©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Soluble Ligands of the Integrin Mediate Enhanced Tyrosine Phosphorylation of Multiple Proteins in Adherent Bovine Pulmonary Artery Endothelial Cells (*)

Sunita Bhattacharya (1) (2)(§), Chenzhong Fu (3), Jahar Bhattacharya (4), Steven Greenberg (3)

From the (1)St. Luke's-Roosevelt Hospital Center, New York, New York 10019 and the Departments of (2)Pediatrics, (3)Medicine, and (4)Physiology and Cellular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York, 10032

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Binding of substrate-bound extracellular matrix proteins to cell surface integrins results in a variety of cellular responses including adhesion, cytoskeletal reorganization, and gene expression. We have previously shown that addition of soluble SC5b-9, the complement-vitronectin complex, resulted in an RGD-dependent increase in lung venular hydraulic conductivity (Ishikawa, S., Tsukada, H., and Bhattacharya, J.(1993) J. Clin. Invest. 91, 103-109). To identify specific integrin(s) and signal transduction pathways that are responsive to soluble vitronectin-containing ligands, we exposed confluent bovine pulmonary artery cells to purified soluble human mono- or multimeric vitronectin, or SC5b-9, and determined the extent of endothelial cell protein tyrosine phosphorylation. Monomeric vitronectin (Vn) did not induce enhanced protein tyrosine phosphorylation. However, multimeric Vn and SC5b-9 elicited time- and concentration-dependent increases in tyrosine phosphorylation of numerous proteins. Antiserum against vitronectin, RGD peptides, and monoclonal and polyclonal antibodies against the integrin blocked the vitronectin- or SC5b-9-induced enhanced accumulation of tyrosine phosphoproteins, while antibodies against integrins and the integrin did not. Clustering of the integrin using monoclonal antibody LM609 caused a pattern of enhanced tyrosine phosphorylation similar to that caused by multimeric Vn and SC5b-9, suggesting that aggregation of was critical for signaling. Among the proteins that underwent enhanced tyrosine phosphorylation in response to vitronectin were the cytoskeletal proteins paxillin, cortactin, and ezrin, as well as the SH2 domain-containing protein Shc, and p125. We conclude that ligation of the integrin by soluble ligands promotes enhanced phosphorylation of several proteins implicated in tyrosine kinase signaling and suggest that this pathway may be important in inflammatory states which are accompanied by accumulation of SC5b-9.


INTRODUCTION

Integrins are cell surface receptor proteins which exist as transmembrane heterodimers. The extracellular domains of integrins bind a variety of proteins found in the extracellular matrix (ECM),()such as laminin, fibronectin, and vitronectin (reviewed in Ref. 1-4). Upon adhesion to the ECM, cells bearing integrins demonstrate a diverse array of responses, such as increases in [Ca](5, 6, 7) and intracellular pH (8), activation of protein kinase C (9) and mitogen-activated protein kinase(10) , activation of gene expression (reviewed in Ref. 11), and enhanced protein tyrosine phosphorylation (reviewed in Ref. 2, 12). Among the few substrates identified that undergo enhanced tyrosine phosphorylation in response to integrin ligation are p125(13, 14, 15, 16) , pp60(17) , paxillin(18) , and tensin(19) .

Most studies designed to test the capacity of integrins to promote transmembrane signaling utilize substrate-bound ligands, thus mimicking their distribution in the ECM. Interestingly, Conforti et al.(20) demonstrated the presence of the integrin at the luminal aspect of endothelial cells in vivo, which suggests that this integrin may be accessible to soluble stimulii. Although the identity of soluble ligands for this receptor is unknown, one likely candidate is SC5b-9, the complement-vitronectin complex which is found in the serum during a variety of inflammatory states accompanied by activation of complement (21, 22). Substrate-bound SC5b-9 has been shown to promote the adherence of myoblasts via Vn contained within SC5b-9 and the integrin(23) .

Several integrins capable of binding Vn promote transmembrane signaling in a variety of cells. For example, mediates elevations in [Ca] in osteoclasts (6), enhanced invasiveness and protection against apoptosis of M21 melanoma cells(24, 25) , and angiogenesis in response to various agonists(26) . Intravenous administration of mAb LM609, which blocks binding of ligands to , promotes regression of several tumors by inducing apoptosis of angiogenic blood vessels in chick embryos(27) . In contrast, mediates endocytosis of Vn in fibroblasts (28) and migration on Vn-containing matrices in keratinocytes (29) and pancreatic carcinoma cells(30) . The capacity of , another Vn-binding integrin(31) , to initiate transmembrane signaling has not been reported.

There have been relatively few studies to date reporting integrin-mediated signaling in endothelial cells. One study demonstrated -mediated elevations in [Ca] in human umbilical vein endothelial cells (5). A more recent study demonstrated enhanced protein tyrosine phosphorylation in human umbilical vein endothelial cells migrating on fibronectin, although the precise receptor(s) mediating this effect was not identified(32) . Although untested, the ability of the integrin to mediate enhanced protein tyrosine phosphorylation is suggested by two lines of evidence. First, binding of fibrinogen to the platelet integrin (GPIIb-IIIa), which is structurally similar to (33) , leads to enhanced phosphorylation of several tyrosine kinase substrates(34) . Second, insulin stimulation of Rat-1 fibroblasts transfected with DNA encoding the human insulin receptor was shown to induce the association of the integrin with several molecules implicated in tyrosine kinase-mediated signaling pathways, such as phosphatidylinositol-3 kinase and insulin receptor substrate-1(35) .

We recently demonstrated that serum containing the activated complementVn complex SC5b-9 increased lung endothelial hydraulic conductivity (Lp) through an integrin-dependent mechanism (36). In this study, we examined the effects of soluble Vn-containing ligands on the pattern of protein tyrosine phosphorylation in adherent bovine pulmonary artery endothelial cell monolayers and identified as the integrin responsible for mediating enhanced phosphorylation of several cytoskeletal-associated tyrosine kinase substrates, as well as p125.


EXPERIMENTAL PROCEDURES

Cells and Materials

Bovine pulmonary artery endothelial cells (BPAECs), purchased from American Type Culture Collection (Rockville, MD), were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were passed upon attaining confluence. Their identity was confirmed by noting typical cobblestone morphology and positive immunofluorescent staining for Factor VIII antigen. Polyclonal antiserum to SC5b-9, and an enzyme-linked immunosorbent assay kit to detect SC5b-9, were from Quidel (San Diego, CA). Polyclonal antiserum against vitronectin and mAb P1F6 against the integrin were from Chemicon (Temecula, CA). mAb Z035 against paxillin was from Zymed Laboratories (San Francisco, CA). Polyclonal and monoclonal antibodies against Shc were from Transduction Laboratories (Lexington, KY). Affinity purified rabbit IgG against phosphotyrosyl-containing proteins was from ICN Biomedicals (Costa Mesa, CA). Horseradish peroxidase- and alkaline phosphatase-conjugated secondary antibodies were from Jackson Immunoresearch (West Grove, PA). Sulfosuccinimidobiotin was from Pierce. Protein A-agarose and Protein A/G PLUS-agarose were from Santa Cruz (Santa Cruz, CA). Heparin was from Elkins-Sinn (Cherry Hill, NJ), and peptides were from Peninsula Laboratories (Belmont, CA). Antiserum 838 against the integrin, and polyclonal antiserum against integrins, were generously provided by S. Albelda (University of Pennsylvania, Philadelphia, PA). mAb LM609 against the integrin was kindly provided by D. Cheresh (Scripps Clinic and Research Foundation, La Jolla, CA). mAb 4F11 against cortactin was kindly provided by J. T. Parsons (University of Virginia, Charlottesville, VA). Polyclonal antiserum against p125 and ezrin were generously provided by S. K. Hanks (Vanderbilt University, Nashville, TN) and A. Bretscher (Cornell University, Ithaca, NY), respectively.

Purification of SC5b-9 and Vitronectin

SC5b-9 was purified by the method of Gawryl et al.(37) . In brief, SC5b-9 was generated by zymosan activation of human serum (37) followed by ammonium sulfate precipitation, polyethylene glycol fractionation, DEAE-Sephacel chromatography, and lyophilization. The presence of purified SC5b-9 was confirmed by enzyme-linked immunosorbent assay. The presence of vitronectin in the complex was confirmed by SDS-PAGE and immunoblotting using antiserum against Vn.

The following preparations of Vn were used: native (monomeric) Vn (38, 39) kindly supplied by K. T. Preissner (Haemostasis Research Unit, Bad Nauheim, Germany) and D. Mosher (University of Wisconsin, Madison, WI); multimeric Vn, prepared by incubation of monomeric Vn with 6 M urea followed by dialysis and lyophilization (supplied by K. T. Preissner); conformationally altered Vn, purified from human plasma as described by Yatohgo et al.(40) and further purified by gel filtration. To obtain conformationally altered Vn(40) , we allowed 100 ml of human plasma to clot in glassware and added 0.2 M phenylmethylsulfonyl fluoride to the resultant serum. After centrifugation, the supernatant was applied to a Sepharose 4B precolumn, and then to a heparin-Sepharose column. The flow-through fractions were subjected to repeated denaturation with 8 M urea, to reduction with -mercaptoethanol, and to heparin affinity chromatography. Samples were dialyzed against PBS and lyophilized. Verification of purification was assessed by Coomassie Blue staining of SDS-PAGE under reducing conditions, and further verified by immunoblotting using polyclonal anti-Vn, which showed the expected doublet corresponding to a M of 65 and 75. In some experiments, Vn was fractionated by gel filtration using either a Bio-Gel P-100 column ((1.6 100 cm); Bio-Rad) equilibrated with Tris-buffered saline, pH 7.4, containing 0.5% polyethylene glycol and eluted with Tris-buffered saline, or a Sephacryl S-300 column of the same dimensions (Pharmacia) equilibrated with the above buffer supplemented with 0.5 M NaCl, and eluted with the same.

Stimulation of Endothelial Cells

BPAECs were plated on 6-well tissue culture plates in DM10F. After 5-7 days, confluent monolayers were washed in PBS containing 1 mg/ml bovine serum albumin and incubated with various agonists for the indicated time intervals. Cells were lysed in ice-cold buffer containing 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 20 mM Tris-HCl, pH 7.4. Lysates were cleared by centrifugation at 14,000 G for 15 min, and protein concentrations were determined using the DC Protein Assay (Bio-Rad).

Immunoprecipitation of , , and Integrins

Cell-surface lysyl residues of confluent BPAECs were derivatized with 0.2 mg/ml sulfosuccinimidobiotin (0.2 mg/ml) in PBS, pH 8.0, for 30 min at 4 °C. Monolayers were washed in PBS and incubated at 4 °C for 1 h with either mAb LM609, mAb P1F6, a polyclonal antiserum against integrins, or a rabbit serum control. After detergent lysis and collection of immune complexes on Protein A/Protein G-PLUS-agarose, detection of immunoprecipitated proteins was performed following SDS-PAGE and immunoblotting with streptavidin-horseradish peroxidase.

Immunoblotting and Immunoprecipitation of Tyrosine Kinase Substrates

For detection of Vn, samples were electrophoresed onto 7% SDS-polyacrylamide gels under reducing or non-reducing conditions and transferred onto nitrocellulose. Vn was visualized following addition of anti-Vn antiserum followed by alkaline phosphatase-conjugated goat-anti rabbit IgG and development with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. Anti-phosphotyrosine immunoblotting was performed as described previously(41) . In brief, following detergent lysis, equal amounts of protein were electrophoresed onto 10% SDS-polyacrylamide gels under reducing conditions. After electrophoretic transfer onto nitrocellulose, phosphotyrosyl-containing proteins were detected using an affinity purified anti-phosphotyrosine antibody that was previously derivatized with sulfosuccinimidylbiotin, followed by addition of streptavidin-horseradish peroxidase. For immunoprecipitation, cells were lysed in a buffer containing 1% Nonidet P-40, incubated with Protein A-agarose or Protein A/Protein G-agarose preadsorbed with the appropriate antibodies, washed and subjected to SDS-PAGE, and tranferred onto nitrocellulose. Detection of immunoprecipitated and/or phosphotyrosyl-containing proteins was performed by adding the appropriate antiserum/mAbs followed by horseradish peroxidase-conjugated secondary antibodies. Blots were developed using enhanced chemiluminescence.


RESULTS

Vitronectin and SC5b-9 Cause a Time- and Concentration-dependent Increase in Protein Tyrosine Phosphorylation in Bovine Pulmonary Artery Endothelial Cells

Addition of either SC5b-9 or conformationally altered Vn to adherent BPAECs led to a similar pattern of enhanced tyrosine phosphorylation of multiple proteins that peaked at 3 min after the addition of agonist (Fig. 1). Proteins that demonstrated marked enhancement of tyrosine phosphorylation corresponded to M values of 28, 34, 38, 46, 52, 54, 69, and 125. At the lower concentrations used, SC5b-9 was somewhat more effective at inducing enhanced protein tyrosine phosphorylation than Vn; however, for both agonists, 400 µg/ml produced maximal enhancement of the accumulation of phosphotyrosyl-containing proteins (Fig. 2). A similar concentration-dependent increase in the extent of protein tyrosine phosphorylation was seen using conformationally altered Vn further purified by gel filtration (not shown).


Figure 1: SC5b-9 and Vn induce a dynamic enhancement of protein tyrosine phosphorylation in BPAECs. 600 µg/ml of either agonist was applied to confluent BPAEC monolayers for the indicated time intervals at 37 °C, and cells were lysed and subjected to SDS-PAGE and immunoblotting using anti-phosphotyrosine as described under ``Experimental Procedures.'' A, effect of SC5b-9; B, effect of purified conformationally-altered Vn. Molecular weight markers appear at the left.




Figure 2: SC5b-9 and Vn induce a concentration-dependent enhancement of protein tyrosine phosphorylation in BPAECs. The indicated concentrations of either agonist was applied to confluent BPAEC monolayers for 3 min at 37 °C, and cells were lysed and subjected to SDS-PAGE and immunoblotting using anti-phosphotyrosine as described under ``Experimental Procedures.'' A, effect of SC5b-9; B, effect of purified conformationally altered Vn. Molecular weight markers appear at the left.



Multimeric Vn, but Not Monomeric Vn, Induces Enhanced Protein Tyrosine Phosphorylation in Adherent BPAECs

Native Vn obtained from two sources appeared as monomers on native SDS-PAGE (Fig. 3A, lane 1 and data not shown). Addition of this monomeric Vn to adherent BPAECs did not induce a change in the pattern or extent of protein tyrosine phosphorylation (Fig. 3B, lane 1). However, addition of multimeric Vn, obtained by subjecting native Vn to treatment with urea, which induces multimerization of individual Vn monomers(38) , led to a striking increase in the extent of protein tyrosine phosphorylation (Fig. 3B, lane 2). A similar, though less marked, increase in the extent of protein tyrosine phosphorylation was observed in BPAECs treated with Vn purified by the method of Yatohgo et al. ((40); Fig. 3B, lane 3). There was some variability in the extent of responsiveness of BPAECs to different preparations Vn purified by this method, which may have been due to their age and/or the duration of dialysis; these variable have been shown to influence the extent of aggregation of Vn monomers(42) . Consequently, we further fractionated purified conformationally altered Vn by gel filtration, and obtained fractions enriched in high molecular weight complexes of Vn, as assessed by SDS-PAGE under native conditions and immunoblotting using anti-Vn antiserum. Migration of Vn through gel filtration columns was somewhat anomalous in that peaks of purified multimers appeared both in early and very late fractions, which likely reflects ionic and/or hydrophobic interactions between Vn and the bead matrix. We therefore used several different gel filtration media, but obtained similar results (Fig. 3A, lanes 4, 5, and 7). Regardless of the purification scheme, fractions enriched in multimers of Vn were more potent per unit weight at inducing enhanced protein tyrosine phosphorylation than unfractionated conformationally altered Vn (compare lanes 4, 5, and 7 with lane 3 in Fig. 3B). To verify that BPAECs responded to the Vn contained in both SC5b-9 and high molecular weight Vn complexes, rather than to contaminating proteins, we depleted Vn from these samples using saturating concentrations of anti-Vn antiserum, and applied the resultant supernatants to BPAEC monolayers. Immunodepletion of Vn from SC5b-9 or multimeric Vn completely blocked enhanced tyrosine phosphorylation in BPAECs due to either agonist (Fig. 4), confirming that Vn was responsible for eliciting this effect. Together, these data indicate that Vn contained with either multimeric complexes of Vn or SC5b-9 induces enhanced protein tyrosine phosphorylation in adherent BPAECs.


Figure 3: Effect of different preparations of Vn on inducing enhanced protein tyrosine phosphorylation in BPAECs. A, equal amounts of Vn prepared as described under ``Experimental Procedures'' were subjected to SDS-PAGE under native conditions and immunoblotted using anti-Vn antiserum. Lane 1, monomeric Vn; lane 2, multimeric Vn prepared by urea treatment of monomeric Vn; lane 3, unfractionated conformationally altered Vn; lane 4, late Bio-Gel P-100 fraction of conformationally-altered Vn; lane 5, early Sephacryl S-300 fraction of conformationally altered Vn; lane 6, middle Sephacryl S-300 fraction of conformationally altered Vn; lane 7, late Sephacryl S-300 fraction of conformationally altered Vn. B, Vn (400 µg/ml) derived from each of the above fractions was added to confluent BPAEC monolayers at 37 °C for 3 min. Cells were lysed and subjected to SDS-PAGE followed by immunoblotting using anti-phosphotyrosine antibodies. C, control monolayer incubated with PBS alone; lanes 1-7, monolayers incubated with Vn preparations as described above in A. Molecular mass markers appear at left.




Figure 4: Vn contained within SC5b-9 and high molecular weight complexes of Vn is responsible for mediating enhanced protein tyrosine phosphorylation in BPAECs. 600 µg/ml of either SC5b-9, unfractionated conformationally altered Vn, or gel-purified high molecular weight Vn complexes were applied to Protein A-agarose preadsorbed with either control non-immune rabbit serum, or anti-Vn antiserum. Resulting supernatants were added to confluent BPAEC monolayers for 3 min at 37 °C, and cells were lysed and subjected to SDS-PAGE and immunoblotting using anti-phosphotyrosine as described under ``Experimental Procedures.'' Molecular weight markers appear at the left.



RGD-containing Sequences within Multimeric Vitronectin and SC5b-9, but Not Heparin-binding Domains, Are Responsible for Mediating Enhanced Protein Tyrosine Phosphorylation

Several epitopes within Vn have been implicated in binding to cell surface receptors, including an RGD site (28) and a heparin-binding domain(43, 44) . To assess whether either epitope contributed to the promotion of enhanced protein tyrosine phosphorylation, we incubated adherent BPAECs with either SC5b-9 or multimeric Vn in the presence or absence of RGD or control RGE-containing peptides, or heparin. Neither heparin, GRGDSP, or GRGESP alone altered the pattern of protein tyrosine phosphorylation. However, GRGDSP, but neither GRGESP nor heparin blocked the enhanced protein tyrosine phosphorylation induced by either SC5b-9 or multimeric Vn (Fig. 5). Similar results were obtained when conformationally altered Vn purified by the method of Yatohgo et al.(40) was used as a stimulus (not shown). Although this experiment does not address which epitope(s) on Vn is responsible for the binding of these Vn-containing ligands to BPAECs, it does show that the RGD domain of Vn is necessary for inducing enhanced protein tyrosine phosphorylation.


Figure 5: The RGD domain of Vn mediates enhanced protein tyrosine phosphorylation in BPAECs. Confluent BPAECs were exposed to either 300 µg/ml GRGDSP (RGD) or GRGESP (RGE), 500 µg/ml heparin, 400 µg/ml SC5b-9 (S), or multimeric Vn (Vn) or combinations of the above, as indicated, for 3 min at 37 °C. Cells were lysed and subjected to SDS-PAGE and immunoblotting using anti-phosphotyrosine as described under ``Experimental Procedures.'' Molecular weight markers appear at the left.



The Integrin Mediates Enhanced Protein Tyrosine Phosphorylation Due to SC5b-9 and Vitronectin

We first determined whether known Vn-binding integrins were expressed at the cells' surfaces. Immunoprecipitation of biotin-derivatized cell surface proteins by mAbs against the and integrins revealed the presence of these proteins, as assessed by the expected pattern of migration of their subunits (45) following SDS-PAGE (Fig. 6). The apparent faster mobility of the subunit derived from (compare two tops bands in Fig. 6) was similar to the findings of Smith et al.(45) , and is of unknown significance. Although this method does not provide an accurate assessment of the precise numbers of integrins expressed on the cells' surfaces, their relatively equivalent intensity of staining suggests a similar level of surface expression. One or more integrins were also expressed at the surfaces of BPAECs (not shown), although we did not determine whether was one of these.


Figure 6: Immunoprecipitation of surface-labeled and integrins derived from BPAECs. Two 9.6-cm wells of confluent BPAEC monolayers were incubated with sulfosuccinimidobiotin, immunoprecipitated with mAbs against the indicated integrin, or with an isotype-matched control, and subjected to SDS-PAGE and immunoblotting with streptavidin-horseradish peroxidase as described under ``Experimental Procedures.'' Molecular weight markers appear at the left.



To identify which of these integrins is responsible for mediating enhanced protein tyrosine phosphorylation due to soluble Vn-containing agonists, we added antibodies that recognize these integrins to BPAECs during addition of either SC5b-9 or Vn. Polyclonal and monoclonal antibodies against completely blocked enhanced protein tyrosine phosphorylation due to either agonist, whereas anti- antisera or anti- mAbs did not (Fig. 7).


Figure 7: Antibodies against the integrin, but not against or integrins, block enhanced protein tyrosine phosphorylation due to SC5b-9 and Vn in BPAECs. Unstimulated BPAECs or BPAECs stimulated at 37 °C for 3 min with 400 µg/ml of either SC5b-9 (A and C) or high molecular weight Vn (B and D) were coincubated with either mAb LM609 directed against (20 µg/ml) or with mAb P1F6 directed against (20 µg/ml) (A and B), or with a 1:50 dilution of either antiserum 838 against or anti- antiserum (C and D), and cells were lysed and subjected to SDS-PAGE and immunoblotting using anti-phosphotyrosine as described under ``Experimental Procedures.'' Molecular weight markers appear at the left.



Clustering the Integrin on Adherent BPAECs Mediates Enhanced Protein Tyrosine Phosphorylation

To further confirm that the integrin is capable of mediating enhanced protein tyrosine phosphorylation in BPAECs, we incubated adherent BPAECs with mAb LM609 followed by cross-linking of surface-bound mAb with anti-mouse IgG. Clustering of using mAb LM609 caused a marked increase in protein tyrosine phosphorylation of multiple proteins in a pattern similar to that seen after addition of SC5b-9 or Vn (compare Fig. 8with Fig. 1). Interestingly, the addition of either mAb LM609 or polyclonal antiserum against in the absence of a cross-linking antibody did not induce a detectable increase in the extent of protein tyrosine phosphorylation (Fig. 7). Taken together, the above data suggest that clustering of the integrin mediates enhanced protein tyrosine phosphorylation in BPAECs.


Figure 8: Clustering of the integrin is sufficient to trigger enhanced protein tyrosine phosphorylation in BPAECs. Adherent BPAEC monolayers were incubated at 4 °C for 30 min with 20 µg/ml of either mAb LM609 or isotype-matched control mAb, washed, and further incubated with 30 µg/ml donkey anti-mouse IgG at 37 °C for the indicated time intervals, and cells were lysed and subjected to SDS-PAGE and immunoblotting using anti-phosphotyrosine as described under ``Experimental Procedures.'' Molecular weight markers appear at the left.



The Tyrosine Kinase Substrates Paxillin, Cortactin, Ezrin, Shc, and p125, Undergo Enhanced Tyrosine Phosphorylation in Bovine Pulmonary Artery Endothelial Cells in Response to Vn

To identify specific proteins that undergo enhanced protein tyrosine phosphorylation in response to Vn, we incubated adherent BPAECs with multimeric Vn, lysed the cells, and performed immunoprecipitation using antisera against various tyrosine kinase substrates followed by immunoblotting using anti-phosphotyrosine. The cytoskeletal-associated proteins cortactin, paxillin, and ezrin, the SH2 domain-containing protein Shc, and p125, underwent enhanced tyrosine phosphorylation in response to Vn (Fig. 9). The enhanced tyrosine phosphorylation of cortactin is similar to findings in brain microvascular endothelial cells stimulated by activating surface-expressed ICAM-1(46) .


Figure 9: Immunoprecipitation and anti-phosphotyrosine immunoblotting of paxillin, cortactin, Shc, ezrin, and p125 following addition of Vn in BPAECs. 400 µg/ml of multimeric Vn complexes were applied to confluent BPAEC monolayers for 3 min at 37 °C, and cells were lysed and subjected to immunoprecipitation followed by SDS-PAGE and immunoblotting using anti-phosphotyrosine or the indicated antibodies as described under ``Experimental Procedures.'' Immunoblotting using antibodies against the precipitated proteins was performed to compare extent of recovery of immunoprecipitated proteins. Molecular weight markers appear at the left.




DISCUSSION

This study demonstrates that soluble Vn-containing ligands are capable of triggering enhanced protein tyrosine phosphorylation in bovine pulmonary artery endothelial cell monolayers. Several lines of evidence suggest that clustering integrins by multimeric Vn was required for this effect. 1) Fractions enriched in Vn multimers were more potent than unfractionated Vn in mediating enhanced protein tyrosine phosphorylation, whereas monomeric Vn was incapable of triggering enhanced protein tyrosine phosphorylation. 2) SC5b-9, a supramolecular complex which contains several Vn molecules(47, 48) , was equally, if not more potent in mediating protein tyrosine phosphorylation in these cells. 3) A similar pattern of protein tyrosine phosphorylation was seen in endothelial cell monolayers whose cell surface was aggregated using mAb LM609 followed by goat anti-mouse IgG. Addition of mAb LM609 or anti- antiserum alone was ineffective in altering the extent of protein tyrosine phosphorylation. 4) Addition of mAb LM609 or polyclonal antiserum 838, directed against , blocked enhanced tyrosine phosphorylation mediated by either agonist. While these results are consistent with the hypothesis that the valency of Vn is critical for mediating enhanced protein tyrosine phosphorylation, we cannot discount the possibility that receptor occupancy contributes to this response, as well. However, in a recent study, clustering of integrins in the absence of receptor occupancy was sufficient to induce enhanced protein tyrosine phosphorylation in fibroblasts(49) . Since a natural ligand for has yet to be identified that induces receptor clustering in the absence of receptor occupancy, the importance of demonstrating a contributory role for receptor occupancy in this response is questionable.

Vn in its monomeric form is present in the serum in concentrations similar to those used in this study. Only a small percentage of Vn isolated from healthy volunteers is found in high molecular weight complexes, such as ternary complexes containing thrombin and antithrombin III (reviewed in Ref. 50). The method of isolation of Vn has considerable influence on its physical properties. For example, incubation of Vn with 6-8 M urea and -mercaptoethanol (40) exposes a cryptic heparin-binding domain within the Vn monomer, allowing its recognition by several conformation-specific mAbs(42) , and predisposes Vn molecules to multimerization(39, 42) . Stabilization of the multimeric structure is mediated, in part, by disulfide bonding since the resultant Vn multimers migrate as Vn monomers when applied to SDS gels under reducing conditions(42) . Although this conformationally altered Vn preparation is very different from native Vn found in serum, its physical properties are shared by substrate-bound and presumably ECM-derived Vn, as well as Vn bound to the terminal complement complex ((SC5b-9)(42) ).

Despite the exposure of a heparin-binding domain by urea, the heparin-binding domain did not detectably contribute to the enhancement of protein tyrosine phosphorylation in BPAECs, whereas the RGD site was required for this effect. Although these findings seem to conflict with those showing an inhibition of binding of multimeric Vn to endothelial cells by heparin(43, 44) , there are several plausible explanations for this apparent discrepancy. First, the extent to which heparin inhibits the binding of multimeric Vn to endothelial cells is dependent on the source of the endothelial cells. Heparin inhibited more than 90% of the binding of multimeric Vn to human umbilical vein endothelial cells (43), whereas it only inhibited 60% of the binding to porcine aortic endothelial cells(44) . Second, we did not assess binding of Vn to BPAECs. It is certainly possible that more than one epitope within multimeric or aggregated Vn contributes to its binding to endothelial cells of diverse origins. From our data, however, we conclude that the RGD site within multimeric Vn is critical for mediating enhanced protein tyrosine phosphorylation in BPAECs.

It is highly unlikely that luminal integrins are under a state of constant stimulation in vivo. This raises the question of how these receptors signal enhanced tyrosine phosphorylation in vivo, given the high concentrations of Vn present in the plasma (0.1-0.5% of total plasma protein; 50). In a recent study using different preparations of Vn, Zanetti et al.(43) found that monomeric Vn bound poorly, while aggregated Vn bound well, to endothelial cell monolayers. In addition, our results suggest that native monovalent Vn is unlikely to trigger enhanced protein tyrosine phosphorylation, even if a small fraction of it were to bind to the endothelium in vivo. Situations in which a multivalent configuration of Vn is likely to be present in the bloodstream include bacterial sepsis, which is accompanied by the formation of SC5b-9(21) . During sepsis, multivalent Vn complexes within SC5b-9 may bind tightly to luminal receptors and initiate enhanced tyrosine phosphorylation. This mode of signaling is reminiscent of at least one other class of tyrosine kinase-mobilizing receptors, those for the Fc portion of IgG (Fc receptors). Despite the presence of high circulating levels of monomeric IgG, these receptors are optimally ligated by IgG aggregates or by IgG bound to surfaces, such as bacterial cell walls (reviewed in Ref. 51). The analogy to Fc receptors is further underscored by the ability of Vn to bind to a variety of Gram-positive and Gram-negative bacteria(52) . It is possible that during bacteremia due to these organisms, bacterial-bound Vn induces ligation and clustering of endothelial integrins, thus promoting a highly localized stimulus for enhanced protein tyrosine phosphorylation.

The functions of the particular tyrosine kinase substrates identified in this study are only partially understood. Paxillin, a 68-kDa vinculin-binding protein(53) , undergoes enhanced tyrosine phosphorylation in response to a variety of agonists(18, 41, 54, 55) . Paxillin has been shown to bind with high affinity to the SH3 domain of c-Src (56) and v-Crk(57) . Ezrin and cortactin are also associated with the cytoskeleton, and in fact bind F-actin directly(58, 59) . Alterations in the cytoskeleton of endothelial cells have been demonstrated for a variety of stimulii, including those that mediate alterations in monolayer barrier function (reviewed in Ref. 60), and preliminary evidence suggests that alterations in the state of tyrosine phosphorylation affects endothelial cell barrier function.()The roles of these particular tyrosine kinase substrates in effecting changes in the cytoskeleton or contributing to the barrier function of endothelial cell monolayers are unknown. It is particularly interesting that Shc, an SH2 domain-containing ``adaptor'' protein implicated in the Ras signaling pathway(61, 62, 63) , undergoes enhanced tyrosine phosphorylation by Vn-containing ligands. Since Shc is a mediator of growth and differentiation in a variety of cell types, and is required for angiogenesis in chorioallantoic membranes(26) , it is possible that tyrosine phosphorylation mediated by this integrin is essential for its angiogenesis-promoting capacity and that Shc plays a central role in this process. In this respect, Shc may function in integrin-mediated signaling pathways similar to its role in growth factor receptor-mediated pathways. This is consistent with recent studies demonstrating the association of a Shc-binding protein implicated in Ras-mediated signaling, Grb2, with p125 in fibronectin-stimulated fibroblasts (64) or with in insulin-stimulated Rat-1 fibroblasts transfected with DNA encoding the human insulin receptor (35).

It is particularly interesting to note that we demonstrated enhanced tyrosine phosphorylation of p125 using soluble stimulii whose average size is large (720 nm for SC5b-9(48) ), compared with that of inter-endothelial junctional pores (6.5-7.5 nm in size estimated from tracer studies(65) ). Sieving properties of endothelial monolayers grown on membrane filters show restricted diffusion of molecules whose size is equal to, or greater than, fibrinogen (10.6 nm(66) ). It is therefore unlikely that the Vn-containing ligands we used in this study ligated integrins located on the abluminal surface of the endothelium, at sites of focal adhesion. Since p125 is concentrated in areas of focal adhesion to the underlying substrate(13, 16) , it is possible that there is considerable spatial separation between ligated receptors and phosphorylated p125 molecules. Alternatively, a small fraction of p125 may be freely diffusible in the cytosol or associated with the subset of receptors that are accessible to soluble Vn.

We did not address in this study the capacity of soluble Vn ligands to mediate alterations in endothelial cell barrier function. We would predict, however, that may mediate changes in endothelial cell monolayer permeability to macromolecules in vitro, similar to results we have obtained in isolated lung venules. In contrast, a previous study examining endothelial cell monolayer barrier function in human umbilical vein endothelial cells reported that antibodies against the integrin, but not against integrins, increased transendothelial flux of macromolecules(67) . Since the expression and location of is very likely dependent upon cell type and culture conditions, and the authors showed a relative paucity of staining for subunits at intercellular borders, their inability to demonstrate -mediated changes in monolayer permeability to macromolecules in these cells is not surprising. In any case, the relationship between -mediated enhanced protein tyrosine phosphorylation and endothelial cell barrier function awaits further study.


FOOTNOTES

*
This work was supported in part by Grants HL 36024, HL 02641, and HL02483 from the National Institutes of Health, a grant-in-aid from the American Heart Association, a grant-in-aid from the American Heart Association, New York City affiliate, a grant from the Stony Wold-Herbert Foundation, and a generous gift from Dr. George A. Carden, Jr. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Roosevelt Hospital, 428 West 59th St., New York, NY 10019.

The abbreviations used are: ECM, extracellular matrix; BPAECs, bovine pulmonary artery endothelial cells; [Ca], cytosolic-free calcium concentration; DM10F, Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum; PBS, phosphate-buffered saline; Vn, vitronectin; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis.

H. Tsukada, X. Ying, C. Fu, S. Ishikawa, P. McKeown-Longo, S. Albelda, S. Bhattacharya, B. A. Bray, and J. Bhattacharya, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Rashmi Patel for technical assistance.


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