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Endocrinology Vol. 147, No. 10 4646-4654
Copyright © 2006 by The Endocrine Society

Role of Tyrosine Kinase Receptors in Angiotensin II AT2 Receptor Signaling: Involvement in Neurite Outgrowth and in p42/p44mapk Activation in NG108-15 Cells

Bianca Plouffe, Marie-Odile Guimond, Hélène Beaudry and Nicole Gallo-Payet

Service of Endocrinology and Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4

Address all correspondence and requests for reprints to: Nicole Gallo-Payet, Service of Endocrinology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4. E-mail: Nicole.Gallo-Payet{at}USherbrooke.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
NG108–15 cells, which have a rounding-up morphology when cultured in serum-supplemented medium, extend neurites when stimulated for 3 d with angiotensin II (Ang II). The aim of the present study was to investigate whether growth factor receptors are necessary for mediating the effects of Ang II. A 3-d treatment with AG879, an inhibitor of nerve growth factor receptor TrkA, strongly affected neurite outgrowth and phosphorylation of p42/p44mapk induced by Ang II. PD168393, an inhibitor of epidermal growth factor (EGF) receptor slightly decreased Ang II-induced neurite outgrowth, whereas AG213, an inhibitor of both platelet-derived growth factor receptor and EGF receptor, stimulated neurite outgrowth and p42/p44mapk phosphorylation on its own, without affecting further stimulation with Ang II. Moreover, Ang II induced the phosphorylation of TrkA (maximum at 5 min of incubation in the presence of serum or at 20 min in cells depleted in serum for 2 h) and a rapid increase in Rap1 activity, both effects abolished in cells preincubated with 10 µM AG879. In summary, the present results demonstrate that AT2 receptor-induced sustained activation of p42/p44mapk and corresponding neurite outgrowth are mediated by phosphorylation of the nerve growth factor TrkA receptor. However, the results also point out that the presence of other growth factors, such as EGF or PDFG, may interfere with the effect of Ang II. Altogether, the current findings clearly indicate that the effects of the AT2 receptor on neurite outgrowth dynamics are modulated by the presence of growth factors in the culture medium.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ANGIOTENSIN II (Ang II) octapeptide, the active component of the renin-angiotensin system, binds and activates two major types of seven transmembrane domain G protein-coupled receptors, namely AT1 and AT2. Much of the classical actions of Ang II, including modulation of blood pressure, control of fluid/electrolyte balance, and cellular proliferation, are associated with activation of the AT1 receptor (1, 2). In contrast, activation of the AT2 receptor is known to negatively modulate the effects associated with AT1 receptor activation. Indeed, in the cardiovascular system, AT2 receptor activation induces vasodilation, inhibition of cell proliferation, induction of programmed cell death, and remodeling of the extracellular matrix (for reviews see Refs.2, 3, 4, 5, 6, 7). In contrast, Ang II, through the AT2 receptor, increases glucose intolerance and elevates hepatic triglyceride, thus participating in diet-induced obesity and insulin resistance (8, 9). Activation of the AT2 receptor is also implicated in cellular differentiation in fetal tissues (for review see Ref. 10) and in axonal regeneration after injury in the adult (11, 12).

Induction of neurite outgrowth and elongation is one of the best characterized roles of the AT2 receptor in cells of neuronal origin. Specific activation of the AT2 receptor induces neurite outgrowth in neuronal cell lines, in NG108–15 cells (13) and PC12W cells (14) as well as in primary cultures of rat cerebellar granule cells (15), cortical neurons, and striatum, in which respective colocalization of the AT2 receptor with ß-tubulin as well as with the microtubule-associated protein, MAP2, has been shown (16).

Although significant progress has been achieved during the past 5 yr, the precise nature of the signaling pathways activated by the AT2 receptor is still poorly understood. This receptor is not coupled to any of the classical, well-established second messengers, such as cAMP or inositol phosphates (for reviews see Refs. 1 , 10 , and 17, 18, 19, 20). In our endeavor to elucidate AT2 receptor involvement in Ang II-induced neuronal differentiation, we and others have investigated signaling mechanisms activated by this receptor. In NG108–15 cells, we have found that specific activation of the AT2 receptor induces an increase in p42/p44mapk activities (21). Similar results were also obtained in PC12W (22) as well as in nonneuronal COS-7 (23) and NIH3T3 cells overexpressing the AT2 receptor (24). In both NG108–15 (21) and PC12W cells (22), Ang II-induced p42/p44mapk activation appears essential for inducing neurite outgrowth. We also have shown that the related Ras/Raf-1 cassette of signaling, namely Rap1/B-Raf, is involved in AT2 receptor signaling mechanisms leading to morphological differentiation of NG108–15 cells (10), although the exact nature of these mechanisms remains to be established. Because these experiments were conducted in the presence of serum in the culture medium (21, 23), two hypotheses could be proposed: either the AT2 receptor induces transactivation of a receptor tyrosine kinase (RTK) or the AT2 receptor acts in coordination with a RTK signaling pathway initiated by activation of a differentiating factor present in the serum. Among the possible candidates present in the serum are nerve growth factor (NGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF), all described as modulators of neurite outgrowth (25, 26, 27, 28, 29).

Therefore, the present study was conducted to identify the possible involvement of RTK in the effect of the AT2 receptor in promoting neurite outgrowth and p42/p44mapk activation by Ang II; and, whether such is the case, determine whether or not Ang II-induced Rap1 activation is mediated through activation of this RTK.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
The chemicals used in the present study were obtained from the following sources: DMEM, fetal bovine serum (FBS), HAT supplement (hypoxanthine, aminopterin, thymidine), and L-glutamine from Invitrogen Life Technologies, Inc. (Burlington, Ontario, Canada); [Val5]-Ang II and Taxol from Sigma (Oakville, Ontario, Canada); CGP42112A from CIBA-GEIGY (Basel, Switzerland); PD168393, AG213, AG879, and NGF from Calbiochem-Novabiochem (La Jolla, CA); Kaleidoscope prestained standards from Bio-Rad (Mississauga, Ontario, Canada); PD98059, antiphosphorylated p42/p44mapk (1:1000), anti-p42/p44mapk (1:1000), anti-phosphorylated TrkA (directed against tyrosine 490 of TrkA) (1:500) antibody from New England Biolabs (Beverly, MA); anti-TrkA (1:500) antibody and anti-Rap1 (1:200) from Santa Cruz Biotechnology (Santa Cruz, CA); horseradish peroxidase-conjugated antimouse, antirabbit antibodies, enhanced chemiluminescence (ECL) detection system, glutathione Sepharose beads, and Hyperfilms ECL from GE Healthcare (Baie d’Urfe, Quebec, Canada); NJ ß-tubulin (1:500) antibody from Chemicon International (Temecula, CA); complete protease inhibitor and polyvinylidene difluoride membranes from Roche Laboratories (Montreal, Quebec, Canada). All other chemicals were of grade A purity.

Cell culture
NG108–15 cells (initially provided by Drs. M. Emerit and M. Hamon; Institut National de la Santé et de la Recherche Médicale, Unité 238, Paris, France) were cultured (passages 15–29) in DMEM culture medium with 10% FBS, HAT supplement, L-glutamine, and 50 mg/liter gentamycin at 37 C in 75-cm2 Nunclon Delta flasks in a humidified atmosphere of 93% air and 7% CO2, as described previously (21, 30, 31). Subcultures were performed at subconfluency. Under these conditions, cells express only the Ang II AT2 receptor subtype (13).

Cell treatments
According to experiments, cells were stimulated directly in culture medium for intervals ranging from minutes to 3 d (treated daily beginning 24 h after plating). Cells were incubated without NGF (control cells), with NGF (100 ng/ml), or with Ang II (100 nM) in the absence or in the presence of various inhibitors of growth factor receptor activity. PD168393 is a selective inhibitor of EGF receptor (EGFR) tyrosine kinase activity, which acts by binding to the catalytic domain of the EGFR (32); AG213 is an inhibitor of EGF and PDGF receptor (PGDFR) tyrosine kinase activity (33, 34, 35, 36, 37, 38), whereas AG879 inhibits NGF-dependent p140c-trk tyrosine phosphorylation and blocks NGF-induced neurite outgrowth in PC12 cells (36, 39). AG879 does not affect tyrosine phosphorylation of the EGFR or PGDFR (40). Concentrations of inhibitors used were chosen according to their EC50 values and concentrations documented in publications. Cells were treated in the absence or in the presence of PD168393 (0.3 µM for both 3-d treatments and short-term experiments) (41, 42, 43), AG213 (4 µM for 3-d treatments or 30 µM for short-term experiments) (44, 45), AG879 (2 µM for 3-d treatments or 10 µM for short-term experiments) (46, 47, 48), or PD98059 (10 µM), as previously described (21). Cells were treated daily with inhibitors applied 30 min before Ang II or NGF (100 ng/ml) treatment. At the concentrations used, cells remained attached to the substratum and maintained the same morphological appearance as control or Ang II-stimulated cells.

Determination of neurite-exhibiting cells
NG108–15 cells were plated at a density of 6 x 104 cells in 35-mm Petri dishes and incubated for 3 d in 10% FBS containing medium without (control) or in the presence of 100 nM Ang II alone or with various inhibitors, as mentioned above. Three different experiments were conducted. For each experiment, three Petri dishes were used for each condition, and between 168–225 cells from each of the triplicate dishes were examined under a phase contrast microscope. Cells with at least one neurite longer than a cell body were counted as positive for neurite outgrowth, as previously described (49).

Extraction of polymerized microtubules
Preparations enriched in microtubules were obtained from cells grown in 100-mm Petri dishes (equivalent to 8 x 103 cells) as described previously (13). Cells were pretreated with 1 µM Taxol for 2 h before extraction of microtubules. The culture medium was then aspirated and replaced by PM2G buffer (1,4-piperazinediethanesulfonic acid, 0.1 M; glycerol, 2 M; MgCl2, 5 mM; EGTA, 2 mM, aprotinin, 20 µM; phenylmethylsulfonyl fluoride, 2 mM; benzamidine, 1 mM, pH 6.9) containing Taxol (1 µM). Cells were scraped from the substratum and counted, and the cell pellet was extracted with PM2G buffer containing 1% Nonidet P-40 and 1 µM Taxol. After a 15-min incubation at 37 C, the resulting suspension was centrifuged and the pellet containing the microtubules was solubilized in a specific volume of Tris buffer 125 mM, pH 6.8, containing 4% SDS (wt/vol), 20% glycerol (vol/vol), and 10% ß-mercaptoethanol (vol/vol) to obtain an equivalent number of cells per milliliter and heated to 100 C for 5 min. After centrifugation at 10,000 x g for 5 min, the supernatant was stored at –20 C until Western blot analysis was performed as described above.

Western blotting
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/or inhibitors incubated 30 min before addition of Ang II. The reaction was stopped by aspiration of the medium. Cell lysis was performed at 4 C by adding 200 µl of lysis buffer per 35-mm Petri dish (in 50 mM HEPES, pH 7.8, 1% Triton X-100, 0.1 µM staurosporine, 1 mM Na3VO4, and Complete cocktail of protease inhibitors) (for phosphorylated p42/44mapk) or by adding 300 µl of lysis buffer per 100-mm Petri dish [62.5 mM Tris-HCl (pH 6.8), 2% wt/vol SDS, 10% glycerol, 50 mM dithiothreitol, and 0.01% wt/vol bromophenol blue] (for phospho-TrkA). Cell extracts were centrifuged at 8000 x g for 10 min at 4 C (for phosphorylated p42/44mapk) or sonicated and boiled for 5 min (for phospho-TrkA). Equal amounts of protein (12–20 µg) were separated on SDS-polyacrylamide gels. Western blotting was performed as previously described (21, 31). Samples were loaded on 8 or 10% SDS-polyacrylamide gels. After transfer onto polyvinylidene difluoride membranes, membranes were blocked with 1% gelatin, 0.05% Tween 20 in TBS buffer, pH 7.5, or with 5% milk, 0.1% Tween 20 in TBS buffer for phospho-TrkA and TrkA, and subsequently incubated with primary antibodies at the following dilutions and incubation times in blocking buffer or TBS-Tween 20 (0.05%): 1/1000 for 2 h for phospho-p42/44mapk, 1/1000 overnight for p42/44mapk, 1/500 for 2 h for ß-tubulin, 1/500 overnight for phospho-TrkA, and 1/500 for 2 h for TrkA. Membranes were washed six times for 10 min each and incubated with the appropriate secondary antibody conjugated to horseradish peroxidase in blocking buffer for 1 h. After subsequent washing, detection was performed by chemiluminescence with an ECL system on Hyperfilms ECL. Stripping of membranes was achieved by incubation in a solution comprised of 0.2 M glycine (pH 2.5), 0.05% Tween 20 for 2 h at 70 C.

Rap1 activity measurements
The activated form of p21rap was pulled down with glutathione S-transferase (GST)-RalGDS fusion protein from cell lysates as described previously (10). NG108–15 cells cultured for 3–4 d in 35-mm Petri dishes were stimulated with NGF 100 ng/ml or with 100 nM Ang II with or without the inhibitor AG879 10 µM for intervals ranging from 0–30 min. Extraction was subsequently performed as described by Gendron et al. (10). Cells were washed rapidly and lysed for 30 min in 100 µl of ice-cold lysis buffer containing 2% CHAPS in 50 mM Tris-HCl (pH 7.6), 140 mM NaCl, 5 mM MgCl2, 1 mM Na3VO4, and Complete cocktail of inhibitors. After centrifugation, lysates were incubated with 20 µl of 50% slurry glutathione-Sepharose beads precoupled with GST-Ral-GDS for 1 h at 4 C with gentle agitation. Activated p21rap protein (GTP-bound form) was eluted with Laemmli buffer at 95 C and resolved by Western blotting on 15% SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride membranes followed by revelation of p21rap (21 kDa) with a Rap1 polyclonal antibody (1/200).

Data analysis
The data are presented as means ± SE of the number of experiments indicated in parentheses. Statistical analyses of the data were performed by one-way ANOVA for Figs. 5Go and 6Go and two-way analysis for Figs. 2–4GoGoGo using the Sigma Stats software package. Homogeneity of variance was assessed by Bartlett’s test. F values were determined using Fisher’s test, and P values were determined using Tukey’s posttest for significant differences. At least three different experiments were conducted for each figure, each performed in triplicate.


Figure 5
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  		FIG. 5. Effect of Ang II on the phosphorylation of the TrkA receptor. NG108–15 cells were cultured in 100-mm Petri dishes until 80% confluency (5 x 106 cells per dish) in DMEM containing 10% FBS and then stimulated in the presence of serum (A and C) or in the absence of serum (B and D) without or with 100 nM Ang II, alone or in the presence of 10 µM AG879, or with 100 ng/ml NGF for the indicated times. A and B, TrkA phosphorylation (pTrkA) was revealed by Western blot using an anti-phosphorylated TrkA and anti-TrkA antibodies, as described in Materials and Methods. B and D, Comparative densitometric analyses of six independent experiments (mean ± SE). *, P < 0.001, indicates comparison between control and Ang II stimulation.

 

Figure 6
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  		FIG. 6. Effect of NGFR inhibition on Ang II-induced Rap1 activity in NG108–15 cells. NG108–15 cells were cultured for 3–4 d in 35-mm Petri dishes until 80% confluency (1 x 106 cells per dish) in DMEM containing 10% FBS and subsequently stimulated without or with 100 nM Ang II, alone or in the presence of 10 µM AG879 or with 100 ng/ml NGF for the indicated times. A, Cells were then extracted and the activated form of Rap1 was pulled down by using GST-RalGDS followed by Western blotting of Rap1, as described in Materials and Methods. B, Densitometric analysis of four independent experiments (mean ± SE). *, P < 0.01; **, P < 0.001, difference compared with control value, as calculated by two-way ANOVA followed by Bartlett’s test.

 

Figure 2
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  		FIG. 2. Quantification of the effect of tyrosine kinase receptor inhibitors on Ang II-induced morphology of NG108–15 cells. NG108–15 cells were plated at a density of 6 x 104 cells in 35-mm Petri dishes and cultured for 3 d in DMEM containing 10% FBS in the absence or in the presence of 100 nM Ang II alone or with inhibitors (0.3 µM of the EGFR inhibitor, PD168393; 4 µM of the EGFR and PDGFR inhibitor, AG213; 2 µM of the NGFR inhibitor, AG879; or 10 µM of the MEK1 inhibitor, PD98059), as described in Materials and Methods. Cells with at least one neurite longer than a cell body were counted as positive for neurite outgrowth. Data are mean ± SE of three different experiments, each in duplicate. Differences between various groups were analyzed by two-way ANOVA followed by Bartlett’s test. §, P < 0.001, indicates comparison with corresponding conditions (basal ± inhibitors or Ang II ± inhibitors); and **, P < 0.001, indicates comparison between control and Ang II stimulation.

 

Figure 3
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  		FIG. 3. Effect of tyrosine kinase receptor inhibitors on Ang II-induced ß-tubulin polymerization in NG108–15 cells. NG108–15 cells were plated at a density of 6 x 104 cells in 35-mm Petri dishes and cultured for 3 d in DMEM containing 10% FBS in the absence (control, C) or in the presence of 100 nM Ang II alone or with inhibitors (0.3 µM of the EGFR inhibitor, PD168393; 4 µM of the EGFR and PDGFR inhibitor, AG213; 2 µM of the NGFR inhibitor, AG879; or 10 µM of the MEK1 inhibitor, PD98059), as described in Materials and Methods. A, Representative Western immunoblotting of polymerized ß-tubulin performed on microtubule-enriched cell preparations. B, Densitometric analysis of experiments conducted from four different experiments (mean ± SE). Differences between various groups were analyzed by two-way ANOVA followed by Bartlett’s test. §, P < 0.001, indicates comparison with corresponding conditions (basal ± inhibitors or Ang II ± inhibitors); and *, P < 0.05, indicates comparison between control and Ang II stimulation.

 

Figure 4
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  		FIG. 4. Effect of tyrosine kinase receptor inhibitors on Ang II-induced p42/p44mapk activity in NG108–15 cells. NG108–15 cells were cultured for 3 d in 35-mm Petri dishes until 80% confluency (1 x 106 cells per dish) in DMEM containing 10% FBS and subsequently stimulated without or with 100 nM Ang II for the indicated times. Western blot experiments were performed using an anti-phosphorylated p42/p44mapk and anti-p42/p44mapk antibodies, as described in Materials and Methods. A, Effect of Ang II alone (control condition) or after a 30-min preincubation with 0.3 µM of the EGFR inhibitor, PD168393, or 30 µM of the EGFR and PDGFR inhibitor, AG213, or 10 µM of the NGFR inhibitor, AG879, or 10 µM of the MEK1 inhibitor, PD98059. B, Comparative densitometric analyses of experiments conducted after 30 min of treatment. Data are the mean ± SE of eight independent experiments. Differences between various groups were analyzed by two-way ANOVA followed by Bartlett’s test. **, P < 0.001, indicates comparison between control and Ang II stimulation.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The presence of NGF receptors (NGFR) is necessary for Ang II-induced neurite outgrowth
To determine which growth factor receptor could modify morphological changes induced by Ang II, cells were stimulated for 3 d without or with Ang II and various growth factors receptors inhibitors. Control cells exhibited a rounded cell body, without or with only a few thin processes (Fig. 1AGo). When treated for 3 d with 100 nM Ang II, cells exhibited one or two neurite processes, often turning and branching, with varicosities along its rod (Fig. 1Go, B and b). Cells cultured for 3 d in the presence of 0.3 µM PD168393 (an inhibitor of EGFR kinase activity) displayed similar morphology to control cells (Fig. 1CGo). However, in the presence of Ang II, the cell body became flattened and many cells, although not all, had shorter neurites than with Ang II alone (Fig. 1Go, D and d). By contrast, cells treated with 4 µM AG213 (an inhibitor of EGFR and PDGFR) exhibited extended neurites (Fig. 1EGo); coincubation with Ang II further increased this effect (Fig. 1FGo), with neurites appearing straighter and more rigid than with Ang II and without varicosities (Fig. 1fGo). Treatment with 2 µM AG879 (an inhibitor of NGFR) did not modify control morphology (Fig. 1GGo), but reduced the effect of Ang II to very small neurites (Fig. 1Go, H and h). Preincubation with 10 µM PD98059 (a MEK1 inhibitor) induced a rounding-up appearance of cells, even more pronounced (Fig. 1Go, J and j) than in control cells (Fig. 1AGo).


Figure 1
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  		FIG. 1. Effect of tyrosine kinase receptor inhibitors on Ang II-induced morphology of NG108–15 cells. NG108–15 cells were plated at a density of 6 x 104 cells in 35-mm Petri dishes and cultured for 3 d in DMEM containing 10% FBS in the absence (A, C, E, G, and I) or in the presence of 100 nM Ang II alone (B) or in the presence of 0.3 µM of the EGFR inhibitor, PD168393 (C and D), 4 µM of the EGFR and PDGFR inhibitor, AG213 (E and F), 2 µM of the NGFR inhibitor, AG879 (G and H), or 10 µM of the MEK1 inhibitor, PD98059 (I and J), as described in Materials and Methods. Cells were examined by phase-contrast microscopy with a x20 objective (scale bars, 28 µm). b, d, f, h and j, Magnifications of representative neurites appearing in the corresponding panels are shown (scale bars, 80 µm).

 
Neurite outgrowth was quantified (Fig. 2Go) and analyzed by two-way ANOVA. As shown in Table 1Go, there was a statistically significant difference between basal values in the control group compared with cells treated with PD168393 or AG213 (1.2- and 4.8-fold increase, respectively), hence corroborating morphological data. Treatment with PD168393 or AG213 decreased Ang II responsiveness, but nevertheless, remained statistically different from their own control values (2.6- and 1.2-fold increase respectively, compared with 4.7-fold increase in control cells). In contrast, neurite elongation was abrogated in AG879- and PD98059-treated cells.


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  		TABLE 1. Effect of tyrosine kinase receptor inhibitors on angiotensin II (Ang II)-induced neurite outgrowth in NG108–15 cells

 
Western blot analyses of polymerized ß-tubulin exhibited the same profile and statistical results as observed for morphological analyses (Fig. 3Go, A and B). Indeed, results confirmed that the increase in the level of polymerized tubulin observed with Ang II treatment (1.8 ± 0.1-fold increase over control) was abolished in cells preincubated with AG879 and PD98059. Results also confirmed that incubation with AG213 alone was sufficient to increase polymerization of tubulin, whereas PD168383 had no significant effect.

NGFR but not EGFR or PDGFR is involved in Ang II-induced p42/p44mapk phosphorylation
Stimulation with Ang II leads to sustained p42/p44mapk phosphorylation, with maximal effect after a 30-min incubation with Ang II, a condition necessary for inducing neurite outgrowth in NG108–15 cells as shown in Fig. 1JGo and as previously described. To investigate the involvement of RTK in p42/p44mapk activation by the AT2 receptor, cells were stimulated without or with inhibitors of RTK or PD98059 for 30 min before addition of 100 nM Ang II. As illustrated in Fig. 4AGo and quantified in Fig. 4BGo, incubation with AG879 and PD98059 significantly decreased the basal value of p42/p44mapk phosphorylation, although preincubation with AG213 alone significantly increased this phosphorylation by 2-fold. Stimulation with Ang II induced p42/p44mapk phosphorylation, which was unaffected by the presence of 0.3 µM PD168393 or 30 µM AG213. In contrast, preincubation of cells with 10 µM AG879 or with 10 µM PD98059 produced a strong inhibition of p42/p44mapk phosphorylation. These results indicate that NGFR plays a significant role in mediating the effect of Ang II on p42/p44mapk phosphorylation, whereas EGFR and PDGFR do not appear to interfere with this Ang II-induced phosphorylation process.

Ang II induces phosphorylation of the TrkA receptor
Because the above results suggest that activation of NGFR is necessary for p42/p44mapk phosphorylation and subsequent neurite outgrowth, we therefore investigated whether Ang II stimulation could activate the TrkA (tropomyosin-related kinase) receptor of NGF using an antibody targeted toward tyrosine 490 phosphorylation of the TrkA receptor. This site is critical for Shc binding and hence for activation of the MAPK signaling cascade (50). As shown in Fig. 5AGo, Ang II-induced phosphorylation of TrkA with a maximum observed after 5 min of incubation (1.8 ± 0.08%-fold increase over basal level). Preincubation of cells with 10 µM of AG879 inhibited TrkA phosphorylation induced by Ang II (Fig. 5CGo).

Because these experiments were performed in the presence of serum, we also tested whether Ang II was able to increase TrkA phosphorylation in absence of serum. As shown in Fig. 5Go, B and D, stimulation with Ang II on cells depleted in serum for 2 h still increased TrkA phosphorylation, an effect inhibited by AG879. However, this stimulation was delayed (maximum at 20 min), compared with NGF or Ang II stimulation in the presence of serum (Fig. 5AGo) (maximum at 5 min).

Inhibition of TrkA interferes with Rap1 activation by Ang II
The increase in p42/p44mapk phosphorylation by Ang II has previously been shown to be initiated by Rap1 activation (10), similarly to NGF-induced differentiation. To verify whether Rap1 activation by Ang II is dependent or not on TrkA phosphorylation, NG108–15 cells were incubated with 100 nM Ang II alone or in the presence of 10 µM AG879 or with 100 ng/ml NGF for various time intervals ranging from 0–30 min. As illustrated in Fig. 6Go, Ang II induced a rapid increase in Rap1 activity, which was maximal between 1–5 min and returned to basal values after 30 min. This stimulation was abolished in cells preincubated with AG879, indicating that TrkA is necessary for activation of Rap1 by Ang II. NGF also stimulated Rap1 activity, although along a different stimulation profile. The initial transient increase was followed by a more sustained level that remained higher than the basal value.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The hybrid neuroglial cell line NG108–15, which actively divides when cultured in serum-supplemented medium, is able to switch into a neuron-like phenotype upon AT2 receptor stimulation by Ang II. Over the past 10 yr, with the aim of elucidating the manner in which the Ang II AT2 receptor induces neuronal differentiation, we and others have identified sustained activation of p42/p44mapk as essential for neurite outgrowth. The present study provides evidence that the AT2 receptor requires interaction with the NGF TrkA receptor to initiate this increase in p42/p44mapk activation and neurite outgrowth.

Experiments conducted herein revealed that incubation with AG879, a specific inhibitor of the TrkA receptor of NGF, had a strong inhibitory effect on neurite outgrowth and on p42/p44mapk phosphorylation stimulated by Ang II. On the contrary, when cells were treated for 3 d with AG213, an EGFR and PDGFR inhibitor, a significant increase of neurite elongation was observed, with a concomitant increase in microtubule polymerization. In contrast, inhibition of EGFR with PD168393 slightly decreased the effect of Ang II, indicating that the effects observed with AG213 are probably a consequence of PDGFR inhibition. Inhibitors also produce specific informative effects on cell morphology. As previously described (49), cells stimulated with Ang II exhibited three or four neurites and had a bright and round cell body. The longer process exhibited varicosities with no linear trajectories. By contrast, cells treated with AG213 exhibit one straight neurite, probably resulting from stable interaction between microtubule-associated proteins and tubulin (51).

The observation that inhibition of the PDGFR enhances neurite outgrowth and increases the basal level of phosphorylation state of p42/p44mapk in NG108–15 cells is in agreement with 1) results indicating that PDGFß receptor mutants defective for mitogenesis promote neurite outgrowth in PC12 cells (29), 2) results reported by Seidman et al. (52), in which the differentiation of NG108–15 cells can be induced in cells cultured in a serum-free medium, and 3) our recent observation that a decrease in proliferation is associated with promotion of neurite outgrowth in NG108-cells (53). However, mechanisms involved in PDGF action are different from those involved in Ang II because coincubation of Ang II with AG213 increases both neurite outgrowth and p42/p44mapk phosphorylation, in comparison to cells stimulated with Ang II alone. This observation indicates that inhibition of PDGFR by AG213 does not interfere with AT2 receptor-induced p42/p44mapk phosphorylation. An alternative hypothesis may be that AT2 receptor is able to inhibit the growth-promoting effect of PDGFR. Indeed, overexpression of AT2 receptor in mouse decreases the expression of PGDF-BB chain (PDGF-BB) (54).

Morphologically, PD168393- and Ang II-treated cells became flattened, and many cells, but not all, exhibited shorter neurites than under Ang II stimulation alone. Meanwhile, cells preincubated with AG879 were flattened and displayed only very short extensions, but did exhibit branching. Finally, PD98059 induced a rounded-up appearance of cells, without the presence of neurites. These differences indicate that morphological differentiation induced by Ang II involves several complementary pathways, some of which only are mediated by growth factor receptors. Indeed, only the MEK inhibitor, PD98059 abolished Ang II-induced neurite outgrowth and bright cell body morphology. AG879 interfered with the process of elongation, but not with the capacity to exhibit branching. Such observations support our previous observations that indeed, elongation was mediated through p42/p44mapk, whereas branching was dependent of cGMP-PKG activation (49). Such observations strongly support the hypothesis that inhibition of p42/p44mapk phosphorylation inhibits the capacity of microtubule-associated proteins (such as MAP2, MAP1B, or {tau}) to induce tubulin polymerization and extension (13, 51).

EGF and PDGF are well-known growth factors, acting through Ras/Raf-1-transient activation of p42/p44mapk to induce proliferation. By contrast, at least in NGF-stimulated PC12 cells, initiation of neuronal differentiation is associated with a reduction in cell proliferation and a sustained activation (through Rap1/B-Raf) of p42/p44mapk (for review see Refs. 55 and 56). Our results also indicate that Ang II-induced TrkA phosphorylation and Rap1 activation through TrkA receptor is delayed, thus consistent with the observation that phosphorylation of Trk receptors by G protein-coupled receptors is slow, in comparison to NGF receptor activation, which occurs rapidly (57). The results also suggest that growth factors present in the serum (such as PDGF) could interfere with or accelerate the effect of the AT2 receptor on TrkA phosphorylation.

Why does Ang II require TrkA to activate Rap1? We previously have demonstrated that, in contrast to NGF, activation of Rap1 by Ang II occurs through a cAMP- and protein kinase A-independent pathway (31). Rap1, in contrast to Ras, is tightly associated with Ral-GDS in intracellular membranes (58, 59, 60). In NGF-stimulated cells, York et al. (61) have shown that Rap1 activation is mediated by TrkA internalization and phosphatidylinositol 3-kinase activation. The AT2 receptor cannot activate Rap1 through this process, because in contrast to many G protein-coupled receptors, the AT2 receptor does not internalize (62, 63). Thus, from the present results, it could be hypothesized that AT2 uses the internalization property of the activated TrkA receptor to mediate Rap1 activation.

What is the basis of this interaction between Ang II and TrkA? One possibility is that the AT2 receptor transactivates the TrkA receptor. Such interaction between TrkA receptor and G protein-coupled receptor has recently been observed in PC12 cells stimulated with lysophosphatidate receptor 1 (LPA1 receptor) (64), adenosine via adenosine 2A receptors (65), and pituitary adenylate cyclase activating polypeptide (66, 67), in which transactivation could take place on intracellular membranes (67). There are a few examples in the literature citing interactions between the AT2 receptor and tyrosine kinase receptors. Recently, a group of investigators have reported the molecular cloning of a novel protein, AT2-interacting protein, which interacts with the C-terminal tail of the AT2 receptor, but not AT1. Ectopic expression of this protein in eukaryotic cells leads to inhibition of EGFR-induced ERK2 activation indicating that the AT2 receptor is capable of interacting with EGFR and inactivating the latter via AT2-interacting protein (68). Other investigators have found that stimulation of the AT2 receptor induces tyrosine dephosphorylation of many proteins with apparent molecular masses between 80–180 kDa in N1E-115 neuroblastoma cells (69). These molecular weights are associated with tyrosine kinase receptors and support a possible role for AT2 receptor in the negative regulation of cell proliferation. Furthermore, Tsuzuki et al. (70) observed that AT2 receptor stimulation inhibits basic fibroblast growth factor-stimulated cell proliferation via activation of protein tyrosine phosphatase in R3T3 fibroblast cells expressing the AT2 subtype. In fact, phosphatases and particularly Src homology 2 domain-containing tyrosine phosphatase-1 (SHP-1) are often associated with the AT2 receptor signaling cascade (71, 72, 73, 74). The AT2 receptor, by activating SHP-1, is well known for inactivating EGFR in AT2-overexpressing smooth muscle cells (73), the insulin receptor in AT2-transfected CHO cells (75), and insulin-induced phosphatidylinositol 3-kinase activation in PC12 cells (76). A possible mechanism leading to the transactivation of TrkA receptor by the AT2 receptor in our model may be via the capacity of SHP-1 to induce Src activation (77). Recent findings suggest that Src facilitates phosphorylation of TrkA in SK-N-MC cells (78).

In summary, the results herein indicate that AT2 receptor-induced sustained activation of p42/p44mapk is mediated by phosphorylation of the TrkA receptor of NGF probably through a process of transactivation. However, the mechanisms linking the AT2 receptor to the TrkA receptor remain to be elucidated. The present results also confirm that a large portion of AT2 receptor-induced neurite outgrowth, but not all, requires p42/p44mapk activation. In contrast, the presence of other growth factors, such as EGF or PDFG, may interfere with the effect of Ang II. Altogether, these findings clearly indicate that the effects of the AT2 receptor may be modulated by growth factors present in the medium.


   Acknowledgments
 
We are grateful to Lucie Chouinard for her expert experimental assistance and Dr. Marcel D. Payet (Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada) for stimulating discussions.


   Footnotes
 
N.G.-P. is a recipient of a Canada Research Chair in Endocrinology of the Adrenal Gland. This work was supported by grants from the Canadian Institute for Health Research to N.G.-P. and Marcel D. Payet (MOP27912).

This work was presented as an oral communication at the "Société de Neuroendocrinologie," 32nd meeting, September 16–18, 2004, La Grande Motte, France.

Author disclosure summary: B.P., M.-O.G., H.B., and N.G.-P. have nothing to declare.

First Published Online June 29, 2006

Abbreviations: Ang II, Angiotensin II; ECL, enhanced chemiluminescence; EGF, epidermal growth factor; EGFR, EGF receptor; FBS, fetal bovine serum; GST, glutathione S-transferase; NGF, nerve growth factor; NGFR, NGF receptor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; RTK, receptor tyrosine kinase; SHP-1, Src homology 2 domain-containing tyrosine phosphatase-1.

Received October 17, 2005.

Accepted for publication June 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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