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Endocrinology Vol. 142, No. 8 3502-3511
Copyright © 2001 by The Endocrine Society

ARTICLES

Characterization of Signal Transduction Pathway in Neurotropic Action of Angiotensin II in Brain Neurons

Hong Yang1, Xiangyu Wang2 and Mohan K. Raizada

Department of Physiology, College of Medicine, and McKnight Brain Institute, University of Florida, Gainesville, Florida 32610

Address all correspondence and requests for reprints to: Mohan K. Raizada, Ph.D., University of Florida College of Medicine, P.O. Box 100274, Gainesville, Florida 32610-0274. E-mail: mraizada{at}phys.med


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Materials and Methods
 Results
 Discussion
 References
 
Interaction of angiotensin II with the neuronal angiotensin type 1 receptor stimulates the PI3K signaling pathway. Our objective in this study was to investigate the hypothesis that the PI3K cascade regulates the neurotropic actions of angiotensin II in rat brain neurons. We followed growth associated protein-43 expression and neurite extension as markers of neurotropic activity. Angiotensin II, through its interaction with the angiotensin type 1 receptor, increased growth associated protein-43 expression and neurite extension. These effects were abolished by pretreatment of neurons with wortmannin and rapamycin, but not by PD 98059. Antisense oligonucleotides specific for p70S6 kinase also inhibited angiotensin II-stimulated neurotropic activity. These data confirm the involvement of PI3K and p70S6 kinase in angiotensin II-mediated neurotropic action. Further support for this was provided by the observation that angiotensin II caused a time-dependent stimulation of p70S6 kinase by an angiotensin type 1 receptor-mediated process. We also found that the neurotropic actions of angiotensin II are mediated by plasminogen activator inhibitor-1. Evidence for this includes 1) angiotensin II-stimulated neuronal plasminogen activator inhibitor-1 gene expression, 2) potent neurotropic action of exogenous plasminogen activator inhibitor-1, and 3) inhibitory neurotropic effect of angiotensin II by antisense oligonucleotide-mediated depletion of plasminogen activator inhibitor-1. Finally, we found that the neurotropic action of plasminogen activator inhibitor-1 is not blocked by either angiotensin type 1 receptor antagonist or inhibitors of PI3K or p70S6 kinase, indicating that the plasminogen activator inhibitor-1 step is downstream from the p70S6 kinase. These observations demonstrate that angiotensin II is a neurotropic hormone that engages a distinct PI3K-p70S6 kinase-plasminogen activator inhibitor-1 signaling pathway for this action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Materials and Methods
 Results
 Discussion
 References
 
ANGIOTENSIN II (Ang II) exerts diverse physiological actions in both peripheral and neural tissues. In the periphery, its actions include vasoconstriction, hypertrophy, hyperplasia, tissue remodeling, and hormone secretion (1, 2, 3, 4, 5, 6, 7, 8). In the central nervous system, angiotensin II (Ang II) regulates the secretion of neuroendocrine hormones, such as vasopressin, sympathetic activity, and dampening of baroreceptor reflexes (2, 3, 5, 6, 7, 8). In addition, Ang II is involved in glial cell division, tissue remodeling involving neurons and astroglial cells, and neuronal apoptosis (9, 10). Although it is well established that these diverse actions of Ang II are mediated by the type 1 Ang II (AT1) receptor, the mechanism or the cellular basis of such diversity is poorly understood. It has been suggested that coupling of the AT1 receptor to multiple signal transduction pathways may hold the answer to the myriad of cellular effects elicited by Ang II (11, 12). For example, in the periphery, coupling of the AT1 receptor to the Janus kinase-signal transducer and activator of transcription signaling system is involved in DNA replication and hypertrophy in vascular smooth muscle cells, whereas its interaction with the pp60src-PLC{gamma}1 pathway is associated with vascular tissue remodeling (12, 13, 14). In addition, coupling of the AT1 receptor to PI3K and p70S6 kinase results in stimulation of these signal transduction pathways and endothelial growth and proliferation (15). In brain neurons, Ang II stimulation of the Ras-Raf-MAPK signaling pathway regulates phosphorylation of myristoylated alanine-rich C kinase substrate through activation of the PKCß subtype (16, 17), which participates in regulation of the neuromodulatory actions of Ang II (16, 17, 18). Recent studies have established that in addition to the Ras-Raf-MAPK pathway, Ang II stimulates PI3K and protein kinase B in neuronal cells (19). The PI3K signaling pathway is not involved in the neuromodulatory actions of Ang II, and thus, its cellular and physiological roles in brain neurons have yet to be understood.

In the present study we investigated the hypothesis that Ang II stimulation of neurotropic activity in brain neurons is mediated through a PI3K-plasminogen activator inhibitor-1 (PAI-1) signaling system. The rationale for this hypothesis is based on the following: 1) Ang II stimulates PAI-1 gene expression in neurons (20), and this protease inhibitor is linked to neuronal survival and trophic activity both in vitro and in vivo (21, 22); 2) Ang II induces neurite-like growth in neural cell lines, PC12 and NG-108 cells (23); and 3) Ang II stimulates PI3K and p70S6 kinase in cardiac myocytes, and this stimulation is implicated in the hypertropic actions of Ang II in the heart (24). The observations presented in this study demonstrate that Ang II is a potent neurotropic hormone that recruits a PI3K-p70S6 kinase-PAI-1 pathway for this action.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Materials and Methods
Results
 Discussion
 References
 
Materials
One-day-old normotensive Wistar Kyoto (WKY) rats were obtained from our breeding colony, which originated from Harlan Sprague Dawley, Inc. (Indianapolis, IN). DMEM, plasma-derived horse serum (PDHS), and 1 x crystallized trypsin were purchased from Central Biomedia (Irwin, MO). [{gamma}-32P]ATP (3000 Ci/mmol), and chemiluminescence assay reagents were obtained from NEN Life Science Products (Boston, MA). Monoclonal antibody to growth-associated protein-43 (GAP-43) and tubulin, Ang II, and wortmannin were purchased from Sigma (St. Louis, MO). Rapamycin and recombinant rat PAI-1 were from Calbiochem- (La Jolla, CA). Superscript II ribonuclease H- reverse transcriptase and deoxynucleotide mixture were purchased from Life Technologies, Inc. (Grand Island, NY). Dynal beads and other reagents for polyadenylated [poly(A)+] RNA isolation were purchased from Dynal (Lake Success, NY). Oligo(deoxythymidine) and Taq DNA polymerase were obtained from Promega Corp. (Madison, WI). Polyclonal anti-p70S6 kinase was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal antineurofilament was a gift from Dr. Gerry Shaw (University of Florida College of Medicine, Gainesville, FL). All other reagents were purchased from Fisher Scientific (Pittsburgh, PA) and were the highest quality available.

Primers for the measurement of PAI-1 and ß-actin messenger RNA by RT-PCR were synthesized in the DNA synthesis facility of the Interdisciplinary Center for Biotechnology Research, University of Florida (Gainesville, FL). The sequences of these primers have been published previously (16).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Materials and Methods
Results
 Discussion
 References
 
Preparation of neuronal cultures of the WKY rat brain
Neuronal cultures were prepared essentially as described previously (6, 25, 26). In brief, hypothalamus-brainstem areas of 1-d-old WKY rat brains were dissected and cut into small pieces, and cells were dissociated by 0.25% trypsin. Cells were collected by centrifugation at 1500 rpm for 5 min, and the cell pellet was resuspended in DMEM and 10% PDHS. Cells were plated onto poly-L-lysine-precoated tissue culture dishes (3 x 106 cells/35-mm dishes; 2 x 107 cells/100-mm diameter dishes) in DMEM containing 10% PDHS. Three days after plating the cells, the culture medium was removed, and DMEM and 10% PDHS containing 1% cytosine B-arasino furanoside were added. Two days later, medium was removed from the plates, and cultures were fed with DMEM and 10% PDHS. The cultures were allowed to establish for the indicated time periods before being used for experiments. These cultures contain about 85–90% neuronal cells and 10–15% astrocytic glial cells and have been used extensively by us as an in vitro model system to investigate Ang II regulation of neuromodulation and in elucidation of the interaction of this hormone with the catecholaminergic system (6, 7).

Western blot
Neuronal cultures, established in 100-mm dishes, were incubated in the lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 10% glycerol, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 2 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml aprotinin, and 2 µg/ml leupeptin], and lysates were centrifuged at 6000 x g for 10 min at 4 C. The protein content of the supernatant was determined by the Bradford protein assay kit (Bio-Rad Laboratories, Inc., Richmond, CA). The same amount of protein was subjected to SDS-PAGE, essentially as described previously (19). Proteins were transferred to nitrocellulose membrane, and nonspecific binding was blocked by 5% nonfat dry milk in TBST [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20] for 1 h. This was followed by incubation with various antibodies at proper dilution for 1 h at room temperature. Protein bands were detected by chemiluminescence assay reagent after incubation with antimouse or antirabbit IgG Fab conjugated with horseradish peroxidase secondary antibody and quantitated essentially as described previously (19).

RT-PCR measurements of PAI-1 and ß-actin mRNA levels
The mRNA levels for PAI-1 and ß-actin were measured by RT-PCR essentially as described previously (16). The validity of this procedure and optimal conditions for both RT and PCR reactions also have been established previously (16, 27). In brief, the procedure includes isolation of poly(A)+ RNA from neuronal cultures with the use of Dynal beads and running the RT reaction directly with the poly(A+)-Dynal beads complex. This is followed by using 5 µl RT solution for radioactive PCR and specific primers for PAI-1 or ß-actin in separate tubes. Our previous studies established that the PCR reaction is linear with the number of PCR cycles as well as with the RT reaction volumes, whether the PCR for PAI-1 and ß-actin is carried out in a single tube or in separate tubes. Thus, we used the PCR reactions for PAI-1 and ß-actin in parallel tubes with the use of 5 µl RT reaction. PCR products were separated on nondenaturing PAGE essentially as described previously (27). The gel was decasted, wrapped in a plastic bag, and exposed to an x-ray film overnight at 70 C. Bands representing PCR products on the x-ray film were scanned with the UVP Imagestore 5000 system (Ultraviolet Products, Cambridge, UK), and the density of each PCR product was then quantitated by the SW 5000 gel analysis program. Data were presented as the fold increase compared with the control value after normalization with ß-actin mRNA.

Immunofluorescence staining of neurofilaments
Monoclonal antibody directed against neurofilament protein was used to determine neurite extension and growth in hypothalamic-brainstem neurons in culture. Neuronal cultures, established in 35-mm diameter tissue culture dishes and grown for 5 d, were treated with Ang II for 3 d, rinsed twice with PBS, pH 7.4, and fixed in neutral buffered 4% paraformaldehyde for 1 h at room temperature. Nonspecific binding of the antibody was blocked by incubation with 5% normal goat serum containing 1% BSA and 0.03% Triton X-100 in PBS, pH 7.4, and incubated with monoclonal antibody to neurofilament at a 1:100 dilution in 1% BSA and 0.03% Triton X-100 in PBS, pH 7.4/1% BSA for 1 h at room temperature. After removal of excess primary antibody and staining with the fluorescein isothiocyanate-conjugated antimouse IgG Fab, the images were captured by a fluorescent microscope using a digital camera and analyzed essentially as described previously (27, 28, 29).

Effect of PAI-1 on neuronal survival and neurite extension and growth
Neuronal cells were plated onto 35-mm diameter poly-L-lysine- precoated tissue culture dishes at a density of 3 x 106 cells in DMEM containing 10% PDHS. Five days after growth, cultures were treated with the indicated concentrations of PAI-1 for 3 d. Cells were fixed and subjected to immunofluorescence analysis with the use of a neurofilament-specific antibody essentially as described above for the assessment of neurite extension and growth. In addition, cell nuclei were stained with 4,6 diamidine-2 phenylindole-dihydrochloride for the determination of total number of neurons after each treatment (30).

Immunoprecipitation and p70S6 kinase assay
An in vitro kinase assay was carried out essentially as described previously for determination of the activity of p70S6 kinase (31). Briefly, neuronal cell lysates were prepared in a phosphorylation lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 0.5% sodium deoxycholate, 2 mM EDTA, 1 mM phenylmethylsulfonylfluoride, and 10 µg/ml aprotinin]. After 20 min, lysates were centrifuged for 10 min at 14,000 x g at 4 C, and postnuclear supernatants containing 200 µg protein were immunoprecipitated with anti-p70S6 kinase antibody for 1 h (19). Immune complexes were precipitated with protein A/G Plus agarose and washed three times with lysis buffer and once with the kinase assay buffer [50 mM 3-[N-morpholino]propanesulfonic acid (pH 7.4), 10 mM MgCl2, and 1 mM dithiothreitol]. In vitro kinase assays were performed at 30 C in a 50-µl reaction volume of the kinase assay buffer containing 20 µM S6 kinase substrate peptide. The kinase reaction was initiated by the addition of 5 µM ATP, 10 mM MgCl2, and 10 µCi [{gamma}-32P]ATP mixture. The reactions were stopped with 5 x SDS-PAGE sample buffer, and reaction products were separated by SDS-PAGE and subjected to autoradiography (19). Phosphorylation of S6 was quantitated using a UVP Imagestore 5000 digital gel documentation system (16).

Depletion of neuronal cultures of p70S6 kinase and PAI-1 by specific antisense oligonucleotides (AON)
Neuronal cultures were established in 100-mm diameter culture dishes. Five days after establishment, cultures were treated with 2 µM AON or sense-oriented oligonucleotides (SON) for p70S6 kinase or with PAI-1 using lipofectin reagent for 48 h at 37 C essentially as described previously (18). Cells were collected and analyzed for p70S6 kinase protein and PAI-1 mRNA levels, whereas culture medium was used to measure PAI-1 activity using a PAI-1 activity assay kit (Chromogenix, Inc., Mölndal, Sweden). Initial experiments were carried out to establish the time course and concentrations of AON that produced optimal depletion of their homologous proteins. Sequences for the AONs and SONs were as follows: p70S6 kinase: AON, 5'-GTCAAACACTCCTGCCATAAC-3'; SON, 5'-GTTATGGCAGGAGTGTTTGAC-3'; and PAI-1: AON, 5'-GGCTGAAGACATCCTGCATCCT-3'; SON, 5'-AGGATGCA- GATGTCTTCAGCC-3'.

Experimental group and data analysis
Each data point in the measurement of p70S6 kinase, GAP-43 immunoreactivity, and PAI-1 mRNA was obtained from three neuronal culture dishes, derived from multiple brains of 1-d-old rats. Each experiment was repeated at least three times unless stated otherwise. Images from autoradiograms were captured with the UVP Imagestore 5000 system, and bands were quantitated, corrected for equal loading by normalization with a standard protein or ß-actin mRNA, and presented as the mean ± SE. Comparisons between the control and experimental groups were made using t test and ANOVA with Statistica software (Tulsa, OK). All immunofluorescence experiments were repeated at least four times. Images of cultures were captured on a digital camera, and a representative field of immunofluorescence was presented.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Materials and Methods
 Results
Discussion
 References
 
Effect of Ang II on neurotropic activity in brain neurons
Changes in neuronal GAP-43 immunoreactivity by immunofluorescence of neurofilament were used to measure neurotropic activity. Untreated neurons expressed limited numbers of neurites that were poorly stained with the neurofilament antibody (Fig. 1AGo, a and b). Ang II caused a dose-dependent increase in neurite extension and the number of neurites extended by each neuronal cell body (Fig. 1AGo, b–d). An extensive network of neurites was observed when cultures were treated with Ang II for 3 d. Neurite extension and growth were significantly attenuated by the treatment of cultures with losartan, an AT1 receptor-specific antagonist (Fig. 1AGo, e), but not by PD 123,319, an AT2 receptor-specific antagonist (Fig. 1AGo, f). The total numbers of neurons, as judged by the number of DAPI-stained nuclei, were comparable in the control and the Ang II-treated cultures (data not shown). Ang II also caused a 3.5-fold increase in GAP-43 immunoreactivity (Fig. 1BGo). Losartan, but not PD 123,319, inhibited Ang II stimulation of GAP-43 levels, indicating the involvement of the AT1 receptor subtype in this process. These observations demonstrate that Ang II is a trophic hormone to hypothalamic-brainstem neurons.



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  		Figure 1. Effect of Ang II on neurite extension and growth and GAP-43 expression. A, Effect of Ang II on neurite extension and growth. Neuronal cultures were prepared from 1-d-old rats, essentially as described in Materials and Methods. On d 5, cultures were incubated with DMEM plus 10% PDHS (a, b) or with DMEM and 10% PDHS containing 10 nM (c) or 100 nM (d) Ang II in the presence of 10 µM losartan (e) or 10 µM PD123319 (f) for 72 h at 37 C. Culture was fixed in fresh 4% paraformaldehyde and subjected to immunofluorescence analysis with a neurofilament-specific antibody as described in Materials and Methods. Bar, 20 µm. B, Effect of Ang II on GAP-43 immunoreactivity. Experimental conditions were essentially as described in A. Neuronal cultures were lysed, and lysates were used for Western blot to determine levels of GAP-43 as described in Materials and Methods. Top, A representative autoradiogram. Bottom, GAP-43 immunoreactivity was normalized with tubulin for equal loading. Data from three experiments are shown (mean ± SE). *, Significantly different (P < 0.05) from control. #, Significantly different (P < 0.05) from Ang II-treated neurons. Los, Losartan; PD, PD123,319.

 
Involvement of p70S6 kinase in the regulation of Ang II neurotropic action
To characterize the signal transduction pathway that mediates Ang II’s potent effect on neurotropic activity, we studied the effect of Ang II on p70S6 kinase, a signaling molecule that is downstream of PI3K. Ang II caused a time-dependent transient 8-fold increase in p70S6 kinase activity. Maximal stimulation was evident by 5 min, persisted for 30 min, and returned to control levels by 60 min (Fig. 2AGo). The stimulation was Ang II concentration dependent (Fig. 2BGo) and was completely blocked by 10 µM losartan, but not by PD123,319 (Fig. 2CGo). In addition, Ang II stimulation of p70S6 kinase was blocked by 1 nM rapamycin (Fig. 2CGo). This concentration of rapamycin did not have any effect on either basal or Ang II-stimulated MAP kinase or PI3K activities (data not shown).



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  		Figure 2. Effects of Ang II on p70S6 kinase activity. A, Time dependence. After incubation of neuronal cultures with 100 nM Ang II, cells were collected at the indicated time periods, enzyme was immunoprecipitated, and p70S6 kinase activity was measured as described in Materials and Methods. Top, Representative autoradiogram. Bottom, Quantification of radioactive bands (mean ± SE; n = 3). *, P < 0.05 vs. control. B, Dose dependence. Experimental conditions were essentially as described in A. A 10-min incubation with Ang II was carried out. Top, Representative autoradiogram. Bottom, Quantification of radioactive bands (mean ± SE; n = 3). *, P < 0.05 vs. control. C, Effect of inhibitors. Neuronal cultures were incubated without or with 100 nM Ang II containing 10 µM PD123319 or 1 nM rapamycin, and p70S6 kinase activity was measured after immunoprecipitation with the anti-p70S6 kinase antibody as described in Materials and Methods. Top, Representative autoradiogram. Bottom, Quantification of radioactive bands (mean ± SE; n = 3). *, P < 0.05 vs. control. #, P < 0.05 vs. Ang II-treated neurons. Los, Losartan; PD, PD123,319; Rapa, rapamycin.

 
In the next series of experiments we determined the effects of various inhibitors of PI3K and p70S6 kinase activities on Ang II-stimulated neurotropic action. Treatment of neuronal cultures with 100 nM Ang II for 3 d caused a 3-fold increase in GAP-43 levels. This stimulation was completely attenuated by cotreatment of cultures with 100 nM wortmannin or 1 nM rapamycin (Fig. 3AGo). In contrast, PD 98059, an inhibitor of MAPK kinase, failed to cause any significant effect on GAP-43 levels. The specificity of this effect was further established with the use of AON for p70S6 kinase. Pretreatment of neurons with 2 µM AON for p70s6 kinase for 48 h showed an 80% decrease in p70S6 kinase immunoreactivity (Fig. 3BGo). This treatment had no effect on MAP kinase immunoreactivity, verifying the specify of AON to its homologous kinase. The conditions that optimally depleted p70s6 kinase were used in subsequent experiments to determine the role of p70s6 kinase in the Ang II stimulation of GAP-43 levels. Depletion of p70S6 kinase resulted in a significant attenuation of Ang II stimulation of GAP-43 (Fig. 3AGo). Treatment with SON caused no significant changes in p70s6 kinase levels, nor any effects on Ang II-stimulated GAP-43 levels. Similar to the effect on GAP-43, depletion of p70S6 kinase by AON resulted in a significant inhibition of Ang II stimulation of neurite extension (Fig. 3CGo). These data confirmed that both PI3K and p70S6 kinase are involved in Ang II regulation of neurotropic activity.



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  		Figure 3. Effects of various inhibitors and antisense oligonucleotides on Ang II stimulation of GAP-43 levels and neurite extension and growth. A, Effects on GAP-43. Neuronal cultures were incubated without (control) or with 100 nM Ang II in the presence of 100 nM wortmannin, 1 nM rapamycin, or 50 mM PD98059 for 72 h at 37 C. After treatment with inhibitors, GAP-43 levels were determined by Western blot as described in Materials and Methods. For depletion of p70s6 kinase, neuronal cultures were treated with 2 mM AON or SON for p70s6 kinase for 48 h at 37 C as described in Materials and Methods. This was followed by incubation of cells with 100 nM Ang II for 72 h. Cells were lysed, and lysates were used for GAP-43 analysis. Top, Representative autoradiogram. Bottom, Mean ± SE (n = 3). *, P < 0.05 vs. control. #, P < 0.05 vs. Ang II treatment. Wort, Wortmannin, Rapa, rapamycin, PD, PD 98059. B, Effect of p70S6 kinase-specific AON and SON on immunoreactive of p70S6 kinase. Neuronal cultures were established in 100-mm diameter culture dishes. Control cultures and cultures transfected with 2 µM AON or SON for p70S6 kinase for 48 h at 37 C were used for Western blot analysis with the use of p70S6 kinase-specific antibody as described in Materials and Methods. C, Effects of p70S6 kinase depletion by the AON on neurite extension and growth. Neuronal cultures were transfected with 2 µM AON (A) or SON (B) to p70S6 kinase for 48 h. After treatment, 100 nM Ang II was added, and incubation was continued for an additional 72 h. Bar, 20 µM.

 
PAI-1 as a link between PI3K-p70S6 kinase in the neurotropic actions of Ang II
Previous studies have shown that Ang II stimulates the release of PAI-1 in brain neurons by a transcription-translation-dependent mechanism (20). This observation coupled with other studies describing the involvement of PAI-1 in the brain extracellular matrix formation and neurite outgrowth (22, 32, 33), led us to hypothesize that the neurotropic action of Ang II is mediated by PAI-1, and that the PI3K signaling pathway plays a key role in this process. Figure 4AGo shows that Ang II at 100 nM caused a 4-fold stimulation of neuronal PAI-1 mRNA levels. The stimulation was significantly blocked by coincubation of neuronal cultures with 100 nM wortmannin or 1 nM rapamycin, but not by 50 mM PD98059 (Fig. 4BGo).



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  		Figure 4. Effects of Ang II and various inhibitors on PAI-1 mRNA levels. A, Dose-response of Ang II. Neuronal cultures were incubated with the indicated concentration of Ang II for 4 h at 37 C. Levels of PAI-1 mRNA were measured as described in Materials and Methods. Top, Representative autoradiogram. Bottom, Quantification of radioactive bands that were normalized by actin mRNA (mean ± SE; n = 3). *, P < 0.05 vs. control. B, Effects of various inhibitors. Cells were treated without or with 100 nM Ang II in the presence of 100 nM wortmannin (Wort), 1 nM rapamycin (Rapa), or 50 µM PD98059 (PD) for 4 h at 37 C. Levels of PAI-1 mRNA were measured as described in Materials and Methods. Top, Representative autoradiogram. Bottom, Quantification of radioactive bands after normalized by actin mRNA (mean ± SE; n = 3). *, P < 0.05 vs. control. #, P < 0.05 vs. Ang II-treated neurons.

 
A direct effect of PAI-1 on Ang II regulation of GAP-43 was studied to further confirm the involvement of this protease inhibitor as a mediator of Ang II-stimulated neurotropic activity. Treatment with 20 and 40 nM PAI-1 for 3 d resulted in 2- and 3.5-fold increases in GAP-43 levels, respectively (Fig. 5Go). This effect was comparable to that seen with Ang II. Treatment of neuronal cultures with 40 nM PAI-1 resulted in an increase in GAP-43 and was associated with the development of an extensive network of neurites (data not shown). Although wortmannin and rapamycin attenuated Ang II-stimulated GAP-43 levels (Fig. 3Go), they had little effect on the PAI-1 induced increase in GAP-43 levels (Figs. 5Go).



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  		Figure 5. Effect of wortmannin and rapamycin on PAI-1-induced levels of GAP-43. Neuronal cultures were prepared as described in Materials and Methods. On d 5, cultures were incubated with DMEM containing 10% PDHS or with DMEM and PDHS containing PAI-1 in the presence of 100 nM wortmannin (Wort) or 1 nM rapamycin (Rapa). Cells were lysed and lysates were used for Western blot to determine levels of GAP-43 as described in Materials and Methods. Top, Representative autoradiogram. Bottom, Data from three experiments after normalized with tubulin (mean ± SE). *, P < 0.05 vs. control.

 
The specificity of the effect of PAI-1 on neurite extension and growth was further studied with the use of PAI-1 AON. Neuronal cultures were treated with 2 mM PAI-1-specific AON or SON for 48 h. This resulted in an 80% decrease in PAI-1mRNA and immunoreactive PAI-1 in the culture medium (Fig. 6Go, A and B). The decrease was specific for AON, as PAI-1 SON had no effect. Depletion of PAI-1 by AON resulted in a significant decrease in Ang II-stimulated neurite extension and growth (Fig. 6CGo).



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  		Figure 6. Effect of PAI-1 AON on PAI-1 mRNA and secreted PAI-1 levels. A and B, Neuronal cultures were incubated with 2 µM PAI-1 SON or AON for 48 h at 37 C, followed by 72-h treatment with 100 nM Ang II. PAI-1 mRNA levels (A) and PAI-1 activity released into the culture medium (B) were measured as described in Materials and Methods. Data are the mean ± SE (n = 3). *, P < 0.05 vs. control. This resulted in a decrease in PAI-1 mRNA by 80% and in medium PAI-1 by 65%. C, Neuronal cultures were incubated without (a and b) or with 2 mM PAI-1 SON (c) or AON (d) for 48 h at 37 C. After nucleotide incubation, cultures were treated without (a) or with 100 nM Ang II (b–d) for an additional 72 h as described in Materials and Methods. Cultures were subjected to neurofilament staining as described in Materials and Methods.

 
Finally, to quantitate the involvement of the p70S6 kinase-PAI-1 pathway in the neurotropic actions of Ang II, immunoblotting was carried out with the use of the neurofilament antibody. The data in Fig. 7Go confirm the immunofluorescence data. They show that Ang II caused a 6-fold increase in neurofilament protein. This stimulation was blocked by inhibition or depletion of p70S6 kinase or PAI-1.



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  		Figure 7. Effects of various inhibitions on neurofilament protein levels. A, Neuronal cultures were treated for 72 h with Ang II or PAI-1 in the absence or presence of various inhibitors: losartan (Los; 10 mM), PD 123319 (PD; 10 mM), wortmannin (Wort; 100 nM), and rapamycin (Rapa; 1 nM). Cell lysates were used to determine the levels of neurofilament protein by Western blot analysis as described in Materials and Methods. B, Neuronal cultures treated without or with 100 nM Ang II in the absence or presence of 100 nM wortmannin, 1 nM rapamycin, or 50 mM PD98059 in certain experiments. Cells were pretreated with AON to p70S6 kinase for a 48-h period before treatment with Ang II. Cell lysates were used to quantitate neurofilament protein. Top panel, Representative autoradiogram; bottom panel, quantitation of bands. *, P < 0.05 vs. control. #, P < 0.05 vs. Ang II-treated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Materials and Methods
 Results
 Discussion
References
 
This study provides support for a novel neurotropic role of Ang II, a hormone that is well known for its role in norepinephrine neuromodulation, regulation of ion channels, and central control of blood pressure (34, 16, 27). In addition, it establishes a signal transduction pathway involved in this action by the AT1 receptor, a G protein-coupled receptor.

There are numerous studies in the literature describing the cellular events associated with the neurotropic actions of many growth factors (35, 36, 37). However, most of these studies used neurally derived cell lines from peripheral tissues such as PC12 cells. As a result, it has been difficult to extrapolate the mechanism of neurotropic activity from the cell lines to normal neurons and the central nervous system. Our study circumvents this problem by using brain neurons in primary culture. It demonstrates that Ang II stimulation of the AT1 receptor regulates neurotropic activity. This novel action of a G protein-coupled receptor could not have been revealed with the use of neural cell lines such as PC12 cells, because Ang II fails to exert neurotropic actions in these and other cell lines.

Previous studies have suggested a link between the neurotropic and apoptotic pathways, intimating that turning off one pathway stimulates the other. For example, nerve growth factor stimulation of neurotropic action involves activation of PI3K. Inhibition of PI3K attenuates the neurotropic action of nerve growth factor, but stimulates apoptosis (38, 39). This process does not seem to occur in these primary brain neurons. Inhibition of PI3K does not stimulate neuronal apoptosis by Ang II. Further confirmation of this view is provided by the fact that the AT2 receptor subtype, not the AT1 receptor subtype, mediates apoptosis in these neurons (10).

The involvement of the PI3K signaling pathway in various physiological and cellular responses, such as membrane ruffling, receptor internalization, neural survival, neurite extension and growth, and other processes requiring cytoskeletal reorganization, is fairly well established (40, 41). However, characterization of downstream signaling kinases and other mediators of neurotropic action is poorly understood. This study highlights the involvement of a PI3K-p70S6 kinase-PAI-1 signaling cascade in the neurotropic actions of Ang II. It appears that there is little or no cross-over between this signaling system and that of the neuromodulatory signaling pathway of Ang II that involves the Ras-Raf-MAP kinase. This assertion is supported by the following. 1) Inhibition of the MAP kinase pathway by MAPKK inhibitor, PD 98059, had little effect on Ang II-induced neurotropic activity (current study). However, PD 98059 completely attenuated the effect of Ang II on NE neuromodulation (18). 2) Inhibition of PI3K and/or p70S6 kinase attenuated the effect of Ang II on neurotropic activity (current study), without affecting NE neuromodulation (19). Control studies established that inhibitors of PI3K (wortmannin) and p70S6 kinase (rapamycin) exerted no significant effect on MAP kinase activity at concentrations that effectively inhibited PI3K and p70S6 kinase. Similarly, PD 98059 showed no inhibitory effect on Ang II stimulation of PI3K activity (19). Finally, the involvement of these signaling kinases was confirmed by the use of AON-induced depletion of p70S6 kinase. The precise mechanism by which p70S6 kinase activation is achieved in these neurons remains to be elucidated.

This study demonstrates that the neurotropic action of Ang II is mediated by PAI-1. Although PAI-1 has been previously implicated in neurotropic action (21), the link between Ang II and PAI-1 involving the PI3K-p70S6 kinase signaling system is a novel observation. The following evidence strongly support this link: 1) Ang II, exclusively via the AT1 receptor subtype, stimulates PAI-1 gene expression and its secretion into the medium (current study and Ref. 16); 2) exogenous PAI-1 is a potent neurotropic agent in the stimulation of both neurite extension and GAP-43 expression; 3) depletion of PAI-1 by AON results in significant attenuation of Ang II regulation of neutropic action; 4) inhibition of PI3K and p70S6 kinase, but not MAPK kinase, inhibits Ang II stimulation of PAI-1 mRNA; and 5) losartan, the AT1 receptor subtype-specific antagonist, blocks Ang II-induced, but not PAI-1-induced, neurotropic activity. Although the involvement of PAI-1 in Ang II-induced signaling of neurotropic action is unique, the mechanism by which this pathway regulates PAI-1 gene expression and subsequent PAI-1 stimulation of neurotropic action remains speculative. Based on our earlier observations, it is tempting to suggest that the extracellular PAI-1 modifies the nature of extracellular matrix resulting in the rearrangement of neuronal cytoskeletal elements and the promotion of neurite extension and growth (21).

The study raises a number of important issues relevant to the role of Ang II in neuronal growth in vivo. For example, is the neurotropic action of Ang II unique for hypothalamic-brainstem neurons? Our data support the view that this is not the case. Cortical neurons in which the level of AT1 receptor is 20–30% that in hypothalamic-brainstem neurons show a similar effect of Ang II on neurite extension and growth and GAP-43 (unpublished data). Is our finding in in vitro neurons of any physiological relevance in vivo? There is no direct evidence, although the following data support the role of Ang II in the growth, development, and differentiation of the brain. 1) Our preliminary experiments indicated that intracerebroventricular injection of Ang II in the rat brain stimulates PI3K and p70S6 kinase activities in the hypothalamus, indicating that this signaling pathway remains intact in the adult brain and is not the result of establishment of neurons in vitro (19). Expression of the AT1 receptor is developmentally regulated in various brain areas (42). There is a high abundance of these receptors in many anatomical areas, such as the hippocampus; cingulate; retrospleaial, penrhinal, and infralimbic cortex; olivocerebellar system; and hypothalamic nuclei early in development, with a significant decrease in adulthood. Based on these and other observations, the role of Ang II in postnatal development, synapse formation, and neuronal maturation has been proposed (43). Studies with knockout animals have indicated an important role of AT1 receptor in the animal development. There are two subtypes of the AT1 receptor, AT1a and AT1b (44, 45). Initial knockout experiments with one AT1b receptor subtype resulted in a normal phenotype (46). However, double knockout mice produced by mating single gene mutants of AT1a and AT1b resulted in a significant decrease in the survival rate (47). The ones that survived expressed significant developmental, morphological, and physiological deficiencies in various Ang II target tissues (47, 48). It would be pertinent to study the brain developmental pattern in both nonsurviving and surviving double knockouts to determine whether neuronal development was impaired. These data are by no means conclusive, but support the view that the AT1 receptor is important in development.

In conclusion, our observations establish a unique signal transduction pathway for stimulation of the neurotropic actions of Ang II in brain neurons. Ang II stimulates GAP-43 expression and neurite extension by recruiting the PI3K-p70S6 kinase-PAI-1 signaling pathway.


   Acknowledgments
 
We thank Marya Fancy and Mary Spivey for help with the preparation of the manuscript, and Ling Liu for her expert assistance with the preparation of neuronal cultures. Dr. Sharon Francis’s editorial assistance is gratefully acknowledged.


   Footnotes
 
This work was supported by NIH Grant HL-33610 and Grant 98-10149 from the American Heart Association, Florida affiliate.

1 Postdoctoral fellow of the American Heart Association, Florida affiliate. Back

2 Visiting scholar from Fujian Medical University, Division of Cardiology and Hypertension, Fuzhou, People’s Republic of China. Back

Abbreviations: Ang II, Angiotensin II; AON, antisense oligonucleotides; AT1, angiotensin type 1; GAP-43, growth associated protein-43; PAI-1, plasminogen activator inhibitor-1; PDHS, plasma-derived horse serum; poly(A)+, polyadenylated; SON, sense-oriented oligonucleotides; WKY, Wistar Kyoto.

Received January 31, 2001.

Accepted for publication April 27, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Materials and Methods
 Results
 Discussion
 References
 

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