Protein tyrosine phosphatase-dependent proteolysis of focal adhesion complexes in endothelial cell apoptosis

Elizabeth O. Harrington, Anthony Smeglin, Julie Newton, Gajarah Ballard, and Sharon Rounds

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Adenosine and/or homocysteine causes endothelial cell apoptosis, a mechanism requiring protein tyrosine phosphatase (PTPase) activity. We investigated the role of focal adhesion contact disruption in adenosine-homocysteine endothelial cell apoptosis. Analysis of focal adhesion kinase (FAK), paxillin, and vinculin demonstrated disruption of focal adhesion complexes after 4 h of treatment with adenosine-homocysteine followed by caspase-induced proteolysis of FAK, paxillin, and p130CAS. No significant changes were noted in tyrosine phosphorylation of FAK or paxillin. Pretreatment with the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone prevented adenosine-homocysteine-induced DNA fragmentation and FAK, paxillin, and p130CAS proteolysis. Asp-Glu-Val-Asp-ase activity was detectable in endothelial cells after 4 h of treatment with adenosine-homocysteine. The PTPase inhibitor sodium orthovanadate did not prevent endothelial cell retraction or FAK, paxillin, or vinculin redistribution. Sodium orthovanadate did block adenosine-homocysteine-induced FAK, paxillin, and p130CAS proteolysis and Asp-Glu-Val-Asp-ase activity. Thus disruption of focal adhesion contacts and caspase-induced degradation of focal adhesion contact proteins occurs in adenosine-homocysteine endothelial cell apoptosis. Focal adhesion contact disruption induced by adenosine-homocysteine is independent of PTPase or caspase activation. These studies demonstrate that disruption of focal adhesion contacts is an early, but not an irrevocable, event in endothelial cell apoptosis.

cell biology; structural biology; vascular biology


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOTHELIAL CELL INJURY is a hallmark of vascular injury caused by sepsis or associated with trauma. Apoptosis is a form of endothelial cell injury that controls the removal of cells during development and in response to injury. Evidence for apoptosis has been reported in lungs from patients with acute respiratory distress syndrome by Polunovsky et al. (34). Endothelial cell apoptosis also occurs in atherosclerosis (5), hyperoxia-induced lung injury (3), progressive pulmonary hypertension (25), and allograft rejection of heart transplants (41). Furthermore, apoptosis is the mechanism by which angiogenesis is inhibited by angiostatin and endostatin, peptide inhibitors of neovascularization (8, 11). Thus the balance between endothelial cell apoptosis and proliferation is crucial in vascular injury and repair and in angiogenesis.

Endothelial cell apoptosis is initiated by extracellular factors and by the loss of cell adhesion to the extracellular matrix (13, 22). Dawicki et al. (9) and Harrington et al. (19) have demonstrated that extracellular ATP or adenosine in concentrations of 100 µM causes endothelial cell apoptosis as assessed by DNA ladder formation, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, and tritiated thymidine release. These elevated levels of ATP or adenosine may occur in vivo during exocytotic release of nucleotides from stimulated platelet granules, by cytolytic release from cells undergoing necrosis, or from endothelial cells by means of membrane transporters (12). Apoptosis requires ectonucleotidase-mediated hydrolysis of extracellular ATP, with the subsequent uptake of adenosine into endothelial cells (9). Adenosine-induced apoptosis was potentiated by homocysteine and was mimicked by inhibitors of S-adenosyl-L-homocysteine hydrolase (38). An additional inhibitor study (38) indicated that purinergic receptors, adenosine deaminase, and adenosine kinase activity were not involved in extracellular ATP-induced apoptosis.

Focal adhesion complexes are protein aggregates linking actin filaments to the cytoplasmic domain of integrins. Disruption of cell-substratum association has been reported to cause apoptosis of anchorage-dependent cells such as endothelial cells (30, 35). Focal adhesion complexes consist of 1) heterodimeric integrin proteins; 2) the tyrosine kinases Src and focal adhesion kinase (FAK); 3) actin-binding structural proteins such as vinculin, talin, and alpha -actinin; and 4) the adaptor proteins paxillin and p130CAS. Tyrosine phosphorylation of FAK, paxillin, and p130CAS has been shown to be important for the formation of focal adhesion complexes (reviewed in Refs. 24, 39).

In the present studies, we determined the effects of adenosine-homocysteine on endothelial cell focal adhesion contacts. Immunofluorescence analysis of FAK, paxillin, and vinculin demonstrated disruption of focal adhesion contacts in cells treated with adenosine-homocysteine. In addition, adenosine-homocysteine caused caspase-induced proteolysis of selected protein components of focal adhesion contacts but did not affect the tyrosine phosphorylation level of these proteins. Although caspase inhibition prevented apoptosis, it did not prevent adenosine-homocysteine-induced disruption of focal adhesion contacts. In a previous study, Harrington et al. (19) reported that phosphatase activity is increased early in the course of adenosine-homocysteine-induced apoptosis and that phosphatase inhibitors blunt adenosine-homocysteine-induced apoptosis. Because phosphorylation has been found to be crucial to the maintenance of normal interactions between protein components of focal adhesion contacts, we determined the effects of the phosphatase inhibitor sodium orthovanadate (Na3VO4) on adenosine-homocysteine-induced disruption of focal adhesion contacts and on caspase-induced proteolysis of FAK, paxillin, and p130CAS. We found that the phosphatase inhibitor did not prevent adenosine-homocysteine-induced disruption of focal adhesion contacts but that it did inhibit caspase activation; FAK, paxillin, and p130CAS proteolysis; and apoptosis. These results indicate that disruption of focal adhesion contacts is an early event in adenosine-homocysteine-induced endothelial cell apoptosis and that phosphatase action is critical in precipitating caspase-induced proteolysis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell lines and reagents. Endothelial cells were obtained from bovine main pulmonary arteries (PAECs) as previously described (9).

Adenosine, homocysteine, Na3VO4, and 7-amino-4-methylcoumarin (AMC) were purchased from Sigma. Antibodies directed against vinculin and paxillin were obtained from Sigma, and the p130CAS antibody was purchased from Transduction Laboratories. FAK antibodies were purchased from Santa Cruz Biotechnology and Transduction Laboratories. The phosphotyrosine antibody PY99 was obtained from Santa Cruz Biotechnology. Protein G Sepharose was obtained from Pierce.

The Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) inhibitor was obtained from Calbiochem-Novabiochem. The fluorogenic peptide N-acetyl-Asp-Glu-Val-Asp-AMC (Ac-DEVD-AMC) was purchased from PharMingen.

Immunoprecipitations and immunoblot analysis. PAECs were scraped and lysed in FAK buffer (50 mM Tris-Cl, pH 7.5, 250 mM NaCl, 0.5% Nonidet P-40, 10% glycerol, 5 mM EDTA, 50 mM NaF, 500 µM Na3VO4, 10 mM beta -glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml of leupeptin, and 20 µg/ml of aprotinin) as previously described (28). Insoluble cellular debris was removed by centrifugation at 15,000 g for 10 min at 4°C. Protein determinations were performed on the cleared cell lysates.

For immunoprecipitations, 250 µg of cell lysate were incubated with 1 µg of antibody with shaking at 4°C for 1 h. The immunocomplexes were captured with protein G agarose. Immunoprecipitates were washed twice with FAK buffer and suspended in Laemmli buffer. The proteins were resolved on SDS-PAGE and transferred to Immobilon-P membranes supplied by Millipore. Immunoblot analyses were performed as previously described (18).

Immunofluorescent staining of PAECs. PAECs grown on coverslips were rinsed with HEPES buffer and then incubated for 4 h under the indicated incubation conditions. The cells were washed once with PBS, fixed with 4% paraformaldehyde, and rendered permeable with 0.1% Triton X-100 in 0.1% sodium citrate. The cells were then incubated with the primary antibodies in a PBS-5% serum solution for 1 h at 37°C. The cells were washed and then incubated with the fluorescently tagged secondary antibody in PBS-serum solution for 1 h at 37°C. The cells were washed with PBS, and the coverslips were placed facedown on a slide with antifade solution (5% n-propyl gallate, 0.25% 1,4-diazabicyclo[2.2.2]octane, and 0.0025% 2,5-diphenyl-1,3,4-oxadiazole in glycerol). Images were viewed and recorded with a laser scanning confocal microscope.

DNA fragmentation assay. PAECs were cultured in six-well dishes. Fragmented DNA was detected as previously described (38).

Assay of caspase activity. PAECs were harvested, and the washed pellets were resuspended in caspase lysis buffer (10 mM HEPES, pH 7.5, 40 mM beta -glycerophosphate, 50 mM NaCl, 2 mM MgCl2, and 5 mM EGTA) (43). The cells were lysed with freeze-thaw cycles, and insoluble cellular debris was removed by centrifugation. Caspase activity was measured by incubating 25 µg of cell lysate with 50 µM Ac-DEVD-AMC in Asp-Glu-Val-Asp-ase (DEVDase) buffer (20 mM HEPES, pH 7.5, 10% glycerol, and 2 mM dithiothreitol) (21) for 1 h at 37°C. Caspase activity was quantitated as the release of the fluorescent conjugate (AMC) from the peptide substrate with a fluorescent plate reader (Molecular Devices) with a 355-nm/460-nm filter pair.

Statistical analysis. Data are presented as means ± SE. Analysis of variance followed by the least significant difference was used to analyze differences among groups. Differences among means were considered significant when P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Adenosine-homocysteine induced endothelial cell focal adhesion complex disruption and degradation but not dephosphorylation. We investigated whether adenosine-induced endothelial cell apoptosis is mediated through the disruption of cell-extracellular matrix adhesion complexes. PAECs immunofluorescently stained for the focal adhesion complex proteins FAK, paxillin, and vinculin demonstrated the expression of these proteins at discrete focal adhesion contacts (Fig. 1, A-C, respectively). There were no consistent differences in FAK or paxillin immunofluorescent staining in cells treated with buffer and adenosine or adenosine-homocysteine after 30 min, 1 h, or 2 h (data not shown). Incubation with 1 mM adenosine or 100 µM adenosine-100 µM homocysteine for 4 h caused disruption of FAK (Fig. 1A) and paxillin (Fig. 1B) from these focal adhesion contact points, with concomitant redistribution of these proteins in the retracting cells. After 4 h of incubation with 1 mM adenosine or 100 µM adenosine-100 µM homocysteine, there were fewer discrete focal adhesion complexes, and immunofluorescent staining of FAK and paxillin was more diffusely distributed within the cytoplasmic space. Vinculin remained localized around the cell periphery (Fig. 1C) after any treatment, and fewer discrete focal adhesion complexes were stained.


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Fig. 1.   Immunofluorescence analysis of focal adhesion complex proteins in adenosine-homocysteine-treated pulmonary artery endothelial cells (PAECs). PAECs were incubated in HEPES buffer with and without 1 mM adenosine or 100 µM adenosine-100 µM homocysteine for 4 h. The cells were immunofluorescently stained for focal adhesion complex-associated proteins focal adhesion kinase (FAK; A), paxillin (B), or vinculin (C) and visualized with laser scanning confocal microscopy. Arrows, representative focal adhesion complexes immunofluorescently stained for respective proteins. Images are representative of 4 independently performed experiments.

To determine whether the redistribution of FAK and paxillin from the focal contacts was due to degradation, we next analyzed these proteins by immunoblot analysis at various times after treatment with adenosine-homocysteine. Figure 2 demonstrated time-dependent proteolysis of FAK, paxillin, and p130CAS after exposure to adenosine or adenosine-homocysteine. Diminished full-length FAK (125-kDa) and paxillin (68-kDa) protein products were noted 8 h after treatment, with an increase in proteolytic products seen (Fig. 2, A and C, respectively). Analysis of p130CAS demonstrated the degradation of the 130-kDa protein as early as 4 h after adenosine or adenosine-homocysteine treatment (Fig. 2E). Densitometric analyses of the immunoblots demonstrated significantly less full-length FAK, paxillin, and p130CAS after 8 and 14 h of exposure to adenosine-homocysteine compared with the control levels (Fig. 2, B, D, and F, respectively). No significant changes were noted in the protein quantity of vinculin in cell lysates incubated with adenosine or adenosine-homocysteine compared with that in buffer-treated cell lysates (Fig. 2, G and H). Of note, Fig. 2D reveals a diminished level of p130CAS in control cultures over time, although the mean densitometric values in Fig. 2E do not reveal a significant decrease compared with values at 1 h. The absence of serum from these incubations may contribute to a low level of proteolysis in buffer control cultures.



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Fig. 2.   Proteolysis of FAK, paxillin, and p130CAS after adenosine-homocysteine treatment. PAECs were incubated in buffer with and without adenosine or adenosine-homocysteine, and cells were harvested after indicated incubation times. Equivalent protein quantities were resolved on 10% SDS-PAGE and immunoblotted for FAK (A), paxillin (C), p130CAS (E), and vinculin (G). Open arrows, degradation products; solid arrows, intact protein. Nos. at left, molecular mass in kDa. Immunoblots are representative images of 3 (FAK and p130CAS) or 4 (paxillin and vinculin) experiments. The immunoblot signals were quantitated by densitometry. B: FAK (125 kDa). D: paxillin (68 kDa). F: p130CAS (130 kDa). H: vinculin (116 kDa). Values are means ± SE of relative protein content. * P < 0.05 compared with protein content after respective treatment at 1 h.

The level of tyrosine phosphorylation of focal adhesion contact proteins has been shown to be critical in determining cellular adhesion to the extracellular matrix, apoptosis, and migration (1, 4, 23). We assessed FAK and paxillin immunoprecipitates for changes in tyrosine phosphorylation by immunoblot analysis with a phosphotyrosine antibody. No significant changes in FAK (Fig. 3, A and B) or paxillin (Fig. 3, C and D) tyrosine phosphorylation levels were noted even after 8 h of exposure to adenosine-homocysteine. Thus it seems that the adenosine-homocysteine-induced focal adhesion contact disruption and proteolysis occurs independently of changes in the state of tyrosine phosphorylation of these protein components.


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Fig. 3.   PAEC FAK and paxillin tyrosine phosphorylation levels were not affected by adenosine-homocysteine. PAECs were incubated in buffer with and without adenosine or adenosine-homocysteine, and cells were harvested after indicated incubation times. Equivalent protein quantities were immunoprecipitated with FAK (A) or paxillin (C) antibodies and harvested with protein G Sepharose. The immunoprecipitated proteins were resolved on SDS-PAGE and immunoblotted (IB) with a phosphotyrosine antibody (PY99). The blots were stripped and reprobed with the immunoprecipitating antibody. Images are representative of 3 (paxillin) or 4 (FAK) independently performed experiments. B and D: immunoblot signals of FAK and paxillin, respectively, were quantitated by densitometry and are presented as the ratio of tyrosine phosphorylation to total immunoprecipitated protein. Values are means ± SE.

The role of caspases in adenosine-homocysteine-induced apoptosis. The caspases are a family of proteases involved in the cleavage of cellular substrates, which ultimately leads to apoptosis (42). To assess the requirement for caspases in adenosine-homocysteine-induced endothelial cell apoptosis and focal adhesion contact disruption, adenosine-homocysteine-treated PAECs were pretreated with 100 µM Z-VAD-FMK, a broad-spectrum caspase inhibitor. Adenosine- and adenosine-homocysteine-induced apoptosis were completely blocked in endothelial cells preincubated with Z-VAD-FMK (Fig. 4A). Analysis of caspase activation demonstrated significantly elevated DEVDase activity in endothelial cells after 4 and 8 h of treatment with adenosine or adenosine-homocysteine (Fig. 4B). Preincubation of the endothelial cells with Z-VAD-FMK blocked adenosine (371.5 ± 61.0 pmol · mg-1 · min-1)- and adenosine-homocysteine (452.8 ± 87.3 pmol · mg-1 · min-1)-induced DEVDase activity (5.2 ± 3.3 and 2.7 ± 1.7 pmol · mg-1 · min-1, respectively).


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Fig. 4.   Adenosine-homocysteine-induced endothelial cell apoptosis requires a Z-Val-Ala-Asp-fluoromethylketome (Z-VAD-FMK)-sensitive protease. A: PAECs were pretreated for 1 h before induction of apoptosis with 100 µM Z-VAD-FMK. Soluble DNA was isolated and quantitated. +, With; -, without. Values are means ± SE; n = 6 experiments. * P < 0.05 compared with control treatment without inhibitor. B: Asp-Glu-Val-Asp-ase (DEVDase) activity was determined in equivalent quantities of PAEC lysate after incubation with buffer, adenosine, and adenosine-homocysteine. Values are means ± SE; n = 12 experiments. * P < 0.005 vs. control treatment.

We assessed whether the Z-VAD-FMK inhibitor was blocking adenosine- and adenosine-homocysteine-induced apoptosis by preventing focal adhesion complex disruption or by cellular proteolysis of the focal adhesion complex proteins. Preincubation of endothelial cells with Z-VAD-FMK did not block cellular retraction or the redistribution of FAK from focal adhesion complexes in adenosine-homocysteine-treated cells (Fig. 5). Also, cells immunofluorescently stained for paxillin or vinculin demonstrated similar cellular retraction and focal adhesion complex disruption. Similar results were seen in cells treated with adenosine and immunofluorescently stained for FAK, paxillin, or vinculin (data not shown). Next, we compared FAK, paxillin, and p130CAS protein content in buffer-, adenosine-, or adenosine-homocysteine-incubated endothelial cells that had been either pretreated or not pretreated with the caspase inhibitor. Figure 6 demonstrates that Z-VAD-FMK blocked FAK, paxillin, and p130CAS proteolysis in endothelial cells treated with adenosine or adenosine-homocysteine for 14 h (P < 0.05). These results were consistent with the DNA fragmentation experiment and suggest that the degradation of proteins important for focal adhesion contact integrity is required for apoptosis to occur in adenosine- or adenosine-homocysteine-treated PAECs.


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Fig. 5.   Effect of Z-VAD-FMK on adenosine-homocysteine-induced focal adhesion complex protein relocalization. PAECs were not pretreated or were pretreated with Z-VAD-FMK for 1 h and then incubated in buffer or adenosine-homocysteine with and without Z-VAD-FMK for 4 h. The cells were immunofluorescently stained for FAK and visualized with laser scanning confocal microscopy. A: buffer control. B: buffer supplemented with 100 µM Z-VAD-FMK. C: 100 µM adenosine-100 µM homocysteine. D: 100 µM adenosine-100 µM homocysteine supplemented with 100 µM Z-VAD-FMK. Arrows, representative focal adhesion complexes immunofluorescently stained for respective proteins. Representative images are presented for experiments performed 3 times.



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Fig. 6.   Z-VAD-FMK blocks adenosine-homocysteine-induced FAK, paxillin, and p130CAS degradation. PAECs were incubated with buffer, adenosine, or adenosine-homocysteine. In parallel, PAECs were treated as described in Fig. 5 and coincubated with Z-VAD-FMK. Cell lysates were collected from cells incubated as described in Fig. 4 after 14 h, and immunoblot analyses were done for FAK, paxillin, and p130CAS. A: representative images from experiments performed at least 3 times. B: immunoblot signals were quantitated by densitometry. Values are means ± SE of relative protein content. * P < 0.05 compared with control treatment not coincubated with Z-VAD-FMK. ** P < 0.05 compared with respective treatment not coincubated with Z-VAD-FMK.

The role of protein tyrosine phosphatases in focal adhesion complex disruption. Harrington et al. (19) have demonstrated that treatment of endothelial cells with adenosine enhanced intracellular protein tyrosine phosphatase (PTPase) activity. In addition, Na3VO4, a PTPase inhibitor, blunted the apoptotic effect of adenosine or adenosine-homocysteine (19). Thus we investigated whether the early event of FAK or paxillin relocalization induced by adenosine or adenosine-homocysteine was dependent on PTPase activity.

The amount of endothelial cell retraction and focal adhesion contact disruption was not significantly different in cells coincubated with Na3VO4 and adenosine-homocysteine for 4 h (Fig. 7D) compared with adenosine-homocysteine-incubated cells not treated with Na3VO4 (Fig. 7C). Similar results were seen in cells immunofluorescently labeled with antibodies directed against paxillin or vinculin. Additionally, no significant differences were noted in PAECs coincubated with Na3VO4 and adenosine and immunofluorescently stained for FAK, paxillin, or vinculin (data not shown). Next, we examined whether adenosine- and adenosine-homocysteine-induced FAK, paxillin, and p130CAS protein degradation was dependent on PTPase activity. PAECs were incubated with adenosine or adenosine-homocysteine in the presence and absence of Na3VO4. Figure 8, A and B, demonstrates significantly less proteolysis of FAK, paxillin, and p130CAS in cells coincubated with Na3VO4 and adenosine for 14 h compared with respective treatments without Na3VO4 (P < 0.005). Although endothelial cells coincubated with Na3VO4 and adenosine-homocysteine had a diminished level of FAK, paxillin, and p130CAS proteolysis, the protection was significant for p130CAS. Coincubation with Na3VO4 also decreased adenosine- and adenosine-homocysteine-induced increases in DEVDase activity (Fig. 8C). Na3VO4 had no effect on the DEVDase activity of adenosine-homocysteine-treated cell lysates when added during the in vitro caspase assay. These data suggest that adenosine-homocysteine-activated PTPase(s) is required for the initiation of caspase-dependent FAK, paxillin, and p130CAS proteolysis.


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Fig. 7.   Effect of sodium orthovanadate (Na3VO4) on adenosine-homocysteine-induced focal adhesion complex protein relocalization. PAECs were incubated in buffer, adenosine, or adenosine-homocysteine with and without 25 µM Na3VO4 for 4 h. The cells were immunofluorescently stained for FAK and visualized with laser scanning confocal microscopy. A: buffer control. B: buffer supplemented with Na3VO4. C: 100 µM adenosine-100 µM homocysteine. D: adenosine-homocysteine supplemented with Na3VO4. Arrows, representative focal adhesion complexes immunofluorescently stained for respective proteins. Representative images are presented for experiments performed 3 times.



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Fig. 8.   Protein tyrosine phosphatase inhibition blocks FAK, paxillin, and p130CAS proteolysis and DEVDase activity. A: PAECs were incubated with buffer, adenosine, or adenosine-homocysteine. In parallel, PAECs were treated as described in Fig. 7 and coincubated with Na3VO4. Cell lysates were collected from cells incubated as described in Fig. 2 after 14 h, and immunoblot analyses were done for FAK, paxillin, and p130CAS. Lanes 1 and 4, buffer-treated lysate; lanes 2 and 5, 1 mM adenosine-treated lysate; lanes 3 and 6, 100 µM adenosine-100 µM homocysteine-treated lysate. Representative images are presented for experiments performed at least 3 times. B: immunoblot signals were quantitated by densitometry. Values are means ± SE of relative protein content. * P < 0.05 compared with control treatment not coincubated with Na3VO4. ** P < 0.001 compared with respective treatment not coincubated with Na3VO4. C: PAECs were treated with buffer (open bars; control), adenosine (hatched bars), or adenosine-homocysteine (solid bars) in the presence and absence of Na3VO4 for 4 h, and cell lysates were analyzed for DEVDase activity. Values are means ± SE; n = 7 experiments. * P < 0.005 vs. control treatment. ** P < 0.05 vs. respective treatment without Na3VO4.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Several studies (13, 30, 35, 43) have shown that disruption of cell-extracellular matrix interactions induced apoptosis in adherent cell types. Our results indicate that disruption of focal adhesion complexes is an early event in adenosine-homocysteine-induced apoptosis, occurring after 4 h of treatment. However, disruption of focal adhesion complexes is not an irrevocable step in the pathway of programmed cell death because agents such as a caspase inhibitor, Z-VAD-FMK, and a PTPase inhibitor, Na3VO4, did not prevent focal adhesion complex disruption but did inhibit adenosine-homocysteine-induced DNA fragmentation; caspase activation; and FAK, paxillin, and p130CAS proteolysis.

Dawicki et al. (9) have previously shown that extracellular ATP or adenosine in a concentration of 100 µM causes endothelial cell apoptosis. Sources of extracellular ATP and adenosine release include exocytosis in platelet granules, cytolytic release from cells undergoing necrosis, and noncytolytic release from endothelial cells by means of membrane transporters. The intracellular concentration of ATP in most cells is 3-5 mM (15). Thus cytolytic release due to rhabdomyolysis and hemolysis or platelet degranulation (15) can be a significant source of extracellular ATP. The action of endothelial and blood cell surface ectonucleotidases on ATP generates adenosine. Thus the concentration of adenosine used in these studies is achievable in vivo.

Homocysteinemia is associated with arterial and venous thrombosis (17) and atherosclerosis (29). The mechanism(s) by which homocysteine causes vascular injury is not known, but studies have shown that homocysteine injures cultured endothelial cells by a prooxidant effect (40) and that exposure to homocysteine changes endothelial cell gene expression (7, 27, 32). However, these effects required millimolar concentrations of homocysteine. Plasma homocysteine levels of 20-30 µM are associated with vascular injury in humans as assessed by endothelial cell detachment (20). Levels > 12 µM are associated with vascular disease (29). Genetic absence of cystathione beta -synthase activity, a cause of homocysteinuria, results in plasma levels > 100 µM (26). Thus the concentration of homocysteine that we used in these studies is within the range associated with disease in vivo.

Adenosine-homocysteine-induced disruption of focal adhesion complexes was followed at 8-14 h by proteolysis of FAK, paxillin, and p130CAS, focal adhesion complex protein components. This proteolysis was prevented by the broad-spectrum caspase inhibitor Z-VAD-FMK, which was also effective in preventing adenosine-homocysteine-induced apoptosis. Others have described relocalization of FAK from focal adhesion complexes during apoptosis (43) and caspase-induced proteolysis of FAK (2, 14, 28, 43, 45) and p130CAS (2) in various cell types and models of apoptosis. In these studies, we demonstrated caspase-induced proteolysis of FAK, paxillin, and p130CAS but not of vinculin. The reason for the sparing of vinculin is not clear but may relate to a relatively protected site in the cellular cytoskeleton.

In addition, we have demonstrated that the PTPase inhibitor Na3VO4 prevented both caspase activation and caspase-induced proteolysis of protein components of focal adhesion complexes. In a previous study, Harrington et al. (19) have shown that increased PTPase activity occurs within 1 h of exposure of endothelial cells to adenosine-homocysteine, that Na3VO4 is an effective PTPase inhibitor in the concentration used in this study, and that Na3VO4 inhibits adenosine-homocysteine-induced apoptosis.

The level of tyrosine phosphorylation of the components of focal adhesion complexes has been shown to be important for regulating the assembly, and possibly the disassembly, of focal adhesion contacts. On activation, FAK autophosphorylates Tyr397, creating a binding site for the SH2 domain of the Src family kinases Src (16). Binding of Src, in turn, results in the further tyrosine phosphorylation of FAK, paxillin, tensin, and p130CAS (37, 44). FAK directly interacts with paxillin and p130CAS (33). In addition, phosphorylation of FAK on Tyr925 results in the association with Grb2 and activation of the Ras/mitogen-activated protein kinase signal transduction pathway (16). Similar interactions have been demonstrated for paxillin and p130CAS (1, 4, 31). The balance between phosphorylation and dephosphorylation of focal adhesion complex components is important for the maintenance of cell-extracellular matrix interactions and in intracellular signal transduction.

PTPase inhibitors, including Na3VO4, have been shown to enhance phosphorylation of FAK and paxillin in endothelial cells (10, 46) and fibroblasts (36). Thus we considered the possibility that FAK dephosphorylation might be the mechanism of adenosine-homocysteine-induced disruption of focal adhesion complexes. However, the results of this study demonstrate that the disruption of focal adhesion complexes was not prevented by the PTPase inhibitor and that tyrosine dephosphorylation of FAK or paxillin is not caused by adenosine-homocysteine. Thus the mechanism of Na3VO4 inhibition of adenosine-homocysteine-induced apoptosis does not involve changes in tyrosine phosphorylation of FAK or paxillin. The dependence of caspase activation, proteolysis, and apoptosis on PTPase activity suggests that tyrosine dephosphorylation is crucial in the activation of caspases. Addition of Na3VO4 did block caspase activation, suggesting a possible requirement for PTPase-dependent DEVDase activation. Cardone et al. (6) have demonstrated that procaspase-9 is serine phosphorylated by Akt, a posttranslational modification that conferred inactivation of this caspase. However, there have been no studies describing the caspase tyrosine phosphorylation level as being crucial for the activation of these proteases. Thus it is probable that PTPase(s) does not act directly on the caspases but indirectly on the upstream targets, which then mediate the activation of caspases.

In vitro assays have demonstrated FAK to be a suitable substrate for caspase-3, caspase-6, caspase-7, and caspase-8 (14, 28, 45). It is possible that adenosine-homocysteine produces tyrosine, serine, or threonine dephosphorylation of other focal adhesion complex protein components, causing diminished protein-protein interactions between the focal adhesion complex proteins. The adenosine-homocysteine-induced alteration(s) of the focal adhesion complex protein(s) may, in turn, expose protein domains containing consensus sequences recognized and proteolysed by the caspases. Studies (2, 43) have shown that the levels of tyrosine-phosphorylated FAK and paxillin diminish on induction of apoptosis in other models. However, our results indicate that tyrosine dephosphorylation of FAK and paxillin does not occur in endothelial cells exposed to adenosine-homocysteine. Decreased interactions between FAK and paxillin, vinculin, or p130CAS have been noted in endothelial cells after apoptosis induced by growth factor deprivation (28) and lipopolysaccharide (2); thus it is possible that this is occurring in our system.

While this manuscript was in preparation, another group (43) demonstrated that FAK dephosphorylation and loss of focal adhesion contacts preceded caspase-induced FAK proteolysis in renal epithelial cells exposed to a nephrotoxicant. Van de Water et al. (43) showed that PTPase activity was not required for caspase-dependent FAK proteolysis or epithelial cell apoptosis. Also, that study presented data in which apoptosis, focal adhesion complex disruption, and cell detachment occurred in the absence of PTPase activity. We also show that focal adhesion complex disruption is not dependent on PTPase or caspase activation. However, in contrast to the van de Water et al. study, we show a requirement for PTPase for the complete induction of endothelial cell caspase activation, FAK, paxillin, and p130CAS proteolysis and DNA degradation on exposure to adenosine-homocysteine. Thus it is likely that multiple pathways are utilized for the transmission of apoptotic signals, which both share common steps but are also divergent depending on the apoptosis-inducing agent and cell type.

In summary, these studies demonstrate that disruption of focal adhesion complexes is an early event in programmed cell death and that caspase-induced proteolysis and apoptosis are dependent on PTPase activation. There appear to be at least three sequential stages in adenosine-homocysteine-induced apoptosis: 1) focal adhesion complex disruption, 2) caspase-induced proteolysis, and 3) DNA fragmentation. Although activation of an as yet unknown PTPase(s) occurs early in this sequence, it is not required for focal adhesion complex disruption.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-64936, Veterans Affairs (VA) Merit Review, and VA/Department of Defense Collaborative Research Project grants (to S. Rounds), American Heart Association, Rhode Island Affiliate Beginning Grant-in-Aid (to E. O. Harrington), VA Merit Review Type II grant (to E. O. Harrington), and American Cancer Society Grant IN-45-39 (to E. O. Harrington).


    FOOTNOTES

Some of the reported studies were presented at the Annual Meeting of the American Thoracic Society in May 1999 and were published in abstract form (Am J Respir Crit Care Med 159: A217, 1999).

Address for reprint requests and other correspondence: S. Rounds, Providence VA Medical Center, Pulmonary/Critical Care Medicine Section, 830 Chalkstone Ave., Providence, RI 02908 (E-mail: Sharon_Rounds{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 11 July 2000; accepted in final form 7 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abedi, H, and Zachary I. Vascular endothelial growth factor stimulates tyrosine phosphorylation and recruitment to new focal adhesions of focal adhesion kinase and paxillin in endothelial cells. J Biol Chem 272: 15442-15451, 1997[Abstract/Free Full Text].

2.   Bannerman, DD, Sathyamoorthy M, and Goldblum SE. Bacterial lipopolysaccharide disrupts endothelial monolayer integrity and survival signaling events through caspase cleavage of adherens junction proteins. J Biol Chem 273: 35371-35380, 1998[Abstract/Free Full Text].

3.   Barazzone, C, Horowitz S, Donati YR, Rodriguez I, and Piguet PF. Oxygen toxicity in mouse lung: pathways to cell death. Am J Respir Cell Mol Biol 19: 573-581, 1998[Abstract/Free Full Text].

4.   Burridge, K, Turner CE, and Romer LH. Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J Cell Biol 119: 893-903, 1992[Abstract].

5.   Cai, W, Devaux B, Schaper W, and Schaper J. The role of Fas/APO 1 and apoptosis in the development of human atherosclerotic lesions. Atherosclerosis 131: 177-186, 1997[ISI][Medline].

6.   Cardone, MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, and Reed JC. Regulation of cell death protease caspase-9 by phosphorylation. Science 282: 1318-1321, 1998[Abstract/Free Full Text].

7.   Chacko, G, Li Q, and Hajjar KA. Induction of acute translational response genes by homocysteine. Elongation factors-1alpha , -beta , and -delta . J Biol Chem 273: 19840-19846, 1998[Abstract/Free Full Text].

8.   Claesson-Welsh, L, Welsh M, Ito N, Anand-Apte B, Soker S, Zetter B, O'Reilly M, and Folkman J. Angiostatin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD. Proc Natl Acad Sci USA 95: 5579-5583, 1998[Abstract/Free Full Text].

9.   Dawicki, DD, Chatterjee D, Wyche J, and Rounds S. Extracellular ATP and adenosine cause apoptosis of pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 273: L485-L494, 1997[Abstract/Free Full Text].

10.   Defilippi, P, Retta SF, Olivo C, Palmieri M, Venturino M, Silengo L, and Tarone G. p125FAK tyrosine phosphorylation and focal adhesion assembly: studies with phosphotyrosine phosphatase inhibitors. Exp Cell Res 221: 141-152, 1995[ISI][Medline].

11.   Dhanabal, M, Ramchandran R, Waterman MJF, Lu H, Knebelmann B, Segal M, and Sukhatme VP. Endostatin induces endothelial cell apoptosis. J Biol Chem 274: 11721-11726, 1999[Abstract/Free Full Text].

12.   Dubyak, G, and el-Moatassim C. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am J Physiol Cell Physiol 265: C577-C606, 1993[Abstract/Free Full Text].

13.   Frisch, SM, Vuori K, Ruoslahti E, and Chan-Hui PY. Control of adhesion-dependent cell survival by focal adhesion kinase. J Cell Biol 134: 793-799, 1996[Abstract].

14.   Gervais, FG, Thornberry NA, Ruffolo SC, Nicholson DW, and Roy S. Caspases cleave focal adhesion kinase during apoptosis to generate a FRNK-like polypeptide. J Biol Chem 273: 17102-17108, 1998[Abstract/Free Full Text].

15.   Gordon, J. Extracellular ATP: effects, sources, and fate. Biochem J 233: 309-319, 1986[ISI][Medline].

16.   Hanks, SK, and Polte TR. Signaling through focal adhesion kinase. Bioessays 19: 137-145, 1997[ISI][Medline].

17.   Harpel, PC, Zhang X, and Borth W. Homocysteine and hemostasis: pathogenic mechanisms. J Nutr 126: 1285S-1289S, 1996[Medline].

18.   Harrington, EO, Loffler J, Nelson PR, Kent KC, Simons M, and Ware JA. Enhancement of migration by protein kinase Calpha and inhibition of proliferation and cell cycle progression by protein kinase Cdelta in capillary endothelial cells. J Biol Chem 272: 7390-7397, 1997[Abstract/Free Full Text].

19.   Harrington, EO, Smeglin A, Parks A, Newton J, and Rounds S. Adenosine induces endothelial cell apoptosis by protein tyrosine phosphatase activation: a possible role for p38alpha inactivation. Am J Physiol Lung Cell Mol Physiol 279: L733-L742, 2000[Abstract/Free Full Text].

20.   Hladovec, J, Sommerova Z, and Pisarikova A. Homocysteinemia and endothelial damage after methionine load. Thromb Res 88: 361-364, 1997[ISI][Medline].

21.   Howard, AD, Kostura MJ, Thornberry N, Ding GJF, Limjuco G, Weidner J, Salley JP, Hogquist KA, Chaplin DD, Mumford RA, Schmidt JA, and Tocci MJ. IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1beta precursor at two distinct sites and does not cleave 31-kDa IL-1alpha . J Immunol 147: 2964-2969, 1991[Abstract/Free Full Text].

22.   Hoyt, DG, Mannix RJ, Gerritsen ME, Watkins SC, Lazo JS, and Pitt BR. Integrins inhibit LPS-induced DNA strand breakage in cultured lung endothelial cells. Am J Physiol Lung Cell Mol Physiol 270: L171-L177, 1996[Free Full Text].

23.   Ilic, D, Almeida E, Schlaepfer D, Dazin P, Aizawa S, and Damsky C. Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. J Cell Biol 143: 547-560, 1998[Abstract/Free Full Text].

24.   Ilic, D, Damsky CH, and Yamamoto T. Focal adhesion kinase: at the crossroads of signal transduction. J Cell Sci 110: 401-407, 1997[Abstract/Free Full Text].

25.   Jones, PL, and Rabinovitch M. Tenascin-C is induced with progressive pulmonary vascular disease in rats and is functionally related to increased smooth muscle cell proliferation. Circ Res 79: 1131-1142, 1996[Abstract/Free Full Text].

26.   Kluijtmans, LAJ, Boers GHJ, Stevens EMB, Renier WO, Kraus JP, Trijbels FJ, van den Heuvel LP, and Blom HJ. Defective cystathionine beta -synthase regulation by S-adenosylmethionine in a partially pridoxine responsive homocystinuria patient. J Clin Invest 98: 285-289, 1996[Abstract/Free Full Text].

27.   Kokame, K, Kato H, and Miyata T. Homocysteine-respondent genes in vascular endothelial cells identified by differential display analysis. GRP78/ BiP and novel genes. J Biol Chem 271: 29659-29665, 1996[Abstract/Free Full Text].

28.   Levkau, B, Herren B, Koyama H, Ross R, and Raines EW. Caspase-mediated cleavage of focal adhesion kinase pp125FAK and disassembly of focal adhesions in human endothelial cell apoptosis. J Exp Med 187: 579-586, 1998[Abstract/Free Full Text].

29.   McCully, K. Homocysteine and vascular disease. Nat Med 2: 386-389, 1996[ISI][Medline].

30.   Meredith, JE, Jr, Fazeli B, and Schwartz MA. The extracellular matrix as a cell survival factor. Mol Biol Cell 4: 953-961, 1993[Abstract].

31.   Nakamoto, T, Sakai R, Honda H, Ogawa S, Ueno H, Suzuki T, Aizawa S, Yazaki Y, and Hirai H. Requirements for localization of p130CAS to focal adhesions. Mol Cell Biol 17: 3884-3897, 1997[Abstract].

32.   Outinen, PA, Sood SK, Liaw PCY, Sarge KD, Maeda N, Hirsh J, Ribau J, Podor TJ, Weitz JI, and Austin RC. Characterization of the stress-inducing effects of homocysteine. Biochem J 332: 213-221, 1998[ISI][Medline].

33.   Polte, TR, and Hanks SK. Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130CAS. Proc Natl Acad Sci USA 92: 10678-10682, 1995[Abstract].

34.   Polunovsky, VA, Chen B, Henke C, Snover D, Wendt C, Ingbar DH, and Bitterman PB. Role of mesenchymal cell death in lung remodeling after injury. J Clin Invest 92: 388-397, 1993[ISI][Medline].

35.   Re, F, Zanetti A, Sironi M, Polentarutti N, Lanfrancone L, Dejana E, and Colotta F. Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells. J Cell Biol 127: 537-546, 1994[Abstract].

36.   Retta, SF, Barry ST, Critchley DR, Defilippi P, Silengo L, and Tarone G. Focal adhesion and stress fiber formation is regulated by tyrosine phosphatase activity. Exp Cell Res 229: 307-317, 1996[ISI][Medline].

37.   Richardson, A, and Parsons T. A mechanism for regulation of the adhesion-associated protein tyrosine kinase pp125FAK. Nature 380: 538-540, 1996[ISI][Medline].

38.   Rounds, S, Yee WL, Dawicki DD, Harrington E, Parks N, and Cutaia MV. Mechanism of extracellular ATP- and adenosine-induced apoptosis of cultured pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 275: L379-L388, 1998[Abstract/Free Full Text].

39.   Schlaepfer, DD, and Hunter T. Integrin signaling and tyrosine phosphorylation: just the FAKs? Trends Cell Biol 8: 151-157, 1998[ISI][Medline].

40.   Starkebaum, G, and Harlan JM. Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest 77: 1370-1376, 1986[ISI][Medline].

41.   Szabolcs, M, Michler RE, Yang X, Aji W, Roy D, Athan E, Sciacca RR, Minanov OP, and Cannon PJ. Apoptosis of cardiac myocytes during cardiac allograft rejection. Relation to induction of nitric oxide synthase. Circulation 94: 1665-1673, 1996[Abstract/Free Full Text].

42.   Thornberry, NA, and Lazebnik Y. Caspases: enemies within. Science 281: 1312-1316, 1998[Abstract/Free Full Text].

43.   Van de Water, B, Nagelkerke JF, and Stevens JL. Dephosphorylation of focal adhesion kinase (FAK) and loss of focal contacts precede caspase-mediated cleavage of FAK during apoptosis in renal epithelial cells. J Biol Chem 274: 13328-13337, 1999[Abstract/Free Full Text].

44.   Vuori, K, Hirai H, Aizawa S, and Ruoslahti E. Introduction of p130CAS signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases. Mol Cell Biol 16: 2606-2613, 1996[Abstract].

45.   Wen, LP, Fahrni JA, Troie S, Guan JL, Orth K, and Rosen GD. Cleavage of focal adhesion kinase by caspases during apoptosis. J Biol Chem 272: 26056-26061, 1997[Abstract/Free Full Text].

46.   Yuan, Y, Meng FY, Huang Q, Hawker J, and Wu HM. Tyrosine phosphorylation of paxillin/pp125FAK and microvascular endothelial barrier function. Am J Physiol Heart Circ Physiol 275: H84-H93, 1998[Abstract/Free Full Text].


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