an increase in MAPK activity, which is essential for neurite outgrowth.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effect of Ang II on Protein Tyrosine Phosphorylation
Figure 1AGo shows that stimulation of NG108–15 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. 1BGo, 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.



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Figure 1. Western Blot Analysis of the Effect of Ang II on Tyrosine Phosphorylation in NG108–15 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{gamma}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 NG108–15 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. 2Go, 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. 2Go, 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. 2Go, 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. 2EGo).



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Figure 2. Effect of Ang II on p21ras Activity in NG108–15

NG108–15 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. 3Go). 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. 3Go, 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. 3BGo). 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 3DGo shows that after blockade of the AT2 receptor by PD123319, Ang II induced a rapid and transient increase in MAPK phosphorylation.



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Figure 3. Effect of Ang II on MAPK Activity in NG108–15 Cells

NG108–15 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 NG108–15 cells after Ang II treatment, we aimed at verifying whether MAPK activation was necessary for the differentiation of NG108–15 cells. As previously published (10), a 3-day treatment with Ang II caused an increase in neurite formation (Fig. 4BGo vs. 4A). Coincubation of Ang II with 10 µM PD98059 for 3 days, a dose at which MAPK is inhibited (Fig. 3BGo), caused a decrease in the length and number of neurites (Fig. 4CGo), an effect further amplified using 50 µM of PD98059 (Fig. 4DGo), 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. 4EGo). 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. 4EGo). 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. 4FGo).



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Figure 4. Effect of PD98059 on Neurite Outgrowth in NG108–15 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 NG108–15 Cells Transfected with RasN17
To further investigate the involvement of p21ras on Ang II-induced neurite outgrowth, we produced a stably transfected NG108–15 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. 5AGo) 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. 5BGo), 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. 5CGo, where the basal level of GTP-bound Ras was decreased by 47% in RasN17 cells, compared with control transfected cells (Fig. 5CGo). Figure 6Go shows the morphological consequences of a 3-day expression of RasN17 in NG108–15 cells. The control transfected cells were polygonal, actively dividing and extended one or two small processes, much like untransfected NG108–15 cells (compare Fig. 6AGo with 4A). After 3 days of IPTG induction, the RasN17-transfected cells exhibited several long processes (Fig. 6BGo). The neurites appeared longer and more branched than that of untransfected NG108–15 cells treated for 3 days with Ang II (Fig. 6BGo vs. Fig. 4BGo). Immunofluorescence for ß-tubulin exemplified the morphological appearance of neurites. One or two thin processes were observed in control transfected cells (Fig. 6CGo), while long neurites with distinct growth cones at each tip were observed in IPTG-induced RasN17 cells (Fig. 6DGo). These morphological changes were correlated with an increase in the level of polymerized tubulin (Fig. 6EGo).



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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 {alpha}-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 NG108–15 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.

 


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Figure 6. Morphology of RasN17-Transfected NG108–15 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. 7AGo) or in IPTG-induced RasN17 cells (Fig. 7BGo) induced the same time-dependent increase in MAPK activity, as described in NG108–15 cells (Fig. 3Go). As for NG108–15 cells, the maximal increase was 7.92-fold over control for p42mapk and 10.5 over control for p44mapk in control transfected cells (Fig. 7CGo) and 3.92- and 7.15-fold over control for p42mapk and p44mapk, respectively, in IPTG-induced RasN17 cells (Fig. 7DGo). Moreover, IPTG induction of RasN17 increased the basal activity of p42mapk/p44mapk compared with control transfected cells or NG108–15 cells (Fig. 7EGo, c vs. a).



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Figure 7. Effect of Ang II on MAPK Activity in Stably RasN17-Transfected NG108–15 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 NG108–15 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. 8CGo vs. Fig. 8BGo), adopting morphology similar to control cells (Fig. 8CGo vs. 8A). Application of 50 µM PD98059 had a major inhibitory effect on cell morphology (Fig. 8DGo). 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. 8EGo). 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. 8EGo). 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).



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Figure 8. Effect of PD98059 on Neurite Outgrowth in RasN17-Transfected NG108–15 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 NG108–15 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 NG108–15 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 NG108–15 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 NG108–15 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 NG108–15 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 NG108–15- 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 NG108–15 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 NG108–15 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 NG108–15 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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), [{alpha}-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
NG108–15 cells (provided by Drs M. Emerit and M. Hamon; INSERM, U. 238, Paris, France) were cultured (passage 12–21) 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). NG108–15 cells (1 x 106 cells) were incubated for various time intervals at 37 C in Hank’s 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
NG108–15 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
NG108–15 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. 2CGo; 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 95–100 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, NG108–15 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 81–131 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, NG108–15 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 NG108–15 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 NG108–15 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.


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