INTRODUCTION

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DURING VASCULAR INJURY, stressed and damaged cells and platelets release ATP (9). In vascular smooth muscle cells, ATP has been shown to induce changes in both contraction and proliferation (4, 17). ATP acts via multiple P2 nucleotide receptor subtypes to increase intracellular calcium concentration ([Ca²⁺]_i) (4, 13, 15). Extracellular ATP also stimulates vascular smooth muscle cell proliferation through activation of P2 nucleotide receptors (15, 17, 28). ATP appears to coordinately regulate protein synthesis, DNA synthesis, and expression of immediate-early and delayed-early genes (15, 17). The ATP-induced increase in vascular smooth muscle cell proliferation is synergistic with growth factors such as insulin, insulin-like growth factor-1, epidermal growth factor, and platelet-derived growth factor (PDGF) (28). However, little is known about the intracellular signaling pathway(s) utilized by ATP and how these pathways are similar to or different from those activated by other receptors.

The Ras/extracellular signal-regulated kinase (ERK) signal transduction pathway has been firmly implicated in regulation of cell proliferation and differentiation (22). ATP activates ERK1/ERK2 in cardiac myocytes, kidney, and PC12 cell lines (10, 11, 20, 23). Recently, a UTP-sensitive P2 nucleotide receptor has been shown to be involved in initiation of ERK1/ERK2 activity in rat renal mesengial cells and PC12 cells (10, 23). Little is known about the intracellular signaling pathways lying between the binding of ATP to its receptor and activation of ERK1/ERK2 and cell proliferation; however, phosphorylation and activation of some protein kinase C (PKC) isoforms have been implicated in PC12 cells (23). In this study, we present evidence that ATP binding to a UTP-sensitive P2Y nucleotide receptor activates ERK1/ERK2 in coronary artery smooth muscle cells, which is necessary for cell proliferation. In addition, we demonstrate that ATP stimulates activation of ERK1/ERK2 in a phosphatidylinositol 3-kinase (PI3K)-independent manner, and cell proliferation requires both ERK1/ERK2 and PI3K activity. These data demonstrate that ATP-stimulated coronary artery smooth muscle cell proliferation requires signals via both ERK and PI3K.

	METHODS

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Isolation and culture of porcine coronary artery smooth muscle cells. Porcine hearts were obtained from Yorkshire farm pigs. The myocardial surface was rinsed in sterile physiological saline that contained (in mM) 2 CaCl₂, 138 NaCl, 1 MgCl₂, 5 KCl, 10 HEPES, pH 7.4, 10 glucose, as well as 1% penicillin and 1% streptomycin. Coronary arteries were dissected from the heart and denuded of endothelium using aseptic techniques and placed in physiological buffer. Smooth muscle cells were isolated from the coronary arteries by enzymatic digestion of the connective tissue with 294 U/ml of collagenase, 0.2% bovine serum albumin (BSA), 0.1% soybean trypsin inhibitor, and 0.04% DNase. Tissues were incubated in a shaking water bath at 37°C, and the collagenase solution was replaced every 60 min for 3 h. Dispersed cells were isolated and placed into culture (5). Cultures of porcine coronary artery smooth muscle cells were grown in DMEM/high-glucose medium that contained 10% fetal bovine serum (FBS) at 37°C with 5% CO₂. Smooth muscle cell lineage was confirmed by smooth muscle actin immunocytochemistry. Stock cell cultures were maintained in a subconfluent state and used before passage 10.

Determination of phosphotyrosyl-ERK1/ERK2. Cells were serum deprived for 24 h before treatment with either ATP or PDGF at the indicated concentration or time. For experiments using the PI3K inhibitors wortmannin (Sigma Chemical; St. Louis, MO) and LY-294002 (Bio-Mol; Plymouth Meeting, PA) or the mitogen-activated protein (MAP) kinase/ERK kinase (MEK) inhibitor PD-98059 (New England BioLabs; Beverly, MA), cells were incubated for 60 min at 37°C in DMEM/high-glucose medium that contained the inhibitor before the addition of agonists. After treatment, cells were washed with ice-cold phosphate-buffered saline, solubilized in 2× Laemmli sample buffer with 200 mM dithiothreitol, and boiled. Lysates were sonicated to disrupt DNA, and proteins were separated on 10% SDS-PAGE gels. The proteins were electrophoretically transferred to nitrocellulose in 25 mM Tris, 192 mM glycine, 20% methanol, and 0.02% SDS. The nitrocellulose was blocked with 5% nonfat dry milk in 20 mM Tris, pH 7.4, 150 mM NaCl, and 0.01% Tween 20. The membranes were probed with a polyclonal phosphotyrosyl-MAP kinase-specific antibody (New England BioLabs) in 20 mM Tris, pH 7.4, 150 mM NaCl, 3% BSA, and 0.01% Tween 20. This antibody recognizes only the tyrosine-phosphorylated (active) form of p44 MAP kinase (ERK1) and p42 MAP kinase (ERK2). The blots were washed in 20 mM Tris, pH 7.4, 150 mM NaCl, and 0.01% Tween 20, and bound antibody was detected by a horseradish peroxidase-conjugated anti-rabbit IgG and enhanced chemiluminescence (Pierce, Rockford, IL).

ERK1/ERK2 in-gel kinase assay. Cells were stimulated and isolated as described above. Proteins were separated on 10% SDS-PAGE gels that contained 0.5 mg/ml myelin basic protein. SDS was removed from the gels in 50 mM Tris, pH 8.0, with 20% isopropanol, and proteins were denatured in 50 mM Tris, pH 8.0, 5 mM -mercaptoethanol, and 6 M guanidine-HCl. The proteins were renatured in 50 mM Tris, pH 8.0, 50 mM -mercaptoethanol, and 0.04% Triton X-100. The kinase reaction was run for 1 h at 23°C in 40 mM HEPES, pH 8.0, 10 mM MgCl₂, 2 mM dithiothreitol, and 40 µM [-³²P]ATP (10 µCi/ml). The gels were then washed, dried, exposed to film, and quantified by either phosphorimager or cutting and counting the bands.

DNA synthesis assay. Incorporation of [³H]thymidine into DNA was carried out as we have previously described (29) with minor modification. Cells were cultured as described above on 12-well culture plates. As cells reached confluence they were treated without or with ATP for 24 h at 37°C in DMEM/high-glucose medium containing 0.5% FBS before the addition of 1 µCi of [methyl-³H]thymidine (NEN, Wilmington, DE) for an additional 24 h at 37°C. The cells were washed three times in ice-cold PBS and solubilized in 0.1% SDS. Trichloroacetic acid was added to a final concentration of 10%, and the precipitate was collected by filtration on glass-fiber disks for determination of radioactivity by liquid scintillation counting.

Cell proliferation assay. Cells were seeded at 1 × 10⁴ cells per well in six-well dishes in DMEM/high glucose containing 10% FBS for 16 h at 37°C. Cells were then serum deprived for 24 h at 37°C. Cells were then treated as described in the legend of Fig. 6. Cells were fed with the same media every 2 days, and cells were counted every second day. Eight determinations were made by hemocytometer from each of three wells.

Data interpretation. Unless noted in the legends of Figs. 1-6, all experiments were conducted a minimum of three times in triplicate with similar results. Representative gels are depicted in Figs. 1-5, and numerical values represent means ± SE from at least three separate experiments.

	RESULTS

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Over the past several years MAP kinases (ERK1/ERK2) have been the subject of intensive investigation (22). We were interested in determining whether extracellular ATP could stimulate activation of ERK1/ERK2 isoforms of MAP kinase in CASMC and in beginning to define the signaling events involved in ERK1/ERK2 activation and its role in smooth muscle cell proliferation. To approach this question we have used a phosphotyrosyl-ERK1/ERK2-specific antibody that reacts only with the tyrosine-phosphorylated, active form of ERK1/ERK2 and in-gel kinase assays to measure ERK activation. We have used thymidine incorporation and cell counts as a measure of cell proliferation (21). Thus all results were confirmed by a second experimental design.

ATP stimulation of ERK1/ERK2 activation. CASMC were treated with increasing concentrations of ATP (10⁸ to 10⁴ M) for 10 min at 37°C (Fig. 1, left). There was a low level of phosphotyrosyl-ERK1 (p44 MAP kinase) in the absence of ATP treatment, and the level of phosphotyrosyl-ERK2 (p42 MAP kinase) was higher than that for ERK1 in the absence of ATP. There was little change in the level of phosphotyrosyl-ERK1 and -ERK2 with concentrations of ATP <1 µM ATP. However, we observed a dose-dependent increase in phosphotyrosyl-ERK1 and -ERK2 at higher ATP concentrations (Fig. 1, top left). To confirm that ERK1/ERK2 activity is correlated with the tyrosine phosphorylation of ERK1/ERK2, we conducted in-gel kinase assays on similar samples. ATP stimulated a dose-dependent increase in ERK1/ERK2 activity with a half-maximal effective concentration (EC₅₀) of 2-3 µM ATP, and a maximal response was observed at 10 µM (Fig. 1, bottom left). In addition, ATP (10⁴ M) treatment produced a time-dependent increase in the level of phosphotyrosyl-ERK1 and -ERK2 (Fig. 1, top right) and ERK1/ERK2 activity (Fig. 1, bottom right). Maximal phosphorylation of the ERK1/ERK2 was achieved at 5 min and was maintained through 10 min followed by a decline with treatment times of 20 min. ATP treatment of the smooth muscle cells for 20 min decreased the level of phosphotyrosyl-ERK1 and -ERK2 and ERK1/ERK2 activity below basal, suggestive of increased protein phosphatase activity toward ERK1/ERK2. Thus ATP stimulates the activation of both the ERK1 and ERK2 isoforms of MAP kinase in a time- and dose-dependent manner.

View larger version (14K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   Fig. 1.   Dose response (left) and time course (right) of ATP-stimulated tyrosine phosphorylation of extracellular signal-regulated kinase (ERK) 1/ERK2 (top) and ERK1/ERK2 activity (bottom) in coronary artery smooth muscle cells (CASMC). Confluent cultures of CASMC were treated with the indicated concentrations of ATP for 10 min (left) or with 100 µM ATP for indicated times (right). Phosphospecific ERK1/ERK2 Western blotting was used to determine effect of ATP on tyrosine phosphorylation of ERK1/ERK2 (top), and in-gel kinase assays were utilized to quantify ATP-stimulated ERK1/ERK2 activity (bottom). After treatment, cells were lysed and processed for phospho-ERK1/ERK2 blotting or in-gel kinase assay as described in METHODS.

UTP stimulation of ERK1/ERK2 activation. To determine the class of nucleotide receptor responsible for ATP-stimulated ERK1/ERK2 activation, we have used the P2Y nucleotide receptor-selective agonist UTP. CASMC were treated with increasing concentrations of UTP (10⁸ to 10⁴ M) for 10 min at 37°C (Fig. 2, left). There was a progressive increase in phosphotyrosyl-ERK1 and -ERK2 (Fig. 2, top left) and ERK1/ERK2 activity (Fig. 2, bottom left) from 1 to 100 µM UTP. UTP stimulated ERK1/ERK2 activation with an EC₅₀ of 1 µM and reached a maximum at 10 µM. In addition, when cells were treated for increasing lengths of time with UTP (10⁴ M), there was a transient increase in the level of phosphotyrosyl-ERK1 and -ERK2 (Fig. 2, top right) and ERK1/ERK2 activity (Fig. 2, bottom right). Maximal activation of the ERK1/ERK2 was achieved between 5 and 10 min, which subsequently declined with treatment times of 20 min or longer. UTP appears to stimulate the activation of ERK1/ERK2 with a time course similar to that for ATP, and UTP appears equipotent to ATP in stimulating activation of ERK1/ERK2. Thus ATP stimulation of ERK1/ERK2 appears to utilize a P2Y class of nucleotide receptor for activation of ERK1/ERK2 (1).

View larger version (15K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   Fig. 2.   Dose response (left) and time course (right) of UTP-stimulated tyrosine phosphorylation of ERK1/ERK2 (top) and ERK1/ERK2 activity (bottom) in CASMC. Confluent cultures of CASMC were treated with various concentrations of UTP for 10 min (left) or with 100 µM UTP for various times (right). ERK tyrosine phosphorylation and activity were assayed as in Fig. 1.

Inhibition of MEK activity blocks ATP-stimulated ERK1/ERK2 activation. The dual-specificity kinases MEK1 and MEK2 phosphorylate ERK1 and ERK2 on a threonine and a tyrosine residue, leading to their activation. However, other MAP kinase kinases (MKK) involved in activation of the p38 MAP kinase and stress-activated protein (SAP) kinases have been shown to phosphorylate ERK1 and ERK2 in vitro (8). To determine if MEK1/MEK2 are the upstream kinase(s) responsible for phosphorylation of ERK1/ERK2 in CASMC, we have used PD-98059, a MEK1/MEK2-specific inhibitor (2). Cells were stimulated with ATP (10⁴ M) for 5 min at 37°C after pretreatment with PD-98059 for 60 min at 37°C. In the absence of pretreatment with the MEK inhibitor, ATP stimulated an increase in the level of phosphotyrosyl-ERK1 and -ERK2 (Fig. 3, top) and ERK1/ERK2 activity (Fig. 3, bottom). Pretreatment of the cells with PD-98059 reduced the level of ATP-stimulated phosphotyrosyl-ERK1 and -ERK2 and ERK1/ERK2 activity in a dose-dependent manner with a half-maximal inhibitory concentration (IC₅₀) of 5 µM. Thus MEK1/MEK2 appear(s) to catalyze the phosphorylation and activation of ERK1/ERK2 in coronary artery smooth muscle cells.

View larger version (16K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   Fig. 3.   Inhibition of ATP-stimulated tyrosine phosphorylation of ERK1/ERK2 (top) and ERK1/ERK2 activity (bottom) by the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor PD-98059. Confluent cultures of CASMC were treated for 60 min at 37°C with various concentrations of PD-98059 before ATP (104 M) treatment for 5 min at 37°C. ERK tyrosine phosphorylation and activity were assayed as in Fig. 1.

Effect of the PI3K inhibitors wortmannin and LY-294002 on ATP- and PDGF-stimulated ERK1/ERK2 activation. We have used wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY-294002), two PI3K inhibitors with distinct mechanisms of action, to determine if activation of the PI3K is required for ATP-stimulated ERK1/ERK2 activation. Whereas wortmannin can inhibit myosin light-chain kinase (24) with an ID₅₀ of 200 µM, wortmannin at lower concentrations inhibits the PI3K without effect on myosin light-chain kinase (25), and LY-294002 appears specific for PI3K (19, 27). Cells were stimulated with ATP (10⁴ M) for 5 min at 37°C after pretreatment with wortmannin or LY-294002 for 60 min at 37°C. ATP stimulated an increase in phosphotyrosyl-ERK1 and -ERK2 and ERK1/ERK2 activity in the absence of PI3K inhibitor as was observed above (Fig. 4, left). This ATP-stimulated increase in phosphotyrosyl-ERK1 and -ERK2 and ERK1/ERK2 activity was not affected by pretreatment with wortmannin at concentrations as high as 100 nM (Fig. 4, left). Thus ATP-stimulated activation of ERK1/ERK2 appears independent of PI3K activity. To confirm this result, we used a second PI3K specific inhibitor, LY-294002 (Fig. 4, right). Pretreatment of cells with LY-294002 before ATP stimulation also had no effect on the ATP-stimulated increase in phosphotyrosyl-ERK1 and -ERK2 or ERK1/ERK2 activity (Fig. 4, right). At these concentrations, wortmannin and LY-294002 block cellular PI3K activity (19, 25, 27). Therefore, ATP stimulated ERK1/ERK2 by a PI3K-independent mechanism.

View larger version (9K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effect of phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin (left) and LY-294002 (right) on ATP-stimulated tyrosine phosphorylation of ERK1/ERK2 (top) and ERK1/ERK2 activity (bottom). Confluent cultures of CASMC were treated for 60 min at 37°C with various concentrations of wortmannin or LY-294002 before ATP (104 M) treatment for 5 min at 37°C. ERK tyrosine phosphorylation and activity were assayed as in Fig. 1.

8

View larger version (9K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   Fig. 5.   Effect of PI3K inhibitors wortmannin (left) and LY-294002 (right) on platelet-derived growth factor (PDGF)-stimulated tyrosine phosphorylation of ERK1/ERK2 (top) and ERK1/ERK2 activity (bottom). Confluent cultures of CASMC were treated for 60 min at 37°C with various concentrations of wortmannin or LY-294002 before PDGF (108 M) treatment for 5 min at 37°C. ERK tyrosine phosphorylation and activity were assayed as in Fig. 1.

Effect of the MEK inhibitor PD-98059 and the PI3K inhibitor LY-294002 on ATP-stimulated DNA synthesis and cell proliferation. We stimulated CASMC with ATP (10⁴ M) to test whether ATP has the ability to increase DNA synthesis, a marker of cell proliferation. ATP treatment increased DNA synthesis by threefold (Fig. 6, left), which was 45% of the response stimulated by 10% FBS. Pretreatment of cells with the MEK inhibitor PD-98059 (100 µM) completely blocked the ATP-stimulated increase in DNA synthesis. In addition, pretreatment of cells with the PI3K inhibitor LY-294002 (10 µM) completely blocked ATP-stimulated DNA synthesis. To confirm the effect of the MEK and PI3K inhibitors on cell growth, we conducted cell proliferation assays (Fig. 6, right). Freshly plated cells were untreated (control), cultured with 10% FBS, or cultured with 100 µM ATP in the absence or presence of 10 µM LY-294002 or 100 µM PD-98059. Serum treatment led to a 5.5-fold increase in cell number after 8 days. ATP treatment led to a threefold increase in cell number after 8 days. The ATP-stimulated increase in cell number was completely inhibited by either LY-294002 or PD-98059 without inducing cell death. These data support the hypothesis that ATP-stimulated CASMC proliferation requires activation of both the ERK1/ERK2 and PI3K signaling pathways.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effect of MEK and PI3K inhibitors on ATP-stimulated CASMC DNA synthesis and proliferation. Left: confluent cultures of CASMC were treated without () or with (+) 10 µM MEK inhibitor (PD-98059) or 10 µM PI3K inhibitor (LY-294002) for 1 h at 37°C before ATP (100 µM) stimulation or with 10% fetal bovine serum (FBS; Serum) for 24 h at 37°C. [3H]thymidine incorporation into DNA was measured as described in METHODS. Error bars represent SD of 6 determinations from 1 experiment. Similar experiments were conducted 3 times. Right: subconfluent cultures of CASMC were continually treated without or with 10 µM PD-98059 or 10 µM LY-294002 in presence of 100 µM ATP or with 10% FBS as a positive control. Results presented are representative of 2 similar experiments having the same result.

	DISCUSSION

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The platelet secretory product ATP is a candidate mediator of smooth muscle responses to vascular injury. Treatment of smooth muscle cells from various vascular beds with ATP has been shown to evoke both short- and long-term responses, i.e., contraction and proliferation, respectively (4, 6, 11, 17, 28). Of these two responses, the intracellular signaling events leading to contraction are better understood. ATP stimulates phospholipase C activity, leading to an increase in D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] (11). Ins(1,4,5)P₃ binds to the Ins(1,4,5)P₃ receptor, initiating the release of calcium from intracellular stores (13, 15, 20). This increase in [Ca²⁺]_i alters the contractile state of the cell. In contrast to the ATP-stimulated intracellular signaling pathway leading to control of contractile state, little is known about the intracellular signaling pathways activated by ATP that regulate proliferation. Recently, Soltoff et al. (23) have demonstrated a role for the calcium-independent PKC- isoform in ATP-stimulated activation of ERK1/ERK2 in PC12 cells, although they did not rule out the possibility that other PKC isoforms may also be involved in this response (23). In preliminary experiments, clamping [Ca²⁺]_i at the level seen with ATP treatment (300 nM) by using a defined extracellular Ca²⁺ concentration and specific amounts of ionophore had no effect on ERK1/ERK2 activation; however, by clamping [Ca²⁺]_i at > 600 nM using the same method, ERK1/ERK2 activity was increased (data not shown). The present study shows that ATP activates the ERK signaling cascade, which has previously been implicated in the control of cell proliferation (8, 12, 22).

The Ras/ERK signal transduction cascade is involved in determining the proliferative and differentiation state of a wide variety of cell types (8, 12, 22). At this time we do not have a clear understanding of the relationship between the activation state of ERK1/ERK2 and cellular differentiation and proliferation (8). Three parallel but interrelated MAP kinase cascades have to date been defined (for review, see Ref. 8). The first MAP kinase cascade described is initiated by growth factor receptor tyrosine kinases and includes activation of Ras, Raf, MEK, and ERK. More recently, inflammatory cytokines, such as tumor necrosis factor- and interleukin-1, and cell stresses, such as ultraviolet and ionizing radiation, hyperosmolarity, heat, and oxidative stress, have been documented to activate the ERK1/ERK2 homologs SAP kinase and p38 MAP kinase. The upstream signaling steps involved in activation of these additional MAP kinase cascades are less well defined but have been shown to include small GTP-binding proteins (Rac and Cdc42) and homologs of MEK1/MEK2, such as SAP/ERK kinase (SEK) 1, MKK3, and MKK4 (8, 16). Whereas activation of parallel MAP kinase cascades in Saccharomyces cerevisiae has a high degree of kinase fidelity with little cross talk between pathways (8), it is unclear whether the mammalian kinases at each level in the respective cascades can phosphorylate and activate the downstream kinases in each of the other cascades. We have demonstrated that activation of MEK is required for ATP-stimulated activation of ERK1/ERK2. Thus either ATP does not activate the MEK-equivalent kinases (i.e., SEK and MKK) in the SAP kinase or p38 MAP kinase signaling pathways, or SEK and MKK cannot phosphorylate ERK1/ERK2, or both. The data only show that ATP stimulation is blocked by the MEK inhibitor. The possibility remains that other stimuli could activate ERK1/ERK2 via other MAP kinase kinases. It appears that ATP utilizes at least some of the signaling events of the Ras, Raf, and MEK signaling cascade in activation of ERK1/ERK2.

For some receptors, activation of ERK1/ERK2 by agonists requires PI3K activity, whereas its activation by other receptors is independent of PI3K (7, 8, 14, 18, 26). ATP-stimulated activation of ERK1/ERK2 is PI3K independent. This demonstrates that in CASMC the ERK signaling cascade and the signaling pathway(s) in which PI3K is involved are independent. ATP-stimulated proliferation of CASMC requires the activation of both the ERK1/ERK2 and PI3K signaling pathways. In addition, neither of these pathways alone appears to be sufficient to generate all of the signals necessary to support ATP-stimulated proliferation. We hypothesize that the ERK1/ERK2 signaling pathway may lead to alteration in gene transcription necessary for proliferation, whereas the PI3K signaling pathway may be responsible for increased protein synthesis required in cells to allow progression through the cell cycle. If this hypothesis proves to be true, this could explain why both signaling pathways are required for ATP-stimulated cell proliferation but neither pathway alone is sufficient.