NO regulates PDGF-induced activation of PKB but not ERK in A7r5 cells: implications for vascular growth arrest

Lakshman Sandirasegarane, Roger Charles, Nicole Bourbon, and Mark Kester

Department of Pharmacology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In addition to the well-documented role of nitric oxide (NO) as a vasodilator, NO has also been implicated in vascular smooth muscle cell (VSMC) growth arrest. Signaling mechanisms responsible for growth factor receptor-mediated VSMC proliferation include the extracellular signal-regulated kinase (ERK) and possibly the protein kinase B (PKB) cascade. Thus the present study was designed to test the hypothesis that, in A7r5 vascular smooth muscle-derived cells, platelet-derived growth factor (PDGF)-induced activation of either ERK or PKB is regulated by NO, which then modulates cellular proliferation and/or apoptosis. PKB-alpha was the predominant isoform of PKB expressed in A7r5 cells and was also expressed in rabbit carotid arteries and aortae. Phosphorylation of PKB-alpha and ERK induced by PDGF-BB was maximal within 5-15 min in A7r5 cells. Preincubation of A7r5 cells with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) resulted in a biphasic regulation of PDGF-stimulated PKB-alpha phosphorylation and bioactivity. Acute exposure to SNAP significantly augmented PDGF-induced activation of PKB-alpha , whereas prolonged incubation led to a marked diminution in PDGF-induced activation of PKB-alpha . In contrast, SNAP did not affect PDGF-induced activation of ERK at any time point. The cGMP-independent effects of SNAP on PDGF-induced activation of PKB-alpha were established with the use of an inhibitor of soluble guanylyl cyclase, ODQ, as well as a cell-permeable analog of cGMP, 8-bromo-cGMP. Prolonged treatment of A7r5 cells with SNAP led to a significant decrease in DNA synthesis without an appreciable increase in apoptosis. These data suggest that, after prolonged exposure to SNAP, NO selectively attenuates PDGF-induced increase in PKB-alpha activation, which in turn may contribute to diminished VSMC proliferation by mechanisms involving growth arrest but not apoptosis.

protein kinase B; extracellular signal-regulated kinase; nitric oxide; growth factors; proliferation; platelet-derived growth factor; vascular smooth muscle cell


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PLATELET-DERIVED GROWTH FACTOR (PDGF) is a mitogen responsible for vascular smooth muscle cell (VSMC) proliferation and is intimately involved in the development of vascular proliferative lesions observed in atherosclerosis and in restenosis after angioplasty (11, 32). In vivo administration of neutralizing antibodies to PDGF before and after balloon catheter deendothelialization results in a considerable decrease in neointimal smooth muscle accumulation (11). The endothelial layer in the vascular wall is known to exert a regulatory influence on vascular tone and VSMC proliferation under normal physiologic conditions, in part, by its ability to release nitric oxide (NO) upon activation of endothelial NO synthase (eNOS) (37). After balloon angioplasty, NO production via eNOS is shown to be diminished due to endothelial denudation (36), which may contribute to abnormal VSMC proliferation. In vivo transfer of the eNOS gene in denuded rat carotid arteries (37) and the administration of an NO donor (34) after balloon angioplasty result in suppression of abnormal VSMC proliferation, thus supporting the antiproliferative effects of NO. However, the cellular mechanisms governing the antiproliferative effects of NO are not completely understood (34, 37).

The role of extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase cascade, in growth factor receptor-induced increases in DNA synthesis and proliferation of VSMC has been extensively studied (12, 17, 21). The signaling events following activation of PDGF receptors include sequential activations of ras GTPase, raf, MEK1 (mitogen-activated protein kinase kinase), and subsequently ERK1/ERK2. In addition to ERK activation, recent studies have demonstrated that PDGF-induced increases in DNA synthesis and proliferation of bovine tracheal smooth muscle cells may involve activation of additional kinase cascades linked ostensibly to cell survival, including phosphatidylinositol 4,5-bisphosphate 3-kinase (PI-3K) and protein kinase B (PKB) (38). Thus a novel signaling pathway involving activation of PKB may also play a pivotal role toward cell survival and proliferation in VSMC. However, as yet, there are no reports documenting which, if any, of the PKB isoforms are expressed or activated by growth factors in VSMC.

PKB (also called Akt) is a serine/threonine protein kinase that exists as three distinct isoforms: PKB-alpha , PKB-beta , and PKB-gamma (4, 24, 25). PKB is clearly established as one of the downstream targets of the lipid kinase PI-3K (4, 13). Studies involving transient expression of dominant-negative mutants of PI-3K, and also pretreatments of cells with two chemically different PI-3K inhibitors, LY-294002 and wortmannin, have confirmed the obligatory involvement of PI-3K in the growth factor-induced activation of PKB (4, 13, 19). The known substrates for phosphorylation by PKB include Bad and caspase-9, which when phosphorylated prevent apoptotic cell death (4). A recent study indicates that activation of PKB is intimately involved in the activation and phosphorylation of eNOS, thereby facilitating NO production (10, 15). However, there are no reports documenting a putative negative-feedback regulatory effect of NO on growth factor receptor-induced activation of PKB in any cell type, including VSMC.

NO is known to stimulate cytosolic soluble guanylyl cyclase to increase cGMP formation with the accompanying activation of cGMP-dependent protein kinase (2, 5, 23, 31, 33). It has been shown that the NO donors, including S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside, exert an inhibitory effect on VSMC proliferation by cGMP-dependent (40) and cGMP-independent (2, 5, 20, 27) mechanisms. The putative role of NO/cGMP to modulate PKB-dependent mitogenesis has not yet been investigated.

The present study has examined PKB isoform protein expression in A7r5 vascular smooth muscle-derived cells and intact rabbit vascular tissues. In addition, the differential effects of the NO donor SNAP on PDGF-induced activation of both PKB and ERK have been studied before and after selective inhibition of soluble guanylyl cyclase with H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). To determine whether the inhibitory effects of SNAP on PDGF-induced activation of PKB lead to diminished proliferation or to induction of apoptosis, the alterations in DNA synthesis and the number of cells undergoing apoptotic cell death were monitored after prolonged exposure to SNAP.


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Materials. PDGF-BB, insulin-like growth factor I (IGF-I), and DMEM were purchased from GIBCO (Grand Island, NY). Endothelin (ET)-1 was obtained from the Peptide Institute (Tokyo, Japan). The antibodies to PKB-alpha , PKB-beta , and phospho-ERK as well as the protein kinase A inhibitor peptide were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody to PKB-gamma and the apoptosis detection kits were from Upstate Biotechnology (Lake Placid, NY). (Ser-473)phospho-PKB-alpha antibody was purchased from New England Biolabs (Beverly, MA). Histone H2B was from Boehringer Mannheim (Indianapolis, IN). SNAP and N-acetylpenicillamine were from Sigma Chemical (St. Louis, MO). ODQ and LY-294002 were from Calbiochem (San Diego, CA). The MEK1 inhibitor PD-98059 was from Biomol Research Laboratories (Plymouth Meeting, PA). [gamma -32P]ATP (sp act 4,500 Ci/mmol) and [3H]thymidine (sp act 6.7 Ci/mmol) were obtained from ICN Pharmaceuticals (Costa Mesa, CA). All other chemicals were from either Fisher Scientific (Fair Lawn, NJ) or Sigma Chemical (St. Louis, MO).

Cell culture and treatments. Rat embryonic thoracic aorta smooth muscle-derived A7r5 cells were obtained from American Type Culture Collection (Rockville, MD). The cells were incubated in DMEM, supplemented with 10% FBS at 37°C in a humidified atmosphere of 95% air-5% CO2. After attainment of confluency (~80%), the cells were incubated in DMEM devoid of serum for 20 h. The serum-starved cells were then exposed to PDGF-BB, IGF-I, ET-1, SNAP, LY-294002, PD-98059, ODQ, and 8-bromo-cGMP at the indicated concentrations and time intervals as described in the legends to the respective figures.

Preparation of tissue extracts from rabbit aortae and carotid arteries. New Zealand White rabbits weighing ~3 kg were used in this study. Animal living conditions are consistent with standards required by the Association for Assessment and Accreditation of Laboratory Animal Care International. The rabbits were killed by injecting Nembutal (100 mg/kg). The right carotid arteries as well the aortae were dissected free of adventitial fat and washed in ice-cold PBS. The tissues were then homogenized, using a polytron, under ice-cold conditions.

Western blot analyses. The control and treated A7r5 cells were washed twice with ice-cold PBS and exposed to lysis buffer [20 mM HEPES (pH 7.5), 40 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, 20 mM beta -glycerophosphate, 0.5 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 10 µg/ml aprotinin] for 20 min. The cell lysates or the tissue homogenates were vortexed for 10 s and centrifuged at 14,000 g at 4°C for 10 min. The supernatants (20 µg) from A7r5 cell lysates or from the homogenates of rabbit aortae and carotid arteries were mixed with the sample buffer [0.062 M Tris · HCl (pH 6.8), 2% SDS, 5% beta -mercaptoethanol (vol/vol), 10% glycerol, and 0.01% bromphenol blue] and boiled for 5 min. The proteins were separated on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membrane (Hybond-C, Amersham). The membranes were incubated overnight with the blocking buffer consisting of 5% nonfat dry milk in Tris-buffered saline (TBS, pH 7.5). After washing the membranes with TBS (10 min × 3), the membranes were probed with appropriate primary antibodies (1 in 1,000 dilution) for 2 h at room temperature. After being washed three times with TBS, the membranes were incubated in TBS containing the appropriate secondary antibody (1 in 6,000 dilution) for 2 h at room temperature. After three more washes with TBS, the protein bands were detected by the enhanced chemiluminescence method and quantified by laser densitometry. For some experiments, the membranes were incubated in the stripping buffer [62.5 mM Tris (pH 6.7), 2% SDS, and 100 mM beta -mercaptoethanol] for 35 min at 55°C, blocked with nonfat dry milk, and reprobed with a different primary antibody. The efficiency of the stripping procedure was determined by excluding the primary antibody and verifying for the absence of signals on the stripped membrane. The protein concentration was determined by the Bradford method.

Assay of PKB-alpha activity. The control and treated cells were washed twice with ice-cold PBS and exposed to lysis buffer (pH 7.5) for 20 min. The cell lysates were vortexed for 10 s and centrifuged at 14,000 g at 4°C for 10 min, after which the supernatants (protein concentration 100 µg) were incubated overnight at 4°C with a primary antibody specific for the PKB-alpha isoform. Subsequently, IgG agarose was added to the respective samples and incubated at 4°C for an additional 4-5 h. After centrifugation, the respective immunoprecipitates of PKB-alpha were washed with the lysis buffer, followed by a wash with the kinase buffer [50 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 10 mM beta -glycerophosphate, and 0.5 mM sodium orthovanadate]. The bioactivity of PKB-alpha was determined (in vitro kinase assay) by incubating the immunoprecipitates of PKB-alpha at room temperature for 20 min in 30 µl of kinase buffer in the presence of 1 µM protein kinase A inhibitor peptide, 50 µM unlabeled ATP, and 6 µCi [gamma -32P]ATP, using exogenous histone H2B (1.5 µg/assay tube) as the substrate (26). The reactions were terminated by adding 10 µl of 4× Laemmli sample buffer, after which the samples were heated at 95°C for 5 min. The proteins in the samples were resolved by 12% SDS-PAGE and transferred to nitrocellulose membrane, after which autoradiography was performed for determining the incorporation of 32P-labeled phosphate into histone H2B. As a negative control, supernatants of lysates from the control group of cells were subjected to mock immunoprecipitation without the addition of anti-PKB-alpha antibody. These mock immunoprecipitates did not reveal any bands in the in vitro kinase assay.

Measurement of nitrite. A7r5 cells were incubated in DMEM in the absence or presence of SNAP (100 µM) for 10 min or 18 h, after which media were collected and used for nitrite measurements. Nitrite levels in the media were quantitated colorimetrically by the Griess reaction according to the manufacturer's instructions (Calbiochem, San Diego, CA).

Determination of cGMP. Serum-starved A7r5 cells were preincubated in the absence or presence of ODQ (10 µM) at 37°C for 1 h. Subsequently, both untreated and ODQ-treated cells were exposed to SNAP (100 µM) for 10 min or 18 h. At the appropriate time points, media were aspirated, and the cells were washed twice with ice-cold PBS and extracted with acidified methanol (50% methanol and 0.1 N HCl). After lyophilizing these extracts, the samples were processed for cGMP measurements by RIA (cGMP 125I scintillation proximity assay system, Amersham). The protein was determined by the Bradford method.

[3H]thymidine incorporation. A7r5 cells were grown to confluency (~80%) in DMEM supplemented with 10% FBS, after which they were deprived of serum by incubation in DMEM devoid of FBS for 20 h. Subsequently, the cells were supplied with the fresh DMEM devoid of FBS and pretreated with the following agents: SNAP (100 µM, 1 h), ODQ (10 µM, 1 h), LY-294002 (10 µM, 2 h), and PD-98059 (50 µM, 2 h). Both control and treated cells were then exposed to PDGF-BB (10 ng/ml) or IGF-I (50 ng/ml) for an additional 20 h in the presence of the above agents. During the last 4-h incubation period, the cells were labeled with [3H]thymidine (0.5 µCi/ml). After labeling, the cells were washed three times with ice-cold PBS and then exposed to 10% trichloroacetic acid (TCA; 10 min × 3). After complete removal of the TCA, the acid-insoluble material was extracted with 0.1 N NaOH, and the incorporation of [3H]thymidine into DNA was determined with a liquid scintillation counter (6).

Cell counts. Serum-starved A7r5 cells were preincubated in the absence or presence of SNAP (50 µM, 1 h), ODQ (10 µM, 1 h), LY-294002 (1 µM, 2 h), or PD-98059 (5 µM, 2 h), after which the cells were stimulated with PDGF-BB (10 ng/ml) for 3 days. The cells were supplied with fresh serum-free DMEM containing the appropriate agents every 24 h. The cells were then collected using 0.06% trypsin and counted in a hemocytometer.

Measurement of apoptosis. A7r5 cells were grown on eight-well Lab-Tek slides (Nalge Nunc International). Apoptotic cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick end labeling (TUNEL) method (Upstate Biotechnology). Briefly, the cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.05% Tween for 15 min, incubated with TdT end-labeling cocktail for 60 min, blocked for 20 min, and then incubated with avidin-FITC in the dark for 30 min. As a positive control, the permeabilized cells were incubated with DNase I for 60 min. To quantitate apoptotic cell death, the percentage of TUNEL-positive cells to total cells was calculated after counting the number of TUNEL-positive cells and total cells in five random fields, at a magnification of ×100, using a fluorescent microscope.

Statistical analyses. The results are expressed as means ± SE of 3 or more experiments. The data were analyzed by repeated-measures one-way ANOVA followed by Bonferroni t-test or Kruskal-Wallis one-way ANOVA followed by Dunn's method. The differences between means were considered statistically significant when P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Detection of PKB isoform(s) in A7r5 cells, rabbit aortae, and carotid arteries. Initial experiments characterized PKB isoform(s) protein expression in A7r5 cells (Fig. 1A). Western blot analyses revealed a marked expression of PKB-alpha protein with more modest expressions of PKB-gamma and PKB-beta proteins in A7r5 cells. PC12 (pheochromocytoma) cells expressed all three isoforms of PKB (1) and thus served as a positive control. To confirm PKB-alpha protein expression in intact vascular tissues, the homogenates of rabbit aortae and carotid arteries were probed with anti-PKB-alpha antibody (Fig. 1B). In comparison with aortic tissues, carotid arteries were characterized by relatively more abundant PKB-alpha protein levels.


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Fig. 1.   Detection of protein kinase B (PKB) isoforms in A7r5 cells, rabbit aortae, and carotid arteries. A: serum-starved A7r5 cells were lysed, the cell lysates subjected to 12% SDS-PAGE, and the immunoblots probed with polyclonal antibodies specific for PKB-alpha , PKB-beta , or PKB-gamma . The protein bands are representative of 3 separate experiments. B: homogenates of rabbit aortae and carotid arteries were electrophoresed, and the immunoblots were probed with the primary antibody specific for PKB-alpha .

Comparison of the stimulatory effects of PDGF-BB, IGF-I, and ET-1 on the phosphorylation of PKB-alpha and ERK in A7r5 cells. Although the ERK signaling cascade has been implicated in mitogenesis, the role of PKB in cell proliferation is not clearly established. Hence, we assessed PKB-alpha and ERK1/2 phosphorylations in A7r5 cells after stimulation with the following mitogens: PDGF-BB or IGF-I, which activate receptors possessing intrinsic tyrosine kinase activity, or ET-1, which activates G protein-coupled receptors (Fig. 2, A-C). Preliminary experiments revealed that maximal phosphorylations of either PKB-alpha or ERK1/2, by PDGF-BB, IGF-I, or ET-1, occurred between 5 and 15 min. Hence, a time point of 5 min was chosen in this study. The relative increases in PKB-alpha and ERK phosphorylations on stimulation with PDGF-BB (10 ng/ml) were much higher in comparison with that observed with the other two agonists, IGF-I (50 ng/ml) and ET-1 (100 nM). The increases in PKB-alpha phosphorylations in A7r5 cells after stimulation with PDGF-BB, IGF-I, and ET-1 were ~163-fold, 24-fold, and 5-fold, respectively, in comparison with the unstimulated cells (n = 4), as measured after 5 min. Under similar assay conditions, the increases in ERK2 phosphorylations after stimulation with PDGF-BB, IGF-I, and ET-1 were ~34-fold, 4-fold, and 4-fold, respectively, when compared with the unstimulated cells (n = 3). The phosphorylation profile for ERK1 in A7r5 cells after stimulation with PDGF-BB, IGF-I, and ET-1 remained essentially the same as that for ERK2.


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Fig. 2.   Comparison of the stimulatory effects of platelet-derived growth factor (PDGF)-BB, insulin-like growth factor-I (IGF-I) and endothelin (ET)-1 on PKB-alpha and extracellular signal-regulated kinase (ERK) phosphorylations (A-C) and of PDGF-induced phosphorylations of PKB-alpha and ERK in the absence and presence of phosphatidylinositol 4,5-bisphosphate 3-kinase (PI-3K) and MEK1 inhibitors, respectively, in A7r5 cells (D and E). A-C: serum-starved cells were exposed to either PDGF-BB (10 ng/ml), IGF-I (50 ng/ml), or ET-1 (100 nM) for 5 min, after which the cell lysates were electrophoresed and the immunoblots probed with anti-phospho-PKB-alpha or anti-phospho-ERK. The blots shown (A) are representative of 3 or 4 separate experiments, the data for which are shown in B and C. Note that although the representative blots for PKB-alpha and ERK did not show an apparent basal phosphorylation under control conditions, densitometric analyses of the data from several experiments showed that the basal phosphorylations of PKB-alpha and ERK were 0.015 ± 0.007 and 0.048 ± 0.023, respectively (obtained after subtraction of the blank). D and E: serum-starved cells were pretreated with the PI-3K inhibitor LY-294002 (10 µM) or the MEK1 inhibitor PD-98059 (50 µM) for 2 h, after which both control and treated cells were stimulated with PDGF-BB (10 ng/ml) for 5 min and probed for PKB-alpha and ERK2 phosphorylations. Data are means ± SE of 3 separate experiments. * P < 0.05 compared with PDGF-stimulated phosphorylations of PKB-alpha (D) or ERK2 (E) in the absence of inhibitors (repeated-measures one-way ANOVA followed by Bonferroni t-test).

Effects of PI-3K inhibitor and MEK1 inhibitor on the basal and PDGF-BB-induced phosphorylation of PKB-alpha and ERK. Figure 2, D and E, shows the effects of the PI-3K inhibitor LY-294002 and the MEK1 inhibitor PD-98059 on PDGF-BB-induced increases in phosphorylation of PKB-alpha and ERK2. After preincubating A7r5 cells with 10 µM LY-294002 for 2 h, the PDGF-BB-induced increase in PKB-alpha phosphorylation was completely abolished (P < 0.05). Pretreatment of A7r5 cells with 50 µM PD-98059 for 2 h also led to a significant attenuation of PDGF-induced increase in ERK2 phosphorylation (P < 0.05). LY-294002 and PD-98059 did not produce significant changes in the basal phosphorylations of PKB-alpha and ERK2, respectively.

Time dependency for PDGF-BB-induced phosphorylation of PKB-alpha and ERK. We next assessed PDGF-augmented PKB and ERK phosphorylation as a function of time (Fig. 3A). Maximal PDGF-induced PKB-alpha phosphorylation was observed at the time points ranging between 5 and 15 min, followed by gradual decline to basal levels. Similar time-dependent effects on ERK phosphorylation were also observed on stimulation of A7r5 cells with PDGF (Fig. 3B).


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Fig. 3.   Time dependency for PDGF-induced phosphorylations of PKB-alpha and ERK in A7r5 cells. Serum-starved cells were exposed to PDGF-BB (10 ng/ml) for the indicated time intervals, after which the cell lysates were probed for phospho-PKB-alpha (A) and phospho-ERK2 (B). The bar graphs for PKB-alpha (A) and ERK2 (B) are depictive of the densitometric analyses (means ± SE) of the respective blots from 3 separate experiments. * P < 0.05 compared with control (Kruskal-Wallis one-way ANOVA followed by Dunn's method). Con, control.

Differential effects of the NO donor SNAP on PDGF-BB-induced phosphorylation of PKB-alpha and ERK. Because we hypothesized that exposure of VSMC to NO leads to growth arrest, we next examined the effects of the NO donor SNAP on PDGF-BB-induced increases in PKB-alpha and ERK2 phosphorylations in A7r5 cells (Fig. 4, A and B). A biphasic response to PDGF-BB-stimulated increases in PKB-alpha phosphorylations was observed on exposure of A7r5 cells to 100 µM SNAP. Acute pretreatments of A7r5 cells with SNAP for 10 and 30 min were found to cause a significant enhancement in PDGF-BB-stimulated increases in PKB-alpha phosphorylations by 46 ± 16% (P < 0.05) and 85 ± 32% (P < 0.05), respectively, in comparison with that observed in the control cells. Although a slight increase in PDGF-BB-stimulated increase in PKB-alpha phosphorylation was still observed upon pretreatment with SNAP for 2 h, this increase was not statistically significant. When the preincubation time with SNAP was further extended to 6 h, there was an attenuation of PDGF-BB-stimulated increase in PKB-alpha phosphorylation by ~21%, which however is not statistically significant. Prolonged incubation with SNAP for 18 h resulted in a significant attenuation (P < 0.05) of PDGF-BB-induced increase in PKB-alpha phosphorylation by 57 ± 16%. Similarly, a 24-h preincubation with SNAP caused a significant attenuation (P < 0.05) of PDGF-BB-induced increase in PKB-alpha phosphorylation by 63 ± 3%. In contrast to PKB-alpha , pretreatment of A7r5 cells with 100 µM SNAP did not significantly alter PDGF-BB-induced increases in the phosphorylation of ERK2 at any of the time points shown in Fig. 4, A and B. Although SNAP, by itself, produced a marginal increase in the basal phosphorylation of ERK2 (Fig. 4A), statistical analyses of the data from several experiments (n = 4) revealed that there were no significant changes in the basal phosphorylations of ERK or PKB-alpha following exposure to SNAP alone. As a control, pretreatment of A7r5 cells with the inactive analog of SNAP, N-acetylpenicillamine (100 µM), for up to 18 h did not lead to any changes in PDGF-BB-induced increases in PKB-alpha phosphorylation (n = 3). Furthermore, SNAP (100 µM)-induced release of NO was assessed by measuring nitrite levels in the incubation medium. After exposure of A7r5 cells to SNAP (100 µM) for 10 min and 18 h, nitrite levels in the media were found to be 1.9 ± 0.14 and 25.2 ± 1.7 µM, respectively (n = 4). There were no detectable amounts of nitrite in the media collected from the control group of cells. These data suggest that NO released from SNAP is responsible for the observed effects on PKB-alpha phosphorylation.


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Fig. 4.   Time-dependent effects of the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) on PDGF-induced increases in PKB-alpha and ERK2 phosphorylations in A7r5 cells. Serum-starved cells were preincubated with 100 µM SNAP or 100 µM N-acetylpenicillamine for the indicated time intervals, after which both control and treated cells were stimulated with PDGF-BB (10 ng/ml) for 5 min. The cell lysates were electrophoresed, and the immunoblots were probed with anti-phospho-PKB-alpha or anti-phospho-ERK. A: representative blots comparing the time-dependent effects of SNAP on PKB-alpha and ERK phosphorylations and of N-acetylpenicillamine on PKB-alpha phosphorylation are shown. B: time course experiments for SNAP-induced changes in the phosphorylations of PKB-alpha and ERK2 are shown. Data are means ± SE of 3 or 4 separate experiments. * P < 0.05 compared with the respective controls (repeated-measures one-way ANOVA followed by Bonferroni t-test).

Effects of SNAP on PDGF-BB-induced activation of PKB-alpha , as assessed by in vitro kinase assay. The biphasic effects of SNAP on PDGF-BB-induced increases in PKB-alpha phosphorylations were verified by immunoprecipitating PKB-alpha from unstimulated and PDGF-BB-stimulated cells after prior treatment with SNAP and by observing the changes in the phosphorylation states of the exogenous substrate histone H2B (Figs. 5, A-D). These in vitro kinase assays utilizing immunoprecipitated PKB-alpha showed that the PDGF-BB-induced increase in PKB-alpha activity was subject to inhibition by LY-294002, thus supporting the obligatory role of PI-3K in PDGF-BB-induced activation of PKB (Fig. 5, A and B). It should be noted that the increase in the activation of PKB-alpha by PDGF, as assessed by the in vitro kinase assay, is less pronounced than that seen in immunoblotting procedures using phosphospecific PKB-alpha antibodies. Recently, Summers et al. (35) have shown that the in vitro kinase assay, utilizing anti-PKB antibody, produces a much higher basal kinase activity than that observed using anti-phospho-PKB antibody (35). The observation of a relatively less pronounced increase in PKB-alpha activity after stimulation with PDGF may be due to an increase in the assay background in the present experimental conditions. Nevertheless, confirming the Western blot experiments utilizing phosphospecific antibodies, acute exposure of A7r5 cells to 100 µM SNAP for 10 min caused a significant augmentation of PDGF-BB-stimulated increase in PKB-alpha activity (Fig. 5C). In addition, prolonged treatment of A7r5 cells with 100 µM SNAP for 18 h led to a significant attenuation in PDGF-BB-stimulated increase in PKB-alpha activity. Pretreatment of A7r5 cells with the inactive analog of SNAP, N-acetylpenicillamine (100 µM), for either 10 min or 18 h did not affect PDGF-BB-induced increases in PKB-alpha activity (data not shown). Figure 5D shows that exposure of A7r5 cells to increasing concentrations of SNAP (10 µM, 100 µM, and 1 mM) for 18 h resulted in a significant dose-dependent attenuation in PDGF-BB-stimulated increases in PKB-alpha activities.


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Fig. 5.   In vitro kinase assay with immunoprecipitated PKB-alpha after acute and prolonged treatments of A7r5 cells with SNAP. Serum-starved cells were preincubated with the indicated concentrations of SNAP for 10 min or 18 h or with 100 µM LY-294002 for 18 h, after which both control and treated cells were exposed to PDGF-BB (10 ng/ml) for 5 min. The respective PKB-alpha immunoprecipitates were then used for in vitro kinase assay. A: representative autoradiogram for the changes in histone H2B phosphorylations is shown. B: effects of LY-294002 on PDGF-BB-induced increase in PKB-alpha activity. C: effects of a fixed concentration of SNAP (100 µM, 10-min and 18-h pretreatment) on PDGF-BB-induced increases in PKB-alpha activity. D: concentration dependency for SNAP-induced diminutions in PDGF-BB-stimulated increase in PKB-alpha activity. Note that PDGF-stimulated increase in PKB-alpha activity is normalized to 100% in C and D. Data are means ± SE of 3 separate experiments. * P < 0.05 compared with control (repeated-measures one-way ANOVA followed by Bonferroni t-test).

Effects of ODQ and 8-bromo-cGMP on SNAP-induced changes in PDGF-BB-stimulated phosphorylation of PKB-alpha . Pretreatment of A7r5 cells with a selective inhibitor of NO-sensitive soluble guanylyl cyclase, ODQ, resulted in a complete inhibition of SNAP-induced increase in cGMP levels (Table 1). Hence, experiments involving the use of ODQ and 8-bromo-cGMP (a cell-permeable, nonhydrolyzable stable analog of cGMP) were performed to determine whether cGMP mediates the biphasic effects of SNAP on PDGF-BB-stimulated PKB-alpha phosphorylations. As shown in Fig. 6A, the attenuation of PDGF-BB-stimulated increase in PKB-alpha phosphorylation, after exposure to 100 µM SNAP for 18 h, was still observed in the presence of 10 µM ODQ. ODQ, by itself, did not affect PDGF-BB-stimulated increase in PKB-alpha phosphorylation. The augmentation of PDGF-BB-stimulated increase in PKB-alpha phosphorylation, observed after exposure of A7r5 cells to 100 µM SNAP for 10 min, also remained unaffected by prior treatment of these cells with 10 µM ODQ for 1 h (data not shown). In addition, preincubation of A7r5 cells with 100 µM 8-bromo-cGMP for 1 h did not affect PDGF-BB-induced increase in PKB-alpha phosphorylation. Figure 6B shows that PDGF-BB-stimulated increase in ERK2 phosphorylation was not altered significantly after prior treatments with SNAP, ODQ, or 8-bromo-cGMP. These data suggest that both acute and chronic SNAP regulations of PDGF-stimulated PKB-alpha activity are independent of cGMP.

                              
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Table 1.   SNAP-induced increase in cGMP in the absence or presence of 10 µM ODQ



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Fig. 6.   Effects of SNAP, H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and 8-bromo-cGMP (8-BrcGMP) on PDGF-induced phosphorylations of PKB-alpha and ERK2 in A7r5 cells. Serum-starved cells were pretreated with 100 µM SNAP in the absence or presence of 10 µM ODQ for 18 h, or with 100 µM 8-bromo-cGMP for 1 h, after which the cell lysates were electrophoresed and the immunoblots probed with anti-phospho-PKB-alpha (A) or anti-phospho-ERK (B). Data are means ± SE of 3 separate experiments. * P < 0.05 compared with PDGF-stimulated phosphorylations of PKB-alpha (A) in the absence of inhibitors (repeated measures one-way ANOVA followed by Bonferroni t-test).

Effects of SNAP on PDGF-BB- or IGF-I-induced increase in [3H]thymidine incorporation. We next investigated if chronic SNAP treatment altered DNA synthesis as assessed by [3H]thymidine incorporation into acid-insoluble DNA. Figure 7A shows that prolonged (21-22 h) treatments with 100 µM SNAP, 10 µM LY-294002, or 50 µM PD-98059 reduced both basal as well as PDGF-BB-induced increases in [3H]thymidine incorporation. ODQ did not reverse the inhibitory effects of SNAP upon PDGF-BB-induced [3H]thymidine incorporation, thereby suggesting that SNAP-induced increase in cGMP is not responsible for this inhibition. The inhibitory effects of SNAP, independent of cGMP, were also observed with another growth factor, IGF-I. These data demonstrate that SNAP could exert an inhibitory effect not only for PDGF-induced increase in DNA synthesis, but also for IGF-I-induced increase in DNA synthesis.


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Fig. 7.   Effects of SNAP, ODQ, LY-294002, and PD-98059 on [3H]thymidine incorporation (A) and proliferation (B) in A7r5 cells. A: serum-starved cells were pretreated with the following agents: SNAP (100 µM, 1 h), ODQ (10 µM, 1 h), LY-294002 (10 µM, 2 h), and PD-98059 (50 µM, 2 h). Subsequently, both control and treated cells were exposed to PDGF-BB (10 ng/ml) or IGF-I (50 ng/ml) for an additional 20 h in the presence of the above agents. [3H]thymidine incorporation was determined as described in MATERIALS AND METHODS. Data shown are means ± SE of 4-7 separate experiments. *,# P < 0.05 compared with control cells without PDGF-BB or IGF-I (repeated measures one-way ANOVA followed by Bonferroni t-test). B: serum-starved cells were pretreated with the following agents: SNAP (50 µM, 1 h), ODQ (10 µM, 1 h), LY-294002 (1 µM, 2 h), and PD-98059 (5 µM, 2 h). Subsequently, both control and treated cells were exposed to PDGF-BB (10 ng/ml) for 3 days. Cell number was determined as described in MATERIALS AND METHODS. Data shown are means ± SE of 3 separate experiments. * P < 0.05 compared with control (repeated measures one-way ANOVA followed by Bonferroni t-test).

Effects of SNAP on PDGF-BB-induced increase in cell proliferation. Stimulation of A7r5 cells with PDGF (10 ng/ml) for 3 days led to an increase in cell number by 71 ± 18% (P < 0.05) in comparison with the control group of cells (Fig. 7B). The total number of cells in the control group was 32,250 ± 3,403 per well (n = 3). Pretreatment of cells with SNAP followed by stimulation with PDGF led to an increase in cell number by 11 ± 10%, which was not significantly different from control. Inclusion of ODQ and SNAP, followed by stimulation of cells with PDGF, resulted in an increase in cell number by 8 ± 22%. These data further demonstrate that the inhibitory effect of SNAP on PDGF-induced proliferation of A7r5 cells was not reversed by ODQ. Additionally, PDGF-induced increases in cell number after pretreatment with LY-294002 and PD-98059 were 1 ± 8 and 13 ± 12%, respectively.

Effects of SNAP on apoptotic cell death. We next asked if the effects of SNAP or LY-294002 on proliferation correlated with the induction of apoptosis. As shown in Fig. 8, exposure of A7r5 cells to 100 µM SNAP for 18 h did not lead to appreciable apoptosis. The percentage of TUNEL-positive cells after SNAP treatment was 3.25 ± 0.15% in comparison with a control value of 0.95 ± 0.15%. When A7r5 cells were subjected to coincubation with 100 µM SNAP and PDGF-BB (10 ng/ml), for 18 h, the percentage of TUNEL-positive cells was 1.15 ± 0.05%. The exposure of A7r5 cells to the PI-3K inhibitor LY-294002 (10 µM) in the absence and presence of PDGF-BB (10 ng/ml) for a similar time interval produced significant apoptosis to the extent of 34 ± 6 and 22 ± 3%, respectively. The permeabilized cells incubated with DNase I served as positive control, wherein nearly 96-98% of the cells were TUNEL positive. Trypan blue exclusion studies showed that exposure of A7r5 cells to 100 µM SNAP for 18 h did not induce cellular necrosis (data not shown). The lack of apoptotic or necrotic damage suggests that NO released from SNAP under these conditions promotes growth arrest without cytotoxicity.


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Fig. 8.   Effects of SNAP and LY-294002 on apoptosis in A7r5 cells. Serum-starved cells were exposed to SNAP (100 µM) or LY-294002 (10 µM) in the absence or presence of PDGF-BB for 18 h. The magnitude of apoptosis was quantified using the TUNEL method. The percentage of green fluorescent TUNEL-positive cells was calculated by counting both TUNEL-positive cells and total cells in 5 random fields. * P < 0.05 compared with control (repeated measures one-way ANOVA followed by Bonferroni t-test).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NO released from endothelial cells may affect VSMC proliferation under normal and diseased states. The present study using A7r5 cells documents that exogenous NO diminishes PDGF-induced proliferation without producing appreciable apoptosis. The antimitogenic actions of NO are mediated through a novel mechanism involving regulation of PKB-alpha , but not ERK phosphorylation. The regulation of PKB-alpha by NO appears to be independent of cGMP. These data implicate the PKB signaling cascade as an important mitogenic pathway that is subject to modulation by NO in VSMC.

PKB, which has earlier been shown to be expressed in different cell types such as human umbilical vein endothelial cells (9) and rat fibroblasts (13), is also expressed in vascular smooth muscle-derived A7r5 cells. The predominant isoform of PKB expressed in A7r5 cells is PKB-alpha , which is followed by PKB-gamma and PKB-beta . To know whether the A7r5 cell line would provide a model for the vascular tissues in vivo, attempts were also made to determine the relative expressions of PKB-alpha in two different intact vascular tissues from rabbits. PKB-alpha protein was detected in freshly isolated aortae as well as in carotid arteries from rabbits. The apparent difference in the expression levels of PKB-alpha between the aortae and carotid arteries may simply be due to a difference in the cellularity of the two vessels. This is the first report demonstrating the relative protein expression levels of PKB isoform(s) in A7r5 cells, and of PKB-alpha in intact vascular tissues. However, the preponderance of the cell types in the intact vascular tissues that express distinct PKB isoforms is not known at the present time. Further studies involving histochemical techniques with the cryosections of vascular tissues would delineate the relative distributions of PKB isoforms in the vascular wall in normal and diseased states.

Previously, Graves et al. (17) demonstrated that PDGF-BB, but not IGF-I, stimulates ERK activity in human newborn aortic VSMC. In line with these studies, our data suggest that, in addition to ERK activation, PKB-alpha also undergoes preferential activation by PDGF-BB in rat A7r5 cells. A transient maximal increase in the PDGF-induced phosphorylation of both PKB-alpha and ERK2, within a time frame of minutes, suggests that the phosphorylated forms of these kinases are subject to rapid dephosphorylation by cell type-specific phosphatases. However, in human umbilical vein endothelial cells, shear stress-induced increases in PKB-alpha phosphorylation remain elevated for up to 6 h (9).

There is little information on the potential biological agents that facilitate or antagonize PKB activation in VSMC. The effects of the vasodilator NO on VSMC proliferation have been extensively studied (2, 5, 27, 34, 40). However, none of these previous studies has examined the possible regulatory effects of NO on PKB activation in VSMC. The present study shows that acute exposure of A7r5 cells to SNAP resulted in marked augmentation of PDGF-induced increases in PKB-alpha phosphorylation and enzyme activity. In contrast, prolonged exposure of A7r5 cells to SNAP led to marked diminutions in PDGF-induced increases in PKB-alpha phosphorylation and activity. It is known that SNAP, which has a half-life of ~5 h, releases NO spontaneously (20). The accumulation of the stable oxidation product of NO, nitrite, in the media, after exposure of A7r5 cells to SNAP for 18 h, was ~13-fold higher than that observed at the 10-min exposure time to SNAP. Additionally, the inactive analog of SNAP, N-acetylpenicillamine, which does not possess the S-nitroso group, did not produce any alterations in the basal or PDGF-induced activation of PKB-alpha for up to 18 h. These data suggest that NO, released from SNAP, is responsible for the observed dual effects under either acute or prolonged treatment conditions. Further studies involving immunoprecipitation of PKB-beta or PKB-gamma , followed by in vitro kinase assay, would delineate the likely activation of PKB-beta and/or PKB-gamma by PDGF and the possible regulation of these activated isoforms by NO.

The biphasic/dual effects of NO donors have earlier been reported in numerous cell types, including rat cardiomyocytes and human mesangial cells (8, 23). NO donors, including SNAP, have been shown to initially augment and then diminish cyclooxygenase-2 expression in mesangial cells as a function of time (8). In addition, Kojda et al. (23) have shown that low concentrations of NO donors, including SNAP, stimulate cardiac contractility, whereas high concentrations of SNAP cause a depression of cardiac contractility. The physiological significance of enhanced PDGF-induced PKB-alpha activation after acute exposure to SNAP is not known. Nevertheless, the diminished PDGF-induced PKB-alpha activation after prolonged exposure to NO may adversely affect VSMC proliferation.

The mechanism responsible for the enhanced activation of PKB with PDGF on acute exposure to SNAP is unknown. A potential mechanism may involve activation of ras, because acute exposure of human Jurkat T cells to an NO donor, sodium nitroprusside, causes S-nitrosylation of the cysteine118 residue in ras (7, 28). This in turn leads to activation of PKB by a PI-3K-dependent, wortmannin-sensitive mechanism (7). However, in A7r5 cells, it appears that this enhanced activation by SNAP occurs downstream of ras for the following reasons. 1) SNAP has no effect on basal PKB phosphorylation, and 2) SNAP does not affect the ERK cascade, which is ras activated.

Studies were performed to investigate whether cGMP mediates the diminution in PDGF-induced activation of PKB-alpha after prolonged exposure (18 h) of A7r5 cells to SNAP. A selective inhibitor of NO-sensitive soluble guanylyl cyclase, ODQ, has previously been shown to block SNAP-induced elevations in cGMP levels in rat aortic VSMC (27), human umbilical vein endothelial cells (18), and rat cardiomyocytes (33). Our data show that ODQ also inhibits SNAP-induced increase in cGMP in A7r5 cells. Furthermore, the present experiments involving the use of ODQ and 8-bromo-cGMP suggest that the attenuation of PDGF-induced activation of PKB-alpha , following prolonged exposure to SNAP, occurs by a mechanism independent of cGMP. It is possible that, on prolonged exposure of A7r5 cells to SNAP, the NO released from SNAP may exert inhibitory effects on PI-3K or directly on PKB by mechanisms involving S-nitrosylation of cysteine residues and/or nitration of tyrosine residues in these enzyme proteins. Irrespective of the mechanisms involved, it appears that NO modulation of PDGF signaling affects only the PKB and not the ERK pathway.

[3H]thymidine incorporation and cell proliferation studies suggest that prolonged exposure of A7r5 cells to NO would result in inhibition of PDGF-induced increase in DNA synthesis and proliferation, respectively, and that these effects could occur independent of cGMP. In support of these present observations, recent studies utilizing ODQ have also demonstrated that several NO donors including SNAP could exert an inhibitory effect on DNA synthesis and proliferation in rat aortic VSMC (27) and human umbilical vein endothelial cells (18) by mechanisms independent of cGMP. Furthermore, it has been demonstrated by Ishida et al. (20) that SNAP can induce the expression of cyclin-dependent kinase (Cdk) inhibitor, p21 Cip1, which in turn can inhibit the G1/S transition phase of VSMC proliferation by inhibiting cdk2. The present observations of diminished activation of PKB-alpha on prolonged exposure to SNAP suggest that NO may exert a negative regulatory effect on PKB signaling pathway which may, in part, be accountable for increased induction of Cdk inhibitor, p21 Cip1, eventually leading to diminished VSMC proliferation.

Previous studies have shown that PDGF is a competence growth factor, whereas IGF-I, ET-1, and ANG II act as progression growth factors in VSMC (3, 22, 30). It has also been reported that the mitogenic effect of IGF-I is relatively less than that produced by PDGF in human VSMC (30), whereas the role of ET-1 on the mitogenic response in VSMC remains controversial (39). Because, in the present study, IGF-I also stimulated PKB-alpha phosphorylation (although of a lesser magnitude in comparison with PDGF), we verified that NO exerts an inhibitory effect on IGF-I-induced mitogenesis in A7r5 cells. Similar to the data observed with PDGF, SNAP also exerted an inhibitory effect on IGF-I-induced DNA synthesis independent of cGMP. IGF-I-induced increases in PKB-alpha phosphorylation and DNA synthesis were relatively less compared with responses produced by PDGF, thereby suggesting a possible correlation between the extent of PKB-alpha activation and the extent of proliferation. Thus the antagonistic effects of NO on growth factor-induced activation of PKB-alpha may have potential implications for VSMC proliferation.

We tested whether the antagonistic effect of SNAP on the proliferation of A7r5 cells is coincident with the induction of apoptosis. Previous studies have shown that exposure of VSMC to a high concentration of an NO donor, sodium nitroprusside (500 µM), for 48 h results in apoptosis (14). However, in the present study, exposure of A7r5 cells to 100 µM SNAP for 18 h did not lead to appreciable apoptosis. The present data are consistent with the reports of Ishida et al. (20) in that a similar concentration of SNAP did not produce apoptosis in human umbilical VSMC, as determined by flow cytometry. The fact that LY-294002, and not SNAP, could induce apoptosis is intriguing. This suggests the possibility that NO has the potential to protect against apoptosis even when PKB is inhibited. In this regard, NO has been shown to directly inhibit caspase-3 activation via S-nitrosylation (29, 36).

Taken together, our data suggest that prolonged exposure of A7r5 cells to NO results in marked attenuations of PDGF-induced activation of PKB-alpha , which in turn diminishes cell proliferation by a mechanism involving growth arrest and not apoptosis. Future studies with animal models subjected to angioplasty may reveal the role of activated PKB isoform(s) in the genesis of neoplasia. Treatment with NO-generating agents under these conditions has the potential to ameliorate this abnormal smooth muscle proliferation by exerting a chronic inhibitory effect on the PKB signaling pathway.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-53715 and a grant from the W. W. Smith Charitable Trust (to M. Kester) as well as by a beginning grant-in-aid from the American Heart Association, Pennsylvania-Delaware Affiliate (to L. Sandirasegarane).


    FOOTNOTES

Address for reprint requests and other correspondence: L. Sandirasegarane, Dept. of Pharmacology, The Pennsylvania State University College of Medicine, Hershey, PA 17033 (E-mail: lxs51{at}psu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 15 July 1999; accepted in final form 24 January 2000.


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DISCUSSION
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