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(Circulation Research. 2004;94:1227.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Urokinase-Type Plasminogen Activator Receptor Is Involved in Mediating the Apoptotic Effect of Cleaved High Molecular Weight Kininogen in Human Endothelial Cells

Dian J. Cao, Yan-Lin Guo, Robert W. Colman

From the Sol Sherry Thrombosis Research Center (D.J.C., Y.-L.G., R.W.C.), Department of Medicine (R.W.C.), Temple University School of Medicine, Philadelphia, Pa.

Correspondence to Robert W. Colman, MD, Temple University School of Medicine, 3400 N Broad St, Philadelphia, PA 19140. E-mail colmanr{at}temple.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cleaved high molecular weight kininogen (HKa) has been shown to inhibit in vivo neovascularization and induce apoptosis of endothelial cells. We have shown that HKa-induced apoptosis correlated with its antiadhesive effect and was regulated by extracellular matrix (ECM) proteins. In this study, we identified the urokinase-type plasminogen activator receptor (uPAR) as a target of HKa activity at the endothelial cell surface. Anti-uPAR antibodies blocked the apoptotic effect of HKa. Further studies revealed that uPAR formed a signaling complex containing integrin _vß₃ or ₅ß₁, caveolin, and Src kinase Yes in endothelial cells. HKa physically disrupted the formation of this complex in a manner that paralleled its apoptotic effect. For the first time, our results provide a mechanistic explanation for the previous observation that HKa selectively induces apoptosis of endothelial cells grown on vitronectin, but not cells grown on fibronectin. These data also resolve the controversial role of uPAR in mediating the apoptotic and antiadhesive activities of HKa.

Key Words: urokinase-type plasminogen activator receptor • high molecular weight kininogen • endothelial cells • apoptosis • angiogenesis

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Angiogenesis, the formation of new capillaries from existing blood vessels, is a cellular event crucial for both physiological and pathological processes such as embryogenesis and cancer development,¹ respectively. A positive balance in favor of angiogenic factors leads to new blood vessel formation, whereas the predominance of angiogenic inhibitors will switch the equilibrium to vessel quiescence or vessel regression.^2–5

High molecular weight kininogen (HK) is a plasma protein that was first identified as a precursor of the bioactive peptide bradykinin. We now recognize that HK is a multifunctional protein that plays important roles in many pathophysiological processes, such as fibrinolysis, thrombosis, and inflammation.⁶ HK is a 120-kDa single-chain glycoprotein consisting of six domains (designated as D1 to D6, respectively) with each having distinct functions.⁷ After proteolytic cleavage of HK by kallikrein and release of bradykinin contained within D4, the remaining portion of the molecule, HKa, undergoes major conformational changes that lead to a greater surface exposure of the D5 region. As a result, HKa acquires new properties. In comparison with HK, HKa shows an increased antiadhesive effect due to domain rearrangements.^8,9 HK specifically and reversibly binds to endothelial cells in a Zn²⁺-dependent manner. Thus, the endothelial cell surface is an important site for the generation of both bradykinin and HKa, each of which regulates the functions of endothelial cells. Although the involvement of bradykinin in the regulation of many physiological and pathophysiological processes has been intensively studied, the functional implications of the generation of HKa are incompletely understood. Recent studies from our laboratory and other investigators indicate that HKa may act as a naturally occurring angiogenic inhibitor.^10,11 Interestingly, it has recently been shown that bradykinin stimulates angiogenesis.^12,13 Therefore, HK is a precursor of both an angiogenic stimulator (bradykinin) and an angiogenic inhibitor (HKa).

After our initial discovery of HKa and its domain 5 as angiogenic inhibitors,¹⁰ we have further shown that HKa inhibits human umbilical vein endothelial cell (HUVEC) proliferation and induces apoptosis,^14,15 which together may represent a critical step of their antiangiogenic activity. In our most recent study,¹⁶ we demonstrated that the apoptotic effect of HKa is related to its antiadhesive activity and its ability to disrupt signaling pathways required for cell adhesion. Furthermore, the apoptotic effect of HKa is highly regulated by interactions with different extracellular matrix proteins; it strongly induces apoptosis of endothelial cells grown on vitronectin (Vn) but has no significant effect on the viability of cells grown on fibronectin (Fn).^16,17 However, a fundamentally important unanswered question is how the effects of HKa and D5 are mediated at the endothelial cell membrane.

In the present study, we provide substantial evidence to support the hypothesis that urokinase-type plasminogen activator receptor (uPAR), a glycophosphatidylinositol (GPI)-anchored cell surface protein that plays an important role in cell adhesion and signal transduction,¹⁸ is a target of HKa action at the endothelial cell surface. We have previously shown that HKa physically binds to uPAR in endothelial cells.¹⁹ We now demonstrate the specific downstream events: HKa disrupts the signaling complex by decreasing uPAR-integrin and/or uPAR-caveolin association. Thus, this study constitutes the first report that provides a mechanistic explanation for the previous observation that HKa selectively induces apoptosis of endothelial cells grown on Vn but not cells grown on Fn.

	Materials and Methods

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Apoptosis Analysis
Apoptotic cell death induced by HKa has been characterized by a combination of DNA and nuclear fragmentation and annexin V staining, which have been described in our previous studies.^14,20 The present study used nuclear fragmentation analysis with Hoechst 33285 staining to identify apoptotic cells at the cellular level. HUVECs were seeded on Vn- (0.053 mmol/L) or Fn- (0.023 mmol/L) coated coverslips in EGM. After the cells attached to the coverslips, the medium was changed to EBM (EGM minus growth factors and FBS) plus 0.59 µmol/L bFGF in the presence or absence of 100 nmol/L HKa. P-D2D3 (the anti-serum against the D2 and D3 domains of uPAR) or the same amount of control preimmune serum (PIS) was added to the medium at a concentration of 30 µL/mL. After incubation for 48 hours, cells were analyzed for apoptosis according to methods described previously.¹⁴

Apoptosis was quantified by a Cell Titer Aqueous kit (Promega) as described previously.¹⁴ The cells were incubated with bFGF (0.59 µmol/L) in the presence or absence of 100 nmol/L HKa. Monoclonal anti-uPAR antibodies (E33, E174, and E180) or control mouse IgG, P-D2D3, or rabbit PIS, were added to the medium at different concentrations as indicated. Apoptosis assay was also performed using anti-cytokeratin-1 and anti-gC1qR (clone 74.5.2) antibodies at concentrations indicated.

Immunoprecipitation and Immunoblot Analysis
HUVECs were plated on cell culture dishes coated with Vn or Fn in EGM. After the cells attached to the dishes, the culture medium was changed to EBM plus 0.59 µmol/L bFGF. Cells were treated with or without 100 nmol/L HKa for 25 to 30 hours at 37°C. After treatment, the cells attached to the dishes and those from the medium were collected and lysed with modified M-per mammalian cell protein extraction buffer (Pierce) containing 5 mmol/L Ca²⁺, 2 mmol/L Mg²⁺, and protease inhibitor cocktail (Chemicon International). After incubation for 10 minutes at room temperature, cell lysates were collected by centrifugation at 21 000g for 15 minutes at 4°C.

The complex formation of uPAR with other signaling molecules was determined by immunoprecipitation (IP) according to the methods described by Wei et al²¹ with some modifications. Cell lysate was incubated with antibodies to _vß₃, ₅ß₁, uPAR, or PECAM-1 followed by incubation of protein A/G beads. The immunoprecipitates were subjected to SDS-PAGE under reduced conditions, and immunoblot analysis was performed as described.¹⁴

For a description of other methods, see the expanded Materials and Methods available in the online data supplement at http://circres.ahajournals.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Anti-uPAR Antibodies Block Apoptotic Effect of HKa in HUVECs
A previous investigation from this laboratory has shown that HKa directly binds to uPAR because an anti-uPAR D2D3 (domains 2 and 3) antibody blocked the binding of HKa to endothelial cells.¹⁹ We postulated that uPAR may be involved in mediating the apoptotic effect of HKa. To test this hypothesis, we used P-D2D3, an antibody raised against the D2D3 region of uPAR, to examine if this antibody can block the apoptotic effect of HKa. HUVECs were cultured on Vn or Fn and incubated with HKa in the presence or absence of P-D2D3. We choose to use 100 nmol/L HKa based on our previous report.¹⁶ As shown in Figure 1, bFGF sustained the viability of HUVECs within a 48-hour incubation period, with cells displaying normal nuclei morphology (Figure 1A, bFGF). HKa caused condensation/fragmentation of nuclei (apoptotic nuclei) in many cells grown on Vn (Figure 1A, bFGF+HKa). The effect of HKa was reversed by P-D2D3 (Figure 1A, bFGF+HKa+P-D2D3), whereas preimmune serum (PIS) had no effect (Figure 1A, bFGF+HKa+PIS). Consistent with our previous observation that HKa did not induce apoptosis of HUVECs grown on Fn,¹⁶ neither HKa nor P-D2D3 and PIS exhibited an apparent effect on cell viability when cells were grown on Fn (Figure 1B). P-D2D3 alone did not show apparent effect on the viability of cells grown on Vn and Fn (data not shown).

View larger version (46K): [in this window] [in a new window]   Figure 1. The anti-uPAR D2D3 antibody blocks apoptosis induced by HKa. HUVECs grown on Vn (A) or Fn (B) were treated with 100 nmol/L HKa in the absence or presence of P-D2D3 (30 µL/mL) (bFGF+HKa, bFGF+HKa+P-D2D3, respectively). After 48 hours of treatment, cells were stained with Hoechst 33258. Typical nuclear morphology of apoptotic cells is indicated with white arrows.

We have previously shown that apoptosis of endothelial cells induced by HKa was largely due to its antiadhesive effect.¹⁶ As a result, most of late apoptotic cells are released into the medium. The number of viable cells still attached to the culture plate after treatment, which is inversely related to the number of apoptotic cells in the medium, was quantified to reflect HKa-induced apoptosis. When cells were grown on Vn, viable cells left in the HKa-treated cells was 30% of the control (in the absence of HKa), representing 70% of the cells death from apoptosis. The apoptotic effect of HKa under this condition was defined as 100%. As shown in Figure 2A, the apoptotic effect of HKa was significantly inhibited by antibody P-D2D3 in a concentration-dependent manner, whereas PIS had no effect (Figure 2A). The same experiments were performed using three monoclonal anti-uPAR antibodies (E33, E174, and E180) at a concentration of 10 µg/mL. All of these antibodies significantly blocked the apoptotic effect of HKa but with different potency (Figure 2B). Because both the polyclonal and monoclonal antibodies were made against D2D3 domains of uPAR, it is likely the D2D3 domains of uPAR are the targeted regions by HKa. A nonrelevant mouse IgG (m-IgG) as a control for the monoclonal antibodies did not affect the effect of HKa (Figure 2B). Similar experiments were also performed using anti-cytokeratin-1 and anti-gC1qR antibodies (Figure 2A, Ab1 and Ab2, respectively). The results showed that the apoptotic effect of HKa was blocked by anti-cytokeratin-1, but not by anti-gC1qR antibodies (Figure 2A). Consistent with the results presented in Figure 1B, HKa did not induce apoptosis of endothelial cells grown on Fn (data not shown).

View larger version (27K): [in this window] [in a new window]   Figure 2. Effect of anti-uPAR antibodies on HKa-induced HUVEC apoptosis. HUVECs were treated with or without 100 nmol/L HKa for 48 hours. HKa-treated cells were incubated with increasing amounts (0, 10, and 30 µL/mL) of P-D2D3 (A) or with E33, E174, and E180 at a concentration of 10 µg/mL (B). Same amount of PIS was used as controls for P-D2D3. Nonspecific mouse IgG (m-IgG) served as controls for E33, E174, and E180. Similarly, HKa-treated cells were incubated with anti-cytokeratin-1 (Ab1, 30 µg/mL) or anti-gC1qR (Ab2, 10 µg/mL) antibodies (A). Apoptosis in bFGF+HKa-treated groups is expressed as 100% (control, C). Results are mean±SEM from 3 to 6 individual experiments. *P<0.01 vs control.

uPAR Forms Complexes With Integrins and Caveolin
Because uPAR is a GPI-anchored cell surface protein, the signal received by uPAR must be transduced through its interaction with other signaling molecules. uPAR has been shown to interact with integrins and caveolin in other cell types.^21–24 We then tested whether this is the case in endothelial cells. Cell lysates from HUVECs were incubated with anti-_vß₃ or anti-₅ß₁ antibodies. As shown in Figures 3A and 3B, both uPAR and caveolin were detected in the samples precipitated either by anti-_vß₃ or by anti-₅ß₁ antibodies, indicating that uPAR, _vß₃, or ₅ß₁, and caveolin form a complex in HUVECs grown on gelatin (Figure 3A, Gel), Vn (Figure 3B, Vn), or Fn (Figure 3B, Fn). The presence of integrin _vß₃ and ₅ß₁ was confirmed by probing the immunoprecipitates with anti-integrin _v or ß₁ subunits, respectively. Further results showed that the complex precipitated with anti-₅ß₁ antibody contained _v subunit (Figure 3C), whereas the complex precipitated with anti-uPAR antibody contained both _v and ß₁ subunits (Figure 3D). In a control experiment, cell lysate was incubated with anti-PECAM-1 antibodies. The immunoprecipitate contained abundant PECAM-1, as expected, but not uPAR (Figure 3E). These results indicate that uPAR form a complex that contains specific signaling components.

View larger version (29K): [in this window] [in a new window]   Figure 3. uPAR forms complexes with integrins vß3, 5ß1, and caveolin. A, Cell lysate from HUVECs grown on Gel was immunoprecipitated with anti-vß3 or anti-5ß1 antibodies (IP). Components of the immunoprecipitates were probed with the indicated antibodies (IB). B, Same experiments as described in A were performed using cell lysates from cells grown on Vn or Fn. C, Cell lysates from cells grown on Gel were immunoprecipitated with anti-5ß1 antibodies. Immunoprecipitates were probed for ß1 or v subunit. D, Cell lysates from cells grown on Gel were precipitated with anti-uPAR antibodies (E33) and then the immunoprecipitates were probed for uPAR and integrin v or ß1 subunits. E, Cell lysate prepared as described above was immunoprecipitated with an anti–PECAM-1 antibody and probed with antibodies against PECAM-1 and uPAR.

HKa Disrupts the Complex Formation Among uPAR, Integrin, and Caveolin
We have shown that HKa-induced apoptosis in endothelial cells is ECM protein dependent.¹⁶ HKa exhibited a maximum apoptotic effect on HUVECs grown on Vn, whereas its effect on cells grown on Fn was minimal. We further analyzed the effect of HKa on complexes formed by uPAR, _vß₃/₅ß₁, and caveolin in HUVECs grown on Vn and Fn. As shown in Figure 4A, HKa treatment reduced the amount of uPAR associated with _vß₃ to a similar extent in cells grown on Vn and Fn; however, the amount of caveolin in the complex was markedly reduced in cells grown on Vn compared with cells grown on Fn (Figure 4A). Figures 4B and 4C quantified the relative amount of uPAR and caveolin associated with _vß₃ determined by densitometry from three independent experiments. In Figure 4G, total cell lysates from control and HKa-treated groups before IP were probed with anti-caveolin and anti-uPAR antibodies. No significant differences in total amount of these proteins were detected among groups.

View larger version (46K): [in this window] [in a new window]   Figure 4. HKa dissociates uPAR and caveolin from integrin vß3 or 5ß1 in cells grown on Vn and Fn. Cell lysates from HUVECs grown on Vn or Fn were precipitated with anti-vß3 (A) or anti-5ß1 antibodies (D), and the immunoprecipitates were probed with the antibodies as indicated. Total cell lysates before IP were analyzed for actin to show equal amount of protein used for IP experiments. B, C, E, and F, Summarized results of the relative amount of uPAR and caveolin in the complexes quantified from 3 individual experiments by densitometry and are presented using relative units normalized to control (without HKa treatment). G, Cell lysates from control and HKa-treated groups before IP were subjected to SDS-PAGE and probed with anti-caveolin and anti-uPAR antibodies. *P<0.05 vs control.

In parallel experiments, cell lysates were incubated with anti-₅ß₁ antibodies as shown in Figure 4D. Interestingly, in cells cultured on Vn, HKa almost completely disassociated uPAR and caveolin from the complex precipitated with anti-₅ß₁ antibody. On the other hand, when cells were grown on Fn, although HKa also disassociates uPAR from ₅ß₁ to some extent, it had no apparent effect on the association of caveolin with ₅ß₁ (Figures 4D through 4F). Cell lysates before IP were also analyzed for actin. As shown in Figure 4A and 4D, the amount of actin was similar among groups, indicating that the same amount of proteins was used for IP experiments. HKa disassociates uPAR with a higher potency from the complex formed by ₅ß₁ than it does from the complex formed with _vß₃. HKa almost completely dissociates caveolin from both complexes in cells grown on Vn, indicating that disassociation of caveolin from the complex may play an important role in mediating the apoptotic and antiadhesive effects of HKa.

HKa Disrupts the Association of Src Kinase Yes From Integrin _vß₃-Assembled Complex
Src family tyrosine kinases are nonreceptor tyrosine kinases that have been reported to interact with caveolin and integrins.²³ Therefore, Src kinases are downstream effectors of integrin pathways in some cells.^24,25 To test whether this is the case in endothelial cells, we chose to examine the presence of Yes as a representative of this kinase family. The immunocomplex precipitated by anti-_vß₃ antibodies was probed with an anti-Yes antibody. Results shown in Figure 5A demonstrate that Yes was associated with _vß₃ in endothelial cells, and that HKa caused almost complete loss of Yes from the _vß₃ complex in cells grown on Vn, and to a lesser extent, in cells grown on Fn. In Figure 5B, a dose-response was performed regarding HKa-induced dissociation of Yes from the _vß₃ complex in cells grown on Vn. These data suggest that the HKa not only disrupts the uPAR-integrin-caveolin signal complex formation at the cell membrane, it also effectively blocks signal transduction at downstream uPAR-integrin signaling pathways.

View larger version (34K): [in this window] [in a new window]   Figure 5. Src kinase Yes is associated with vß3-uPAR-caveolin complex. A, Cell lysates prepared as described in Figure 4 were precipitated with anti-vß3, and the resolved precipitates were probed with anti-v and anti-Yes antibodies. B, HKa dose-response for the relative amount of Yes in the vß3-uPAR-caveolin complex in cells grown on Vn.

uPAR Mediates HUVEC Adhesion to Vn
Given the fact that Vn is a well-known adhesion molecule for many types of cells including endothelial cells and that uPAR is well-recognized adhesion receptor for Vn in epithelial cells²⁶ and leukocytes,²⁷ the contribution of uPAR-mediated cell adhesion in endothelial cells is not clear. A report by Kanse et al²⁸ indicated that uPAR is a major Vn binding protein. Surprisingly, uPAR-mediated cell adhesion was not detected in that study, an observation made from the indirect experiments using uPA as an uPAR ligand. However, it has been reported that uPAR mediates several cellular processes in a ligand (uPA)-independent manner.²⁹ In an effort to resolve this uncertainty in endothelial cells, we treated cells with PI-PLC to cleave uPAR from the cell surface. The result, as shown in Figure 6, demonstrated that uPAR is responsible for 50% of HUVEC binding to Vn under the conditions tested. PI-PLC treatment, however, was not able to significantly affect the cell adhesion on Fn (Figure 6).

View larger version (17K): [in this window] [in a new window]   Figure 6. PI-PLC treatment reduced HUVEC adhesion to immobilized Vn and Fn. HUVECs treated with or without PI-PLC were seeded on Vn- or Fn-coated 96-well plates in triplicates followed by further incubation at 37°C for 30 minutes. After washing, the attached cells were fixed, stained with 0.5% crystal violet, and counted (6 fields per well). Cell number in control group (without PI-PLC treatment) was set as 100%. *P<0.05 vs control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
HKa and its domain 5 (D5) have been shown to inhibit in vivo neovascularization in different animal models independently by this group¹⁰ and other investigators.¹¹ However, the signaling mechanism that mediates their activity has not been elucidated. For the first time, we demonstrate that uPAR, an adhesion and signaling receptor, is a target of HKa action at the endothelial cell surface. Together with our previously published results,¹⁶ we demonstrate that HKa can disrupt the uPAR-_vß₃/₅ß₁-caveolin-yes-FAK-paxillin signaling pathway at each level of these signaling components. The results described in this study represent major progress in our effort to characterize HKa as an angiogenic inhibitor.

Several endothelial cell membrane proteins that interact with HK or HKa have been identified, including uPAR,¹⁹ the receptor for the globular head of C1q (gC1qR),³⁰ and cytokeratin-1.^30,31 It has been shown that cytokeratin-1 binds to both uPAR and gC1qR, but uPAR and gC1qR do not bind to each other.³² A recent study revealed that uPAR and cytokeratin-1 colocalize on endothelial cells and may form a receptor complex that mediates HK binding and the subsequent release of bradykinin.³³ Among these proteins, uPAR, a GPI-anchored cell surface protein that contains three domains, is particularly relevant to cell adhesion and angiogenesis.^18,34 Although initially identified as a receptor for urokinase plasminogen activator (uPA) to localize proteolytic activity on the cell surface during invasion,^18,35 it is now known that uPAR also interacts with integrins at the cell membrane and binds to Vn in a uPA-dependent or independently manner.²⁹ Vn is a major provisional ECM protein and plays an important role in angiogenesis during vascular remodeling and vessel repair.³⁶ As a result of these interactions, uPAR acts as an adhesion receptor for Vn and transduces signals through integrin pathways.³⁷

The initial notion that uPAR may mediate the apoptotic effect of HKa came from our previous study,¹⁹ which showed that HKa binds to endothelial cells through D2 and D3 of uPAR. The binding of HKa to endothelial cells can be completely blocked by an anti-uPAR D2 and D3 antibody, or by soluble recombinant uPAR.¹⁹ We, therefore, postulated that uPAR could be a logical target of HKa for its antiangiogenic activity. However, a recent study¹¹ by Zhang et al indicated that none of the three antibodies against gC1qR, cytokeratin-1, or uPAR, which had previously been shown to inhibit HK or HKa binding to endothelial cells,^19,30,31 affected the ability of HKa to induce apoptosis of endothelial cells. Our examination of the other two antibodies was demonstrated in Figure 2A. We were able to show that the anti-cytokeratin-1 antibody blocked the apoptotic effect of HKa on cells grown on Vn. As demonstrated by Joseph et al,³² cytokeratin-1 forms a complex with uPAR. Thus, the antibody against cytokeratin-1 could interfere with the association of HKa with uPAR-cytokeratin-1 complex and block the apoptotic effect of HKa. We also performed similar experiments using the anti-gC1qR antibody. We were unable to detect any blocking effect of this antibody to HKa-induced cell apoptosis on Vn. This finding indicates that uPAR plays more significant role under our experimental condition in which Vn is used as a coating ECM protein and consistent with previous finding that gC1qR does not form a complex with uPAR.³²

Although the results from Zhang’s report argue against a role of uPAR in mediating the effect of HKa,¹¹ in collaboration with Chavakis et al,²⁷ we were able to show that HKa or D5 completely inhibited uPAR-mediated cell adhesion to Vn, thereby promoting cell detachment in U937 and uPAR-transfected BAF-3 cells. HKa or D5 failed to elicit a similar effect when cells were plated on Fn under the same conditions.²⁷ We recently demonstrated in endothelial cells that HKa and D5 exhibit dramatic apoptotic activity on the cells grown on Vn, but its effect on cells grown on Fn was minimal.¹⁶ These studies suggested that the effect of HKa and D5 depends on the nature of ECM proteins, and that uPAR could be a target of HKa but may require the presence of Vn. We now provide strong evidence to support this hypothesis. First, four different anti-uPAR D2/D3 antibodies blocked the apoptotic effect of HKa on cells grown on Vn but not on Fn. Secondly, we demonstrated that uPAR formed a signaling complex with _vß₃/₅ß₁, caveolin, and Yes kinase. Although this phenomenon and the functional implication of this signaling cluster formation has been studied in other cell systems,^21–24 it has not been reported in endothelial cells in previous studies. The most important finding of these experiments, however, is that HKa could disrupt the association of uPAR with these signaling components in a manner that parallels its antiadhesive and apoptotic effects. Although HKa also disrupted uPAR-₅ß₁/_vß₃ complex formation in cells on Fn to some extent, it did not result in cell detachment or apoptosis. This result could be explained by the fact that Fn is the ligand for at least 12 integrins, whereas Vn is a ligand for only four (_vß₁, _vß₃, _vß₅, and _IIbß₃).³⁸ The larger number of integrins binding to Fn may contribute to the resistance of cells to the antiadhesive effect of HKa on cells grown on Fn. This hypothesis may not only explain the ECM protein dependency of antiadhesive and apoptotic effect of HKa but also the failure of the anti-uPAR antibodies to block the apoptotic effect of HKa reported by Zhang et al,¹¹ where the experiment was conducted in cells grown on gelatin (in the absence of Vn).

We previously demonstrated that HKa directly binds to Vn.²⁷ The present study extends that report by demonstrating specific downstream events. The hypothesis that HKa binds to Vn and subsequently blocks cell adhesion to Vn does not contradict the results presented in the current study. Whether the upstream event is HKa binding to Vn and/or HKa competing with Vn for binding to uPAR, the downstream event might be the same. These two steps will finally lead to interference of uPAR/integrin interaction and subsequently endothelial cell apoptosis

Because ₅ß₁ is not a ligand of Vn, we are not certain how HKa treatment also caused disassociation of uPAR and caveolin from ₅ß₁ complex. Because uPAR can form complexes with several integrins, including ₃ß₁ and ₅ß₁, and _vß₃,²¹ it is possible that these complexes may consist of more than one integrins. As a result of these associations, the integrins can modulate each other’s function. For example, Chapman and colleagues²¹ have shown that, in 293 cells, uPAR complex with ₃ß₁, promoting direct (to Vn) and indirect (to Fn) cell adhesion through modulating the function of ₅ß₁ and ₂ß₁. Cross-talk between _vß₃ and ₅ß₁ is also implicated in the regulation of cell migration.³⁹ We speculate that collaboration between _vß₃ and ₅ß₁ may also occur in endothelial cells. Indeed, the _v subunit was detected in the uPAR-₅ß₁-complex. Therefore, HKa-induced disruption of uPAR-₅ß₁-caveolin may indirectly affect Vn-_vß₃ interaction.

In our recent study,¹⁶ we have shown that the antiadhesive effect and the apoptotic effect of HKa are associated with the inhibition of activation of focal adhesion kinase (FAK) and paxillin, two important signal molecules required for cell adhesion and cell survival.^40,41 We can now establish a signaling cascade, Vn-uPAR-_vß₃-caveolin-Src-FAK-paxillin, that mediates adhesion, proliferation, and survival of endothelial cells. HKa disrupts this signaling cascade by at least two mechanisms: first, through its direct binding to the somatomedin B (SMB) domain of Vn, HKa prevents the binding of Vn to integrin _vß₃²⁷; and secondly, HKa directly binds to the D2 and D3 domains of uPAR through its D5 region, thus preventing the binding of Vn to uPAR and its interaction with integrins as well as other signaling molecules, such as caveolin. As a result of these interactions of HKa with the cascade molecules, it is apparent that cells will not be able to adhere properly and will eventually undergo apoptosis, as we have found with cells grown on Vn in the presence of HKa. HKa can directly bind to Mac-1 integrin found in leukocytes, thus blocking adhesion of HEK293 cells to fibrinogen and ICAM-1.⁴² Therefore, HKa may potentially disrupt integrin-mediated adhesion in a similar manner through its direct association with certain integrins present in endothelial cells. It was recently reported that the apoptotic activity of HKa may be mediated through its interaction with tropomyosin.⁴³ Apparently, the interaction between HKa and cell surface proteins is highly complex.

Tarui et al demonstrated that integrin ₅ß₁ is pivotal to the signal transduction of uPAR.⁴⁴ Together with the evidence that uPAR binds integrin with high affinity and facilitate integrins’ function in cell adhesion and migration as discussed in this study, dissociation of uPAR from integrins diminishes these signaling events. Thus, by providing evidence that anti-uPAR antibodies blocked the apoptotic effect of HKa and HKa mediated dissociation of uPAR-integrin, the present study provides a mechanistic explanation of HKa-induced human endothelial cell apoptosis.

	Acknowledgments

 
This study was supported by R01 CA63938 (R.W.C.) and a grant from the American Heart Association 0265404U (Y.-L.G.). Dian J. Cao is a recipient of a postdoctoral fellowship supported by NIH Training Grant T32 HL 07777 (R.W.C.).

	Footnotes

 
Original received December 3, 2003; resubmission received January 29, 2004; revised resubmission received March 16, 2004; accepted March 16, 2004.

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