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(Circulation Research. 1997;81:249-257.)
© 1997 American Heart Association, Inc.

Articles

Angiotensin II Activates Phosphatidylinositol 3-Kinase in Vascular Smooth Muscle Cells

Laura Saward Peter Zahradka

From the Institute of Cardiovascular Sciences, St Boniface General Hospital Research Centre, and the Department of Physiology, University of Manitoba, Winnipeg, Canada.

Correspondence to Peter Zahradka, Institute of Cardiovascular Sciences, St Boniface General Hospital Research Centre, 351 Tache Ave, Winnipeg, MB, Canada R2H 2A6. E-mail peterz{at}sbrc.umanitoba.ca

	Abstract

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Abstract Phosphatidylinositol 3-kinase (PI3K) is an important component of the signal transduction systems activated by tyrosine kinase receptors. It has not been established, however, whether PI3K is also an essential mediator for G protein–coupled receptors. The potential involvement of PI3K in G protein–linked angiotensin II (Ang II)–dependent signaling was assessed in a primary cell culture system of porcine coronary artery smooth muscle cells (SMCs). Treatment of quiescent SMCs with Ang II (10^-5 to 10^-8 mol/L) resulted in a dose-dependent activation of PI3K when assayed in vivo and in vitro. The Ang II receptor antagonists losartan and PD123319 were used to establish that Ang II stimulates PI3K through the Ang II type-1 (AT₁) receptor. Immunofluorescent microscopy revealed that Ang II (10^-6 mol/L) stimulated the translocation of p85, the regulatory subunit of PI3K, from the perinuclear region to distinct foci throughout the cell within 15 minutes. Western blot analysis of p85 subcellular distribution demonstrated that p85 concentrations were also increased within 15 minutes in the membrane fraction and concomitantly decreased in the cytoskeletal and nuclear fractions. These changes in PI3K location and activity were paralleled by increased tyrosine phosphorylation of p85. A potential correlation between angiotensin-mediated PI3K activation and SMC growth was found using LY294002, a specific inhibitor of PI3K, which blocked the increase in DNA and RNA synthesis as well as cellular hyperplasia generated by Ang II (10^-6 mol/L) stimulation of quiescent SMCs. These data indicate that PI3K may operate as a mediator of vascular SMC growth after stimulation with Ang II.

Key Words: phosphatidylinositol 3-kinase • angiotensin II • smooth muscle cell • LY294002 • immunofluorescent microscopy

	Introduction

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Angiotensin II is a biologically active peptide that operates both as a hemodynamic regulator and as a growth factor. The mitogenic effects of Ang II in vascular tissues have been linked to specific pathophysiological conditions, such as hypertension, atherosclerosis, and restenosis.¹ For this reason, defining the signal transduction systems that mediate Ang II–dependent cell growth could assist in the search for new therapeutic agents. The cellular response to Ang II is directed by specific G protein–coupled receptors^{2 3} located on the cell surface. Protein tyrosine phosphorylation has recently been identified as an additional signal transduction component of the growth response to G protein receptor activation.⁴ Recent studies have established that Ang II stimulates the tyrosine phosphorylation of a distinct set of cellular proteins,^{5 6} including p125^FAK,⁷ Stat91 (from the STAT family of transcription factors),⁶ and phospholipase C.⁸ Although the precise role of this signaling pathway remains to be established, evidence is mounting to suggest that tyrosine phosphorylation plays an important role in the growth-promoting effect of G protein receptor agonists like Ang II. Thus, significant overlap exists in the signaling pathways coupled to tyrosine kinase and G protein–coupled receptors.⁹ This observation is supported by recent reports demonstrating that activation of the MAP kinase cascade by insulin-like growth factor 1, a tyrosine kinase receptor–coupled hormone, has been linked to the release of G protein ß-subunits.¹⁰ Cross talk between G protein and cytokine-dependent processes via the STAT pathway has also been observed.^{11 12}

PI3K, a heterodimeric protein composed of 85- and 110-kD subunits that catalyzes the synthesis of 3-phosphorylated phosphoinositides, is a key intermediate in receptor-stimulated mitogenesis.¹³ The regulatory p85 subunit of PI3K has no intrinsic catalytic activity but forms complexes with activated growth factor receptors as well as adapter proteins such as IRS-1 and Shc through SH2 domains after phosphorylation of the tyrosine moiety.^{14 15} The resultant association leads to a translocation of the catalytic p110 subunit from the soluble fraction to a vesicular fraction enriched in Golgi membranes.¹⁶ This maneuver enhances the catalytic activity of p110 and leads to the production of PI3P, PI(3,4)P₂, and PIP₃. These lipids subsequently serve as intermediates for specific downstream signal transduction events that determine the cellular response to a particular growth factor. In fact, they may serve an autoregulatory function, since they have been shown to modulate the association of p85 with other proteins by directly binding to SH2 domains.¹⁷ In addition, a unique function for PI3K has been established in receptor endocytosis and intracellular vesicular trafficking, which may also control the downstream effects.^{18 19}

LY294002, a compound synthesized by modification of the broad spectrum tyrosine kinase inhibitor quercetin, has been identified as a highly specific inhibitor of PI3K.^{20 21 22} Studies conducted with this compound have demonstrated its ability to inhibit a variety of metabolic processes, including glucose transporter translocation, DNA synthesis, MAP kinase activation, pp70 S6 kinase phosphorylation, protein synthesis, and glucose uptake.^{22 23 24 25} On the basis of these results, it is evident that PI3K is an important component of the intracellular signaling cascades activated by growth factors that operate through tyrosine kinase receptors.

Treatment of vascular SMCs with Ang II results in receptor-dependent stimulation of cell proliferation that requires the tyrosine phosphorylation of various proteins.^{5 6 7 8} Since PI3K has been reported to respond to both tyrosine kinase and G protein receptor–dependent agonists,^{26 27} this enzyme could serve as the crossover point for these distinct systems. Support for this premise was recently provided by Saad et al,²⁸ who reported that Ang II stimulated both the tyrosine phosphorylation of IRS-1 and the formation of an IRS-1/p85 complex in rat heart. This information, however, does not indicate whether there is a functional correlation between PI3K activity and the cellular response to Ang II. We have therefore used the PI3K inhibitor LY294002 to evaluate the role of PI3K in Ang II–dependent cell proliferation. Our data demonstrate that this enzyme is activated in vascular SMCs after treatment with Ang II and that its activity may be crucial for cell growth.

	Materials and Methods

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Materials
Tissue culture medium, antibiotics, and multiwell culture dishes (Linbro) were obtained from ICN Flow. FBS and culture plates were purchased from GIBCO. Radiolabeled compounds ([methyl-³H]thymidine, [5,6-³H]uridine, [-³²P]ATP, and [³²P]orthophosphate) were from Dupont-NEN. Immobilon-P PVDF membranes were supplied by Millipore, the ECL detection system was purchased from Amersham, and protein G–Sepharose was from Pharmacia-LKB. Antibodies were acquired from Transduction Laboratories (anti-phosphotyrosine PY20), Upstate Biotechnology Inc (anti-PI3K p85), or Santa Cruz (anti-PI3K p110). Phosphatidylserine, PI, and PI(4,5)P₂ were provided by Calbiochem. LY294002 and wortmannin were purchased from Bio-Mol and Sigma Chemical Co, respectively. Insulin, MTT, pyruvate, transferrin, ascorbic acid, selenium, Cy3-conjugated anti-rabbit and FITC-coupled anti-mouse antibodies, and Hoescht No. 33258 nuclear stain were obtained from Sigma. The Ang II receptor antagonists losartan and PD123319 were kindly provided by DuPont-Merck and Warner-Lambert Parke-Davis, respectively. Whatman Silica G TLC plates (20x20 cm, 250 µm thickness) were from Fisher Scientific.

Cell Culture
Vascular SMCs were prepared by migration from free-floating explants of porcine left descending coronary artery segments and propagated in DMEM containing 20% FBS, 2 mmol/L glutamine, 86 µmol/L streptomycin, and 140 µmol/L penicillin.²⁹ Cells (at passage 2) were plated and grown to 65% to 75% confluence, rinsed with PBS, and incubated in serum-free DMEM supplemented with 65.8 nmol/L transferrin, 1 nmol/L selenium, 200 µmol/L ascorbate, and 10 nmol/L insulin for 7 days to induce a quiescent state.²⁹ Cell number was established after trypsinization by hemocytometer counts.

Metabolic Labeling of PI Pools
Quiescent cells in four-well culture dishes were labeled with 200 mCi/L [³²P]orthophosphate for 4 hours in phosphate-free DMEM. Cells were preincubated for 10 minutes with receptor antagonists or LY294002 before stimulation with agonists. At 15 minutes after the addition of Ang II, the medium was decanted, replaced with ice-cold 5% perchloric acid, and incubated on ice for 20 minutes. The precipitated material was collected by scraping and transferred to 15-mL conical tubes. Extraction of the phosphatidylinositides³⁰ and analysis by TLC on Silica G plates³¹ was conducted as described previously.

Immunoprecipitation
Cell lysates were prepared from 100-mm culture dishes by the addition of 1 mL lysis buffer (1% NP-40, 20 mmol/L Tris-HCl [pH 7.5], 10% glycerol, 137 mmol/L NaCl, 1 mmol/L MgCl₂, 1 mmol/L PMSF, and 0.4 mmol/L orthovanadate). The lysates were cleared by centrifugation, and their protein concentrations were determined by the BCA method (Pierce). A 100-µg aliquot (1 µg/µL) was then mixed for 2 hours at 4°C with protein G–Sepharose, which was subsequently removed by centrifugation at 12 000g for 5 minutes. Each aliquot was then mixed over 2.5 hours at 4°C with 4 µg of anti-phosphotyrosine antibody, anti-p85 antibody, or anti-p110 antibody. Protein G–Sepharose was added for an additional 2 hours, and the beads were collected by centrifugation. The beads were washed four times with 1 mL lysis buffer and either resuspended directly in 50 µL of 2x SDS/gel loading buffer (1x buffer contains 62.5 mmol/L Tris-HCl [pH 6.8], 1% SDS, 10% glycerol, 0.005% bromophenol blue, and 5% ß-mercaptoethanol) for Western blot analysis or treated further before measuring PI3K activity (see below).

Subcellular Fractionation
Quiescent SMCs were prepared in 100-mm plates as previously described. After stimulation with Ang II (10^-6 mol/L) for various times (0 to 30 minutes), the cells were treated with 1 mL of buffer A (50 mmol/L ß-glycerophosphate, 1 mmol/L EDTA, 2 mmol/L EGTA, 0.34 mmol/L CaCl₂, 250 mmol/L sucrose, 0.05% digitonin, 1 mmol/L PMSF, and 0.1 mmol/L leupeptin) to isolate the cytoplasmic fraction. After removal of buffer A, the cells were incubated in 1 mL buffer B (50 mmol/L ß-glycerophosphate, 1 mmol/L EGTA, 1% Triton X-100, 1 mmol/L PMSF, and 0.1 mmol/L leupeptin), and the lysate was centrifuged for 15 minutes at 12 000g to separate the membrane fraction (supernatant) and the nuclear fraction (pellet, resuspended in 100 µL of 2x SDS/gel loading buffer). The cytoskeletal fraction was prepared by the addition of 1 mL of 2x SDS/gel loading buffer to the plate, followed by vigorous scraping. Note that all steps were performed at 4°C and fractions were stored at -20°C for Western blot analysis. Equivalent amounts of total protein were loaded for each group of samples on the basis of densitometric analysis after gel electrophoresis and staining.

Western Blotting
The immunoprecipitates or cell lysates in 2x SDS/gel loading buffer were heated at 95°C for 5 minutes. The sample was loaded onto a 7.5% polyacrylamide gel and electrophoresed at 6-mA constant current over 2 to 3 hours, and protein was transferred to PVDF membrane at 90 V (0.5 A) over 90 minutes in 20% methanol, 25 mmol/L Tris, and 130 mmol/L glycine. Membranes were blocked by a 60-minute treatment at room temperature with blocking solution (3% BSA in TBS-T containing 10 mmol/L Tris-HCl [pH 7.5], 0.1 mol/L NaCl, 1 mmol/L EDTA, and 0.1% Tween 20). Anti-p85 antibody or anti-phosphotyrosine antibody (1:5000 dilution) was added in fresh blocking buffer and incubated for 60 minutes at 37°C. Similar conditions were used for the secondary antibody (horseradish peroxidase–conjugated anti-rabbit IgG). The membranes were washed five times over 30 minutes with TBS-T, and horseradish peroxidase was detected using the ECL system.

In Vitro PI3K Activity
The immunoprecipitates obtained with the anti-p85 antibody were assayed according to Whitman et al,³² with minor modifications.³³ The protein G–Sepharose pellets were washed three times with assay buffer (20 mmol/L Tris-HCl [pH 7.6], 10 mmol/L MgCl₂, and 100 mmol/L NaCl) and resuspended in 90 µL assay buffer containing 20 µmol/L [³²P]ATP. The reaction was initiated by adding 10 µL of a phosphoinositide mixture. The lipid substrates were prepared by sonicating equal quantities of phosphatidylserine, PI, and PI(4,5)P₂ in assay buffer to yield a final concentration of 200 µg/mL. After a 20-minute incubation at 37°C, the reaction was stopped by the addition of 200 µL of 1 mol/L HCl-methanol (1:1), and the samples were extracted twice with 200 µL chloroform. The lipids were recovered from the combined organic phases by evaporation, suspended with 10 µL chloroform, and analyzed by TLC on Silica G plates.³¹

Immunofluorescent Microscopy
Cells were grown on glass slides (Superfrost Plus, Fisher) and incubated in serum-free defined media for 7 days to induce quiescence as described above. After Ang II (10^-6 mol/L) stimulation for varying time periods (0 to 30 minutes), cells were washed twice with PBS, fixed in methanol (-20°C), and air-dried. After rehydrating in PBS containing 0.1% BSA and 0.05% sodium azide, cells were incubated with anti-p85 antibody (1/100 dilution) overnight at 4°C. The primary antibody was detected using a Cy3-coupled anti-rabbit antibody (1/200 dilution). The nuclear stain Hoescht No. 33258 was used to visualize the nuclei. For the actin costain, the slide was then incubated with anti–SM -actin antibody (1/200 dilution) overnight at 4°C and then detected after incubation with FITC-coupled anti-mouse antibody.

DNA and RNA Synthesis Assays
Cultures of quiescent SMCs, in 24-well dishes containing 1 mL of serum-free medium, were stimulated by direct addition of the indicated compounds (volumes of addition were 10 µL or less) without replacing the medium. Tracer amounts of labeled precursor were added for the specified time of the assay.³⁴ The appropriate dilution of inhibitor LY294002 was preincubated with the cells for 10 minutes before stimulation with Ang II. Incorporation of [³H]uridine (2 µCi) over the 6-hour time period following Ang II stimulation was used to measure RNA synthesis. After a 24-hour incubation with Ang II, the cells were incubated an additional 48 hours with [³H]thymidine (2 µCi) to measure DNA synthesis. The cells were subsequently lysed with 1.0 mL of solution containing 10 mmol/L Tris-HCl (pH 7.4), 100 mmol/L NaCl, 1 mmol/L EDTA, and 0.5% SDS, and the nucleic acids, precipitated with an equal volume of cold 20% trichloroacetic acid, were collected on Whatman GF/A glass fiber filters. The filters were washed four times with 5% trichloroacetic acid and once with ethanol, and the radioactivity was determined by liquid scintillation counting.

Cytotoxicity
Quiescent cells, prepared in 96-well culture dishes, were maintained in serum-free supplemented medium with varying concentrations of LY294002 over a total of 72 hours. Cell number was measured after a 4-hour incubation with MTT (603 µmol/L) according to Shi et al.³⁵ Color development was quantified using a Molecular Devices ThermoMAX plate reader with a 550-nm filter.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
To explore the role of PI3K in the signal transduction pathways activated by Ang II, we measured PI3K activity through the production of phosphoinositides modified at the 3 position. Metabolic labeling with [³²P]orthophosphate was used to evaluate the activity of this enzyme in vivo. After treatment of quiescent SMCs with Ang II, the labeled phosphoinositides were extracted and analyzed by TLC. Because of the inability of TLC analysis to resolve PI3P and PI4P as well as PI(3,4)P₂ and PI(4,5)P₂, the most accurate measure of PI3K activity correlates with the level of PIP₃.³¹ Quantification of the labeled phosphoinositides demonstrated that Ang II (10^-5 to 10^-10 mol/L) stimulated a dose-dependent increase in PIP₃ at 15 minutes, which could be inhibited by the PI3K inhibitor LY294002 (10^-5 to 10^-8 mol/L) (Fig 1).

View larger version (34K): [in this window] [in a new window]   Figure 1. Ang II stimulation of PI3K activity and the inhibition by LY294002. Quiescent vascular SMCs were prelabeled with [32P]orthophosphate and then treated with Ang II (with or without inhibitor) for 15 minutes. Phosphoinositides were extracted from the cell lysates, and the phosphorylated forms of phosphoinositol were resolved by TLC as described in "Materials and Methods." In panel A, a full autoradiogram of the TLC plate is shown for quiescent SMCs treated with Ang II (10-6 mol/L) in the presence of increasing amounts of LY294002 at 10-5 to 10-8 mol/L. Panel B illustrates Ang II (10-5 to 10-10 mol/L) dose-dependent stimulation of PIP3 formation in SMCs, which was quantified by densitometric analysis of the autoradiogram. Results were normalized to control (no stimulation). These results were confirmed in three independent experiments with different SMC isolations.

To further substantiate that Ang II stimulates PI3K activity, a complementary measure was performed in vitro on cell lysates. Quiescent SMCs were stimulated with Ang II (10^-6 mol/L) for the specified time and lysed, and PI3K was immunoprecipitated with either anti-p85 or anti-p110 antibody antibodies (Fig 2). PI3K activity in the immunoprecipitates was subsequently measured by the amount of labeled phosphate transferred from [-³²P]ATP to PI and PI(4,5)P₂ to form PI3P and PIP₃. In agreement with the assessment of PI3K activity in vivo, Ang II treatment resulted in a rapid increase in labeled PIP₃ at 15 minutes in p85 immunoprecipitates. It should be noted that PI3K prefers PI(4,5)P₂ over PI as its substrate and preferentially converts the PI(4,5)P₂ to PIP₃. The formation of labeled PI(3,4)P₂ in the assay represents the phospholabeling of PI4P contaminants in the substrates. The efficacy of LY294002 as an inhibitor of PI3K activity was confirmed using this assay, since only PI3K is expected in the immunoprecipitated sample. A parallel evaluation of PI3K activity in p110 immunoprecipitates conclusively demonstrated that Ang II stimulation resulted in increased PI3K activity (Fig 2).

View larger version (45K): [in this window] [in a new window]   Figure 2. Enzymatic analysis of PI3K activity in vitro after immunoprecipitation (IP). Cell lysates of SMCs after Ang II (10-6 mol/L) treatment were immunoprecipitated with either anti-p85 antibody or anti-p110 antibody and assayed for PI3K phosphorylation of PI and PI(4,5)P2 as described in "Materials and Methods." LY294002 (LY, 10-5 mol/L) was added to the assay reaction containing the Ang II–stimulated immunoprecipitate before the addition of the lipid substrates. These results were confirmed in four independent experiments with different SMC isolations.

To verify that the activation of PI3K by Ang II was receptor dependent and to determine which Ang II receptor subtype mediates this stimulation, the ability of Ang II to stimulate PI3K was evaluated in the presence of either losartan (AT₁ receptor antagonist) or PD123319 (AT₂ receptor antagonist). In agreement with the data in Figs 1 and 2, treatment of quiescent SMCs with Ang II (10^-6 mol/L) for 15 minutes resulted in enhanced PI3K activity. Pretreatment with losartan (10^-5 to 10^-6 mol/L) significantly inhibited the synthesis of PIP₃ in vivo and in p85 immunoprecipitates in vitro (Fig 3). In contrast, PD123319 did not inhibit Ang II stimulation of PI3K in vivo or in vitro. These results demonstrate that Ang II–dependent stimulation of PI3K is mediated by the AT₁ receptor.

View larger version (70K): [in this window] [in a new window]   Figure 3. Ang II receptor subtype contribution to PI3K activation. In panel A, an autoradiogram of the TLC plate is shown for the in vitro analysis of PI3K activity following p85 immunoprecipitation. Anti-p85 immunoprecipitates from quiescent SMCs after stimulation with Ang II (10-6 mol/L) in the presence of the AT1 receptor antagonist losartan (Los) or the AT2 receptor antagonist PD123319 (PD, 10-5 mol/L) were assayed for PI3K phosphorylation of PI and PI(4,5)P2, as described in "Materials and Methods." In panel B, an autoradiogram of the TLC plate is shown for the in vivo analysis of PI3K activity. Quiescent SMCs prelabeled with [32P]orthophosphate for 4 hours were treated with Ang II (10-6 mol/L) in the presence of varying concentrations of Los or PD (10-5 to 10-8 mol/L), and the phosphoinositides were extracted and analyzed as described in "Materials and Methods."

The activation of PI3K has been shown to correlate with changes in protein tyrosine phosphorylation. More specifically, it has been observed that the p85 regulatory subunit of PI3K either becomes phosphorylated directly or it associates through its SH2 domain with polypeptides that have been tyrosine-phosphorylated after growth factor stimulation.^{13 14} Although Ang II operates through G protein–coupled receptors, changes in tyrosine phosphorylation of several cytoplasmic proteins have been observed.^{6 7 8} Thus, the phosphorylation state of the p85 subunit of PI3K in SMCs may respond to Ang II stimulation. Quiescent SMCs were incubated with Ang II (10^-6 mol/L) for various times (0 to 30 minutes), and cell extracts were immunoprecipitated with an anti-p85 antibody. Western blot analysis with antiphosphotyrosine antibody clearly demonstrated that this PI3K subunit is modified directly by tyrosine phosphorylation in response to Ang II treatment (Fig 4A). The kinetics of p85 phosphorylation revealed that the action of Ang II was rapid and transient, peaking at 10 to 15 minutes and returning to control levels by 30 minutes. A subsequent Western blot analysis with anti-p85 antibody was used to normalize the levels of p85 in all samples (Fig 4B) and demonstrated that the increase in tyrosine phosphorylation of p85 mediated by Ang II was 2-fold after 10 minutes. Confirmation that the target of this antibody was p85 was established by a similar pattern of staining reproduced with a different polyclonal antibody to p85 (data not shown). A complementary analysis of anti-phosphotyrosine antibody immunoprecipitates followed by Western blot analysis with an anti-p85 antibody demonstrated an identical time course for phosphorylation, with a 2.5-fold increase after 10 to 15 minutes (Fig 4C). Thus, the stimulation of quiescent SMCs with Ang II results in the tyrosine phosphorylation of the regulatory p85 subunit of PI3K.

View larger version (76K): [in this window] [in a new window]   Figure 4. Assessment of p85 PI3K regulatory subunit tyrosine phosphorylation by Ang II. Quiescent SMCs were treated with Ang II (10-6 mol/L) for the indicated times, and cell lysates were prepared for immunoprecipitation. A, Extracts were incubated with anti-p85 antibody, and Western blot analysis of the immunoprecipitated proteins was performed with anti- phosphotyrosine PY20 antibody. B, The relative amounts of p85 immunoprecipitated in each sample are shown by Western blot analysis with anti-p85 antibody. C, The immunoprecipitation was conducted with the anti-phosphotyrosine antibody PY20, and the blot was analyzed with anti-p85 antibody to verify the time course of phosphorylation. These results were confirmed in three independent experiments with different SMC isolations. IP indicates antibody used for immunoprecipitation; IB, antibody used for Western blotting.

It has been proposed that changes in the subcellular pools of PI3K can operate as an additional mechanism for regulating PI3K activity. Using indirect immunofluorescent microscopy, we were able to monitor the intracellular distribution of p85 before and after treatment with Ang II. In quiescent SMCs, p85 exhibits a diffuse pattern of staining concentrated in the perinuclear region (Fig 5A) with distinct focal areas of p85 scattered throughout the cytoplasm. After a 5-minute stimulation with Ang II (10^-6 mol/L), a dramatic shift in p85 localization is evident, as shown by the shift from the diffuse perinuclear pattern to a punctate staining pattern throughout the entire cytoplasm (Fig 5B). After 15 minutes of Ang II treatment, p85 staining has completely vacated the nuclear region (Fig 5C). Interestingly, the perinuclear staining pattern characteristic of quiescent cells reappears after 30 minutes (Fig 5D). Examination at a higher magnification confirmed that Ang II induces a rapid (5- to 15-minute) translocation of p85 from the perinuclear cytoplasm to specific foci throughout the cytoplasm and possibly the cytoskeleton (Fig 6). As shown in Fig 7A and 7B, the perinuclear pattern of p85 in quiescent cells did not colocalize with the nuclear stain; however, this comparison cannot confirm the complete absence of p85 in the nuclei of these cells. In addition, it was possible to show that the pattern of p85 distribution following Ang II treatment does not colocalize with the actin filaments (Fig 7C). The polyclonal antibody of PI3K used to obtain the information presented in Figs 5 through 7 has been previously used to establish the subcellular distribution of p85 by other laboratories.^{36 37} Further confirmation that the target of this antibody is p85 was established by a similar pattern of staining reproduced with a different polyclonal antibody to p85 (data not shown). Furthermore, cells treated with Cy3-conjugated anti-rabbit antibody or anti-p85 antibody alone as controls exhibited no detectable staining (data not shown).

View larger version (160K): [in this window] [in a new window]   Figure 5. Examination of the subcellular localization of p85 after Ang II stimulation. Quiescent SMCs were treated with Ang II (10-6 mol/L) and prepared on glass slides as outlined in "Materials and Methods." p85 was detected using indirect immunofluorescent microscopy at a magnification of x33. The pattern of p85 staining is depicted in untreated cells (A) and after 5 minutes (B), 15 minutes (C), and 30 minutes (D) of Ang II treatment. The results shown represent the pattern consistently observed in five independent experiments.

View larger version (128K): [in this window] [in a new window]   Figure 6. Pattern of p85 distribution and translocation to focal sites after Ang II stimulation. The pattern of p85 distribution is depicted in quiescent untreated SMCs (A) and after 5 minutes (B) and 15 minutes (C) of Ang II treatment (10-6 mol/L). Indirect immunofluorescent microscopy of p85 was conducted as described in "Materials and Methods." Samples were examined at a magnification of x100.

View larger version (60K): [in this window] [in a new window]   Figure 7. Analysis of p85 association with the nucleus and the cytoskeleton. A and B, The perinuclear pattern of p85 distribution (detected with a Cy3-coupled secondary antibody, red) in quiescent SMCs (A) relative to the location of the nuclei (stained with Hoescht 33258, blue; B) was examined by indirect immunofluorescent microscopy as outlined in "Materials and Methods." C, The pattern of p85 (Cy3, red) and SM -actin (FITC, green) is shown after Ang II (10-6 mol/L) treatment for 15 minutes. Samples were examined at x66 magnification.

To complement the visual analysis of p85 translocation in response to Ang II stimulation, a subcellular fractionation of quiescent SMCs at various time points (0 to 30 minutes) after Ang II (10^-6 mol/L) stimulation was performed, and the relative content of p85 was assessed by Western blot analysis (Fig 8). Ang II treatment resulted in an increase in the p85 content of the membrane fraction at 15 minutes (2.1-fold) and a parallel decrease in p85 levels in the cytoskeletal and nuclear fractions (2.2-fold at 15 minutes and 2.7-fold at 10 minutes, respectively), as well as a 5-fold increase in the nuclear p85 content after 30 minutes. It should be noted that the amount of p85 detected in the nuclear fractions was significantly less than in the other cellular fractions (<5%), as indicated by the extended exposure time required to detect nuclear p85, and may reflect contamination of this fraction by other cell components. No significant changes in the levels of p85 were observed in the cytoplasmic fraction.

View larger version (25K): [in this window] [in a new window]   Figure 8. Ang II–dependent stimulation of p85 translocation. Quiescent SMCs were stimulated with Ang II (10-6 mol/L) for varying periods of time, lysed, and separated into cytoplasmic, membrane, nuclear, and cytoskeletal fractions as described in "Materials and Methods." Equivalent amounts of protein were loaded in each well, and the relative amounts of p85 in each fraction were determined by Western blot analysis with anti-p85 antibody. Exposure time after ECL treatment for the cytoplasmic, membrane, and cytoskeletal fractions was 15 minutes, whereas the data for the nuclear fraction were obtained after a 90-minute exposure.

PI3K has been identified as an essential factor for cell proliferation in a number of cell systems after tyrosine kinase receptor activation. However, its potential contribution to G protein receptor–mediated growth has not been established. Ang II has been defined as a growth factor in this SMC system and results in the stimulation of both RNA and DNA synthesis.³⁸ The role of PI3K in the downstream activation of cell growth by Ang II was examined in the present study by measuring RNA and DNA synthesis as determined by the rate of [³H]uridine and [³H]thymidine incorporation, respectively, in conjunction with the PI3K inhibitor LY294002. Typically, Ang II (10^-6 mol/L) stimulates the synthesis of both RNA (184±13%) and DNA (172±17%), but inclusion of LY294002 (10^-5 to 10^-10 mol/L) blocked the stimulation in a concentration-dependent manner (Fig 9A and 9B). In addition, LY294002 (10^-5 mol/L) blocked the increase in cell number that is observed 96 hours after stimulation with Ang II (10^-6 mol/L) (Fig 9C). Thus, PI3K appears to be necessary for the induction of cell growth and proliferation by Ang II. To eliminate the possibility that the observed inhibition of cell growth could be attributed to cellular toxicity of LY294002, cell survival was monitored over a range of LY294002 concentrations. As demonstrated in Fig 9D, no cell death was evident at the concentration of LY294002 (10^-5 mol/L) used in the present study over a 72-hour period.

View larger version (42K): [in this window] [in a new window]   Figure 9. Effect of PI3K inhibitor LY294002 (LY) on Ang II–dependent stimulation of RNA and DNA synthesis and cell number. A, Effect of varying concentrations of LY on the stimulation of RNA synthesis by Ang II (10-6 mol/L), based on [3H]uridine incorporation over a 6-hour period. B, Inhibition by LY of Ang II–stimulated DNA synthesis, based on [3H]thymidine incorporation over a 48-hour period. Each data point represents the mean±SE of at least three separate experiments. In both panels, the value of the Ang II–stimulated control was set at 100%. These results were confirmed in five independent experiments with different SMC isolations. C, Increase in total cell number after Ang II (10-6 mol/L) treatment of quiescent SMCs for 96 hours compared with 5% FBS treatment and the ability of LY to prevent Ang II–dependent hyperplasia. D, Cytotoxicity of LY (10-4 to 10-6 mol/L) on quiescent SMCs over 24, 48, and 72 hours was evaluated by the MTT assay, which reflects the number of active mitochondria. Each data point represents the mean±SE of at least three separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The most important finding of the present study is the demonstration that Ang II activates PI3K in vascular SMCs, and this report is the first study to document this connection. Because PI3K has been traditionally associated with receptor tyrosine kinase activation,¹⁵ the contribution of this enzyme to G protein–mediated signaling pathways has received little attention. Nevertheless, substantial evidence has accumulated to show that tyrosine phosphorylation occurs rapidly upon stimulation of SMCs by Ang II, a G protein–coupled system.^{6 7 8} To evaluate the role of PI3K in Ang II–dependent signal transduction pathways, the formation of PIP₃ was measured as a direct indication of PI3K activity. Labeling of SMCs in vivo with [³²P]orthophosphate in the presence and absence of Ang II, followed by TLC analysis of the total extracted phosphoinositide pool, was used as a measure of PI3K activity, since PIP₃ can be resolved from other phosphoinositide products. The results of this assay indicate that Ang II produces a concentration-dependent increase in PI3K activity after 15 minutes that can be prevented by the PI3K inhibitor LY294002 (Fig 1). To further substantiate the validity of this assay, PI3K activity was measured in vitro with extracts from Ang II–stimulated SMCs. In agreement with the in vivo assay, Ang II stimulated PI3K activity, as demonstrated with both p85 and p110 immunoprecipitates (Fig 2). It should be noted that the PIP₃ detected in control (unstimulated) cells represents the basal level of PI3K activity required for various growth-independent processes active in quiescent cells.³³

Ang II mediates its cellular effects through at least two distinct receptor subtypes, AT₁ and AT₂. In order to verify that the activation of PI3K by Ang II was receptor-mediated and to determine the receptor subtype involved in this SMC system, the non–peptide receptor antagonists losartan and PD123319 were used to selectively block the AT₁ and AT₂ receptors, respectively. The observed increase in PI3K activity immediately following Ang II treatment was blocked by losartan but not PD123319 in both the in vivo and in vitro PI3K assays (Fig 3). Thus, stimulation of PI3K by Ang II occurs through the AT₁ receptor.

Two potential mechanisms for the regulation of PI3K activity by growth factors were investigated in the present study: changes in subcellular localization and modification of p85 by tyrosine phosphorylation. The kinetics of both PI3K activation and tyrosine phosphorylation of the regulatory subunit p85 in response to Ang II suggest these events are coupled (Fig 4). In addition, Ang II treatment of quiescent SMCs resulted in distinct changes in the subcellular localization of p85 within a similar time course. In quiescent SMCs, staining for p85 exhibited a diffuse pattern concentrated in the perinuclear region. Our results are in agreement with previous reports that indicate the presence of a large pool of PI3K in resting cells.³³ Ang II stimulation resulted in the translocation of p85 from the perinuclear area to specific foci throughout the cytoplasm and possibly the cytoskeletal apparatus (Figs 5 through 7). Furthermore, a subcellular fractionation study of p85 in SMCs after Ang II stimulation demonstrated an increase in the membrane fraction and a parallel decrease in the cytoskeletal and nuclear fractions within a time frame consistent with p85 tyrosine phosphorylation and immunofluorescent translocation (Fig 8). Although the present study represents the first analysis of the distribution pattern of p85 in SMCs in response to a growth factor such as Ang II, the patterns observed are very similar to those previously described by Kapeller et al³⁶ in platelet-derived growth factor–stimulated 3T3-L1 fibroblast cells. Their study describes an association of p85 with the microtubule network that is consistent with a role for PI3K in microtubule-based motility. Since it has been postulated that PI3K mediates the reorganization of the cytoskeletal apparatus in cell growth, Ang II–dependent tyrosine phosphorylation of adhesion factors such as paxillin³⁹ and p125^FAK40 may be important for defining the subcellular localization of PI3K. An examination of PI3K colocalization with SM -actin established that PI3K is not associated with SM -actin filaments (Fig 7); however, the potential interaction of p85 with other cytoskeletal factors is currently under investigation. Another established role for PI3K is the control of endosome trafficking and receptor endocytosis.^{18 19} The punctate staining observed in our SMC system and the increase in the p85 content of the membrane fraction with Ang II stimulation may reflect an increase in PI3K association with endosomes. A similar pattern of immunofluorescence has been observed for src kinase, a protein also associated with endosomes.^{41 42}

In addition to establishing a direct link between PI3K activity and Ang II, the functional contribution of PI3K to Ang II–dependent SMC growth was investigated. The present study clearly demonstrates that LY294002 prevents the stimulation of SMC growth by Ang II, as determined with both RNA and DNA synthesis as well as cellular hyperplasia (Fig 9). Because of the high concentrations of LY294002 used to inhibit PI3K activity in our system, the possibility that this compound may also be regulating an additional step in the signaling pathways of Ang II cannot be ruled out. Although wortmannin, a popular PI3K inhibitor that is structurally distinct from LY294002,⁴³ has been found to affect a variety of unrelated kinases, such as myosin light chain kinase,⁴⁴ as well as phospholipases C, D, and A₂,^{45 46} LY294002 is considered highly selective for PI3K.^{20 21 22} These factors led to the selection of LY294002 as the inhibitor of choice for our experiments, although wortmannin effectively yielded identical results (data not shown). Unfortunately, LY294002 cannot distinguish between the p85-associated p110/ß and G_ß-activated p110 isoforms of PI3K.⁴⁷ Also, it has recently been demonstrated that LY294002 inhibits the PI3K homologue mTOR (target of rapamycin) proteins that are essential for cell cycle progression.⁴⁸ Thus, although it has been possible to establish that the p85/p110 PI3K isoform is activated in SMCs after AT₁ receptor stimulation with Ang II, it is not possible to determine whether suppression of SMC growth and proliferation by LY294002 is mediated by this PI3K isoform. Nevertheless, it is likely that inhibition of one or more of these enzymes accounts for the long-term effects of LY294002.

To date, the functional contribution of PI3K to the mitogenic pathways of Ang II has not been established. It is clear that the treatment of SMCs with Ang II results in the immediate stimulation of protein phosphorylation and gene expression, eventually leading to DNA synthesis and cell division. Furthermore, these metabolic changes are accompanied by a reorganization of the cytoskeletal apparatus, an event that alters the cell shape and provides the spatial clues needed for mitosis. A recent study by Saad et al²⁸ reported a correlation between Ang II stimulation and the association of minute quantities of tyrosine-phosphorylated p85 with IRS-1 in cardiac tissue; however, no direct evidence of Ang II regulation of PI3K activity or contribution was provided. The increase in PIP₃ and concomitant translocation of p85 described in the present report indicate that PI3K is activated within 15 minutes of Ang II addition (Figs 1 through 8). With this criterion, we have clearly demonstrated that both the catalytic activity and the subcellular localization of PI3K respond to Ang II and that these events correlate with tyrosine phosphorylation of the p85 regulatory subunit. Furthermore, significant amounts of p85 are recruited to the plasma membrane and to distinct sites throughout the cytoplasm within a 15-minute time period, consistent with the observed increase in PI3K activity. Experiments to identify the intracellular mediators of Ang II receptor activation involved in the recruitment of PI3K are ongoing. Since PI3K apparently participates in the early tyrosine phosphorylation cascades leading to activation of MAP kinase and S6 kinase,^{23 24 25} as well as membrane traffic processes and cytoskeletal organization,^{18 19 40} it is plausible that activation of PI3K by Ang II is necessary for both intracellular signaling and structural reorganization. Recent reports showing that pp60^src is activated by Ang II⁴⁹ suggest a possible mechanism by which PI3K integrates these events. It has become evident that c-src is a critical mediator of cell proliferative processes, influencing a variety of intracellular events. A recent publication by Karnitz et al⁵⁰ reports that members of the Src-kinase family control PI3K activation by interleukin-2. Thus, the binding and phosphorylation of proteins such as p125^FAK by c-src may influence both the intracellular location and activity of PI3K. This association is currently being investigated.

The changes in PI3K activity and p85 translocation clearly indicate that PI3K is an important signal transduction intermediate for Ang II. Furthermore, the inhibition of Ang II–dependent RNA and DNA synthesis and cellular hyperplasia by LY294002 (Fig 9) establish that early activation of PI3K may be necessary for cell cycle progression. Given that Ang II is a growth factor for vascular tissue and potentially contributes to the pathophysiology of several cardiovascular diseases, the identification of PI3K as a potential mediator of SMC growth in vitro warrants further investigation with in vivo models of vascular growth.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II AT1, AT2 = Ang II type 1 and 2 receptors ECL = enhanced chemiluminescence MAP = mitogen-activated protein MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide PI = phosphatidylinositol PI(3,4)P2 = phosphatidylinositol 3,4-bisphosphate PI(4,5)P2 = phosphatidylinositol 4,5-bisphosphate PI3K = phosphatidylinositol 3-kinase PI3P, PI4P = phosphatidylinositol 3- and 4-phosphate PIP3 = phosphatidylinositol 3,4,5-triphosphate PMSF = phenylmethylsulfonyl fluoride SH2 = src homology 2 SM -actin = smooth muscle -actin SMC = smooth muscle cell STAT = signal transducers and activators of transcription TLC = thin-layer chromatography

	Acknowledgments

 
This study was supported by grants from the Medical Research Council Group in Experimental Cardiology and the Juvenile Diabetes Foundation International. Laura Saward is the recipient of a scholarship from the Medical Research Council of Canada.

	Footnotes

 
Previously published in part in abstract form at the International Vascular Biology Meeting VI, Seattle, Wash, September 4-8, 1996.

Received August 22, 1996; accepted May 27, 1997.

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