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Biochemistry |
vß3 as Two Receptors for the 3(IV) Chain of Type IV Collagen on Tumor Cells1
Department of Medicine, University of Pennsylvania School of Medicine [T. A. S., N. A. K.] and The Connective Tissue Research Institute, University City Science Center, Philadelphia, Pennsylvania 19104 [T. A. S., Z. Z., N. A. K.]; and the Laboratoíre de Biochimie Medicale et Biologie Moleculaire, University of Reims-Champagne-Ardenne, CNRS UPRESA 6021, IFR 53 Biomolecules, UFR Médicine, 51095 Reims, France [S. P., A. F., G. B., J-C. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 chain of basement membrane collagen (COL IV), comprising residues 185–203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation independently of its ability to promote cell adhesion; these properties require the presence of the triplet -SNS- at residues 189–191 (J. C. Monboisse et al., J. Biol. Chem., 269: 25475–25482, 1994; J. Han et al., J. Biol. Chem., 272: 20395–20401, 1997). More recently, we demonstrated that native COL IV and -SNS-containing synthetic peptides (10 µg/ml) added to culture medium inhibit the proliferation of not only melanoma cells but also breast, pancreas, and stomach tumor cells up to 82% and prostate tumor cells by 15%. This inhibition was shown to be dependent on a COL IV- or peptide-induced increase in intracellular cAMP (T. A. Shahan et al., Connect. Tissue Res., 40: 221–232, 1999). Attempts to identify the putative receptor(s) on tumor cells led to the isolation of five proteins (Mr 33,000, 52,000, 72,000, 95,000, and 250,000) from melanoma and prostate cells by affinity purification with the 3(IV)179–208 peptide. The Mr 52,000, 95,000, and 250,000 proteins were shown to be CD47/integrin-associated protein(IAP), the integrin ß3 subunit, and the vß3 integrin complex, respectively. The Mr 33,000 and 72,000 proteins have not yet been identified. To confirm the specificity of ligand binding to the receptors, cell membranes from either melanoma or prostate tumor cells were pretreated with the unlabeled ligand 3(IV)187–191 (-YYSNS-); alternatively, the peptide was pretreated with a peptide-reactive monoclonal antibody (A5D7) before receptor isolation. These treatments inhibited the purification of CD47/IAP, the integrin ß3 subunit, and the vß3 integrin complex from tumor cells. Furthermore, cells treated with CD47/IAP- or the vß3 integrin-reactive antibodies prevented the 3(IV)185–203 peptide from inhibiting cell proliferation and the subsequent rise in intracellular cAMP. Pretreating cells with the 3(IV)187–191 (-YYSNS-) peptide also inhibited their adhesion to the 3(IV)185–203 peptide substrate, whereas the inactive 1(IV)185–203 peptide, from the same region of the 1 chain as the 3(IV)185–203 peptide, had no effect. Incubation of cells with either CD47/IAP and/or vß3 integrin-reactive antibodies inhibited their adhesion to the 3(IV)185–203 peptide, whereas antibodies to the ß1 and ß2 integrin subunits were without effect. These data suggest that ALC-COL IV, through its 3(IV) chain, inhibits tumor cell proliferation using the receptors CD47/IAP and the vß3 integrin. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1–6) different chains. COL IV molecules interact at their NH2 terminus to form tetrameres. Such tetrameres come together at their noncollagenous domains (NC1) and by side-to-side associations to form complex polymeric structures that interact with other basement membrane components such as laminins, entactin, and proteoglycans (1
, 2) .
The cell regulatory nature of ALC-COL IV was demonstrated in our laboratories (3)
by showing that ALC-COL IV treatment inhibited the activation of phorbol myristate acetate- or f-Met-Leu-Phe-stimulated polymorphonuclear leukocytes, through an increase in icAMP. The inhibitory activity of ALC-COL IV was shown to reside within residues 185–203 of the NC1 domain of the 3(IV) chain. Using synthetic peptides in which the sequence of amino acids were altered, it was demonstrated that the triplet -SNS- was an absolute requirement for the inhibitory activity. COL IV from the EHS tumor, which lacks the 3(IV) chain (4)
, failed to inhibit phorbol myristate acetate-stimulated PMN activation (3)
. Later, Han et al. (5)
demonstrated that a substrate composed of the 3(IV)185–203 peptide promoted melanoma cell adhesion. The same peptide also inhibited cell proliferation but in a manner independent of adhesion. Both cell adhesion and the inhibition of proliferation depended on the presence of the triplet -SNS-, and these processes were also inhibited by treatment of the peptides with the 3(IV)185–203 reactive mAb (A5D7). In a recent study, Shahan et al. (6)
demonstrated that ALC-COL IV and its synthetic peptides differentially inhibited breast, pancreas, and stomach tumor cell proliferation. The inhibition of tumor cell proliferation by ALC-COL IV and the synthetic peptide was shown to be mediated through an increase in icAMP and a cAMP-dependent protein kinase (6)
. More recently, 3(IV)185–203 binding proteins, isolated from PMN leukocytes, were identified as CD47/IAP and the vß3 integrin and shown to be essential to the inhibition of PMN activation.4
Using affinity chromatography, we have identified receptors for the 3(IV)179–208 and the 3(IV)185–203 synthetic peptides as CD47/IAP and the vß3 integrin, respectively, from tumor cells. Pretreatment of tumor cells with receptor-specific antibodies inhibited the biological effects of the 3(IV)185–203 peptide. This work describes a novel function for CD47/IAP and the vß3 integrin in the control of tumor cell proliferation by the 3(IV) chain of COL IV.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vß3 integrin (MAB1976), and to CD47 (MAB1796) were purchased from Chemicon (Temecula, CA.). The CD47/IAP reactive mAb, B6H12, was obtained from Dr. E. Brown at the University of California, San Francisco. A hybridoma producing mAb B6H12 was latter obtained from American Type Culture Collection (Bethesda, MD; Ref. 7
). The integrin ß2 subunit-reactive mAb (YM1110) was purchased from Accurate (Westbury, NY). The A5D7 mAb, which reacts with the 3(IV)185–203 peptide, and the D12H5 mAb, which is peptide or COL IV unreactive, were prepared in our laboratory (5)
. In this study, it was demonstrated that the A5D7 mAb was highly specific for the -SNS- triplet at position 189–191 on the 3 chain (5)
. The polyvinylidene fluoride transfer membrane was purchased from MSI Separations (Westboro, MA.), and all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO.).
Tumor Cell Culture.
The melanoma cell line W-164 was obtained from Dr. Meenhard Herlyn at the Wistar Institute (Philadelphia, PA; Refs. 8
and 9
). The prostate (DU-145) tumor cell line was obtained from the American Type Culture Collection (Bethesda, MD). The melanoma cell line was cultivated in medium MCDB-153 (78%) with L-15 medium (10%), tryptose phosphate broth (10%), FBS (2%), and 50 units/ml insulin, as described previously by Herlyn et al. (8
, 9)
. The HT-144 melanoma cell line was obtained from Dr. P. Braquet (Bioinova, France) and cultivated in McCoys 5a medium. The prostate tumor cell line was cultivated in Eagle’s MEM with 10% FBS. The ovarian tumor cell lines (OV10) were obtained from Dr. Eric Brown at the University of California, San Francisco. The OV10 cell line, which is CD47/IAP negative, was made CD47/IAP positive by transfection. The line designated OV10(-) is CD47/IAP- and vß3+, and the OV10(+) is CD47/IAP+ and vß3+ (10)
. The above cells were cultivated in Iscove’s modified Dulbecco’s medium, 10% FBS, and Genticin (10 µg/ml) and counted before plating using either a Coulter counter (Model ZM; Hialeah, FL) or a Neubauer hemocytometer.
Type IV Collagen Isolation from Bovine ALC and Preparation of Its Synthetic Peptides.
COL IV was extracted from ALC according to the protocol of Brinker et al. (11)
. EHS-COL IV, which contains only the 1 and 2 chains, was a gift from Dr. Hynda Kleinman of the NIH (12)
. Synthetic peptides corresponding to the primary sequences of regions from the NC1 domain of COL IV (13)
were synthesized and purified at the Protein Core Facility at the University of Pennsylvania (Philadelphia, PA) essentially according to the method of Barany and Merrifield (14)
. The peptides were synthesized using an Applied Biosystems automated peptide synthesizer (Foster City, CA) and dissolved in medium without FBS (overnight at 4°C) or with 10 µl DMSO and diluted to the appropriate concentration in medium with FBS (2%). Vehicle control consisted of only medium in which the peptides were diluted. Primary sequences of synthetic peptides used in this study are listed in Table 1
. The primary sequence for the TSP peptide 7N3, which was demonstrated to bind to CD47/IAP, was obtained from Gao et al. (15)
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To further clarify the effect of CD47/IAP and the vß3 integrin as receptors for the 3(IV) chain peptides, reactive antibodies on the antiproliferative activity of the 3(IV)185–203 peptide, cells were incubated with the respective antibodies for 30 min at 37°C and washed two times with medium before the addition of the peptide.
Intracellular Measurement of cAMP.
Isolation of icAMP from tumor cells was performed using a modified procedure of Mayer et al. (17)
and measured using a cAMP-specific ELISA as described previously (6)
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Isolation of Specific Receptors for the 3(IV)179–208 Peptide from Tumor Cells.
Receptors specific for the 3(IV)179–208 peptide were isolated by peptide-specific affinity chromatography using a modified procedure of Wennogle et al. (18)
. Cells were released from adherence by scraping with a rubber policeman in HBSS, centrifuged (15,000 rpm for 30 min at 10°C), and resuspended in ice-cold RIB containing 20 mM Tris base (pH 7.4), 1 mM polymethylsulfonyl fluoride, 2 mM benzamidine, 1 mM aprotinin, 4 mM chymostatin, and 20 µM leupeptin. Cell membranes were prepared by sonication on ice (3 x 1 min, 40% power) and centrifuged (as described above); the supernatant was discarded, and the membranes were resuspended in HBSS. The peptide was biotinylated by adding 100 µg of the peptide and 50 µl of DMSO to 2 mg of NHS-biotin, followed by the addition of 200 µl of RIB (30 min at 37°C). The biotinylated peptide was triple purified using a 3000 NMW column (Millipore, Bedford, MA) and resuspended in 200 µl of HBSS. To 3 ml of the cell membrane preparation (5 x 108 cells/ml) was added 50 µg of the biotinylated 3(IV)179–208 synthetic peptide and incubated for 1 h at 34°C. Alternatively, the same biotinylated peptide was also pretreated with the peptide reactive mAb (A5D7, undiluted) and the D12H5 mAb (data not shown); likewise, cell membranes were also pretreated, either with the unlabeled 3(IV)187–191 (-YYSNS-) or the 1(IV)185–203 peptides before addition of the biotinylated peptide to determine their effect on receptor isolation. The receptor-ligand complexes were then released from the cell membrane preparation by incubation with RIB + detergent, which contained RIB appended with 50 mM N-dodecyl-N,N(dimethylammonio)butyrate, 1.5% glycerol, and 2% NP-40 for 1 h at 37°C and centrifuged at 95,000 rpm for 3 h at 15°C. The complexes were first precleared with washed Sepharose beads alone (1 h at 37°C). Then 15 µl of washed streptavidin-coated Sepharose beads were added and incubated overnight with rotation at 4°C and centrifuged (2000 rpm for 20 min). The beads were washed three times with HBSS and incubated with 4.5 M KCN for 45 min at 37°C to detach streptavidin-Sepharose beads from the biotinylated ligand-receptor complex. The isolated proteins were further purified with a 5000 NMW column to a final volume of 145–175 µl. The ligand receptor complex was then separated by nonreducing SDS-PAGE (8.5%) and transferred to polyvinylidene fluoride membranes. The blots were probed with either HRPO-labeled streptavidin or integrin-specific antibodies; antibodies were detected with class-specific HRPO-labeled antibodies, developed with enhanced chemiluminescence, and exposed to X-ray film (Fuji, Tokyo, Japan).
Adhesion Analysis.
Untreated high protein binding 96-well plates (3590; Corning-Costar Scientific, Aston, MA) were treated with COL IV or bioactive synthetic peptides (25 µg/ml) suspended in coating buffer (0.1 M Tris-HCl, pH 8.0), incubated for 24 h (4°C), and washed two times with PBS. To each well was added 150 µl of blocking buffer (HBSS with 2% BSA, 10 mM HEPES at pH 7.4) and incubated for 1 h at room temperature. Melanoma cells near confluence were collected by treatment with 3.0 mM EDTA in HBSS, washed twice in medium with 1% BSA, and incubated for 30 min before use. Cells were then plated (1 x 105 cells/well) in tumor medium with 1% BSA and 25 µM HEPES (pH 7.2). To test the effects of the anti-integrin antibodies or peptide treatment, cells were incubated for 20 min at 37°C and washed prior to addition of cells and then incubated for 60 min at 37°C after addition to the wells. To remove unattached cells, each well was filled with PBS and incubated on a shaking platform for 30 min. Attached cells were fixed with 3% paraformaldehyde in PBS (15 min at room temperature) and stained with 0.5% crystal violet in 20% methanol/H2O (30 min at room temperature). Wells were washed (four times) with H2O to remove excess dye, and the bound dye was solubilized in 1% SDS. The dye content was measured on a Bio-Tek 96-well plate reader (Model EL 340; Winooski, VT) at 605 nm.
Detection of 3 Peptide Binding to CD47/IAP and the vß3 Integrin on Melanoma Cells by Flow Cytometry.
To further prove that the peptide binds to CD47/IAP and the vß3 integrin, melanoma cells (1 x 106 cells) were incubated with either unlabeled 3(IV)187–191 peptide (-YYSNS-) or the CD47/IAP (1:100) and/or vß3 integrin-reactive mAbs (1:75), followed by incubation with the biotinylated 3(IV)185–203 peptide (20 µg/ml) that was produced in the same manner as described in "Materials and Methods." After 30 min, the cells were washed three times and then incubated with the FITC-labeled anti-biotin mAb (1:75). Cells were analyzed by flow cytometry.
Statistical Analysis.
Data were analyzed using Sigmaplot (Jandel Scientific Software, San Rafael, Ca) and were expressed as mean ± SD. To evaluate the effect of peptide-reactive mAbs on cell proliferation and icAMP levels, comparisons were performed using the Tukey’s honest significant difference test. Data were considered statistically significant if qs were <0.05. The OV10 adhesion data were analyzed using a modified multiple inference Student’s t test. Data were considered statistically significant if ps were <0.05 (19)
.
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RESULTS |
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3(IV)179–208 Peptide-specific Receptors on Melanoma Cells.3(IV)185–203 binds to specific receptors on tumor cells. Receptors specific for the 3(IV)179–208 peptide were isolated by affinity chromatography as described in "Materials and Methods." Five proteins (Mr 33,000, 52,000, 72,000, 95,000 and 250,000) were detected in the receptor preparation (Fig. 1
, blot 1). Using Western blot analysis, the Mr 250,000 protein was identified as the vß3 integrin (Fig. 1
, blot 2), the Mr 95,000 protein as the ß3 integrin subunit (Fig. 1
, blot 3), and the Mr 52,000 protein as CD47/IAP (Fig. 1
, blot 4); the other two bands have not been identified to date. As reported by Fawzi et al.,4
both CD47/IAP and the vß3 integrin have been identified recently as the receptors for the 3(IV)185–203 peptide on human PMN leukocytes. Monoclonal antibodies reactive with the ß1 and ß2 integrin subunits failed to react by Western blot analysis with any of the proteins isolated in the membrane preparations; however, we have demonstrated their presence on melanoma and pancreatic tumor cells by Western blot analysis of whole-cell lysates (data not shown). To confirm that the isolated proteins bound specifically to the 3(IV)185–203 peptide and required the presence of the -SNS- triplet, the cell membranes were pretreated with either unlabeled 3(IV)187–191(-YYSNS-) (Fig. 1
, Lane C) or with the 1(IV)185–203 peptide (Fig. 1
, Lane D). Pretreatment of the cell membranes with the unlabeled 3(IV)187–191-(-YYSNS-) peptide inhibited the purification of CD47/IAP, the vß3 integrin, and the ß3 integrin subunit but had no effect on the Mr 33,000 or Mr 74,000 bands. Furthermore, pretreatment with the 1(IV)185–203 peptide, which does not inhibit proliferation, had no effect on the purification of any bands. To further confirm that the proteins were binding specifically to the 3(IV)179–208 peptide, the latter was incubated with the peptide-reactive A5D7 mAb prior to receptor isolation. Treatment with the mAb inhibited the isolation of all bands (Fig. 1
, Lane G). In addition, the D12H5 control mAb, which does not react with the above peptide, had no effect on the purification of any of these proteins (data not shown). Receptor purification and identification were also performed using native ALC-COL IV, as ligand, and the HT-144 melanoma cell line. Both CD47/IAP and the vß3 integrin were identified from a number of proteins that were isolated (data not shown).
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3(IV)179–208 synthetic peptide, receptor-specific mAbs were tested for their ability to diminish the inhibitory activity of ALC-COL IV and the 3(IV)185–203 peptide. Table 2
shows that pretreatment of melanoma cells with either CD47/IAP- and/or the vß3 integrin-reactive mAbs significantly reduced the ability of ALC-COL IV or the 3(IV)185–203 synthetic peptide to inhibit cell proliferation or cause an increase in icAMP. Incubation of cells with either the integrin ß1 or ß2 subunit-reactive mAbs had no effect on the biological activities of ALC-COL IV or the bioactive peptide (data not shown). Receptor-specific Abs by themselves had no effect on proliferation. The TSP-derived peptide 7N3 (FIRVVMYEGKK), which was shown to bind to CD47/IAP (15)
, was tested for its ability to inhibit melanoma cell proliferation. This peptide inhibited proliferation on the average of 22% with an increase in icAMP of 0.97 ± 0.31 from control levels of 0.76 ± 0.17 pmol/l x 106 cells ± SD. However, pretreatment of melanoma cells with anti-CD47/IAP and -vß3 mAbs alone or in combination did not significantly alter the effect of the TSP peptide on cell proliferation or the rise in icAMP. To further investigate the effect of CD47/IAP and vß3 on the ability of ALC-COL IV and its peptides to inhibit tumor cell proliferation, the CD47/IAP+ and CD47/IAP- ovarian tumor cell lines (OV10+ and OV10-) were treated with receptor reactive mAbs before analysis. OV10- cells treated with the CD47/IAP-reactive mAb had no effect on the ability of the 3(IV)185–203 peptide to inhibit cell proliferation (Fig. 2A)
; however, treatment of the cells, which are vß3 integrin positive, with the vß3 integrin-reactive mAb prevented the peptide from inhibiting proliferation, allowing near normal proliferation (Fig. 2A)
. As expected, treatment with the 3(IV)185–203 peptide alone inhibited cell proliferation (Fig. 2A)
, whereas treatment with the inactive peptide 3(IV)L5 1–15 had no effect (Fig. 2A)
. Treatment of the OV10+ cell line with either CD47/IAP- or vß3 (Fig. 2B)
-reactive mAbs partially inhibited the antiproliferative effect of the 3(IV)185–203 peptide, whereas treatment of the cells with both mAbs completely suppressed its inhibitory activity (Fig. 2B)
. As with the other tumor cell lines, ALC-COL IV and 3(IV)185–203 differentially increased icAMP in both OV10- and OV10+ cell lines. Increases in the levels of icAMP in the OV10 cell lines were inversely proportional to cell proliferation levels in the presence of COL IV or peptides (data not shown), as was seen with melanoma cell proliferation (6)
. Proliferation of the OV10+ cell line was inhibited 47% by 3(IV)185–203 (10 µg/ml) as compared with the OV10- cell line, which was only inhibited 28% (Fig. 2)
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3(IV)187–191 Peptide or Receptor-specific Antibodies Inhibit Cell Adhesion to the 3(IV)185–203 Peptide.3(IV) peptides specifically bind to CD47/IAP and the vß3 integrin, we studied the ability of different peptides and receptor-reactive antibodies to inhibit melanoma cell adhesion to the 3(IV)185–203 synthetic peptide. Fig. 3A
shows that melanoma cells pretreated with the 3(IV)187–191 peptide (-YYSNS-) at 100 ng/ml almost completely inhibited cell adhesion to the 3(IV)185–203 substrate. In comparison, melanoma cells treated with the 3(IV)190–203 peptide (-NS- at 190–191, without -S- at 189) failed to inhibit adhesion (Fig. 3A)
. The TSP-derived peptide 7N3, which has been shown to bind specifically to CD47/IAP by Gao et al. (15)
, inhibited cell adhesion to the 3(IV)185–203 substrate by 43% at 100 ng/ml (Fig. 3A)
. Also tested was the ability of receptor-reactive antibodies to influence melanoma cell adhesion to the 3(IV)185–203 substrate. Cells treated with the CD47/IAP-reactive mAb B6H12, at a 1:10 dilution, inhibited adhesion on average 31% (Fig. 3A)
, and treatment with the vß3 integrin-reactive antibody inhibited adhesion by 42% (Fig. 3A)
, whereas pretreatment of the cells with both antibodies inhibited adhesion on average 92% (Fig. 3A)
. All active agents were shown to inhibit adhesion in a concentration-dependent manner.
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3(IV)185–203 peptide inhibited proliferation of the prostate tumor cell line (DU-145) at levels seen with the melanoma cell lines (6)
. For comparison, the same adhesion studies were performed on the prostate tumor cell line. Cells pretreated with the 3(IV)187–191 peptide at 10 ng/ml inhibited cell adhesion to the 3(IV)185–203 peptide 93% (Fig. 3B)
, whereas treatment of the cells with the 3(IV)190–203 peptide had no effect on cell adhesion (Fig. 3B)
. At 1000 ng/ml, the TSP peptide inhibited adhesion 48% (Fig. 3B)
. Treatment with the CD47/IAP reactive antibody at a 1:100 dilution inhibited cell adhesion 41% (Fig. 3B)
, and at 1:10, the vß3 integrin-reactive antibody inhibited adhesion 56% (Fig. 3B)
. In comparison, treatment of the cells with both vß3 integrin- and the CD47/IAP-reactive antibodies, each at a 1:100 dilution, inhibited adhesion 82% (Fig. 3B)
. In both cases, the use of antibody dilutions higher than 1:100 failed to significantly change adhesion.
As shown in Fig. 2
, proliferation of the ovarian tumor cell lines OV10+ and OV10- was differentially inhibited by treatment with the ALC-COL IV or the 3(IV)185–203 peptide. This inhibition was blocked by both CD47/IAP (in the OV10+ cell line only) and vß3-reactive mAbs.
To further clarify the interaction of the peptide with both CD47/IAP and vß3, an adhesion assay similar to the one described above was designed. The OV10- cells were shown to bind to the peptide on the average 28% less efficiently than the OV10+ cells. As expected, the CD47/IAP mAb had no effect on cell adhesion of the OV10- cell line to the 3(IV)185–203 peptide; however, it did inhibit adhesion of the OV10+ cell line (Table 3)
. Incubation of OV10- cells with the vß3-reactive mAb alone almost completely inhibited cell adhesion and partially inhibited adhesion of the OV10+ cells, whereas addition of the CD47/IAP mAb completely inhibited adhesion. As observed with the melanoma cells, pretreatment of the OV10 cells with the 3(IV)187–191 (-YYSNS-) peptide inhibited adhesion of both cell lines by about 86% (Table 3)
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vß3 integrin, a competition binding assay was designed in which a biotinylated 3(IV)185–203 peptide competed for binding on melanoma cells with other related unlabeled synthetic peptides or receptor-specific antibodies as described in "Materials and Methods." As seen in Fig. 4D
, melanoma cells bound little biotinylated 3(IV)185–203 peptide when they were pretreated with unlabeled soluble 3(IV)187–191 (-YYSNS-) peptide. In contrast, cells pretreated with the 3(IV)190–203 (containing only -NS-) still bound biotinylated peptide, as is evident from the increase in mean fluorescent intensity (Fig. 4E)
. Cells pretreated with the receptor-reactive antibodies to CD47/IAP and/or to vß3 integrin bound considerably less biotinylated peptide (Fig. 4, F–H)
. For a negative control, cells were run alone (Fig. 4A)
and with the biotinylated peptide alone (Fig. 4B)
; for positive control, cells were incubated with the biotinylated 3(IV)185–203 peptide, followed by incubation with the FITC-labeled, biotin-reactive mAb (Fig. 4C)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 chain of COL IV promoted melanoma cell adhesion and inhibited proliferation of melanoma cells independent of its ability to promote cell adhesion (3)
. More recently, we have demonstrated that ALC-COL IV and the 3(IV) peptides inhibit tumor cell proliferation by increasing icAMP (6)
. In the present study, we have extended this work by identifying the receptors that bind the 3(IV)179–208 synthetic peptide, and presumably the -SNS- triplet at position 189–191, on tumor cells as CD47/IAP and the integrin vß3 (Fig. 1)
. In parallel studies using human neutrophils, we have demonstrated that the 3(IV)185–203 peptide binds the same two proteins (Fawzi et al.4
). In our laboratory, two other proteins of Mr 72,000 and Mr 33,000 were also isolated, and the Mr 72,000 protein has been recently identified as matrix metalloproteinase 2 (unpublished data). The binding of matrix metalloproteinase 2 to the 3(IV)179–208 peptide was shown to be nonspecific because the 3(IV)189–191 peptide does not inhibit the binding and isolation of the protein. In addition to the Mr 72,000 band, we also identified an Mr 85,000 band as the integrin ß3 subunit alone, part of the integrin Vß3. The mechanism by which the vß3 integrin dissociates is unknown; however, studies are being designed to answer this question. To date, we have identified the Mr 250,000 band as the integrin Vß3 by both monoclonal antibody and NH2-terminal sequence analysis.
To further investigate the involvement of these receptors in the regulation of cell function by COL IV and its peptides, a number of functional assays were performed using various receptor-specific antibodies and synthetic peptides (Fig. 2
; Table 2
). We have demonstrated that ALC-COL IV and synthetic peptides derived from the NC1 domain of the 3(IV) chain differentially inhibit proliferation of several epithelial tumor cell types, including tumor cell lines from breast, stomach, pancreas, and prostate. It was also noted that COL IV from EHS tumor failed to inhibit tumor cell proliferation (6)
, likely because it lacks the 3(IV) chain (4)
. Although the prostate tumor cell line expresses both CD47/IAP and the vß3 integrin, it responds poorly to the peptide in proliferation experiments, which may be due to a lower receptor density on these cells, as evidenced from adhesion data, which suggest that the prostate tumor cells express less CD47/IAP and the vß3 integrin than the melanoma cell line (Fig. 3)
. Competition binding studies with the 3(IV)187–191 peptide (-YYSNS-) have demonstrated that the amount of peptide required to inhibit melanoma cell adhesion to the 3(IV)185–203 peptide was 
10 times more than what was needed to inhibit prostate tumor cell adhesion (Fig. 3)
, suggesting a higher density of receptor in the former cells. In the same study, a similar observation was made when cells were pretreated with CD47/IAP and the vß3 integrin-reactive antibodies (Fig. 3)
. Although the differences in receptor densities could explain the differences in adhesion between melanoma and prostate tumor cells, are they great enough to explain the observed differences in the effect of the peptide on cell proliferation? Work is presently being performed to answer this question more in depth and will be the subject of a future paper.
CD47/IAP was initially copurified with vß3 from human placenta by Brown et al. (7)
. It has been described as a receptor for the COOH-terminal domain of TSP. The cell binding domain was narrowed down to two peptides, FIRVVMYEGKK and RFYVVMKQVTQS, each having the VVM triplet in common (15)
. Work described in this report indicates that the 7N3 TSP peptide (FIRVVMYEGKK) partially inhibited melanoma cell adhesion to the 3(IV)185–208 peptide and partially inhibited melanoma cell proliferation. Because both the 3(IV)185–203 peptide and the TSP peptide bind CD47/IAP, to explain the differences in activity, it is suggested that both peptides may bind to different locations on CD47/IAP. Because TSP does not bind to the vß3 integrin and the 3(IV)185–203 peptide does, it is possible that binding of the latter to both CD47/IAP and the vß3 integrin is needed for complete activity.
A number of studies suggest that CD47/IAP interacts functionally with vß3 (7
, 20)
. Work by Lindberg et al. (10)
indicates that both CD47/IAP and vß3 are required for optimal vitronectin binding. Our data suggest that both receptors are also needed for optimal adhesion to the 3(IV)185–203 peptide, as well as the inhibition of cell proliferation and the subsequent rise in icAMP (Table 2)
. This was demonstrated by the difference in inhibitory activity of the peptide when used alone and when used after pretreatment of cells with either CD47/IAP- and/or vß3-reactive mAbs. To rule out the possibility that the mAbs are stimulating tumor cell proliferation in the presence of the 3(IV)185–203 peptide, additional studies were performed with receptor-negative cell lines. To answer this question as well as investigate the role of CD47/IAP and vß3, we studied the effects of the peptide on the ovarian tumor cell line OV10-, which is naturally CD47-/vß3+, and on the transfected OV10+ cell line, which is CD47+/vß3+. Our data indicate that the 3(IV)185–203 peptide (10 µg/ml) inhibits cell proliferation of the OV10+ cell line by 47%, compared with 28% with the OV10- cell line. These data further suggest the codependence of CD47/IAP and vß3 in the inhibitory activity of the peptide and discounts the idea that mAbs alone are influencing cell proliferation.
In our studies, several lines of evidence implicate CD47/IAP and the vß3 integrin as specific receptors for the 3(IV) peptides: (a)the peptides have a reasonably high affinity for the CD47/IAP and vß3 integrin receptors, as demonstrated by the fact that receptor purification was performed without receptor-ligand cross-linking, and the proteins were further separated in nonreducing SDS polyacrylamide gels; (b) the biotin-labeled 3(IV)179–208 peptide, pretreated with the peptide-specific mAb (A5D7) before affinity chromatography, failed to bind either CD47/IAP or the vß3 integrin; (c) melanoma cell adhesion to the 3(IV) peptides was partially inhibited when cells were pretreated with the TSP-derived peptide 7N3, which specifically binds CD47/IAP (15)
. However, the binding site of the TSP peptide must be different from that of the 3(IV) peptide because the use of both CD47/IAP and vß3 mAbs did not significantly alter the effect of the TSP peptide on cell proliferation, whereas their use did inhibit the adhesion to the 3(IV) peptide. These data suggest that both peptides are sharing a common receptor; (d) melanoma and prostate tumor cell adhesion to the 3(IV) peptides was inhibited by pretreatment of the cells with the 3(IV)187–191 peptide (-YYSNS-) in a concentration-dependent manner as described in Fig. 3
; (e) treatment of cells with CD47/IAP- or the vß3 integrin-reactive mAbs blocked cell adhesion to the 3(IV)185–203 substrate as well as inhibition of the cell proliferation and the subsequent rise in icAMP (Table 3)
; and (f) melanoma cell adhesion was inhibited by 92% when the substrate composed of the 3(IV)185–203 peptide was pretreated with the peptide-specific A5D7 mAb (5)
.
In conclusion, we have demonstrated that peptides from the NC-1 domain of the 3 chain of type IV collagen bind specifically to the CD47/IAP and vß3 integrin receptors. We have also demonstrated that pretreatment of cells with receptor-reactive mAbs inhibits the antiproliferative effect of the peptide as well as the significant increase in icAMP. Furthermore, by using adhesion and competition binding assays with 3(IV)-derived peptides and receptor-reactive mAbs, we have further demonstrated that the 3(IV)185–203 peptide binds specifically to these receptors. These data imply that basement membranes through COL IV and the 3 chain have a regulatory role on cell proliferation through CD47/IAP and the vß3 integrin.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by Grants AR-20553, HL-29493, and AR07490 from the NIH (to N. A. K.), Grant UPRESA 6021 from the Centre National de la Recherche Scientifique to the University of Reims and Ligue Nationale Contre le Cancer, and a NATO Collaborative Research grant. Peptide synthesis was provided by the Protein Chemistry Laboratory of the Medical School of the University of Pennsylvania, which is supported by core grants of both the Diabetes and Cancer Centers. 
2 To whom requests for reprints should be addressed, at Connective Tissue Research Institute, University of Pennsylvania, 3624 Market Street 5-E, Philadelphia, PA 19104. Phone: (215) 382-7840; Fax: (215) 382-1749; E-mail: kefalide{at}mail.med.upenn.edu
3 The abbreviations used are: COL IV, collagen type IV; ALC, anterior lens capsule; icAMP, intracellular 3',5' cyclic AMP; EHS, Engelbreth-Holm-Swarm; PMN, polymorphonuclear; mAb, monoclonal antibody; IAP, integrin-associated protein; FBS, fetal bovine serum; BrdUrd, 5-bromo-2'-deoxyuridine; TSP, thrombospondin; RIB, receptor isolation buffer; HRPO, horseradish peroxidase.
4 A. Fawzi, C. Perreau, J. C. Monboisse, N. Ohno, F. X. Maquart, N. A. Kefalides, and G. A. Bellon. A peptide of the 3(IV) chain of type IV collagen modulates human neutrophil activity through the IAP/CD47 receptor, submitted for publication.
Received 2/19/99. Accepted 7/16/99.
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) or synthetic peptides or pretreated with receptor-reactive mAbs before the addition of peptides. Cell proliferation was assessed using the BrdUrd incorporation proliferation assay as described in "Materials and Methods." The CD47/IAP mAb was B6H12, and the 
), 
), 
), 
). Results are the mean of triplicate determinations for at least three experiments for each ligand expressed as percentage of inhibition relative to control-stimulated proliferation.
), CD47/IAP-reactive mAb (