Pulmonary and Critical Care Medicine Section, Providence Veterans Affairs Medical Center, Brown University School of Medicine, Providence, Rhode Island 02908
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ABSTRACT |
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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 p38
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 p38
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 p38
activity.
vascular endothelium; signal transduction; nucleotides; homocysteine
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INTRODUCTION |
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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-
(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 p38 activity, a mechanism
requiring p38
tyrosine dephosphorylation.
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MATERIALS AND METHODS |
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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, p38Terminal 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%
-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, p38Immunoblot 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%
-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).
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RESULTS |
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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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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 p38
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Attenuation of p38 is mediated by PTPase(s) in
adenosine-treated ECs.
To test whether the attenuation of p38
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 p38
kinase activity was determined. Figure 5
demonstrates that adenosine significantly decreased p38
kinase
activity in ECs by 52% compared with buffer-treated cells by 1 and
2 h after treatment. The PTPase inhibitor significantly increased
p38
kinase activity in both buffer- and adenosine-treated ECs by
2 h. Coincubation of adenosine-treated ECs with PTPase inhibitor
enhanced the p38
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 p38
kinase activity at both 1 and
2 h. No significant differences in p38
kinase activities were
noted between the buffer- and adenosine-treated lysates with and
without the PTPase inhibitor (data not shown). The p38
and p38
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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DISCUSSION |
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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
p38 activity as demonstrated by diminished kinase activity in
adenosine-treated cell lysates and enhanced DNA fragmentation on p38
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-
. In their model, if
p38 kinase activity was blocked, the cells underwent apoptosis. In
contrast, other studies (33, 51) have demonstrated a
requirement for p38
for the induction of myocyte apoptosis during
ischemia. In our study, diminished p38
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 p38
kinase activity. However, we cannot exclude the
possibility that inhibitors of p38
and adenosine and/or homocysteine
are causing apoptosis by different but complementary pathways. Further studies in which p38
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 p38 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 p38
kinase activity by SB-203580 altered measures of
DNA fragmentation. Thus our results suggest that PTPase activation, and
possibly p38
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 p38.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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