Ligation of Integrin alpha 5beta 1 Is Required for Internalization of Vitronectin by Integrin alpha vbeta 3*

(Received for publication, March 28, 1996, and in revised form, November 4, 1996)

Vivian Pijuan-Thompson and Candece L. Gladson Dagger

From the Department of Pathology, Division of Neuropathology, University of Alabama at Birmingham, Birmingham, Alabama 35294

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Remodeling of the matrix by tumor cells is necessary for tumor invasion. We have shown previously that malignant astrocytomas, in contrast to normal astrocytes, synthesize vitronectin and express integrins alpha vbeta 3 and alpha vbeta 5. The activity states of these two integrins are differentially controlled. Thus, we investigated the regulation of the activity of integrins alpha vbeta 3 and alpha vbeta 5 with regard to their role in vitronectin internalization in U-251MG astrocytoma cell monolayers adherent to fibronectin, collagen, or laminin in serum-free conditions. Binding of [125I]vitronectin occurred in a specific, saturable manner that was partially inhibitable by monoclonal antibodies (mAbs) specific for integrins alpha vbeta 3 or alpha vbeta 5. Specific, lysosomally-mediated degradation of [125I]vitronectin was detectable at 1 h and increased over the 24-h assay period. The cell substrate affected the rate of turnover of [125I]vitronectin, which was 3.0 ng/min for cells plated on fibronectin but 0.35 ng/min for cells plated on collagen. Furthermore, although mAbs specific for either integrin alpha vbeta 3 or alpha vbeta 5 inhibited degradation (30%; combined effect 70%) of [125I]vitronectin by cells plated on fibronectin, only mAb anti-alpha vbeta 5 inhibited degradation (70-90%) by cells plated on collagen or laminin. To determine the requirement for integrin alpha 5beta 1 ligation in order for integrin alpha vbeta 3 to internalize its ligand, cells were plated on mAbs anti-integrin alpha 5 or anti-integrin alpha 3. When plated on mAb anti-alpha 5, mAbs anti-alpha vbeta 3 and anti-alpha vbeta 5 both inhibited degradation. However, when plated on mAb anti-alpha 3, mAb anti-alpha vbeta 3 had no effect whereas mAb anti-alpha vbeta 5 inhibited degradation. These data indicate that a signal from integrin alpha 5beta 1 is necessary for integrin alpha vbeta 3 to internalize vitronectin, whereas integrin alpha vbeta 5 constitutively internalizes vitronectin.


INTRODUCTION

We have previously reported that two vitronectin receptors, integrins alpha vbeta 3 and alpha vbeta 5, are expressed on malignant astrocytoma cells both in vivo and in vitro and that they are markers of the malignant phenotype (1, 2). These two integrins differ in their ligand specificity and mechanism of regulation. Integrin alpha vbeta 5 recognizes only vitronectin and osteopontin as ligands, whereas integrin alpha vbeta 3 is a promiscuous receptor that recognizes vitronectin, fibrinogen, von Willebrand factor, fibronectin, thrombospondin, thrombin, osteopontin, bone sialoprotein, laminin, and degraded collagen types I and IV (reviewed in Refs. 3-5; 6-8). The activity state of integrin alpha vbeta 5 varies according to the cell type (2, 9-11), whereas it is thought that integrin alpha vbeta 3 is expressed in a constitutively active state on all nucleated cells (reviewed in ref. 4) and is regulated by an integrin-associated protein, phosphorylation of associated proteins, and growth factors (reviewed in Ref. 12; 13-17). Ligation of integrin alpha vbeta 3 has been shown to act as a signal for integrin alpha 5beta 1, inhibiting fibronectin internalization by integrin alpha 5beta 1 (16, 17). There is also indirect evidence that the expression of integrin alpha 5beta 1 can modulate the activity of integrin alpha vbeta 3 (18, 19). Thus, for example, unligated integrin alpha 5beta 1 is important for integrin alpha vbeta 3-mediated motility in Chinese hamster ovary cells, and absence of beta 1 integrins in embryonal carcinoma cells reduces integrin alpha vbeta 3-mediated motility (18, 19).

Previous studies have indicated that in fibroblast monolayers, alpha vbeta 5 mediates the internalization of vitronectin in a protein kinase C-dependent manner (20, 21). In granulation tissue, matrix proteins, such as vitronectin, are removed as part of the tissue remodeling; however, the fate of extracellular vitronectin in tumors is unclear. Remodeling of the extracellular matrix is necessary for tumor invasion (22). We have shown previously that malignant astrocytomas have the potential to remodel the matrix because, in contrast to normal astrocytes, these cells synthesize vitronectin (1, 2).

In vitro, vitronectin exists in two conformers, known as native and altered, both of which promote cell attachment (23); however, only altered vitronectin is internalized by fibroblasts (24). Vitronectin purified by heparin-Sepharose chromatography after urea denaturation is referred to as altered or denatured vitronectin (23). Vitronectin is an adhesive glycoprotein that promotes cell adhesion and migration through its Arg-Gly-Asp (RGD) peptide cell adhesion domain (23). Altered vitronectin, in contrast to native vitronectin, is a multimeric protein and possesses a functional heparin-binding domain through which it regulates the activity of several other proteins, such as the thrombin-anti-thrombin III complex and the C5b-9 terminal complement complex (23).

We have investigated the fate of extracellular vitronectin when astrocytoma cells are adherent to protein constituents of the pial-glial and endothelial cell basement membranes because these membranes are extensively remodeled in this tumor and matrix protein internalization is thought to occur as part of matrix remodeling (22). These studies are of physiologic significance, as malignant astrocytoma cells are known to invade throughout the brain, in part, by adhesion and migration on pial-glial and endothelial cell basement membranes (22). We demonstrate that integrin alpha vbeta 3-mediated internalization of altered vitronectin requires ligation of integrin alpha 5beta 1.


MATERIALS AND METHODS

Reagents and Antibodies

Heparin, chondroitin sulfate A, chloroquine, and mouse IgG were purchased from Sigma. Peptides were synthesized by the University of California at San Diego Peptide Synthesis Facility. Neutralizing mAbs1 anti-integrin alpha vbeta 3 (LM609), anti-integrin alpha vbeta 5 (P3G2), and anti-integrin beta 1 (P4C10) were generous gifts from Dr. David A. Cheresh (Scripps Research Institute, La Jolla, CA). Neutralizing mAbs anti-integrin alpha 3, alpha 5, and alpha 6 were purchased from Chemicon (Temecula, CA). Affinity-isolated rabbit anti-integrin alpha 5beta 1 antiserum, neutralizing mAbs anti-integrin alpha 2, and anti-integrin alpha 3 ascites were purchased from Life Technologies, Inc. The IgG fraction of mAbs anti-integrin alpha 2, anti-integrin alpha 3, and anti-integrin alpha 5beta 1 were purified as described (1). Fluorescein isothiocyanate-conjugated goat anti-rabbit and goat anti-mouse IgG antibodies were purchased ( Jackson Laboratory, West Grove, PA).

Purification and Iodination of Vitronectin

Altered vitronectin was purified from human plasma as described (25) and migrated as a 75/65 kDa doublet on disulfide-reduced 10% SDS-PAGE and as a high molecular weight aggregate in nonreduced SDS-PAGE, as expected. Vitronectin was iodinated by the iodogen method (average specific activity of 10 µCi/µg) and free iodine was removed by gel filtration, as described (26, 27). The migration of iodinated vitronectin on 12% disulfide-reduced SDS-PAGE was identical to that of the cold protein. Native vitronectin was a gift from Dr. Deanne Mosher (University of Wisconsin, Madison, WI) and migrated on nonreduced SDS-PAGE as a doublet at 75/65 kDa.

Degradation and Binding Assays

Human malignant astrocytoma cells (U-251MG) from the ATCC were cultured in complete medium, as described (1), and were Mycoplasma-free. For the vitronectin degradation and binding experiments, 12-well culture plates were coated with 10 µg/ml fibronectin (Boehringer Mannheim) collagen type I (ICN Biomedicals, Costa Mesa, CA), or laminin (Collaborative Biomedical Products, Bedford, MA) at 37 °C for 6 h or overnight, washed, and then blocked for 1 h at 25 °C with 1% bovine serum albumin after heat denaturation (98 °C for 10 min). Cells (105 per well) aliquoted onto fibronectin, collagen, or laminin-coated wells attached and spread, and their morphology was similar. All experiments were performed with cells that had been plated for 1 h as well as overnight in serum-free Dulbecco's modified Eagle's medium/1% bovine serum albumin. 125I-Labeled altered vitronectin (600 ng/ml, 1 × 106 cpm) in 0.2% bovine serum albumin was added to the wells, followed by incubation at 37 °C and timed removal of medium aliquots, as described (20, 21, 24, 28). 125I-Labeled altered vitronectin degradation was quantitated as the radioactivity in a 50-µl aliquot of medium that was soluble in 20% trichloroacetic acid. Background radioactivity obtained from substrate-coated wells not containing cells that were incubated in parallel to the experimental wells was subtracted from the total trichloroacetic acid-soluble radioactivity. In experiments done in the presence of heparin, heparin was preincubated with the monolayer prior to [125I]vitronectin addition. For degradation experiments in which wells were coated with mAbs prior to the plating of the cells, wells were coated for 1 h at 25 °C with goat anti-mouse IgG (20 µg/ml), washed, blocked with 1% heat-denatured bovine serum albumin for 1 h at 37 °C, washed, incubated with 10 µg/ml mAbs anti-integrin alpha 3 or alpha 5 IgG (Chemicon, Temecula, CA) for 1 h at 25 °C, as described (29, 30). After the wells were washed, cells were plated for 1 h. Medium aliquots at various time points were subjected to 12% disulfide-reduced SDS-PAGE and autoradiography to confirm the absence of proteolytic degradation of [125I]vitronectin in the media. To measure bound [125I]vitronectin, labeled medium was removed, the wells were washed with phosphate-buffered saline, and the cells were solubilized in 1 N NaOH and scintillation counted. Nonspecific cell binding obtained from wells containing 100 µg/ml unlabeled vitronectin was subtracted from all determinations. Binding assay data represent the mean ± S.E. from triplicate wells of one experiment. Binding assay experiments were performed three times with similar results. Degradation assay data represent the mean ± S.E. from three or four separate experiments, where in each experiment the condition was assayed as a single well or in duplicate.

FACS Analysis

U-251MG cells were plated onto fibronectin, collagen, or laminin-coated flasks in serum-free media, and after a 1 h or overnight incubation harvested with buffered EDTA, reacted with primary antibody, followed by goat anti-mouse or anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody, and then subjected to FACS analysis, as described (27).

Cell Adhesion Assays

96-well plates were coated overnight at 25 °C with 10 µg/ml fibronectin, collagen, or laminin in phosphate-buffered saline. Cells were harvested with buffered EDTA, suspended in adhesion assay buffer, and incubated with 50 µg/ml of the indicated antibody for 30 min (1, 29). Subsequently, cells were plated onto coated wells (40,000 cells/well), allowed to attach for 45 min at 37 °C, washed, crystal violet stained, and quantitated by spectrophotometric absorbance at 570 nm, as described (1, 29).


RESULTS

[125I]Vitronectin Binding and Degradation by a Human Malignant Astrocytoma Cell Monolayer Plated on Fibronectin

We initially characterized the binding of altered vitronectin to a human astrocytoma cell monolayer. 125I-Labeled altered vitronectin (600 ng/ml, 1 × 106 cpm) was added to a subconfluent monolayer of serum-starved U-251MG cells that had been plated on fibronectin. Labeled vitronectin was found to bind maximally to the cell monolayer after 3-6 h, at which time approximately 7% (42 ng) of the added [125I]vitronectin was specifically bound (Fig. 1A). Cold vitronectin (100 µg/ml) inhibited binding by 85% (data not shown), indicating that binding was specific. To determine whether integrins alpha vbeta 3 or alpha vbeta 5 mediated vitronectin binding, as we have reported their expression on these cells (1, 2), mAbs anti-alpha vbeta 3 and anti-alpha vbeta 5 were preincubated with the monolayer, and each inhibited binding by 30% at 6 h. Studies were also done in the presence of heparin to eliminate a heparan sulfate proteoglycan-mediated event, and heparin (100 µg/ml) significantly inhibited [125I]vitronectin binding to the cell monolayer (70% at 6 h), in contrast to chondroitin sulfate. Vitronectin binding was identical whether cells were allowed to attach for 1 h or overnight. [125I]Vitronectin binding was determined at the end of all subsequent degradation assays and was nearly identical to the 6 h time point in Fig. 1A.


Fig. 1. [125I]Vitronectin binds to and is degraded by an astrocytoma cell monolayer adherent to fibronectin. Heparin or chondroitin sulfate (100 µg/ml), mAbs (50 µg/ml), unlabeled vitronectin (100 µg/ml), or chloroquine (100 µM) were added to a subconfluent cell monolayer plated on fibronectin in serum-free media prior to addition of [125I]vitronectin (600 ng/ml). Control denotes [125I]vitronectin binding or degradation in the absence of inhibitor. [125I]Vitronectin binding (A) was determined by solubilization of the monolayer with NaOH and scintillation counting. Data points are the average of triplicate wells ± S.E. [125I]Vitronectin degradation (B-D) was determined from the radioactivity present in medium aliquots obtained at the indicated times. Data is shown as the mean ± S.E. from four separate experiments. Nonspecific binding was determined from wells containing 100 µg/ml cold vitronectin and was subtracted from the total vitronectin bound. Background radioactivity determined from wells coated with fibronectin but lacking cells was subtracted from the total vitronectin degraded.
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To determine the characteristics of vitronectin internalization, medium aliquots over time were subjected to trichloroacetic acid precipitations and the degradation of vitronectin was calculated. Degradation was detected as early as 1 h and the amount of vitronectin degraded continued to increase over the 24-h assay period (Fig. 1B). At 24 h, approximately 30% (187 ng) of the added [125I]vitronectin was degraded. Degradation was specific and required integrin-mediated internalization because either cold vitronectin (Fig. 1B) or an RGD-containing peptide (GRGDSP, 200 µM) (data not shown) markedly inhibited [125I]vitronectin degradation. The random hexapeptide (SPGDRG, 200 µM) failed to inhibit degradation. Chloroquine, a lysosomal inhibitor, greatly inhibited degradation throughout the 24-h assay period (Fig. 1B), indicating that degradation of the [125I]vitronectin occurred through a lysosomal pathway. 125I-Labeled native vitronectin was not degraded to any significant extent (data not shown), as has been reported for fibroblasts (24). To determine whether integrin alpha vbeta 3 or alpha vbeta 5 mediated vitronectin internalization, the astrocytoma cell monolayer was incubated (30 min) with neutralizing mAbs specific for integrin alpha vbeta 3 or alpha vbeta 5 prior to the addition of [125I]vitronectin (Fig. 1C). mAb anti-alpha vbeta 3 or alpha vbeta 5 inhibited vitronectin degradation to a similar extent (30%) throughout the 12-h assay period. When added together, the antibodies markedly inhibited (70%) vitronectin degradation throughout the time course, suggesting that both integrins alpha vbeta 3 and alpha vbeta 5 mediate vitronectin internalization by astrocytoma cells plated on fibronectin. Studies were done in the presence of heparin to eliminate a heparan sulfate proteoglycan-mediated event, and heparin (100 µg/ml) inhibited degradation after 6 h by 40% (Fig. 1D), consistent with the results of other investigators (24).

Integrins Mediating Astrocytoma Cell Adhesion to Fibronectin, Collagen or Laminin

To determine the integrin receptor(s) mediating U-251MG cell adhesion to fibronectin, cell adhesion assays were performed as described (1, 29). Neutralizing mAb anti-integrin beta 1 completely blocked astrocytoma cell attachment to a purified fibronectin substrate, and neutralizing mAb anti-integrin alpha 5 and polyclonal anti-integrin alpha 5beta 1 IgG inhibited attachment to fibronectin by 80 and 70%, respectively, indicating that integrin alpha 5beta 1 is the major receptor mediating fibronectin attachment of these cells (Fig. 2A). In contrast, neutralizing mAbs anti-integrin alpha vbeta 3 or anti-integrin alpha vbeta 5 (data not shown) failed to inhibit cell adhesion to fibronectin. As controls, we determined the integrin receptor(s) mediating U-251MG astrocytoma cell attachment to collagen type I and laminin. Similar to the results of the fibronectin adhesion assay, neutralizing mAb anti-integrin beta 1 completely inhibited cell adhesion to collagen (Fig. 2B). Neutralizing mAb anti-integrin alpha 3 inhibited adhesion by 50%, and neutralizing mAb anti-integrin alpha 2 as well as the combination of mAbs anti-integrin alpha 2 and anti-integrin alpha 3 completely inhibited adhesion. However, mAb anti-integrin alpha 5 had no effect on cell attachment to collagen, indicating that beta 1 integrins, but not integrin alpha 5beta 1, mediate astrocytoma cell adhesion to collagen. Also, mAb anti-integrin alpha vbeta 3 failed to inhibit cell attachment to collagen. Similar to fibronectin- and collagen-adherent cells, mAb anti-integrin alpha vbeta 3 did not inhibit cell attachment to laminin (Fig. 2C). mAbs anti-integrin alpha 2, anti-integrin alpha 3, and anti-integrin alpha 5 also failed to inhibit cell attachment to laminin. In contrast, mAbs anti-integrin beta 1 and anti-integrin alpha 6 inhibited cell attachment to laminin by 70 and 60%, respectively (Fig. 2C). Integrins alpha 2beta 1, alpha 3beta 1, alpha 5beta 1, and alpha 6beta 1 were expressed on U-251MG cells assayed by FACS analysis (data not shown), consistent with the reported results of other investigators (31).


Fig. 2. Integrins mediating astrocytoma cell adhesion to fibronectin, collagen, or laminin. Cells were harvested with buffered EDTA, suspended in adhesion assay buffer, and incubated with 50 µg/ml of mouse IgG1(control) or the indicated antibodies for 30 min. Cells were then plated onto fibronectin (A), collagen (B) or laminin (C)-coated 96-well plates and allowed to attach for 45 min, followed by washing, fixation, and crystal violet staining as described under "Materials and Methods." Results are the mean ± S.E. of quadruplicate wells for each condition.
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[125I]Vitronectin Degradation by a Human Malignant Astrocytoma Cell Monolayer Plated on Collagen

Because integrin alpha 5beta 1 has been reported to collaborate with integrin alpha vbeta 3 in cell attachment and migration (18, 19), we investigated whether the absence of integrin alpha 5beta 1 ligation affected vitronectin internalization by integrin alpha vbeta 3. Similar to the results described for fibronectin-adherent cells, it was found that on adherence of U-251MG cells to collagen, 6-7% of the added [125I]vitronectin bound specifically to the cell monolayer and that this binding was inhibited by heparin and mAbs anti-integrin alpha vbeta 3 and anti-integrin alpha vbeta 5 (data not shown). The pattern, but not the rate, of [125I]vitronectin degradation on collagen was similar to that observed in cells plated on fibronectin. Degradation of [125I]vitronectin by cells plated on collagen was specific and integrin-dependent because it was inhibited by cold vitronectin or an RGD-containing peptide (data not shown). Chloroquine inhibited vitronectin degradation (Fig. 3A), indicating that, similar to cells plated on fibronectin, vitronectin degradation in cells plated on collagen occurred through a lysosomal pathway. [125I]Vitronectin degradation was identical whether cells were plated on collagen for 1 h or overnight. To determine whether integrin alpha vbeta 3 or alpha vbeta 5 mediated vitronectin internalization, studies with mAbs anti-alpha vbeta 3 and anti-alpha vbeta 5 were performed. In contrast to cells plated on fibronectin (Fig. 1C), mAb anti-alpha vbeta 3 had no effect, whereas mAb anti-alpha vbeta 5 inhibited degradation by 70% (Fig. 3B). Furthermore, when cells were treated with both antibodies, there was no increased inhibition over that obtained with mAb anti-alpha vbeta 5 alone. These results indicate that on collagen, astrocytoma cell integrin alpha vbeta 3 is not capable of mediating vitronectin internalization.


Fig. 3. Degradation of [125I]vitronectin by astrocytoma cells plated on collagen. In A, chloroquine (100 µM) was preincubated with a collagen-adherent cell monolayer prior to [125I]vitronectin addition. Control denotes degradation in the absence of inhibitor. Degradation was determined as described in Fig. 1. In B, cells were plated on collagen in serum-free media for 1 h, and treated as described in Fig. 1C. Data are shown as the mean ± S.E. from three separate experiments.
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[125I]Vitronectin Binding and Degradation by a Human Malignant Astrocytoma Cell Monolayer Plated on Laminin

In order to demonstrate that ligation of integrin alpha 3beta 1 or alpha 2beta 1 by collagen-adherent cells does not negatively regulate the activity state of integrin alpha vbeta 3, binding and degradation studies were performed on laminin-adherent cells. As shown in Fig. 4A, [125I]vitronectin bound maximally to a laminin-adherent cell monolayer after 6 h, at which time approximately 4% of the added label was specifically bound. Cold vitronectin (100 µg/ml) inhibited binding by 80% (data not shown), indicating that binding was specific. Similar to fibronectin-adherent cells, mAbs anti-integrin alpha vbeta 3 and anti-integrin alpha vbeta 5 inhibited binding to laminin by 40 and 50%, respectively, at 24 h, and heparin (100 µg/ml) also inhibited binding. Similar to the results seen on fibronectin and collagen, [125I]vitronectin degradation by laminin-adherent cells was detected within 1 h and the amount of vitronectin degraded continued to increase over the 24 h assay period (Fig. 4B). Cold vitronectin markedly inhibited [125I]vitronectin degradation, indicating this is a specific process. Degradation by laminin-adherent cells was also inhibited by chloroquine and an RGD-containing peptide (GRGDSP, 200 µM) (data not shown), indicating that degradation was lysosomal and integrin-mediated, respectively. A random hexapeptide (SPGDRG, 200 µM) failed to inhibit degradation. To determine the integrin(s) mediating vitronectin internalization by laminin-adherent cells, studies with mAbs anti-integrin alpha vbeta 3 and anti-integrin alpha vbeta 5 were performed. Similar to collagen-adherent cells, mAb anti-integrin alpha vbeta 3 failed to inhibit [125I]vitronectin degradation by laminin-adherent cells. mAb anti-integrin alpha vbeta 5 or the combination of mAbs anti-integrin alpha vbeta 3 and anti-integrin alpha vbeta 5 nearly abolished degradation, indicating that on laminin-adherent cells, integrin alpha vbeta 5 mediates the major portion of vitronectin internalization.


Fig. 4. Binding and degradation of [125I]vitronectin by astrocytoma cells plated on laminin. Heparin or chondroitin sulfate (100 µg/ml), mAbs (50 µg/ml), or unlabeled vitronectin (100 µg/ml) were added to a subconfluent cell monolayer plated on laminin in serum-free media prior to [125I]vitronectin addition. Control denotes [125I]vitronectin binding in the absence of inhibitor. In A, [125I]vitronectin binding was determined as described in Fig. 1. Data points are the average of quadruplicate wells ± S.E. [125I]Vitronectin degradation (B) was determined as described in Fig. 1. Data are shown as the mean ± S.E. from three separate experiments.
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Expression of Integrins alpha vbeta 3 and alpha vbeta 5 on Malignant Astrocytoma Cells Adherent to Fibronectin, Collagen, or Laminin

To determine whether the inability of integrin alpha vbeta 3 to mediate vitronectin internalization on collagen or laminin was due to differential expression of integrins alpha vbeta 3 and alpha vbeta 5 on interaction with the two substrates, FACS analysis was performed. Approximately 40% of the cells expressed integrin alpha vbeta 3 and greater than 90% of the cells expressed integrin alpha vbeta 5 with a similar mean fluorescent intensity, whether cells were plated on fibronectin (Fig. 5A), on collagen (Fig. 5B), or in 10% serum (data not shown). The cell attachment time (1 h or overnight) did not affect the results of the FACS analysis. When cells were adherent to laminin, 70% of the cells expressed integrin alpha vbeta 3 and 80% expressed integrin alpha vbeta 5 (Fig. 5C). These results indicate that differential expression of integrin alpha vbeta 3 cannot account for the inability of integrin alpha vbeta 3 to internalize vitronectin when these cells are plated on collagen or laminin.


Fig. 5. Expression of integrins alpha vbeta 3 and alpha vbeta 5 by astrocytoma cells adherent to fibronectin, collagen type I, or laminin. Cells previously plated on fibronectin in serum-free media (12 h) were reacted with 10 µg/ml anti-integrins alpha vbeta 3, alpha vbeta 5, mouse IgG (negative control), or rabbit anti-integrin alpha 5beta 1 IgG (A). Cells were washed and subsequently reacted with goat anti-mouse or goat anti-rabbit fluorescein isothiocyanate-conjugated IgG and analyzed on a FACS Sort analyzer. Identical results were obtained for cells plated on fibronectin for 1 h. B and C demonstrate integrin alpha vbeta 3 and alpha vbeta 5 receptor expression on cells previously plated on collagen in serum-free medium (1 h) or laminin in serum-free medium (12 h), respectively, and treated as described in A. The fluorescence above background is represented to the right of the bar. The data are presented as a histogram.
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Ligation State of Integrin alpha 5beta 1 Regulates Integrin alpha vbeta 3-Mediated [125I]Vitronectin Internalization

To definitively determine whether ligation of integrin alpha 5beta 1 was required for integrin alpha vbeta 3 to internalize vitronectin, the ability of astrocytoma cells plated on neutralizing mAbs anti-alpha 5 or anti-alpha 3 to degrade vitronectin was determined (Fig. 6). Attaching and spreading of cells on neutralizing anti-integrin mAbs has been described previously (30). The adhesion characteristics of the cells plated on the mAbs were similar. When astrocytoma cells were plated on mAb anti-alpha 5, mAb anti-integrin alpha vbeta 3 and alpha vbeta 5 each inhibited vitronectin degradation by 60% at 24 h (Fig. 6A) and in combination produced 80-100% inhibition; these findings were similar to those of fibronectin-adherent cells (Fig. 1C). At 24 h, the specific binding of [125I]vitronectin to the mAb anti-alpha 5-adherent monolayer was 5 ± 0.48%, and thus did not significantly differ from specific [125I]vitronectin binding to the fibronectin-adherent cell monolayer (Fig. 1A). In contrast, when cells were plated on mAb anti-alpha 3, only mAb anti-alpha vbeta 5 inhibited degradation (70% at 24 h; Fig. 6B). Furthermore, degradation in the presence of both mAb anti-alpha vbeta 3 and mAb anti-alpha vbeta 5 was similar to that obtained with anti-alpha vbeta 5 alone, indicating that integrin alpha vbeta 3 was unable to mediate vitronectin internalization when the astrocytoma cells were adherent to mAb anti-alpha 3.


Fig. 6. Degradation of [125I]vitronectin by an astrocytoma cell monolayer plated on mAbs anti-integrin alpha 5 or anti-integrin alpha 3. Wells were coated with mAb anti-integrin alpha 5 IgG (A) or mAb anti-integrin alpha 3 IgG (B), and cells were plated as described under "Materials and Methods." The cell monolayers were preincubated for 30 min with the indicated antibodies prior to [125I]vitronectin addition. Degradation was determined as described in Fig. 1. Results are the mean ± S.E. from three separate experiments.
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Rate of [125I]Vitronectin Degradation in Astrocytoma Cells Adherent to Fibronectin or Collagen

To determine the turnover rate of vitronectin by malignant astrocytoma cells plated on fibronectin or collagen, increasing concentrations of cold vitronectin were added along with [125I]vitronectin to the monolayer. [125I]Vitronectin degradation by cells adherent to fibronectin was saturable at 50 µg/ml vitronectin, and the rate of degradation determined from medium aliquots obtained between 5 and 60 min was approximately 3 ng/min (Fig. 7A). The rate of vitronectin degradation in fibronectin-adherent cells preincubated with mAb anti-integrin alpha vbeta 5 was similar to that obtained in the absence of antibody (Fig. 7A). In contrast, the rate of vitronectin degradation in fibronectin-adherent cells preincubated with mAb anti-integrin alpha vbeta 3 was significantly lower (0.10 ng/min (Fig. 7)) and was comparable to the rate of [125I]vitronectin degradation on collagen (0.35 ng/min (Fig. 7)). These data suggest that integrin alpha vbeta 3 internalizes and degrades vitronectin at a significantly higher rate than integrin alpha vbeta 5.


Fig. 7. Rate of [125I]vitronectin degradation by an astrocytoma cell monolayer plated on fibronectin or collagen. In A and B, cold vitronectin at the indicated concentration was added to a subconfluent cell monolayer of 100,000 cells plated on fibronectin or collagen in serum-free medium as described in Fig. 1. A shows the rate of degradation by cells adherent to fibronectin in the absence of antibody, cells adherent to fibronectin with added mAb anti-integrin alpha vbeta 5 (50 µg/ml), cells adherent to fibronectin with added mAb anti-integrin alpha vbeta 3 (50 µg/ml), and cells adherent to collagen in the absence of antibody. The rate of degradation for collagen-adherent cells in the absence of antibody and for fibronectin-adherent cells with added mAb anti-integrin alpha vbeta 3 are shown on an expanded scale in B. Trichloroacetic acid precipitations were performed on medium aliquots obtained between 5 and 60 min after [125I]vitronectin addition for fibronectin-adherent cells and between 1 and 3 h after [125I]vitronectin addition for collagen-adherent cells. Degradation was determined as described in Fig. 1. Results are the mean ± S.E. from three separate experiments.
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DISCUSSION

In this report, we demonstrate that altered [125I]vitronectin binds an astrocytoma cell monolayer adherent to fibronectin, collagen type I, or laminin in serum-free conditions in a specific and saturable manner. Subsequent to binding, astrocytoma cells specifically internalize and degrade vitronectin by a lysosomal pathway. Integrins alpha vbeta 3 and alpha vbeta 5 both mediate vitronectin internalization by cells plated on fibronectin; however, in cells adherent to collagen type I or laminin, integrin alpha vbeta 3 fails to mediate vitronectin internalization. Other investigators have recently shown that on melanoma cells, integrins alpha vbeta 3 and alpha vbeta 5 both mediate internalization of adenovirus (32). This internalization was mediated by cells in suspension, and it was recently suggested that integrin internalization occurs by different pathways depending on whether the cells are adherent or in suspension (32, 33). Our observations cannot be accounted for by substrate-dependent differential expression of alpha vbeta 3 on astrocytoma cells because FACS analysis demonstrated no decrease in integrin alpha vbeta 3 expression when the cells were adherent to collagen or laminin. Also, vitronectin was available on the cell surface of collagen-adherent cells for internalization because [125I]vitronectin binding to astrocytoma cells plated on collagen was comparable to that of cells plated on fibronectin (6-7% at 6 h). Less [125I]vitronectin bound to the laminin-adherent astrocytoma cell monolayer (4% at 6 h), which probably accounts for the reduced [125I]vitronectin degradation (13%) seen at 24 h. Integrin alpha 5beta 1 mediated astrocytoma cell attachment to fibronectin in large part, whereas it failed to participate in astrocytoma cell attachment to collagen type I or laminin, suggesting that ligation of integrin alpha 5beta 1 is necessary for integrin alpha vbeta 3 to internalize vitronectin.

To confirm that hypothesis, we performed vitronectin degradation assays on cells plated on mAb anti-alpha 5 or anti-alpha 3. We found that integrin alpha vbeta 3 mediated vitronectin internalization when the cells were adherent to mAb anti-alpha 5; however, integrin alpha vbeta 3 failed to mediate internalization when the cells were adherent to mAb anti-alpha 3, demonstrating that ligation of integrin alpha 5beta 1 is necessary for integrin alpha vbeta 3 to internalize vitronectin. The degradation assays on purified substrates, taken together with those on the purified mAbs, provide direct evidence that ligation of integrin alpha 5beta 1 positively regulates the activity state of integrin alpha vbeta 3. The fact that integrin alpha vbeta 3 failed to internalize vitronectin when cells were adherent to laminin and that laminin attachment is mediated largely by integrin alpha 6beta 1 without a detectable contribution from integrin alpha 3beta 1 indicate that our results are not due to a negative regulation of integrin alpha vbeta 3 by integrin alpha 3beta 1 or collagen. It is unclear why we observed less inhibition of vitronectin degradation with anti-integrin mAbs at early time points when the cells were plated on fibronectin or collagen. This has also been reported by other investigators (20). However, when the cells were plated on mAb anti-integrin alpha 5 and laminin, complete inhibition of vitronectin degradation was demonstrated at early time points with combined mAbs anti-integrin alpha vbeta 3 and alpha vbeta 5 and mAb anti-integrin alpha vbeta 5, respectively. Other investigators have shown that integrin alpha vbeta 3 ligation and complex formation with integrin-associated protein regulates integrin alpha 5beta 1-mediated fibronectin phagocytosis in K562 cells (16, 17). We have yet to determine whether integrin-associated protein regulates integrin alpha vbeta 3 internalization by the astrocytoma cells. Taken together, the results suggest that "cross talk" between integrins alpha 5beta 1 and alpha vbeta 3 can potentially occur in both directions, depending on the cell type, the ligation status of the receptor, complex formation with integrin-associated protein, and probably other conditions.

The rate of vitronectin degradation inhibitable by mAbs anti-integrin alpha vbeta 3 and alpha vbeta 5 on astrocytoma cells adherent to fibronectin was approximately 3 ng/min, comparable to that reported in fibroblasts (24) and approximately 10-fold higher than the rate of vitronectin turnover by astrocytoma cells plated on collagen and inhibitable by mAb anti-integrin alpha vbeta 5. This suggests that integrin alpha vbeta 3 internalizes vitronectin at a significantly higher rate than integrin alpha vbeta 5 on astrocytoma cells. These data suggest that integrins alpha vbeta 3 and alpha vbeta 5 are regulated differently on astrocytoma cells, consistent with their different cytoplasmic tail sequences and the differences in regulation reported by other investigators on other cell types (4, 9-11, 34, 35). The data also are consistent with the hypothesis that integrins alpha vbeta 5 and alpha vbeta 3 mediate different vitronectin-dependent functions in malignant astrocytomas in vivo.

In summary, these studies demonstrate that malignant astrocytoma cells are capable of remodeling their extracellular matrix through the internalization and lysosomal degradation of altered vitronectin. In astrocytoma cells this process is constitutively mediated by integrin alpha vbeta 5, and integrin alpha vbeta 3 participation in vitronectin internalization is dependent on ligation of integrin alpha 5beta 1. These results are physiologically relevant because beta 1 integrins are predominantly expressed by perivascular astrocytoma cells (1), and in these experiments, the cells were adherent to matrix proteins recognized by beta 1 integrins. Our data indicate that integrin alpha vbeta 3 internalization of its ligand(s) contributes to the remodeling of the matrix by astrocytoma cells and that integrin alpha vbeta 3 requires a signal from integrin alpha 5beta 1 to mediate this process.


FOOTNOTES

*   This work was supported by Grant CA59958 from the National Institutes of Health-National Cancer Institute (to C. L. G.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: University of Alabama at Birmingham, LHRB 567, 701 South 19th St., Birmingham, AL 35294. Tel.: 205-934-4243; Fax: 205-975-9927.
1    The abbreviations used are: mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; FACS, fluorescence-activated cell sorter.

Acknowledgments

We thank J. Robert Grammer for technical assistance and Dr. John R. Couchman (University of Alabama at Birmingham, Birmingham, AL) for reviewing the manuscript.


REFERENCES

  1. Gladson, C. L., and Cheresh, D. A. (1991) J. Clin. Invest. 88, 1924-1932 [Medline] [Order article via Infotrieve]
  2. Gladson, C. L., Wilcox, J. N., Sanders, L., Gillespie, G. Y., and Cheresh, D. A. (1995) J. Cell Sci. 108, 947-956 [Abstract/Free Full Text]
  3. Hynes, R. O. (1992) Cell 69, 11-25 [Medline] [Order article via Infotrieve]
  4. Gladson, C. L., and Cheresh, D. A. (1994) in Integrin: The Biologic Problem (Takada, Y., ed), pp. 83-89, CRC Press, Boca Raton, FL
  5. Faull, R. J., and Ginsberg, M. H. (1995) Stem Cells 13, 38-46 [Abstract]
  6. Liaw, L., Skinner, M. P., Raines, E. W., Ross, R., Cheresh, D. A., Schwartz, S. M., and Giachelli, C. M. (1995) J. Clin. Invest. 95, 713-724 [Medline] [Order article via Infotrieve]
  7. Montgomery, A. M., Reisfeld, R. A., and Cheresh, D. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8856-8860 [Abstract]
  8. Smith, J. W., Vestal, D. J., Irwin, S. V., Burke, T. A., and Cheresh, D. A. (1990) J. Biol. Chem. 265, 11008-11013 [Abstract/Free Full Text]
  9. Klemke, R. L., Yebra, M., Bayna, E. M., and Cheresh, D. A. (1994) J. Cell Biol. 127, 859-866 [Abstract]
  10. Wayner, E. A., Orlando, R. A., and Cheresh, D. A. (1991) J. Cell Biol. 113, 919-929 [Abstract]
  11. Conforti, G., Calza, M., and Beltran-Nunez, A. (1994) Cell Adhes. Commun. 1, 279-293 [Medline] [Order article via Infotrieve]
  12. Clark, E. A., and Brugge, J. S. (1995) Science 268, 233-239 [Medline] [Order article via Infotrieve]
  13. Bartfeld, N. S., Pasquale, E. B., Geltosky, J. E., and Languino, L. R. (1993) J. Biol. Chem. 268, 17270-17276 [Abstract/Free Full Text]
  14. Sepp, N. T., Li, L.-J., Lee, K. H., Brown, E. J., Caughman, S. W., Lawley, T. J., and Swerlick, R. A. (1994) J. Invest. Dermatol. 103, 295-299 [Abstract]
  15. Vuori, K., and Ruoslahti, E. (1994) Science 266, 1576-1578 [Medline] [Order article via Infotrieve]
  16. Blystone, S. D., Graham, I. L., Lindberg, F. P., and Brown, E. J. (1994) J. Cell Biol. 127, 1129-1137 [Abstract]
  17. Blystone, S. C., Lindberg, F. P., LaFlamme, S. E, and Brown, E. J. (1995) J. Cell Biol. 130, 745-754 [Abstract]
  18. Bauer, J. S., Schreiner, C. L., Giancotti, F. G., Ruoslahti, E., and Juliano, R. L. (1992) J. Cell Biol. 116, 477-487 [Abstract]
  19. Stephens, L. E., Sonne, J. E., Fitzgerald, M. L., and Damsky, C. H. (1993) J. Cell. Biol. 123, 1607-1620 [Abstract]
  20. Panetti, T. S., and McKeown-Longo, P. J. (1993) J. Biol. Chem. 268, 11492-11495 [Abstract/Free Full Text]
  21. Panetti, T. S., Wilcox, S. A., Horzempa, C., and McKeown-Longo, P. J. (1995) J. Biol. Chem. 270, 18593-18597 [Abstract/Free Full Text]
  22. Russell, D. S., and Rubinstein, L. J. (eds) (1989) Pathology of Tumors of the Central Nervous System, 5th Ed., pp. 83-289, Williams and Wilkins, Baltimore
  23. Tomasini, B. R., and Mosher, D. F. (1991) Prog. Hemostasis Thromb. 10, 269-306 [Medline] [Order article via Infotrieve]
  24. Panetti, T. S., and McKeown-Longo, P. J. (1993) J. Biol. Chem. 268, 11988-11993 [Abstract/Free Full Text]
  25. Yatohgo, T., Izumi, M., Kashiwagi, H., and Hayashi, M. (1988) Cell Struct. Funct. 13, 281-292 [Medline] [Order article via Infotrieve]
  26. Fraker, P. J., and Speck, J. C. (1978) Biochem. Biophys. Res. Commun. 80, 849-857 [Medline] [Order article via Infotrieve]
  27. Schrappe, M., Klier, F. G., Spiro, R. C., Waltz, T. A., Reisfeld, R. A., and Gladson, C. L. (1991) Cancer Res. 51, 4986-4993 [Abstract]
  28. McKeown-Longo, P. J., Hanning, R., and Mosher, D. F. (1984) J. Cell Biol. 98, 22-28 [Abstract]
  29. Filardo, E. J., Brooks, P. C., Deming, S. L., Damsky, C., and Cheresh, D. A. (1995) J. Cell Biol. 130, 441-450 [Abstract]
  30. Werb, Z., Tremble, P. M., Behrendtsen, O., Crowley, E., and Damsky, C. H. (1989) J. Cell Biol. 109, 877-889 [Abstract]
  31. Vogel, B. E., Tarone, G., Giancotti, F. G., Gailit, J., and Ruoslahti, E. (1990) J. Biol. Chem. 265, 5934-5937 [Abstract/Free Full Text]
  32. Wickham, T. J., Mathias, P., Cheresh, D. A., and Nemerow, G. R. (1993) Cell 73, 309-319 [Medline] [Order article via Infotrieve]
  33. Dalton, S. L., Scharf, E., Briesewitz, R., Marcantonio, E. E., and Assoian, R. K. (1995) Mol. Biol. Cell 6, 1781-1791 [Abstract]
  34. Ramaswamy, R., and Hemler, M. E. (1990) EMBO J. 9, 1561-1568 [Abstract]
  35. McLean, J. W., Vestal, D. J., Cheresh, D. A., and Bodary, S. C. (1990) J. Biol. Chem. 265, 17126-17131 [Abstract/Free Full Text]

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