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Endocrinology, doi:10.1210/en.2006-0411
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Endocrinology Vol. 147, No. 9 4263-4272
Copyright © 2006 by The Endocrine Society

Involvement of Protein Kinase C{alpha} (PKC{alpha}) in the Early Action of Angiotensin II Type 2 (AT2) Effects on Neurite Outgrowth in NG108–15 Cells: AT2-Receptor Inhibits PKC{alpha} and p21ras Activity

Hélène Beaudry, Louis Gendron, Marie-Odile Guimond, Marcel D. Payet and Nicole Gallo-Payet

Service of Endocrinology (H.B., L.G., M.-O.G., N.G.-P.) and Department of Physiology and Biophysics (L.G., M.-D.P.), Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4

Address all correspondence and requests for reprints to: Nicole Gallo-Payet, Service d’endocrinologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4. E-mail: nicole.gallo-payet{at}usherbrooke.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The aim of the present study was to investigate whether protein kinase C (PKC) isoforms may be among the putative candidates implicated in the primary effects of the Ang II type 2 (AT2) receptor. Western blot analyses revealed the presence of PKC{alpha},{epsilon}, {iota}, and {zeta} in NG108–15 cells. After a 3-d treatment with 3 nM Gö6976, a specific inhibitor of classical PKC isoforms, cells were characterized by the presence of one elongated process similar to that observed after treatment with Ang II or with CGP42112, a selective AT2 receptor agonist. Similar findings were observed in cells expressing a dominant-negative mutant of PKC{alpha} (K368A). Inhibition of PKC{alpha} in NG108–15 cells also decreased cell number and proliferation. In conditions of acute stimulation, Ang II induced a time-dependent and transient inhibition of PKC{alpha} activity, as well as a decrease in PKC{alpha} levels associated with the membrane. Treatment of cells with Gö6976 was also found to inhibit p21ras (between 1–10 min) but stimulated Rap1 activity (1–5 min) in a time-course similar to that of Ang II. Incubation of NG108–15 cells with Gö6976 (3 nM) inhibited basal p42/p44mapk phosphorylation, but failed to interfere with its activation by the AT2 receptor, indicating that inhibition of PKC{alpha} is not directly involved in the Rap1-MEK-p42/p44mapk cascade. Taken together, these results indicate that PKC{alpha} is a primary target of the AT2 receptor. Inhibition of PKC{alpha} leads to a decrease in both p21ras activity and cell proliferation, which may facilitate AT2 receptor signaling through p42/p44mapk, thereby leading to neurite outgrowth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ANGIOTENSIN II (Ang II) type 2 (AT2) receptor is a member of the seven-transmembrane domain G protein-coupled receptor family. One of the most striking features of the AT2 receptor is its high level of expression in most fetal tissues (1, 2) including the brain (3, 4). Some neuronal cell lines such as NG108–15 (5, 6, 7), PC12W (8), and N1E-115 cells (9) also express the AT2 receptor at high levels. The AT1 to AT2 receptor ratio increases dramatically after birth (4, 10), suggesting an involvement of the AT2 receptor in fetal development. Indeed, 3-d treatment of cultured NG108–15 cells, PC12W cells, as well as rat cerebellar granule cells with Ang II induced neurite outgrowth (8, 11, 12). Moreover, this Ang II-induced morphological differentiation of cells from neuronal origin was accompanied by an increase in polymerized ß-tubulin levels (8, 11, 12) as well as in the modulation of neuronal excitability and in the promotion of cell migration (13).

In the adult, AT2 receptor expression is limited to only scattered tissues such as the adrenal gland and specific areas of the brain where its activation is involved in various cellular processes including vasodilation, inhibition of cell proliferation, induction of programmed cell death, remodeling of the extracellular matrix, and axonal regeneration (for reviews see Refs. 13, 14, 15). Interestingly, under certain pathological conditions (heart and renal failure, myocardial infarction, brain lesions, vascular injury, and wound healing), the AT2 receptor was shown to be reexpressed, thus suggesting a key role for Ang II and the AT2 receptor in tissue repair and regeneration (16, 17, 18).

The precise nature of the signaling pathways activated by the AT2 receptor is still poorly understood. This seven-transmembrane domain receptor is not coupled to any of the classical, well-established second messengers such as cAMP or inositol phosphates (for reviews see Refs. 13 and 19, 20, 21, 22). However, various mediators that, individually, can exert opposite cellular and physiological effects, have been associated with activation of the AT2 receptor, depending on cell type and experimental conditions. More precisely, a sustained increase in p42/p44mapk activity was found to be associated with neuronal differentiation. Hence, stimulation of the AT2 receptor with Ang II or CGP42112 induced a delayed but sustained activation of the p42/p44mapk cascade in NG108–15 (23) and in PC12W (24) cells, as well as in COS-7 (25) and NIH3T3 cells overexpressing the AT2 receptor (26). Concomitant with this AT2-induced p42/p44mapk activation, which is essential for the induction of neurite outgrowth, a decrease in p21ras activity was also observed (23). Moreover, we have recently 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 (27). Nitric oxide and cGMP are also involved in the effect of the AT2 receptor on neurite outgrowth. However, cGMP is not involved in Ang II-induced activation of p42/p44mapk. Indeed, blockade of any protein from this signaling cascade (nitric oxide synthase, soluble guanylyl cyclase, cyclic GMP-dependent protein kinase) failed to interfere with the effects of Ang II on p42/p44mapk signaling (28).

What remains enigmatic at this point are the initial events linking AT2 receptor activation with various signaling cascades, in addition to the link between the various steps initiating each pathway. Among the putative candidates for the missing components of this AT2 signaling puzzle are protein kinase C (PKCs). PKC isoforms are a family of enzymes implicated in the regulation of growth, survival, and differentiation of several cell types. At least 11 genes coding for 12 different isoforms have been subgrouped into classical (PKC{alpha}, ßI, ßII, and {gamma}), novel (PKC{delta}, {epsilon}, {eta}, and {theta}), and atypical (PKC{iota}/{lambda} and {zeta}) isoforms in addition to PKCµ and {upsilon}, which have completely different characteristics. This classification is based on the regulatory and structural properties of each isoform (29, 30, 31). It is well known that different isoforms of PKC can have opposite effects on growth or differentiation within the same cell system. Thus, specific targeting of a particular PKC isoform may be sufficient to influence cell growth. Several publications have described the involvement of some PKC isoforms, particularly PKC{epsilon} and {zeta}, in nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells (32, 33), aplysia neurons (34, 35), and in various cell lines (36). Such possibilities have not been investigated for the AT2-mediated effects of Ang II. Therefore, the present study was conducted to test the hypothesis that PKCs may be involved in the signaling cascade regulating neuritogenesis by Ang II. In a first instance, we sought to determine the expression patterns of classical, novel, and atypical PKCs in undifferentiated and Ang II-treated cells. Secondly, using selective PKC inhibitors, we aimed at delineating the role of PKC on Ang II-induced neurite elongation as well as on cell growth and activation of the p42/p44mapk signaling pathway. Results reveal that among the isoforms present in NG108–15 cells, PKC{alpha} plays a pivotal role in signaling and that Ang II inhibits activity of PKC{alpha}, leading to a decrease in both p21ras activity and proliferation. These effects facilitate AT2 receptor signaling through p42/p44mapk, hence resulting in neurite outgrowth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
The chemicals used in this study were obtained from the following sources: DMEM, fetal bovine serum, and gentamycin from Life Technologies, Inc. (Burlington, Ontario, Canada); hypoxanthine, aminopterin, thymidine supplement, anti-PKC{alpha}, anti-PKC{delta}, and anti-PKC{zeta} from Sigma (Oakville, Ontario, Canada); anti-PKCß, anti-PKC{gamma}, anti-PKC{epsilon}, anti-PKC{eta}, anti-PKC{theta}, and anti-PKC{iota} from BD Biosciences (Mississauga, Ontario, Canada); anti-PKCµ and anti-Rap1 from Santa Cruz Biotechnology (Santa Cruz, CA); Ang II from Bachem (Marine, Delphen, CA); CGP42112 from Ciba-Geigy (Basel, Switzerland); and Gö6976 and monoclonal antibody to p21ras (Pan-Ras Ab-3 antibody) from Calbiochem-Novabiochem (La Jolla, CA). The dominant-negative PKC{alpha}DN (K368A) was a kind gift from Drs. Jean-Guy LeHoux and Gilles Dupuis (Université de Sherbrooke, Sherbrooke, Quebec, Canada) (37). Four Fast-flow G-Sepharose protein, horseradish peroxidase-conjugated antimouse and antirabbit antibodies were from Amersham Pharmacia Biotech (Oakville, Ontario, Canada); Complete protease inhibitor, polyvinylidene difluoride (PVDF) membranes and enhanced chemiluminescence (ECL) system from Roche (Montréal, Quebec, Canada); nitrocellulose membranes from Bio-Rad (Mississauga, Ontario, Canada); antirabbit Alexa Fluor 594 from Molecular Probes (Eugene, OR); Vectashield from Vector Laboratories (Burlingame, CA); Bio-Rad detergent compatible protein assay from Bio-Rad Laboratories (Hercules, CA); and antiphosphorylated p42/p44mapk and anti-p42/p44mapk from New England Biolab (Beverly, MA). Glutathione S-transferase (GST)-RalGDS fusion protein was a kind gift from Dr. Johannes L. Bos (Utrecht University, Utrecht, The Netherlands) and GST-RafRBD fusion protein was from Dr. Nathalie Rivard (Université de Sherbrooke), respectively. All other chemicals were of grade A purity.

Cell culture and morphological studies
NG108–15 cells were cultured (passages 7–30) in DMEM with 10% fetal bovine serum, hypoxanthine, aminopterin, thymidine supplement, and 50 mg/liter gentamycin at 37 C in 75 cm2 Nunclon {delta} flasks in a humidified atmosphere of 7% CO2 and 93% air, as described previously (5). Under these conditions, NG108–15 cells express only the AT2 receptor of Ang II (11, 38). According to experiments, cells were stimulated directly in the medium for time intervals ranging from minutes to 3 d. For 3-d treatments, cells were stimulated once daily, 24 h after plating, without (control) or with Ang II (100 nM) or Gö6976, which inhibits PKC{alpha} at a concentration of 3 nM (39). Cells were examined under a phase-contrast Leica Corp. microscope (Deerfield, IL) equipped with a x20 objective. Cells with at least one neurite longer than the cell body were counted as positive for neurite outgrowth (28).

Western blotting
After 3 d of culture, without (control) or with 100 nM Ang, cells were washed with Hanks’ buffered saline (HBS) (130 mM NaCl, 3.5 mM KCl, 2.3 mM CaCl2·2H2O, 0.98 mM MgCl2·6H2O, 5 mM HEPES, 0.5 mM EGTA) and lysed in 100 µl of 1% SDS solution in ice-cold HBS. The extracts were sonicated, heated at 95 C, and processed for Western blot analyses as previously described (23, 27). Detection of PKC proteins was achieved using the primary antibodies at the following dilutions: anti-PKC{alpha} (1:20,000), anti-PKCß (1:250), anti-PKC{gamma} (1:1,000), anti-PKC{delta} (1:10,000), PKC{epsilon} (1:1,000), anti-PKC{eta} (1:250), anti-PKC{theta} (1:250), anti-PKC{iota} (1:250), anti-PKC{zeta} (1:20,000) or anti-PKCµ (1:200). Detection was performed by reaction with horseradish peroxidase-conjugated secondary antibody and visualized by ECL according to the manufacturer’s instructions.

Cell transfections
NG108–15 cells were transfected with pcDNA3/PKC{alpha}DN (K368A) or with pcDNA3. Briefly, 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 (50–60%) in 35-mm Petri dishes and incubated for 3 h at 37 C with the DNA-lipid complex. Transfection medium was replaced thereafter with complete, fresh medium, after which 400 µg/ml of Geneticin (G-418) was added at 24 h posttransfection. Morphological studies were conducted on stably transfected cells, grown in a G-418-containing medium (200 µg/ml). Cells with at least one neurite longer than the cell body were counted as positive for neurite outgrowth (28).

Proliferation assay
Cell proliferation was measured using fluorescence BrdU incorporation, as described elsewhere (40). Briefly, after 24 h of culture, 10 µM BrdU was added to the culture medium. Cells were then treated for 4 h without or with Ang II and in combination of Gö6976. Cells were fixed with 3.7% (wt/vol) formaldehyde in HBS for 10 min at room temperature and permeabilized for 10 min with 0.2% Triton X-100 in HBS. Cells were incubated with anti-BrdU Alexa Fluor-594 (1:500). Fluorescence intensity was determined using a FL600 microplate fluorescence reader (Bio-Tek) (excitation 560 ± 40 nm; emission 645 ± 40 nm). Results are expressed as percent changes from basal conditions using a six-well culture plate for each experimental condition.

PKC{alpha} activity measurements
The kinase activity of PKC{alpha} was measured essentially as described by Ohmichi et al. (41). Briefly, cells were grown in 100-mm dishes for 3 d without treatment, until 80% confluence. Cells were then treated with Ang II (100 nM) for 0–30 min and extracted for measurement of kinase activity. After washing with ice-cold HBS buffer, cells were harvested in 500 µl of lysis buffer (HEPES 50 mM pH 7.5, 150 nM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, 10 mM sodium pyrophosphate, and 100 µM Na3VO4) containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and protease inhibitors. PKC{alpha} was immunoprecipitated and incubated for kinase assay in the presence of 1 µg myelin basic protein (MBP) and 10 µCi [{gamma}-32P]ATP in kinase reaction buffer (20 mM HEPES pH 7.4, 5 mM MgCl2, 1.5 mM CaCl2). After 15 min of incubation at 37 C, the reaction was stopped with 20 µl Laemmli buffer 2x (150 mM Tris pH 6.8, 1% SDS, 30% glycerol, 15% ß-mercaptoethanol, 0.025% bromophenol blue) and samples were loaded on a 15% polyacrylamide gel. The amount of phosphorylated MBP was determined by densitometry analysis and expressed in arbitrary units.

Subcellular fractionation
Cells were grown in 100-mm Petri dishes for 3 d without treatment, until 80% confluence. Cells were then treated with Ang II (100 nM) for 0–30 min and washed with ice-cold HBS buffer. Thereafter, cells were harvested in 500 µl HBS. Subcellular fractionation was performed as previously described (32). After centrifugation, cells were sonicated in extraction buffer [20 mM Tris-HCl (pH 7.5), 10 mM EGTA, 2 mM EDTA, and a mixture of protease inhibitors] and centrifuged at 100,000 x g for 1 h at 4 C. The membrane fraction in the pellet was resuspended in extraction buffer containing 1% Triton X-100, sonicated, incubated on ice, and centrifuged under the same conditions. The resulting supernatant corresponded to membrane proteins, whereas the pellet was composed of cytoskeletal proteins. Soluble and membrane fractions were then resolved by SDS-PAGE (8%) and transferred onto nitrocellulose membranes. The amount of PKC{alpha} was determined by Western blot analysis as described above. Immunoreactive bands were scanned and densitometry expressed in arbitrary units as described previously.

Immunofluorescence studies
Cells were plated on glass coverslips in 35-mm Petri dishes for 3 d until subconfluence. Plated cells were then treated with Ang II (100 nM) for 30 sec to 60 min. Culture medium was first removed by aspiration, then cells fixed with –20 C methanol and blocked for 1 h with 5% nonfat dry milk in HBS. Anti-PKC{alpha} (1:2000) was added and incubated for 2 h at room temperature. Cells were washed and subsequently incubated for 1 h with antirabbit Alexa-Fluor 594 (1:1000) followed by 4',6-diamidino-2-phenylindole (DAPI) staining (1:1000) for 1 min. After several washes, coverslips were mounted with Vectashield Mounting Solution and examined on a Nikon Eclipse 300 upright microscope (Mississauga, Ontario, Canada) equipped with a CoolSnap fx digital camera (Roper Scientific, Hinsdale, IL). Images were acquired using a x100 oil immersion objective.

MAPK activity measurements
Cells were cultured in 35-mm Petri dishes for 3 d and incubated with Gö6976 (3 nM) 30 min before Ang II (100 nM) or treated with Ang II alone for durations ranging from 0–120 min. Measurement of p42/p44mapk activity was done as previously described (23). The reaction was stopped by washing cells with HBS, extraction was performed, and after 10% SDS-PAGE gel and protein transfer, PVDF membranes were incubated with anti-phospho p42/p44mapk (1:1000). Detection was performed by reaction with horseradish peroxidase-conjugated secondary antibody and visualized by ECL according to the manufacturer’s instructions. To determine the total amount of p42/p44mapk, membranes were stripped and reprobed with an anti-p42/p44mapk antibody.

Ras and Rap1 activity measurements
The activated form of p21ras was pulled down with GST-RafRBD (the Ras-binding domain of Raf) fusion protein from cell lysates as described previously (23). NG108–15 cells cultured for 4 d in 35-mm Petri dishes were stimulated with 100 nM Ang II or incubated with 3 nM Gö6976 for intervals ranging from 0–30 min. Extraction was subsequently performed as described by Gendron et al. (23). Cells were rapidly washed and lysed for 30 min in 1 ml of ice-cold lysis buffer containing 1% Triton X-100 and 1% N-octyl-glucopyranoside in 50 mM Tris-HCl (pH 7.5), 15 mM NaCl, 20 mM MgCl2, 5 mM EGTA, and Complete mixture of inhibitors. After centrifugation, lysates were incubated with 20 µl of a 50% slurry of glutathione Sepharose beads precoupled with GST-RafRBD for 1 h at 4 C with gentle agitation. Activated p21ras 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 PVDF membranes followed by revelation of p21ras (21 kDa) with a Pan-Ras monoclonal antibody (1:100).

The activated form of Rap1 was pulled down with GST-RalGDS fusion protein from cell lysates as described previously (27). At the end of the stimulation period, cells were washed with ice-cold HBS and lysed in 100 µl of ice-cold lysis buffer containing 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in 50 mM Tris-HCl (pH 7.6), 140 mM NaCl, 5 mM MgCl2, 1 mM Na3VO4, and Complete mixture of inhibitors. Lysates were centrifuged and total proteins were incubated with glutathione Sepharose beads precoupled with GST-RalGDS for 1 h at 4 C with gentle agitation. Beads were then washed twice with the lysis buffer and activated Rap1 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 PVDF membrane and Rap1 revealed with a Rap1 polyclonal antibody (1:200).

Data analysis
Unless indicated otherwise, data are presented as the mean ± SEM of the number of experiments indicated in the text. Statistical analyses of the data were performed using the one-way ANOVA test. Homogeneity of variance was assessed by Bartlett’s test and P values were obtained from Dunnett’s tables.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of PKC isoforms in NG108–15 cells
The expression of PKC isoforms was first analyzed in cellular extracts from both undifferentiated and differentiated NG108–15 cells using specific antibodies to PKC isoforms. As shown in Fig. 1Go, all antibodies recognized an immunoreactive band in rat cerebellum extracts (Cb) used as a positive control. In undifferentiated NG108–15 cells (C), PKC {alpha}, {epsilon}, {iota}, and {zeta} isoforms were present (Fig. 1AGo), whereas the ß, {gamma}, {delta}, {theta}, and µ isoforms were not detected (Fig. 1BGo). This PKC expression pattern was not modified in cells treated for 3 d with 100 nM Ang II (Fig. 1Go, A and B).


Figure 1
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  		FIG. 1. Presence of PKC isoforms in NG108–15 cells. Cells were cultured for 3 d in 35-mm Petri dishes, stimulated with or without Ang II for 3 d. Proteins were extracted as described in Materials and Methods. Protein content was determined using the Bio-Rad DC method, and equal protein amounts were separated on 8% polyacrylamide gel. Proteins were subsequently transferred onto nitrocellulose membranes and subjected to Western blot analysis with appropriate antibodies against the PKC isoform. A, PKC isoforms present in NG108–15 cells. B, PKC isoforms absent in the NG108–15 cells. Cb, Cerebellum extract of 2-d-old rat. C, Cellular extracts from control NG108–15. Ang II, Cellular extracts from NG108–15 cells treated for 3 d with 100 nM Ang II.

 
Effect of Ang II and CGP42112 on PKC{alpha} activity and localization
NG108–15 cells were treated with or without 100 nM Ang II for periods ranging from 0–30 min, followed by measurement of PKC activity using the phosphorylation of MBP as substrate. Morphological results led us to investigate for PKC{alpha} activity because PKC{epsilon} and PKC{zeta} did not seem to be involved in neurite outgrowth (data not shown). As shown in Fig. 2AGo, a strong basal phosphorylation of MBP was observed in nonstimulated cells. After application of Ang II to NG108–15 cells, PKC{alpha} activity was decreased by approximately 30% within the first 5 min, as indicated by a reduction in MBP phosphorylation level. Inhibition of PKC{alpha} after Ang II treatment was found to be rapid and transient, with maximal inhibition observed at 3 min (Fig. 2BGo).


Figure 2
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  		FIG. 2. Measurement of PKC{alpha} kinase activity in NG108–15 cells. Cells were plated in 100-mm dishes for 3 d without treatment. Cells were then subsequently treated for intervals ranging from 0–30 min with Ang II (100 nM). PKC{alpha} kinase activity was measured as described in Materials and Methods by an immune complex kinase assay using the anti-PKC{alpha} antibody, with MBP as a substrate and [{gamma}-32P]ATP as the phosphate donor. A, The reaction product was resolved by SDS-PAGE and detected by autoradiography. B, Densitometry analysis of one representative experiment of four is shown.

 
For the majority of PKC isoforms, activity is associated with cell membrane localization (30). To verify the possible correlation between Ang II-induced modulation of PKC{alpha} activity and its cellular localization, spatial localization of PKC{alpha} was assessed by immunofluorescence labeling. In control cells (Fig. 3AGo) and within the first 3–5 min of stimulation with Ang II (Fig. 3Go, B and C, respectively), PKC{alpha} immunoreactivity was found both in the cytosol and at the plasma membrane level (arrows). After 10 min of treatment with Ang II, the labeling pervaded the cytoplasm and was virtually absent from the membrane (Fig. 3DGo). Membrane localization of PKC{alpha} immunoreactivity then reappeared 15 and 30 min after Ang II application (Fig. 3Go, E and F, respectively). These results demonstrate that Ang II-induced inhibition of PKC{alpha} is accompanied by its translocation into the cytoplasm.


Figure 3
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  		FIG. 3. Immunofluorescence localization of PKC{alpha} in NG108–15 cells. Cells were plated on glass coverslips in 35-mm dishes for 3 d without treatment. Cells were then stimulated without (A) or with Ang II (100 nM) for 3 (B), 5 (C), 10 (D), 15 (E), and 30 min (F). Cells were fixed and permeabilized with methanol as described in Materials and Methods. Immunolabeling was performed using an antibody against PKC{alpha}. Antirabbit coupled to Alexa-Fluor 594 as well as DAPI were added and coverslips mounted with Vectashield Mounting Solution. Blue, Nucleus stained with DAPI; red, PKC{alpha} stained with Alexa-Fluor 594. Images are representative of at least 30 cells originating from three different experiments. Scale bar, 30 µm.

 
To further substantiate the aforementioned data, the distribution of PKC{alpha} was measured in cytosol and membrane fractions, after stimulation of NG108–15 cells with the selective AT2 receptor agonist CGP42112 or with Ang II. As shown in Fig. 4Go, A and C, the expression of PKC{alpha} was elevated in the membrane fraction of control cells. Stimulation with 10 nM CGP42112 or 100 nM Ang II was sufficient to decrease the amount of protein associated with the membrane. This decrease in PKC{alpha} associated with the membrane was transient and observed only over 5 min (Fig. 4Go, A and C). Corresponding quantifications are presented in Fig. 4Go, B and D. Altogether, these results indicate that the AT2 receptor exerts a rapid and transient effect on PKC{alpha} activity.


Figure 4
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  		FIG. 4. Subcellular localization of PKC{alpha} in NG108–15 cells. Cells were plated in 100-mm dishes for 3 d without treatment. Cells were treated subsequently for the indicated time periods with CGP42112 (10 nM), the specific agonist of AT2 receptor (A), or with Ang II (C). Membranes and soluble proteins were extracted as detailed under Materials and Methods after determination of protein content by the Bio-Rad DC method. Equal amounts of proteins were submitted to Western blot analysis using anti-PKC{alpha} antibody. Corresponding densitometry analysis is shown for CGP42112 (B) and Ang II (D).

 
Role of PKC{alpha} in the p21ras/Rap1/ p42/p44mapk cascade
In a previous report, we had shown that activation of the AT2 receptor inhibits p21ras (23) and increases Rap1 activity (27). Because proliferation and neurite outgrowth are associated, respectively, with specific activation of these proteins, the effect of PKC{alpha} inhibition on both protein activities was assessed. NG108–15 cells were incubated with 100 nM Ang II or with 3 nM Gö6976 for various time intervals ranging from 0–30 min. As illustrated in Fig. 5Go, A and B, inhibition of PKC{alpha} with Gö6976 was sufficient to inhibit p21ras to the same extent as that observed with Ang II. The effect was rapid and maximal after 10 min of incubation, reaching more than 40% of p21ras inhibition. In contrast, Gö6976, similarly to Ang II, rapidly activated Rap1 (within 1 min) (Fig. 5Go, C and D).


Figure 5
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  		FIG. 5. Effect of PKC{alpha} inhibition on p21ras and Rap1 activation. NG108–15 cells were cultured during 4 d without treatment. Cells were subsequently stimulated for intervals ranging from 0–30 min with 100 nM Ang II or 3 nM Gö6976. Activated forms of p21ras (A) and Rap1 (C) were measured by Western blot analysis after GST-pull down precipitation as described in Materials and Methods. Densitometric analysis of activated p21ras (B) and activated Rap1 (D) of the experiments shown in upper panels. Data shown are representative results from three independent experiments.

 
A sustained increase in p42/p44mapk activity by Ang II is necessary for the induction of neurite elongation (23). Hence, experiments were conducted to verify whether the inhibition of PKC{alpha} could affect p42/p44mapk phosphorylation. As shown in Fig. 6AGo, 30 min of stimulation with Gö6976 induced a dose-dependent decrease in basal phosphorylation of p42/p44mapk; however, preincubation with Gö6976 did not interfere with Ang II-induced p42/p44mapk phosphorylation (Fig. 6BGo). Together, these results suggest that inhibition of PKC{alpha} by the AT2 receptor inhibits p21ras activity, decreases the basal level of phosphorylation of p42/p44mapk, but does not directly interfere with the capacity of Ang II to stimulate p42/p44mapk phosphorylation.


Figure 6
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  		FIG. 6. Effect of Gö6976 on p42/p44mapk activity in NG108–15 cells. Cells were plated in 35-mm dishes for 3 d without treatment. Cells were subsequently stimulated for 30 min with various concentrations of Gö6976 (A) or for the indicated time periods without or with Ang II (100 nM) alone or with Gö6976, added during a 30-min preincubation period to inhibit PKC{alpha} (B). Western blot analysis of phosphorylated p42/p44mapk (pp42mapk and pp44mapk) was performed as described in Materials and Methods. The total amount of p42/p44mapk (shown in the lower portion of each panel) was determined after reprobing the membrane with an anti-total p42/p44mapk antibody (upper panel). Results shown are from one representative experiment of a total of four independent experiments.

 
Effect of PKC inhibition on NG108–15 cell morphology
To assess the putative involvement of PKC{alpha} in the induction of neurite outgrowth, NG108–15 cells were cultured in the absence or presence of PKC{alpha} inhibitor, Gö6976. As described previously, nontreated cells (control) exhibited a rounded cell body, without or with few thin and short processes only (Fig. 7AGo). By contrast, cells treated with 100 nM Ang II or with 10 nM CGP42112 exhibited one or two extended neurite processes, whereas the cell body retained a rounded appearance (Fig. 7Go, B and C, respectively). After a 3-d treatment with 3 nM Gö6976, an inhibitor of classic PKC isoforms (39) and namely PKC{alpha} in NG108–15 cells, cells exhibited the morphology of differentiated cells, characterized by at least one long process and two or more shorter neurites (Fig. 7DGo). As illustrated in Fig. 8AGo, 20.9 ± 0.3% of Ang II-treated cells and 23.0 ± 0.4% of Gö6976-treated cells exhibited at least one neurite longer than a cell body (P < 0.05, n = 3). This proportion was significantly increased when Ang II and Gö6976 were added concomitantly (30.4 ± 0.4%, P < 0.05, n = 3). To confirm the results obtained with pharmacological agent, cells were transfected with a dominant-negative mutant of PKC{alpha} (K368A). As shown in Fig. 7EGo and quantified in Fig. 8BGo, cells expressing the dominant-negative kinase presented neurite elongation (27.9 ± 0.9%, P < 0.001, n = 3) at about the same extent as with Ang II. This effect on neurite outgrowth was not significantly modified when PKC{alpha} (K368A)-transfected cells were incubated with Ang II (Figs. 7FGo and 8BGo).


Figure 7
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  		FIG. 7. Comparative effect of PKC inhibition, Ang II, and CGP42112 on neurite outgrowth in NG108–15 cells. Cells were plated in 35-mm dishes as described in Materials and Methods and were treated once daily for 3 d, 24 h after plating. Cells were cultured in the absence (A) or presence of Ang II (100 nM) (B), CGP42112 (10 nM), a specific agonist of the AT2 receptor (C), or Gö6976 (3 nM), an inhibitor of PKC{alpha} (D). Cells transfected with dominant-negative PKC{alpha} (K368A) in the absence (E) or presence of 100 nM Ang II (F). Phase-contrast microscopy was conducted after 3 d in culture. Images were acquired with a x20 objective. Scale bars, 120 µm.

 

Figure 8
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  		FIG. 8. Effect of Ang II and PKC inhibition on neurite outgrowth in NG108–15 cells. Cells were plated in 35-mm dishes as described in Materials and Methods and were treated daily for 3 d, 24 h after plating. Cells were cultured in the absence or presence of Ang II (100 nM), Gö6976 (3 nM), an inhibitor of PKC{alpha}, or with the combination of Gö6976 and Ang II (A). A, Percentage of cells exhibiting at least one neurite longer than the cell body. Results are expressed as means ± SE of at least 50 cells from three independent experiments. B, Transfected cells with dominant-negative PKC{alpha} (K368A) were cultured in the absence or presence of Ang II (100 nM). Results are expressed as the mean ± SE of three independent experiments, each conducted in triplicate. Statistical significance: *, P < 0.05, compared with control.

 
As evidenced in Figs. 7DGo and 9AGo, Gö6976 treatment for 3 d decreased the number of cells by 47.4 ± 20.4% (P < 0.05, n = 3), compared to nontreated cells. This decrease in cell number was mostly due to an arrest in cell proliferation because, when measured over a 4-h incubation period, Gö6976 decreased BrdU incorporation (12.8 ± 6.2%, P < 0.02, n = 3) (Fig. 9BGo) without affecting apoptosis (data not shown).


Figure 9
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  		FIG. 9. Measurement of cell proliferation in NG108–15 cells. A, Cells were cultured in the absence or presence of Ang II (100 nM), Gö6976 (3 nM), an inhibitor of PKC{alpha}, or with the combination of Gö6976 and Ang II, as in morphological experiments and counted as described in Materials and Methods. B, Cells were plated in 96-well plates for proliferation studies (30 x 103 cells per well) measured by BrdU incorporation. Results are expressed as the mean ± SE of three experiments, with each experimental condition containing six individual samples. Statistical significance: *, P < 0.01 compared with control.

 

   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 investigated the signaling mechanisms associated with this receptor. The present study provides evidence that PKC{alpha} is involved in the transition from proliferation to neurite outgrowth after AT2 stimulation.

Role of PKC{alpha} in neurite outgrowth
The role of PKC in differentiation has been documented in a number of studies. However, there is no consensus as to the nature of the isoforms involved and on their potential intracellular targets. Pioneer studies on the role of PKC isoforms in neurite outgrowth were conducted using phorbol ester as a stimulus, although conflicting results have been reported. Although some studies indicate that depletion of PKC by chronic treatment with phorbol esters does not inhibit neurite outgrowth (42, 43), others have clearly shown that these compounds induce neurite elongation (42, 43, 44), decrease cell growth, and enhance expression of differentiating markers (45, 46). Several groups have described the involvement of PKC{epsilon} and PKC{zeta} in NGF-induced neurite outgrowth in PC12 cells (32, 33, 47, 48), in aplysia neurons (34, 35), and in other various cell lines (36).

To understand whether and how Ang II signaling could interact with PKC, we first compared expression of PKC isoforms in control and Ang II-treated cells. Results reveal that PKC {alpha}, {epsilon}, {iota}, and {zeta} are present in NG108–15 cells, whereas the isoforms ß, {gamma}, {delta}, {theta}, and µ are not. Furthermore, the expression of all isoforms remained the same in control and in Ang II-treated cells exhibiting neurites. Similarly to previous reports, with the exception that PKC{delta} was also found in NG108–15 cells (49, 50), these results indicate that neurite outgrowth in NG108–15 cells occurs independently of a change in PKC expression. With the current availability of relatively specific PKC inhibitors, we then investigated whether PKC isoforms expressed by the NG108–15 cells could play a role in the induction of neurite outgrowth. Hence, Gö6976, a specific inhibitor of classical PKC{alpha} and PKCßI (not expressed in our cell model) when used at a concentration of 3 nM (39), was able to promote neurite elongation in NG108–15 cells. Moreover, coincubation of Gö6976 with Ang II further enhanced neurite outgrowth elicited by Ang II or Gö6976 alone. Similar results have also been reported by Zeidman et al. (36) in a study conducted on SH-SY5Y cells, whereby a decrease in cellular proliferation was observed when these cells were treated with Gö6976, as well as a potentiation of morphological differentiation induced by growth factors.

Relationship between PKC{alpha} inhibition and neurite outgrowth in NG108–15 cells
In response to agonists, classical and novel PKCs are recruited at the plasma membrane, where they become activated (30). Using an in vitro kinase assay and MBP as a substrate, we were able to measure PKC activity. Our observations reveal that Ang II induced a transient inhibition of PKC{alpha} kinase activity, which was further substantiated by both biochemical and immunofluorescence studies. Indeed, Ang II and CGP42112 were found to induce a transient translocation of PKC{alpha} from the membrane to intracellular compartments. Hence, these results strengthen our hypothesis that a transient inhibition of PKC{alpha} is involved in the AT2-induced signaling cascade promoting neurite elongation. In the present study, a correlation was observed between PKC{alpha} inhibition and a decrease in proliferation, which is in agreement with several studies describing the involvement of PKC{alpha} in growth stimulation in several cell types (36, 51, 52, 53). Moreover, the fact that activation of the AT2 receptor leads to a reduction in cell proliferation corroborates well with the initial steps required for neuronal differentiation.

It is well known that proliferation is associated with an activation of both p21ras and p42/p44mapk (for reviews see Ref. 54). In control conditions, NG108–15 cells actively divide. Results in this study show that PKC{alpha} inhibition by Gö6976 decreases p21ras with the same time-course profile as that observed with Ang II (23, 27). Such results provide some explanation as to why the inactivation of PKC{alpha} is accompanied by a decrease in the basal state of p42/p44mapk phosphorylation, supporting the now well-established notion that p21ras activation is associated with transient activation of p42/p44mapk pathway and proliferation, whereas Rap1 and sustained activation of p42/p44mapk are associated with neurite outgrowth (27, 55). Studies have indeed shown that p21ras may be modulated by PKC (56, 57).

In contrast, the finding that inhibition of PKC{alpha} failed to interfere with Ang II-induced p42/p44mapk activation further strengthens the hypothesis that two complementary signaling cascades are involved in the AT2-receptor effect on neurite outgrowth in NG108–15 cells. The first cascade involves the inhibition of PKC{alpha} and p21ras (23), whereas the second cascade involves the activation of Rap1/B-Raf and p42/p44mapk (27). The fact that inhibition of PKC{alpha} mimics Ang II-induced Rap1 activation, while, at the same time, decreasing basal activity of the downstream p42/p44mapk may appear paradoxical at first. The link between these proteins remains unknown, but several studies have shown that p21ras and Rap1 pathways act independently (58, 59, 60, 61) (for review see Ref. 62). Moreover, a balance between activation of Ras and Ral is under the control of PKC activity (63). In our proposed model (Fig. 10Go), it is possible that the inhibition of PKC{alpha} could lead to inactivation of p21ras, thus promoting the activation of Rap1. It has also been shown by Hagemann and Rapp (64) that Rap1 and Raf-1 can indeed interact, a process that decreases the association of Ras and Raf-1. In line with this hypothesis, active PKC{alpha} may allow association between p21ras with B-Raf, which cannot be activated by Rap1. Once the AT2 receptor has been activated, p21ras/B-Raf dissociation would occur, allowing the Rap1/B-Raf/p42/p44mapk cassette to be activated. This hypothesis may also explain why increased in p42/p44mapk phosphorylation by Ang II is not affected by preincubation with Gö6976, p42/p44mapk activation needing additional partners to be complete. An alternative hypothesis could be that PKC{alpha} is upstream of a RasGRP protein in the regulation of p21ras activity. As described by many groups, PKC can phosphorylate RasGEF or RasGAP to control ERK activation (65, 66, 67). In our model, it may be possible that this pathway is inhibited by the AT2 receptor, thus explaining both the inhibition of p21ras as well as the effect of Gö6976 on basal p42/p44mapk activity.


Figure 10
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  		FIG. 10. Proposed model for AT2 receptor signaling through PKC{alpha}. We have previously shown that neurite elongation of NG108–15 cells induced by Ang II AT2 receptor activation was associated with the inhibition of p21ras and activation of Rap1/B-Raf. These steps were necessary for promoting p42/p44mapk activation under two separate pathways. The present results indicate that AT2-induced p21ras inhibition is preceded by PKC{alpha} inhibition. PKC{alpha} could act through stimulation of a RalGEF protein to inhibit p21ras. Inhibition of PKC{alpha} and of p21ras decreases proliferation and stimulates neurite outgrowth, a result reproduced in cells transfected with the dominant-negative PKC{alpha} isoform, K368A. However, inhibition of PKC{alpha} does not interfere with p42/p44mapk activation by Ang II, indicating that stimulation of the AT2-receptor activates a second pathway, more directly implicated in the activation of Rap1/B-Raf and p42/p44mapk. Together, these results suggest that the AT2-receptor effect on neurite outgrowth in NG108–15 cells involves two complementary cascades of signaling, the first being the inhibition of PKC{alpha} and p21ras (23 ), whereas the second involves the activation of Rap1/B-Raf and p42/p44mapk. Both these cascades are complementary in the sense that inhibition of PKC{alpha} and of p21ras facilitates AT2 receptor signaling through Rap1/B-Raf.

 
The current data demonstrate that PKC{alpha} is the primary protein implicated in AT2 receptor signaling in NG108–15 cells. However, because PKCs are not known to interact directly with G protein-coupled receptor, some receptor binding proteins may be necessary to recruit PKC{alpha}. For example, recent studies have documented that the AT2 receptor likely interacts with two proteins, one identified as ATIP (for Ang II AT2 receptor-interacting protein) (68) and the other as CNK1 (for connector enhancer of Ksr) (69). Supporting these hypotheses is the fact that CNK2 interacts with Raf1 and is implicated in NGF-induced Rap1/B-Raf activation leading to neuronal differentiation (70, 71). In light of this, one could raise the hypothesis that the AT2 receptor possibly recruits other partners to form a complex with p21ras, Rap1, PKC{alpha}, and other regulatory proteins to activate p42/p44mapk. Hence, the role of PKC{alpha} would be facilitated due to the proximity of all the primary effectors of the pathway.

Conclusions
As summarized in Fig. 10Go, the present findings demonstrate that inhibition of PKC{alpha}, after stimulation of the AT2 receptor, inhibits p21ras, decreases proliferation, and stimulates neurite outgrowth. However, while decreasing its basal level of phosphorylation, inhibition of PKC{alpha} does not interfere with p42/p44mapk activation induced by Ang II. Finally, although the present study focuses on PKC{alpha}, we cannot exclude the possibility that the PKC{epsilon} and PKC{zeta} isoforms may also be involved in AT2 receptor action on neurite outgrowth as shown previously, for example, in aplysia neurons (34, 35) and for NGF in PC12 cells (32, 33, 47, 48).


   Acknowledgments
 
We are grateful to Dr. Johannes L. Bos (Utrecht University) for the GST-RalGDS fusion protein and Dr. Nathalie Rivard (Université de Sherbrooke) for the GST-RafRBD fusion protein. We also thank Drs. Jean-Guy LeHoux and Gilles Dupuis for the PKC{alpha}DN (K368A) cDNA (Department of Biochemistry, University of Sherbrooke). We gratefully acknowledge Lucie Chouinard and Claude Roberge for their invaluable experimental assistance and stimulating discussions.


   Footnotes
 
Present address for L.G.: Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280.

L.G. is a recipient of a fellowship from the Canadian Institute for Heath Research (MFE-63497). 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 M.D.P. (MOP27912).

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

First Published Online June 1, 2006

Abbreviations: Ang II, Angiotensin II; AT2, Ang II type 2; DAPI, 4',6-diamidino-2-phenylindole; ECL, enhanced chemiluminescence; GST, glutathione S-transferase; HBS, Hanks’ buffered saline; MBP, myelin basic protein; NGF, nerve growth factor; PKC, protein kinase C; PVDF, polyvinylidene difluoride.

Received March 30, 2006.

Accepted for publication May 25, 2006.


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 Discussion
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B. Plouffe, M.-O. Guimond, H. Beaudry, and N. Gallo-Payet
Role of Tyrosine Kinase Receptors in Angiotensin II AT2 Receptor Signaling: Involvement in Neurite Outgrowth and in p42/p44mapk Activation in NG108-15 Cells
Endocrinology, October 1, 2006; 147(10): 4646 - 4654.
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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals