Angiotensin II stimulates rat astrocyte mitogen-activated protein kinase activity and growth through EGF and PDGF receptor transactivation

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Regulatory Peptides 144 (2007) 115 – 122 www.elsevier.com/locate/regpep

Angiotensin II stimulates rat astrocyte mitogen-activated protein kinase activity and growth through EGF and PDGF receptor transactivation Michelle A. Clark ⁎, Noelvy Gonzalez Department of Pharmaceutical and Administrative Sciences, College of Pharmacy, Cardiovascular and Metabolic Research Unit, Nova Southeastern University, 3200 South University Drive, Fort Lauderdale, FL 33328, United States Received 15 May 2007; received in revised form 3 July 2007; accepted 4 July 2007 Available online 13 July 2007

Abstract We showed that the intracellular tyrosine kinases src and pyk2 mediate angiotensin II (Ang II) stimulation of growth and ERK1/2 mitogenactivated protein (MAP) kinase phosphorylation in astrocytes. In this study, we investigated whether the membrane-bound receptor tyrosine kinases platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors mediate Ang II stimulation of ERK1/2 and astrocyte growth. Ang II significantly stimulated PDGF and EGF receptors in a dose- and time-dependent manner. The PDGF receptor and the EGF receptor were maximally stimulated with 100 nM Ang II (0.98 ± 0.18- and 4.4 ± 1.4-fold above basal, respectively). This stimulation occurred as early as 5 min, and was sustained for at least 15 min for both receptor tyrosine kinases. Moreover, 1 μM AG1478 and 0.25 μM PDGFRInhib attenuated Ang II stimulation of the EGF and PDGF receptors, respectively. Ang II-induced phosphorylation of ERK1/2 and astrocyte growth was mediated by both PDGF and EGF receptors. This report also provides novel findings that co-inhibiting EGF and PDGF receptors had a greater effect to decrease Ang II-induced ERK1/2 (90% versus 49% and 71% with PDGF receptor and EGF receptor inhibition, respectively), and astrocyte growth (60% versus 10% and 32% with PDGF receptor and EGF receptor inhibition, respectively). In conclusion we showed in astrocytes that the PDGF and the EGF receptors mediate Ang II-induced ERK1/2 phosphorylation and astrocyte growth and that these two receptors may exhibit synergism to regulate effects of the peptide in these cells. © 2007 Elsevier B.V. All rights reserved. Keywords: Angiotensin II; PDGF; EGF; Astrocytes; Growth; Mitogen-activated protein kinase

1. Introduction Angiotensin II (Ang II) is a potent mitogen, stimulating the growth and proliferation of numerous cells located in the periphery as well as in the central nervous system (CNS). The classic role of Ang II in controlling blood pressure as a result of its proliferative and vasoconstrictive effects on vascular smooth muscle cells (VSMCs) and other peripheral cells is well known [1]. In recent years, the importance of the intrinsic brain renin angiotensin system in influencing blood pressure has been established [2]. Well-defined actions of Ang II in the CNS include regulation of blood pressure by controlling sympathetic outflow, water intake, sodium appetite, and secretion of ACTH, vasopressin and other pituitary hormones [3–6]. Ang II interacts with AT1 and AT2 receptors to mediate its effects. Studies using ⁎ Corresponding author. Tel.: +1 954 262 1340; fax: +1 954 2622278. E-mail address: [email protected] (M.A. Clark). 0167-0115/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2007.07.001

selective AT1 and AT2 receptor antagonists and gene targeting studies suggest that most of the functions of the renin angiotensin system are mediated by AT1 receptors [7,8]. AT2 receptors are important in fetal development, and can be induced later in adult life under pathological conditions [9]. Indeed, central effects of Ang II primarily occur by its interaction with AT1 receptors located in specific brain regions known to be involved in sensory and motor functions. AT1 receptors have been clearly identified in the central nervous system and their stimulation is coupled to the activation of phosphoinositide specific phospholipase C (PLC), calcium release, and prostaglandin release [10–17]. The AT1 receptor has also been shown to mediate Ang II regulation of neuronal activity via a series of events that include reactive oxygen species (ROS) generation, and inhibition of the delayed rectifier potassium current (I(Kv)) suggesting that these signaling pathways are critical cellular events in central Ang II regulation of cardiovascular function [18].

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The AT1 receptor-mediated vasoconstrictive and growth promoting effects in nonneural cells occur in large part via stimulation of ERK1 and ERK2 MAPK (ERK1/2) which are terminal serine/threonine kinases in the MAPK pathway. In the CNS, neuronal AT1 receptors also signal via ERK1/2, and are essential for Ang II-mediated increase in tyrosine hydroxylase, dopamine hydroxylase, and norepinephrine transporter gene transcription [19]. In previous studies, we showed that Ang II induces ERK1/2 in a concentration- and time-dependent manner in astrocytes cultured from the brainstem and the cerebellum [20]. Moreover, ERK1/2 were involved in Ang IIinduced proliferation of astrocytes since blocking Ang II stimulation of ERK1/2 using PD98059, the MAP kinase kinase (MEK) inhibitor, prevented Ang II stimulation of astrocyte growth. This effect was more apparent in brainstem cultures which reflect higher levels of AT1 binding sites as compared to AT2 binding sites in these regions [14]. Interestingly, Ang II induction of ERK1/2 was blocked with the nonselective tyrosine kinase inhibitor, genistein, suggesting that Ang IIinduced astrocyte growth was partially dependent on an upstream, as yet unknown, tyrosine kinase. Recently, we showed that the endogenous tyrosine kinases src and pyk2 are essential intracellular signals that are involved in Ang II-induced ERK1/2 phosphorylation and astrocyte growth [21]. However, numerous studies have shown that crosstalk between the AT1 receptor and the membrane-bound tyrosine kinases, platelet-derived growth factor (PDGF) receptor and epidermal derived growth factor (EGF) receptor, is an important factor in the initiation of intracellular signals after Ang II binds to the AT1 receptor [22–24]. However, the involvement of these transmembrane tyrosine kinase receptors in Ang II-mediated effects in astrocytes has not been studied and was the focus of our study. In the current study, we determined whether Ang II stimulated these membrane-bound receptors with tyrosine kinase activity in brainstem astrocytes and investigated whether these tyrosine kinase receptors act in concert to mediate Ang II-induced ERK1/2 phosphorylation and Ang II stimulation of astrocyte growth. 2. Materials and methods 2.1. Materials 3

H-thymidine (2000 Ci/mmol) was purchased from ICN Biomedical (Irvine, CA). Dulbecco's modified Eagles Medium (DMEM)/F12 (1:1), fetal bovine serum (FBS), penicillin, streptomycin, and trypsin/EDTA, were obtained from VWR (Suwanee, GA). The phospho-specific ERK1/ ERK2 antibody and the phospho-specific PDGF beta receptor antibody were purchased from Cell Signaling Technology (Beverly, MA). The phospho-specific EGF receptor antibody (EGFR [pY1068]) was purchased from Biosource International (Camarillo, CA). Anti-actin was purchased from Sigma (St Louis, MO). ECL chemiluminescent reagents were purchased from GE Healthcare (Piscataway, NJ). Ang II was obtained from Bachem (Torrance, CA). The PDGF

receptor inhibitor ((4-(6,7-Dimethoxy-4-quinazolinyl)-N-(4phenoxyphenyl)-1-piperazine-carbox-amide; PDGFRInhib), and the EGF receptor inhibitor (AG1478) were purchased from Calbiochem, Inc. (La Jolla, CA). All other chemicals were purchased from VWR International (Suwanee, GA) or Sigma (St Louis, MO). 2.2. Preparation of astrocytes Timed, pregnant Sprague–Dawley rats were obtained from Charles River Laboratories (Wilmington, MA) and maintained in the ALAAC-accredited animal facility of Nova Southeastern University. Primary cultures of astrocytes were prepared from the brainstem of 2–3 days old neonatal pups by physical dissociation as previously described [14]. Cells were maintained in DMEM/F12 with 10% FBS, 100 μg/mL penicillin, and 100 units/mL streptomycin at 37 °C in a humidified CO2 incubator (5% CO2 and 95% air). Cultures were fed every 3 to 4 days until confluent. Confluent monolayers were placed in DMEM/F12 containing 10 mM Hepes, pH 7.5, 10% FBS and antibiotics and shaken overnight to remove oligodendrocytes. Astrocytes were detached with trypsin/EDTA (0.05% trypsin, 0.53 mM EDTA), replated at a ratio of 1 to 10, and grown to confluence prior to use (approximately 7 days). Isolated cells showed a positive immunoreactivity with an antibody against glial fibrillary acidic protein and negative immunoreactivity with markers for neurons, fibroblasts or oligodendrites. 2.3. Cell preparation Cultured astrocytes were made quiescent by a 48 hour treatment with serum-free media and treated for 10 min with Ang II in the presence and absence of the EGF receptor inhibitor (1 μM AG1478) or the PDGF receptor inhibitor (0.25 μM PDGFRInhib). Basal and stimulated levels of the proteins were determined in the presence of DMSO which was used to dissolve both PDGFRInhib and AG1478. Cells were also treated with increasing concentrations of Ang II (0.1 nM to 1 μM) or with 100 nM Ang II for 1 min to 30 min. Cell lysates were prepared by washing the monolayers with phosphate-buffered saline containing 0.01 mM NaVO4 to prevent the dephosphorylation of activated phosphorylated proteins. Cells were solubilized in supplemented lysis buffer (100 mM NaCl, 50 mM NaF, 5 mM EDTA, 1% Triton X-100, 50 mM Tris–HCl, 0.01 mM NaVO4, 0.1 mM PMSF and 0.6 μM leupeptin, pH 7.4) for 30 min on ice. The supernatant was clarified by centrifugation (12,000 ×g for 10 min, 4 °C) and the protein concentration was measured by the Lowry method [25]. 2.4. Western blot analysis Solubilized proteins were separated in 10% polyacrylamide gels and transferred to nitrocellulose membranes. Nonspecific binding to the membranes was blocked by incubation in 5% Blotto (5% evaporated milk, 1% Tween-20 in Tris-buffered saline). Subsequently, membranes were probed with the

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Fig. 1. Effect of Ang II on PDGF receptor protein phosphorylation. Quiescent monolayers of brainstem astrocytes were incubated for 10 min with increasing concentrations of Ang II (A) or with 100 nM Ang II for 1 min to 30 min (B). PDGF receptor protein phosphorylation was measured by Western blot analysis using an antibody specific for the phosphorylated form of the PDGF receptor. Protein loading was visualized using an actin antibody. The data were analyzed by densitometry and the amount of phosphorylation was calculated as the fold-increase above basal in the presence of vehicle. Each value represents the mean±SEM of preparations of brainstem astrocytes from 5 to 6 litters of neonatal rat pups. ★ and # denotes pb 0.05 as compared to basal levels for PDGF receptor phosphorylation in astrocytes prepared from the brainstem.

following antibodies that specifically recognized the activated phosphorylated form of the proteins: ERK1/2 (1:5000 dilution in 5% Blotto); PDGF receptor (1:1000 in Tris-buffered saline

containing 5% BSA); EGF receptor (1:1000 in Tris-buffered saline containing 1% BSA). After incubating with primary antibodies, the membranes were probed with goat anti-rabbit

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antibody coupled to horseradish peroxidase: 1:5000 dilution in 5% Blotto for ERK1/2 and PDGF receptor and 1:2000 in 5% Blotto for EGF receptor. The immunoreactive bands were visualized using ECL reagents and the data quantified by densitometry. To visualize protein loading, actin was measured on solubilized proteins that were separated by electrophoresis in 10% polyacrylamide gels. The proteins were subsequently transferred to nitrocellulose membranes and then nonspecific binding was minimized by blocking with 5% Blotto. The membrane was then incubated with an anti-actin antibody (1:1000 in Tris-buffered saline containing 5% BSA) and subsequently probed with goat anti-rabbit antibody coupled to horseradish peroxidase (1:5000 dilution in 5% Blotto). The immunoreactive bands were visualized using ECL reagents and quantified by densitometry. 2.5. Measurement of DNA synthesis Subconfluent monolayers of cells growing in 24-well culture dishes were made quiescent by a 48 hour treatment with serum-free media. Individual wells were then treated for 48 hour with 100 nM Ang II in the presence and absence of various inhibitors, and 3 H-thymidine (0.25 Ci/mL culture medium) was added during the last 24 hour of treatment. Basal and Ang II-induced DNA synthesis was measured in the presence of DMSO which was used to dissolve the tyrosine kinase inhibitors. Newly synthesized DNA was precipitated with 5%TCA, dissolved in 0.25 N NaOH, and quantified by liquid scintillation spectrometry as previously described [26]. 2.6. Statistics All data are expressed as the mean ± SEM of 4 or more experiments, as indicated. T-tests or repeated measures one-way analysis of variance (ANOVA) with Dunnett's post-test was used to compare treatment groups with control, using PRISM (GraphPad). The criterion for statistical significance was p b 0.05. 3. Results 3.1. PDGF receptor activation by Ang II Cultured neonatal brainstem astrocytes were incubated with increasing concentrations of Ang II (0.1 nM to 1 μM) to determine whether the peptide activates the PDGF receptor in these cells. Ang II increased the phosphorylation of the PDGF receptor in brainstem astrocytes in a concentration-dependent manner (Fig. 1A). Maximal phosphorylation was observed with 100 nM Ang II (0.98± 0.18-fold above basal) and the EC50 value for Ang II-induced PDGF receptor phosphorylation was 0.2 nM. This Ang II effect was time-dependent and occurred as early as 5 min and continued for up to 30 min (Fig. 1B). Quiescent astrocytes were also pretreated for 15 min with a selective PDGF receptor inhibitor (0.25 μM 4-(6,7-Dimethoxy4-quinazolinyl)-N-(4-phenoxyphenyl)-1-piperazine-carbox-

amide) and then stimulated with 100 nM Ang II for 10 min. Pretreatment of astrocytes with the selective PDGF receptor inhibitor attenuated Ang II-induced receptor phosphorylation by 67% suggesting that this was a selective effect of Ang II to activate the PDGF receptor (Table 1). 3.2. EGF receptor activation by Ang II To determine whether Ang II stimulated the EGF receptor in brainstem astrocytes, quiescent cells were treated with increasing concentrations of Ang II (0.1 nM to 1 μM) for 10 min. Fig. 2A shows that Ang II stimulated phosphorylation of the EGF receptor with a maximal effect occurring with 100 nM Ang II (4.4 ± 1.4 above basal levels). The EC50 value for Ang II-induced EGF receptor phosphorylation was 1.1 nM. Although lower doses of Ang II produced a 50% increase in the PDGF receptor, the EGF receptor was more sensitive to the effects of Ang II with a 4-fold higher level of stimulation. The time pattern for increases in phosphorylation of the EGF receptor by Ang II was similar to that observed with the PDGF receptor. Phosphorylation occurred as early as 5 min, and peaked at about 15 min (Fig. 2B). Further, treatment of astrocytes with a combination of 100 nM Ang II and the selective EGF receptor inhibitor AG1478 (1 μM) prevented more than 90% Ang II induction of the EGF receptor suggesting that Ang II selectively phosphorylated this receptor (Table 1). 3.3. Mitogen-activated protein kinase regulation by PDGF and EGF receptors To delineate the role of the PDGF receptor and the EGF receptor in Ang II stimulation of ERK1/2 MAPKs, quiescent brainstem astrocytes were pretreated with 0.25 μM of the PDGF receptor inhibitor, or with 1 μM AG1478, the selective EGF receptor inhibitor, for 15 min. Subsequently, the cells were treated with 100 nM Ang II for 10 min. As shown in Table 2, Ang II stimulated the phosphorylation of ERK1/2. The selective PDGF receptor inhibitor attenuated by 62% Ang II-induced ERK1/2 activities. Likewise, pretreatment of astrocytes with the EGF receptor inhibitor decreased Ang II-induced ERK1/2 phosphorylation by 82%. These findings suggest that Ang II transactivates both the PDGF receptor and the EGF receptor

Table 1 Effects of Ang II on PDGF and EGF receptors phosphorylation Treatment

100 nM Ang II +0.25 μM PDGFRInhib +1 μM AG1478

PDGF receptor protein

EGF receptor protein

(Fold above basal)

(Fold above basal)

1.1 ± 0.18 0.36 ± 0.15⁎

3.8 ± 1.1 0.22 ± 0.25⁎

⁎ Denotes p b 0.05 as compared to Ang II stimulation of the PDGF or EGF receptor proteins. Basal and stimulated astrocytes were treated with DMSO, the vehicle for the inhibitors (PDGFRInhib; (4-(6,7-Dimethoxy-4-quinazolinyl)-N(4-phenoxyphenyl)-1-piperazine-carbox-amide). Values are calculated based on the individual experiments of 5 or more preparation of astrocytes.

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Fig. 2. Effect of Ang II on EGF receptor protein phosphorylation. Quiescent monolayers of brainstem astrocytes were incubated with increasing concentrations of Ang II for 10 min (A) or with 100 nM Ang II for 1 min to 30 min (B). EGF receptor protein phosphorylation was measured by Western analysis using an antibody specific for the phosphorylated form of the EGF receptor. Protein loading was visualized using an actin antibody. The data were analyzed by densitometry and the amount of stimulation was calculated as the fold-increase above basal in the presence of vehicle. Each value represents the mean ±SEM of preparations of brainstem astrocytes from 5 or more litters of neonatal rat pups. ★ and # denotes pb 0.05 as compared to basal levels for EGF receptor phosphorylation in astrocytes prepared from the brainstem.

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Table 2 Effects of EGF receptor and PDGF receptor on Ang II-induced ERK1/2 MAP kinase phosphorylation Treatment

Treatment

EGF receptor protein

MAP kinase activity (Fold above basal)

100 nM Ang II +1 μM AG1478 +0.25 μM PDGFRInhib +1 μM AG1478 and 0.25 μM PDGFRInhib 100 nM Ang II +500 nM AG1478 +125 nM PDGFRInhib +500 nM AG1478 and 125 nM PDGFRInhib

Table 3 Effect of the PDGF receptor on Ang II-induced EGF receptor phosphorylation

24.7 ± 7.0 4.4 ± 1.7⁎ 9.4 ± 2.1⁎ 2.3 ± 1.2⁎ 22.8 ± 6.0 6.5 ± 2.0⁎ 1.7 ± 3.5⁎ 0.70 ± 0.5⁎

⁎Denotes p b 0.05 as compared to Ang II stimulation of ERK1/2 MAP kinase. Basal and stimulated astrocytes were treated with DMSO, the vehicle for the inhibitors. Values are calculated based on the individual experiments of 6 or more preparation of astrocytes.

before stimulating this MAP kinase pathway. However, the EGF receptor played a more significant role than the PDGF receptor in this Ang II effect and may reflect a higher sensitivity of the EGF receptor to Ang II. Since both receptor tyrosine kinases seem to be involved in Ang II induction of ERK1/2, we tested whether concomitant blockade of both receptors would prevent Ang II-induced ERK1/2 phosphorylation. Astrocytes were pretreated with both the EGF and PDGF receptor inhibitors for 15 min followed by treatment with 100 nM Ang II for 10 min. As shown in Table 2, the combination treatment decreased by 91% Ang II-induced ERK1/2 MAP kinase phosphorylation, an effect that was greater than treating with each individual inhibitor. To determine if lower doses of the inhibitors would cause a similar effect, the concentrations of AG1478 and the PDGF receptor inhibitor were decreased by 50% (500 nM and 125 nM, respectively). Although the lower doses of the individual blockers decreased Ang II-induced ERK1/2 MAP kinase phosphorylation to a smaller extent (49% and 71% inhibition with the PDGF and EGF receptor inhibitors, respectively), the combination treatment resulted in a similar inhibition pattern of 92% inhibition (Table 2). These findings suggest that the two receptors may act in concert to control Ang II induction of ERK1/2 activity. Since inhibition of the EGF receptor had a greater effect in reducing Ang II induction of ERK1/2, astrocytes pretreated with the PDGF receptor inhibitor followed by treatment with 100 nM Ang II were probed with the EGF receptor antibody. As shown in Table 3, blocking the PDGF receptor had no effect on Ang II-stimulated EGF receptor phosphorylation. These findings suggest that the greater inhibition of Ang II-induced ERK1/2 MAP kinase observed by blocking this receptor is due to a selective effect of Ang II on the EGF receptor and not through sequential stimulation of the PDGF receptor then the EGF receptor. Although we cannot rule out transactivation of other tyrosine kinase receptors in this Ang II effect, our findings suggest that the EGF and PDGF receptors play major roles in Ang II activation of ERK1/2 in brainstem astrocytes through discrete mechanisms of action.

(Fold above basal) 100 nM Ang II +0.25 μM PDGFRInhib

3.9 ± 0.8 3.8 ± 0.7

Basal and stimulated astrocytes were treated with DMSO, the vehicle for PDGFRInhib. Values are calculated based on the individual experiments of 4 or more preparation of astrocytes.

3.4. Effect of PDGF and EGF receptors on Ang II-induced astrocyte DNA synthesis Since the PDGF and EGF receptors are involved in Ang II phosphorylation of ERK1/2, studies were done to determine whether these receptor tyrosine kinases are involved in Ang IIinduced astrocyte DNA synthesis. As shown in previous studies, Ang II significantly increased brainstem astrocyte growth (Table 4). This effect was attenuated about 57% and 77% in the presence of the PDGF and EGF receptor inhibitors, respectively, suggesting that both receptors participate in Ang II stimulation of astrocyte growth. To determine whether concomitant blockade of both the EGF and PDGF receptor prevented Ang II-induced astrocyte growth, quiescent astrocytes were treated for 48 hour with both inhibitors in the presence and absence of 100 nM Ang II. In the presence of both inhibitors, no additional growth inhibition was observed as compared to the individual inhibitors (Table 4). However, the combination treatment may have been toxic since the cell growth observed in the presence of the inhibitors alone was significantly lower than control (8120 ± 1200 CPM in controls versus 5000 ± 670 CPM combination treatments). To determine whether using lower doses of the inhibitors would overcome this toxicity, the concentrations of the inhibitors were decreased to 500 nM AG1478 and 125 nM of the PDGF receptor inhibitor. As shown in Table 4, the lower concentrations of AG1478 and the PDGF receptor inhibitor only blocked Ang II-induced astrocyte growth by 32% and 10%, respectively. Interestingly, the combination of the two inhibitors at the lower doses prevented Ang II-induced astrocyte Table 4 Effects of Ang II on astrocyte DNA synthesis: role of the PDGF receptor and EGF receptor Treatment

3

H-thymidine incorporation

(Fold above basal) 100 nM Ang II +0.25 μM PDGFRInhib +1 μM AG1478 +0.25 μM PDGFRInhib and 1 μM AG1478 100 nM Ang II +125 nM PDGFRInhib +500 nM AG1478 +125 nM PDGFRInhib and 500 nM AG1478

2.8 ± 0.5 1.2 ± 0.4⁎ 0.64 ± 0.2⁎ 0.98 ± 0.1⁎ 3.1 ± 0.8 2.8 ± 0.8 2.1 ± 1.0 1.2 ± 0.4⁎

⁎Denotes p b 0.05 as compared to Ang II-stimulated thymidine incorporation. Basal and stimulated astrocytes were treated with DMSO, the vehicle for the inhibitors. Values are calculated based on the individual experiments of 6 or more preparation of astrocytes.

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growth by over 60% suggesting that the two inhibitors may have a synergistic effect to prevent Ang II-induced astrocyte growth. Moreover, this effect was not due to toxicity of the inhibitors since the combination inhibitor treatment did not affect astrocyte growth (10,330 ± 3200 CPM in controls versus 9300 ± 2670 CPM combination treatment). Thus, in brainstem astrocytes, the EGF and the PDGF receptors may have synergistic effects to mediate Ang II-induced astrocyte growth. 4. Discussion The brain contains an intrinsic renin angiotensin system that participates in Ang II regulation of cardiovascular functions. In particular, astrocytes contain various components necessary for Ang II synthesis and, in fact, serve as the major source of angiotensinogen, the precursor molecule for Ang II in the CNS [27–29]. Ang II is a mitogen that stimulates the growth of numerous cells in the periphery, and as shown by our group and others, also stimulates growth of astrocytes in the CNS [20,30]. We demonstrated that Ang II-induced astrocyte growth was attenuated by PD98059, a selective MEK kinase inhibitor. Furthermore, genistein, a nonselective tyrosine kinase inhibitor prevented Ang II stimulation of ERK1/2 as well as Ang II stimulation of astrocyte growth [20]. Recently, we showed that the endogenous tyrosine kinases src and pyk2 are involved in this Ang II effect [21]. In the current study, we showed for the first time that the membrane-bound tyrosine kinases PDGF and EGF receptors mediate Ang II phosphorylation of ERK1/2 and DNA incorporation in astrocytes. In peripheral cells such as VSMCs and other cells, crosstalk between Ang II and these membrane-bound tyrosine kinases mediates several important effects of Ang II including protein synthesis of growth promoting molecules [22–24]. Furthermore, mitogen stimulation of EGF and PDGF receptors has been shown in astrocytes suggesting that astrocytes express these membrane-bound tyrosine kinase receptors and are potential targets for Ang II transactivation in astrocytes [31–33]. Ang II stimulated both PDGF and EGF receptors in a concentration- and time-dependent manner (Figs. 1 and 2). Maximal effects of Ang II on both the EGF receptor and the PDGF receptor were observed with 100 nM Ang II and as early as 5 min in brainstem astrocytes. These findings are consistent with previous studies showing that 100 nM Ang II is ideal for stimulation of Ang II intracellular effects such as PLC stimulation and calcium release [13–15]. The effects of Ang II on the EGF and PDGF receptors were sensitive to selective blockers (Table 1), and although this is a novel finding for astrocytes, it has been previously described for other cell types [34]. Further, pretreatment of astrocytes with the selective receptor tyrosine kinase antagonists not only prevented the Ang II-induced ERK1/2 phosphorylation but also Ang II induction of astrocyte growth (Table 4); thus, it is clear that the pathways leading to ERK1/2 phosphorylation from the AT1 receptor in astrocytes involve at least both tyrosine kinase receptors, EGF and PDGF. Interestingly, when ERK1/2 activation is viewed in the context of Ang II transactivation of membrane-bound receptor tyrosine kinases,

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it appears that Ang II may act at both receptors to activate ERK1/2 in astrocytes since inhibition of the EGF receptor or the PDGF receptor affected Ang II MAPK phosphorylation (see data in Table 2). This observation was substantiated in further experiments using a combination treatment with both inhibitors. The co-addition of both receptor inhibitors prevented by over 90% Ang II induction of ERK1/2 phosphorylation (Table 2). This effect was still apparent even when the concentrations of the inhibitors were decreased by 50% and the inhibition was greater than that observed with the individual receptor blockers (Table 2). In addition, Ang II discretely activates the EGF receptor to cause this effect since inhibiting the PDGF receptor was ineffective in preventing the stimulation of the EGF receptor (Table 3). Although other tyrosine kinase receptors may be transactivated by Ang II in brainstem astrocytes, we suggest that the PDGF and the EGF receptors are playing major roles in Ang II stimulation of ERK1/ 2 MAP kinases phosphorylation in these cells. Transactivation of PDGF and EGF receptors participates in ERK1/2 activation and cellular proliferation following stimulation with agonists of certain G-protein-coupled receptors [35–38]. The studies presented clearly show that in the CNS there is crosstalk between membrane-bound tyrosine kinase receptors and Ang II receptors accounting for a portion of the stimulation of astrocyte growth thus supporting the aforementioned findings observed with other G-protein-coupled receptor agonists. Moreover, the two receptor tyrosine kinases may have an additive effect to block Ang II-induced astrocyte proliferation since the combination treatment caused at least a 50% greater inhibition of Ang II-induced astrocyte proliferation as compared to the individual inhibitors (Table 4). Synergism between the EGF and the PDGF receptors has been demonstrated in peripheral systems. For example, coinhibition studies have demonstrated that in human lens epithelial cells these two receptors control signaling through the PDGF receptor [39] and they are also involved in hypotonic stress-induced phosphorylation of ERK1/2 and JNK/SAPK [40]. However, this report is the first to demonstrate that these two receptors have an additive effect in controlling intracellular and physiological effects of Ang II. In addition, the results of this study are novel in the finding that these two receptors may have additive effects in astrocytes. Our experimental findings suggest that Ang II (which can be produce by astrocytes) may stimulate receptor tyrosine kinases on astrocytes to stimulate astrocyte growth. This increase in the number of astrocytes may lead to an increase in central Ang II levels since astrocytes contain high levels of angiotensinogen, the precursor molecule for Ang II [27–29]. This may be an endogenous regulatory mechanism to control central Ang II levels and its effects. It is now well established that a local or paracrine renin angiotensin system exists in a number of tissues and that these systems may play a significant role in regulating blood pressure [4]. Dysregulation of the central renin angiotensin system signaling mechanisms is associated with numerous pathological conditions (e.g., hypertension and heart failure) making elucidation of intracellular second messenger systems of Ang II in the brain critical to our understanding of these Ang II-dependent

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diseases [41]. Indeed, transgenic rats with low brain glial angiotensinogen have lower blood pressure throughout life and do not develop the age-related deficits in cardiovascular and metabolic function seen during normal aging of the control Sprague–Dawley line [42]. In conclusion, these studies are important in establishing the role of the endogenous brain renin angiotensin system and the interplay between this system and others that may contribute to cardiovascular diseases such as hypertension. Acknowledgements This work was supported in part by National Heart, Lung and Blood Institutes Grant HL-077199 and a President's Faculty Research & Development Grant from Nova Southeastern University. We acknowledge the excellent technical assistance of Ms. Jumirlet Ortega. We are grateful to Drs. E. Ann Tallant and Debra Diz for proofreading of this manuscript. References [1] Touyz RM. Recent advances in intracellular signalling in hypertension. Curr Opin Nephrol Hypertens 2003;12:165–74. [2] Veerasingham SJ, Raizada MK. Brain renin–angiotensin system dysfunction in hypertension: recent advances and perspectives. Br J Pharmacol 2003;139:191–202. [3] Morimoto S, Sigmund CD. Angiotensin mutant mice: a focus on the brain renin–angiotensin system. Neuropeptides 2002;36:194–200. [4] Bader M, Peters J, Baltatu O, Muller DN, Luft FC, Ganten D. Tissue renin–angiotensin systems: new insights from experimental animal models in hypertension research. J Mol Med 2001;79:76–102. [5] Matsukawa S, Keil LC, Reid IA. Role of endogenous angiotensin II in the control of vasopressin secretion during hypovolemia and hypotension in conscious rabbits. Endocrine 1991;128:204–10. [6] Reid IA. Interactions between ANG II, sympathetic nervous system, and baroreceptor reflexes in regulation of blood pressure. Am J Physiol 1992;262:E763–78. [7] Timmermans PB, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JA, Smith RD. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev 1993;45:205–51. [8] Tharaux PL, Coffman TM. Transgenic mice as a tool to study the renin– angiotensin system. Contrib Nephrol 2001:72–91. [9] Shanmugam S, Corvol P, Gasc JM. Angiotensin II type 2 receptor mRNA expression in the developing cardiopulmonary system of the rat. Hypertension 1996;28:91–7. [10] Raizada MK, Stenstrom B, Phillips MI, Sumners C. Angiotensin II in neuronal cultures from brains of normotensive and hypertensive rats. Am J Physiol 1984;247:C115–9. [11] Sumners C, Zhu M, Gelband CH, Posner P. Angiotensin II type 1 receptor modulation of neuronal K+ and Ca2+ currents: intracellular mechanisms. Am J Physiol 1996;271:C154–63. [12] Sun C, Sumners C, Raizada MK. Chronotropic action of angiotensin II in neurons via protein kinase C and CaMKII. Hypertension 2002;39:562–6. [13] Tallant EA, Diz DI, Ferrario CM. Identification of AT1 receptors on cultured astrocytes. Adv Exp Med Biol 1996;396:121–9. [14] Tallant EA, Higson JT. Angiotensin II activates distinct signal transduction pathways in astrocytes isolated from neonatal rat brain. Glia 1997;19:333–42. [15] Tallant EA, Jaiswal N, Diz DI, Ferrario CM. Human astrocytes contain two distinct angiotensin receptor subtypes. Hypertension 1991;18:32–9. [16] Zimmerman MC, Sharma RV, Davisson RL. Superoxide mediates angiotensin II-induced influx of extracellular calcium in neural cells. Hypertension 2005;45:717–23. [17] Zhu M, Gelband CH, Posner P, Sumners C. Angiotensin II decreases neuronal delayed rectifier potassium current: role of calcium/calmodulindependent protein kinase II. J Neurophysiol 1999;82:1560–8.

[18] Sun C, Sellers KW, Sumners C, Raizada MK. NAD(P)H oxidase inhibition attenuates neuronal chronotropic actions of angiotensin II. Circ Res 2005;96:659–66. [19] Yang H, Lu D, Vinson GP, Raizada MK. Involvement of MAP kinase in angiotensin II-induced phosphorylation and intracellular targeting of neuronal AT1 receptors. J Neurosci 1997;17:1660–9. [20] Clark M. Angiotensin II activates mitogen-activated protein kinases and stimulates growth in rat medullary astrocytes. FASEB J 2001:A1169. [21] Clark MA, Gonzalez N. Src and Pyk2 mediate angiotensin II effects in cultured rat astrocytes. Regul Pept 2007, doi:10.1016/j.regpep.2007.02.008. [22] Daub H, Weiss FU, Wallasch C, Ullrich A. Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors. Nature 1996;379:557–60. [23] Eguchi S, Inagami T. Signal transduction of angiotensin II type 1 receptor through receptor tyrosine kinase. Regul Pept 2000;91:13–20. [24] Saito Y, Berk BC. Transactivation: a novel signaling pathway from angiotensin II to tyrosine kinase receptors. J Mol Cell Cardiol 2001;33:3–7. [25] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. [26] Freeman EJ, Chisolm GM, Ferrario CM, Tallant EA. Angiotensin-(1–7) inhibits vascular smooth muscle cell growth. Hypertension 1996;28:104–8. [27] McKinley MJ, Albiston AL, Allen AM, Mathai ML, May CN, McAllen RM, Oldfield BJ, Mendelsohn FA, Chai SY. The brain renin–angiotensin system: location and physiological roles. Int J Biochem Cell Biol 2003;35:901–18. [28] Stornetta RL, Hawelu-Johnson CL, Guyenet PG, Lynch KR. Astrocytes synthesize angiotensinogen in brain. Science 1988;242:1444–6. [29] Genain C, Bouhnik J, Tewksbury D, Corvol P, Menard J. Characterization of plasma and cerebrospinal fluid human angiotensinogen and desangiotensin I-angiotensinogen by direct radioimmunoassay. J Clin Endocrinol Metab 1984;59:478–84. [30] Sumners C, Tang W, Paulding W, Raizada MK. Peptide receptors in astroglia: focus on angiotensin II and atrial natriuretic peptide. Glia 1994;11:110–6. [31] Hutchins JB. Platelet-derived growth factor receptors of mouse central nervous system cells in vitro. J Comp Neurol 1995;360:59–80. [32] Kapoor GS, O'Rourke DM. Mitogenic signaling cascades in glial tumors. Neurosurgery 2003;52:1425–34. [33] Luo J, Miller MW. Platelet-derived growth factor-mediated signal transduction underlying astrocyte proliferation: site of ethanol action. J Neurosci 1999;19:10014–25. [34] Shah BH, Neithardt A, Chu DB, Shah FB, Catt KJ. Role of EGF receptor transactivation in phosphoinositide 3-kinase-dependent activation of MAP kinase by GPCRs. J Cell Physiol 2006;206:47–57. [35] Daub H, Wallasch C, Lankenau A, Herrlich A, Ullrich A. Signal characteristics of G protein-transactivated EGF receptor. EMBO J 1997;16:7032–44. [36] Luttrell LM. Activation and targeting of mitogen-activated protein kinases by G-protein-coupled receptors. Can J Physiol Pharm 2002;80:375–82. [37] Miura S, Zhang J, Matsuo Y, Saku K, Karnik SS. Activation of extracellular signal-activated kinase by angiotensin II-induced Gq-independent epidermal growth factor receptor transactivation. Hypertens Res 2004;27:765–70. [38] Wetzker R, Bohmer FD. Transactivation joins multiple tracks to the ERK/ MAPK cascade. Nat Rev Mol Cell Biol 2003;4:651–7. [39] Chen KC, Zhou Y, Zhang W, Lou MF. Control of PDGF-induced reactive oxygen species (ROS) generation and signal transduction in human lens epithelial cells. Mol Vis 2007;13:374–87. [40] Taruno A, Niisato N, Marunaka Y. Hypotonicity stimulates renal epithelial sodium transport by activating JNK via receptor tyrosine kinases. Am J Physiol 2007;293:F128–38. [41] Phillips MI, Sumners C. Angiotensin II in central nervous system physiology. Regul Pept 1998;78:1–11. [42] Kasper SO, Carter CS, Ferrario CM, Ganten D, Ferder LF, Sonntag WE, Gallagher PE, Diz DI. Growth, metabolism, and blood pressure disturbances during aging in transgenic rats with altered brain renin– angiotensin systems. Physiol Genomics 2005;23:311–7.