Adenosine induces endothelial apoptosis by activating protein tyrosine phosphatase: a possible role of p38alpha

Elizabeth O. Harrington, Anthony Smeglin, Nancy Parks, Julie Newton, and Sharon Rounds

Pulmonary and Critical Care Medicine Section, Providence Veterans Affairs Medical Center, Brown University School of Medicine, Providence, Rhode Island 02908


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelial cell (EC) apoptosis is important in vascular injury, repair, and angiogenesis. Homocysteine and/or adenosine exposure of ECs causes apoptosis. Elevated homocysteine or adenosine occurs in disease states such as homocysteinuria and tissue necrosis, respectively. We examined the intracellular signaling mechanisms involved in this pathway of EC apoptosis. Inhibition of protein tyrosine phosphatase (PTPase) attenuated homocysteine- and/or adenosine-induced apoptosis and completely blocked apoptosis induced by the inhibition of S-adenosylhomocysteine hydrolase with MDL-28842. Consistent with this finding, the tyrosine kinase inhibitor genistein enhanced apoptosis in adenosine-treated ECs. Adenosine significantly elevated the PTPase activity in the ECs. Mitogen-activated protein kinase activities were examined to identify possible downstream targets for the upregulated PTPase(s). Extracellular signal-regulated kinase (ERK) 1 activity was slightly elevated in adenosine-treated ECs, whereas ERK2, c-Jun NH2-terminal kinase-1, or p38beta activities differed little. The mitogen-activated protein kinase-1 inhibitor PD-98059 enhanced DNA fragmentation, suggesting that increased ERK1 activity is a result but not a cause of apoptosis in adenosine-treated ECs. Adenosine-treated ECs had diminished p38alpha activity compared with control cells; this effect was blunted on PTPase inhibition. These results indicate that PTPase(s) plays an integral role in the induction of EC apoptosis upon exposure to homocysteine and/or adenosine, possibly by the attenuation of p38alpha activity.

vascular endothelium; signal transduction; nucleotides; homocysteine


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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APOPTOSIS IS A HIGHLY REGULATED process that controls the removal of cells during development and tissue homeostasis and in response to injury. Endothelial cell (EC) apoptosis occurs in atherosclerotic lesions (7), hyperoxia-induced lung injury (4), and primary pulmonary hypertension (28). Additionally, allograft rejection of heart transplants was shown to be partly due to the induction of apoptosis in the endothelium (48). Furthermore, one of the biological effects of angiostatin, a potent peptide inhibitor of neovascularization, is induction of EC apoptosis (9). Thus the balance between EC apoptosis and proliferation is crucial in vascular injury and repair, angiogenesis, and tumor progression.

EC apoptosis is initiated by extracellular factors including lipopolysaccharide (8), tumor necrosis factor-alpha (41), oxidized low-density lipoprotein (14), and angiotensin (15). In previous studies (13, 46, 56), our laboratory and others have demonstrated that exposure to extracellular ATP or adenosine causes EC apoptosis. The inhibitor of S-adenosylhomocysteine hydrolase (SAHH), MDL-28842, mimicked the effects of adenosine and homocysteine, suggesting that SAHH inhibition is the mechanism of apoptosis (46). Elevated levels of extracellular ATP or adenosine occur during exocytotic release of nucleotides from stimulated platelet granules, during the cytolytic release from cells undergoing necrosis, or from ECs by means of membrane transporters (16). The release of adenosine due to cell lysis occurs during hemolysis and rhabdomyolysis, events associated with trauma and multiorgan system failure. The apoptotic effect of adenosine was potentiated by homocysteine (46). Hyperhomocysteinemia is associated with atherosclerotic vascular disease (35). Thus it is important to understand the mechanism of vascular injury associated with these reagents.

In this study, we further explored the mechanism of adenosine-induced apoptosis in ECs by examining intracellular signaling pathways. The mitogen-activated protein kinases (MAPKs) are a diverse family of enzymes that transduce intracellular signals that are important for many biological processes including cell growth, migration, differentiation, and apoptosis (29, 44). The MAPKs are activated by dual phosphorylation of threonine and tyrosine residues (10). The state of protein phosphorylation, determined by protein kinases and phosphatases, is a fundamental means of regulation of the intracellular signaling necessary for a wide range of cellular functions. Protein phosphorylation by protein tyrosine kinases has been associated with many growth-enhancing cellular functions (25), normal as well as neoplastic. Protein tyrosine phosphatases (PTPases) reverse this action. The PTPases comprise a family of cytoplasmic and membrane-associated enzymes, the biological functions of which are less clear (38). A few PTPases have been identified as important in regulating cellular proliferation (19), differentiation (47), and apoptosis (24, 52). It has been suggested that EC apoptosis that results from withdrawal of either growth factors (3) or extracellular matrix (36) is mediated by PTPases.

Together, the current data demonstrate that several pathways are responsible for transmitting apoptotic signals. In this study, we show a requirement for early activation of membrane-associated PTPase activity for the complete induction of apoptosis in ECs exposed to homocysteine and/or adenosine. We further show that adenosine-induced apoptosis is associated with diminished p38alpha activity, a mechanism requiring p38alpha tyrosine dephosphorylation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Cell lines and reagents. Bovine main pulmonary arterial ECs (BPAECs) were obtained with the use of a standard scraping technique without enzymes and were grown in MEM containing 10% fetal bovine serum, 1 mM sodium pyruvate, 10 U/ml of penicillin, 10 U/ml of streptomycin, and 250 ng/ml of Fungizone. BPAECs were identified by typical phase-contrast morphology, by immunofluorescence to factor VIII antigen, and by uptake of acetylated low-density lipoprotein.

Adenosine, sodium orthovanadate (Na3VO4), p-nitrophenylphosphate, genistein, and myelin basic protein were purchased from Sigma (St. Louis, MO). MDL-28842 was a gift from Hoechst Marion Roussel (Cincinnati, OH). SB-203580 was a gift from SmithKline Beecham Pharmaceuticals (King of Prussia, PA). PD-98059 was purchased from New England Biolabs (Beverly, MA). Dose-response curves were performed to determine the most effective concentrations of SB-203580, PD-98059, or Na3VO4 for inhibition of p38, extracellular signal-regulated kinase (ERK) 1 and ERK2, or PTPase activity, respectively.

Antibodies directed against phosphotyrosine, ERK1/2, p38alpha , p38beta , and c-Jun NH2-terminal kinase (JNK) 1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies specific for the phosphorylated forms of ERK1/2 and p38 were purchased from Santa Cruz Biotechnology and New England Biolabs, respectively. The c-Jun1-79 and activating transcription factor (ATF)1-110 peptides were also purchased from Santa Cruz Biotechnology. The p38 substrate, glutathione S-transferase (GST)-ATF-21-109, was expressed and purified from Escherichia coli with the vector pGEX-ATF-21-109 (a generous gift from Roger J. Davis, University of Massachusetts Medical Center, Worcester, MA) as previously described (22). Antibodies directed against Bcl-2 protein were obtained from Transduction Laboratories (Lexington, KY). Protein G agarose was purchased from Pierce Chemicals. Microcystin-LR was obtained from Alexis (San Diego, CA).

Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. BPAEC monolayers grown on coverslips were rinsed with PBS and then incubated for 20-24 h under the indicated incubation conditions. Cells were washed once with PBS, fixed with 4% paraformaldehyde, and rendered permeable with 0.1% Triton X-100 in 0.1% sodium citrate. Fragmented DNA was detected as previously described (46). Briefly, cells used as a positive control were pretreated with 10 µg/ml of DNase I for 15 min. Cells were subsequently washed with terminal deoxynucleotidyltransferase (TdT) buffer (30 mM Tris, pH 7.2, 140 mM cacodylic acid, 1 mM cobalt chloride, and 0.05% BSA) and incubated in TdT buffer supplemented with 0.3 U/ml of TdT and with 1 nmol/ml of biotinylated dUTP at 37°C for 1 h. Cell samples that served as negative controls for staining were incubated in the absence of biotinylated dUTP. The cells were washed, fluorescein-conjugated streptavidin probe was added, and the cell samples were incubated at room temperature for 1 h. The stained cells were washed, and the slides were placed facedown on a slide with Fluor-save. Cultures were examined with phase and fluorescence microscopy.

DNA fragmentation assay. BPAECs were cultured in six-well dishes. Fragmented DNA was detected as previously described (13). Before reaching confluence, the BPAECs were radiolabeled overnight with 0.5 µCi/ml of [3H]thymidine in complete medium at 37°C in 95% air-5% CO2. The cells were washed twice with buffer I (10 mM HEPES, pH 7.35, 135 mM NaCl, 2 mM CaCl2, 2 mM MgSO4, 5 mM KCl, and 10 mM glucose) and incubated with the various agents in buffer I for 20-24 h at 37°C. Treatments were performed in duplicate. Adherent and nonadherent cells were harvested and pelleted at 2,000 g at 4°C for 10 min. From the resulting supernatant, 200 µl were counted (culture supernatant). The cellular pellet was lysed in 500 µl of ice-cold 0.2% Triton X-100, 10 mM Tris, and 2 mM EDTA, pH 7.35, for 20 min and then centrifuged at 12,000 g for 20 min at 4°C. The supernatant was counted (100 µl; lysate supernatant). The resulting pellet, which consisted of large DNA fragments and intact chromatin, was solubilized with 100 µl of 1 N NaOH, and the total volume was counted (pellet). The radioactivity of each sample was determined. The percent DNA fragmentation was calculated with the following formula, taking into account various dilution factors: percent DNA fragmentation = [(culture supernatant counts/min)(25) + (lysate supernatant counts/min)(5)][100]/[(culture supernatant counts/min)(25) + (lysate supernatant counts/min)(5) + (pellet counts/min)].

PTPase assays. PTPase activity was determined essentially as described by Tsuzuki et al. (50). BPAECs were grown in 10-cm2 dishes in complete medium until 80-90% confluence was reached. The cells were rinsed once with buffer I and incubated in buffer I with the appropriate agent. To collect the cells at the indicated times, they were rinsed once with ice-cold PBS, then scraped into 2 ml of ice-cold PBS. The cells were pelleted at 1,000 g for 5 min. The cell pellet was resuspended in 500 µl of hypotonic buffer [50 mM Tris-Cl, pH 7, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% beta -mercaptoethanol, 20 µg/ml of aprotinin, 20 µg/ml of leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)] and lysed with four strokes in a cooled Dounce homogenizer. The cell lysate was pelleted at 3,000 g for 5 min at 4°C. The pellet was resuspended in 500 µl of hypotonic buffer supplemented with 0.1% Triton X-100 and 10% glycerol. The suspension was incubated on ice for ~15 min. The Triton-insoluble fraction was removed by centrifugation at 15,000 g for 15 min at 4°C. The supernatant was taken and the protein concentration was determined. Equivalent amounts of protein (50 µg) were assayed in a final volume of 200 µl containing PTPase reaction buffer (40 mM Tris-Cl, pH 7, 10 mM dithiothreitol, 5 mM EGTA, 10 µM ZnCl2, 0.1 µM microcystin-LR, and 10 mM p-nitrophenylphosphate) for 10 min at 30°C. The reactions were quenched with 1 ml of 0.4 N NaOH, and the samples were read at an optical density of 410 nm with a spectrophotometer.

Kinase assays. BPAECs were plated in 100-mm2 dishes and grown in complete medium until they were 70-90% confluent. The cells were rinsed once with buffer I and incubated in buffer I with the appropriate agent. The cells were collected at the indicated times by rinsing once with ice-cold PBS and incubating in 500 µl of radioimmunoprecipitation assay (RIPA) buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 20 mM NaF, 20 mM sodium pyrophosphate, 1% Triton X-100, 1 mM Na3VO4, and 1 mM PMSF) at 4°C for 30 min on a rocking platform. The cell lysates were collected, and the protein concentrations were determined.

The MAPKs (ERK1 and ERK2) and the stress-activated protein kinases (SAPKs; JNK1, p38alpha , and p38beta ) were immunoprecipitated from equivalent amounts of protein (250 µg) by incubating with 1 µg of the appropriate antibody at 4°C for 1 h on a rocking platform. The immune complexes were harvested with protein G agarose by incubating at 4°C overnight on a rocking platform. The immune complexes were pelleted and washed twice with RIPA buffer and once with RIPA buffer containing 350 mM NaCl. The ERK1 and ERK2 immune complexes were then washed once in MAPK buffer (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, and 10 mM MgCl2) and resuspended in 50 µl of MAPK buffer. The JNK1 immune complexes were washed once in SAPK buffer (50 mM HEPES, pH 7.6, and 10 mM MgCl2) and resuspended in 50 µl of SAPK buffer. The p38alpha and p38beta immune complexes were washed with the p38 kinase buffer (0.1 M Tris-Cl, pH 7, 0.4 mM EGTA, 0.4 mM Na3VO4, 40 mM magnesium acetate, 1 mM dithiothreitol, 1 mM PMSF, 20 µg/ml of aprotinin, and 20 µg/ml of leupeptin) and resuspended in 50 µl of p38 kinase buffer.

ERK1 and ERK2 activities were assayed by incubating 20 µl of each sample with 20 µl of the MAPK reaction mixture [8 µg of myelin basic protein, 0.5 µCi of [gamma -32P]ATP (3,000 Ci/mmol), and 10 µM ATP] for 30 min at 25°C. JNK1 activity was assayed by incubating 20 µl of each sample with 20 µl of the JNK1 kinase reaction mixture [1 µg of c-Jun1-79 peptide, 0.5 µCi of [gamma -32P]ATP (3,000 Ci/mmol),and 10 µM ATP] for 20 min at 30°C. Activities of p38alpha and p38beta were assayed by incubating 20 µl of each sample with 20 µl of the p38 kinase reaction mixture [1 µg of ATF1-110 peptide or 1 µg of GST-ATF-21-109 peptide, 0.5 µCi [gamma -32P]ATP (3,000 Ci/mmol), and 10 µM ATP] for 20 min at 30°C. Each reaction was quenched with 15 µl of 4× Laemmli buffer. The phosphorylated myelin basic protein c-Jun1-79 peptide and ATF1-110 peptide were resolved on 15 or 10% SDS-polyacrylamide resolving gels, respectively, with 4% stacking gels. The gels were washed in a 5% trichloroacetic acid-1% sodium pyrophosphate solution and dried, and the radiolabeled proteins were visualized by autoradiography.

Immunoblot analysis. BPAECs were scraped from the culture dish and washed with PBS. The cell pellet was resuspended in RIPA buffer and incubated on ice for 30 min. Insoluble cellular debris was removed by centrifugation at 15,000 g for 15 min at 4°C. Protein determinations were performed, and equivalent quantities of protein were suspended in Laemmli buffer (62.5 mM Tris-Cl, pH 6.8, 2% SDS, 10% glycerol, 2.5% beta -mercaptoethanol, and 0.001% bromphenol blue) and resolved on a 10% SDS-polyacrylamide separating gel with a 4% polyacrylamide stacking gel. The proteins were subsequently transferred to Immobilon-P membranes supplied by Millipore, (Bedford, MA) according to manufacturer's recommendations.

Statistical analyses. Data are reported as means ± SE. Differences among the means were tested for significance with Student's t-test (two groups) or ANOVA plus Fisher's least significance difference test (three or more groups).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PTPase involvement in EC apoptosis. To assess the requirement for PTPases in adenosine-induced endothelial programmed cell death, BPAECs were treated with adenosine and/or homocysteine in the presence and absence of 25 µM Na3VO4, an inhibitor of PTPases. Figure 1 shows the results of TdT-mediated dUTP nick end labeling (TUNEL) staining. Few ECs stained positive for apoptosis when incubated with buffer (Fig. 1A) or with 25 µM Na3VO4 alone (Fig. 1B). Figure 1C shows a population of BPAECs that stained positive for apoptosis when cultured in the presence of 1 mM adenosine. This effect was reversed by the addition of Na3VO4 (Fig. 1D). The coincubation of 100 µM adenosine with 100 µM homocysteine enhanced the number of BPAECs that became apoptotic (Fig. 1E), as previously described (45). This effect was also blunted on the addition of Na3VO4 (Fig. 1F).


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Fig. 1.   Sodium orthovanadate (Na3VO4) reduces endothelial cell (EC) apoptosis. Bovine main pulmonary artery ECs (BPAECs) were grown on coverslips and incubated for 16 h in HEPES buffer alone (A) or with 25 µM Na3VO4 (B), 1 mM adenosine (C), 1 mM adenosine plus 25 µM Na3VO4 (D), 100 µM adenosine plus 100 µM homocysteine (E), or 100 µM adenosine plus 100 µM homocysteine plus 25 µM Na3VO4 (F). The cells were fixed, and DNA fragmentation was assessed by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining. Arrows, positively stained apoptotic cells. Insets: corresponding phase-contrast view of immunofluorescent image.

Figure 2 shows the results of DNA fragmentation assays performed to quantitate the effect of the PTPase inhibition on adenosine-induced EC apoptosis. Coincubation of 100 µM adenosine with 100 µM homocysteine (Fig. 2A) or 1 mM adenosine (Fig. 2B) increased DNA fragmentation. Addition of Na3VO4 to these culture conditions resulted in significantly less DNA fragmentation. The amount of DNA fragmentation produced in ECs cultured in the presence of 100 µM adenosine, 100 µM homocysteine, or 25 µM Na3VO4 alone was not significant compared with those cultured under control conditions. A previous study (46) suggested that intracellular adenosine acts by inhibiting SAHH. Thus we assayed the effect of PTPase inhibition on apoptosis caused by MDL-28842, a SAHH inhibitor. The percent DNA fragmentation produced by coincubation of ECs with Na3VO4 and 100 µM MDL-28842 was significantly diminished compared with cells cultured in 100 µM MDL-28842 alone (Fig. 2B). Thus the PTPase inhibitor attenuated the apoptotic effects of adenosine and homocysteine and completely blocked the effect of MDL-28842. These data demonstrate involvement of PTPases in apoptosis of ECs exposed to adenosine and/or homocysteine.


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Fig. 2.   Inhibition of protein tyrosine phosphatase (PTPase) blunts EC apoptosis. [3H]thymidine-labeled BPAECs were incubated with the indicated agents in the presence (+) and absence (-) of Na3VO4 for 20 h. Soluble DNA was isolated and quantitated. A: BPAECs were incubated in buffer I as described in MATERIALS AND METHODS with and without 100 µM adenosine, 100 µM homocysteine, or 100 µM adenosine plus 100 µM homocysteine. Control, n = 13 experiments; control plus Na3VO4, n = 8 experiments; 100 µM adenosine, 100 µM homocysteine, and 100 µM adenosine plus 100 µM homocysteine, n = 4 experiments. B: BPAECs were incubated in buffer I with and without 1 mM adenosine or 100 µM MDL-28842. Control (buffer I alone), n = 13 experiments; control plus Na3VO4, n = 8 experiments; 1 mM adenosine and 100 µM MDL-28842, n = 6 experiments. Values are means ± SE. * P < 0.05 compared with control; ** P <=  0.001 compared with the respective treatment without Na3VO4.

In parallel experiments, inhibition of tyrosine kinases with 50 µM genistein significantly augmented the percent DNA fragmentation produced in 1 mM adenosine-treated ECs (adenosine coincubated with genistein, 56.2 ± 5.2% vs. adenosine alone, 44.4 ± 7.4%, n = 8 samples; P < 0.05). Addition of genistein did not cause DNA fragmentation in ECs in the absence of adenosine (control, 10.0 ± 2.0 vs. control with genistein, 11.5 ± 3.0). These data further demonstrate the importance of the degree of phosphorylation of tyrosine residues for protection from apoptosis in ECs exposed to adenosine.

Programmed cell death requires that many intracellular enzymes be activated or inactivated at critical times for cellular destruction to proceed. DNA cleavage, detected by the TUNEL and DNA fragmentation methods, is a late event in apoptosis. Many PTPases are localized close to the cellular membrane and are activated shortly after cellular receptors bind their agonists (2, 42). Thus if PTPase activation is critical for adenosine-induced endothelial programmed cell death, we hypothesized that upregulation of PTPase activity would occur shortly after adenosine exposure. In vitro phosphatase assays performed on cell lysates isolated from buffer-treated or adenosine-treated ECs demonstrated an enhanced level of PTPase activity as early as 1 h after adenosine treatment, and the level remained elevated for 15 h compared with that in buffer-treated ECs (Fig. 3A). This phosphatase activity was suppressed on addition of Na3VO4. Fractionated cell homogenates demonstrated an enhanced PTPase activity in the membrane fraction (Fig. 3B), suggesting that the PTPase(s), which is activated upon early exposure to adenosine, is membrane bound.


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Fig. 3.   Enhanced PTPase activity in adenosine-treated ECs. A: BPAECs were incubated in buffer I with (open circle ) and without (black-triangle) 1 mM adenosine or 25 µM Na3VO4 (dotted lines). Cells were harvested at the indicated times. Equivalent amounts of total protein were taken, and the PTPase activity was determined. B: BPAECs were incubated in buffer I with (solid bars) and without (open bars) 1 mM adenosine, and cells were harvested after 4 h of treatment. The cytosolic and membrane cellular protein fractions were isolated, equivalent amounts of total protein were taken, and PTPase activity was determined. Values are means ± SE; n = 3 experiments/group in A and 6 experiments/group in B. * P < 0.05 compared with control. OD410nm, optical density at 410 nm.

The protooncogene Bcl-2 has been shown to repress apoptosis under a variety of conditions (30). Both the protein level and phosphorylation state of Bcl-2 have been suggested as important determinants for the induction of apoptosis (30, 43). Immunoblot analysis of cell lysates retrieved from ECs treated with 1 mM adenosine for 20 h did not display significantly diminished levels of the 25-kDa Bcl-2 protein compared with those in control ECs (data not shown). In addition, the level of Bcl-2 tyrosine phosphorylation was not altered by the addition of adenosine to the ECs. Thus adenosine-induced EC apoptosis was not likely dependent on modulation of Bcl-2.

Role of MAPK pathways in adenosine-induced EC apoptosis Next, we sought to identify downstream intracellular mediators on which the adenosine-activated PTPase(s) might be acting. The MAPKs comprise a family of signaling molecules important for coordinating a wide variety of cellular responses (reviewed in Refs. 26, 44). Members of the MAPK family include ERK1, ERK2, JNK1, and p38. Each of these MAPKs are reversibly activated by the phosphorylation of a conserved threonine and tyrosine motif (TXY) (10). Thus it is possible that one mechanism by which adenosine is evoking endothelial programmed cell death is by the activation of a PTPase, resulting in the tyrosine dephosphorylation and inactivation of MAPK(s).

MAPK activities have been shown to be transient or sustained depending on the stimuli and biological pathways being initiated (11, 34, 45). Therefore, BPAECs were collected at various times after treatment with adenosine or buffer alone. The EC lysates were assayed for ERK1, ERK2, JNK1, or p38alpha and p38beta kinase activities. Adenosine-treated cell cultures demonstrated an initial decrease in ERK1 activity by 10 min after the treatment, with a slight elevation of ERK1 activity by 1-4 h postexposure (Fig. 4A). However, none of the activities differed significantly from buffer control lysates. The activities of ERK2, JNK1, and p38beta were not significantly elevated in adenosine-exposed cell lysates compared with control levels (Fig. 4, B-D). Figure 4E shows that the p38alpha kinase activity of adenosine-treated EC lysates was diminished below control values as early as 10 min, reaching significance by 1-2 h of exposure.


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Fig. 4.   Altered mitogen-activated protein kinase (MAPK) activities in adenosine-treated ECs. BPAECs were washed once with buffer I and incubated with () and without (control; ) 1 mM adenosine for the indicated times (in min). Equivalent amounts of cell lysates were immunoprecipitated with antibodies against extracellular signal-regulated kinase (ERK) 1 (A), ERK2 (B), c-Jun NH2-terminal kinase (JNK) 1 (C), p38beta (D), or p38alpha (E). Kinase activities of the immunoprecipitates were measured by determining the quantity of radiolabeled phosphorylated myelin basic protein (for ERK1 and ERK2), glutathione S-transferase (GST)-c-Jun peptide (for JNK1), or GST-activating transcription factor (ATF)-2 protein (for p38alpha and p38beta ) as described in MATERIALS AND METHODS. The autoradiographs (representative of at least 3 experiments) were quantitated with scanning densitometry, and the results are means ± SE. * P < 0.05 compared with control.

To further analyze whether ERK1/2 are downstream mediators of adenosine-induced endothelial programmed cell death, we tested the effect on adenosine-induced apoptosis of chemical inhibitors for MAPK kinase (MEK1), the activating enzyme of ERK1 and ERK2 (1, 17) and p38. ECs pretreated with PD-98059, a MEK1 inhibitor, enhanced the percent DNA fragmentation in the presence of 1 mM adenosine compared with ECs treated with 1 mM adenosine alone (Table 1). The MEK1 inhibitor produced a similar trend in ECs treated with 100 µM adenosine. These results suggest that the ERK1/2 activities are not required for the onset of apoptosis but that the slight increase in ERK1 activity may be involved in protecting the ECs from the proapoptotic effects of adenosine.

                              
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Table 1.   Effect of MEK-1 and p38 inhibitors on endothelial cell DNA fragmentation

Preincubation of ECs with SB-203580, an inhibitor specific for p38alpha and p38beta (27, 32) but not for p38gamma and p38delta (12, 21), significantly enhanced the percent DNA fragmentation in ECs treated with buffer (Table 1). These results suggest that the inhibition of p38alpha and p38beta alone is sufficient to cause a degree of apoptosis in BPAECs. In addition, ECs preincubated with SB-203580 produced an approximately twofold greater amount of DNA fragmentation in 100 µM adenosine- or 100 µM homocysteine-treated cells compared with cells incubated with either treatment in the absence of the p38 inhibitor (Table 1). SB-203580 produced no additional DNA fragmentation in ECs coincubated with 100 µM adenosine plus 100 µM homocysteine or with 1 mM adenosine. It is possible that significant inhibition of p38 kinase activity occurs in the presence of adenosine and/or homocysteine and that an additional inhibitor of p38 would not cause any enhancement of DNA fragmentation under these conditions. Consistent with the data presented in Fig. 4E, these data suggest that adenosine-induced EC apoptosis occurs in part because of lowered p38alpha kinase activity.

In vitro assays were performed to determine whether 10 µM PD-98059 or 10 µM SB-203580 was an effective concentration of the inhibitors for blocking ERK1/2 and p38 activity, respectively. Addition of 10 µM PD-98059 produced 3.1- to 2.1-fold lower levels of phosphorylated ERK1/2, whereas 10 µM SB-203580 produced 2.3-fold lower p38alpha kinase activity in these ECs compared with buffer treated-ECs.

Attenuation of p38alpha is mediated by PTPase(s) in adenosine-treated ECs. To test whether the attenuation of p38alpha was a result of adenosine-enhanced PTPase activity, ECs were incubated with buffer or adenosine for 1 or 2 h in the presence or absence of Na3VO4. Cell lysates were harvested, and p38alpha kinase activity was determined. Figure 5 demonstrates that adenosine significantly decreased p38alpha kinase activity in ECs by 52% compared with buffer-treated cells by 1 and 2 h after treatment. The PTPase inhibitor significantly increased p38alpha kinase activity in both buffer- and adenosine-treated ECs by 2 h. Coincubation of adenosine-treated ECs with PTPase inhibitor enhanced the p38alpha kinase activity by 1 h; however, it did not reach significance (P = 0.08 compared with adenosine alone). The addition of Na3VO4 prevented adenosine-induced decreases in p38alpha kinase activity at both 1 and 2 h. No significant differences in p38beta kinase activities were noted between the buffer- and adenosine-treated lysates with and without the PTPase inhibitor (data not shown). The p38alpha and p38beta immunoprecipitates were also analyzed by immunoblot analysis to ensure that equivalent amounts of the p38 protein were assayed within each experiment.


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Fig. 5.   Inactivation of p38alpha is dependent on PTPase activity. BPAECs were washed once with buffer I and incubated with and without adenosine or Na3VO4 for 1 or 2 h. Equivalent amounts of cell lysates were immunoprecipitated with antibodies against p38alpha . Kinase activities of the p38alpha immunoprecipitates were measured by determining the quantity of radiolabeled phosphorylated GST-ATF-21-109 protein as described in MATERIALS AND METHODS. The autoradiographs were quantitated with scanning densitometry. Values are means ± SE; n = 8 experiments for control and 1 mM adenosine; n = 4 experiments for control plus Na3VO4; and n = 5 experiments for 1 mM adenosine plus Na3VO4. * P < 0.05 compared with 1-h control. dagger  P < 0.01 compared with 2-h control. ** P < 0.001 compared with 2-h control or 1 mM adenosine without Na3VO4.

Thus the data suggest that diminished activity of p38alpha may be one of the mechanisms induced by adenosine during EC apoptotsis. These results suggest that the adenosine-activated PTPase(s) may mediate EC apoptosis by the dephosphorylation, hence, the lowered activity, of p38alpha . The slight activation of the ERK1 kinase may represent a defense mechanism of the cell to protect itself from destruction, albeit one insufficient to prevent EC apoptosis upon exposure to adenosine.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrated that homocysteine- and/or adenosine-induced EC apoptosis is dependent on the level of cellular PTPase activity. Inhibitors of PTPases blunted the amount of DNA fragmentation that occurred in homocysteine- and/or adenosine-treated cells. Consistent with this finding, protein tyrosine kinase inhibition increased the level of apoptosis induced by these agonists. PTPase activity was upregulated as early as 1 h after adenosine treatment and remained elevated up to 15 h. The early response of the PTPase(s) suggested that the enzyme is present in the cell in a less active state and does not require protein synthesis. In addition, the early activation of the PTPase(s) on exposure to adenosine suggested that the PTPase(s) may be involved in the initial intracellular signaling pathways necessary for committing ECs to undergo DNA fragmentation. These data demonstrated that homocysteine- and/or adenosine-induced EC apoptosis is mediated, in part, by a PTPase-dependent pathway.

In other cell systems, activation of p38 or JNK1 has been shown to be an important determinant in the mediation of a variety of apoptotic signals (5, 18, 54). Our results, however, suggest that adenosine-induced EC apoptosis may be mediated by the attenuation of p38alpha activity as demonstrated by diminished kinase activity in adenosine-treated cell lysates and enhanced DNA fragmentation on p38alpha inhibition. In fibroblasts, Roulston et al. (45) showed a requirement for early p38 and JNK1 kinase activities to promote cell survival in response to tumor necrosis factor-alpha . In their model, if p38 kinase activity was blocked, the cells underwent apoptosis. In contrast, other studies (33, 51) have demonstrated a requirement for p38alpha for the induction of myocyte apoptosis during ischemia. In our study, diminished p38alpha kinase activity was noted within 1 h of adenosine treatment, and this decrease was blunted by the PTPase inhibitor Na3VO4. In addition, inhibition of p38 kinase activity by SB-203580 enhanced adenosine-induced apoptosis. Thus our data suggest that adenosine-activated PTPase(s) promotes EC apoptosis, in part, by the attenuation of p38alpha kinase activity. However, we cannot exclude the possibility that inhibitors of p38alpha and adenosine and/or homocysteine are causing apoptosis by different but complementary pathways. Further studies in which p38alpha is overexpressed in ECs may help to clarify the requirement for this kinase in the adenosine-induced apoptotic pathway.

ERK1 activity has been shown to be associated with cell survival signaling in response to both fibroblast growth factor (20) and insulin-like growth factor (39). Additionally, overexpression of ERK1 blocked ultraviolet-induced apoptosis in fibroblasts (6). Thus if adenosine mediated apoptosis by means of inhibiting the ERK1 pathway, then we would have expected to see diminished ERK1 kinase activity on addition of adenosine. In contrast, exposure of ECs to adenosine caused a slight enhancement of ERK1 kinase activity. Also, the MEK-1 inhibitor PD-98059 increased DNA fragmentation. These results suggest that the ERK1 activation may be an EC survival response, a way to resist undergoing apoptosis on adenosine exposure. Similarly, activation of ERK1 due to growth factor withdrawal (54) or of ERK2 due to H2O2 exposure (23) in PC12 cells has been suggested to serve a protective function during cellular stresses. Furthermore, these experiments demonstrate that adenosine does not mediate apoptosis by ERK1 or ERK2 kinase inactivation.

We have identified the adenosine-activated PTPase(s) as a membrane-associated enzyme; thus it is possible that the phosphatase is activated during cellular uptake of homocysteine and/or adenosine into the ECs. We report that PTPase inhibition blunts adenosine- or homocysteine-induced EC apoptosis. A number of studies have been published reporting that inhibition of PTPase protected ECs from apoptosis signaled by other agents. For example, Meredith et al. (37) demonstrated that Na3VO4 blocked calpain-induced EC apoptosis. Additionally, PTPase inhibition blocked EC apoptosis induced by loss of cell-extracellular matrix interactions (36, 55).

The PTPases constitute a family of at least 75 enzymes, many of the biological functions and substrate specificities of which are unknown (38). Three membrane-associated PTPases have been identified as having a role in apoptosis in other cell types. First, overexpression of leukocyte common antigen-related (LAR) gene induced apoptosis in COS-7 cells by a mechanism involving caspase-3 (52). The second membrane-associated PTPase, SHP-1, was shown to dephosphorylate CD72 in B cells, which, in turn, promoted B cell antigen receptor-induced apoptosis (53). Finally, enhanced PTP1B enzyme activity appeared to sensitize ME-180 tumor cells to tumor necrosis factor-induced apoptosis (40). It is unclear whether LAR gene, SHP-1, PTP1B, or an as yet to be identified PTPase is expressed in ECs or whether any of these PTPases are responsible for transmitting the apoptotic signals in homocysteine- and/or adenosine-exposed ECs.

The time courses of changes in PTPase(s) activity and p38alpha kinase activity indicate that these events occur early in the course of adenosine-induced apoptosis. In contrast, DNA fragmentation is a late event in apoptosis and requires 16-20 h to become evident as assessed by TUNEL staining and the release of radiolabeled DNA (13). Nevertheless, inhibition of PTPase(s) activity by Na3VO4 and inhibition of p38alpha kinase activity by SB-203580 altered measures of DNA fragmentation. Thus our results suggest that PTPase activation, and possibly p38alpha kinase inhibition, are early events in adenosine-induced apoptosis.

Extracellular ATP and adenosine have been reported to cause apoptosis of a variety of cell types, including lymphocytes (55), HL60 cells (49), and sympathetic neurons (31). The mechanisms of these effects appear to differ among cell types. Previous studies by this laboratory (13, 46) indicate a mechanism of EC apoptosis involving inhibition of the intracellular enzyme SAHH. Because of the potential importance of adenosine and homocysteine as causes of vascular injury, it is important to understand the mechanism of adenosine- and/or homocysteine-induced EC apoptosis.

In summary, our results demonstrate that homocysteine and/or adenosine promote EC apoptosis, in part by upregulating intracellular PTPase activity. Also, our findings suggest that enhanced PTPase activity mediates the apoptotic signals by decreasing the activity of p38alpha .


    ACKNOWLEDGEMENTS

We thank Dr. Ekkehard H. W. Bohme (Hoechst Marion Roussel), R. Fajardo (SmithKline Beecham Pharmaceuticals), and Dr. Roger J. Davis (University of Massachusetts Medical Center) for the kind gifts of MDL-28842, SB-203580, and pGEX-ATF-21-109, respectively.


    FOOTNOTES

This work was supported by grants to S. Rounds from the Veterans Affairs (VA) Merit Review, the Cystic Fibrosis Foundation, and the VA/Department of Defense Collaborative Research Project. It was also supported by an American Heart Association, Rhode Island Affiliate Beginning Grant-in-Aid; a VA Merit Review Type II grant, and American Cancer Society Grant IN-45-39, all to E. Harrington.

Some of the reported studies were presented at the annual meeting of the American Thoracic Society in May 1998 and published in abstract form (Am J Respir Crit Care Med 157: A207, 1998).

Address for reprint requests and other correspondence: E. O. Harrington, Providence VA Medical Center, Research Services 151, 830 Chalkstone Ave., Providence, RI 02908 (E-mail: Elizabeth_ Harrington{at}brown.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. Section 1734 solely to indicate this fact.

Received 26 October 1999; accepted in final form 11 April 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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