From the
Department of Oncology, Albert Einstein College of Medicine, Bronx, New York 10467,
Department of Medicine (Cardiology), Albert Einstein College of Medicine, Bronx, New York 10467
Received for publication, December 12, 2002
, and in revised form, February 6, 2003.
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ABSTRACT |
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INTRODUCTION |
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Integrins function as both outside-in and inside-out mediators of cell signaling due to their ability to regulate the activities of cytoplasmic kinases, growth factor receptors, and ion channels (reviewed in Refs. 4, 8, and 9). The cytoplasmic tails of integrins lack enzymatic activity; therefore, they transduce signals by stimulating the association of adapter proteins that link cytoplasmic and transmembrane kinases with the cytoskeleton. Interactions with other cellular signaling pathways have also been described. The multiple complex and overlapping integrin-ECM interactions are a consequence of the 20 or more different and
subunits that dimerize in many different combinations and are differentially expressed on various cell types (9). Different components of the ECM also serve as substrates for single or in some cases multiple integrins. Expression of specific integrins can be up-regulated by cytokines and angiogenic factors, thereby altering the nature of the interaction of the cell with the ECM. Integrins also appear to be necessary for the optimal activation of growth factor and angiogenic factor receptors, including receptors for VEGF, insulin, fibroblast growth factor, tumor necrosis factor-
, and platelet-derived growth factor (4, 10, 11). As such, cross-talk between integrins and growth factor receptors amplifies the angiogenic response on extracellular matrices that engage those integrins.
Upon binding to ECM, integrins directly activate cell signaling pathways (4, 8, 12). Focal adhesion kinase (FAK) is a 125-kDa nonreceptor protein-tyrosine kinase that is recruited to focal adhesions by integrin engagement with ECM (6, 13). Concurrent with these events, FAK is tyrosine-phosphorylated, and thereby its own kinase activity is increased (14). This activation is an early event associated with the assembly of focal adhesions and is accompanied by the recruitment of other cytoplasmic and cytoskeletal proteins to the focal adhesions, including talin, paxillin, vinculin, and ultimately actin (6). It is these interactions that place FAK in a central role in mediating cell attachment and migration.
The angiogenic factor platelet-derived endothelial cell growth factor is identical to human thymidine phosphorylase (TP), an enzyme that catalyzes the reversible conversion of thymidine to thymine and 2-deoxyribose-1-phosphate; the latter is subsequently converted to 2-deoxyribose (2dR) (15, 16, 17, 18). TP is chemotactic for endothelial cells and has angiogenic activity in several in vivo assays, although it did not directly stimulate endothelial cell proliferation (16, 17, 18, 19, 20). Studies have established a role for TP in experimental and clinical tumor angiogenesis, including transfection studies in which the TP gene increased the vascularization and growth of tumors growing in nude mice (20, 21), and immunohistochemical studies of primary human solid tumors, in which TP was often found to be elevated in the tumors when compared with the corresponding nonneoplastic regions of the same organs (22, 23, 24, 25, 26). The mechanism by which TP mediates angiogenesis is unknown; we have used endothelial cell migration as an in vitro model to address this question. No cell surface receptor for TP has been identified; indeed, the protein lacks a signal sequence required for cell secretion (20). Our previous studies of the mechanisms by which tumor cells expressing high levels of TP induce EC migration have demonstrated that it is mediated by the intracellular synthesis and extracellular release of the thymidine metabolite 2dR to form a chemotactic gradient; the effects of 2dR on EC were identical to those of TP (27). That the angiogenic actions of TP are mediated by a diffusible metabolite clearly distinguishes it from other angiogenic factors (e.g. VEGF), which work, as do most cytokines, by specific binding to cell surface receptors. That TP and 2dR can stimulate endothelial cell chemotaxis indicates that they must engage, in an unknown manner, intracellular signaling pathways that lead to migration.
The aim of the present study was to determine the effect of TP/2dR on focal adhesion formation, to define the role of extracellular matrix proteins and their cognate integrin receptors in TP/2dR-mediated migration, and, in the absence of a known receptor for TP/2dR, to identify specific signaling pathways engaged by TP/2dR to mediate endothelial cell migration and focal adhesion formation. This report documents these events and also provides evidence that there are both similarities and differences in aspects of the activation of cell surface integrins and focal adhesion formation in EC between TP and VEGF.
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EXPERIMENTAL PROCEDURES |
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Isolation of Endothelial Cells from Human Umbilical VeinsHuman venous endothelial cells (HUVEC) were isolated from umbilical cords as previously described (28). Culture medium consisted of Medium 199 (M199) supplemented with 20% (v/v) newborn calf serum, 5% (v/v) pooled human serum, 2 mM L-glutamine, 5 units/ml penicillin G, 5 µg/ml streptomycin sulfate, 10 units/ml heparin, and 7.5 µg/ml EC growth supplement (Invitrogen). Primary cultures of HUVEC were passaged with 0.05% trypsin, 0.02% EDTA. Confluent HUVEC monolayers (passages 14) were used in the experiments described below.
Boyden Chamber Assay of Endothelial Cell MigrationConfluent HUVEC monolayers were cultured with media lacking growth supplement for 48 h prior to harvesting with cell dissociation solution (Sigma). Harvested cells were suspended at 106/ml in M199 with 1% serum, and 105 cells were seeded into precoated transwell inserts (8-µm pore; Costar). Inserts containing HUVEC were placed into a 24-well plate containing M199 with 1% serum and incubated for 1 h at 37 °C. HUVEC migration was stimulated by the addition of a purified factor (TP, VEGF, or 2dR) to the lower chamber. After 5 h, HUVEC were stained with 10 µM Cell Tracker Green (Molecular Probes, Inc., Eugene, OR) for 30 min at 37 °C, and the upper membrane of the insert was swabbed to remove nonmigrated cells. Inserts were washed three times with PBS, fixed in 3.7% formaldehyde in PBS for 10 min at room temperature, and mounted on microscope slides. HUVEC migration was quantitated by counting the number of cells in three random fields (x100 total magnification) per insert. The data are expressed as the average number of cells/field.
Where indicated, migration of HUVEC on alternate substrates was examined by coating the inserts with 0.2% (w/v) gelatin or fibronectin, thrombospondin, or vitronectin (10 µg/ml). In addition, the involvement of integrins in TP-induced migration was determined through inclusion of monoclonal antibodies. For these experiments, cells were treated for 15 min with antibodies (10 µg/ml) against specific integrin subunits and heterodimers (Chemicon International) prior to plating (along with the antibody) for 1 h in the Boyden chamber. Antibody was also added to the lower wells upon the addition of the chemotactic stimulus. Nonspecific IgG was used as the control in these experiments.
Immunoprecipitation and Western BlotsConfluent HUVEC, seeded on fibronectin, were treated with TP (100 ng/ml), VEGF (10 ng/ml), or 2dR (1 µM) for up to 4 h. Monolayers were washed twice in PBS containing 1 mM phenylmethylsulfonyl fluoride and scraped into immunoprecipitation lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% (v/v) Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mg/ml leupeptin). Cell suspensions were incubated on ice for 30 min and clarified by centrifugation. For immunoprecipitation, protein content was determined, and 300 µg of total protein was incubated overnight at 4 °C with protein G-agarose beads coated with saturating amounts of antibodies to integrins 3,
v
3, and
5
1 (Chemicon International, CA) or p125FAK (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The resulting immune complexes were recovered after centrifugation by boiling for 10 min in SDS-PAGE loading buffer.
For immunoblotting, aliquots of whole cell lysates (30 µg) or isolated immunocomplexes were separated by SDS-PAGE under reducing conditions using 10% polyacrylamide gels. Proteins were transferred onto polyvinylidene difluoride membrane and analyzed by immunoblotting as previously described (28) using antibodies against phosphotyrosine, FAK, integrin 5 (Santa Cruz Biotechnology), vinculin (Sigma), integrin
1 (Chemicon), and FAK phosphorylated on various tyrosine residues (including Tyr397, Tyr407, Tyr576, Tyr861, and Tyr925) (Biosource International). Antibodies against
-tubulin and unphosphorylated FAK were used to control for loading when using whole cell lysates, whereas antibodies against integrins
1,
3, and
3 were used to quantitate protein loading for the immunoprecipitation reactions.
Immunofluorescent Staining for Focal Adhesion ComplexesHUVEC were seeded on Fn and stimulated with TP (100 ng/ml), 2dR (1 µM), VEGF (10 ng/ml), or media (control) for 4 h. HUVEC were fixed with 4% paraformaldehyde in PBS for 10 min and rendered permeable by incubation with 0.1% Triton X-100 in PBS for 20 min. Cells were washed three times with 1% bovine serum albumin in PBS (PBS-B) and blocked with 3% preimmune goat serum in PBS-B for 90 min at room temperature. For staining focal adhesions, cells were incubated with antibodies against vinculin (Sigma), FAK (Santa Cruz Biotechnology), or integrin 5
1 (Chemicon) for 1 h. Staining was detected with either Cy3-or FITC-conjugated second antibodies, and images were collected with a Bio-Rad MRC 600 krypton/argon laser-scanning confocal microscope on a Nikon Eclipse 200 microscope with a x60 numerical aperture 1.4 planapo infinity corrected objective. Controls were imaged to ensure that there was no background fluorescence and no cross-talk from the FITC channel to the Cy3 channel. For co-localization analysis, Cy3 and FITC images were overlaid using Photoshop 6.0 imaging software (Adobe Systems). The analysis of focal adhesion size and number was performed using automated image analysis software (Scion Image).
Flow Cytometric Analysis of Cell Surface Integrin ExpressionConfluent HUVEC monolayers on fibronectin-coated plates were treated with media alone (control), TP (100 ng/ml), VEGF (10 ng/ml), or 2dR (1 µM) for up to 4 h. Cells were washed twice with PBS and harvested using cell dissociation solution. Cells were suspended in PBS-B for 30 min at 4 °C. At the end of the incubation, antibodies to integrin 5
1 or
v
3 (5 µg/ml; Chemicon) were added. Negative controls were stained with preimmune mouse serum. Cells were incubated with antibodies for 90 min at 4 °C and washed three times with PBS-B before incubation with an FITC-conjugated secondary antibody (1:100 in PBS-B) for 60 min. Analysis of integrin surface expression was performed on a flow cytometer using an argon ion laser (
ex 488 nm). Data are expressed as-fold change in mean fluorescence intensity (Gm) of stimulated HUVEC compared with control unstimulated HUVEC.
Statistical AnalysisData were pooled, and statistical analysis was performed using the Mann-Whitney U test.
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RESULTS |
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The simplest, although not exclusive, explanation for these observations is that TP and VEGF utilize the same or substantially overlapping pathways to induce HUVEC migration. These pathways are not likely to be identical in all aspects, however, as VEGF acts by binding to a family of well defined, high affinity cell surface receptors with which TP most likely does not interact with in a similar manner.
TP, 2dR, and VEGF All Induce Focal Adhesion Formation and FAK Phosphorylation in HUVEC in VitroWhereas the effects of VEGF on EC are well described, the signaling mechanisms induced by TP/2dR to elicit a migratory response are undocumented. Thus, we examined the signaling pathways used by VEGF to compare them with those of TP/2dR. One of the primary mechanisms used by VEGF to induce migration is the activation of integrins and their respective signaling pathways. We first examined whether TP/2dR could enhance focal adhesion formation in HUVEC. Focal adhesion content of EC monolayers stimulated for 4 h with TP (100 ng/ml), 2dR (1 µM), or VEGF (10 ng/ml) was examined using vinculin immunostaining (Fig. 2). Focal adhesions in HUVEC in media alone (control) were few in number and small in size. Focal adhesion formation was significantly induced by treatment with either TP or 2dR and was equivalent to the induction by VEGF (Fig. 2A). In all cases, the number of focal adhesions/cell was increased 3.5-fold, and the average size of the focal adhesions was increased 2-fold in HUVEC treated with the chemotactic agents (Fig. 2, B and C).
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Since FAK is important for the formation of focal adhesions by multiple growth factors (6), we assessed whether FAK Tyr phosphorylation, as a marker of activation, was elevated in response to TP/2dR. FAK was immunoprecipitated from lysates of HUVEC stimulated with TP (100 ng/ml), VEGF (10 ng/ml), or 2dR (1 µM), and the levels of Tyr phosphorylation were examined by immunoblotting (Fig. 3A). Total Tyr phosphorylation of FAK was stimulated by all factors with similar kinetics. For each stimulus, FAK phosphorylation was increased at 1 h and maximally stimulated at 4 h, and phosphorylation persisted for 24 h (Fig. 3A). FAK can be phosphorylated on multiple Tyr residues, and we next defined which residues were involved using site-specific antibodies. As can be seen in Fig. 3B, TP and 2dR induced significant phosphorylation of residues Tyr397, the autophosphorylation site, and Tyr925. VEGF also induced phosphorylation of these sites. In contrast, TP and 2dR suppressed the phosphorylation of Tyr576 of FAK, which remained unaffected by VEGF stimulation. Thus, TP and 2dR appear to activate FAK to regulate focal adhesion formation and enhance endothelial cell migration.
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TP-and VEGF-stimulated HUVEC Migration Was Optimal on Fibronectin and Mediated through Two Distinct Integrins The observed increase in focal adhesion formation and FAK activation by TP and 2dR was strong evidence that these agents activated signaling pathways that regulate cell attachment and migration. Focal adhesions and, in particular, their integrin constituents are the point at which extracellular and intracellular events interface. Integrin heterodimers mediate the attachment of cells to different extracellular matrix components via their extracellular domains, with the attachment to each matrix protein mediated through a specific subset of integrins (29). To begin to explore the nature of the activation of these processes, we next determined the matrix protein(s) on which the response of HUVEC to TP and 2dR was optimal. Transwell inserts were coated with either fibronectin, vitronectin, thrombospondin, or gelatin (10 µg/ml), and HUVEC migration in response to TP, 2dR, and VEGF was examined (Fig. 4). Stimulation of HUVEC migration with TP was optimal on fibronectin, compared with the other matrix proteins examined. Whereas the induction of migration by VEGF was also maximal on fibronectin, in contrast to TP, optimal migration by VEGF was also observed when HUVEC were plated on vitronectin.
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HUVEC contain a distinct complement of integrin receptors. Of the 18 and 11
integrin subunits identified to date, HUVEC express
subunits
2,
3,
5,
6,
V,
L,
M, and
and
subunits
1,
3,
5, and
(9, 30). Using neutralizing antibodies against individual integrin subunits, chemotaxis in response to TP on fibronectin-coated inserts was found to be dependent upon the integrins
5,
V,
1, and
3 but not integrins
2,
3, and
6 (Fig. 5A). The migration of VEGF was only sensitive to inhibition by
V and
3 antibodies, and in contrast to TP, it was not inhibited by antibodies to
5 or
1 (Fig. 5A). Of the integrins known to mediate attachment to fibronectin, HUVEC potentially contain
5
1,
v
3,
V
1, and
3 heterodimers, of which
5
1 and
V
3 would be predicted to be involved in mediating the chemotactic effects. Using heterodimer-specific antibodies, we confirmed the data obtained using antibodies to individual integrin subunits by observing that the migration of HUVEC in response to TP (100 ng/ml) was inhibited by antibodies to either
5
1 or
V
3 integrins (Fig. 5B). As expected, chemotaxis in response to 2dR was inhibited by antibodies to the same integrins. In contrast, VEGF-induced migration was only sensitive to the inhibition of
V
3 (Fig. 5B).
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TP and 2dR Stimulated Increased Association of Integrins 5
1 and
v
3 with FAK and VinculinTo further substantiate the involvement of these integrins in TP/2dR- and VEGF-induced migration, we examined the extent to which they became associated with the forming focal adhesions that were observed in Fig. 2. The appropriate integrin heterodimers (either
5
1,
V
3, or
3) were immunoprecipitated from lysates of HUVEC that had been plated on fibronectin and treated with either TP, 2dR, or VEGF for up to 4 h. The resulting complexes were subject to PAGE and were probed for the presence of FAK and vinculin, two components of the focal adhesion complex. In Fig. 6, it can be seen that stimulation of HUVEC with TP or 2dR resulted in an increased association of p125FAK and vinculin with
5
1 and
v
3 (Fig. 6, A and B, respectively). The association was maximal at 1 h and persisted for up to 4 h. Thus, the association of FAK and vinculin with the integrins preceded the onset of migration, which was not detectable before 2 h and was maximal at 5 h (data not shown). However, VEGF increased the association of p125FAK and vinculin with only
v
3 (Fig. 6, A and B). None of the stimuli induced significant association of either focal adhesion components with the
3 integrins (Fig. 6C). These data were consistent with the migration data obtained in the presence of integrin-blocking antibodies, which showed the requirement of these integrins for TP/2dR-mediated chemotaxis (Fig. 5). The data further demonstrated that TP/2dR activates the signaling pathways associated with the same subset of integrin receptors that are important for TP/2dR-mediated HUVEC chemotaxis.
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To confirm that the 2dR/TP-induced association of 5
1 with FAK and vinculin occurred in vivo in intact cells and to determine whether it was associated with focal adhesion formation in response to 2dR/TP, we used immunofluorescent analysis and confocal microscopy to examine the co-localization of
5
1 and FAK. HUVEC, plated on fibronectin, were incubated with either VEGF (10 ng/ml), TP (100 ng/ml), or 2dR (1 µM) for 1 h and immunostained for FAK and
5
1 with Cy3- and FITC-labeled antibodies (Fig. 7). Integrin
5
1 and p125FAK were observed to co-localize in focal adhesions of HUVEC treated with TP and 2dR but not VEGF. These data were consistent with our studies on HUVEC migration utilizing neutralizing antibodies (Fig. 5) and the immunoprecipitation experiments (Fig. 6).
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Having established that integrins 5
1 and
v
3 were required for migration in TP/2dR-stimulated cells on fibronectin and that TP/2dR stimulated the phosphorylation of FAK and its association with
5
1 and
v
3, we next wanted to link these components of TP/2dR action by demonstrating that these same integrins were required for TP/2dR-induced signal transduction, as mediated via FAK phosphorylation. TP, 2dR, and VEGF-induced cells were pretreated with neutralizing antibodies to integrins
5
1 and
v
3, in a manner analogous to their use in the migration assay shown in Fig. 5. Neutralizing antibodies to either
5
1 or
v
3 abrogated the Tyr397 phosphorylation of FAK in response to TP/2dR (Fig. 8, lanes 3, 4, 7, and 8), demonstrating that the function of both integrins was required for TP/2dR-stimulated FAK phosphorylation and, presumably, focal adhesion formation. Thus, inhibition of the function of either integrin abrogated both TP/2dR-signaling and, as shown previously, subsequent migration. As predicted from our data, the phosphorylation of FAK in response to VEGF stimulation was only sensitive to the inhibition of
v
3 (Fig. 8, lanes 11 and 12). No inhibition occurred with either nonspecific mouse IgG or an antibody to integrin
3.
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TP and 2dR Increased 5
1 Integrin Cell Surface ExpressionThe formation of focal adhesions is a multistep process in which the initial lateral clustering of integrins promotes the reorganization of actin filaments into large stress fibers, which subsequently causes further integrin clustering (4). In addition to their clustering at the cell membrane, other changes accompany integrin activation, including increases in their overall cellular expression, dynamic changes resulting in their increased abundance and/or persistence at the cell surface, and changes in their affinity as a consequence of conformational alterations (4, 9). A stimulation of clustering of
v
3 and
5
1 could account for the increases in focal adhesion size observed with TP/2dR, but it would not necessarily explain the observed increase in the number of focal adhesions per cell. We sought to examine the cell surface expression of
5
1 and
V
3 integrins on HUVEC stimulated with TP, 2dR, or VEGF. All three chemotactic agents induced a 1.5-fold increase in
5
1 surface expression at 1 h, which was enhanced to a 3-fold increase at 4 h (Fig. 9B). The 3-fold increase in the expression of
5
1 correlated well with the 3-fold increase in the number of focal adhesions observed in TP/2dR-treated HUVEC observed in Fig. 2. However, no change in the expression of
v
3 was observed (Fig. 9A). Surprisingly, neither
5 nor
1 total cellular protein levels were increased with TP, 2dR, or VEGF treatment for 4 h (Fig. 9C). Taken together, these data suggest that the initial (i.e. up to 1 h) TP/2dR-induced formation of focal adhesions was due to the clustering of integrins already present at the cell surface, without apparent changes in the levels of their expression at the cell surface or in their total cellular levels. This was subsequently followed (at 4 h) by an increase in
5
1 integrins on the cell surface, which also occurred in the absence of detectable changes in total cellular
5
1 levels. It would be of interest to further define the functional and other changes the
5
1 integrins undergo in response to their stimulation by TP/2dR.
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DISCUSSION |
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TP and VEGF appeared to differ, however, in the degree to which specific integrins participated in these cellular events. For example, in our experiments, VEGF-mediated HUVEC migration was blocked by an V
3 integrin antibody but not by an
5
1 antibody, in agreement with previous reports (37). Our experiments with VEGF were also consistent with previous studies, which have shown that the
v
3 integrin is directly involved in embryonic, physiologic, and tumor angiogenesis (11, 38, 39). Expression of
v
3 is much higher in blood vessels undergoing angiogenesis compared with resting vessels and can be induced by hypoxia; inhibitors of
v
3 block angiogenesis in the CAM assay without affecting preexisting blood vessels (40, 41, 42). VEGF can induce a severalfold increase in both mRNA and surface expression of
V
3 integrin; other integrin subunits can also be induced by VEGF, with the specific types induced reflecting the extracellular matrix upon which the cells are plated (38, 39, 42). In addition to increased mRNA levels, integrin activity is also mediated by cycling between cytoplasmic compartments and the cell surface (43). Interestingly, although
v
3 inhibitors reduced tumor angiogenesis in mouse models, the opposite was observed in
v
3-null mice, which exhibited extensive developmental angiogenesis and showed enhanced pathological angiogenesis and more rapid tumor growth than wild type mice (44, 45). These findings raised questions as to the degree to which
v
3 plays a central role in normal and tumor angiogenesis (44, 45).
Our studies found that HUVEC migration induced by TP was maximal on fibronectin, and in contrast to VEGF, migration was blocked by antibodies to either the v
3 or
5
1 integrins. Consistent with this was our observation that TP and 2dR increased the association of FAK and vinculin with both
v
3 or
5
1 integrins in focal adhesions, whereas VEGF only increased the association of FAK and vinculin with
v
3. It has been shown that attachment of HUVEC and other cells to fibronectin occurs through integrin
5
1, and antibodies to
5
1 blocked basic fibroblast growth factor-, tumor necrosis factor-
-, and IL-8-stimulated angiogenesis (37, 46, 47). Gene knockout studies with fibronectin- or
5
1-deficient mice as well as antibody inhibitory studies have shown that both molecules were required for pre- and postnatal vasculogenesis and angiogenesis in mice, including tumor growth in syngeneic mice (37, 48, 49, 50, 51). Expression of fibronectin and
5
1 were found to be increased on blood vessels of tumors and in growth factor-stimulated vessels (37). Taken together, these studies suggest that
5
1 plays an important role in EC migration and angiogenesis.
Despite the differences noted above, data suggest that 5
1 and
v
3 influence the same angiogenesis pathways (37). Although we found that both
v
3 and
5
1 antibodies were inhibitory to TP-mediated chemotaxis, the combination of the two did not produce additive inhibition, suggesting that
v
3 and
5
1 do not act independently (data not shown). Since it has been reported that the
v
3 integrin as well as the
5
1 integrin can mediate cell interactions with fibronectin (52, 53), one explanation for our observations is that both integrins are activated separately by TP/2dR, and both are required for the TP/2dR effect on HUVEC migration. Alternatively, there are recent studies that suggest that there is cross-talk between different integrin heterodimers within the same cell in which the occupancy of one integrin by its ligand modulates the functions of other integrins (37, 52, 53, 54, 55, 56). For example,
v
3 integrin was able to inhibit the
5
1-mediated cell migration of human embryonic kidney cells and the phagocytosis of human macrophages, in both instances on cells plated on fibronectin (52, 54, 55), whereas other studies demonstrated that
5
1 regulates the function of integrin
v
3 on HUVEC migration in vitro and angiogenesis in vivo (56). Integrin cross-talk depends upon the actions of signal transduction pathways, suggesting that cross-talk is not solely a result of integrin interactions at the cell surface (54, 55, 56). This could provide a basis for understanding how the induction of migration by TP/2dR in our studies could be blocked by an antibody to
v
3 without there being a concomitant increase in the cell surface expression of
v
3 in TP/2dR-treated cells.
FAK is activated by phosphorylation at potentially six tyrosines, and it has one site (Tyr397) for autophosphorylation, which must occur before phosphorylation on its other tyrosine residues can occur (12, 57). Tyr397 is not strictly an autophosphorylation site, since it can also be phosphorylated by Src (14). This creates a binding site for the Src homology 2 domain, which allows for the recruitment of Src kinase and other protein-tyrosine kinases, which then can phosphorylate other tyrosine residues in FAK, and other components of the focal adhesion, including the cytoskeleton proteins paxillin and tensin, and a docking protein, p130cas (8, 12). The tyrosine phosphorylation of FAK occurs in response to a variety of different extracellular and intracellular stimuli, both integrin- and nonintegrin-dependent, including polypeptide growth factors, extracellular matrix proteins, bioactive lipids, and oncogenes, and can be inhibited by protein kinase C inhibitors and agents that disrupt actin (8, 58, 59). Data further suggest that different stimuli can induce phosphorylation on distinct tyrosine residues, linking specific phosphorylations with subsequent biological events (60).
In the present study, both VEGF and TP/2dR induced persistent FAK Tyr397 phosphorylation, indicating that TP/2dR probably activates one or more downstream signaling pathways. That the TP/2dR-induced FAK phosphorylation was blocked by neutralizing antibodies to the same integrins that blocked cell migration confirms the importance of these cell surface components to TP/2dR signaling pathways. The extents of FAK phosphorylation at the other tyrosine residues examined were similar with VEGF and TP/2dR treatment, except for a difference in Tyr576 phosphorylation of FAK, a site located within the kinase domain. Tyr576 is a c-Src phosphorylation site and is intrinsically linked to the elevated kinase activity observed upon FAK "activation" (14). FAK phosphorylated on Tyr397 complexes to multiple proteins through Src homology 2 domains, including Src family kinases (61). It has been proposed that many downstream targets of FAK are actually substrates of associated Src kinases and that FAK serves mainly as a scaffold for assembly of enzyme and substrate. Interestingly, Tyr576 was dephosphorylated in response to TP and 2dR under conditions in which focal adhesion formation was dramatically enhanced. If this dephosphorylation proves to be indicative of the lack of activation of the c-Src pathway by TP/2dR, this system may aid in elucidating the role of the kinase activity of FAK in the regulation of focal adhesions separate from associated Src kinase activities and its cross-talk with the Src signaling cascade. Moreover, decreased phosphorylation of Tyr576 may indicate alternate pathways of FAK activation between TP/2dR and VEGF. Tyr576 also lies within the catalytic domain of FAK, and studies suggest that its phosphorylation might alter the conformation of the catalytic domain and modulate the binding and phosphorylation of protein substrates (12, 14). If this modulation affected the specificity of the protein substrates recognized by FAK, the difference observed in Tyr576 between VEGF and TP/2dR could result in variations in the downstream signaling pathways being activated.
Whereas the studies reported here begin to elucidate the signal transduction pathways that are activated by TP/2dR in HUVEC, the proximal site of the action of 2dR within the cell remains unknown. Cell surface receptors for ribose and related sugars in bacteria are known and have been shown to mediate chemotaxia (62, 63); thus, it is possible that a similar receptor exists in eukaryotic cells. Other effects of 2dR on cells, including the modulation of apoptosis, have been reported to be mediated by effects on glutathione, caspase 3, or mitochondrial function, and examination of their relevance to the effect of 2dR on EC migration would be useful (64, 65).
Mild reducing conditions have been shown to be sufficient to bring about integrin activation, the process by which the integrin ligand binding site is uncovered and its affinity increased (66). 3 integrins, especially
IIb
3, were especially sensitive to activation by reducing conditions, since they contain a redox site within the extracellular domain, and sustained activation of
3-containing heterodimers required reduction of extracellular free sulfhydryls and disulfide exchange (67, 68). Since 2dR is a strong reducing sugar, it theoretically could act as a physiological redox agent that regulates cellular functions through, for example, an effect on disulfide bonds at key integrin cysteine residues (64, 66, 67). FAK has also recently been shown to be sensitive to redox conditions and can be activated by changes in redox state (68). Such direct activation of integrins and/or FAK could elicit the enhanced focal adhesion formation observed by treating HUVEC with 2dR/TP. If this hypothesis is correct, the question remains as to whether and how 2dR selectively reduces the
5
1 and
v
3 heterodimers, in preference to other
1-and
3-containing heterodimers.
Cell motility and cell matrix attachment require the assembly of focal adhesion complexes with actin stress fibers, processes that are regulated by the Rho family of GTPases (5). Other regulators of HUVEC migration, such as sphingosine 1-phosphate, have been shown to activate integrins and cause the formation of focal contacts in a Rho-dependent manner, and this was attributed to the ability of sphingosine 1-phosphate to rapidly activate Rho activity (69). It would be of interest to determine the effects of 2dR on these aspects of HUVEC migration.
In conclusion, by demonstrating an effect on focal adhesions and integrins, these studies provide the first insights into the mechanism by which the angiogenic factor TP activates endothelial cell migration. Despite the likelihood that they have distinct "receptors," our data suggest that TP and VEGF have some overlap in the signaling pathways by which they activate HUVEC migration. The molecular actions of TP/2dR and VEGF were not identical, however, with the most prominent difference being the contribution of the 5
1 integrin to the actions of TP/2dR. In addition to their effects on EC migration and angiogenesis, TP and 2dR have been shown to modulate other cellular functions, including apoptosis, inflammation, neuronal survival, and neurite outgrowth (65, 70). The extent to which the pathways described in this paper contribute to these cellular actions would be important to explore.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Oncology, Albert Einstein Cancer Center, Montefiore Medical Center, 111 E. 210th St., Bronx, NY 10467. Tel.: 718-920-4015; Fax: 718-882-4464; E-mail: eschwart{at}aecom.yu.edu.
1 The abbreviations used are: EC, endothelial cell(s); 2dR, 2-deoxyribose; FAK, focal adhesion kinase; HUVEC, human umbilical vein endothelial cell(s); TP, thymidine phosphorylase; VEGF, vascular endothelial growth factor; ECM, extracellular matrix; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate.
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REFERENCES |
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