1Section of Pulmonary and Critical Care Medicine, The University of Chicago, Chicago, Illinois 60637; 2Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111; and 3University of Rochester Medical Center, Rochester, New York 14642
Submitted 4 December 2003 ; accepted in final form 7 April 2004
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ABSTRACT |
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adenosine triphosphate; purinergic receptors; protein kinase A; serum response factor; proliferation; -actin; SM22
The expression of P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors in VSMC was shown at the mRNA and protein levels (29, 41). P2Y1, P2Y2, P2Y4, and P2Y6 receptors stimulate inositol 1,4,5-trisphosphate (IP3) production and Ca2+ release (20). P2Y11 receptors are linked to both IP3 and cAMP pathways (4, 5). The downstream signaling of P2Y receptors includes various protein kinases (PKC, MAPKs, PKB/Akt) whose activation is critical for P2Y-induced VSMC proliferation (11). P2Y1, P2Y2, P2Y4, and P2Y6 receptors were recently linked to the activation of a small GTPase RhoA and to the stress fiber formation in VSMC (33). We (7) found that ATP stimulates the activity of serum response factor (SRF), a transcription factor commonly activated by a RhoA-dependent mechanism (18) and implicated in the induction of many smooth muscle (SM)-specific genes in VSMC (25, 35).
In the present study, we found that ATP stimulates a profound but transient activation of protein kinase A (PKA), an effector enzyme commonly activated by cAMP (37). In VSMC, the cAMP-raising agents inhibit cell proliferation (2) and SRF activity (7). Therefore, we sought to determine how PKA is implicated in ATP-induced VSMC proliferation and SM gene expression. Our data suggest that the functional significance of the transient PKA activation by ATP is at least twofold: 1) it is positively implicated in ATP-induced VSMC proliferation, and 2) it negatively regulates the SRF-dependent expression of SM genes.
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METHODS |
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Adenovirus-mediated gene transduction. The cells were plated on a 12-well plate at a density of 100,000 cells per well and were grown for 24 h, followed by serum deprivation for 24 h. The cells were then incubated with adenovirus at desired concentrations (see figure legends) for 24 h in the media containing 0.1% BSA.
Nonradioactive in vitro assay for PKA activity. After stimulation with desired agonists, the cells (grown in 12-well plates) were lysed in 0.15 ml/well lysis buffer containing 25 mM HEPES (pH 7.5), 0.5% Nonidet P-40, protease inhibitors (1 mg/ml leupeptin, 1 mg/ml aprotinin, and 1 mM PMSF), and phosphatase inhibitors (1 mM NaF and 200 µM Na-orthovanadate). The lysates were cleared from insoluble material by centrifugation at 20,000 g for 10 min, and 5 µl of cleared lysates were subjected to a kinase reaction with the fluorescence-labeled PKA substrate kemptide (Promega), according to the manufacturer's protocol. The reaction was stopped by boiling the samples for 10 min. The phosphorylated kemptide was separated from nonphosphorylated kemptide by 0.8% agarose electrophoresis. The fluorescent images were taken with the use of a luminescent image analyzer LAS-3000 (Fujifilm) and were analyzed quantitatively.
[3H]thymidine uptake assay. The WKY7 cells were plated on 48-well plates at a density of 5,000 cells per well, grown for 24 h, and serum starved for 48 h. The cells were then stimulated with desired agonists for 24 h. [3H]thymidine (1 µCi/ml) was added 6 h after cell stimulation for 18 h. The cells were then washed twice with ice-cold PBS, precipitated with 10% trichloroacetic acid (TCA) for 30 min, washed once with 10% TCA, and lysed in a solution containing 0.1% NaOH and 0.1% SDS for 15 min. The lysates were analyzed for radioactivity by using scintillation spectrometry.
Western blot analysis. After stimulation with desired agonists, cells were lysed in modified radioimmunoprotection assay buffer containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 1% Triton-X100, 0.1% SDS, 2 mM EDTA, 2 mM EGTA, 10% glycerol, 1 mM NaF, 200 µM Na-orthovanadate, and protease inhibitors (1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM PMSF). The lysates were cleared from insoluble material by centrifugation at 20,000 g for 10 min, boiled in Laemmli buffer, subjected to polyacrylamide gel electrophoresis, analyzed by Western blotting with desired primary antibodies followed by horseradish peroxide-conjugated secondary antibodies (Calbiochem), and developed using an enhanced chemiluminescence reaction (Pierce).
Luciferase assay.
The cells grown in 24-well plates at a density of 50,000 cells per well were transfected with 100 ng/well luciferase reporter plasmid and 100 ng/well cytomegalovirus (CMV)-driven LacZ plasmid under the serum-free conditions for 24 h, using Lipofectamine Plus reagent (Invitrogen). Cells were then stimulated with desired agonists for 6 h, washed twice with PBS, and lysed in protein extraction reagent. The cleared lysates were assayed for luciferase and -galactosidase activity using the corresponding assay kits (Promega, Madison, WI). To account for differences in transfection efficiency, we normalized the luciferase activity of each sample to the
-galactosidase activity and expressed relative to the control.
Reagents.
An E1, E3 replication-deficient adenovirus (Ad5) vector encoding the complete sequence of the PKA inhibitor (PKI) gene under the control of CMV promoter (AdPKI) was described previously (23) and was kindly provided by Dr. Richard Green (University of Illinois at Chicago, Chicago, IL). The control adenovirus encoding CMV-driven LacZ gene was a gift from Dr. Michael Dunn (Medical College of Wisconsin, Milwaukee, WI) (12). The adenoviruses encoding cDNAs for the dominant negative mutant of SRF (dnSRF) and for the LacZ gene under the control of SM22 promoter was described previously (26). The SRF-luciferase reporter plasmid (SRE.L-Luc) was described previously (7, 18). The polyclonal anti-SM22 antibodies (15) were kindly provided by Dr. Julian Solway (University of Chicago). Anti-ERK1/2 was a gift from Dr. Michael J. Dunn (Medical College of Wisconsin); anti-phospho-ERK1/2 was obtained from Cell Signaling; and anti-SM--actin and anti-
-actin were obtained from Sigma. ATP, adenosine 5'-O-(3-thiotriphosphate) (ATP
S), ADP, and UTP also were obtained from Sigma.
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RESULTS |
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Inhibition of PKA by adenovirus-mediated transduction of PKI.
To examine the role of PKA in cell responses to ATP, it is necessary to inhibit PKA effectively and specifically in the total population of cells. The pharmacological agents H-89 and KT5720 are widely used for PKA inhibition. However, recent evidence suggests that these inhibitors elicit nonspecific effects on several other protein kinases (6) that are critical for VSMC proliferation and gene expression. We (7) have previously used the PKA dominant negative mutant (dnPKA) cDNA by transient cotransfection with the desired reporter constructs. However, because of moderate transient transfection efficiency in VSMC (10%), this approach cannot be used in the total cell population assays. Therefore, we accommodated another approach, a high-efficiency (close to 100%) adenovirus-mediated expression of the PKA inhibitor PKI (AdPKI), which was previously used for PKA inhibition in a total population of human microvascular endothelial cells (23). As shown in Fig. 3A, the transduction of AdPKI resulted in a dose-dependent inhibition of ATP-induced PKA activity in VSMC. The transduction of a control adenovirus carrying the LacZ gene (AdLacZ) had no effect on PKA activity (Fig. 3B). Neither AdPKI nor AdLacZ affected the ATP-induced ERK1/2 phosphorylation, as determined by Western blotting of cell lysates with phospho-specific ERK1/2 antibodies (Fig. 3C) or by electrophoretic mobility shift assay for phosphorylated ERK1 and ERK2 (Fig. 3D). Together, these data demonstrate the effectiveness and the specificity of AdPKI as a tool for PKA inhibition in intact VSMC.
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Because our results were not in accord with the commonly accepted view of PKA as a negative regulator of VSMC growth (2), we next examined the effect of the -agonist ISO and of the adenylyl cyclase activator forskolin (FSK), whose regulatory effects on VSMC proliferation are well established. As shown in Fig. 5C, isoproterenol and FSK inhibited the ATP-induced [3H]thymidine uptake, which was expected. Together, these data suggest that the role of PKA in VSMC proliferation is agonist specific: PKA activation by ATP or UTP promotes VSMC proliferation, whereas PKA activation by ISO or FSK plays a regulatory role.
To begin to understand the agonist-dependent role of PKA in VSMC proliferation, we compared the time course of PKA activation in response to ATP with the time courses induced by ISO or FSK. As shown in Fig. 6A, ATP stimulated a transient PKA activation, reaching its maximum at 5 min and declining to the basal level by 30 min. By contrast, the effects of ISO (Fig. 6B) and FSK (Fig. 6C) were more sustained and lasted for up to 2 h. This suggested that PKA might play a dual role in the proliferation of VSMC dependent on the duration of its activity; a transient PKA activation (induced by ATP) might promote VSMC proliferation (cooperating with MAPK and other signaling pathways), whereas a sustained PKA activation (induced by ISO or FSK) might play a negative role.
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DISCUSSION |
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The vascular wall is represented by at least two phenotypes of VSMC: the contractile and the synthetic ones (1). The contractile phenotype is characterized by a profound expression of SM-cytoskeletal proteins (34, 35), whereas the synthetic phenotype is characterized by a decrease in their expression and by an increased rate of proliferation and migration (9). The relative amount of VSMC with synthetic phenotype is increased in the intimal thickening during atherosclerosis and restenosis (1, 32).
Our results, summarized in Table 1, suggest that VSMC phenotypic responses to ATP are dependent on the concentration of the agonist. At low micromolar concentrations (13 µM), corresponding to normal levels in plasma (38), ATP stimulates SRF activity and SRF-dependent expression of SM cytoskeletal proteins, such as SM--actin and SM22 (Figs. 8 and 9), thus fostering the contractile phenotype of VSMC. The low concentrations of ATP also stimulate a maximal phosphorylation (and, presumably, activation) of the MAPKs ERK1/2 (Figs. 2C and 4C). However, the latter does not result in a profound DNA synthesis (Fig. 4A). In contrast, at high micromolar concentrations, which may mimic pathological conditions such as atherosclerosis and restenosis (8), ATP elicits a dual effect on VSMC. First, it stimulates the proliferation of VSMC, the response reported by several laboratories (3, 11, 40) and confirmed by us (Fig. 4A). Second, it stops stimulating the expression of SM cytoskeletal proteins (Fig. 8). Together, these two responses may lead to a shift of VSMC from the contractile to the proliferative phenotype. According to our results, PKA plays a critical role in such a phenotypic transformation of VSMC by high ATP concentrations.
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The regulatory role of PKA in ATP-induced expression of SM--actin and SM22 proteins is consistent with our previous publication (7) reporting the negative regulation of SRF activity and SM-
-actin expression by PKA at the promoter level. Our present results indicate that at low doses, ATP stimulates a profound SRF activity, whereas at high doses, this effect is negated (Fig. 8). Furthermore, such a biphasic activation of SRF by ATP parallels the regulation of SM-
-actin and SM22 expression at the protein level (Fig. 8, B and C). The initial increase in SM-
-actin and SM22 protein levels by low doses of ATP is mediated by SRF (Fig. 9), which is consistent with the SRF dependency of SM-
-actin and SM22 gene transcription (17, 36). The decrease of SRF activity and of SM-
-actin and SM22 protein levels at higher doses of ATP (Fig. 8) is mediated by PKA activation, as judged by 1) comparison of the dose responses of PKA activation (Fig. 4) to those of SRF activity and SM-
-actin and SM22 protein levels (Fig. 8); 2) downregulation of low ATP-induced SM-
-actin and SM22 protein expression by ISO (Fig. 10); and 3) upregulation of SM-
-actin and SM22 protein levels in response to high doses of ATP, when PKA is inhibited by PKI overexpression (Fig. 11).
Several important questions arise from this study and are currently being addressed in our laboratory. First, what is the mechanism by which ATP stimulates PKA activity, and what determines such a transient response? Several types of purinergic receptors are expressed in VSMC (29, 41), providing a number of possibilities. Our data suggest that ATP-induced PKA activation is partially (but not entirely) mediated by P2Y2 or P2Y4 receptors, but not by P2Y1, P2Y12, or P2Y13 receptors, because 1) UTP is as potent as ATP in PKA activation (Fig. 2), implicating P2Y2 or P2Y4 receptors (39); 2) at high doses, UTP is less effective than ATP in PKA activation (Figs. 1C and 2, A and B), suggesting that the other ATP-selective receptors are recruited; 3) ADP, which is a potent agonist for P2Y1, P2Y12, and P2Y13 receptors (24, 27, 39), has little effect on PKA activation; and 4) the P2Y1-selective antagonist MRS-2179 has no effect on PKA activation induced by various concentrations of ATP (data not shown). In addition, ATP may partially mediate the effect of UTP, because the latter can be used by ectonucleoside diphosphokinase to convert ADP (present in the media) to ATP by transphosphorylation (22). Regarding the UTP-insensitive, ATP-selective component of PKA activation, the P2Y11 receptors, which are sensitive to ATP but not to ADP or UTP and which are coupled to cAMP and IP3 signaling (4, 5), could be likely candidates. However, this possibility is still open, because 1) no P2Y11-selective antagonists are available; and 2) the rodent (in our case, rat) P2Y11 receptor cDNAs have not been cloned. Alternatively, it is possible that the ATP-selective component of PKA activation is mediated by P2X receptors (19, 28).
The second important question relates to the mechanism by which the transient PKA activation promotes VSMC proliferation. We previously demonstrated (7) that cAMP response element (CRE)-dependent gene transcription was profoundly activated by ATP, entirely in a PKA-dependent manner. Our additional data also suggest that PKA is partially involved in the stimulation of the activator protein-1 (AP-1) by ATP (data not shown). Given that these transcription factors are implicated in the induction of early response genes such as c-fos, cyclin D1, and others, it is conceivable that these genes are upregulated by ATP in a PKA-dependent manner. Finally, the mechanism by which PKA regulates the SRF activity and the expression of SM genes remains to be determined. SRF activation is mediated by the small GTPase RhoA (18), whose membrane localization is regulated by its PKA-dependent phosphorylation (21). It is likely that the inhibition of SRF activity by PKA occurs as a result of RhoA regulation, but other possibilities also exist. VASP is implicated in SRF activation by promoting polymerization of actin (14). Phosphorylation of VASP by PKA results in its release from the filamentous actin (16) and may ablate its ability to promote SRF activation. We previously showed (7) that ATP stimulates a complete, sustained, and PKA-dependent phosphorylation of VASP, suggesting an additional mechanism for SRF regulation and SM gene expression by PKA. In addition, the regulation of SM gene expression by PKA may occur at a posttranscriptional level. These possibilities are currently being evaluated in our laboratory.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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